Chetan Sharma, Anil K. Sharma, K. R. Aneja-Frontiers in Food Biotechnology (2016)

BIOTECHNOLOGY IN AGRICULTURE, INDUSTRY AND MEDICINE
FRONTIERS IN FOOD BIOTECHNOLOGY
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BIOTECHNOLOGY IN AGRICULTURE, INDUSTRY AND MEDICINE
FRONTIERS IN FOOD BIOTECHNOLOGY
CHETAN SHARMA
ANIL K. SHARMA
AND
K. R. ANEJA
EDITORS
New York
Copyright © 2016 by Nova Science Publishers, Inc.
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CONTENTS
Preface vii
Chapter 1 Lactic Acid Bacteria (LAB) in Food and Fermented Products 1
Namita Rokana and Harsh Panwar
Chapter 2 Probiotics, Prebiotics and Synbiotics: Current Status and
Future Prospects 25
Akshay Joshi, Vikram B. Lanjekar, Prashant K. Dhakephalkar
and Sumit S. Dagar
Chapter 3 Indigenous Fermented Foods and Beverages 47
Niharika Singh, Sampan Attri, Kavita Sharma and Gunjan Goel
Chapter 4 Bacteriocins as Potential Biopreservatives in Foods: An Overview 75
Kamal Rai Aneja, Romika Dhiman, Neeraj Aggarwal
and Ashish Aneja
Chapter 5 Genetically Modified Foods: Current Overview 95
Jatinder Kaur and Priya Katyal
Chapter 6 Biocolours: An Insight into Production, Applications,
Stability and Regulation 115
Sunakshi Rastogi, Chetan Sharma, Anil K Sharma,
Himanshu Aggarwal and Vikas Beniwal
Chapter 7 Food Allergens: Chemistry, Detection and Future Implications
on Human Health 155
Priti Mudgil
Chapter 8 Rapid Methods for the Detection of Food Borne Pathogens 187
Divya Arora, Nisha Sharma, Vishal Sharma, Vidushi Abrol
and Sundeep Jaglan
Chapter 9 Role of Enzymes in Food Industries 219
Gaurav Garg, Nirmala Sehrawat and Mukesh Yadav
Chapter 10 Role of Nanotechnology in Food Processing 253
Sunita Dalal and Vidushi Malhan
vi Contents
Chapter 11 Single Cell Protein: Microbial Production and Analysis 271

Simran Preet Kaur
Chapter 12 Baker‘s and Brewer‘s Yeast: Production, Applications
and Genetic Manipulations 283
Priya Katyal, Parampal Sahota, Gurvinder Singh Kocher
and Jatinder Kaur
Chapter 13 Nutraceuticals: A Potent Therapeutic Agent 311
Sachin Gulati, Anita Yadav and Neeraj Kumar
Chapter 14 Nutritional Genomics: Nutrigenomics and Nutrigenetics 331
Eshu Singhal Sinha, Radhika Mohan and Anu Mehta
Index 343
PREFACE
The application of biotechnology in food sciences has subsequently increased the

production of food and enhanced its quality and safety. Food biotechnology is a dynamic field
and the continual progress and advances in the field has not only dealt effectively with the
issues relating to food security but also augmented the nutritional and health aspects of food.
In the past decade, major breakthroughs have happened and enormous progress has been
made in field of food biotechnology, including improvements in industrial process
technology, farming system for growing and harvesting food, genetic improvements to
organisms used in the food supply, and use of advanced techniques to monitor food safety,
nutritional quality, flavor, texture and their shelf life. Food biotechnology, begins with
exploring the role of microbes in food fermentation, has now progressed to increasing the
shelf life of food and enhancing its flavor. Presently, there has been a shift in the focus of
biotechnological progress to find out new approaches in food fermentation and develop
multifunctional microorganisms to improve the nutritional and health benefits of food. Major
challenges facing the world today are not just those of food production and food quality for
meeting the protein and calorie needs but also those related for better health. So
implementation of any food produced through biotechnology should have to cross the
environmental and ethical issues barrier. Today, it is expected from food biotechnologists that
they satisfy many requirements related to health benefits, sensory properties and possible long
term effects associated with the consumption of food produced by biotechnology. Therefore,
several researchers across the globe are investigating novel, biological, molecular and
biochemical strategies by using cutting-edge technologies and state-of-the-art techniques for
improving the food production and processing, for enhancing the safety and quality of food
ingredients for better human health. The progress in food biotechnology is a ray of hope to
tackle the food security of the over explosive population especially in developing countries.
Thus, with these issues in mind, this book has been assembled with the hope of being an
authoritative, comprehensive, conceptually sound and highly informative compilation of
recent advances in various important areas of food biotechnology. So, in writing this book,
we are honored to have 14 chapters written by eminent authors in their respective fields. The
book begins with a general introduction of lactic acid bacteria along with fermented products
which is followed by the concept of probiotics, prebiotics, synbiotics and indigenous
fermented food and beverages. The concept of bacteriocins, GM foods, biocolors and
different food allergens along with rapid methods for the detection of food borne pathogens
have also been covered in this book. Then we switch our focus to the role of enzymes and
viii Chetan Sharma, Anil K. Sharma and K.R. Aneja
nanotechnology in food industry along with industrial production of Single Cell Proteins
(SCP), bakers and brewing yeast. Lastly, we emphasized upon some upcoming trends in food
biotechnology as nutraceuticals and nutritional genomics. The continued success of the books
published under the banner of esteemed NOVA publishing house is the result of a joint effort
of a dedicated editorial and publishing team and we will continue to evolve progressively for
the benefit of our worthy contributors and readers. While thanking all the contributors, we
reiterate our commitment for ethical and quality work published through this book on food
biotechnology and microbiology. With great pleasure and respect, we extend our sincere
gratitude to all the authors who have put considerable effort into their contributions and for
their timely responses and consistent cooperation. This book is a valuable reference material
for graduate and post graduate students, researchers, scientists and food policy makers. We
anticipate that this book is intended to equip the readers with the basics and applied research
in food biotechnology. The basic aim behind this book is to develop an authentic account of
food biotechnology in the food industry and stimulate research in this area. Unlike past, the
present food industry is profitably deriving benefits from bioengineering. It is intended that
this book addresses various disciplines of food microbiology, food biotechnology and food
engineering.
Finally, we acknowledge the Almighty God and our family members, who provided all
the channels to work in cohesion and coordination right from the conception of the idea to the
development of the final version of this book Frontiers in Food Biotechnology.
Dr. Chetan Sharma

Assistant Professor
Department of Biotechnology
M.M. University, Mullana (Ambala), Haryana
India-133207
Tel.+91-9812287101
E-mail: chetanmicro147@gmail.com
Dr. Anil K. Sharma

Professor and Head
Department of Biotechnology
M.M. University, Mullana (Ambala), Haryana
India-133207
Tel.+91-8059777758
E-mail: anibiotech18@gmail.com
Prof. K.R. Aneja

Formerly, Head, Department of Microbiology
Kurukshetra University, Kurukshetra, Haryana
India-136119
Tel.+91-9466241532
E-mail: anejakr@yahoo.ca
In: Frontiers in Food Biotechnology ISBN: 978-1-63484-671-4
Editors: Chetan Sharma, Anil K. Sharma et al. © 2016 Nova Science Publishers, Inc.
Chapter 1
LACTIC ACID BACTERIA (LAB)

IN FOOD AND FERMENTED PRODUCTS
Namita Rokana1, and Harsh Panwar2

1
School of Life Sciences (SLS), Jawaharlal Nehru University (JNU), New Delhi, India
2
Department of Dairy Microbiology, College of Dairy Science and Technology,
Guru Angad Dev Veterinary and Animal Sciences University (GADVASU),
Ludhiana, Punjab, India
ABSTRACT
Fermented food preparations of milk, cereals, meat and vegetables have been largely
adapted in the human diet since ancient times. Fermentation of raw material provides a
safe way to preserve food without losing its nutritive and organoleptic properties.
Moreover, it rather improves the bioavailability of essential components by increasing the
digestibility and palatability of the raw material. Lactic acid bacteria (LAB) are the
diverse group of phylogeneticaly related lactic acid producing Gram-positive bacteria.
Because of their metabolic specificities, LAB are largely associated with the fermented
foods of diverse range. Besides acid production, LAB supplements beneficial minerals,
vitamins, antimicrobial substances, sweeteners and aromatic components to the food
matrix. In this chapter we summarize the role of LAB in fermented foods popular in
different geographical and ethical communities worldwide. The analysis of functional
LAB culture in fermented foods may be used for the technological and nutraceutical
advancements in the process of product development.
Keywords: lactic acid bacteria (LAB), fermented foods, homofermentation,

heterofermentation

Corresponding author: School of Life Sciences (SLS), Jawaharlal Nehru University (JNU), New Delhi-110067,
India. Email: drnamita.rokana@gmail.com, tel: +91-9521613431.
2 Namita Rokana and Harsh Panwar
INTRODUCTION
Lactic acid bacteria (LAB) have been associated with human life and their food related
habitats since the beginning of time. It is a diverse group of phylogenetically related Gram-
positive genera which produces primarily lactic acid as a product of anaerobic glycolysis. A
clear definition of LAB was given by Orla-Jensen (1919) which describes that the ‗lactic acid
bacteria‘ denoted for a group of Gram-positive, nonmotile, non-sporeforming, rod- and
coccus-shaped organisms that ferment carbohydrates and higher alcohols to form chiefly
lactic acid. Thereby, the term LAB more refers to a biological group than a taxonomical
group of microorganisms. These bacteria predominantly belong to Carnobacterium,
Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus,
Streptococcus, Tetragenoccus, Vagococcus, Weissella genera. The genus Bifidobacterium is
also included with LAB because of their physiological similarity with the group.
The basic defining feature of LAB is metabolism of hexose sugars yielding lactic acid as
a main product. On the basis of metabolic mechanism of lactic acid production, LAB is
divided into two distinct groups. The homo-fermentative group utilizes the Embden-
Meyerhof-Parnas (glycolytic) pathway and produce lactic acid as a major end product. On the
other hand, Hetero-fermentative bacteria produce equimolar amounts of lactate, CO2, ethanol
or acetate by using phosphoketolase pathway. Lactococcus, Pediococcus, Enterococcus,
Streptococcus is homofermentative genera whereas Leuconostoc, Oenococcus and Weisella
are completely heterofermentative groups. A complete description of homo and
heterofermentative LAB groups is given in Table 1.
Table 1. Characteristic of homo and heterofermentative lactic acid bacteria
Characterization Homofermentative LAB Heterofermentative LAB

Products Lactic acid Lactic acid, ethanol, diacetyl, formate,
acetoin or acetic acid, and carbon dioxide
Metabolic pathways Hexose: Embden- Hexose: phosphogluconate and
Meyerhof pathway phosphoketolase pathway
Pentose: pentose phosphate Pentose: phosphoketolase pathway
pathway
Theoretical yield of Hexose: 1.0 g/g Hexose: 0.5 g/g (1.0 mol/mol)
lactic acid to sugars (2.0 mol/mol) Pentose: 0.6 g/g (1.0 mol/mol)
Pentose: 1.0 g/g
(1.67 mol/mol)
Genera Lactococcus, Leuconostoc, Oenococcus, some
Streptococcus, Lactobacillus species
Pediococcus,
Enterococcus, some
Lactobacillus
Adapted from Abdel-Rahman et al., 2013.
Lactic Acid Bacteria (LAB) in Food and Fermented Products 3
Adapted from Modler et al., 1990.
Figure 1. Carbohydrate metabolism and end products in homo, heterofermentation and bifid shunt
metabolic pathways.
Bacteria that follow the homofermentative pathway theoretically produce two molecules
of lactic acid from one molecule of hexose sugar molecule. The terminal electron acceptor in
this pathway is pyruvate which is reduced to lactic acid. In heterofermentative LAB, alternate
pentose monophosphate pathway converts 6-carbon sugars (hexoses) to 5-carbon sugars
(pentoses) and results in two main end products, lactic acid and ethanol or acetic acid and
carbon dioxide as a by-product. These bacteria preferentially produce lactate and ethanol in a
anaerobic environment and lactate and acetate in an aerated environment. Bifidobacteria
metabolise hexose sugar with the help of fructose-6-phosphoketolase enzyme through a
characteristic ―bifid shunt‖ metabolic pathway (Figure 1). The total energy yield in
homofermentative LAB is 2 mol of ATP and 2 mol of lactic acid from 1 mol of glucose and
heterofermentative LAB is 1 mol each of lactic acid, ethanol and ATP per 1 mol of fermented
glucose. Whereas, in comparison to LAB bifidobacteria produce more energy due to ―bifid
shunt.‖ They yield 2.5 ATP, 1.5 mol of acetate and 1 mol of lactate molecules from 1 mol of
fermented glucose (Pokusaeva et al., 2011).
In addition to sugar fermentation, some additional metabolic pathways of LAB which
produce bioactive peptides, bacteriocins and flavour components have also significant role in
the development of different fermented foods (Panwar, 2014). We will discuss these aspects
in different parts of chapter.
FERMENTED FOODS
Fermentation is a process of food preservation that has been used by humans going back
thousands of years. Since the dawn of civilization man has learned to ferment milk, meat and
vegetables. With developing knowledge of fermentation practice, many traditional
methodologies were handed down from generation to generation around the world to handle
and store the raw material in the form of safe and organolaptically improved foods.
Traditionally fermented foods have a diverse range of raw materials as milk, cereals,
vegetables, legumes and meat which indicated the adaptation of fermenter microflora under
diverse environmental conditions. The microbiological bases of fermentation of variety of
substrates in these fermented foods predominantly involve LAB. Descriptions of different
kinds of LAB fermented foods and their constituents are summarized in Table 2.
MICROBIOLOGY OF DIFFERENT TYPES OF FERMENTED PRODUCTS

As illustrated in Table 2, a diverse range of fermented foods is popular in different parts
of word. These large varieties of fermented food products are made by the combination of
different raw material, starter culture and fermentation condition. Starter culture plays a key
role in fermentation process by bringing up desirable changes in food stuff. The activity of
microbial culture starts with conversion of carbohydrates in desired substances such as lactic
acid, acetic acid, alcohol and/or CO2. Some products require fermentation by secondary
fermentation by additional bacteria for addition of flavour and texture. Production of some
additional secondary metabolites contributes to particular aroma and flavour of fermented
foods. Here we will discuss the microbiology and processing of major types of fermented
foods which are popular in different parts of the world.
1. Dairy Products
1.1. Yoghurt
Yogurt is a fermented milk product, similar to Indian curd/dahi. Fat content of yogurt
varies between 0 to 5 per cent and solids between 9 to 20 per cent. The nutritional
composition and consistency of yogurt is affected by many factors including the substrate
material (type of milk); microbes responsible for fermentation; additives (milk solids, solids
non-fat, sweeteners, fruits, flavor, etc.) along with the incubation temperature and time.
Different factors responsible for affecting the yogurt quality are discussed here in brief. Milk,
serving as the substrate/base material for yogurt is the primary factor affecting the
consistency of final product. Varieties of milk viz. whole, skimmed, semi-skimmed,
evaporated or powdered forms can be used for production of yogurt with characteristic
flavour. Microbes‘ being used for yogurt preparation specifically explores symbiotic
relationship between Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) and
Streptococcus salivarius subsp. thermophilus (S. thermophilus). However, beside these, other
additional strains may be added for slight variations in flavour and texture. In few scenarios,
organisms like lactose fermenting yeasts (Torula cremoris), Leuconostoc sp., L. acidophilus
and S. diacetylactis are being included with the traditional strains (Kneifel et al., 1993). High
antimicrobial sensitivity of the dual strains makes it imperative to select raw milk free from
any antimicrobial residues. Yogurt may be supplemented with several additives, such as
fruits, fruit pulp, flavours, etc. for enhancing acceptability of the final product. The market is
now flooded with a vast array of yogurts differing in their textures (e.g., liquid, set, smooth,
stirred), fat contents (e.g., luxury, low-fat, fat-free), flavours (e.g., natural, fruit, cereal), taste
(sweet and savoury) and colours (white, rosemary, etc.).
Table 2. Types of fermented foods and their main constituent
Raw material Products Starter culture

Milk Yoghurt, Cheese, L. bulgaricus, L. helvaticus, L. plantarum, L. lactis subsp.
Beverages lactis, L. lactis subsp. cremoris, L. lactissubsp. lactis var.
diacetylactis L. casei, L. kefir, L. kefiranofacies, S.
thermophilus
Cereals and Sourdough, Miso, L. sanfransiscensis, L. farciminis, L. fermentum, L. brevis,
legumes Mahewu, Idli, L. plantarum, L. amylovorus, L. reuteri, L. pontis, L.
Dosa Oncom panis, L. alimentarius, W. cibaria, Leuconostoc
mesenteroides
Fruits and Sauerkraut, L. plantarum, Leuconostoc mesenteroides, P. acidilactici,
vegetables Kimchi, Pickles L. brevis, L. pentosus, L. fermentum
Cereals/fruits Wine, Beer O. oeni, L. sakei
Meat Sausages Lactobacillus sakei, L. plantarum, L. curvatus, L. casei, L.
leichmanii, L. brevis, L. buchneri, L. gasseri or
Pediococcus pentosaceus, P. acidilactici
Others Coffee, cocoa Lactobacillus plantarum, Lactobacillus fermentum,
beans Leuconostoc mesenteroides, Leuconostoc
pseudomesenteroides, Weissella cibaria
Yogurt has also been proposed as a bio-therapeutic with potential health benefits.
Different varieties of yogurt with suggested health benefits are Live yogurts, which contain
living bacteria having GRAS status; Probiotic yogurts, which contain live micro-organisms,
earlier established as probiotic with bio-therapeutic potential; Bio yogurts, consisting of
Bifidobacteria (B. bifidum) and/or Lactobacillus acidophilus, having a milder, less acidic
flavour.
Preparation of Yogurt
Milk is first pre-heated (60°C), homogenised (2500 psi), heated (85°C/30 min) and
cooled (43°C), followed by addition of required percent (1 percent of each) of fresh starter
culture (S. thermophilus and L. bulgaricus). Heating the milk is one of the crucial steps
responsible for change in the milk proteins, so that they set together instead of curdling and
wheying off. Non fat dry milk powder may be added to cooled milk to increase the firmness
of final product. Milk inoculated with starter cultures is incubated at 41-42°C (105-110°F) for
4-6 hours (Figure 2). The milk proteins coagulate and set to form yogurt at pH around 4.6
(0.75% acidity), with a distinct acetaldehyde flavour. S. thermophilus normally attains acidity
levels of around 0.85 to 0.95 per cent, whereas, L. bulgaricus continues to raise acidity levels
to around 1.5 percent. A final acidity of at least 0.95% is desired in the product. Set yogurt
can be stored at refrigerated conditions for around 2-3 weeks without any considerable loss of
flavour and taste. Depending upon the post-preparation treatments, yogurts of following types
can be made viz. Set yogurt, A solid textural, undisturbed product; Stirred yogurt, set yogurt
broken up with a spoon and dispensed into secondary serving containers; Drinking yogurt,
Stirred yogurt to which additional milk and flavours are added resulting in raised pH. Other
common variations include fruit yogurt and frozen yogurts.
Figure 2. Schematic presentation of the procedure of yogurt preparation.
Given the right conditions, i.e., correct temperature and moisture, the bacteria are able to
ferment the milk sugar (lactose), producing lactic acid. S. thermophilus grows faster than L.
bulgaricus and produces formic acid and carbon-di-oxide, which further stimulates the
growth of L. delbrueckii subsp. bulgaricus. The proteolytic potential of L. delbrueckii subsp.
bulgaricus produces stimulatory peptides and amino acids for use by S. thermophilus.
Streptococci drops the initial pH of yogurt to around 5, followed by pH 4, with the metabolic
activity of Lactobacilli. Rate of acid production is considerably high, when both the strains
are used in symbiotic relationship. The yogurt curdles due to acid production and drop down
in pH, with typical flavour and texture.
1.2. Fermented Milk Beverages
Kefir
Kefir can be explained as a self carbonated, fermented milk product also included in the
beverage family, having lactic acid (1%) and alcohol (1%). It is an attractive, refreshing
fermented product providing nutrition along with long shelf life. Raw cow or goat milk
usually acts as the base for kefir production. Kefir grains, the primary requirement for kefir
holds its roots back in history, with a common belief that the grains were gifted to people
living in Caucasian mountains by the Prophet Mohammed, and hence the grains are often
represented as ‗gift of the gods.‘ Microbiology of kefir grains is inconsistent and harbours
undefined number and species of bacteria and yeasts embedded in a gummy matrix held
together by a polysaccharide gum termed as kefiran-a polymer made up of equal proportions
of glucose and galactose. The common flora identified and having role as starter cultures
involves Acetobacter aceti, Lactobacillus sp. (L. kefiranofaciens, L. kefir, L. brevis, L.
acidophilus, L. helveticus, L. delbreuckii subsp. bulgaricus, L. rhamnosus, L. gasseri, L.

fermentum, etc.); Lactococci (L. lactis, L. cremoris, L. diacetylactis); Geotrichum candidum
(white mould); Torula sporadelbrueckii; Saccharomyces sp. (S. cerevisiae, S. fragilis);
Kluyveromyces sp. (K. marxianus, K. lactis); Candida sp. (C. kefyr, C. tenius, C. holmii, C.
friedrichii); Torulopsis sp., Mycotorula sp., S. thermophilus; Betabacterium caucasicum, kefir
bacilli; lactose fermenting yeasts, etc. (Simova et al., 2002). The microbial content of kefir
grains vary with their source.
Preparation of Kefir
Traditionally kefir is being produced by continuous fermentation using raw cow or goat
milk along with kefir grains for inoculation, as a source of starter culture in a leather sack.
The milk is filled in leather sacks, inoculated with kefir grains and placed in sun during day
time followed by storage at room temperature at night time. The leather sack is normally left
hanging near a door and anyone passing by has to touch the sack for proper and frequent
mixing of components. The final product is regularly taken out from the sack, followed by the
addition of the equivalent amount of fresh milk. The acid, ethanol, CO2 content and flavour
varies with the holding temperature. Commercial cow milk is warmed (70°C), homogenized
(12.5-20 MPa), heated (85 C 1 min or 9 -95 C 2-3 min), cooled (22 C) and inoculated with
kefir grains at a ratio of 1:30 to 1:50 and allowed to incubate for 8-12 hours until final acidity
reaches 1 per cent lactic acid (Figure 3). The coagulum is agitated followed by slow cooling
for 10-12 hours. Before packaging, the product is agitated again, filled in containers and
ripened in cold storage. Slow cooling of the coagulum is desirable for allowing development
of required flavour and aroma. During fermentation, kefir grains rise to coagulum surface due
to CO2 production, and are collected for fresh batch inoculation.
Figure 3. Flow diagram of kefir preparation.

Koumiss
Koumiss is an effervescent acidic, alcoholic fermented bio-therapeutic product made
primarily from mare‘s milk. The name Koumiss has been derived from Kumane tribe, who
inhabited along the river Kumane in the Asiatic steppes. Mare‘s milk does not curdle at the
iso-electric point of casein and hence is included in beverage family. Mare‘s milk or cow‘s
milk or skim milk with added sucrose may be exploited for Koumiss production (Yadav et al.,
1993).
Traditionally, Koumiss was prepared from raw mare‘s milk, which was allowed to
ferment in bags made up of smoked horse hide or lamb skin. Similar to kefir, koumiss
production involves continuous fermentation. As koumiss is consumed, fresh milk is added to
the bags. Microorganisms involved in fermentation are not well defined but comprises mainly
of thermophilic lactobacilli (L. delbrueckii subsp. bulgaricus); L. acidophilus; lactose
fermenting (S. lactis; Torula koumiss), non lactose fermenting (S. cartilaginosus) yeasts and
non-carbohydrate fermenting (Mycoderma sp.) yeasts (Law, 1997).
The major end products in koumiss are lactose (2.3%), fat (1.5%), proteins (2%), lactic
acid (0.7-1.8%), ethanol (1.3%) and carbon-di-oxide (0.5-0.88%) (Yadav et al., 1993a). On
the basis of acid and alcohol content, koumiss has been represented as Low acidic and Low
alcoholic (0.6% lactic acid and 0.7% alcohol); medium acidic and medium alcoholic (0.8%
lactic acid and 1.1-1.7% alcohol); and high acidic and high alcoholic (1% lactic acid and 1.7-
2.5%) koumiss.
For commercial preparation, mare‘s or cow‘s milk is heated (9 C 5 min), cooled (26-
28°C) followed by inoculation with 3.0% starter consisting of 1 part of L. bulgaricus (37°C
for 7 hours) plus 2 parts of Torula spp. (30°C for 15 hours). The mixture is incubated at 28°C
until acidity reaches 0.70.8%. After desired fermentation, product is agitated after every 1- 2
hours followed by cooling (20°C), packaging and refrigerated storage for 24 hours (Figure 4).
Figure 4. A general diagram of procedure of koumis preparation.

1.3. Cheese
The art of cheese making was learned by human beings about 8000 years ago. The
accidental innovation of cheese production was probably made by wandering nomads, who
left some raw milk in a pouch made by the stomach of a domestic animal. Now, more than
1000 cheese varieties are popular among different regions of the world. The general
processing of cheese making involves curdling of milk by enzymatic or acid fermentation
method, whey expulsion followed by maturation (Figure 5). pH, salt concentration, moisture
content and presence of antimicrobial components of starter and nonstarter bacteria are the
factors that influence the extended shelf life of the cheese. That‘s why most of the ripened
and unripened soft cheese are consumed fresh whereas, some of hard cheese (e.g., Parmesan)
can be kept for more than two years for maturation. Thereby, variation in processing and
ripening conditions are the key factors impelling the distinctive texture, aroma and taste of the
different cheese varieties.
The very first stage of cheese making is coagulation of milk by rennet or culturing of acid
producer bacterial culture. Rennet is a natural complex of chymosin, pepsin and lipase
enzyme. Enzyme chymosin specifically hydrolyzes the peptide link between Phe105-Met106of
κ-casein (Delfour et al., 1965). The cleavage causes destabilization of casein micelles by
releasing hydrophilic C-terminal peptides into the whey. Remaining N terminal parts of κ-
casein aggregates and form the cheese curd. However, in acid coagulated cheese curd, certain
species of LAB produce lactic acid from lactose and cause acid coagulation of casein protein
around 3 to 4 pH range. Commonly Lactococcus lactis, Streptococcus thermophilus,
Leuconostoc spp., Lactobacillus delbrueckii subsp. bulgaricus and L. helveticus are the
associated primary LAB and thereby called as starter culture of cheese (Table 3).
Figure 5. Schematic diagram of cheese preparation and ripening.

Table 3. Different types of cheese varieties and associated microflora
Category Cheese Starter cultures Nonstarter lactic Other

acid bacteria nonstarter
(NSLAB) organisms
Very hard/ Parmesan L. lactis, L. L.bulgaricus -
Hard cremoris, S.
thermophilus
Cheddar L. cremoris, L. L.diacetylactis, L. -
lactis casei, L. plantarum
Hard Emmental, L. lactis, S. Propionibacterium -
(with eyes) Gruyere, thermophilus, shermanii,
Maasdam L. helveticus Propionibacterium
freudenreichi
Semi hard Brick L. lactis, L. - Brevibacterium
cremoris, S. linens
thermophilus
Limburger L. lactis, L. - Brevibacterium
cremoris linens
Semi hard Edam, Gouda L. lactis Leuconostoc -
(with eyes) cremoris, L.
diacetylactis
Soft, ripened Roquefort, Stilton L. lactis - Penicillium
(internal mould) roqueforti
Camembert L. cremoris, L. Leuconostoc Penicillium
(surface mould) lactis cremoris camemberti
Brie (surface L. cremoris, L. Leuconostoc Penicillium
mould) lactis cremoris camemberti,
Geotrichum
candidum
Soft/ Cottage L. cremoris, L. - -
Unripened lactis,
Leuconostoc
cremoris
Mozzarella S. - -
thermophilus,
L. bulgaricus
Cream L. cremoris, L.
diacetylactis, S.
thermophilus,
L. bulgaricus
L. lactis: Lactococcu slactis subsp lactis, L. diacetylactis: Lactococcus lactis subsp diacetylactis, L.
cremoris: Lactococcus lactis subsp cremoris, Leuconostoc cremoris: Leuconostoc mesenteroides
subsp cremoris, L. bulgaricus: Lactobacillus delbrueckii subsp bulgaricus.
Cheese curd is subjected to a ripening process ranging from few days to a year in most of
the cheese varieties. During this period, some advantageous contaminants grow at internal or
external surface of cheese blocks. These organisms are called as secondary culture or
nonstarter organisms (NSOs). The main NSOs are Leuconostoc spp., Lactococcus lactis
subsp diacetylactis, Lactobacillus casei, L. plantarum, L. helveticus, Propionibacterium
freudenreichii, P. shermanii, Brevibacterium linens, Penicillium roqueforti and P.
camemberti, Geotrichum candidum. These cultures do not contribute in acid production but
play a major role in development of organoleptic properties of the cheese variety.
Secondary culture involves in a complex series of biochemical reactions including citrate
metabolism, proteolysis, lipolysis and catabolism of fatty acids and amino acids. For
example, surface or internally grown molds contribute in development of aroma by their good
proteolytic and lipolytic activity. Whereas, the nonstarter LAB (NSLAB) grow internally in
most of the cheese varieties and are dominated by mesophilic, heterofermentative LAB
(Beresford et al., 2001). Few NSLAB such as Luconostoc and Lactococcus spp. metabolize
citric acid in fresh cheese and enhance the flavour intensity by the production of diacetyl, CO2
and other small molecules. Moreover, L. plantarum and L. paracasei strains have glutamate
dehydrogenase activity which ends up with the production of aroma compound (Tanous et al.,
2002). Also, Propionibacteria are associated with swiss type of cheese and form
characteristic eyes into cheese by the production of CO2 and from lactate metabolism. Hence,
we can say that the composition of secondary culture is the core element of cheese
preparation which could convert a rather similar kind of curd into a cheese variety of unique
texture and flavour.
2. Cereal and Legume Based Fermented Foods
Among all food fermentations, cereals form the highest mass and most divert range of
fermented foods in different parts of world. The worldwide presence of countless types of
cereal-based fermented foods shows the concern of human population for the nutritive and
preservative values of these products. A large proportion of traditional fermented preparation
is produced by blending of cereals and legumes. Blending and fermentation of these two
types of grains in the diet fulfil the deficiency of sulphur containing amino acids of legumes
as well as lysine deficiency of cereals; thereby, improve the overall nutritive value of these
foods. The fermentation of cereal-based foods is performed by natural flora in most of the
traditional processing, which includes LAB (Leuconostoc, Lactobacillus, Streptococcus,
Pediococcus), Micrococcus, Bacillus, yeast and some fungal species of Aspergillus,
Trichothecium, Paecilomyces, Cladosporium, Fusarium and Penicillium (Blandino et al.,
2003). Here we have listed the series of most popular cereal and legume based fermented
food and their predominant microflora in Table 4.
Fermentation of grains starts with soaking in water which initially activates endogenous
cereal specific enzymes (i.e., α and β- amylase) followed by competitive growth of starter
culture. As the raw material is used without pasteurization, a significant number of Gram-
positive spore formers, micrococci and moulds are naturally present in base material.
Thereby, addition of salt and other additives and maintenance of specific temperature range
(25-30°C) are crucial for the advantageous growth of fermentative starter culture which is
generally dominated with LAB. After generation of fermentable sugar by endogenous
amylase activity, starter flora rapidly lowers the pH to 4.0 and inhibits the growth of spoilage
organisms by production organic acid. The antimicrobial activity further strengthens by
accumulation of free fatty acids, H2O2 and small peptides like bacteriocins. Composition of
developed starter culture relates with texture, flavour, nutrients and inhibition of undesirable
components in a particular food. Starter culture may bring these changes either by acting in a
parallel or in a sequential manner during fermentation and ripening period. Figure 6 shows a
general mechanism of fermentation process of cereal and legume based foods.
Table 4. Worldwide popular traditional cereal/legume based fermented foods
Products Substrates Starter culture Country

Adai, Dosa, Cereal/legume Pediococcus, Streptococcus, Leuconostoc, India
Idli, Dhokla Torulopsis candida, Tricholsporon
pullulans
Rye bread Rye flour L. plantarum, L. bulgaricus, S. Europe, Rossia
thermophilus, Leuconostoc mesenteroides
Soy Sauce Soybeans, wheat Aspergillus oryzae, L. delbrueckii, US
Pediococcus halophilus, Saccharomyces
rouxii, Streptococcus spp.
Boza Wheat, millet, Lactobacillus, Saccharomyces cerevisiae, Albania,
maise and other Leuconostoc Turkey,
cereals Bulgaria,
Romania
Busa Rice or millet Lactobacillus, Saccharomyces Syria, Egypt,
Turkestan
Hamanatto, Wheat and Aspergillus oryzae, Streptococcus, Japan
Soybean milk soybeans Pediococcus
Kecap Wheat, soybeans Aspergillus oryzae, Lactobacillus, Indonesia
Hansenula, Saccharomyces
Khanomjeen Rice Lactobacillus, Streptococcus Thailand
Kishk Wheat and milk L. plantarum, L. brevis, L. casei, Bacillus Egypt, Syria,
subtilis and yeasts Arabian
Mahewu, Maise LAB, yeast South Africa
Mawe
Miso Rice and soy Aspergillus oryzae, Torulopsis etchellsii, Japan, China
beans or other Lactobacillus
cereals
Puto Rice, sugar Leuconostoc mesenteroides, Strepromyces Philippines
faecalis, yeasts
Sourdough Wheat and rye L. sanfransiscensis, L. farciminis, L. Egypt, Europe,
flour fermentum, L. brevis, L. plantarum, L. US
amylovorus, L. reuteri, L. pontis, L. panis,
L. alimentarius, W. cibaria
Uji, Ogi Maise. Leuconostoc mesenteriodes, Lactobacillus Kenia, Uganda,
Sorghum, millet platarum Saccharomyces cerevisiae, Tanganyika,
Candida mycoderma, Corynebacterium, West Africa
Aerobacter, Rhodotorula, Cephalosporium,
Fusarium, Aspergillus and Penicillium
Collected from Blandino et al., 2003; Leroy and De Vuyst, 2004.
Figure 6. A general processing of cereals and legumes during fermentation.
Cereal and legume grains are a good source of dietary proteins, carbohydrates, vitamins,
trace minerals and dietary fibres. However, some components of plant origin i.e.,
polyphenols, phytic acid and tenins also found in these food bases which have an anti-
nutritive value in a food product. The fermentation of cereal-based food improves the
nutritive value by fermentation of carbohydrates, poly and oligosaccharides, production of
volatile fatty acids such as diacetyl and butyrate, synthesis of certain aminoacids and
vitamins. LAB also increases the bioavailability of iron, zinc, magnesium by degrading tennin
and phytate at lower pH condition (Svanberg et al., 1993).
As illustrated in Figure 7, fermentation also improves the texture, taste and flavour of
products by production of organic acids, exopolysccharide, CO2, volatile and non-volatile
small molecules. A particular umami taste of cereal foods is develops due to formation of
predominant amino acid glutamine (Drake, 2007). Some LAB species contain glutaminase
enzyme to produce glutamate from glutamine which creates a complex blend of flavour to
cereal fermented foods.
Thereby, in comparison to other commercialized fermented food products, there is a
range of traditional cereal-based foods that have not completely characterized yet. Nowadays,
when these locally popular foods are also being introduced in food industry, knowledge of
strain selectivity and their relation with food environment is required for the uniform and
optimal process designing of these products. Widespread research is going on to develop the
technology to enhance the safety, sensory quality and health properties of fermented foods
which will remain the main focus area of the food industry in the future.
Figure 7. Biochemical Changes in Cereal-based Fermented Foods.
3. Fermented Vegetables
Fermented vegetables and fruits have been part of human food for about two thousands
years. Earliest evidences for use of fermented vegetables are found in Asia and Egypt and the
technology was then conveyed to Europe and other parts of the new world. Several regional
vegetables and fruits such as cabbage, turnips, radishes, carrots, olives, green mango, lemon
and many others are used for the production of appetizers, pickle or fermented dried
ingredient for other food preparations. The technique of fermentation was simply included
dry salting or brining to inhibit the growth of unwanted spoilage microorganisms and
subsequent storage of vegetables in earthen pot at particular temperature to promote the
growth of predominant flora of LAB. It has been passed on for generations to enhance
preservation, flavour, texture and nutritive value of fruits and vegetables. For example,
sauerkraut, kimichi, Sinki and Gundruk serves a rich source of minerals and vitamin A, C and
B (Cheigh et al., 1994; Nehal, 2013). Likewise, Pulque (a fermented plant sap) is rich source
of thiamine, niacin and riboflavin after fermentation (Steinkraus, 1992). A wide variety of
traditional fermented vegetables are popular in different parts of world which have provided
food security and nutrition to the regional communities (Table 5).
Despite advancements in sophisticated technology in equipment and starter culture in
other types of fermented food preparations, fermentation of vegetables still relay on
contaminating LAB flora of vegetables or equipment surfaces to bring desired changes in raw
material. Surface of fresh vegetables is contaminated with species of aerobic spore formers,
molds, yeasts, Pseudomonas, Enterobactereaceae and very low number of LAB (Heard,
2002). The salt concentration and incubation conditions create the advantageous environment
for the growth of natural LAB flora. Commonly associated LABs with fruits and vegetable
fermentations are homofermentative Lactobacillus species i.e., Lactobacillus delbrueckii, L.
leichmannii, L. plantarum, L. lactis, L. acidophilus and heterofermentative L. brevis, L.
fermentum and L. buchneri, some Gram-positive cocci such as Streptococcus thermophilus, S.
bovis, S. faecalis, Leuconostoc mesenteroides, L. dextranicum, L. paramesenteroides, L.
oenos, Pediococcus cerevisiae, P. acidilactici and P. pentosaceus (Breidt et al., 2007).
The fermentation of vegetables most likely based on homo or hetero-fermentation of
sugars into lactic acid, acetic acid, ethanol, diacetyl, acetaldehyde, mannitol and other volatile
flavour compounds. Other metabolites are essential for the development of flavour and taste
in finished product. CO2 produced by heterofermentative LAB also contributes in
organoleptic properties of these products.
4. Fermented Meat
Preservation of meat by fermentation is done in different parts of world. Several types of

cooked or uncooked base material such as beef, lamb, pork, thyme, hem, and horse are used
for processing to achieve unique properties like palatability, safety, tenderness, colour and
flavour which vary from place to place depending on divergence in processing methods and
time. These fermented products are available in dry or semidry conditions and known with as
diverse names as their processing methods i.e., salami, sausages, saucisse, pepperoni,
ardenner, ole, arles, chorizo, spaeipylsa, nham or metwurst.
Table 5. Worldwide popular fermented vegetables
Product Vegetable/Fruit Other ingredients Place

Sauerkraut Cabbage Salt Europe, US
Kimchi Cabbage Salt, spices Korea
Pickles or Achar Olives, lemon, cabbage, radish, Salt, spices Asia, Africa
carrot, mango, etc.
Khalpi Cucumber Brine Nepal
Pak-Gard-Dong Mustard leaf Salt, sugar Thailand
Tempoyak Durian fruit (Duriozibethinus) Salt Malaysia
Lamounmakbous Lemon Salt, oil, spices Asia, north
and Msir Africa
Nukamiso-zuke Vegetables Rice bran, salt Japan
Gundruk Mustard, radish and cauliflower - Nepal
leaves
Kocho False banana - Ethiopia
(Enseteventricosum)
Sinki Radish - India, Nepal,
Bhutan
Pulque Plant sap - Maxico
Tempe-bongrek Peanut and coconut press-cake - Indonesia
Collected from Battcock and Azam-Ali, 1998.
The processing of meat briefly starts with cooked/uncooked raw meat, then addition of
curing agents and starter followed by smoking, chopping and stuffing in 2-1/2 inch diameter
synthetic or animal intestine casing for fermentation and ripening at particular temperature for
few days. At the initial stage of ripening predominant bacteria are aerobic Gram-negative
psychrotrophic Pseudomonas and Enterobacteriaceae bacteria followed by very small
number of LAB and some Gram-positive. During ripening when oxygen is exhausted, growth
of LAB and other Gram-positive bacteria is encouraged (Hui et al., 2014). Processing and
ripening conditions can influence the rate of acid production and also the ultimate pH in
traditional products. The type of microorganism will also influence fermentation and the final
flavour. Time, temperature, humidity, and smoke are also variables that control the quality of
the final product. Nowadays, for uniform commercial production several homo or hetero
species starters of Lactobacillus sakei, L. plantarum, L. curvatus, L. casei, L. leichmanii, L.
brevis, L. buchneri, L. gasseri or Pediococcus pentosaceus, P. acidilacticiare used at
industrial scale.
Both natural and controlled fermentation involves a significant contribution of LAB.
Fermentation caused by LAB reduces the pH and brings several biochemical and textural
changes in meat. LAB lowers the pH to about 4.8-5.0 which is near to isoelectric point of
small solublized proteins allowing coagulation and release of water from product. Reduced
water activity further lowered by addition of salt and drying process and improve firmness
and slice ability of sausages. Table 6 demonstrates the major characteristic of three types of
fermented meat products.
Table 6. Basic characteristics of three major types of fermented meats
Types of fermented sausage Characteristics

Dry; long ripening, e.g., dry or Chopped and ground meat
hard salami, saucission, Commercial starter culture or back inoculum
pepperoni Fermentation temperature 15-35ºC (59-95ºF) for 1-5 days
Not smoked or lightly smoked
bacterial action reduces pH to 4.7-5.3 (0.5-1.0% lactic acid, total acidity 1.3%
which facilitates drying by denaturing protein resulting in a firm texture,
moisture protein ratio <2.3:1, moisture loss 25-50%, Moisture level <35%
processing time 12-14 weeks
Dried to remove 20-50% of moisture; contains 20-45% moisture, fat 39%,
protein 21%, salt 4.2%, aw 0.85-0.86, yield 64%
Moisture protein ratio no greater than 2.3 to 1.0
Less tangy taste than semi dry
Semi dry; sliceable, e.g., Chopped or ground meat
summer sausage, holsteiner, Bacterial action reduces pH to 4.7-5.3 (lactic acid 0.5-1.3%, total acidity 1%),
cervelat (zervelat), thringer, processing time 1-4 weeks
chorizos Dried to remove 8-30% of moisture by heat; contains 30-50% moisture, 24%
fat, 21% protein, 3.5%, salt, aw 0.92-0.94, yield 90%
Usually packaged after fermentation/heating
Generally smoked during fermentation
Moisture protein ratio no greater than 2.3-3.7 to 1.0
Moist; undried; spreadable Contains 34-60% moisture, production time 3-5 days
e.g., teewurst, mettwurst, Weight loss ~ 10%, aw 0.95-0.96
braunschweig, frische Usually smoked
Highly perishable, refrigerate, consume in 1-2 days
Adapted from Ockerman and Basu, 2007.
Collected from Ockerman and Basu, 2007 and Vignolo et al., 2010.
Figure 8. Flow diagram of processing of meat for dry and semidry sausages production.
As illustrated in Figure 8, the rate and time extent of the processing steps and quality,
quantity and types of added material influence the particular taste and flavour of different
types of sausages. During the fermentation process predominant flora of non-coagulating
Micrococcus sp. contributes in development of anaerobic condition and flavour development
by their lipolytic activity. Lipolytic enzymes attack triglycerides and break down in to
glycerol and fatty acids and sequentially into volatile fatty acids, saturated and unsaturated
aldehydes of a distinct aroma. Some by products like alcohols are responsible for a specific
fruity flavour in the final product. Many non-volatile peptides and amino acids formed during
latter LAB fermentation period also contributes in an overall characteristic flavour and
specific tangy taste of sausages. Thereby, the sensory characteristic of final product depends
upon the combined activities of undefined raw meat flora and defined starter culture during
the fermentation process.
As summarized in Figure 9, the biochemistry of meat processing involves two main
reorganization stages. The first stage is curing of meat in which additives are introduced into
raw meat. Sodium chloride is the major additive that will allow lactic acid bacteria to grow
and will inhibit several unwanted microorganisms. Added sugars during this stage also
influence the growth of LAB at the next step. Nitrite is added for antibacterial, colour, and
antioxidant purposes. The stable red colour (nitrosomyoglobin) of sausages is caused by
nitrosylation and the sequential denaturation of myoglobin at first stage. Nitrite undergoes
chemical reductions to nitrous acid (NO) at pH not less than 5.4 to 5.5. Produced NO binds to
meat myoglobin to form the heat stable, red coloured NO- myoglobin (Honikel, 2008). Some
additional additives like ascorbic acid, spices, vegetable oil basically add flavour in end
product as well as serves as donor of Mn for the promotion of growth of LAB in latter stage.
Figure 9. Flow diagram of biochemical changes during meat processing.
The second stage of biochemical changes includes fermentation and ripening step of meat
processing. In this stage, facultative anaerobic bacteria metabolize sugars by glycolysis
(Embden-Meyerhof-Parnas pathway) or heterofermentative 6-phosphogluconate/
phosphoketolase pathway and produce predominantly lactic acid followed by acetic acid,
alcohol, CO2. The overall lowering of pH by these bacteria results in uncharged undissociated
state of the weak acids which acts as antimicrobial compounds for Gram-negative pathogens.
Lipolytic and proteolytic activities of bacteria during the fermentation process also release
several volatile and non-volatile flavouring components. Thereby, LAB not only strongly
influences the physicochemical, sensory and textural composition of fermented meat foods,
but bacteriocins and organic acids produced by LAB also have an important role in long time
preservation of prepared products.
5. Wine
Wine is among the oldest popular fermented product in human civilization. The
production of wine starts with extraction of several kinds of fruits juice such as grape, berries,
apple, citrus, etc. The raw material of crushed fruits is an adequate source of sugars for the
initial yeast derived fermentation process. In fact, the process of wine making includes two
phases of fermentation two distinct type of microflora. The initial phase starts with
indigenous or inoculated yeast microflora i.e., S. cerevisiae, Kloeckera apiculata, Candida
stellata, Metschnikowia pulcherrimaandIssatchenkia terricola (Granchi et al., 1999) which
spontaneously ferment sugars into ethanol, CO2 and some by products. Accumulation CO2
creates an anaerobic environment which selectively supports the growth of anaerobic
organisms. Moreover, ethanol and organic acids also restricts the growth of lactic acid
bacteriain second phase of fermentation. The phase called as malolactic fermentation (MLF)
in which malic acid, a tart tasting dicarboxylic acid present in grape must, is converted to
softer tasting lactic acid by few species of LAB i.e., Lactobacillus, Pediococcus and
Oenococcus particularly by Oenococcusoeni, (Wibowo et al.,1985). Figure 10 has elucidated
the schematic diagram of the procedure of most popular grape wine preparation.
Yeasts are facultative anaerobic organisms and thereby use both tricarboxylic acid (TCA)
and the Embden-Meyerhoff (EM) pathway for the glycolysis of the sugars. Under aerobic
conditions, the pyruvate is decarboxylated by pyruvate dehydrogenase and the product, acetyl
CoA, feeds directly into the TCA cycle (Hutkins, 2008). In contrast, during anaerobic
metabolism, the enzymes of the TCA cycle are not expressed because the absence of oxygen
causes repression of the genes that code for those enzymes hence, glycolysed by EM
pathway. Additionally, yeasts express pyruvate decarboxylase and alcohol dehydrogenase
enzymes which produce ethanol as an end product (Figure 11).
Besides, MLF by lactic acid bacteria occurs at the end of yeast fermentation. The reaction
is catalysed by malolactic enzyme that decarboxylates malic acid and forms a less acidic
lactic acid (Liu, 2002). In addition to ethanol and lactic acid, very less amounts of citrate,
polyols, aldehydes and phenolic acids are also produced. These components have a significant
impact on the taste and aroma of wine.
Figure 10. Flow diagram of wine manufacture.

Figure 11. Carbohydrate metabolism and ethanol production by yeast.
BENEFITS OF FERMENTATION
In addition to extended shelf-life, fermented foods have specific advantages of improved
taste, texture, flavour, aroma and nutritive values. By virtue of their metabolic activities,
fermenter microorganisms bring several changes in visual appearance and biochemical
composition of food which contributes in the development of palatability and nourishing
properties of raw material. Here we will discuss some important beneficial aspects of food
fermentation by LAB.
1. Improvement of Nutrition Value
Fermented foods have been termed as nutraceuticals due to beneficial action of these
formulations in supply of additional essential nutrients. Here we present several examples
where LAB has enhanced certain nutraceuticals during the fermentation process. LAB such as
Leuconostoc mesenteroides, L. plantarum, L. lactis and Propionibacterium are known for the
production of low calorie sugars like mannitol, sorbitol, tagatose and trehalose via their usual
hetero fermentative pathway. Some vitamins such as riboflavin (B2), folate (B11) and
cobalamin (B12) are essential in the human diet. Although, these vitamins are naturally found
in poultry products, milk, fish and green leafy vegetables, legume and cereals, certain LAB
resynthesizes these components and enhances the bioavailability of essential vitamins in
fermented foods. For example, Streptococcus thermophilus and Propionibacterium are have
the advantage of production of food grade vitamin B11 and B12 during fermentation.
Besides, important vitamin riboflavin exists in milk in the form of co-enzyme. Fermentation
with two LAB strains L. lactis and Propionibacterium freudenreichii ssp. shermanii has been
reported to increase the bioavailability of riboflavin by the production of vitamin in free form.
LAB also used for the bioconversion of certain food components into beneficial
nutraceuticals. Anaerobic bacteria Propionibacterium freudenreichii are reported to convert
linoleic acid into conjugated linoleic acid (CLA) which has beneficial physiological effect.
2. Improvement of Health
Nowadays, the two main genera of LAB Lactobacillus and Bifidobacteria are used as
probiotics to improve the generalized health of human population. Probiotics can improve
intestinal health, innate and adaptive immunity of host with the help of their tolerance
mechanism. Fermented foods are proven to be the most suitable carrier matrix for the delivery
of probiotics in human population as they improve the functionality of probiotics during the
fermentation process (Vinderola et al., 2011). Several evidences suggest the involvement of
fermented foods of different probiotic strains in the enhancement of immunity and reduced
disease burden in the context of leading to a healthy life (Dongarrà et al., 2013; Pot et al.,
2013). LAB also protects our health by degradation or masking of some toxic of harmful
substances in our food. For e.g., these bacteria can breakdown lactose by β-galactosidase
enzyme during fermentation process which produces a more digestible food than milk to the
lactose intolerance population. Some LAB also capable to degradation of some phytic acid
and phenolic compounds in plant origin foods.
3. Preservation and Enhancement of Safety
Fermentation practices for food preservation have advantages over drying and salting
methods due to their sensory attributes. Accumulation of organic acids and alcohols with
reduction of sugar contents preserves fermented food in a general means. Some specific
products such as bacteriocins enhance the preservation effects of fermented foods. A large
number of bacteriocins are produced by lactic acid bacteria and share a common mode of
action in their ability to form pores in the membrane of the target bacteria. An important
bacteriocin, Nisin is produced by strains of L. lactis. It was the first bacteriocin discovered in
LAB and has been used as food grade preservative in several processed foods. An overall
combined effect of different biological and biochemical factors such as lowered pH, depletion
of oxygen as well as accumulation of H2O2 and bacteriocins develops a hurdle system in
fermented foods. Thereby, these food grade bacteria serve a safeguard against pathogens and
spoilage organisms without any nutritive or health risk for the human population.
4. Improvement of Flavour
Technical aspect of starter LAB in the fermented food industry includes the ability of
bacteria to produce desirable sensory qualities in fermented products. Lactococcus lactis
subsp. lactis biovar diacetylactis (L. diacetylactis), and some species belonging to
Leuconostoc and Weissella produce diacetyl by citrate metabolism which gives a pleasant
flavour to dairy products. L. helvaticus produces several small peptides during cheese
ripening period due to its proteolytic activity which results in a particular flavour of aged
cheese. However, lactic fermentation itself is associated with enhanced flavour of cereals and
vegetable products. In the wine industry, combined metabolic activities of LAB and yeasts
improve the flavour during the aging process.
CONCLUSION
Several examples have presented the significance of LAB in diversification of fermented
foods. The benefits gained from LAB supports their greater integration in fermented foods
with increased nutritional values. However, despite the greater contribution in fermented
foods, different strains of LAB have also illustrated their impact on human health and well-
being through probiotic mechanism. Thereby, being an integral part of the human diet, lactic
acid bacteria and their functional food formulations offers a worthy supplement in regular
consumption.
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Chapter 2
PROBIOTICS, PREBIOTICS AND SYNBIOTICS:

CURRENT STATUS AND FUTURE PROSPECTS
Akshay Joshi, Vikram B. Lanjekar, Prashant K. Dhakephalkar

and Sumit S. Dagar
Bioenergy Group, Agharkar Research Institute, Pune, Maharashtra, India
ABSTRACT
Changing lifestyles, dietary habits and environmental conditions have led to an
increase in diseases like diabetes, obesity, inflammatory intestinal diseases, cancer,
cardiovascular disease, osteoporosis, etc. Diet plays a crucial role in maintaining health
and preventing onset of diseases. Probiotic products have enormous potential to benefit
wide spectrum of consumers. Therefore, healthy food products supplemented with
beneficial microbes and/or other compounds are becoming very popular among health
conscious population. These beneficial microbes that either by themselves or through
their products confers health benefits to host are termed probiotics, while prebiotics
arenon-digestible food ingredients that reaches large intestine undigested and beneficially
affects the host by selectively stimulating the growth and/or activities of probiotics/gut
microbes. The synergistic combinations of these probiotics and prebiotics are called
synbiotics. Numerous researchers have validated the suggested health benefits, and
several national and international food companies have come up with various probiotic
and prebiotic products. However, further efforts needs to be invested for enhancing
outreach of probiotic products through cost reduction, development of next generation
functional food products and spreading awareness among consumers.
Keywords: bacteria, health, gut, dairy, functional food, disease, diet

Corresponding author: Bioenergy Group, Agharkar Research Institute, Gopal Ganesh Agarkar Road, Pune,
411004, India. Tel: + 91-20-25325120; Fax: + 91-20-25651542. E-mail: dagarsumit@gmail.com;
ssdagar@aripune.org.
26 Akshay Joshi, Vikram B. Lanjekar, Prashant K. Dhakephalkar et al.
PROBIOTICS
In the Old Testament (Genesis 18:8), fermented milk was recorded as only source of
living organisms. The culture of consumption of fermented milk is followed until the present
day. However, the concept of probiotics was discovered by Elie Metchnikoff at the beginning
of 20th century. He observed that regular consumption of large amount of fermented dairy
products was associated with the longevity of Bulgarians peasants. Subsequently, he linked
this to ‗Bulgarian bacillus‘ named as a Bacillus bulgaricus, now known as ‗Lactobacillus
bulgaricus’ (Fuller, 1992). He believed that microbes present in intestine produce toxic
compounds, hence, these bad microbes should be replaced by useful microbes like lactic acid
bacteria. A French paediatrician named Henry Tissier also observed abundance of Y shaped
bacteria (bifid) in healthy children in comparison to children with diarrhoea (Anukam and
Reid, 2007). He also suggested administration of these bacteria to help restore a healthy gut
flora in diarrhoea patients. Since then, a number of researchers have worked on probiotics and
their associated health benefits. The term ‗probiotics‘ was first used by Lilly and Stillwell
(1965) to describe substances secreted by one organism which stimulate the growth of
another. However, various researchers have changed this definition over the period.
Currently, probiotics are defined as ―live micro-organisms,‖ which, when administered in
adequate amounts confers a health benefit on the host (Ducatelle et al., 2015).
Probiotic Microbes, Their Selection Criterion and Mode of Action
It is well documented that the large intestine is the most densely populated ecosystem in
nature consisting of 500 to 1000 different species of bacteria, of which several strains
belonging to different genera of Bifidiobacterium and Lactobacillus are most commonly used
as a probiotics (Jain et al., 2014). However, several other bacteria and few yeasts are also
known to have probiotic activities. The details of different probiotic bacteria, their
antimicrobial compounds and commercially available strains are given in Table 1.
Selection of the desired strain is one of the most critical parameters to ensure the best
performance of a probiotic. Since, the probiotics are ―live‖ microbes several criteria have
been established that must be followed before their use (Figure 1). The probiotics must be
able to survive (competitive nature, pH and bile tolerance), colonize (adherence to intestinal
mucosa) and grow in the gastrointestinal tract. They should possess beneficial properties like
production of antimicrobial compounds, immunomodulation, anti-genotoxic, anti-
inflammatory, anti-carcinogenic activities, etc. In addition, the probiotics must have good
technological properties like the ability to grow in milk or other food bases for mass
production, good sensory properties, stability of desired attributes, phage resistance,
accessible for genetic modification and better viability. The safety aspects include ―generally
regarded as safe‖ (GRAS) status, human or food origin, non-toxic, non-pathogenic and non-
haemolytic nature, and absence of antibiotic resistance characteristics. The probiotics should
also exert documented health benefits and match up with the printed labels for number and
types of microbes.
Numerous studies have shown that the normal intestinal flora provides efficient barrier
against pathogenic microbes. Probiotics are important in suppressing pathogens, maintaining
Probiotics, Prebiotics and Synbiotics 27
a beneficial pH, and producing bioactive compounds that enhance digestion and exert health
beneficial effects. Although, the precise mechanism of mode of action of probiotics is not
well understood, some of most widely accepted points are:
 Probiotics compete with pathogens for adherence and colonisation, thereby limiting
access of pathogens to the epithelium.
 Probiotics balance the intestinal microflora by producing antimicrobial compounds
and reducing lumen acidity against pathogens.
 Improves intestinal transport system by ion secretion, phosphorylation and prevents
mucosal damage triggered by toxins, antigens and drugs.
 Produces iron binding substances which inhibits growth of pathogens
The information related to modes of action and mechanism of inhibition by probiotics is

summarised in Table 2.
Probiotics are administrated in different forms like tablets, capsules, powder sachets, etc.,
but nowadays their consumption via functional food products like yogurt, curd, lassi, etc. is
generally favored and more popular among consumers. In India, consumption of traditional
dairy products (e.g., dahi, lassi, shrikhand) is very popular among rural masses. However,
with increasing awareness among consumers, several companies have also launched a number
of probiotic products like yoghurt, buttermilk, ice cream, etc. in Indian market (Table 3).
Figure 1. Selection Criteria for Probiotic Microorganisms.

Table 1. List of probiotic bacteria, their antimicrobial compounds and commercially

available strains by various industries
Genus and Species Antibacterial compounds and their Commercial probiotic strains and their
strains (if specific) source companies
Bifidobacterium
B. adolescentis - -
B. animalis - Bifidobacteriumanimalis subsp. lactis HN019
(DR10) (Danisco, Denmark)
B. breve - B. breve strain Yakult (Yakult, Japan)
B. bifidum Bifidocin 1454; Bifidocin B (B. bifdum -
NCFB)
B. essensis - -
B. infantis Bifidin I (B. infantis BCRC 14602) -
B. lactis Bifilact Bb-12 (B. lactis) B. lactisHN019 (DR10) (New Zealand Dairy
Board)
B. lactisBb-12 (Chr. Hansen, Denmark)
B. longum - B. longumSBT-2928 (Snow Brand Milk
Products Co., Ltd., Japan)
B. longumBB536 (Morinaga Milk Industry Co.,
Ltd., Japan)
B. thermophilum - -
Lactobacillus
L. acidophilus Acidolin, Acidophilin, Lactocidin L. acidophilus NCFM (Rhodia, Inc, USA)
(various strains); Acidocin B (L. L. acidophilusDDS-1 (Nebraska Cultures, Inc.,
acidophilus M46); Acidocin D20079 (L. NE)
acidophilus AA11 And L. acidophilus L. acidophilus SBT-2062 (Snow Brand Milk
DSM20079); Acidocin A (L. acidophilus Products Ltd, Japan)
TK9201s); Acidocin CH5 (L. acidophilus L. acidophilus R0011 (InstitutRosell, Montreal,
CH5); Acidocin JI132 (L. acidophilus Canada)
JCM 1132); Lactacin B (L. acidophilus L. acidophilus LB (Lacteol Laboratory, France)
N2)
L. brevis Lactobacillin and Lactobrevin (various L. brevis KB290 (Kagome Co., Ltd., Japan)
strains)
L. casei Lactocin 705 (L. casei CRL 705) L. caseiDN014001 (Immunitas) (Danone Le
Plessis-Robinson, France)
L. casei Shirota (Yakult, Japan)
L. casei DN 114(Danone, France)
L. crispatus - L. crispatusCTV05 (Gynelogix, Boulder, CO)
L. curvatus Curvacin A (L. curvatusLTH1174)
L. delbrueckii Bulgarican L. delbrueckii subsp. bulgaricus2038 (Meiji
Milk Products, Japan)
L. fermentum - L. fermentumRC-14 (Urex Biotech Inc.,
Canada)
L. gasseri Acidocin LF221 (L. gasseri LF221);
Gassericin A (L. gasseri LA39)
L. helveticus Helveticin J (L. helveticus 481);
Helveticin V-1829 (L. helveticus 1829)
Genus and Species Antibacterial compounds and their Commercial probiotic strains and their
strains (if specific) source companies
L. rhamnosus L. rhamnosusGR-1 (Urex Biotech Inc.,
Canada)
L. rhamnosus271 (Probi AB, Sweden)
L. rhamnosusR0052 (InstitutRosell, Montreal,
Canada)
L. johnsonii Lactocin F (L. johnsonii VPI 11088) L. johnsoniiLa1 (same as Lj1) (Nestle,
Switzerland)
L. plantarum Lactolin, Plantaricinplwa, L. plantarum299V (Probi AB, Sweden)
Plantaricinplwb, Plantaricin C (various
strains); Plantaricin EF and JK (L.
plantarum C11); Plantaricin MG (L.
plantarumKLDS1.0391); Plantaricin
NC8 (L. plantarumNC8); Plantaricin S
(L. plantarum LPCO10)
L. paracasei Proteinaceous compounds (L. paracasei L. paracasei F19 (Arla Dairy, Sweden);
subsp. paracasei strain M3) L. paracasei CRL 431 (Chr. Hansen, Denmark)
L. reuteri Reutericyclin L. reuteriSD2112 (same as MM2) BioGaia
(Raleigh, North California, USA)
Lactobacillus
L. rhamnosus - L. rhamnosus GG Valio Dairy (Helsinki,
Finland)
L. rhamnosusLB21 (Essum AB, Sweden)
L. salivarius ABP-118 (L. salivarius subsp. L. salivariusUCC118 (University College,
salivarius Cork, Ireland)
UCC118)
Other
Enterococcus faecalis Enterocin AS-48 (E. faecalis A-48- -
32); Cytolysin CyILL and CyILS
Enterococcus faecium Enterocins A and B (E. faecium -
WHE 81)
Lactococcuslactis Nisin, Nisin Z, Enterocin A and LactococcuslactisL1A (Essum AB, Sweden)
Lacticin 481 (various strains)
Pediococcusacidilacti Pediocin A
Propionibacterium - -
freudenreichii
Streptococcus cremoris - -
S. diacetylactis - -
S. intermedius - -
S. salivarius - -
S. thermophilus Streptophilin Streptococcus thermophiles MN-ZLW-
002(Inner Mongolia Mengniu Dairy Industry
Co. Ltd., China)
Saccharomyces - Saccharomyces boulardii (Biocodex Inc.,
boulardii USA)
(O‘Connor et al., 2 5; de Vreseand Schrezenmeir, 2 8; Nagpal et al., 2 12; 2 13).
Table 2. Different modes of action of probiotics
Action Probiotics Pathogens Mechanism of inhibition Reference

Competition for Lactobacilli Numerous range Secrets siderophores to chelate ferric (Grozdanov
limiting of pathogens and ferrous iron and limiting et al., 2004)
resources resources.
Anti-adhesive BifidobacteriumlactisB Salmonella, Compete with pathogens for (Collado
b12; Clostridiumand adherence and colonisation and et al., 2007)
Lactobacillus E. coli thereby limits access of pathogens to
rhamnosus the epithelium
LGG
Anti-invasive Bifidobacteriumlactis Salmonellaty- Enhances and balance the intestinal (Botes
Bb12 phimurium microflora by producing et al., 2008;
antimicrobial compounds and Ingrassia
reducing lumen acidity against et al., 2005)
pathogens
Anti-toxin 15 different probiotic Shiga toxin 2A Improves intestinal transport system (Carey
lactobacilli strains by ion secretion, phosphorylation et al., 2008)
and prevents mucosal damage
triggered by toxins, antigens and
drugs
Anti-viral Saccharomyces Rotavirus (RV) Soluble metabolites from (Buccigrossi
boulardii Saccharomyces inhibited oxidative et al., 2014)
stress and chloride ion secretion in
intestinal epithelial cells
Lactobacillus HIV Alleviate diarrhea, increases CD4 (Anukam
delbruekiivar- cell count, resolved nausea et al., 2008)
bulgaricus and
Streptococcus-
thermophillus
supplemented with L.
rhamnosusGR-1 and
L. reuteriRC-14
Lactobacillus H9N2Avian Increase in immune response against (Poorbaghi
acidophilus influenza H9N2 AIV by chemotactic migration et al., 2014)
Virus (AIV) of dendritic cells into Peyer‘s
patches and increases potential local
immune response in GI tract.
Table 3. Probiotic dairy products available in Indian market
Product Name Product Type Company Probiotic

Yakult Fermented Milk Yakult Danone India Lactobacillus caseishirota
drink (P) Ltd
b-Activ Plus Probiotic Dahi Dahi Mother Dairy BifidobacteriumlactisBb12
b-Activ Plus Probiotic Lassi Lassi Mother Dairy Not mentioned
Nutrifit (Strawberry and Mango) Milk drink Mother Dairy Lactobacillus acidophilus La5
Amul Prolife Probiotic Buttermilk Buttermilk Mother Dairy Not mentioned
AmulProLife Probiotic Lassee Lassi Amul Not mentioned
AmulProLife Probiotic Dahi Dahi Amul Not mentioned
AmulProLife Probiotic Ice cream Ice cream Amul Not mentioned
AmulFlaavyo Frozen Yoghurt Yoghurt Amul Not mentioned
Nesvita Probiotic Yogurt Yoghurt Nestle Not mentioned
Nestle Actiplus Probiotic Dahi Dahi Nestle Lactobacillus acidophilus
Viability of Probiotics
Viability of probiotics depends upon storage temperature, moisture content, pH, type and
form of food used for supplementation. Different probiotic species and strains exhibit distinct
properties that can affect their survival. Short cells are more stable and viable than longer
ones (Senz et al., 2015). Since, bacterial cells are prone to damage during various industrial
processes; viability of probiotic cultures is a major marketing and technological concern.
Furthermore, probiotics are susceptible to various environmental conditions like preservation
method, presence of oxygen, processing techniques, concentration of food constituents, etc.
Therefore, lots of efforts have been invested for improving the survivability of probiotics
during processing, storage and in vivo administration. Several methods have been
recommended to improve the viability of probiotics (Shah, 2000). Some of them are
mentioned below:
 Protection against oxygen

 Addition of oxygen scavengers
o Ascorbic acid
o Cysteine
o Use of air tight containers
 In fermented products
 Two-stage fermentation
 Addition of micronutrients
 Stress adaptation
 Technological approaches
 Microencapsulation
 Freeze-drying
 Probio-Tec
 LiveBac
PREBIOTICS
Gibson and Roberfroid (1995) initially introduced the word prebiotic. Prebiotics are
―non-digestible food ingredients that beneficially affects the host by selectively stimulating
the growth and/or activity of one or a limited number of bacteria in the colon, and thus
improves host health‖ (Rastall and Gibson, 2015; Bindels et al., 2015; Younis et al., 2015).
Prebiotics are the whole foods with different types of fibers that affect specific bacteria, their
fermentation end-products, and thus exert possible health benefits on the host. Some parts of
prebiotics are fermentable, which in comparison to other fibers are crucial for good health.
Based on their ferment ability, many substances and foods are being considered prebiotic.
Most of the prebiotics are simple or complex saccharides, which selectively promotes growth
of beneficial bacteria (Younis et al., 2015). Several properties of good prebiotic include
ability to resist host digestion, absorption, and adsorption before fermentation by one species
of the resident microbiota.
Table 4. Types, sources composition and method of manufacture of commonly used prebiotics
Type Source Composition Method of manufacture

Inulin Chicory root Linear chains of fructose units linked by β-2,1- Extraction from chicory root
glycosidic bonds
Fructo-oligosaccharides Asparagus, sugar beet, Β-2,1-linked fructans; Combination of kestose, nystose Β-fructofuranosidase based transfructosylation of disaccharide
garlic, chicory, onion, and fructofuranosylnystose sucrose or enzymatic hydrolysis of chicory inulin
honey, barley, etc.
Xylo-oligosaccharides Bamboo, fruits, Β-1,4-linked xylose Enzymatic hydrolysis of xylan
vegetables, milk, honey,
wheat bran, etc.
Galacto- Human‘s and cow‘s milk Oligo-galactose (85%), with Produced from lactose by b-galactosidase
oligosaccharides some glucose and lactose
Cyclodextrins Water-soluble glucans Composed of 5 or more α-D-glucopyranoside units Cyclodextrin glucosyl transferases based hydrolysis of starch
Raffinose Seeds of legumes, lentils, Composed of galactose, glucose, and fructose Water or aqueous methanol or ethanol solutions based
peas, beans, Chickpeas, extraction from plant materials
etc.
Soya-oligosaccharide Soybean Composed of raffinose, Extracted from soya bean whey, a by-product of soy protein
stachyose and verbascose isolate and concentrate
Lactulose Lactose (Milk) Composed of fructose and galactose Alkaline isomerization of glucose moiety of lactose to fructose
Lactosucrose Lactose Composed of galactose, glucose, and fructose. Transfructosylation of lactose and sucrose by β-
fructofuranosidase
Isomaltulose/Palatinose Sucrose Composed of α-1,6 linked glucose and fructose Enzymatic rearrangement of the glycosidic
linkage from a (1, 2)-fructoside to a (1, 6)-fructoside of sucrose
Malto-oligosaccharides Starch Α-1,4 linked glucose monomers From starch by action of debranching enzymes such as
glucose monomers pullulanase and iso-amylase, combined with hydrolysis by
various α-amylases
Isomalto- Starch Composed of α-1,6- and α-1,4-linked oligosaccharides By α-glucosidase based transgalactosylation activity of maltose
oligosaccharides
Gentio-oligosaccharides Starch Composed of 2-5 glucose residues linked by β-1,6 Acid or enzymatic hydrolysis of starch and subsequent
glycosidic transglycosylation of glucose syrup catalysed by β-glycosidase
Arabinoxylo- Wheat bran Composed of β-1,4-linked xylose units to which O-2 Enzymatic removal of starch and proteins from commercial
oligosaccharides and/or O-3 arabinose units are linked wheat bran to arabinoxylan, and subsequent conversion to
arabinoxylo-oligosaccharides using endoxylanase
Pyrodextrins Potato starch Mixture of glucose containing Pyrolysis of potato or maize starch
oligosaccharides
Chito-oligosaccharides Composed of β-1,4-linked N-acetylglucosamine Acid or glycosyl hydrolases based enzymatic hydrolysis of
and/orglucosamine chitosan
(De Vrese and Schrezenmeir, 2008; Al-Sheraji et al., 2013; Macfarlane et al., 2006; Swennen et al., 2006; Aam et al., 2010).
These prebiotic compounds are mainly found in vegetables, where they serve as energy
reserve to be used during germination. Some prebiotics are also present in milk in form of
some oligosaccharides. There are reports that specific prebiotics encourage specific colon
microbial populations both in vitro and in vivo. Amylomaize starch has been reported to
selectively stimulate the growth of bifidobacteria at the cost of bacteroides and coliforms
(Wang et al., 2002). Lactulose has also been reported to support the growth of bifidobacteria
and decrease the number of bacteroides in a continuous culture model (Fadden et al., 1997)
and human colon (Bouhnik et al., 2004). Oral administrations of galactooligosaccharides
arealso reported to increase faecal levels of B. lactis (Malinen et al., 2002). On the contrary,
b-glucans support the growth of lactobacilli more than bifidobacteria (Jaskari et al., 1998).
Functions
Prebiotics acts as a substrate for the useful gut microorganisms, therefore, exert health
benefits to the host. Large population of microorganisms, having specific population at
specific regions, occupies the entire human gut (Macfarlane and Macfarlane, 1997). In the
large intestinal part of the gut, relatively stable microbial population exists because of a
longer transit time (48-70 h) in comparison to the small intestine (4-6 h). The pH and
relatively low absorptive state of the colon further support large microbial colonization and
growth (O‘Sullivan et al., 1996). Prebiotics reach the colon without being digested because of
their chemical nature. A part of the prebiotic material remains undigested by pancreatic and
small intestinal enzymes in the human gut and therefore, reaches the large intestine. Due to
this microflora, the colon has the ability to undertake complex hydrolytic digestive functions
(Cummings and Macfarlane, 1991), which involves breakdown of dietary components,
complex carbohydrates and some proteins that are not hydrolysed or absorbed in the upper
digestion tract. The colonic microflora derives their food from non-digestible
oligosaccharides, dietary fibers, and undigested protein. They also get their substrates from
mucin; the main glycoprotein constituent of the mucus. Therefore, any undigested food that
reaches the colon is a source of prebiotics (Mussatto and Mancilha, 2007).
Selection of the Suitable Prebiotic
 Resistance to gastric acid, enzymatic digestion, and intestinal absorption

 Fermentation by the intestinal microbiota
 Selective stimulation of growth and activity of intestinal bacteria
 Low dosage forms
 Low calorific value
 Multifunctional properties
 Easily incorporated into food
 Target the distal colon
Substances Used as Prebiotics
The substances used as prebiotics should be ‗selective‘ or ‗specific‘ towards ecological

and functional features of the microbiota more likely to be relevant for host physiology.
These substances should resist the digestibility by gastric juice and α-amylase. The most
commonly used prebiotics are inulin, fructo-oligosaccharides (FOS) and galacto-
oligosaccharides (GOS). Details about type, sources, composition and method of manufacture
of commonly used prebiotics are given in Table 4. Among these, high yields of
oligosaccharides of specific chain length from simple raw materials such as inulin and
sucrose is a technical challenge so, the industrial production of short-chain FOS is attracting
the attention of researchers and industrialists. Xylo-oligosaccharides (XOS) are also of
importance due to their lignocellulosic origins.
SYNBIOTICS
Synbiotics are synergistic combinations of probiotics and prebiotics, which impart health
benefits to the consumer (Wasilewski et al., 2015). The prebiotic compound may selectively
favor the probiotic organism, or both work independently towards overall health. For
example, the probiotics colonizes the entire gastrointestinal tract to prevent pathogen
adhesion, while role of prebiotics is confined only to large intestine, where they stimulate gut
microflora. Therefore, despite having separate working sites, they collectively help in
maintaining health. Although this area of research is very promising, only limited studies
have been conducted. Some of the commonly used probiotic and prebiotic combinations and
their applications are listed in Table 5.
Since, the probiotic cells are susceptible to loss of viability and death during processing
and storage of food and during their passage through gastrointestinal tract,
microencapsulation technique has been successfully used to prevent from unfriendly
environmental conditions (Sarao, 2015). In this regard, Pinto et al. (2015) microencapsulated
BifidobacteriumBB-12 and inulin or polydextrose using whey or whey retentate by spray
drying. All the microcapsules showed high count of bifidobacteria, prebiotics improved the
stability of the microcapsules. Lactobacillus plantarum ATCC 8014 was encapsulated with
pectin or inulin as prebiotics in alginate coated chitosan microcapsules and significant
improvement in survival of encapsulated cells was observed under acidic (pH 1.5) and high
bile salt (4.5%) conditions (Pop et al., 2015). Rajam and Anandharamakrishnan (2015),
showed enhanced cell viability of microencapsulated Lactobacillus plantarum MTCC 5422
with fructo-oligosaccharide during storage.
Health Benefits of Probiotics, Prebiotics and Synbiotics
The essential role of the gut microbiota for health has generated tremendous interest in
modulating its composition and metabolic functions. Numerous health benefits are attributed
to probiotics, prebiotics and synbiotics (Table 6) that are reported to modulate gut microbes.
Although the mechanisms for these effects by probiotics are yet to be elucidated, various
suggested mechanisms include antimicrobial activities (acids, bacteriocins, etc.),

agglutination of pathogens, competition with pathogens, production of beneficial compounds
(fatty acids, conjugated linoleic acid, vitamins, etc.) and degradation of toxic or carcinogenic
metabolites.
Table 5. Combination of probiotics and prebiotics as synbiotics and their applications
Probiotic Prebiotic Available Application Reference

Synbiotics
L. acidophilus  Cellobiose  Lactobacilli +  Enhances gut (Van Zanten
NCFM  Dextran Cellobiose microbiota et al., 2014;
L. casei  Lacitol  Lactobacilli +  Increase in Ogawa et al.,
L. bulgaricus  Inulin Dextran production of 2006; Sekhon
L. brevis  Lactulose  Lactobacilli + branched chain fatty and Jairath,
L. cellobiosus  Galacto- lacitol acids 2010)
L. fermentum oligosccharide (GOS)  Lactobacilli +  Also reduced
L. Sporogenes  Xylo-oligosaccharide Inulin allergic reaction in
L. reuteri (XOS)  Lactobacilli + mice
L. lactis
 Fructo- FOS
L. plantarum
oligosaccharides
L. rhamnosus
(FOS)
 Soy-oligosaccharide
 Raffinose
Bifidobacteriuma  Transgalactosylated  Bifidobacteria +  XOS induces (Sekhon and
nimalis oligosaccharide FOS bifidogenesis Jairath, 2010;
B. infantis  XOS  Bifidobacteria +  Enhances immune Childs et al.,
B. longum  FOS GOS system 2014;
B. bifidum  Bifidobacteria +  Anti-infectious Asaharaet al.,
B. animalis XOS activity 2001)
B. breve
Saccharomyces  FOS  Saccharomyces  Used in agricultural (Abdulrahma
cerevisiae + FOS science n and Ahmed,
 Applicable for 2015)
fishes
 Effective on blood
cells and immunity
Enterococcus  Inulin  Enterococcus +  Increased rate of (Awad et al.,
faecium Inulin nutrient absorption 2008)
and change in
intestinal
morphology of
chicken
On the other hand, prebiotics mainly helps in supplementing probiotics and selectively
stimulates the microflora of large intestine. Liu et al. (2015) studied effects of prebiotics (FOS
and inulin) supplementation on fecal microbiota and gut physiology, and found that fructans
reduced the relative abundance of Firmicutes and Bacteroidetes responsible for obesity and
increased Actinobacteria and Verrucomicrobia. Paturi et al. (2015) studied effects of
probiotics Bifidobacteriumanimalis subsp. lactisHN019 and Lactobacillus rhamnosusHN001,
prebiotics (FOS, GOS and inulin), and synbiotics (B. lactisHN019 + FOS; B. lactisHN019 +
GOS; B.lactisHN019 + inulin; L. rhamnosusHN001 + FOS; L.rhamnosusHN001 + GOS;
L.rhamnosusHN001 + inulin) on large bowel health of rats. They observed beneficial effects
of all treatments on the biomarkers of large bowel health, although with some variations.
Chappell et al. (2015) reported increase in production of short chain fatty acids by fecal gut
microbiota on administration of oats and barley. Since, prebiotics increases the number of
beneficial bifidobacteria and lactobacilli that in turn improves the barrier function in IBD and
prevents mucosal colonization by aerobic enterobacteria (Guarner, 2007).
Table 6. Suggested health benefits of probiotics, prebiotics and synbiotics
Gastrointestinal tract related conditions References

 Effect on gut microbes (Liu et al., 2015; Chappell et al., 2015; Butel, 2014; Lin
et al., 2014)
 Irritable bowel syndrome (IBD) (Ford et al., 2014; Verbeke et al., 2014; Guandalini et
 Constipation al., 2014; Aragon et al., 2010; Yoon et al., 2014)
 Bloating
 Abdominal painCrohn‘s disease
 Ulcerative colitis
 Necrotizing enterocolitis
 Diverticulitis
 Inflammation of an ileal pouch
 Infectious diarrhea caused by viruses or (de Vrese and Schrezenmeir, 2006; Lee et al., 2015)
bacteria
 Antibiotic associated diarrhea
 Diarrhea in immunocompromised
subjects
 Relapsing Clostridium difficile colitis
 Traveller‘s diarrhea
 Infant diarrhoea
 Lactose intolerance (Raichandani, 2013; Williams et al., 2014; Szilagyi,
1999)
Other
 Antimicrobial effects (Tejero-Sariñena et al., 2012)
 Atopic dermatitis (Rosenfeldt et al., 2003; Weston et al., 2005; Akatsu et
al., 2015)
 Allergy (Özdemir, 2010; Yang et al., 2013)
 Bacterial vaginosis (Homayouni et al., 2014; Falagas et al., 2007)
 Cancer (Kumar et al., 2010; Chong, 2014; Lee, 2014)
 Colon cancer
 Cervical cancer
 Bladder cancer
 Diabetes (Panwar et al., 2013; Barrett et al., 2012)
 Hepatic encephalopathy (Holte, 2012; Lunia et al., 2014)
 Hypocholesterolaemic and cardioprotective (Ettingeretal., 2014; Vyas and Ranganathan, 2012; Ooi
effects and Liong, 2010)
 Immunomodulation (Hardy et al., 2013; Dong et al., 2013)
 Memory and mental state (Lee, 2014)
 Obesity (Sanz et al., 2013; Delzenne et al., 2011)
 Osteoporosis and calcium metabolism (Kruger and Coetzee, 2013; Parvaneh et al., 2014)
 Urinary tract and upper respiratory tract (Maldonado et al., 2012; Popova et al., 2012)
infections
Probiotic microbes also play a critical role in the development and regulation of
immunity. The performance of immune system is affected by several factors including dietary
habits and gut bacteria. Since probiotics are known to change the community composition of
the gut, therefore, they play important role in maintaining local and systemic immunity and
immune homeostasis (Hardy et al., 2013). It is reported that late colonisation of lactobacilli
and bifidobacteria increases risk of allergic response, while an early colonisation reduces
these risks (West et al., 2015). Treatment with probiotics has been found to prevent atopic
eczema in infants (Kukkonen et al., 2007). In a study, Vieira et al. (2013) showed that dietary
manipulations using organic selenium and marine alga Lithothamniummuelleri with probiotic
Saccharomyces boulardii UFMG 905 and Bifidobacterium sp. could stimulate and manipulate
the gut immune response, inducing intestinal homeostasis. Several recent studies have also
shown positive effects of prebiotics in various immune system related diseases (Gourbeyre,
2011). Ghanei et al. (2015) studied the effect of supplementation of prebiotic (inulin + FOS)
in 7-24 month old children with atopic dermatitis, and reported a decrease in serum IgE
levels, which may play positive role in treatment of dermatitis. In a study, Akatsu et al. (2015)
reported enhanced vaccination effects against influenza on administration of two prebiotics
i.e., GOS and bifidogenic growth stimulator
Cardiovascular diseases are also a major group of diseases that can be prevented or cured
by administration of probiotics and/or prebiotics. There are some reports in which the culture
of Lactobacillus plantarum reduced cholesterol level in vitro by production of bile salt
hydrolase (Bosch et al., 2014). Several species of Lactobacillus have been found to reduce
blood cholesterol level by cholesterol assimilation and inhibition of 3-hydroxy-3-
methylglutaryl coenzyme A (Tomaro-Duchesneau et al., 2014). Administration of several
prebiotics like inulin, FOS, starch, cyclodextrin, etc. has shown significant reduction in
lowering of cholesterol (Anandharaj et al., 2014). Recently, Yasmin et al. (2015) reported
significant reduction in serum total cholesterol, low-density lipoprotein (LDL) and
triglycerides, and an increase in high-density lipoprotein using a FOS supplemented whey
drink. Use of probiotics also reduces inflammatory response and oxidative stress, thereby,
increasing insulin sensitivity and reduces risk of type 2 diabetes. They also increase
expression of adhesion proteins by reducing intestinal permeability and prevent destruction of
β cells by autoimmunity (Gomes et al., 2014).
Prebiotics and probiotics at a particular ratio also provide health benefits to infants. In a
study, Strong et al. (2015) reported that dose of routine cow‘s milk-based formula
supplemented with a prebiotic blend of polydextrose (PDX) and GOS, or the probiotic
Lactobacillus rhamnosus GG in a standardized ratio added in partially or extensively
hydrolyzed formulas, showed a positive effect on the growth of infants. Boehm et al. (2004)
studied the prebiotic effects of oligosaccharide mixture in preterm and term infants, and
reported significant increase in population of bifidobacteria and decrease in number of
pathogens. Dilli et al. (2015) reported the efficacy of probiotic (Bifidobacteriumlactis) and
prebiotic (inulin), alone or as synbiotic (Bifidobacteriumlactis + inulin) for the prevention of
necrotizing enterocolitis (NEC) in very low birth weight infants. However, in very low birth
weight infants only probiotic (Bifidobacteriumlactis) and synbiotic (Bifidobacteriumlactis +
inulin) could decrease NEC.
Cardile et al. (2015) have demonstrated the role of prebiotics and probiotics in pediatric
patients for treating acute viral gastroenteritis and preventing antibiotic-associated diarrhea in
healthy children. They are also considered as an option for recurrent and relapsing antibiotic
sensitive pouchitis and in selected patients with mild ulcerative colitis. Ferrer et al. (2015)
proved that by addition of prebiotic and bioactive microorganisms decreased the bio-
accessibility of mycotoxins, with a concentration-dependent behavior, which may be a
potential strategy for reducing human exposure to these minor mycotoxins.
FUTURE PROSPECTS
Despite several recent advances, several key questions regarding the mechanisms of
action of probiotics and prebiotics are yet to be elucidated. Future research should give major
emphasis on their metagenomics, metabolomics and proteomic aspects for studying host
mucosal response, and ecological relationship with gut microbiota. The enhanced
understanding of the role of probiotics will help in development of new strategies to improve
the health status of consumers and will contribute to overall reduction in healthcare costs.
More clinical trials are also needed to validate their suggested health benefits. Innovative
technologies are required for efficient screening of their functional attributes, and the safety
of probiotics must be ensured during fermentation, downstream processing and consumption.
In addition, development and exploitation of high-throughput screening systems for discovery
of new probiotics; including archaebiotics, psychobiotics, and food grade genetically
engineered probiotics need more attention.
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Chapter 3
INDIGENOUS FERMENTED FOODS

AND BEVERAGES
Niharika Singh, Sampan Attri, Kavita Sharma and Gunjan Goel

Department of Biotechnology and Bioinformatics,
Jaypee University of Inforamtion Technology, Himachal Pradesh, India
ABSTRACT
The awareness of traditional foods harbored by the tribal communities is rich in
terms of its capacity to heal diseases. Fermented foods are encountered universal and are
believed to prolonge the shelf life and enhancement in the nutritive value of the foods.
The food fermentation enriches the food substrate with protein, essential amino acids,
essential fatty acids, and vitamins and eliminates anti-nutrient components in the raw
ingredients. Fermented foods have enhanced flavor and aroma with enhanced
digestibility and health promoting benefits. Fermentation may assist in the destruction or
detoxification of certain undesirable compounds which may be present in raw foods.
Other health benefits include cholesterol control, anti-cancer effects, immunity,
longevity, anti-hypertensive effect, and anti-diabetic effect. Therefore, the chapter throws
light on these technologies and gives a way for further scientific analysis and
authentication of this knowledge.
Keywords: food fermentation, beverages, fermented cereals, dairy products, fermented meat
INTRODUCTION
The enhancement of the qualitative value of human diet comes up through the growth of
a broad diversity of aroma, flavors, and textures in foods; the preservation of foods through
alcohol, lactic acid, acetic acid, and alkaline fermentations; the enrichment of food substrates

Corresponding author: Department of Biotechnology and Bioinformatics, Jaypee University of Information
Technology, Waknaghat, Solan-173234, E-mail: gunjanmicro@gmail.com, Tel: +91-1792239410; Fax No.:
+911792245362.
48 Niharika Singh, Sampan Attri, Kavita Sharma et al.
biologically with proteins, fatty acids, amino acids, and vitamins; the detoxification of
undesirable compounds; and the decrease in cooking times and fuel demands. Fermented
foods and beverages are one of the indispensable components of the dietary culture of every
community in the world. Steinkraus represented indigenous fermented foods as foods where
microorganisms delivered some biochemical advances which occur in the substrates during
fermentation (Steinkraus, 1996). Traditional fermentation, smoking, drying, and salting
processes were developed by ancient people to preserve foods for consumption, a remarkable
step in the food culture history of human beings. The diversity of such fermented products
derives from the heterogeneity of traditions found in the world, cultural preference, different
geographical areas where they are produced and the staple and/or by-products used for
fermentation. In many instances, the process of production is unknown, which is passed down
by cultural and traditional values to subsequent generations. Roughly 50-400 g per capita of
fermented foods and alcoholic beverages are consumed daily worldwide, representing
approximately 5-40% of the total daily nutrient intake. Nearly 5000 varieties of unlisted
major and minor fermented foods and beverages are prepared and devoured by billions of
people belonging to different communities and ethnicities across the globe. Most of the ethnic
fermented foods and beverages are produced by natural fermentation, except the alcoholic
beverages in Asia, which are created by using a consortium of microorganisms in the flesh of
a dry, grain-based dispatcher. Variety within the species or strains of several functional
genera of dominant microorganisms has created ethnic foods with different sensory
characteristics.
Preservation and safeguarding of foods and beverages remain the most significant
objectives of fermentation, with transparency, acceptability and overall quality, having
become ever more valued features to consumers, especially in rural areas where old customs
and cultural particularities in food fermentations are generally well sustained. However, it
must be noted in this context, despite modern food biotechnology making significant
technological advances, limitations in infrastructure and existing low technologies in rural
areas of most countries create challenges to keeping abreast of global developments toward
industrialization and also importantly in terms of quality and safety of products (Holzapfel,
2002).
Origin and Rationale of Fermentation
Fermentation is one of the oldest methods of food preservation, together with drying and
salting, which embedded in traditional cultures and village life. Fermentation processes are
believed to have been developed over the years by women, in order to preserve food for times
of scarcity, to impart desirable flavor to foods, and to reduce toxicity (Rolle et al., 2002).
Today, fermentation is still widely practiced as a household or village-level technology in
many countries (Holzapfel, 2002). As a technology, food fermentation dates back at least
6000 years and probably originated from microbial interactions of an acceptable nature.
Fermentation has enabled our ancestors in temperate and cold regions to survive winter
season and those in the tropics to survive drought periods, by improving the shelf-life and
safety of foods and beverages. The importance of fermentation in modern-day life is
underlined by the wide spectrum of foods marketed both in developing and industrialized
Indigenous Fermented Foods and Beverages 49
countries, not only for the benefit of preservation and safety, but also for their highly
appreciated sensory attributes.
Fermented foods are treasured as major dietary constituents in developing countries
because of their keeping quality under ambient conditions - thereby contributing to food
security - and because they add value, enhance nutritional quality and digestibility, improve
food safety, and are traditionally acceptable and accessible (Rolle et al., 2002; Holzapfel,
2002). Fermentation is a low-input enterprise and provides individuals with limited
purchasing power, access to safe, inexpensive and nutritious foods.
This chapter extends the description of products; fermented foods and beverages of the
Indian subcontinent with cereals, milk and meat as the raw ingredients.
FERMENTED BEVERAGES
Fermentations that involve production of ethanol are among the most ancient processes
known. In India many fermented alcoholic beverages are made from fruits like - apple,
grapes, cashew apple, jackfruit, banana etc. and cereals rice, wheat, barley etc. Indigenous
people are using undefined microorganisms as starters unknowingly for various traditional
processes (Sekar and Mariappan, 2007).
Jhara/Harhia
Jhara or Harhia is rice beer which is prepared by tribal inhabitants of tea garden in Terai
of West Bengal. Various herbal plants are used to modify the taste and odour of Jhara. Starter
material used for preparation of Jhara is Ranu dabai. For consumption, this fermented stock
is diluted with water at the rate of 5 litre water per kilogram of rice (Ghosh and Dass, 2004).
Soor
Soor is a traditional alcoholic beverage prepared in Tons valley in Garhwal Himalaya of

Uttarakhand. Starter culture of Soor is known as Keem and the plants used for preparing
Keem are generally known as Jadiya. Fermentable sugar rich cereals mainly rice, barley or
finger millet and fruits such as apples, pears, peach, apricot etc. are used for its production.
Alcoholic content of Soor lies between 35-40% (Rana et al., 2004).
Jann
Jann is a traditional fermented drink of the Bhotiyas of Uttarakhand and contains very
low concentration of alcohol. It is commonly prepared out of rice. However cereals such as
Koni (Setaria Italica), wheat (Triticum aestivum), jau (Hordeum vulgare), china (Panicum
miliaceum), oowa (Hordeum himalayens), chuwa (Amaranthus paniculatus) and fruits like
apple, banana, pumpkin, orange etc. can be used for jann preparations. For a good quality of
jann slow fermentation at low temperature is a required (Roy et al., 2004).
Daru
Daru is a distilled drink of Uttarakhand which contains ethanol at high concentration.

Commonly used substrates for its preparation are rice, jaggery, koni, chuwa, wheat and oowa.
Starter culture used for Daru is balam. After about a week of fermentation, when the mixture
is in a semi liquid condition which is further distilled. The distillate substance is the Daru.
For making the daru attractive in appearance, a small quantity of turmeric is added in it (Roy
et al., 2004).
Palm Wine or Toddy
Palm wine is a sweetish, heavy, milky white mild alcoholic beverage. In India mainly
three types of palm wines are consumed (i) Palmyra palm wine (ii) Silver date palm wine (iii)
Coconut palm wine. The palm wine undergoes lactic, alcoholic and acetic fermentation
involving lactic acid bacteria, yeast and acetic acid bacteria as well as Zymomonas and
Leuconostoc. Sucrose is the main ingredient of the fresh palm sap, which is about 12-15% by
weight. Alcohol content of palm wine is between 5-8%. Palm wine has a special place in
traditional celebrations and ceremonies such as marriages, burials and settling disputes and it
is believed to be good for the health, eyesight and also serves as a sedative (Chandrasekhar et
al., 2012; Singaravadivel et al., 2012).
Jackfruit Wine
It is the tribal and social drink of people north eastern states of India and it is made via
fermentation of jackfruit (Artocarpus heterophyllus Lam.) pulp. It contains 7-8% alcohol and
has pungent aroma and flavour (Sekar and Mariappan, 2007).\
Zutho
Zutho is a fermented drink obtained from unhulled rice grains and polished rice grains. It
is a drink of the Angami Nagas and is commonly consumed by all Naga tribes in the rural
regions of Nagaland. It contains approximately 5% (v/v) of ethanol, and is known for its
fruity odor, which is partly imparted by the acetyl esters present in generous amounts.
Traditionally, zutho is prepared by allowing starch-rich solutions to ferment. The yeast
Saccharomyces cerevisiae acts on the starch and ferments it. In addition, Rhizopus sp. is
also in process home-made zutho has a white slurry-like appearance (Teramoto et al., 2002).
Feni/Fenny
Feni is an alcoholic beverage which is produced and consumed in Goa. It is registered as

a Geographical Indication (GI). There are two types of feni which are consumed, cashew feni
and coconut feni. Microorganisms involved in fermentation are yeasts and Lactic acid
bacteria (Sekar and Mariappan, 2007; Desai et al., 2012).
Apong
Apong is a traditional alcoholic beverage of Arunachal Pradesh and Assam. Starter

culture used Apong preparation is Ipoh which contains mainly yeast. Rice is used as raw
material for preparation of Apong at room temperature for a period of 3 to 5 days (Tiwari and
Mahanta, 2007).
Kodo Ko Jaanr
Kodo ko Jaanr is prepared from dry seeds of finger millet (Eleusine coracana) in Sikkim.
Murcha is a starter culture used for its preparation. For preparing kodoko jaanr cooked seeds
of finger millet are mixed with Murcha and fermentation take place for 4-7 days (Das and
Deka, 2012).
Xaj Pani
Xaj pani is an aromatic, sweet in taste alcoholic drink of Assam. It is a kind of rice beer
produced by using starter culture Vekur pitha. The fermentation take place in dark condition
for period of 4-5 days in air locked condition (Saikia et al., 2007).
Judima
Judima is prepared by the Dimasa tribe of Assam. Humao is used as starter culture for its
preparation. It is a type of beer prepared from rice as a substrate. Fermentation takes place for
period of 3-7 days (Chakrabarty et al., 2009).
Chhang
Chhang is an alcoholic beverage of Lahaul valley of Himachal Pradesh made by solid

state fermentation. Rice or barley is used as raw material and Phab act as starter culture
(Savitri and Bhalla, 2007).
Yu
Yu is traditionally consumed as drug in Manipur. It is rice based alcoholic drink and

Hamei is used as starter culture. Fermentation takes place in sunlight for 3-4 days in summer
and 5-6 days in winter (Singh and Singh, 2006).
Themsing
Themsing is a cereal based alcoholic drink of Arunachal Pradesh. Finger millet or Barley
or mixture of both is used as raw material and Pham is used as a starter culture. The vessel is
kept air tight and undisturbed for 1-3 year for fermentation (Singh et al., 2007).
Sujen
It is rice based alcoholic drink of West Bengal and Assam. Mod pitha is used as a starter
culture and rice mixed with rice husk as a raw substrate. Fermentation takes 3-4 days during
summers and 6-7 days in winter (Deori et al., 2007).
Kiad
Kiad is a traditional alcoholic drink of Pnar tribe of Jaintia hills district, Meghalaya.
Local red rice as raw material and Thiat as a starter culture are required for its production.
Fermentation takes place for 2-3 days (Samati and Begum, 2007).
Tchang/Jhar
It is a cereal based alcoholic beverage of Sikkim. Millet (Paspalum sp.) and starter
material marcha are the main ingredients of Tchang. Fermentation takes place for 1-2 weeks
in anaerobic conditions. Rice powder and maize flour are added to enhance taste of the
product (Singh and Jain, 1995).
Sura
It is an alcoholic beverage of Himachal Pradesh. It is consumed during marriage

ceremonies and local festivals. Millet flour as a raw material and starter culture Dhehli are
used for its preparations. It takes 8-10 days to ferment (Bhalla, 2007).
Handia
It is a traditional alcoholic drink among the tribes of central India. It is a rice base
beverage and Ranu tablets are used as a starter culture. Some plant parts, e.g., Elephanto
pusscaber L. roots, Argyreia bella (C.B. Clarke) Raizada stem, Casearia graveolens
Dalz.bark, Symplocos racemosa Roxb. bark, etc. are also added to increase intoxication or
decrease the period of fermentation. After 3-5 days, the fermented product is extracted and is
ready for consumption (Kumar and Rao, 2007).
Chulli
Chulli is a fermented alcoholic beverage of Himachal Pradesh. Dried wild apricots are
naturally fermented for its preparation (Bhalla, 2007).
Angoori
Angoori is a traditional fermented beverage consumed during local festivals and marriage
ceremonies by the tribal people of Kinnaur. It is made from red or green grapes (Bhalla,
2007).
Mahua
Mahua is a flower based alcoholic drink of tribal people of central part of India. It is
made from Corollas of Madhuca longifolia var. latifolia (Koen.) Macbr and Ranu Tablets as a
starter culture are used. Juice of Buchanania lanzan Spreng leaves is added to help the
fermentation (Kumar and Rao, 2007).
CEREAL BASED (WITH/WITHOUT PULSES)

FERMENTED FOODS
Idli
A fermented, thick suspension made of rice (Oryza sativum) and dehulled black gram
(Phaseolus mungo) is used in several traditional foods in South India. It is a pulse based food
consumed with sambhar. It is acid leavened steamed product made from rice and dehulled
black gram soaked in buttermilk, ground to a fine paste, mixed with the clear water of curds,
cumin, coriander, pepper, and as afoetida and then allow to ferment for 12-15 hours of at
room temperature (Steinkraus, 1997). Microorganisms involved in the fermentation are
Leuconostoc mesenteroides, Streptococcus faecalis and Pedicoccus cerevisiae.
Dosa, Dhokla and Khaman
Dosa, a traditional fermented pancake food made from rice and black gram, was first
mentioned in Tamil Sangam literature in India about AD sixth century (Das et al., 2012). For
the preparation of dosa, wheat, bajra, maize, sprouted beans, cow peas, field beans, fresh
beans or fresh groundnut oilcakes are used. It is thinner as compare to idli. Dhokla is an
ethnic, fermented spongy-textured product made by Gujaratis that is prepared from Bengal
gram and rice products, and is similar to idli except that dehulled Bengal gram dhal is used in
place of black gram. Khaman, similar to dhokla, is also an ethnic, fermented spongy-textured
product made by Gujaratis and is made solely using seeds of Bengal gram (Sekar and
Mariappan, 2007).
Selroti
Selroti is a popular fermented rice-based ring shaped, spongy, pretzel like, deep fried
food item commonly consumed in Sikkim and Darjeeling hills in India. For the preparation of
selroti, local variety of rice (Oryza sativa L.) attey is sorted, washed, and soaked in cold water
for overnight or 4-8 h at ambient temperature. Water is then decanted from the rice by using
bamboo made sieve called chalni and spread over a woven tray made up of bamboo, locally
called naanglo and dried for 1 h. Soaked rice is pounded into coarse powder in a wooden
mortar and pestle. Larger particles of pounded rice flour are separated from the rest by
winnowing in a bamboo tray. Then the rice flour is mixed with nearly 25% refined wheat
flour, 25% sugar, 10% butter or fresh cream and 2.5% spices/condiments containing large
cardamom (Amomum subulatum Roxb.), cloves coconut, fennel, nutmeg, cinnamon, and
small cardamom, are added to the rice flour and mixed thoroughly. Milk (boiled/unsoiled) or
water is added, kneaded into a soft dough and finally into batter with easy flow. Batter is left
to ferment naturally at ambient temperature (20–28οC) for 2 to 4 h during summer and at 10–
18°C for 6–8 h during winter. The oil is heated in a cast iron frying pan locally called tawa.
The fermented batter is squeezed by hand or metallic serving spoon, deposited as continuous
ring onto hot edible oil and fried until golden brown and is drained out from hot oil by poker
locally called jheer or suiro or also by a spatula. Deep fried selroti is served as confectionary
(Hannah and Tamang, 2009).
Fermented Rice
Fermented rice are prepared by adding water to cooked rice and kept the mixture over
night. The water is drained off and the rice is mixed with curd and salt. The bacteria isolated
after 16 hours of fermentation are Streptococcus faecalis, Pediococcus acidolactici, Bacillus
sp. and Microbacterium flavum. The fermentation results in decrease in pH from 6.4 to 4.0
(Ramakrishnan, 1979).
Ambali
Ambali is a fermented product prepared by combining ragi, millet flour, with water to
make thick batter and ferment for 14-16 h. The bacteria isolated from fermented ragi are
Leuconostoc mesenteroides, Lactobacillus fermentum and Streptococcus faecalis. The pH
decreases from 6.4 to 4.0 indicating some CO2 production as there is increase in the volume
(Ramakrishnan, 1979).
Bhattejaanr and Anarshe
Bhattejaanr and Anarshe both are rice based breakfast in India. Anarshe is sweetened
snack foodstuff fermented with lactic acid bacteria while bhattejaanris sweet and sour
alcoholic paste fermented by Mucorrouxianus and Hansenula anomala (Blandinob et al.,
2003).
Sez
Sez is a traditional semi-fermented food made from rice mainly consumed by the
Bhotiyas in Uttaranchal of India. It is mostly used as snacks. Previously, it was a delicacy and
was prepared only during festivals. Generally, sez is extracted while preparation of rice jann
(local beer) (Roy et al., 2004).
Kulcha, Nan and Bhatura
These are the various kinds of traditional wheat based fermented snack foods like kulcha
(white wheat flour product), bhatura (white wheat flour product) and nan (wheat flour
product) are prepared indigenously in India. The fermentation of these products is mainly
done by Saccharomyces cerevisiae and LAB are used (Sanjeev and Sandhu, 1990).
Siddhu
It is pulse and cereal-based dish of Himachal Pradesh made from wheat flour mixed with
spices and starter material (malera). Khobli is an added name for siddhu (Thakur et al., 2004).
Marchu
Marchu is cereal based snack food of Himachal Pradesh. It is mainly consumed at the
time of festivals, religious and marriage ceremonies. It is prepared from wheat flour and
starter material (malera) (Thakur et al., 2004).
Parotta
The cereal based food of South India practiced by the wheat flour as the chief raw
material blended with salt, sugar, egg, water and oil. It is allowed to ferment natively. It can
be served with vegetarian gravy dishes (Indrani et al., 2007).
Babru
Babru is cereal based food stuff mainly popular in Himachal Pradesh. It is prepared by
combining wheat flour and water (2:1) and finished into semi-solid paste. In addition, salt or
spices are put in for taste and kept for fermentation at 25-30°C for 3-4 h. After fermentation,
the slurry is cooked as smooth pancake with oil (Kanwar et al., 2007).
Taotjo
Taotjo is a sort of condiment made from fermentation of roasted wheat meal by

Aspergillus oryzae, it is mainly well-liked in eastern India (Blandinob et al., 2003).
CEREAL BASED FERMENTED SWEETS AND SNACKS

Jalebi
Jalebi is a sweetened fermented cereal-based product, made from refined wheat flour,
curd and water. The fermented batter is deep fried in oil in spiral shapes and submerged in
sugar syrup for few minutes. This traditional food is prepared during marriage ceremonies
and festivals of North and South India. The bacteria found in fermented batter are
Lactobacillus fermentum, L. buchneri, Streptococcus lactis, S. Faecalis and Saccharomyces
cerevisiae. The pH reduces from 4.4 to 3.3 and there is a 9% volume raise in the batter. Both
free sugar and amino nitrogen decrease during fermentation (Steinkraus, 1996).
Gulgule
It is a cereal-based product from Himachal Pradesh used mainly on religious and social
ceremonies. The usual composition/ingredients are wheat flour and Malera (starter) made into
viscous slurry by adding water, sugar. This sweetened slurry is formed into small oval shape
to be deep fried in oil until brown (Thakur et al., 2004).
Chhuchipatra Pitha
Chhuchipatra pitha is a cereal and pulse type of food which consumed in Orissa. Usual
ingredients include par-boiled rice, black gram, coconut, sugar and curd. The batter is
prepared and native fermentation or inoculum is added. Curd is included as an inoculum. The
process of cooking included the fermented batter is flattened thin over a warm greased pan
using a spatula. The fillings of grated coconut, curd and sugar are taken in the centre of the
pancake. It is then folded into a square shape for frying. Shelf life of food is two days and is
usually taken without any adjunct due to its sweet taste (Roy et al., 2007).
PULSE BASED FERMENTED FOODS

Kinema
Kinema is a bacilli-fermented, sticky soybean food with a slight ammonia flavor

produced by natural fermentation in the Darjeeling hills and Sikkim. During the conventional
preparation of kinema, small-sized ―yellow cultivar‖ dry soybean seeds are favored. Soya
beans are washed and soaked overnight, and soaked soybeans are removed and absorbed in a
container with clean water, and boiled for 2-3 h until they turn easy. Surplus water is drained
away, and the cooked soybean seeds are filled into a wooden mortar, to split the seed leaves.
Approximately 1% of firewood, ash is added directly to the cooked soybeans and mixed
thoroughly to keep the alkaline condition of the product. Soy grits are placed in a bamboo
basket lined with a locally grown fresh fern, Glaphylopteriolopsis erubescens, or Ficus and
banana leaves. The basket is covered in a jute carrier and left to ferment naturally at 25-40°C
for 1-3 days over an earthen oven. During summer time, the fermentation may need 1-2 days
while in winter it may need 2–3 days. The shelf life of freshly prepared kinema is 2-3 days in
summer, and the maximum is a week in winter without refrigeration. It may be prolonged by
drying in the sun for 2–3 days. Dried kinema is stored for several months at room
temperature. Preparation of kinema varies from place to place and is nevertheless confined to
houses. It is fascinating to note that mountain women using their indigenous information of
food production to prepare kinema. Dried kinema is sometimes mixed with leafy vegetables to
make a mixed curry (Singh et al., 2007; Tamang, 2015).
Tungrymbai
Tungrymbai is an ethnic, fermented soybean food popular in Meghalaya. Soybean seeds

are washed and soaked in water for about 4-6 h. The outer skin of soaked soybeans is
removed before cooking by rubbing gently. The soaked soybeans are boiled for about 1-2 h
till water is wholly taken in. Boiled beans are allowed to cool, and are packed with fresh
leaves of Clinogyne dichotoma and are put inside a bamboo basket and covered with a thick
cloth. The covered basket is kept over the fireplace, and fermentation occurs naturally in 3–5
days. Tungrymbai is mashed and put into a container with water and boiled till the water
evaporates, and the container stirred continuously. It is combined with fried onion, garlic,
ginger, chilly, ground black sesame, and salt. A thick curry is made and is served as a side
dish with steamed rice. Pickles are also made from tungrymbai. The microorganisms involved
are Bacillus subtilis, LAB and yeast (Tamang et al., 2009).
Aakhone
Aakhone is a tribal, fermented sticky soybean food prepared in Nagaland. Soybean seeds
are soaked and cooked, and then enclosed in fresh leaves of banana or Phrynium pubinerve or
Macaranga indica and kept over a fireplace for fermentation to occur naturally in 5–7 days.
Fresh aakhone is made into bars and dried above an earthen oven. Sometimes, each fermented
bean is broken up by hand, and is dried in the sun for 2–3 days. Such dried aakhone is stored
in containers for future consumption. The pickle is made from freshly fermented aakhone by
mixing with green chilly, tomato, and salt. The dried aakhone cakes are cooked with pork and
are eaten as a side dish with steamed rice. B. subtilis and other species of Bacillus are present
in aakhone (Tamang et al., 2009; Tamang, 2015).
Bekang
Bekang is an ethnic, fermented soybean food popular in Mizoram. During the preparation
of bekang, small-sized dry seeds of soybean are cleaned and soaked in water for 10-12 h. The
extra water is dewatered and the beans are boiled for 2-3 h in an open container until the
beans become soft. The excess water is drained away and wrapped in fresh leaves of
Calliparpa aroria or leaves of Phrynium sp. The wrapped beans are held within a bamboo
basket and then placed near an earthen oven or a warm place and is set aside to ferment
naturally for 3-4 days. Bekang is consumed as it is, or is made into a curry and eaten as a side
dish with steamed rice. Many species of Bacillus are present in bekang (Singh et al., 2007;
Tamang et al., 2009).
Peruyaan
Peruyaan is a fermented soybean product prepared in Arunachal Pradesh. Dry seeds of

soybeans are rinsed and cooked for 2-3 h till the beans get soft. The excess water is drained
off and is cooled for some time. The cooked soybeans are kept in a bamboo basket generally
covered with fresh ginger leave sand is retained on the wooden rack above a fireplace for
fermentation for 3-5 days. The stickiness of the product is ensured, and if the product is sticky
enough, then the product is all set for consumption. Peruyaan is consumed mostly as a side
dish. It is mixed with hot water, chillies, and salt, and is directly consumed without frying or
cooking. Many species of Bacillus are associated with peruyaan fermentation (Tamang,
2015).
Aagya
It is pulse based food of Arunachal Pradesh. It is a low cost source of high protein
flavored food having soybean as the main component (Singh et al., 2007).
Chukchoro
It is a pulse based food of Arunachal Pradesh. It can be consumed directly after mixing
with varieties of local vegetables. For longer preservation, it is made into paste and stored in
banana leaves. It is used in making chutney with local chilli (solu), tomato, yak cheese and
salt (Singh et al., 2007).
Adai and Vada
Adai and vada both are cereal legume based breakfast or snack food in India.
Streptococcus, Pediococcus, Leuconostoc are leading microorganism involved for the
fermentation of both the products. It is low cost source of high protein flavored food
composed of soybean (Blandinob et al., 2003).
Hawaizaar
Hawaizaar is a soybean based food of Manipur. This food is most often used in the
festival Gobardhan puja, surap (ritual ceremony after 13 days of death of a man) and Asti
(ritual ceremony after 6 days of death of a man) (Singh et al., 2007).
Wadi
It is a pulse based food from Punjab and West Bengal. Black gram and oil are main
ingredients. Microorganisms involved are L. mesenteroides, L. fermentum, S. Cerevisiae and
Trichosporon cutaneum become dominant. Candida vartiovaarae and K. marxianus are also
often found. The development and prevalence of microflora are affected by the seasons,
summer being more favourable for bacteria and winter for yeasts. The production of acid and
gas results in a fall of pH from 5.6 to 3.2 and two-fold raise in the volume of the dough. The
lactic acid bacteria are mainly responsible for the acidification of dough, favourable
conditions for the yeasts to grow and become active for leavening. The fermentation brings
about a significant increase in soluble solids, non-protein nitrogen, soluble nitrogen, free
amino acids, proteolytic activity and vitamins including thiamine, riboflavin and cyano
cobalamine. Conversely, the levels of reducing sugars and soluble protein decrease. It is
reported that amylase activity increases initially, but declines thereafter.
FERMENTED VEGETABLES AND FRUITS

Vegetable and Unripe Fruits Based Fermented Foods
and Pickles
Sinki
It is acidic non-salted fermented radish taproot mainly prepared in Sikkim. It is used as
the base for soup or as a pickle. The fermentation is done by the bacteria Lactobacillus
fermentum, L. Brevis, and L. Plantarum. Sinki is prepared by fermentation in 1m deep pit. It
has acidic flavor containing 14.5% of protein, 2.5% of fat and 11.3% of ash or dry weight. It
has properties like it cures diarrhea, stomach pain etc. (Singh et al., 2007).
Pickles
A pickle is very spicy and hot due to the addition of chillies and spices. Dry salted lime
pickle is a popular homemade fermented product in India. During preparation, the well
ripened lime fruits are washed with water, cut into pieces and mixed with salt, then allowed
for fermentation in clay or earthen pots for a week. By similar methods, cucumber, Indian
gooseberry and mango pickles are prepared. Khalpiis a traditional cucumber pickle used in
the Himalayan region of India (Tamang, 1998).
Sauerkraut
It is a type of preserved fermented cabbage (Brassica oleracea L.) food. After
fermentation, Indians use this as a base of curry. The fermentation is started by L.
mesenteroides and followed by L. plantarum (Steinkraus, 1996).
Kimchi
Kimchi is a generic term used to denote a group of fermented cabbage, radish, and garlic
foods in Korea. It is a spicy fermented pickle. The flavor of kimchi is dependent on the
ingredients, fermentation conditions, and LAB involved in the fermentation process. Kimchi
can be stored for several months, when a lactic fermentation occurs (Cho et al., 2006).
Gundruk
Gundruk is an ethnic fermented, dry, and acidic vegetable product of the Himalayas. The
daily per capita consumption of gundruk is 1.4 g with an annual production of 3.2 kg/house in
the Indian state of Sikkim. Gundruk is prepared from fresh leaves of a local vegetable called
rayo-sag (Brassica rapa subspecies campestris variety cuneifolia), mustard (Brassica
juncea), and cauliflower (Brassica oleracea variety botrytis), which are wilted and shredded
using a sickle. Wilted and shredded leaves are crushed mildly and pressed into an earthen jar
or container, and made airtight by covering with dried bamboo bract sheaths and fern fronds,
and weighted down by stones. The container is kept in a warm place and allowed to ferment
naturally for about 7–10 days. Unlike kimchi and sauerkraut, freshly fermented gundruk is
sun dried for 3–4 days before consumption, and dried gundruk is preserved for more than 2
years. Gundruk is eaten as a soup or pickle. The soup is made by soaking gundruk in water
for 10 min, and then squeezing and frying in edible oil with chopped onions, tomatoes,
chillies, turmeric powder, and salt. It is then boiled for 10–15 min, and served hot with
steamed rice. Gundruk soup is a good appetizer in a bland and starchy diet. Gundruk is sold in
all local markets (Tamang et al., 2007).
Mesu
Mesu is a fermented dish derived from bamboo, mainly prepared in Himalayan regions of
Darjeeling hills and Sikkim. The months of June to September are suitable for the preparation
of mesu when Bamboo shoots sprout. Locally available species of bamboo are used such as
choya bans (Dendrocalamus hamiltonii Nees and Arnott), bhalu bans (D. sikkimensis
Gamble) and karati bans (Bambusa tulda Roxb). The fermentation of mesu is initiated by
Pediococcus pentosaceus followed by Lactobacillus brevis and finally dominated by L.
plantarum. The pH of mesu is declined from 6.4-3.8 due to increase in titrable acidity from
0.04- 0.95% (Tamang and Sarkar, 1996; Sekar and Mariappan, 2007).
Soibum
It is a vegetable based food from Manipur and Nagaland. It is a food with therapeutic
usage. Bamboo shoots are the main ingredients and Lactic acid fermentation is there.
Microorganisms involved in the preparation are Lactobacillus plantarum, L. brevi, L.
corniformis, L. delbrueckii, Leuconostoc fallax, L. lactis, L. mesenteroides, Enterococcus
durans, Streptococcus lactis, Bacillus subtilis, B. licheniformis, B.coagulans and yeast
Candida, Saccharomyces and Torulopsis. These fermented products have been considered to
possess therapeutic properties for the treatment of various ailments including peptic ulcer,
cancer etc. The Manipur Meitei women use soibum along with fermented fish (Singiamis)
against the plague disease (Jeyaram et al., 2009).
Hiring
Hiring is a vegetable based food of Northeast India prepared from Bamboo shoots. Lactic
acid fermentation is there involved Lactobacillus plantarum and Lactococcus lactis. It can be
kept for 2-3 months at ambient temperature (Singh et al., 2007).
FERMENTED MILK PRODUCTS

Fermentation is among the oldest and safest techniques of food conservation, and an easy
way to derive new products. Spontaneous fermentation takes place by constructing up in milk
many types of lactic acid bacteria (LAB) species. Growth of LAB promotes a rapid lowering
of the pH (acidification), which in turn inhibits spoiling microorganisms and pathogens.
Bacterial growth further plays a function in the digestion of milk components, increasing their
bioavailability and in the formation of desirable flavor compounds. Not amazingly, the
fermentation process is primarily brought about by a series of different LAB members
(FAO/WHO 2003). The term LAB embraces a diverse lot of bacteria producing lactic acid as
the major end product from carbohydrate utilization. They are non-sporulated, anaerobic, aero
tolerant microorganisms presenting limited biosynthetic capabilities, therefore taking a rich
medium to grow and a series of growth factors, such as amino acids, vitamins, purines, and
pyrimidines (Carr et al. 2002). Typical LAB members belong to the genera Lactococcus,
Lactobacillus, Leuconostoc, and Pediococcus. Nowadays, although phylogenetically
unrelated, Propionibacterium and Bifidobacterium species are also considered among the
LAB, because they are frequently found in the same ecological niches and used for similar
industrial applications (Parente and Cogan, 2004).
Yogurt
Yogurt represents a very significant dairy product in modern times and its consumption
stands next to whole milk especially during summer. It is a semisolid fermented product made
from a heat-treated standardized milk mix by the activity of a symbiotic blend of
Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus (Clark and
Plotka, 2004; Ozer, 2010). Yogurt is a widely consumed highly nutritious fermented milk,
defined by the Codex Alimentarius (FAO/WHO 2003) as a coagulated milk product resulting
from the fermentation of milk by S. thermophilus and L. delbrueckii subsp. bulgaricus.
Traditional yogurt manufacture involved spontaneous acidification of milk (with or without
boiling) at reasonably high temperatures (between 40-50°C). Either the heating or the high
incubation temperature or both select thermophilic microorganisms, among which S.
thermophilus and Lb. delbrueckii subsp. bulgaricus are dominant. Yogurt cultures are
homofermentative and ferment lactose in a like way, although L.delbrueckii can produce up
to 4% lactic acid, while S. thermophilus produces only between 0.6% and 1.1%. The function
of these two LAB species in yogurt manufacture includes milk acidification and synthesis of
aromatic compounds, but they further influence flavor, texture, appearance and overall quality
attributes of the finished ware. Industrial yogurt manufacture involves a preliminary milk
fortification with non-fat powdered milk, milk protein concentrate, or condensed skim milk to
increase total solids (up to 12–13%) and acquire a thicker consistency. After blending, the
ingredients are homogenized at 15–20 MPa and pasteurized at high temperature (85-88°C for
30 min).
Dahi
Dahi plays an important part in the socio-religious practices of the Indian subcontinent
and is considered as a sacred item in many festivals and religious ceremonies both by Hindus
and Buddhists. Dahi, a curd, is the most popular fermented milk in India which is obtained by
lactic fermentation of cow or buffalo milk. Dahi is well known for its palatability and
nutritive value (Rathi et al., 1990). It resembles plain yoghurt in appearance and consistency,
but differs in having less acidity. Dahi is consumed directly as a refreshing non-alcoholic
beverage and savory. It is used to prepare many ethnic milk by-products such as lassi
(buttermilk) and ghee (butter). This class of fermented milks is undoubtedly the simplest and
probably the oldest. Not surprisingly, the traditional manufacture of non-heated natural
fermented milk (NFM) from raw milk is open worldwide. Milk and fermented milk products
form the major points in Indian diets and have been eaten for more than 5,000 years. It is well
recognized in ancient Indian story that day; buttermilk and ghee were widely used up milk
products during 3000 BC (Tamang, 2010). Some common ethnic fermented products of
modern India are dahi, lassi, mistidahi, dahi, shrikhand, rabadi, kalari, chhurpi, soamr, philu
and chhu (Prajapati and Nair, 2003). Natural fermented milk products of Himalayan region of
India prepared by mountain people from cow and yak milk are dahi, ghee, chhurpi, chhu,
somar, maa and philu (Aneja et al., 2002).
Mishti Doi
Mishti doi (sweetened dahi, mishit doi) is a sweetened fermented milk product with a
cream to light-brown color, firm consistency, smooth texture and pleasant aroma. It holds 2–
9% fat, 10–14% solids-not-fat and 17–19% sugar. The most common sweetener used is cane
sugar. In some special varieties of mishit doi, fresh palm jaggery is used as a sweetener.
Typically, a mix comprising 71.26% milk (3% fat, 9% solids-not-fat), 5.32% cream (35%
fat), 5.42% nonfat dry milk and 18% crystalline sugar is blended. The mix is heat-treated at
85-90°C (185-194°F) for 15 minutes and homogenized. The heating process develops a light-
brown color in the mix. It is then cooled to 42°C (107.6°F). The starter is added at 1% level.
Following dispersion of the starter, mishti-doi mix is dispensed into sanitized cups. The lids
are heat-sealed to make the cups airtight and prevent leakage of the mixture. The sealed cups
are then incubated at 42 ± 1°C (107–108°F) for approximately 6–8 hours until the acidity
develops to 0.7–0.8%. The product is moved to a cold room (4°C/39-40°F) with minimum
disturbance, as the product at this stage has a weak body and an unstable top layer: excessive
shaking will result in undesirable cracks on the top layer or in the curd mass. Mishti doi is
used as a dessert and snack in India and Bangladesh (Gupta and Mann, 2000).
Shrikhand
This is a dahi-based product resembling Greek yogurt. The cultured milk or dahi is
separated from whey to get chakka, which is blended with sugar, color, flavor and spices to
reach a desired level of composition and consistency. The final product contains 8.5% fat,
10% protein, 42% sugar and 60% total solids. The acidity of the product is usually between
1.1 and 1.2%, expressed as lactic acid. Skim milk (9% MSNF, 0.05%fat) is heated to 90°C
(194.4°F)for 10 seconds in a high-temperature–short-time (HTST) pasteurizer, cooled to
30°C (86°F) and inoculated with 0.25–0.50% dahi culture. After 8 h of incubation or at a
titratable acidity of 0.8%, the curd is ready for further processing. Chakka is prepared by
separating the whey from dahi using a basket centrifuge, a quark separator or a desludging
centrifuge. Shrikhand is prepared by adding sugar at a rate of 80% of the amount of chakka
and mixed in a planetary mixer. A predetermined amount of plastic cream (80% fat) is added
to the chakka, along with sugar and flavorings/spices, to obtain at least 8.5% fat in the
finished product. Shrikhand is used primarily as a snack and dessert. Shrikhand is an ethnic
concentrated sweetened fermented milk product of Shrikhandis an ethnic, concentrated
sweetened fermented milk product made by Gujaratis and Rajasthanis in India. Rabadi is
fermented milk-based, thick slurry like product, is made by fermenting grains and pulses in
North and West India (Patel and Ckakraborty, 1988).
Lassi
Lassi, buttermilk, is a by-product obtained during the preparation of clarified butter

(ghee) from dahi by traditional methods, and is the most common nonalcoholic refreshing
beverage for the hot climates in India. Lassi is a refreshing beverage derived from dahi. It is a
popular drink in India, especially North India. Significant advances have been made towards
the industrial production of lassi through the application of ultra-high-temperature (UHT)
treatment of milk (Aneja et al., 2002). Standardized milk (9-10% solids-not-fat and 0.5–1.0%
milk fat) is heated to 85°C (185°F) for 30 min or 91°C (195.8°F) for 2.5–5 min, cooled to
25°C (77°F) and cultured with dahi starter. It is then fermented at 25°C (77°F) to lower the
pH to 4.5. The set curd is broken with the help of a stirrer, while 30% sugar solution is added
to get 8–12% sugar concentration in the blend (Mann, 1986).
Cheese
According to an ancient legend, cheese was accidentally made by an Arabian merchant

when he put his supply of milk into a pouch made of a sheep‘s stomach when he set out on a
long day‘s journey across the desert. The rennet in the lining of the pouch combined with the
heat of the sun caused the milk to separate into curd and whey. This story seems to have
occurred approximately 7000 years B.C. in the Fertile Crescent situated between the rivers
Euphrates and Tigris in Iraq. During fermentation, drainage of whey through cloth or
perforated bowls allowed the collection of curds which, when salted, became cheese
(Kosikowski and Mistry, 1966).
FERMENTED FISH PRODUCTS

Fermented fish products such as fish sauces, fish paste, or salted fish have been
consumed since ancient times. Because of poor roads and other methods of transport, the
provision of fresh fish to potential inland consumers was impossible, and this encouraged
fermentation as a preservation technique.
Fermented fish as sauce or pastes and dried and salted fish products are widely prepared
and consumed in India as seasoning, condiments, and side dish. Fermented fish products are
prepared from freshwater and marine finfish, shellfish, and crustaceans that are possessed
with salt to cause fermentation and, thereby, to prevent putrefaction (Ishige, 1993).
Fermentation of fish was historically associated with salt production, irrigated rice
cultivation, and the seasonal behavior of fish stock. If no vegetables are added, the salt-fish
mixture yields fish sauce, which is commonly used as condiment, and if the product of fish
and salt preserves the whole or partial shape of the original fish, it is called shikora, which is
then made into paste (Ishige and Ruddle, 1987). Fermentation of fish products has been
practiced for many years in many parts of the world. There has been reports of fermented fish
products as far back to ancient Greece, and the trade of this product, garum, was extensive in
the Roman era (Beddows, 1985). There are many varieties of fermented fish products
available today; these include liquid products such as fish sauce also known as nampla in
Thailand, kecapikan or bakasang in Indonesia, patis in the Philippines, nouc-mam in

Vietnam, oyster sauce, hoi-sin sauce, and paste products such as fish and shrimp pastes also
known as belacan or terasi in Indonesia and Malaysia, and dry and semidry fermented fish
(Ishige, 1993).
The Himalayan fish products are slightly different and are mostly dominated by dried and
smoking processes. Fermentation fish products include ngari and hentak in Manipur and
tungtap in Meghalaya in India; the rest of the fish products are dried or smoked that include
sidra, sukuti, and gnuchi in Nepal, Darjeeling hills, and Sikkim, and karati, lashim, and
bordia in Assam, India (Thapa and Pal, 2007; Thapa et al., 2004). Fermented fish foods are
associated with the food culture of the Meiteis in Manipur; the products are prepared and
eaten on every festival and religious occasion. Consumption of fish products in the
Himalayas, though constituting an important part of the diet, is comparatively less than other
fermented products such as vegetables and dairy products. This may be due to the adoption of
the pastoral system of agriculture and the consumption of dairy products in these regions.
Ngari
Ngari is an ethnic fermented fish product of Manipur which is made from Puntius
sophore (‗Phoubu‘) (Tamang, 2010). During its preparation, the fish is rubbed with salt and
dried for 3-4 days, then pressed tightly in an earthen pot, sealed tightly, and ferment for 4-6
months. Ngari is eaten as a side dish with cooked rice (Thapa and Pal, 2007).
Hentak
Hentak is a ball-like thick paste prepared by fermentation of a mixture of sun-dried fish

(Esomus danricus) powder and petioles of aroid plants (Alocasia macrorhiza) in Manipur
(Thapa and Pal, 2007). The processing includes crushing sun-dried fish to powder, an equal
amount of petioles of aroid plants is mixed and a ball-like thick paste is made. The mixture is
kept in an earthen pot and is fermented for 7-9 days. Hentak is consumed as curry as well as a
condiment with boiled rice. Sometimes, it is given to mothers in confinement and patients in
convalescence (Sarojnalini and Singh, 1988).
Tungtap
Tungtap is a fermented fish paste, commonly consumed by the Khasia tribes of

Meghalaya in North-East state of India (Thapa and Pal, 2007). In the traditional methods of
preparation, dried fish (Danio spp.) are mixed with salt, kept in an earthen pot and fermented
for 4–7 days. Tungtap is consumed as pickle and taste enhancer (Rapsang and Joshi, 2012).
Lonailish
Lonailish is a salt fermented fish product from Hilsa. Tenualosa ilisha is very popular in
North eastern parts of India and Bangladesh due to its typical flavor and aroma. During
preparation of Lonailish, fresh or iced Hilsa fish are first washed, descaled and beheaded
without removing the gut and slash diagonally into steaks of about 1.25 to 2.0 cm thickness.
The fish steaks are then dry salted and set aside for 24-48 h in a bamboo basket with a
covering. The fluid from the fish is allowed to drain throughout this time. The salted fish
steaks are then tightly packed in tin containers. When the tin is almost filled, earlier boiled
and cooled saturated brine is poured slowly to pack the voids between the steaks and
maintained a level of brine about 2 cm above the steaks. The tin is then closed with a lid,
which is tied with a wire. The packed tins are stored for fermentation for a period of 4-6
months (Majumdar et al., 2005).
Jaadi
Jaadi is a high-salt fermented fish made from a fish like the Indian mackerel (Rastrelliger
kanagurta), trench sardine (Amblygaster sirm), or skipjack tuna (K. pelamis). The fish
employed are usually gutted and cleaned, then layered in a earthen pot or barrel with a
concoction of salt and the ripe pods of the goraka plant in 3:1 ratio (fish to salt/goraka
mixture). Goraka (Garcinia gamboges) pods contain several organic acids, including citric
and tartaric acids. The fish is set aside to ferment for 2-3 months or longer. The pickle formed
during fermentation is drained out as fish sauce and used in curry or sambol (condiment)
preparations (Subasinghe, 1993).
Fermented fish products are deeply connected with food culture of Meitei in Manipur,
which are cooked and consumed in every festival and religious affairs. Ingestion of fish
products in the Himalayas, though, is significant in the diet and is comparatively less than
other fermented products such as vegetables and dairy products.
FERMENTED MEAT PRODUCTS

Meat is an ingredient of the daily diet for many ethnic people living in the Himalayan
areas of India, Nepal and China. Raw meat gets spoiled at high ambient temperature inside a
few hours due to its high moisture and protein contents, but and are mostly dried or smoked
or worked to prolong the shelf life of perishable raw meat. In developed countries, a wet-
curing process for meat has been evolved which involves the utilization of a solution of salt,
sodium nitrate/nitrite. Nevertheless, in under-developed and developing countries,
preservation of meat is done by curing with salt, followed by drying or smoking or
fermentation. Meat is highly nutritious but, in its fresh state, is perishable and can be an agent
for the transmission of a range of infections and intoxications. Not surprisingly, therefore,
meat was a study of the earliest conservation methods developed by humans. These depended
largely for their consequence on the decrease of water activity (aw) through the removal of
usable water by drying and salting. This had the effect of controlling growth of normal
spoilage micro flora associated with the meat but did not necessarily eliminate microbial
growth entirely. In the absence of the normal spoilage bacteria, lactic acid bacteria (LAB) in
particular were able to grow, especially where there was a moderate reduction in aw. Their
growth enhanced the preservative effect of the reduced aw and improved the sensory
properties of the product in terms of texture and flavor. They also contributed to improved
safety compared with the fresh raw materials.
Kargyong
Kargyongis an indigenous sausage-like product prepared from yak/beef pork meat, and is
mostly consumed by the Bhutia, Lepcha and Sherpa of Sikkim. It is prepared during
November to December. It is soft or hard and brownish color. During traditional method of
preparation of kargyong, the lean meat of yak/cattle/pig with its are chopped finely, and
combined with crushed garlic, ginger and required amount of salt and mixed with little
amount of water. The mixture is stuffed into the segment of gastrointestinal tract of animal
(yak/ox/pig) locally called gyuma, used as natural casings with 3-4 cm in diameter and 40-60
cm in length. One end of the casing is tied up with rope, and other end is sealed after stuffing
and boiled for 20-30 min. Cooked sausages are taken out and hung in the bamboo stripes
above the kitchen oven for smoking and drying for 10-15 days or more to make kargyong.
Three varieties of kargyong are prepared and consumed: yak kargyong (prepared from yak
meat), lang kargyong (prepared from beef meat), and faak kargyong (prepared from pork).
Faak kargyongis also prepared by the non-vegetarian Nepalis/Gorkha of Sikkim and
Darjeeling hills. They use pigs as well as goat intestine as natural casings to stuff the mixture
(chopped pork/chevon). Karyong is eaten after boiling for 10-15 min, sliced and fried in
edible oil by adding onion, tomato powdered chillies, and salt and is made into curry. It is
also consumed as fried sausage with raksi, a distilled liquor or chyaang/kodokajaanr, mild
alcoholic finger-millet-based beverage. Karyong is also eaten as cooked sausage before
fermentation. Yak Karyong is not sold in the market; it is usually prepared for home
consumption, and also during marriage and festivals. However, lang kargyong and faak
kargyong are sold in the local restaurants and food stalls of Sikkim and Darjeeling (Rai et al.,
2009).
Jamma or Geema
Jamma or Geema is a traditional fermented chevon-sausage of the Kumayun Himalyas.

Red goat is chopped into fine pieces; ground finger millet (Eleusine coracana), wil pepper
called timbur (Zanthoxylum spp.) chilli powder and salt are added and mixed. A little amount
of fresh animal blood is also added. Meat mixture is made semi-liquid by pouring water and
stuffed into small intestine of goat of about 2-3 cm in diameter and 100-120 cm in length with
the help of funnel, and the both ends of the long intestine. It is picked randomly to prevent
bursting while boiling. After boiling for 15-20 min, stuffed intestine are smoked above the
kitchen oven for 15-20 days. It is consumed as curry by mixing with onion, garlic, ginger,
tomato and salt. It is also deep-fried and is eaten with local alcoholic beverages. Sometimes,
gemma may be eaten as cooked sausage (Rai et al., 2009).
Arjia
Arjia is sausage-like product made from goat meat of the Kumayun Himalayas consumed
by the Bhutia. The method of preparation involves, a mixture of chopped lungs of goat, salt,
chilli powder, timbur (Zanthoxylum spp.) and fresh blood animal are stuffed into the large
intestine of goat and boiled for about 15-20 min. As in jamma, pricking of large intestine is
necessary to prevent bursting while boiling. It is dried for 15-20 days and smoked above the
kitchen oven. Arjia is consumed as curry or deep fried sausage along with main meal
(Tamang, 2009).
Satchu
Satchu is a dried or smoked meat product of the Himalayas, mostly prepared from
yak/beef meat. Red meat of yak/beef is sliced into several strands of about 60-90 cm is and is
mixed thoroughly with turmeric powder, edible oil or butter and salt. The meat strands are
hung in the bamboo stripes or wooden stick and are kept in an open air in corridor of the
house or are smoked above the kitchen oven for 10-15 days. Satchu can be kept at room
temperature for several weeks. This is a natural type of preservation of perishable raw meat in
absence of refrigerated or cold storage. Satchu is made into curry by washing and soaking in
water briefly, squeeze, and fry in yak/cow butter with chopped ginger, garlic, chilli and salt.
Thick gravy is made which is consumed with thupka and boiled or baked potatoes by the
Bhutia, Tibetan, Dhukpa, Lepcha and Sherpa. Yak satchu (prepared from yak meat) is usually
prepared for home consumption. Lang satchu (made from beef) is sold in the local
restaurants, and food stalls in Darjeeling hills, Sikkim, Bhutan etc. (Tamang, 2009).
Kheuri
Kheuri is a classic indigenous meat (yak/beef) product of Sikkim restricted to Bhutia and
Lepcha. It is usually prepared during winter depending on the availability of meat. During the
preparation of kheuri, yak/beef meat, its fat and intestine are cut into pieces, combined with
salt. The mixture of meat is packed into empty stomach of sheep, locally called khyabo
(previous cleaned sheep stomach), stitched the opening, and pressing with heavy load for 10-
15 h. After pressing, it is kept for 1-2 months in an open portion for fermentation. Currently,
the people of North Sikkim have discontinued preparing kheuri because of unavailability of
the sheep stomach due to ban on slaughtering high altitude sheep. Kheuri dish is prepared by
frying in yak or cow butter, locally called maa, mixed with chopped onion, ginger, garlic,
powdered chillies, salt and made into thick curry. It is also eaten by boiling it for 10-15 min
with salt. Kheuri dish is consumed with main meals by the Bhutia and the Lepcha as side dish
or curry with baked potatoes (Tamang et al., 2012).
Table 1. Indigenous Fermented Products
Raw ingredient Fermented food Nature Use

Ethnic milk products
Cow milk Butter milk Acidic, sour Drink
Cow milk Chhurpi(soft) Less sour, soft, cheese like Curry, pickle
Yak milk Chhurpi (hard) Hard mass Masticator, gum -like
Cow or yak milk Chhu Soft, strong flavored Curry, soup
Yak milk Churyuupa Mildly acidic, soft, flavored Curry, soup
Cow milk Dahi Acidic, viscous Curd, savory
Cow milk Dudhchhurpi Hard mass Masticator, chewing gum -
like
Cow milk Ghee Soft, oily mass, solid Butter
Cow milk Lassi Acidic, butter milk Refreshing beverage
Yak milk Maa Mildly acidic, viscous Butter
Cow milk Mohi Acidic, butter milk Refreshing beverage
Buffalo/cow milk Mistidoi Mildly acidic, thick gel, sweet Curd, savory
Yak milk Phrung Mildly acidic, Hard mass Masticator
Yak/cow milk Philu Cream Curry
Tea-yak butter, salt Pheuja/suja Salt with buttery flavor, liquid Refreshing tea
Buffalo/cow milk Paneer Whey, soft, cheese like product Fried snacks
Buffalo/cow milk Shrikhand Acidic, sweet, viscous Savory
Yak/cow milk Somar Strong flavoured, paste Condiment
Yak milk Shyow Acidic, thick gel, viscous Curd-like, savory
Ethnic fish products
Fish Ayaiba Smoked fish Pickle, curry
Fish Bordia Dried, salted Curry
Hill-river fish Gnuchi Smoked fish Curry
Fish and petioles of aroid Hentak Fermented fish paste Curry
plants
Fish Kararti Dried, salted Curry
Fish Lashim Dried, salted Curry
Fish Mio Dried Curry
Fish Naakangba Dried Pickle, Curry
Fish Ngari Fermented fish Curry
Fish Sidra Dried fish Curry
River fish Sukakamaacha Smoked, dried Curry
Raw ingredient Fermented food Nature Use
Fish Sukuti Dried fish Curry
Fish Tungtap Fermented fish, paste Pickle
Ethnic meat products
Large intestine of chevon Arjia Sausage Curry
Pork Bagjinam Fermented pork Curry
Chevon Chartayasha Dried, smoked meat Curry
Pork Faakkargyong Sausage, soft or hard brownish Curry
Intestine of chevon, finger Jamma Sausage, soft Curry
millet
Beef Lang kargyong Sausage, soft Curry
Beef Lang satchu Dried, smoked meat Curry
Beef fat Lang chilu Hard Edible oil
Sheep fat Likchilu Hard solid, oily Edible oil
Beef Lang kheuri Chopped intestine of beef Curry
Buffalo meat Sukokamasu Dried, smoked Curry
Yak fat Yak chilu Hard, oily Edible oil
Yak Yak kargyong Sausage, soft Curry
Yak Yak kheuri Chopped intestine of yak Curry
Yak meat Yak satchu Dried, smoked meat Curry
Suko Ka Masu
Suko ka masu is a dried/smoked strand like meat product meat like satchu. It is
commonly eaten up by the Nepalis/Gorkha of Darjeeling hills and Sikkim. Suko ka masu is
made by slicing the red meat of goat or buffalo into stripe up to 25-30 cm, into which mustard
oil, turmeric powder and salt are added and mixed thoroughly. Mixed meat stripes are hung
above the earthen kitchen oven and smoked for 7-10 days. The smoked product is called suko
ka masu or sea kuaa fter complete drying, which can be stored at room temperature for
several weeks. It is cleaned and soaked in lukewarm water for 10 min, and then fried in
mustard oil, with chopped ginger, onion, chilli powder and salt (Rai et al., 2009).
Chartaysha
Chartaysha is a traditional and delicious goat meat products of the Kumaon Himalyas,
consumed by the Bhutia of Dharchula and Munsiary in the region of Pithoregarh. It is similar
to Satchu. Red goat meat is slash into tiny pieces of 3-4 cm, blended with salt, sewed in a
long thread and is hung in the wooden stick or bamboo stripes and then kept in an open air for
10-15 days. It can be placed at room temperature for some weeks for future consumption.
Curry is made by frying in edible oil with tomato, ginger, garlic, onion and salt. The tribal
people of the Kumaon Himalayas prepare Chartaysha curry especially during kolatch festival
and offer to ancestors before eating (Tamang, 2012).
Chilu
Chilu is stored animal product prepared in North Sikkim. Fatty portions of freshly
slaughtered meat (yak/lamb/beef) are separated, kneaded by hand and pressed into the clean
stomach of sheep and then stitched. This stuffed meat is pressed with heavy stones for about
5-10 h, and are kept hanging in the corridor of the house in wooden plank for 10-15 days.
Chilu can be used for a year or more. It has been noted during survey that chilu production
has declined in North Sikkim due to unavailability of sheep stomach. Chilu is used in place of
edible oil for cooking by the Bhutia and Lepcha (Rai et al. 2009).
CONCLUSION
Fermented foods play a vital role in human diet in terms of improved shelf-life and
improved nutritional value of the raw ingredients. The flora responsible for the fermentation
in various cases are indigenous and comprise of lactic acid bacteria, yeast and fungi strains.
Besides, information about the implication of the presence of the metabolites and their
chemical interaction is still insufficient. Moreover, little scientific literature is found to date
linked to health relevant constituents and contaminants in these alcoholic beverages and
foods. Currently, the fermentation procedures have been used to develop novel foods with
enhanced health properties, which is a trend likely to pursue in the future.
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Chapter 4
BACTERIOCINS AS POTENTIAL BIOPRESERVATIVES

IN FOODS: AN OVERVIEW
Kamal Rai Aneja1, Romika Dhiman2, , Neeraj Aggarwal2 *
and Ashish Aneja3

1
Vaidyanath Research, Training and Diagnostic Centre, Kurukshetra, India
2
Department of Microbiology, Kurukshetra University, Kurukshetra, India
3
University Health Centre, Kurukshetra University, Kurukshetra, India
ABSTRACT
Biopreservation of foods through the application of bacteriocins that target food
pathogens without toxic or other adverse effects has received greater attention in recent
years. Bacteriocins are ribosomally synthesized antimicrobial peptides or proteins
produced by bacteria that kill or inhibit the growth of other bacteria. Lactic acid bacteria
(LAB) especially lactobacilli have gained particular attention due to the production of
bacteriocins. The role of bacteriocins as a biopreservative into diverse food systems has
been thoroughly studied and found to be effective against pathogenic and spoilage
microorganisms. Although several bacteriocins have been characterized biochemically
and genetically; nisin and pediocin is the two bacterocins that has been officially
employed in the food industry and their use have been approved globally. Combination of
bacteriocin with other technologies such as hurdle technology, non thermal methods may
be effective having preservative potential in various food systems. The aim of this review
is to give an overview on the bacteriocin classification, their mode of action and
applications in various food systems.
Keywords: biopreservative, bacteriocin, antimicrobial, nisin, hurdle technology
*
Correspondence Author: Romika Dhiman. Email-romikadhiman@gmail.com ; Tel: +91-8901086621.
76 Kamal Rai Aneja, Romika Dhiman, Neeraj Aggarwal et al.
INTRODUCTION
Food is a source of nutrients and more vulnerable to be spoiled by the growth of
microorganisms. Although a few microorganisms may bring about desirable changes in food
but some microorganisms can cause undesirable changes that deteriorate flavor, odor, color,
sensory, and textural properties of foods. Food preservation is a challenge for food industries
and health authorities owing to the demands of consumers for safe, minimally processed and
ready-to-eat fresh food products (Devileghere et al., 2004; Lucera et al., 2012; Negi, 2012).
The main aim of food preservation methods includes inactivation, growth delay and
growth prevention of spoilage and pathogenic microorganisms. Low temperature, reduced
water activity, acidification, fermentation, modified atmosphere packaging, addition of
antimicrobials, compartmentalization in water in oil emulsions are some food preservation
methods preventing or slowing down the growth of spoilage microorganisms. On other hand
pasteurization, sterilization, microwave heating, ionization radiation, high pressure, pulsed
electric field and high frequency ultrasound methods act by inactivating microorganisms.
Aseptic handling, centrifugation and filtration prevent the entry of microorganisms into food.
Use of antimicrobial compounds is one of the oldest and most traditional food preservation
techniques but with several adverse effects on humans. Consumer demand for food without
chemical preservatives has promoted the search for biopreservatives from natural sources
such as animals, plants and microorganisms (Vigil et al. 2005; Aneja et al., 2014).
The use of natural antimicrobials such as organic acids, essential oils, plant extracts and
bacteriocins could be good alternatives to ensure food safety. Bacteriocins are generally
recognized as ―natural‖ components able to influence the safety and quality of foods.
Bacteriocins has been consumed since an ancient time as product of lactic acid bacteria and
considered as natural food ingredient. Bacteriocins are low molecular weight, ribosomally
synthesized extracellularly released peptides or proteins which have a bactericidal or
bacteriostatic effect on other bacteria either in same species (narrow spectrum) or across
genera (broad spectrum). The applications of bacteriocins from LAB that target food
pathogens without toxic or other adverse effects has received great attention because food
safety has become an increasingly important concern. Several studies have been conducted to
prove efficacy of bacteriocins (enterocin, pediocin, lactacin and others) as natural
preservatives in various food systems, nisin and pediocins are the only two commercially
used bacteriocins as a food preservative, marked as Danisco‘s Nisaplin™ and ALTATM
respectively, in meat, dairy products as milk and cheese, mashed potatoes, soymilk, fruit and
vegetables juice, fresh cut products and other fermented foods such as olives, sourdough and
cider (Devileghere et al., 2004; Deegan et al., 2006; Ananou, 2007; Gálvez et al., 2007;
Settani and Corsetti, 2008; Tiwari et al., 2009; Lucera et al., 2012).
SOURCES OF BIOPRESERVATIVES
Biopreservation can be defined as the extension of shelf life and food safety by the use of
natural or controlled microbiota and their antimicrobial compounds (Ananou, 2007; Settani
and Corsetti, 2008). The sources of biopreservatives are essential oils derived from plants
(e.g., basil, thyme, oregano, cinnamon, clove, and rosemary), enzymes obtained from animal
Bacteriocins as Potential Biopreservatives in Foods: An Overview 77
sources (e.g., lysozyme, lactoferrin), bacteriocins from microbial sources (nisin), organic
acids (e.g., sorbic, propionic, citric acid) and naturally occurring polymers (chitosan)
(Figure 1).
Plant essential oils are a group of terpenoids, sesquiterpenes, and possibly diterpenes
with different groups of aliphatic hydrocarbons, acids, aldehydes, acyclic esters, or lactones;
gaining a wide acceptance in food industry as decontaminating agent as they are generally
recognized as safe. The antimicrobial activity of essential oil is not attributed to one specific
mechanism, but there are several targets in the cell. Hydrophobicity of essential oil enables
them to partition in the lipids of bacterial cell membrane and mitochondria, disturbing the
structures and rendering them more permeable (Aneja et al., 2014).
Figure 1. Biopreservative - Sources and Compounds.
Vanillin, GRAS (Generally Recogonized As Safe), is commonly used in ice cream,

beverages, biscuits, chocolate, confectionary, desserts, and more (Walton et al., 2003).
Vanillin possesses inhibitory affect against microorganisms owing to its ability to negatively
affect the integrity of the cytoplasmic membrane, with loss of ion gradients, inhibition of
respiratory activity but keeping energy generation largely unaffected (Fitzgerald et al., 2004).
Enzymes from animal origin have also shown potential as preservative in food. Lysozyme
is a protein present in milk and eggs that catalyzes the hydrolysis of the β-1,4 linkages
between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan layer of the
bacterial cell wall. Lysozyme exhibits antibacterial activity exclusively against gram positive
bacteria which is attributed to the presence of lipopolysaccharide layer in the outer
membrane. The FAO/WHO joint and several countries including Austria, Australia, Belgium,
Denmark, Finland, France, Germany, Italy, Japan, Spain, and United Kingdom have approved
its use in some foods when used in accordance with good manufacturing practices (GMP). It
is used as antimicrobial agent in casing for frankfurters, on cooked meat and poultry products,
and to prevent the blowing caused by Clostridium tyrobutyricun in semi hard cheeses (Losso
et al., 2000; Raybaudi- Massilia et al., 2009; Lucera et al., 2012).
Lactoperoxidase, a hemoprotein present in milk and other secretions, exhibits broad
spectrum antimicrobial activity against bacteria, fungi, and viruses. The lactoperoxidase
system exerts its antimicrobial action through short-life oxidation products, mainly
hypothiocyanate (OSCN−) and hypothiocyanous acid (HOSCN), which produce
microbiocidal or microbiostatic effects by the oxidation of thiol groups (-SH) of cytoplasmic
enzymes and damage to the outer membrane, cell wall or cytoplasmic membrane, transport
systems, glycolytic enzymes and nucleic acids. The regulatory organization, Food Standards
Australia New Zealand (FSANZ), has permitted the use of the lactoperoxidase system for the
treatment of meat (including poultry), fish and milk products as an antimicrobial at maximum
levels of 20 mg/kg meat or 30 mg/L milk (Naidu, 2000; Raybaudi-Massilia et al., 2009).
Chitosan, a heteropolysaccharide composed of β−1, 4 linked 2-amino-2-deoxy-β-D-
glucose obtained commercially by deacetylation of chitin, is an abundant constituent of
crustacean shells and fungi. Chitosan got GRAS status in 2005 by USFDA and is marketed as
food additive or supplement in Japan, Korea, England, Italy, Portugal and United States. It is
more active against yeasts, moulds and gram negative bacteria. It may be owing to
polycationic structure at pH 6.3; interaction with anionic components such as
lipopolysaccharide and proteins of the membrane cell surface responsible for the disruption of
the integrity of the outer membrane resulting in loss of barrier function, however lacking
direct bactericidal activity (Rhoades and Roller 2000; Novack et al., 2003; Sebti et al., 2005;
No et al., 2007; Raybaudi-Massilia et al., 2009).
One of the most common forms of food biopreservation is through fermentation, a
process based on the growth of microorganisms in foods, whether natural or added. These
organisms mainly comprise lactic acid bacteria (LAB) which have been used for centuries in
fermentation of a variety of dairy products. Lactic acid and other end products of LAB
metabolism, including hydrogen peroxide, diacetyl, acetoin, reutericyclin, antifungal peptides,
bacteriocins and other organic acids, act as bio preservatives by altering the intrinsic
properties of the food to such an extent as to actually inhibit spoilage microorganisms
(Devlieghere et al., 2004; Deegan et al., 2006; Gálvez et al., 2007). Nisin and pediocin are the
two bacteriocins which received a great deal of attention because of their beneficial effects to
human health as well as the replacement of chemical preservatives that are being
continuously questioned with regard to safety (Zendo, 2013)
DISCOVERY OF BACTERIOCINS
Bacteriocins (Gr. Bacterion = little rod + ios = poison) are ribosomally synthesized
peptides or proteins with antimicrobial activity, currently the term bacteriocins is applied to
describe the small, heat stable cationic peptides synthesized by gram positive bacteria, mainly
LAB, which possess wider spectrum of inhibition. The first bacteriocin was discovered by
Gratia in 1925, principle V was produced by one strain of E.coli against another culture of
E.coli (Clevland et al., 2001; Chen and Hoover, 2003). The term bacteriocin was coined by
Jacob et al. in 1953 as a general term for highly specific antibacterial proteins. Nisin, the most
commonly used bacterocin in food as biopreservative was discovered in 1928 followed by the
discovery of subtulin in 1948. The first use of nisin, the most extensively studied bacteriocin,
in pasteurized processed cheese spreads as food additive was approved by the U.S. food and
drug administration (FDA) in 1988 opened the door for the potential application of other
bacteriocins as preservatives in food. Now nisin is approved for use in 48 countries as a food
preservative (Cleveland et al., 2001; Hoover and Chen, 2005; Settani and Corsetti, 2008;
Garcia et al., 2010).
DIFFERENCES BETWEEN BACTERIOCINS

AND ANTIBIOTICS
Bacteriocins are often confused with antibiotics. Bacteriocins are often considered more
natural because they are thought to have been present in many of the foods eaten since an
ancient time in comparison to antibiotics. The major difference between bacteriocins and
antibiotics is that bacteriocins restrict their activity to strains of species related to the
producing species and particularly to strains of the same species, antibiotics on the other hand
have a wider activity spectrum and even if their activity is restricted this does not show any
preferential effect on closely related strains. In addition, bacteriocins are ribosomally
synthesized and produced during the primary phase of growth, though antibiotics are usually
secondary metabolites. Bacteriocins are inactivated by trypsin and pepsin enzymes found in
the gastrointestinal tract and therefore, they do not alter the microbiota of the digestive tract.
In addition to these, other differences between bacteriocins and antibiotics are given in Table
1 (Cleveland et al., 2001; Zacharof and Lovitt, 2012; Balciunas et al., 2013).
Table 1. Differentiation between Bacteriocins and Antibiotics

(adapted from Cleveland et al., 2001)
Characteristics Bacteriocins Antibiotics

Application Food Clinical
Synthesis Ribosomal Secondary
Activity Narrow Broad
Host cell immunity Yes No
Mechanism of target cell Usually adaptation affecting cell Usually a genetically
transferable
Resistance or tolerance Membrane composition Determinant affecting different
sites depending the mode of
action
Interaction requirements Sometimes docking molecules Specific target
Mode of action Mostly pore formation, but in a Cell membrane or intracellular
few cases possibly cell wall targets
biosynthesis
Toxicity/side effects None known Yes
Table 2. Classification of bacteriocins (adapted from Garcia et al., 2010)
Class General features Examples of bacteriocins

I-Lantibiotics Modified, heat stable, <15 kDa
Ia-Linear Pore forming, cationic Nisin, Lacticin 481, Plantaricin C
Ib-Globular Enzyme inhibitors, nocationic None
Ic-Multi-component Two peptides Lct3147, Plantaricin W
II-Unmodified peptides Heat stable, <15 kDa
IIa-Pediocin-like anti-listeria, YGNGV consensus Pediocin PA1/AcH, Enterocin A,
Sakacin A
IIb-Miscellaneous Non-pediocin-like Enterocin B, L50, Carnobacteriocin A
IIc- Multi-component Two peptides Lactococcin G, Plantaricin S, Lactacin F
III-Large proteins Heat labile, >30 kDa
IIIa-Bacteriolytic Cell wall degradation Enterolysin A, Lcn972a
IIIb-Non-lytic Cytosolic targets Colicinsb E2-E9
IV-Circular peptides Heat stable, tail-head peptide bond AS-48, Gassericin A, Acidocin B
a
Lcn972 binds to the cell wall precursor lipid II and blocks cell wall biosynthesis, 15 kDa.
b
Colicins are synthesised by E. coli.
CLASSIFICATION OF BACTERIOCINS
A ―universal‖ classification of bacteriocins is still a matter of debate (Klaenhammer,
1993; Cleveland et al., 2001; Hoover and Chen, 2005; Garcia et al., 2010; Zacharof and
Lovitt, 2012).
Heng and Tagg (2006) proposed a scheme for classification of bacteriocins. Bacteriocins
are grouped into four main classes. Most LAB bacteriocins which have been applied in food
biopreservation belong to Class Ia, II and IV (Table 2).
Class I bacteriocin or lantibiotics contain 19 to more than 50 amino acids. Class I is

further categorized into Class Ia and Class Ib.
Class Ia bacteriocins constitute cationic and hydrophobic peptides that form pores in
target membranes and have a flexible structure compared to the more rigid class Ib.
Class Ib bacteriocins, which are globular peptides, have no net charge or a net negative
charge. Their antimicrobial activity is attributed to inhibition of specific enzymes
Class II bacteriocin include heat stable, nonlanthionine- containing, unmodified
peptides. In general, they are short cationic peptides with high isoelectric points.
They are further divided into three subgroups:-
Class IIa encompasses Pediocin-like Listeria active peptides with a conserved N-
terminal sequence Tyr–Gly–Asn–Gly–Val and two cysteines forming a S–S bridge in
the N-terminal half of the peptide.
Class IIb involves two different peptides for activity. The primary amino acid sequences
of the peptides are different. Though each is encoded by its own adjacent genes, only
one immunity gene is needed.
Class IIc covers those bacteriocins that are secreted by the general sec-system
Class III bacteriocin comprises large heat labile proteins with modest prospects as food
biopreservatives. Gram negative bacteriocins fall in this class except colicin V and
microcins.
Class IV bacteriocin includes circular peptides characterized by a peptide bond between
the C- and N-terminus are clustered and required carbohydrates and lipids moieties
for activity.
BACTIBASE
Bactibase is a helpful tool in accessing sets of bacteriocins for detailed analysis of a
number of microbiological and physicochemical data. It is data repository of bacteriocin
natural antimicrobial peptides, established by the joint efforts of Functional Proteomics and
Alimentary Bio-preservation Unit at Institute of Applied Biological Sciences Tunis
(ISSBAT), Tunisia with Nutraceuticals and Functional Foods Institute (INAF), Laval
University, Canada (http://bactibase.pfba-lab-tun.org/main.php). The current version of the
BACTIBASE dataset (version 2, July 2009) encompasses 31 genera of bacteriocin sequences
from 156 Gram-positive organisms and 18 of Gram-negative organisms with lactic acid
bacteria constituting the major group of producers of 113 bacteriocins. As per database, the
peptide length among the bacteriocins of Gram-positive organisms varies from 20 to 60
amino acids (84% of cases while Gram-negative bacteriocins have a very broad range of
lengths, the longest (BAC127) being 688 amino acid residues. Bacteriocin Genome Location
(BAGEL) is web based bacteriocin mining tool which helps to determine the presence of
bacteriocins gene from a (non-annotated) genbank file based on a database containing
information of known bacteriocins and adjacent genes involved in bacteriocin activity (Bali et
al., 2014).
Figure 2. Location and mechanism of antimicrobial action of nisin on bacteria (Adapted from
Raybaudi- massilia et al., 2009).
MODE OF ACTION
The mode of action of LAB bacteriocins has been extensively studied particularly nisin,
the first described gram positive bacteriocin, belonging to Class Ia lantibiotics (Hoover and
Chen, 2005; Deegan et al., 2006; Settani and Corsetti, 2008). Class Ia lantibiotics of
bacteriocins acts as antimicrobial agent by combining two modes of action such as pore
formation and inhibiting cell wall synthesis of the target agent ultimately leading to the death
of the organisms. (Figure 2) (Breukink et al., 1999; Wiedemann et al., 2001). This mechanism
is also used by other lantibiotics and non-pore forming bacteriocins such as the non-
lantibiotic Lcn972 (Martıńez et al., 2008). Several class II bacteriocins have been shown to
use the membrane-associated component of the mannose-phosphotransferase system as
specific receptor in target cells (Diep et al., 2007; Raybaudi- massilia et al., 2009; Balciunas
et al., 2013). Class III bacteriocins, i.e., bacteriolysin (e.g., lysostaphin) lyses the target cell
by cell wall hydrolytic activity (Johnsen et al., 2004).
COMMERCIALLY AVAILABLE BACTERIOCINS

For the use of biopreservative on commercial scale, bacteriocins have to fulfill certain
requirements of being non toxic, stable, should not affect the organoleptic properties of the
food products, economical, accepted by recognized authorities, and used in low
concentration. Bacteriocins from Lactic acid bacteria like Lactococcus, Streptococcus,
Pediococcus and Lactobacillus are of special interest as they are considered as GRAS and
commonly used for the production of fermented foods. Several type of bacteriocins have been
isolated and characterized from different bacteriocin producing strains of bacteria but till date
the only commercially produced bacteriocins are nisin (or group N inhibitory substance) and
pediocin PA-1 (Deegan et al., 2006; Chen and Hoover, 2003; Settanni and Corsetti, 2008;
Garcia et al., 2010).
NISIN
Nisin, produced by Lactococcus lactis subsp. lactis, was developed in the early 1960s and
is currently the most researched of all bacteriocins. It is a 34-amino acid post translational
modified polypeptide with a molecular mass of 3510 daltons. It comprises of unusual
thioether amino acids such as lanthionine (Ala-S-Ala), methyllanthionine (Abu-S-Ala), amino
butyric acid (Abu), dehydroalanine (Dha) and dihydrobutyrine (Dhb) (Figure 3). The name
nisin was coined by Mattick and Hirsch in 1947 from N inhibitory substance‖ since L. lactis
was originally classified as Lancefield serological group N Streptococcus. Nisin is the most
commercially important member of a large class of bacteriocins produced by bacteria.
Danisco‘s Nisaplin™ is generally considered to be the most commercially available form of
nisin for food preservative uses (Chen and Hoover, 2003; Deegan et al., 2006). Six different
forms of nisin has been discovered and characterized (designated as A through E and Z), with
nisin A is the most active type. It exhibits broad spectrum activity against various lactic acid
bacteria and other gram-positive bacteria. Moreover, it is particularly effective against heat-
resistant bacterial spores of Clostridium botulinum and against food-borne pathogens such as
Listeria mococytogenes, Staphylococcus aureus and Bacillus cereus. Use of nisin in
conjunction with ethylenediamine tetra-acetic acid (EDTA) may increase the effectiveness.
The effect of nisin can be enhanced by using exposure to chelating agents, sub-lethal heat,
osmotic shock and freezing, because these treatments make the cell wall of gram-negative
microorganisms more permeable and therefore more susceptible to the nisin (Chen and
Hoover, 2003; Gálvez et al., 2007; Lucera et al., 2012).
PEDIOCIN
Pediocin PA-1 is produced by Pediococcus acidilactici and marketed as ALTATM 2431.
The pediocin PA-1-containing fermentate ALTATM 2431 is a commercial food ingredient
reported to extend the shelf life of a variety of foods and, particularly, to inhibit the growth of
Listeria monocytogenes in ready-to-eat meat products (Rodríguez et al., 2002; Deegan et al.,
2006). Pediocin PA-1 is a 44 amino acid peptide with no postranslational modifications. Its
structure has been determined by Edman degradation of the purified peptide and by sequence
analysis of the structural gene (Figure 4). It has molecular weight of 4628 or 4624 Da, in the
absence or presence of two disulfide bonds, respectively. Pediocin PA-1 displays
antimicrobial activity against a wide spectrum of gram-positive bacteria, many of them
responsible for food spoilage or foodborne diseases. Pediocin does not cross the outer
membrane of gram negative bacteria which make it ineffective against gram negative
bacteria; but combination with other technologies such as freezing, gentle heating, exposure
to lactic acid or EDTA, or hydrostatic-pressure pasteurization cause sublethal injuries to the
outer membrane rendering their cytoplasmic membranes accessible to pediocin molecules
(Rodríguez et al., 2002).
USE OF BACTERIOCINS AS BIOPRESERVATIVE

IN VARIOUS FOOD SYSTEMS
LAB and their bacteriocins have been consumed unintentionally for ages, laying down a
long history of safe use. The bacteriocins produced by LAB possess several desirable
characteristics that make them suitable for food preservation such as they are nontoxic on
eukaryotic cells, become inactivated by digestive proteases, having little influence on the gut
microbiota, are usually pH and heat-tolerant, they have a relatively broad antimicrobial
spectrum against many food-borne pathogenic and spoilage bacteria, they are bactericidal in
action, usually acting on the bacterial cytoplasmic membrane and their genetic determinants
are usually plasmid-encoded, facilitating genetic manipulation (Galvez et al., 2007). There are
three basic methods of applying bacteriocins in food:
Figure 3. The structure of nisin. Abu = aminobutyric acid; Dha = dehydroalanine; Dhb dehydrobutyrine ; Ala-S- Ala = lanthionine, Abu -S- Ala- β methyl
lanthionine (adapted from Hoover and Chen, 2005).
Figure 4. Primary structure of pediocin PA-1.

Table 3. Effect of bacteriocins on various food systems
Food group Food Bacteriocin Target Effects References

microorganisms
Meat and Raw buffalo meat Nisin L. monocytogenes Decreased the population 2.4-log10 cycles lower than the Pawar et al., 2000
meat products control samples after 16-d storage at 4°C
Cooked tenderloin pork Nisin L. monocytogenes and inhibited the growth of L. monocytogenes, but not P. fragi. Fang and Lin, 1994
Pseudomonas fragi
Dry fermented Hornád Enterocin CCM L. monocytogenes Reduced the population by 3-og cycles Lauková et al., 1999
salami 4231
Poultry Sakacin K L. innocua Inhibited the growth of L. innocua Hugas et al., 1998
breasts and cooked pork
Raw meat Pediocin L. monocytogenes Inhibitory effect Rodriguez et al.,
2002
Sausages Enterocin AS-48 L. monocytogenes and Inhibitory effect Ananou et al.,
S. aureus 2005a, 2005b
Pork meat Pentocin 311 L. monocytogenes and Inhibitory effect Zhang et al., 2010
Pseudomonas
Sea food CO2 packed cold smoked Nisin L. monocytogenes 1 to 2 log reduction of L. monocytogenes Nilsson, 1999
salmon
Cold smoked salmon Sakacin P L. monocytogenes Inhibitory effect Aasen et al., 2003
VP cold-smoked Nisin L. monocytogenes Inhibitory effect Nykanen et al., 2000
rainbow trout
VP cold-smoked salmon Sakacin P L. monocytogenes Inhibitory effect Katla et al., 2001
Chilled shrimp Nisin Pseudomonas Shirazinejad et al.,
2010
Fruits and Kimchi Nisin L. plantarum, L. brevis The growth of Lactobacillus sp. was inhibited Choi and Park, 2000
Vegetables Lactobacillus more than the growth of Leuconostoc sp. suggested nisin
malefermentans can be used to preserve kimchi by inhibiting lactobacilli
Cider Enterocin B. licheniformis LMG Inactivate B. licheniformis LMG 19409 Gálvez et al., 1989
AS-48 from 19409
Enterococcus
faecalis A-48-32
Cooked mashed potatoes Nisin B. cereus and Reduce the growth of B. cereus and Thomas et al., 2002
B. subtilis B. subtilis up to 27 days at 8°C
Soy milk Enterocin L. monocytogenes and Inhibited the L. monocytogenes for 24 h, reduced the growth Lauková and
CCM 4231 S. aureus of S. aureus up to 3.55 log cycles. Czikková, 1999
Orange and apple juices Enterocin AS-48 A. acidoterrestris No growth was observed in both juices Grande et al., 2005b
DSMZ 2498 until the 15th day
Table 3. (Continued)
Food group Food Bacteriocin Target Effects References

microorganisms
Commercial orange, apple, Enterocin AS-48 A. acidoterrestris No viable cells were observed at 4, 15°C during three month Grande et al., 2005b
pineapple, peach and DSMZ 2498 incubation time, at 37°C growth was observed after 60 days
grapefruit juices
Tomato paste (pH 4.64), Enterocin AS-48 B. coagulans (CECT Effective against vegetative cells of B. coagulans but not Lucas et al., 2006
syrup from canned 561) effective against its spores
peaches (pH 3.97) and juice
from canned pineapple (pH
3.65)]
Apple juice Enterocin AS-48 B. licheniformis Reduced the growth of tested bacteria Lucas et al., 2006
Apple and orange juices Enterocin AS-48 A. acidoterrestris No viable cells wad detested 14 days at 37°C, Grande et al., 2005a
Milk and milk Cheddar cheese Pediocin PA- L. monocytogenes 2 log reduction in microbial population Buyong et al., 1998
products 1(AcH)
Munster cheese Pediocin PA- L. monocytogenes Suppressed growth up to day 11 of Ennahar et al., 1996
1(AcH) ripening and complete kill at day 21
Milk Pediocin 5 L. monocytogenes Reduction in the viable counts Huang et al., 1994
Manchego cheese Enterocin L. monocytogenes Ohio Inhibition of L. monocytogenes Ohio but not of L. Nuñez et al., 1993
and L. monocytogenes monocytogenes Scott A
Scott A
Quarg dessert Nisin Spore forming bacteria Reduction in the number of spore-forming Plocková et al., 1998
bacteria during storage at 4 and 20°C
Cottage cheese Lacticin 3147 L. monocytogenes Scott 99.9% reduction in growth within 5 d at 4°C McAuliffe et al.,
A 1999
Yoghurt lacticin 3147 Listeria Inhibited the growth of Listeria Morgan
and cottage cheese et al., 2001
Skim milk and Enterocin CCM S. aureus Inhibitory effect Laukova, 1999
yoghurt 4231,
yoghurt Enterocin CCM L. monocytogenes Inhibitory effect Laukova, 1999
4231,
Cereal Wheat dough Enterocin AS48 B. subtilis, B. 14 AU/gm effective against vegetative cells of tested Martinez
products licheniformis, B. cereus, bacteria, 23 AU/gm had inhibitory effect on endospore -Viedma et al., 2011
B. pumilus
Boiled rice Enterocin AS48 Bacillus cereus Inhibited the growth of Bacillus cereus Grande et al., 2005b
(-) = not known.
 Directly applied in food in a semi and purified preparation.

 The inoculation of a food product with lactic acid bacteria (LAB) that will
manufacture bacteriocin in the product itself.
 The use of an ingredient in food processing that has been previously fermented with
a bacteriocin producing bacteria (Garcia et al., 2010).
MEAT AND MEAT PRODUCTS

Fresh meat and fermented meat products can provide an ideal environment for the growth
of pathogenic and spoilage microorganisms. The microbial flora associated with meat
environment generally belongs to Entero-bacteriaceae, lactic acid bacteria and Brochothrix
thermosphacata, and pseudomonads. Listeria monocytogenes is present initially in low
numbers but during refrigeration storage it proliferates and cause spoilage in meat and meat
products (Nychas et al., 2008; Galvez et al., 2014). Nitrite commonly used in curing meats, is
to stabilize red meat color and inhibit food spoilage and poisoning organisms, such as
Clostridium botulinum; however, nitrite can react with secondary amines in meats to form
carcinogenic nitrosamines. Keeping in view the adverse health effect on humans, bacteriocins
have been found as an alternative to nitrite (Chen and Hoover, 2003). The most-studied
bacteriocins in meat and meat products include nisin, enterocin AS-48, enterocins A and B,
sakacin, leucocin A, and especially pediocin PA-l/AcH, alone or in combination with several
physicochemical treatments, modified atmosphere packaging, high hydrostatic pressure,
(HHP), heat, and chemical preservatives, as an additional hurdle to control the proliferation of
L. monocytogenes and other pathogens (Table 3) (Annou et al., 2007).
MILK AND MILK PRODUCTS

The microorganisms involved in the spoilage of milk and milk products include aerobic
psychrotrophic Gram negative bacteria, yeasts, molds, heterofermentative lactobacilli, and
spore forming bacteria (Aneja et al., 2008; Ledenbach and Marshall, 2009). LAB have a long
and safe history of use as preservatives in dairy fermentations where they are commonly
employed as starter cultures, especially in the manufacture of cheese. Much of the early work
on the use of bacteriocin-producing cultures during cheese manufacture related to inhibit the
growth of Clostridium which causes gas blowing in Swiss cheese. Nisin-producing lactococci
were applied in these situations and were found to be effective against clostridial spoilage.
While these early examples explained the potential of nisin-producing strains for inhibition of
Clostridium sp. However, nisin interferes with starter performance and ripening of cheese so
that interest in its use as a biopreservative in cheese making diminished. The main aim of
using nisin in cheese products is to provide protection against contamination with L.
monocytogenes creates a problem during cheese manufacture and ripening (Sullivan et al.,
2002). Literature search reveals that there are several other bacteriocins which have been
found as potential biopreservatives in milk and milk products to control pathogenic and
spoilage microorganisms as summarized in Table 3.
FRUITS AND VEGETABLES

Fruits and vegetables create an ideal environment for the survival and growth of many
types of spoilage microorganisms as they are composed of polysaccharide such as cellulose,
hemicelluloses and pectin which act as excellent nutrients for microbes. Spoilage
microorganism especially fungi release extracellular pectinases and hemicellulases that
degrade these polymers and cause spoilage (Barth et al., 2009). Lactic acid bacteria are used
as starter culture in various fermented vegetables such as table olives (Enterococcus faecium
BFE 900, Lactobacillus plantarum LPC010), Sauerkrauts (Leuconostoc mesenteroides
NCK293, L. lactis) and pickles (Settanni and Corsetti, 2008). Along with fermentation, starter
microorganisms can produce a wide range of antimicrobial compounds and proteinaceous
substances which can inhibit or reduce undesirable flora in food products (Ross et al., 2002).
The effectiveness of nisin has not been studied alone in literature against pathogenic
microorganisms in fresh cut fruits; nisin in combination with EDTA, sodium lactate and
potassium sorbate has been found to be effective in reductions of 1 and 1.4 log cfu/g the
Salmonella population in fresh cut cantaloupe (Raybaudi- massilia et al., 2009). In addition to
nisin, several bacteriocins with antimicrobial activity has been reported (Table 3).
SEAFOOD PRODUCTS
The high content of low molecular- weight nitrogenous compounds, neutral pH and high
water activity in seafood products makes them as excellent substrate for microbial spoilage.
Vibrio sp., Aeromonas sp., Shewanella sp., Photobacterium phosphoreum, Pseudomonas
cause spoilage during storage and producing fish like smell in seafood (Gram, 2009; Chahad,
2012). Pathogenic microorganisms such as Aeromonas sp., Clostridium botulinum, Cl.
perfringens, Escherichia coli, L. monocytogenes, Salmonella, Staphylococcusaureus, Vibrio
parahaemolyticus, V. cholera Serovar O1 and O139 are the other pathogenic microbes
reported from seafood products (Ghanbari et al., 2013). The inhibition of Listeria sp. by LAB,
mainly from the Carnobacterium genus has been found in, due to bacteriocin production or
competition mechanisms (Katla et al., 2001; Richard et al., 2003; Brillet et al., 2005; Vescovo
et al., 2006; Pinto et al., 2009).
CEREALS PRODUCTS
Cereal products comprise bakery items (bread, biscuits), refrigerated dough, fresh pasta
products, dried cereal products, snack foods and bakery mixes. Microorganisms responsible
for the spoilage of cereal products encompasses Aspergillus, Candida, Cladosporium,
Claviceps purpurea, Fusarium, Penicillium, Rhizopus, Saccharomyces, Zygosaccharomyces,
Bacillus, Clostridium, Lactobacillus and Leuconostoc (Cook and Johnson, 2009). Lactic acid
bacteria is used as starter culture in various cereal products which include breads (Lb.
sanfrancisco, Lb. brevis and Lb. fermentum), ogi (Lb. plantarum), Idli (Leuconostoc
mesenteriodes, Lb. delbrueckii, E. faecalis and L. lactis) (Caplice and Fitzgerald, 1999).
Corsetti et al. (2004) reported the application of bacteriocins from LAB on the stability of
sourdoughs over consecutive refreshments, reporting on the in situ activity of BLIS 2MF8
from L. pentosus 2MF8. Other bacteriocins, lacticin 3147-like bacteriocin from L. lactis M30
(Settanni et al., 2005) and amylovorin L471 from Lactobacillus amylovorus DCE 471 have
also been examined on the stability of sourdough (De Vuyst et al., 2004).
CONCLUSION
With the growing consumer awareness about the harmful effects of chemical additives to
combat undesired bacterial growth in foods, there is a growing demand for alternative
antimicrobial treatments and bacteriocins are well accepted natural means of selective
microbial inhibition. Moreover, the demand of minimal food processing is also providing an
opportunity for their widespread application. Bacteriocins are a diverse group of antimicrobial
proteins/peptides, many bacteriocins, such as lacticin 3147, pediocin PA-1, and enterocin AS-
48, have been shown to exhibit a large spectrum of food applications (Settani and Corsetti,
2008), however the use of bacteriocins in food is limited owing to its narrow activity spectra,
spontaneous loss of bacteriocinogenicity, limited diffusion in solid matrices, inactivation
through proteolytic enzymes or binding to food ingredients such as lipids, poor adaptation of
the culture to food environments, low production level, and the emergence of bacteriocin
resistant bacteria (Devileghere et al., 2004). The concentration of bacteriocin as a food
additive may exceed the concentration to which a consumer is normally exposed by ingestion
of fermented foods containing bacteriocin producing culture. If bacteriocins from LAB are
used at levels equivalent to that found in cultured foods, then they should be safe for
consumption. Toxicity testing, costs and required levels of bacteriocins must be necessary to
explain the effect of bactericins over the shelf life of product (Hoover and Chen, 2005). The
incorporation of bacteriocin with hurdle technologies develops for food preservation. They
have been included into packaging films and combined with modified atmosphere packaging
(Garcia et al., 2010). Galvez et al. (2007) have also reviewed the synergistic effect of
bacteriocins with emerging preservation technologies like high pressure processing or pulsed
electric fields which offer good opportunities for more effective preservation of foods,
providing an additional barrier to Gram negative bacteria and bacterial endospores. Extensive
research is still needed to invent more bacteriocins, which are commercially viable, have
GRAS status and could be used in biopreservation of shelf life extension of foods often
spoiled by dangerous human pathogenic microorganisms.
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Chapter 5
GENETICALLY MODIFIED FOODS:

CURRENT OVERVIEW
Jatinder Kaur and Priya Katyal

Department of Microbiology, Punjab Agricultural University, Ludhiana, India
ABSTRACT
Genetic engineering has paved way for development of Genetically Modified (GM)
food to satiate the need for high quantity and better nutritional quality food for growing
population. High yielding transgenic crops with disease, pest and stress resistance are
being introduced quite frequently. First world countries like US and third world countries
like India are taking a lead in their popularization. The GM crops are not without risks,
therefore they demand concern of various public awareness organizations which bring
into limelight issues like allergies and toxicity caused by GM food. The nations of world
have different views regarding the acceptance of GM food for their masses. They also
have varied regulations for GM food.
Keywords: genetic modifications, GM regulations, GM labelling, benefits and risks, global

scenario
INTRODUCTION
Food is the prime necessity for humans. There is a need for healthy, nutritious as well as
safe food. By the year 2050, it is assumed that human population would rise to 9 billion. The
increase in population would be prominent in developing nations, especially belonging to
Asian continent. The population in future would demand double the present food production.
The breadbaskets of world can produce only a fraction of the food. Moreover, the problem

Corresponding author: Priya Katyal. Department of Microbiology, Punjab Agricultural University, Ludhiana-
141004, India. E-mail: drpkatyal@pau.edu, tel: +91-9888507787.
96 Jatinder Kaur and Priya Katyal
would exaggerate due to uneven distribution of farmland. For instance, one quarter of world
population reside in China while it has only 7% of world‘s farmland (Kumar 2 12).
The improvement of plants and livestock for food production to combat growing
population has been practiced since last few decades. In the horde of more and better food,
scientists are taking the plant breeding and genetic engineering at the forefront. The last
century has witnessed enormous and valuable developments in the field of agriculture in
general and plant breeding in particular. Selection and cross-pollination remain the standard
choice of breeders, no doubt it has been supplemented with number of new and specialized
techniques which lead to genetic modification resulting in development of novel and better
strains. The world is now in a phase to witness second potential agricultural revolution, the
‗Gene revolution‘ which takes the advantage of genetic engineering to produce genetically
modified (GM) food to overcome the crisis of food. Genetic engineering has been used to
temper with the original gene sequences which leads to both genotypic and phenotypic
changes leading to development of new hybrid GM food. The advent of GMF is closely
linked to genetic engineering. It was in 1986 that first successful field trial of GM organism
was conducted by spraying genetically modified bacteria to protect strawberry from frost
(Juke 1986). In early 9 ‘s supreme court gave approval to GM food and hence the Flavr Savr
tomato (Figure 1) having altered gene for longer shelf life (Redenbaugh et al., 1992) and high
milk producing cows with recombinant Bovine Growth Hormone were available to
consumers. By 2000, 75% of food with ingredients from GM crops flooded US market.
Extensively cultivated GM crops include soybean, corn and cotton.
A steady increase in the total area under GM crop cultivation has been observed but for
the last few years the pace is bit slower. The impact of GM food on human and environmental
health is uncertain. The doubt of its being the sustainable solution to food problem adds to the
controversy. The fundamental aim of GM food is to conquer world hunger but there is a need
to analyse risks and benefits associated with it.
Different countries show verified acceptance to GM food. Europe has shown maximum
resistance to GM crop cultivation and import. US has always been a supporter of GM foods
until the fall of 2000 when a press group revealed the report of usage of GM corn in Taco
Bell® brand taco shells that was not approved for human consumption by the USDA. This
particular report caught the attention of common people all around the world (Pollack, 2000).
Figure 1. Genetically modified Flavr Savr tomato with increased shelf life.
Genetically Modified Foods: Current Overview 97
GENES
Each and every organism, from viruses to humans has a unique set of instructions which
regulates their development, growth and life. These instructions are found in the nucleus of
the cell in form of coiled structures called DNA, which is further divided into functional units
called genes. The genes are the primary structures which control different aspects of growth,
characteristics and functioning of an organism. The number of genes may vary from a few
thousands (in bacteria) to 50,000 (in maize) (Steinbrecher, 1996). Genes are blueprint for
nectar of flower, colour of maize grain, resistance to particular pest by plants and so on and so
forth (Shcmidt and Heslop-Harrison, 1998).
GENETIC ENGINEERING
It was next to impossible to transfer gene among different genera but the technology of
genetic engineering has broken the fundamental genetic barrier-not only between species but
also among humans, animals and plants. Biotechnologists are now snipping, inserting,
recombining, rearranging, editing and programming genetic material. Every now and then
unimaginable transgenic life forms are being created around the world (Gelvin, 1998). With
the staggering advancements in technology, scientists are now able to produce abundant food
at a cheaper and faster pace with improved features by alternating the gene codes. Seedless
grapes are one of the most interesting examples of genetic manipulation. Scientists inserted a
gene which prevented the outer coat formation around the seed of grapes. This alteration
resulted in dissolution of the seed due to absence of protective coat - leading to introduction
of seedless grapes in the modern world (Kumar, 2012) (Figure 2).
Source: United States Department of Agriculture, 2006.
Figure 2. Seedless grapes.

GM FOOD
The term GM foods or GMOs (genetically-modified organisms) is most commonly used
for crop plants created for human or animal consumption using the latest molecular
techniques. The plants are modified in the laboratory to enhance desired traits like increased
resistance to herbicides or improved nutritional content. The enhancement of desired traits
has traditionally been undertaken through breeding, but conventional plant breeding methods
can be time consuming and are often less accurate. Genetic engineering, on the other hand,
can create plants with the exact desired trait very rapidly and with great accuracy. Not only
the genes can be transferred from one plant to another, but genes from non-plant organisms
also can be used. For example, Bt gene introduced in corn and other crops has been isolated
from Bacillus thuringiensis, a naturally occurring bacterium that produces crystal proteins
that are lethal to insect larvae (Trevavus, 1999). Number of GM food are available in market
presently (Table 1). The introduction of GM food in our society has initiated discussion on
various aspects related to GM food like their nature, their safety and place in global world
food production. Concern is related to the GM food, of which some are tested and some are
already used as ingredients in the food we consume.
Table 1. List of Genetically Modified Foods
Genetically modified food Desirable traits incorporated

Rapeseed Pesticide resistant and erucic acid-free
Cotton Pesticide resistant
Sugarcane Pesticide resistant
Canola Pesticide resistant
Flax Resist herbicides
Papaya Virus resistant
Tobacco Reduced nicotine content
Peas Pesticide producers
Cow milk High production of milk
BENEFITS OF GENETICALLY MODIFIED FOODS

Genetic modification of crops mainly focus on four important aspects: increasing yield,
reducing the use of pesticides, herbicides, and fuel, improving the quality of foods, and
disease prevention by developing strains foods that work like edible vaccines. Other aspects
include disease resistance, drought or salinity tolerance and phytoremediation.
Insect Resistance
The soil bacterium Bacillus thuringiensis produce cry protein encoded by cry gene. This
particular protein is effective against many agricultural pests like beetles, moths and flies. Bt
protein specifically binds to epithelial cells of midgut, leading to pore formation and
ultimately result in death of insect (Ramanujan and Blum, 2000). In initial number of years
this proteinaceous compound has been used in thousand tonnes as biopesticide with no
adverse effect on the environment. However, due to its poor stability and high cost, they
didn‘t touch the market with a boom. Therefore to get around with the problem, a large
number of plants resistant to insect pests have been developed by introducing the Bt toxin
gene isolated from the bacterium. Many BT crops have been raised but BT corn (Figure 4)
has been notably accepted as the normal corn is prone to corn borer which used to cause
million dollar loss each year in European market. A drastic decrease in insecticides employed
on corn field in US has been observed on acceptance of BT corn by masses (Figure 3).
Figure 3. Bt corn uptake and insecticide use in US corn fields.
Herbicide Tolerance
Poorly controlled weeds can drastically affect crop yield and quality. Moreover, it is not
feasible to remove the weeds manually by tilling which is quite tedious and less cost
effective. Therefore, farmers prefer to spray large quantities of different herbicides to control
weeds which is an expensive process and can be harmful to crop and environment.
Development of GM crops which are resistant to one powerful dose of herbicide can
overcome the environmental issue by reducing the amount of herbicide employed. Biotech
companies like Monsanto supplies various GM plants that contain gene that makes the plants
tolerant to the herbicide glyphosate (trade name Roundup®). Glyphosate kills the weed by
blocking the amino acids synthesis pathway (Clark and Russell, 2000). Glyphosate is broad
spectrum herbicide and its single application can kill all the plants expect those having
tolerant gene. In addition, herbicides belonging to this class degrade in soil at a quicker pace
thus eliminating the problem of residue carry over in environment.
Disease Resistance
Plants are vulnerable to many viral, bacterial and fungal diseases. Genetic engineers are
working on the aspect to develop disease resistant GM crops Papaya ringspot virus (PRSV)
resistant strains of Papaya have been developed by scientists of University of Hawaii and
Cornell University. The virus takes minimum time for transmission and can destroy entire
transplantation if adequate measures are avoided. The modification used in papaya is one of
the most interesting types in which gene from the pathogen is inserted to fight the pathogen
itself and such modification is known as ‗pathogen derived resistance‘ (Tennant et al., 1994).
Cold Tolerance
Cold tolerant plants can be developed by molecular technology. Unexpected frost can
destroy sensitive seedlings. An antifreeze gene from cold water fish has been introduced into
plants such as tobacco and potato. With this antifreeze gene, these plants are able to tolerate
cold temperatures that normally would kill unmodified seedlings (Domingo, 2000).
Drought Tolerance/Salinity Tolerance
With increase in world population more land is utilized for accommodating human beings
rather than focussing on issue of food production. This would in turn force the farmer to use
unsuitable land for crop cultivation. GM crops tolerate to long period of drought and high salt
condition of soil can help the farmers to grow crop in formerly inhospitable places (Smith,
2003).
Edible Vaccines
Pharmaceutical medicines and vaccines are costly to produce and require defined storage
conditions which are unavailable in the third world countries. Therefore, GM foods are now-
a-days being used as edible vaccines. These vaccines are easier to store, ship and administer
than conventional injectable vaccines. Researchers at Loma Linda University School of
Medicine have succeeded in inserting a gene from the Vibrio cholera into potatoes. The GM
potatoes produce nontoxic cholera toxin that on intake of these potatoes cause triggering of
antibodies production against cholera (Arakawa et al., 1998).
Phytoremediation
Not all GM plants are grown as crops. Soil and groundwater pollution continues to be a
problem in all parts of the world. Plants such as poplar trees have been genetically engineered
to clean up heavy metal pollution from contaminated soil (Bizily, 2000).
BETTER FOOD QUALITY

Genetic modifications have been seen to improve food quality. Food with improved
vitamin, mineral, and nutritional content have been produced. Crops in development include,
higher protein containing soybeans, potatoes with improved amino acid content, pulses
altered to produce essential amino acids and crops with modified fatty acid profile. Melons
and pepper with improved flavour are under field trial. Golden rice (Figure 4) is one of the
examples that are genetically engineered to biosynthesize beta-carotene which in turn is
required for formation of Vitamin A. Plans are underway to develop a golden rice that also
has increased iron content. However, the grant that funded the creation of these two rice
strains was not renewed, perhaps because of the vigorous anti-GM food protesting in Europe,
and so this nutritionally-enhanced rice may not come to market at all (Normile, 1999). Most
importantly, modifications are intended to increase the overall yield of crops eventually
helping to satisfy the growing need of increasing population worldwide.
Genetically modified animals used as food are in news but application of genetic
modification in animals is slow due to social and ethical issues. Inspite of this fact, GM
salmon has been developed which grows to marketable size more rapidly than normal one.
Figure 4. GM golden rise with vitamin A.
RISKS OF GENETICALLY MODIFIED FOODS

Benefits of GM foods are enormous but their risk cannot be overlooked. Risks usually
include: exposure to possible allergens and toxins, harm impact on the environment, antibiotic
resistance development and spread of introduced genes to non-target plants throughout
crossing and pollen drift (Obrycki et al., 2001).
Nestle (1996) and Margulis (2006) reported possibility of toxic and allergic reactions to
GM food. Terra Prima organic corn chip company in Hudson, Wisconsin had to suffer due to
pollen drift of Bt corn planted few miles away from organic corn which was used for
production of Tortilla chips. Terra Prima had to recall and destroy 90,000 bags of chips which
was a significant monetary loss to the small company (Ramanujan and Blum, 2000).
Similar recall was made in 2000 when FDA identified gene product of Cry9C used to
protect plant against insect pests, in StarLink™ corn. The EPA had approved StarLink™ corn
in 1998 but with a condition that it was not to be used for human consumption. Cry9C protein
produced in the modified corn is heat stable and resistant to stomach acids and enzymes
which lead to allergic reactions. Aventis failed to segregate StarLink™ corn from non
genetically modified corn which led to massive recall of products from the market (USDA,
2000).
GM food can cause unintended harm to other organisms. A laboratory study was
published in Nature (Losey et al., 1999) showing that pollen from Bt corn caused high
mortality rates in monarch butterfly caterpillars. Monarch caterpillars consume milkweed
plants, not corn, but the fear is that if pollen from Bt corn is blown by the wind onto
milkweed plants in neighbouring fields, the caterpillars could eat the pollen and perish.
Although the Nature study was not conducted under natural field conditions, the results
seemed to support this viewpoint. GM food may reduce the effectiveness of pesticides. In a
way that some populations of mosquitoes develop resistance to pesticides like DDT, scientists
are concerned that insects will become resistant to Bt or to other toxins that have been
produced in genetically modified crops.
METHODS FOR GENETICALLY MODIFYING FOODS

Foreign DNA can be introduced by three major methods: biological vectors (Ti-plasmid
from Agrobacterium) (Figure 5), physical methods (particle gun and electroporation), and
chemical methods (polyethylene glycol and calcium chloride) (Hemmer, 1997). Out of the
three methods, the biological vector system is most frequently used (McBride and
Summerfelt, 1990).
Addison Wesley Longman, Inc.
Figure 5. Agrobacterium tumefaciens mediated GM crop development.

For efficient working of transgene in the host cell, a promoter and terminator sequence
are required which together along with gene of interest make the gene cassette. Many
potential promoter elements have been identified, but the most commonly used is the
CaMV35S promoter derived from the phytopathogenic cauliflower mosaic virus (Spoth and
Strauss, 2000) while NOS terminator from the Ti plasmid in Agrobacterium tumifaciens is the
most common terminator. There are diverse and complex techniques involved in genetic
engineering but its basic principles are quite simple.
Six major steps are involved in the development of a genetically engineered crop (Hain
and Ehly, 2000) (Figure 6).
1. The DNA is extracted from the organism possessing the trait of interest.
2. Gene of interest is isolated and cloned for further use.
3. Mass-production of the cloned gene is carried out in the host cell.
4. The cloned gene is packed and expressed inside the host plant and thousands of
copies are generated.
5. The packed gene is then introduced into the cells of target plant through a process
called transformation.
6. Finally backcross breeding is carried out, which aids in the transgenic crop breeding
and selection in order to obtain high quality plants that express the inserted gene.
The time duration in developing transgenic plant depends upon number of factors like the
gene, crop species, available resources, and regulatory approval. It may take as many as 6-15
years for a transgenic hybrid to be released for commercial purpose.
Jamiepigjinz, 2003.
Figure 6. Steps involved in development of transgenic crops.

DETECTION OF GENETICALLY MODIFIED FOODS

Methods employed for detection of genetic modifications vary among different
companies and countries, but basically there are two basic means for detecting genetic
modification. The first method determines the product of transgene, usually a protein, while
in another method the DNA fragment from transgene or gene cassette is analysed. Proteins
are assayed using an ELISA (Enzyme Linked ImmunoSorbent Assay) which is comparatively
less costly, quicker when compared with DNA tests and can sometimes be done on site
(Tripathy, 2005). For assay, sample is added to a multi-well plate having immobilized GM
food specific antibody. If GM ingredients are present they will bind to antibody. After
washing, another antibody with tagged enzyme, specific for protein of interest is added to
plate the tagged antibody bind to GM food ingredients. After another rounds of washings, the
substrate for enzyme is added which induces colour change (Holst-Jensen, 2001). The degree
of colour change produced is directly proportional to amount of GM protein (Figure 7).
The biggest drawback of ELISA is that it does not work well on processed foods as the
heat during processing destroys the protein. On other hand, DNA tests are expensive, cannot
be conducted in situ, and take several hours for complete analysis. However, DNA tests are
accurate and can work on processed foods. DNA is extracted and Polymerase Chain Reaction
(PCR) is run with GM specific primes to detect change in DNA structure (Brandner, 2002)
(Figure 8).
Figure 7. Detection of GM food by ELISA.

Kit developed by Biorad.
Figure 8. Detection of GM food by DNA-PCR.

GLOBAL SCENARIO OF TRANSGENIC PLANTS

Though the Government of India has put hold on field trials of 15 genetically modified
(GM) crops, globally, more and more farmers are adopting GM crops to increase
productivity. US is the leading producer of GM crops followed by Brazil and Argentina
(Figure 9). Over 18 million farmers planted GM crops last year in over 181 million hectares
in 28 countries. Over 90% of these are small farmers and GM crops have helped more than
16.5 million small farmers and their families worldwide (Bureau, 2015).
Calculations based on ISAAA, 2014.
Figure 9. Top six countries involved in production of GM crops.
Global land under GM crops cultivation has shown a drastic increase from 20 million
hectares in the mid-nineties to 181 million hectares in 2014 (Clive, 2014; Figure 10). It is
estimated that 81% of the total soybean crop is GM. Third world countries have given
approval to transgenic crops for example Bangladesh has approved Bt brinjal last year and
120 farmers planted the crop in the country. Other developing countries in Asia like Vietnam
and Indonesia have approved cultivation of drought tolerant sugarcane. The success of Bt
cotton in India can be figured out by examining the fact that over 90% of the total cotton
produced in the country is transgenic (Table 2). A report by the International Service for The
Acquisition of Agri-Biotech Applications quoting Brookes and Barfoot‘s latest provisional
estimate indicate that India had enhanced farm income from Bt cotton by $16.7 billion in the
12-year period 2002 to 2013 and $2.1 billion in 2013 alone. The government in India should
put in place a proper regulatory system which ensures that public health concerns are
addressed and farmers‘ interests are taken care of.
Clive James, 2014.
Figure 10. Global Area of Biotech crop in million hectares.
Table 2. Thirteen years of adoption and commercial release of BT cotton in India
Year Adoption Total % Bt Number % of % of Cotton Cotton Total

of Bt cotton cotton of Bt single double production yield insecticides
cotton area area cotton gene gene Bt (M Bales) (Kg/ha) to control
(Mha) (Mha) farmers Bt cotton bollworms
(Million) cotton (Metric tons)
2002-03 0.05 7.7 1 0.05 100 - 13.6 302 4470
2003-04 0.1 7.6 1 0.08 100 - 17.9 399 6599
2004-05 0.5 8.9 6 0.3 100 - 24.3 463 6454
2005-06 1.3 8.9 15 1.0 100 - 24.4 467 2923
2006-07 3.8 9.2 42 2.3 96 4 28 521 1874
2007-08 6.2 9.4 66 3.8 92 8 31.5 567 1201
2008-09 7.6 9.4 81 5.0 73 27 29 525 652
2009-10 8.4 10.3 81 5.6 43 57 30.5 503 500
2010-11 9.4 11.0 85 6.2 30 70 31.2 475 249
2011-12 10.6 12.2 88 7.0 18 82 35.3 493 222
2012-13 10.8 11.6 93 7.2 10 90 33.4 489 -
2013-14 11.6 12.25 95 7.7 4 96 39 541 -
Complied by ISAAA, 2014.
INDIAN SCENARIO OF GM FOODS

India, a developing nation, is highly dependent on agriculture sector which generates
about one quarter of its GDP and help 2/3rd of its people to survive. Researches on transgenic
crops are being conducted by various government and private laboratories of India but none
of them have been commercialized. Many Indian institutes and organisations claim about
development of transgenic plants which are at initial evaluation stages. Department of
Biotechnology holds the responsibility for propagation of research in transgenic plants in the
country. Other national organisations involved in GM crop development include ICAR, DST,
CSIR and other private sectors. National research centre on Plant Biotechnology, New Delhi
and Jawahar Lal Nehru University, New Delhi are working on development of Transgenic
Mustard, Cotton, brinjal, Chick pea, lentil, pigeon pea, tomato, rice, cauliflower, potato, etc.
The rules and procedures for handling GMO are monitored by Institutional Bio-safety
Committee and Review Committee on genetic manipulation. The research institutes or private
Entrepreneurs are required to take approval from the above committees for development,
trials and marketing of DNA products. The Indian government has set out rules, regulations,
guidelines and other legal provisions keeping in mind the safety assessment of GM foods.
GM REGULATIONS
GM foods have been one of the most controversial topics in the few last years. A large
number of European environmental organizations, NGOs and public interest groups have
been constantly protesting against GM foods. Moreover, scientific studies based on effects of
GM on human health and its impact on environment has brought the issue of genetic
engineering into limelight (Fonseca et al., 2012; Losey et al., 1999; Nykiforuk et al., 2012).
Avoiding GM foods is more or less a preventive measure as no prominent evidence related to
negative effect of these have been reported. Across the world, governments have formulated
GMO regulations which are most often country or region specific.
The European GM food regulations are the most stringent and complex in terms of
interpretation therefore they leave no space for GM products. In United States, three different
government agencies, Food and Drug Administration (FDA), US Environmental Protection
Agency (EPA) and United States Department of Agriculture (USDA) have jurisdiction over
GM foods. A combination of regulations from all the three agencies needs to be followed so
as to carry on with GM food. Notwithstanding the regulations, it is estimated that up to 70%
of processed food on US supermarkets shelves contain genetically engineered ingredients.
Approximately 85% of corn, 91% of soybeans and 88% of cotton in cultivated in US is
genetically modified. GM soya bean, canola, corn, potato, sugarbeet and cotton have been
approved for sale in Australia by Food Standards Australia New Zealand (FSANZ), formerly
the Australia New Zealand Food Authority (ANZFA). In many developing countries, though
GMO regulations are formulated but are inconspicuous in case of sudden disaster when the
only aim of the government is to save the starving lot.
In Japan, the Ministry of Health and Welfare has announced that health testing of GM
foods will be mandatory as of April 2001 (Uozum, 1999; Saegusa, 2000). Currently, testing
of GM foods is voluntary. Japanese supermarkets are offering both GM foods and unmodified
foods, and customers are beginning to show a strong preference for unmodified fruits and
vegetables. India's government has not yet announced a policy on GM foods because no GM
crops are grown in India and no products are commercially available in supermarkets yet
(Jayaraman, 1999). India is, however, very supportive of transgenic plant research. It is highly
likely that India will decide that the benefits of GM foods outweigh the risks because Indian
agriculture will need to adopt drastic new measures to counteract the country's endemic
poverty and feed its exploding population.
Table 3. List of countries which have banned GM food
California counties of Mendocino, Trinity and Marin

South Australia
Japan
New Zealand
Germany: Ban on the cultivation or sale of GMO maize (corn).
Austria
Hungary
Greece
Bulgaria
Luxembourg
France
Madeira
Switzerland
India: Ban on GM eggplant
Some states in Brazil have banned GM crops entirely, and the Brazilian Institute for the
Defense of Consumers, in collaboration with Greenpeace, has filed suit to prevent the
importation of GM crops (Neto, 1999). Brazilian farmers, however, have resorted to
smuggling GM soybean seeds into the country because they fear economic harm if they are
unable to compete in the global marketplace with other grain-exporting countries. List of
countries which have banned GM food are provided in Table 3.
GM FOOD AND ENVIRONMENT

Despite the high adoption rates and future promises, there is a multitude of concerns
related to the impact of GM crops on the environment (Paparini and Romano-Spica, 2004). A
wide knowledge gap exists when we talk about effect of GM on our environment. Not many
reports are available on the impact of GM plants on the natural and agro-ecosystems. One of
the major risks associated with GM crops include their impact on non-target soil
microorganisms which play a fundamental role in crop residues degradation and in
biogeochemical cycles (Giovannetti et al., 2005). In addition, GM herbicide tolerant crops
allow the farmers to apply ―Broad spectrum‖ weedicide to their field, which kill other plants.
According to Carpenter and Gianessi (2001) genetically modified crops will lead to
decrease in plant biodiversity, thus reducing sustainability of the planet. In case of use of
insect resistant plants, it can cause the pest population to shift to another plant population
which was unthreatened before.
Transformed Bacillus thuringiensis (Bt) corn plants have been reported to represent a risk
as the modified crop express the Bt toxin in pollen which get further deposited on other plants
near corn fields. A detrimental effect of these is observed on non-target organisms that
consume these plants (Yu and Shepard, 1998). It has been reported that genetically modified
can harm the ecosystem by depleting soil microorganism which are very important for soil
fertility and or influence the micro-environments of other organisms (Giovannetti et al.,
2005). One of the reasonable steps after creating a transgenic plant is to evaluate its potential
benefits and risks to the environment (Poppy, 2004).
GM FOOD AND ECONOMIC ISSUES

GM plants are usually patented which adds to the cost and makes the marketing process
quite lengthy and costly. The farmers are forced to buy the seed from engineering companies
like Monsanto which enforce a high cost to their cultivation. It is a common perception that
patenting the GM crops would widen the gap between the developed and third world
countries. More number of companies and non-profits organizations should follow the
initiative taken by Rockefeller Foundation and offer their GM products at reduced costs to the
developing nations.
IMPORT OF GM FOOD
Rules governing WTO‘s Agreement on Sanitary and Phytosanitary restrict import of
unidentified GM food. Therefore, the government must establish guidelines for importing
GM foods. The guidelines should take into consideration following points (Josling, 2015):
 Type of GM food imported (whole or processed form)

 The country of origin of product
 Certificate of safety assessment of GM food complying with the guidelines of the
regulatory agencies in both the exporting and the importing country
 Labelling of GM food, including genetic information, percentage of GM material
present, possible health hazards associated with modified food and alterations in the
chemical/nutritional composition
GM foods can survive in world if the proper cautionary measures are adopted to ensure
that the consumers are aware of their existence. Every consumer has a right to know what
they are eating therefore in 1998, the European union passed a law, making labelling
mandatory.
LABELING OF GM FOOD
Labeling of GM foods and food products is also an issue of concern. Agribusiness
industries are of the view that labeling should be voluntary and influenced by the demands of
the free market. If consumers show preference for labeled foods over nonlabeled foods, then
industry will taken an initiative to consider the issue. On the other hand consumer interest
groups demand mandatory labeling. The FDA‘s current position on food labeling is governed
by the Food, Drug and Cosmetic Act which is only concerned with food additives, not whole
foods or food products that are considered ―GRAS‖-generally recognized as safe. The FDA
consider GM foods substantially equivalent to non-GM foods, and therefore do not go for
more stringent labeling. In order to label all GM foods and food products, Congress must
enact so as to bring significant changes in the existing food labeling policy. The following
questions must be answered before announcing the labeling of GM foods mandatory.
First of all, will the consumers support the cost of such an initiative? If the food
production industry is required to label GM foods, factories will need to construct two
separate processing streams and monitor the production lines accordingly. Farmers must be
able to keep GM crops and non-GM crops from mixing during planting, harvesting and
shipping. The industry will naturally add extra cost to the product which would be extracted
from the pockets of consumers.
The second question arises, what are the acceptable limits of GM contamination in non-
GM products? The EC has determined that 1% is an acceptable limit of cross-contamination,
yet many consumer interest organizations argue that only 0% is acceptable. Some companies
such as Gerber baby foods (Summit, 1999) and Frito-Lay (Gillins, 2000) have pledged to
avoid use of GM foods in any of their products. In addition, this leads to a connected query
that who would monitor the contamination levels?
What is the level of detectability of GM foods cross-contamination? Current technology
is unable to detect minute quantities of contamination, Therefore 0% contamination using
existing methodologies is not possible. Even 1% threshold is below current levels of
detectability. Finally, who is to be responsible for educating the public about GM food labels
and what would be the cost of such an education? Food labels must be designed so as to
convey accurate information about the product. Educating and informing the public about
GM food or product without hurting the public trust and causing fear is the greatest challenge.
An international trade agreement for labeling GM foods was established in January 2000
(Helmuth, 2000; Macilwain, 2000). More than 130 countries, including the US, the world's
largest producer of GM foods, signed the agreement. The policy states that exporters are
required to label all GM foods and the importing countries have the right to determine the
associated potential risks with the GM food and reject the same if they find it unsuitable. The
agreement may help the US government to resolve the domestic food labeling dilemma more
rapidly.
CONCLUSION
Inspite of the fact that GM foods have potential to overcome the problems related to food
and malnutrition scarcity, there are several challenges for the governments, especially
regarding safety testing, regulation, international policy and food labelling. It is a general
belief that genetic engineering is an inevitable way; however, we must proceed with caution
to avoid harm to the human health and the environment. It is assumed that unintended effects
of genetic modifications are likely to occur due to imprecise nature of the technologies
applied. Transformation will take place in an unpredictable location. Moreover amount
unknown amount of genetic material passed through and the environmental effects on
transferred recombinant DNA and its consequent protein can produce unidentifiable outcome.
This will lead to long term effects which can be as detrimental as the nuclear technology in
the past. It is important to establish an internationally harmonized framework for the safe
handling of recombinant DNA organisms within a few years.
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Chapter 6
BIOCOLOURS: AN INSIGHT INTO PRODUCTION,

APPLICATIONS, STABILITY AND REGULATION
Sunakshi Rastogi, Chetan Sharma, Anil K. Sharma,

Himanshu Aggarwal and Vikas Beniwal*
Department of Biotechnology, Maharishi Markandeshwar University, Mullana,
Ambala, Haryana, India
ABSTRACT
Colour has been the main feature of any food item as it enhances the appeal and
acceptability of food. This also act as an important parameter for sensory analysis and
consumer preference. In addition, the colour of a food substance is important to indicate
its freshness and safety. However, using synthetic colour could be harmful for the health
of a consumer. The scrutiny and negative assessment of synthetic food dyes by the
modern consumer have raised a strong interest in natural colouring alternatives.
Therefore in today‘s progressive world, there is a shift from synthetic to biocolour.
Presently, the demand for biocolour is increasing worldwide due to the increased
awareness of their benefits among public and also because of the recognized toxicity of
synthetic colours. Nature is rich in colours and pigment producing plants, insects,
microalgae and microorganisms as well (fungi, yeasts, bacteria). Among the molecules
produced by these microorganisms are carotenoids, melanins, flavins, quinones,
monascins, violacein, etc. Today the food industry and color suppliers are however
constantly motivated to work towards the improvement of technical and physical
properties of the color preparations. Development of cost-effective, viable technology for
the preparation of a food color and applications thereof in foods remains a major
challenge in current scenario. The Current chapter highlights about biocolours, their
production, extraction, stability, regulation and more importantly about their commercial
applications.
Keywords: biocolorants, pigments, microorganisms, biotechnology, extraction
*
Corresponding author: Department of Biotechnology, Maharishi Markandeshwar University, Mullana-133207,
Haryana, India. Email: beniwalvikash@gmail.com, Tel:+91-8059931063.
116 Sunakshi Rastogi, Chetan Sharma, Anil K. Sharma et al.
INTRODUCTION
Colour is a molecule that absorbs certain wavelengths of visible light and transmits or
reflects others whereas colorant is the activity of that molecule. Colorant becomes the most
sensitive part of any commodity not only for its appeal but also it enhances consumer
acceptability (Chattopadhyay et al., 2008). Being an age old practice, colours are one of the
most significant visual properties of food. The very first characteristic perceived by our
senses is the colour of food which not only determines its appearance, acceptance but also
helps in its recognition. A well-textured food, rich in nutrients and flavour, may not be eaten
unless it has the right colour (Joshi et al., 2003). Marketing strategy of food by major
manufacturers is greatly influenced by colour. Hypothesis is that all individuals are sensitive
to the colour of food. Appetites are also influenced and stimulated by colour and it may
sometimes discourage eating certain foods and diminish the desire for that food. Colours also
suggest the flavours that are anticipated while eating or drinking. Colours from bright green
spinach, ruby red strawberry and deep orange pumpkin that add visual delight are due to
naturally occurring colour compounds. Many studies have emphasized the relation of colour
with the flavour detection threshold, with the sweetness or salinity sensation, and more so
with the susceptibility for preference. Since last 20 to 30 years many food industries are being
processed to create visually appealing food which exhibits good taste and potentially utilized
by the common man (Chattopadhyay et al., 2008; Lakshmi, 2014).
Because of the consumer awareness and concern for a healthy balanced diet, there is an
increased interest in the food colorants of natural origin (Joshi et al., 2003). Despite the fact
that the commercial market is ruled by the synthetic pigments, some of them may be toxic,
carcinogenic or may cause severe damage to vital organs. Therefore one must emphasize
upon the use of natural colouring alternatives. Both natural pigments and synthetic dyes have
been extensively used in everyday life including foods/feeds, textile, paper, printing inks,
cosmetic, pharmaceuticals (Goswami and Bhowal, 2014).
According to Lakshmi (2014), there are different reasons to use colour additives for food
as follows: to restore the original food appearance because of the occurrence of changes
during processing and storage; to assure the colour uniformity; to intensify colours that are
normally found in food; improved colour verses food quality; to protect the flavour and light
susceptible vitamins; to give to food an attractive appearance making it more an appetizing
item; to preserve the identity or character by which food is recognized and finally to help in
the visual assignation of the food quality. The use of food colorants as additives in the food
industry is highly useful for both food manufacturers and consumers in determining the
acceptability of processed food. Currently, the European Union (EU) has authorized
approximately 43 colorants as food additives, whereas in US approximately 30 colour
additives have been approved. In both Europe and the US, most of the colour additives have
been derived from natural sources (Rymbai et al., 2011).
HISTORY
The earliest written record of the use of natural dyes dates back to 2600 BC in China
while addition of colorants to foods was reported in Europe during the Bronze Age. Around
Biocolours: An Insight into Production, Applications, Stability and Regulation 117
1500 BC in Egyptian cities, candy makers used to add natural extracts and wine to improve
the appearance. The first synthetic colour (mauvine) was developed by Sir William Henry
Perkin way back in 1856. The beginning of the 19th century is named for the bulk production
and recovery of synthetic colours from the petroleum derived products like aniline. Hence
they were called ‗coal-tar‘ colours because the starting material was obtained from coal
(Lakshmi, 2014). Historical development of biocolours has been further depicted in Figure 1.
Henna was used even before 2500 BC, while saffron use has been mentioned in the
Bible. Use of natural bio-colorants in food is known from Japan in the shosoin text of the
Nara period (8th century), which contains references regarding colouring soybean and adzuki-
bean cakes. Thus, it appears that coloured processed foods had been taken by the people of
some sections during that period. Other natural foods such as carrots, pomegranates, grapes,
mulberries, spinach, beets, parsley and flowers were also used as food colouring agents. Our
ancestors also used minerals and ores such as azure (copper carbonate), gold and silver leaf
some of which were downright poisonous if used improperly. In the late 18 ‘s the food
industry had a vast array of available synthetic colours. Chemically synthesized colours were
used extensively as they were easy to produce, less expensive, superior in quality and
properties. Moreover they can be easily blended without giving any off-flavour to foods
(Sharma, 2014).
The red beet colour (betanine) is still frequently used to colour fruit yoghurt. The British
Colour Index lists about 10,000 registered colours for certain applications such as food,
cosmetics, pharmaceuticals and toys but the use of chemical colours has been greatly
diminished; these sectors now mainly use natural colours. A number of chemically prepared
colours such as azo-colours plus aromatic amines and their degradation products such as
benzidine-analogue colours (BACs) have been proven to be actually, or potentially toxic and
carcinogenic. EU directives indicate which colours may be used for the colouring of
foodstuffs with no distinctions between synthetic and natural colours. These directives
stipulate how the colours can be produced and which demands of purity must be met.
The concept ―pigment‖ is primarily reserved for inorganic colour compounds which
include white titanium dioxide (E171) and CaCO3 (E170), diverse iron oxides(black, red or
yellow; E172), as well as aluminium (E173), silver (E174), and gold (E175). Some of these
are commonly employed in pharmaceuticals for the colouring of capsules and in cattle feed.
The above mentioned evolution has reintroduced the general use of natural colours of
biological origin. In addition, over the last decade a lot of attention has been paid to the
biotechnological synthesis of colours through microorganisms (Albermann 2011). Genetic or
metabolic engineering of microbial strains involved can lead to economical production on a
larger scale through processes of fermentation of renewable agro-substrates such as starch,
molasses, glucose or sucrose. Bio-colours are also completely biodegradable after their use.
PIGMENTS
Pigments are chemical compounds that absorb light in the wavelength range of the visible
region. Produced colour is due to a molecule-specific structure (chromophore). This structure
captures the energy resulting in excitation of an electron from an external orbital to a higher
orbital. The non-absorbed energy is reflected and/or refracted to be captured by the eye and
generated neural impulses are transmitted to the brain where they could be
interpreted as a colour (Vargas et al., 2000). They are not only present in leaves, fruits,
vegetables, flowers, bacteria and fungi but also in skin, eyes and other animal structures.
Pigments are water insoluble substances that are used to colour articles like ink, paper,
textiles, etc. whereas certain bio-pigments like anthocyanin are water soluble (Rymbai et al.,
2011).
Classification of Pigments on the basis of origin (Vargas et al., 2000)

Pigments can be classified by their origin as natural, synthetic or inorganic.
 Natural pigments are produced by living organisms such as plants, animals, fungi and
microorganisms.
 Synthetic pigments are the organic compounds obtained from laboratories.
 Inorganic pigments can be found in nature or reproduced by synthesis for e.g.,
titanium dioxide, gold, and silver.
Synthetic Colours
Synthetic colours are man-made colours which are not found in nature as azo-dyes.
Synthetic dyes made their advent in India in 18th century and gradually pushed natural dyes
into oblivion due to their superiority in the speed of dyeing or printing and the fastness of
colours. Synthetic dyes are based on toxic raw materials and intermediaries. The continuous
use of synthetic colours in textile and food industry has been found to be detrimental to
human health and also leading to environmental degradation. As the use of synthetic colours
in food increased, the safety concerns were also raised through numerous regulations across
the world. In the USA, only seven synthetic colours have been permitted. In India according
to ―The Prevention of Food Adulteration Act of India‖ the use of eight synthetic colours has
been specified in food commodities at a uniform level of 100 mg kg-1 permitted level.
As the present trends are shifting towards the use of eco-friendly and biodegradable
commodities globally, the demand for natural colorants is increasing day by day. Natural
colours are generally extracted from fruits, vegetables, roots, microorganisms and are often
called ―bio colours‖ because of their biological origin. When the microbial cells are used to
produce the colour, the term refers to microbial pigments.
BIO-COLOURS
Bicolour is a dye obtained from any seeds, fruits, vegetables, leaves, algae and insects
that is capable of colouring food, drugs, cosmetics. According to the application, a suitable
natural colour can be achieved by keeping in mind the factors such as pH, heat, light, storage
conditions and interaction with other ingredients of the formula or recipe. The storage
conditions for natural colours depend on the particular need of the product. A tightly sealed
container is best to store the product in a cold storage to preserve colour strength and quality
along with its degree of cooling point (Sharma, 2014).
BENEFITS OF BIOCOLOURANTS
The use of bio-colorants may show benefits over synthetic colours. They are less toxic,
less polluting, less health hazardous, non-carcinogenic and non-poisonous. Most of them are
water-soluble (anthocyanins) which facilitate their incorporation into aqueous food systems.
These qualities make natural food colorants attractive. Above all, they are environment
friendly and can be recycled after use. Thus, they attribute to food both for aesthetic value and
for quality judgement. Also they tend to yield potential positive health effects, as they possess
potent antioxidants. They have also been observed to possess antineoplastic, radiation-
protective, vasotonic, vasoprotective, anti-inflammtory, chemo and hepatoprotective activities
(Rymbai et al., 2011)
Figure 1. Figure showing the historical developments in biocolours (Joshi et al., 2003).
Natural colours can be obtained from two major sources i.e., plants (Mizukami et al.,
1978) and microorganisms (Ryu et al., 1989; Bhat et al., 2013). The accessible natural
pigments from plants have numerous drawbacks such as instability against light, heat, adverse
pH, low water solubility and often non-availability throughout the year (Mitra et al., 2014).
However, the latter are of great interest owing to the stability of the pigments produced and
the availability of cultivation technology. Microbial pigments are of industrial interest
because they are often more stable and soluble than those from plant or animal sources
(Gunasekaran et al., 2008).
MAIN SOURCES OF BIOCOLOURS

There are mainly three main sources of biocolours as given below:
1. Plants/Fruits/Vegetables (Annatto, Anthocyanin, Astaxanthin, Betanin, β-carotene,
Canthaxanthin, Capsanthin, Caramel, Lutein, Lycopene).
2. Animals/Insects (Cochineal, Carmine)
3. Microorganisms (Bacteria, Yeast, Moulds, Algae)
The scrutiny and negative assessment of synthetic food dyes by the modern consumers
have given rise to a strong interest in natural colouring alternatives. Some companies decided
to colour food with natural colours using mainly plant extracts or pigments from plants e.g.,
red from paprika, beetroots, berries or tomato; yellow from saffron or marigold; orange from
annatto; green from leafy vegetables. Penetration of the fermentation-derived ingredients into
the food industry is increasing year after year. Examples could be taken from the following
fields: thickening or gelling agents (xanthan, curdlan, gellan), flavour enhancers (yeast
hydrolysate, monosodium glutamate), flavour compounds (gamma-decalactone, diacetyl,
methyl ketones), acidulants (lactic acid, citric acid), etc. (Dufosse, 2006). For
biotechnological production of such colorants, plants and microorganisms are more suitable
due to their understanding of proper cultural techniques and processing. Nowadays,
fermentative production of food grade pigments are available in the market for example;
colour from Monascus sp., astaxanthin from Xanthophyllomyces dendrorhous, Arpink red
colour from Penicillium oxalicum, riboflavin from Ashbya gossypii, and β-carotene from
Blakeslea trispora and number of microorganisms produce bio-colors in good amount that
includes Serratia and Streptomyces (Chattopadhyay et al., 2008).
COMMON NATURAL COLORANTS (BIOCOLOUR)

DERIVED FROM PLANTS
1. Purple to Blue Color
Centaurea Cyanus (Cornflower) is used for colouring sugar, confectionaries and one of
the ingredients in tea. The petals of cornflower can be used in salad, cornbread muffins and
also used to garnish food items (Mazza, 2007).
2. Red Color
Annatto Annatto is both the name of the colorant and the tree providing the colorant.
Other names of the tree (Bixa orellana) are lipstick tree and achiote. Seeds of annatto are used
for coloring Gloucester cheese since the 16th century followed by Cheshire, Red Leicester
cheese and cheddar made in Scotland. In Spanish it is called as local saffron. In the European
Union, annatto has been given the E number (E160b) whereas in the US, annatto extract is
listed as a color additive "exempt from certification" which is informally considered to be a
natural coloring (MacDonald, 2000).A yellow to orange colour has been used over two
centuries as a food colour especially in cheese, dairy products and in various other food
products, derived from the outer layer of seeds of the tropical tree, Bixa orellana which are
covered by a red, resinous coating containing the pigments (Chattopadhyay et al., 2008). The
yellow to orange colour is due to the chemical compounds bixin and norbixin, which comes
under apo-carotenoid. The main pigment is bixin which is a cis-isomer and smaller amounts
of trans-bixin, norbixin (demethylated bixin), and trans-norbixin are also present. Norbixin is
used to colour cheese (e.g., cheddar) because it binds to the proteins. It may also be used to
colour beverages with neutral pH, e.g., flavoured milk drinks, but not with low pH because of
precipitation (Mortensen, 2006).
Beta Vulgaris Betanines or betalains, Betacyanins are natural dyes extracted from the red
beet (Beta vulgaris). Beetroot is a variety with a strongly colored root. The purple root owes
its colour to the presence of betalains. Betalains are found in other plants as well, but beetroot
is the only allowed source of betalain colorant in the EU and the USA (Mortensen, 2006).
They are largely used as food colorants in food products. Betanin is the major component
(95%) of the pigments in the extract and have a good flavour. The beet root extract contains
red, yellow and also bluish-red colour pigments depending on the content produced by a
compound known as betanin which is stable at higher pH range than red cabbage extract
(Chattopadhyay et al., 2008). It has wide applications in various food commodities such as
beverages, candy and dairy products, yogurts and ice cream. Besides betanine, another
pigment which is extracted from beetroot is vulgaxanthine (Sturzoiu et al., 2011).
3. Brown Color
Lawsonia Inermis (Henna) The constituents of this plant include essential oils as 1,4
naphthoquinone and 5-10% tannins, gallic acid, flavonoids, lipids, sugars, triacontyl
tridecanoate, mannitol, xanthones, coumarins (5-alkyloxy 7-hydroxycoumarin), 2-3% resins,
and 2% Lawsone (2-hydroxy-1,4-naphthoquinone). Among these, Lawsone has been used as
a coloring agent. The European Commission of Health and Consumer Protection Directorate-
General in 2005 has not approved Henna as a food color. This was agreed by FDA in 2006
because of mild toxicity of Lawsone (Sincich, 2002; Chengaiah et al., 2010; Rajashekaran et
al., 2012).
4. Yellow Orange Colour
Curcuma Longa (Turmeric) Since Harappan civilization, the use of turmeric has been
evidenced. It has been considered as poor man‘s saffron because it imparts yellow coloring
cost effectively and it is also used as an alternative to saffron. Turmeric is bright yellow
colorant can also be obtained from the ground powder of the rhizomes of turmeric plant.
Turmeric contains three pigments; the major one is called curcumin and the two others are
derivatives of curcumin (Delgado-Vargas et al., 2000). Curcumin is the primary pigment of
colour. It is generally used in various food industries for colouring. It is mainly used in dairy
products, beverages, cereal, pickles, sausages, confectionaries, ice cream, bakery and savoury
products. Turmeric oleoresin is water soluble but oil extract can be added to fat based foods
and at high pH, the extract turns orange. In the US, both turmeric oleoresin and curcumin are
allowed colorants whereas only curcumin is recognized as a colorant in the EU (turmeric
oleoresin may be used as a spice, though). Curcumin is insoluble in water and only slightly
soluble in vegetable oil. Curcumin is greenish-yellow, very much like lutein. It is preferred
over lutein because it is cheaper, but where light stability is essential, lutein is preferred
because curcumin is very prone to photo bleaching (Mortensen, 2006).
Tagetes Erecta (Mexican Marigold)Use of Marigold flower as source of food colorants

is known from Aztec civilization. Lutein from Tagetes erecta is a purified extract obtained
from marigold oleoresin. Lutein is extracted from the petals of marigold flowers with organic
solvents which impart yellow to orange color. It is used as a food coloring agent and nutrient
supplement (food additive) in a wide range of baked goods, beverages, breakfast cereals,
chewing gum, dairy product analogs, egg products, fats, oils, sauces, infant and toddler foods.
Marigold flowers are by far the most abundant natural source for commercial lutein
(Chattopadhyay et al., 2008).
Crocus sativus (Saffron) Saffron is the dried stigma of Crocus sativus. The flower is
light purple with thread-like red stigma, has been rated as a valued material. The colour
appears as a powerful yellow in applications such as saffron rice (Chattopadhyay et al., 2008).
In contrast to the other carotenoids, the pigment is not extracted from the raw material; rather
the whole stigma (possibly finely divided) is added to the food. The associated saffron flavour
and the high cost of saffron limit its use as colorant to special applications. In the EU, saffron
is not regarded a colorant but it is considered an ingredient or a spice (Mortensen, 2006).
5. Green Color
Chlorophyll is a green pigment and found in most plants, algae and cyanobacteria.
Chlorophylls, a group of fat soluble natural pigments, are obtained by solvent extraction of
grass material, lucerne and nettle. The principal coloring matters are the phaeophytins and
magnesium chlorophylls, which are highly unstable to light. The green color is due to the
pigments chlorophyll a (blue–green) and chlorophyll b (yellow–green) that occur together in a
ratio of about 3:1.22. Chlorophyll is converted to chlorophyllins in presence of alkali, which
renders it water soluble (Chengaiah et al., 2010). Chlorophylls constitute the most important
subgroup of pigments under tetrapyrrole derivatives. Chlorophyll a and b are found in most of
the plant groups (which only differ in the substitution of the tetrapyrrole ring), except algae
and bacteria. It is used in jam, jelly, candy, ice cream and in several other products
(Chattopadhyay et al., 2008). Being lipid-soluble, chlorophyll is extracted with the use of
organic solvents. Chlorophyll can be made water-soluble by saponification of the oleoresin, in
which case it is called chlorophyllin. Chlorophyll is not allowed in the US butlegal in the EU
when extracted from edible plants, nettle, grass, or alfalfa (Mortensen, 2006; Soltan and
Shehata, 2012).
COMMON NATURAL COLORANTS DERIVED FROM

ANIMALS AND INSECTS
1. Dactylopius Coccus (Cochineal)
The dye extracted from this insect and its eggs is Carminic acid (Carmine), which is red
in colour, which appears as magenta-red upon application. Water insoluble forms of carmine
exhibit a colour range from pink to purple. It is resistant to light, heat and chemical oxidation,
often more stable than synthetic food colorants but unstable at low pH. The water soluble
form is used in alcoholic drinks with calcium carmine but water insoluble form is used in a
variety of products. Together with ammonium, carmine is used in meat, sausages, processed
poultry products, alcoholic drinks, beverages, bakery and dairy products, including desserts
and sweets. Carmine is used as a food dye in juices, ice cream, yogurt, and candy. But as a
food dye it has been known to cause severe allergic reactions and anaphylactic shock in some
people. On an average, people consume one or two drops of carminic acid annually with food
(Chattopadhyay et al., 2008).
2. Sepia Officinalis L (Female Cuttlefish)
It has rich concentrates of orange-red pigment in the accessory nidamental glands. The
pigment is called Sepiaxnthine. The dye is called Sepia ink. The sepia pigment is used in
capsule printing ink which has been patented (European Patent Application EP1361258). It is
used in Spanish cuisine breaded and deep-fried cuttlefish is a popular dish in Andalusia. In
Portugal Chocoscom tinta is served as deep-fried strips cuttlefish in black ink.
3. Cephalopod
It is another member of the molluscan class, Cephalopoda. Cephalopod ink is generally

obtainable from fishmongers or gourmet food suppliers. In cooking, it is used as a food
coloring and flavouring agent in pasta and sauces (de Carvalho et al., 2005).
4. Monascus Purpureus
The red pigments produced by this fungus were traditionally used in oriental countries
due to its potential applications as food additives. The use of this color additive is not yet
regulated in the EU, US and Brazil, Philippines, Taiwan among other regions. Oriental
countries such as Japan make extensive use of these pigments since decades as water soluble
pigments in candies or red pigment for red rice wine (Erdogrul and Azirak, 2004; Shouqin et
al., 2005).
SOME MAJOR BIOCOLOURANTS OF FOOD INDUSTRY

The Common food grade biocolorants have been given in Table 1.
1. Carotenoids
It is one of the most important groups of natural pigments. These are lipid-soluble,
yellow–orange–red pigments found in all higher plants and some animals. Animals cannot
synthesize carotenoids, so their presence is due to dietary intake e.g., the pink salmon flesh
and many birds‘ plumage owe their colour to carotenoids. Plant, algae, fungal and synthetic
(nature-identical) carotenoids are allowed as colorants but not animal carotenoids.
Carotenoids can be divided into carotenes containing only carbon and hydrogen and
xanthophylls made up of carbon, hydrogen and oxygen. Carotenoids owe their name to
carrots (Daucus carota), and xanthophyll is derived from the Greek words for yellow and
leaf. Together with anthocyanins, carotenoids are the most complex class of natural food
colorants with around 750 different structures identified. The most important carotenoids are
carotenes which including (alpha carotene, beta-carotene, betacryptoxanthin, lutein, and
lycopene) and xanthophyll including violaxanthin, neoxanthin, zeaxanthin and canthxanthin
(Zeb and Mehmood, 2004; Rymbai et al., 2011).
1.1. β-Carotene
It is orange-yellow in colour, oil soluble but can be made into a water dispersible
emulsion. Carrot is a good source of β-carotene. It is listed as GRAS compound with no
restriction of usage level. But most β-carotene for commercial use is now derived from algae.
In Dunaliella salina and D.bardawil, higher amount of β-carotene accumulation has been
observed in response to a combination of high light intensity, hypersalinity and under nutrient
stress (Chattopadhyay et al., 2008). Oil palm, orange, apricot, mango, peach and pepper
contributed significantly in increasing β-cryptoxanthin and β-carotene concentrations of foods
(Rymbai et al., 2011). The E-number (E160a) actually comprises four different sources of
carotenes: plants, Dunaliella salina (algae), synthetic and Blakeslea trispora (fungus). Under
the EU legislation, plant carotenes may be derived from edible plants, carrots, vegetable oils,
grass, alfalfa and nettle but only from carrots according to U.S. legislation (Barth et al., 1995;
Mortensen, 2006).
1.2. Lycopene
Being a precursor in the biosynthesis of carotene, it is found in plants containing
carotene, usually at a very low (sometimes undetectable) concentration. It is an expensive
pigment and is very prone to oxidative degradation as compare to carotene, but highly stable
under a wide range of temperature and pH, hence used as common food colorant. It is
available in liquid form or as cold water dispersible powder. Though lycopene is found in
abundance in tomatoes in large proportion, but was also identified in about 70 plant species
including red pepper, onion, Rosa rubiginosa (rose hip), Taxus baccata (yew), Calendula
officinalis (marigold) and Citrullus lanatus (watermelon) but at low concentration (Rymbai et
al.,2011). Under EU legislation, lycopene (E 160d) is considered as food additives. The best-
known sources of lycopene are tomatoes, watermelon, guava. Besides lycopene, a tomato
oleoresin also contains appreciable amounts of β-carotene, phytoene and phytofluene
(Dweck, 2009; Mortensen, 2006).
1.3. Lutein
Marigold (Tagetes erecta) flowers are the most abundant natural source for commercial
lutein. Lutein is primarily found esterified with saturated fatty acids like lauric, myristic,
palmitic and stearic acid. Lutein is more yellowish-green than oil palm carotenes. Lutein is
not allowed as a food colorant in the US except for chicken feed. Lutein is also found in
Zucchini (Cucurbita pepo L. var. giromontia), green vegetables like cabbage, parsley, spinach
and some fruits (Rymbai et al., 2011). Lutein made from Aztec marigold also contains some
zeaxanthin (Muntean, 2005; Jothi, 2008).
1.4. Annatto
A yellow to orange color has been used for over two centuries mainly for colouring dairy
products (mainly cheese) and derived from the outer layer of Bixa orellana seeds (Haila et al.,
1996). The chief coloring principle is the carotenoid, bixin and norbixin. Norbixin is used to
color cheese (e.g., cheddar) as it binds to the proteins. It may also be used to color beverages
with neutral pH e.g., flavored milk drinks. The food colorants obtained from paprika
(Capsicum annuum) including red colour due to red carotenoids which are dominated by
canthaxanthin and capsorubin and yellow colour imparted by xanthophylls includes ß-
cryptoxanthin, zeaxanthin, antheraxanthin and ß-carotene (Perez-Galvez et al., 2003). Their
combination also produces a bright orange to red orange colour in food products. The saffron
coloring matter is crocin which is extracted from the dried stigmas and styles of the saffron
plant. It is water soluble and considered as the most expensive colorant as well as spice. The
yellow color has a powerful application such as saffron rice (Raina et al., 1996; Winterhalter
and Straubinger, 2000).
2. Flavonoids
They are thepolyphenolic compounds and imparts yellow colour of horticultural

products. These are divided into six different major classes (flavonols, flavanones, flavones,
isoflavones, flavonols and anthocyanidins) based on differences in molecular backbone
structure. Flavonols may to fade in strong light but flavones remain more permanent but paler
in colour. The leading representatives of flavone pigments are apigenin, kaempferol,

quercetin, myricetin, luteolin, tricin, and izoramnetin (Patel, 2008).
2.1. Anthocyanidins
They are highly coloured flavonoids. Anthocyanins are the glycosides of anthocyanidins
and are found more in plants than the parent anthocyanidins. Anthocyanins are a class of
compounds belonging to phenolic substances widely distributed in vegetables giving rise to
the blue–purple–red–orange colour of flowers and fruits. The most common anthocyanidins
are cyaniding (red-purple), delphinidin (blue-purple), malvidin (deep purple), peonidin (red),
petunidin (purple) and pelargonidin (orange-red) and the distribution of this pigment in the
horticultural plants is not even (Anderson and Francis, 2004). Some fruits contain a single
type of anthocyanin (e.g., cyanidin in apple, cherry, fig, etc.), some contain two major types
(cyanidin and peonidin as cherry and cranberry); or some with several anthocyanins giving a
variety of colors like red, purple, yellow and blue as in grape or raspberry or strawberry (He,
2010; Rymbai et al.,2011). Anthocyanin is used to color a number of non-beverage foods
including gelatin desserts, fruit fillings and certain confectionaries. Grape peel extract
(enocianina) also imparts reddish purple colour beverages (Chenier, 1994).
3. Betacyanins (Betalains)
They are obtained from the redbeet (Beta vulgaris) extract that are mainly used as a food
colouring agent. It contains red, yellow and also a bluish-red color pigments depending on
their content produced by a compound known as betanin which is stable at higher pH range
than red cabbage extract (Im, 1990). It has wide applications in different food commodities
ranging from beverages to candy and from dairy to cattle products. Turmeric is bright yellow
colorant can also be obtained from the ground powder of the turmeric rhizomes. Turmeric
contains 3–5% volatile oils and 2.5–6% yellow pigments, the curcuminoids, of which
curcumin predominates. Turmeric oleoresin is water soluble but oil extract can be added to fat
based foods and at high pH, the extract turns orange (Marmion, 1991).
4. Corn Endosperm Oil
It is a reddish-brown liquid composed chiefly of glycerides, fatty acids, sitosterols and

carotenoid pigments obtained by isopropyl alcohol and hexane extraction from the gluten
fraction of yellow corn grain. It is used in chicken feed as color additive (Chattopadhyay et
al., 2008).
5. Vegetable Carbon
This colorant is made by heating plant material at a high temperature, leading to

carbonization. Vegetable carbon is a very fine powder that is neither lipid nor water soluble.
The colour in application is gray or black depending on dosage. Vegetable carbon is not
allowed in the US but is allowed in the EU (Mortensen, 2006).
6. Caramel
In terms of volume, caramel is the largest ―natural‖ colorant. Caramel is made by heating
carbohydrates, which produces polymeric, brown, water-soluble pigments. Four different
types of caramels are recognized: plain caramel, sulfite caramel, ammonia caramel and sulfite
ammonia caramel. The sulfite and ammonia caramels are made by the addition of
ammonia/ammonium salts and/or sulfites. The caramels have different isoelectric points,
making them suitable for various applications. Thus, plain caramel is used for beverages with
high alcohol strength (e.g., whiskey) and so is sulphite caramel. Ammonia caramel is used in
beer and bakery goods, whereas ammonia sulfite caramel is primarily used for soft drinks
(e.g., cola), the largest application of caramel (Mortensen, 2006). The Common food grade
biocolorants have been given in Table 1.
MICROBIAL PIGMENTS
In the modern times, industries have resorted to using microbes as a source of many
valuable and industrially important compounds like antibacterials, antitumorals, antifungals,
vitamins, enzymes, etc. and latest addition to this list has been pigments (Palanichamy et al.,
2011). Microbial pigments are a promising alternative source for natural food grade pigments
and have a great potential for food applications due to their natural colour, safety to use,
medicinal properties, nutrients like vitamins, production being independent of season and
geographical conditions with controllable and predictable yield. The advantages of pigment
production from microorganisms include easy and fast growth in the cheaper culture medium,
independent of weather conditions and colours of different shades (Goswami and Bhowal,
2014). They display all the colours from rainbow including light or dark tinges and unusual
colours like black, white, brown, golden, silver and fluorescent green, yellow or blue.
Hence, microbial pigment production is now one of the emerging fields of research. A
wide range of colour rich and pigment producing microorganisms have been reported in
nature (bacteria, fungi, yeast, protozoa) which offer considerable scope for commercial
production of bio-pigments like carotenoids, anthraquinone, chlorophyll, melanin, flavins,
quinones, prodigiosins, monascins, violacein, etc. (Dufosse,2006). The major pigments
produced by microbes are red, yellow and blue. Most research has been focused on yellow
and red pigment production such as monascue produced by Monascus sp., carotenoid from
Phaffia rhodozyma, Micrococcus roseus, Brevibacterium linens and Bradyrhizobium sp., and
xanthomonadin from Xanthomonas campestris. However, study of blue bacterial pigments is
limited because many bacteria are not capable of producing blue pigment. Actinohodine-
related blue pigments are produced by Streptomyces coelicolor A3, mixture of violacein and
deoxybiolacein by Chromobacterium violaceum and Janthinobacterium lividum (Gupta et al.,
2011)
Table 1. Common food grade biocolorants (Chattopachyay et al., 2008; Shafagha

and Salimi, 2008; Giaccio, 2004; Rymbaie et al., 2011)
S.No. Biocolorants (Food grade) Examples

1. Carotenoid Carotene Β-Carotene (C40H56)
Lycopene (C40H56)
Xanthophyll Canthxanthin (C40H52O2)
Zeaxanthin (C40H56O2)
Lutein (C40H56O2)
2. Irodoid Picrocrocin (C6H26O7)
Crocin (C44H64O24)
3. Flavonoids Quercetine (C15H10O7)
Luteolin (C15H10O6)
4. Anthocyanidins Pelargonidin (R1, R2 = H) (C15H11O5+)
Cyanidin(R1 = OH, R2 = H) (C15H11O6+)
Delphinidin(R1, R2 = OH) (C15H11O7)
5. Tetrapyrrole Chlorophyll a (C55H72O5N4Mg)
Pigment diversity is due to differences in their chemical structure compositions and the
presence of specific chromatophores. Pigmented microorganisms have awakened the interest
of the scientific community because their role in taxonomic studies and their biotechnological
potential in processes like fermentation and bioprocess engineering. Expression of the
pigments can be affected by a number of environmental factors including oxygen availability,
nutritional condition, temperature, age of the colony and strain variation. Most of the bacterial
pigment production is still at the R and D stage. Hence, work on the bacterial pigments
should be concerned especially to finding cheap and suitable growth medium in order to
reduce the cost and increase its applicability for industrial production. Fermentation is an
inherently faster and more productive process as compared to other chemical processes so it is
more beneficial to use it for industrial production. Moreover, pigment production from
microbial sources has gained attention owing to public sensitivity regarding ―synthetic food
additives.‖ Microorganisms can be genetically engineered because their relatively large
strands of genes can be easily manipulated. As a result, microbial pigment production can be
increased in geometric proportions through genetic engineering, compared to the scaling up
methods of chemists (Kumar et al., 2015).
The success of any pigment produced by fermentation depends upon its acceptability on
the market, regulatory approval and the size of the capital investment required bringing the
product to market. A few years ago, some expressed doubts about the successful
commercialization of fermentation derived food grade pigments because of the high capital
investment requirements for fermentation facilities and the extensive and lengthy toxicity
studies required by regulatory agencies. Public perception of biotechnology derived products
also had to be taken into account. Nowadays, some fermentative food grade pigments are on
the market: Monascus pigments, astaxanthin from Xanthophyllomyces dendrorhous, Arpink
red from Penicillium oxalicum, riboflavin from Ashbya gossypii, β-carotene from Blakeslea
trispora. The successful marketing of pigments derived from algae or extracted from plants,
both as a food colour and a nutritional supplement, reflects the presence and importance of
niche markets in which consumers are willing to pay a premium for all natural ingredients.
This endeavour has the potential to lead to a more sustainable and environment friendly way
to colour the world with microbial pigments.
Pigments Producing Microorganisms
Different microorganisms (bacteria, moulds, yeasts and algae) have a capability to

produce pigments (Table 2). These microorganisms should have the following criteria to be
explored as biocolourant such as capability to use a wide range of C and N sources;
reasonable colour yield; should have tolerance to pH, temperature, mineral concentration and
possess moderate growth conditions; should be non-toxic and non-pathogenic; must be easily
separable from the cell mass (Joshi et al., 2003).
Table 2. List of pigment producing microorganism (Dufossé, 2006; Gupta et al., 2011;
Joshi et al., 2003; Malik et al., 2012; Venil and Lakshmanaperumalsamy, 2009)
Microorganism(s) Pigments/molecule Colour

Bacteria
Agrobacterium aurantiacum, Paracoccus Astaxanthin Pink-red
carotinifaciens, Xanthophylollomyces
dendrorhous
Bradyrhizobium sp. Canthaxanthin Dark-red
Flavobacterium sp., Paracoccus Zeaxanthin Yellow
zeaxanthinifaciens
Bacillus Riboflavin Brown
Brevibacterium sp. Canthaxanthin Orange yellow
Corynebacterium insidiosum Indigoidine Blue
Rugamonas rubra, Streptoverticillium Prodigiosin Red
rubrireticuli, Vibrio gaogenes, Alteromonas
rubra
Rhodococcus maris β-carotene Bluish- red
Haloferax alexandrines Canthaxanthin Dark red
Staphylococcus aureus Staphyloxanthin Golden yellow
Zeaxanthin
Chromobacterium violaceum Violacein Purple
Serratia marcescens, S. rubidaea Prodigiosin Red
Pseudomonas aeruginosa Pyocyanin Blue green
Xanthomonas oryzae Xanthomonadin Yellow
Janthinobacterium lividum Violacein Purple
Streptomyces sp. Carotenoids Yellow, red, blue
Photosynthetic bacterium, Halobacterium sp. Canthaxanthin Dark –red
Halobacterium salinarium Astaxanthin Pink-red
Bacillus subtillis Riboflavin Yellow
Bacillus thuringienis H-14, Streptomyces Melanin Dark brown
virginiae
Dietzia maris Canthaxanthin Red
Streptomyces echinoruber Rubrolone
Microorganism(s) Pigments/molecule Colour

Streptomyces aureofaciens CCM 323 Indigoidine Blue green
Algae
Dunaliella salina β-carotene Red
Fungi
Monascus purpureus Ankaflavin, Monascin Yellow-red
Penicillium oxalicum Anthraquinone Red
Blakeslea trispora, Fusarium sporotrichioides Lycopene
Monascus sp. Monascorubramin
Cordyceps unilateralis Naphtoquinone Deep blood red
Ashbya gossypi Riboflavin Yellow
Monascus sp. Rubropunctatin Orange
Blakeslea trispora, Fusarium sporotrichioides, β-carotene Yellow–orange,
Mucor circinelloides, Neurospora crassa, cream
Phycomyces blakesleeanus
Aspergillus ruber Physcion Yellow
Fusarium sp. Naphthoquinone Brownish, yellow
Penicillium melinii Atrovenetin Yellow
Haematococcus pluvialis Astaxanthin Red
Monascus roseus Canthaxanthin Orange-pink
Blakeslea trispora Lycopene Red
Pacilomyces farinosus Anthraquinone
Yeast
Cryptococcus sp., Saccharomyces neoformans Melanin Black
var. Nigricans
Yarrowia lipolytica Melanin Brown
Rhodotorula rubra Carotenoids Red
Phaffia rhodozyma Astaxanthin Orange- red
Rhodotorula glutinis Torularhodin
Actinomycetes
Streptoverticillium rubrireticuli Prodiogiosin Red
Streptomyces echinoruber Rubrolone
Some Important Microbial Pigments Used as Food Colorants
1. Riboflavin (Vitamin B2)

It has a variety of applications as yellow food colorant. Its use is permitted in most
countries. It has a specific affinity for cereal based products but its applications are somewhat
limited due to its slight odour and bitter taste. Its applications include dressings, beverages,
ice creams, tablets and other products. There are numerous microorganisms that produce
riboflavin fermentatively. Riboflavin fermentation could be classified into three categories:
weak overproducers (100 mg/L or less, e.g., Clostridium acetobutylicum), moderate
overproducers (up to 600 mg/L, e.g., yeasts such as Candida guilliermondii or Debaryomyces
subglobosus), and strong overproducers (over 1 g/L, e.g., the fungi like Eremothecium ashbyii
and Ashbya gossypi) (Kumar et al., 2015).
2. Monascus
Monascus sp. belongs to the group of Ascomycetes and particularly to the family of
Monascaceae. The genus Monascus can be divided into four species: M. pilosus,
M.purpureus, M. ruberand M. froridanus, which account for the majority of strains isolated
from traditional oriental food.The common names of this fungal product are Red Yeast Rice
(RYR), red rice, angkak, red leaven, benikoji (Japanese), hung-chu, hongqu, zhitai (Chinese),
rotschimmelreis (Europe), red mould (USA) and MFR (Monascus fermented rice). Red rice
has been consumed for centuries in the Far East, where it has been used as a colorant, a
flavour and a preservative, in particular for meat. In red rice, it is fermented with fungi
Monascus and it is most often sold in powdered form. However, the pigments may also be
extracted. At least six pigments are known to be produced by Monascus: the yellow monascin
and ankaflavin, the orange monascorubrin and rubropunctatin, and the red monascorubramine
and rubropunctamine. The levels of yellow, orange and red pigments are controlled by a
number of factors e.g., pH, strain and nitrogen source. The pigments are soluble in ethanol but
poorly soluble in water. However, they react with amines, including amino acids, to produce
water-soluble derivatives. The pigments high affinity for amino acids make them ideal for
colouring meat (instead of nitrite-curing) and surimi. Both red and yellow monascus
preparations are commercially available. Monascus is neither allowed in the EU nor the US as
a food colorant (Mortensen, 2006).
3. Spirulina
Cyanobacteria contain the green chlorophyll a and a blue pigment called phycocyanin.
Just like chlorophyll, phycocyanins are photosynthetic pigments absorbing the red light that
chlorophyll does not strongly absorb. Phycocyanins are water-soluble proteins containing the
covalently bound chromophore phycocyanobilin-a linear tetrapyrrole (compare to
chlorophyll). The blue colorant is known by the name spirulina, which is also the name of a
dietary supplement rich in proteins consisting of dried cyanobacteria (also called microalgae).
Spirulina has also been consumed for centuries as a nutritious food in Africa and South
America. Some confusion exists surrounding the name spirulina. Spirulina (the edible
cyanobacterium) is often described as the species Spirulina maxima and S. platensis.
However, the cyanobacteria used for human consumption is not Spirulina sp. but the related
genus Arthrospira (both of the family Oscillatoriaceae). The name spirulina (for the edible
cyanobacterium and the colorant) is so well established that it will probably persist for some
time to come. Spirulina (the dried cyanobacteria) may be consumed in the Western world as a
dietary supplement, but the (extracted) pigment is allowed neither in the EU nor in the USA
as a food colorant. However, it is used as such in other parts of the world e.g., Australia and
the Far East (Mortensen, 2006). In 2014, the FDA now allows spirulina to be used as a blue
colouring in the following applications: Frostings, Ice cream/frozen desserts, Dessert
coatings/toppings, Beverage mixes/powders, Yogurts, Custards/puddings, Cottage cheese and
Breadcrumbs.
4. Canthaxanthin
Canthaxanthin, a carotenoid, is commercially produced from the algae Haematococcus
lacustris. It is known mainly as the natural pigment of the orange yellow chanterelle
mushroom. It has various physiological functions and can be converted into vitamin A under
stress and used in poultry for the appearance of color shade of the yolk, also in cosmetics and
foods, mainly in dairy products (cheese), confectionary (soft and hard candy), fish and meat
products, fruit products, beverages, snacks, beer and wine (Miller et al., 1996). However,
canthaxanthin is not considered as food additive under EU regulation (Chattopadhyay et al.,
2008).
5. Arpink Red
It is a red coloured pigment produced by the fungus Penicillium oxalicum obtained from
the soil. It contains chromophore of anthraquinone type. The amounts of Arpink Red in
various food products was recommended by Codex Alimentarius Commmisio in and for
different food products amount (mg/kg) are as follows: Meat products (100); Meat and meat
products analogues (200); Milk products and ice cream (150); Confectionery (300) (Kumar et
al., 2015).
Production of Biocolour
The biocolours are used in every type of industry whether it is a food or pharma industry.
They are explored in almost all sort of food industry like beverages, confectionery, processed
foods, bakery and dairy products, pet foods, coloured sugar. Naturally the colour can be
obtained by following three ways: by biotechnological approach; by using certain enzymes
and by using microorganisms.
Biotechnology for the Production of Biocolour
Although there is a large number of biocolorants existing in nature, yet quite a meager
number of them are available from natural extracts. Therefore, biotechnology could be a
solution for providing more number of coloring compounds which are difficult to synthesize
by chemical means or traditional methods of extraction. Over the past few years, there has
been a good afford being made in the studies of biocolorants production through
biotechnology. For biotechnological production of such colorants, plants and microorganisms
are more suitable due to understanding of proper cultural techniques and processing
(Chattopadhyay et al., 2008; Aberoumand, 2011). Biotechnology may play a crucial role for
large fermentation of natural biocolorants. Strategic applications of biotechnology for
production of natural food grade biocolorants are described in Table 3. Biotechnology can be
explored in many ways for improving the natural food colours such as: Genetic modifications
for pigment production; improving the traditional methods for extraction of pigments;
microbial production of pigments; in vivo pigment production by plant tissue culture.
Table 3. Varied applications of biotechnology in the production of Biocolorant

(Chattopadhyay et al., 2008; Gupta et al., 2011)
Food grade bio- Source Large scale

S. No.
colorants Original Biotechnological Production
1 Monascorubramine - Fungus: Monascus purpureus Fermentation and
2 Riboflavin Milk Moulds: Ashbye bioprocess
gossypii, engineering
Eremothecium ashbyii,
Yeast: Candida
gulliermndii,
Debaryomyces
subglbosus
Bacteria: Clostridium
Acetobutylicum
3 Astaxanthin Plants Fungus:
Xanthophyllomyces
dendrorhous
Algae: Haematococcus
lacustris,
H. pluvialis compactin
resistant mutant
4 Canthaxanthin - Algae: Haematococcus
lacustris
Bacteria:
Bradyrhizobium sp.
5 Arpink red - Fungus: Penicillium
oxalicum var. armeniaca
CCM 8242
6 β-Carotene Daucus Fungus: Blakeslea Fermentation and
Carota trispora, Phycomyces bioprocess
blakesleeanus car S mutant engineering
Algae: Dunaliella salina, Organic farming
D. bardwil and Integrated
GM plant: Golden Rice Crop
Management
(ICM)
7 Betanin Beta Higher yielding plant Organic farming
vulgaris generated and ICM
through somaclonal variation
Hairy root culture Fermentation and
bioprocess
engineering
8 Cyanidin and Peonidin Ascherry, Higher yielding plant Organic farming
Canberry generated and ICM
through somaclonal variation
Cell culture Fermentation and
bioprocess
engineering
9 Lycopene Tomato GM fungus: Fusarium Fermentation and
sporotrichioides bioprocess
GM bacteria: Erwnia engineering
Uredovors
10 Zeaxanthin Corn Bacteria: Flavobacterium sp.
Plant tissue culture, microbial fermentation and gene manipulation are being explored
with respect to pigment production for development of new biocolours and improvement of
the existing ones. It can help in altering the metabolic pathways in the micro organism so that
the colour characteristics can be enhanced, resulting in a product with good appearance and
maximum production. Many food colours can be produced through microbial fermentation
with greater efficiency. The genes controlling the production and excretion of indigo have
been transferred into bacteria in order to produce the dye by fermentation rather than
extraction. Plant tissues are often considered to be an effective alternative method for the
production of natural pigments as Carotenoids, anthocyanins and betalins have already been
produced in plant cell cultures. Continuous production using currently available techniques
appears to be impossible because most pigments are not excreted by the cells but are stored
within them so the continuous commercial production of such colours through
microorganisms appear to be a viable proposition (Sharma, 2014).
Genetic Modifications for Pigment Production

In genetic modifications, hereditary apparatus of animal, plant or bacterial cell is altered
so as to produce different chemicals or perform new function. The alteration may be brought
about mainly by two means 1) appropriate mutation through physical and/or chemical means;
2) genetic engineering through recombinant DNA technique. Typically the pigment is
produced intracellularly in the organism. However, by cloning of genes responsible for
pigment production, it is possible to obtain hyperpigment producers. These hyperpigment
producers excrete pigment into the growth medium thus making the process more economical
for example extracellular secretion of the pigment by a mutant of Monascus (Sharma, 2014).
Improving Traditional Methods for Extraction of Pigment

The traditional methods for extraction of colouring agents usually involve disruption of
the source material followed by acidified aqueous extraction for anthocyanins, beet red,
cochineal, etc. or by solvent extraction for chlorophyll, carotenoids, annatto, etc. Various
efforts are being made to eliminate problems encountered in extraction and preparation of the
natural colouring materials by use of enzymes and/or microorganisms aided extraction from
their conventional sources (Sharma, 2014).
Enzymes Aided Extraction

Enzymes are used to facilitate the extraction, increase pigment yield, improve quality and
stability of the result and colour preparation. For the commercial extraction of astaxanthin
from Phaffia rhodozyma, extracellular enzymes produced by Bacillus circulans are used.
These enzymes digest the yeast cell wall and allow easy extraction of the pigment by acetone
or ethanol. Endopolygalacturonase prepared from Aspergillus sp. is used for the extraction of
β-carotene from tissues and juices of various vegetables such as spinach, lettuce, squash,
green pepper, carrot, etc. Glycosidic and proteolytic enzymes such as β-galactosidase and
bromelain are used in extracting pigment. In the case of algal biliproteins, proteins, protease
is used to release the protein bound pigment. Protenase treatment of cochinal extract produces
better quality cochineal. Similarly, treatment of pulped grape gives satisfactory extraction of
anthocyanin and fermentation time is reduced considerably. Addition of invertase to pectinase
treated beet juice increases betalins content by three fold when the juice is purified and
concentrated by membranes processing. Use of these enzymes in extraction of anthocyanin
from strawberries increases the pigment content upto 80%. The maximum liquefaction of
carrot can be achieved by cellulose plus pectinase treatment of the carrot which gives 25%
enhancement of carotenoids concentration in suspension. Pretreatment of orange peels with
enzymes (papain, cellulose or pectinase), enhances the recovery of pigment.
Microorganism Aided Extraction

The natural colouring extracts especially those obtained through aqueous extraction or
direct pressing of plant materials, although they contain required pigments but also known to
have high amount of unwanted dissolved solids like salts, organic acids, phenolics, sugars,
etc. Such high solid extract on concentration produces hygroscopic material which can have
several limitations for its use as powder as beetroot extract has a characteristic odour, high
nitrate and nitrite content, which limits its application in foods. In the same way, sugars in
anthocyanin extract produce furfural during concentration and drying of the extracts which
leads to pigment degradation. Therefore, to rectify such problems application of fermentation
using appropriate microorganisms during and/or after extraction is required. Approx 80% of
beet root juice solids are carbohydrates and nitrogenous compounds. Fermentation of the
juice using yeasts (Saccharomyces sp.) and moulds (Aspergillus sp.) reduces the amount of
solids substantially giving 5-7 fold increase in the yield of pigment on dry weight basis. The
dried product is free from beet root odour and has reduced nitrate content. In this fermentation
process, yeast cells and ethanol are the important by-products where yeast cells may be
harvested and used as a source of biomass for the animal food supplement and ethanol can be
explored for industrial use (Sharma, 2014).
Table 4. Production of food grade colors in plants through

biotechnological approach (PTC)
Food grade colour Method Plant References

Anthocyanin Cells Culture Vitis vinifera Kakegawa et al., 1995;
Aralia cordata Zhang et al., 1997;
Fragaria anansa Suvarnalatha et al., 1994
Perilla frutescens Zhong et al., 1991
Daucus carota Vogelien et al., 1990
Crocin Somatic Crocus sativus Chattopadhyay et al., 2008
Embryogenesis
Carotenoid, bixin and Relationship between Bixa orellana Siva and Krishnamurthy,
Norbixin degree of genetic 2005
diversity using isozymes
Betalain, betacyanin, Cell and Root Cultures Beta vulgaris Leathers et al.,1992; Kino-
Betaxanthins Oka et al., 1996
(portulaxanthin-II
andvulgaxanthin-I),
muscaauri-VII,
dopaxanthin and
indicaxanthin
Plant Tissue Culture (PTC) for Pigment Production
Cells culture is the most common practice for production of plant pigments as culture
ensures uniform quality and continuous production of pigments. Attempts have been made to
produce anthocyanin, betalins, carotenoids, saffron and other pigment through PTC. This
technique offers several advantages over the conventional plant sources. Various studies on
color production in plants are presented in Table 4.
Microbial Production of Biocolour
Fermentation is a process of obtaining products of commercial significance in large

quantities by the use of microorganisms owing to their fast multiplication rate. It is therefore
advantageous to produce natural colours from microorganisms as various pigments are
produced from them as Monascus, Blakeslea, Cordyceps, Euglena, etc. Production of
biocolour by culturing microorganisms has several advantages. The rapid growth of microbes
cuts production time to matter of days and the process leads itself to continuous operation
compared to plant or animal sources. Moreover, production can be obtained by bacteria, yeast
or moulds; the process is flexible and can be controlled very easily. Biocolours produced by
microorganisms include Astaxanthin, Prodigiosin, Napthoquinones, Carotenoids, Monascus
pigments, Phycobiliproteins (Sharma, 2014).
Bacteria
Carotenoids are very well known and highly popular as food colourants. The carotene
production by a pigmented strain of bacteria Bacillus (alkaliphilic yellow) has been
documented(Sharma, 2014). Bradyrhizobium sp. strain are known to produce canthaxanthin
(4, 4‘-diketo-β-carotene) (Lorquin et al., 1997) and the carotenoid gene cluster was fully
sequenced (Hannibal et al., 2000). This keto-carotenoid was also found in another
microorganism, an extremely halophilic bacterium, Halobacterium (Asker and Ohta., 1999).
Isorenieratene and hydroxyl derivatives were produced by Brevibacterium linens,
Streptomyces mediolani and Mycobacterium aurum. Among these, the food grade B.linens (or
Brevibacterium aurantiacum sp. nov) is of particular interest as this microorganism is found
in soft cheese. Its pigments have therefore been consumed by human for a long time. Culture
of Flavobacterium sp. in a nutrient medium containing glucose or sucrose, sulphur-containing
amino acids such as methionine, cystine or cysteine, pyridoxine and bivalent metal ions was
able to produce zeaxanthin 190 mg/L with a cell concentration of 16 mg/g dried cellular mass
(Shepherd et al., 1976).
In another study, apple pomace can be utilized for the production of microbial colours
using solid state fermentation (SSF). About 10-50g/L of apple pomace was incorporated in
the basic medium for the production of Sarcina sp., (dark yellow), Chromobacter sp., (dark
red) and Micrococcus sp., (light yellow). The production of pigment in apple pomace based
medium using SSF gives better yield of biomass and carotenoids. Chromobacter sp.
producing dark red colour, grew best at 35°C with pH of 6.0 for 48hrs of incubation period
(Joshi and Attri, 2006).
Algae
The Microalgae constitute a reservoir of substances of commercial value. The production

is based on exploiting their highly efficient photosynthetic machinery. Red algae (Rhodophta)
and blue-green algae (Cyanophyta) produce a group of highly light absorbing pigments based
on bilin or tetrapyrol skeleton. These phycobilin proteins have potential to be used as natural
colourants for food. The protein bound pigments are separated by protenase treatment and
extracted into dilute alkali. Pigment preparations either in water or alcohol are recommended
for use in ice candies, frozen confections, confectioneries and chewing gum (Sharma, 2014).
The alga Haematococcus lacustris contains large amounts of astaxanthin esters and has
been considered as a potential source of astaxanthin and is commercially produced using
bioreactor (Yuan et al., 1997). Also, echineone and canthaxanthin are identified in
Haematococcus cultures. Other studies, in vivo (Yamane et al., 1997) and in vitro
(Chumpolkulwong et al., 1997a) have shown that high astaxanthin production required high
level of oxygen and high C/N ratio but cell growth requires low C/N ratio and addition of
ethanol during the second stage enhanced the production of astaxanthin 2 times whereas
compactin resistant mutants of H. Pluvialis (compactin inhibits HMGR that strongly blocks
cholesterol formation) showed 2 times enhanced yield (Chumpolkulwong et al., 1997b).
Several species of marine micro algae such as Dunaliella bardawil and D. salina produce β-
carotene as their main carotenoid (Phillips et al., 1995).
Fungi
Fungal carotenoids have also been recently approved as future food colourants by the EU
for the production of polyketide azaphilone pigments. Non-toxigenic fungal strain like
Penicillium and Epicoccum sp. can be used as food colourants (Mapari et al., 2010).
Mold
The pigment production by Monascus genus, i.e., Monascus purpurous and M.anka, for
use as food colour is well known. Its crude pigment is red and marketed as red mold rice in
powder form for use as household as well as industrial food colouring. The pigment is
extensively used in oriental countries like china, Indonesia, Japan, etc. as general food
colorants or as colour additive in wine and also as preservative for meat. The crude pigment is
a mixture of red, yellow and purple pigmented polyketides. It can be made water soluble as
well as oil soluble. The pigment is heat stable and can be autoclaved. The production protocol
involves optimizing organism for growth and pigment production (Sharma, 2014).
Yeast
The Carotenoids astaxanthin produced by Phaffia rhodozyma is considered as an
important source of the natural pigment for colouring foods. The growth of P. rhodozyma on
7-10% B or C grade molasses gives 2-3 times more astaxanthin than with glucose or sugar
blend. Use of grape juice may also be a useful raw material especially where surplus of juice
is available. Temperature is an important factor in the production of type of pigment. At 20°C
mainly astaxanthin (80-85%) is synthesized. In addition to P. rhodozyma even food spoiling
xerophytic yeast produces highly pigmented colonies, which may also likely to contribute in
the natural colouring for food in near future (Sharma,2014).
Process Development with Fungal Cultures
The fungus Blakeslea trispora is a well-known β-carotene producer. The cell growth and
β -carotene production are enhanced in medium containing surfactants such as Span or Triton,
except Triton X-100 (Kim et al., 1997). HPLC analysis, stability tests and microbiological
tests have shown that the β-carotene obtained by fermentation of B. trispora complies with
the EC specifications. Another example of β-carotene production is of Phycomyces
blakesleeanus (Ootaki et al., 1996). B. trispora sexual stimulation leads to carotene
biosynthesis which increase yield up to 35 mg/g (Mehta et al., 1997). However, it is found to
have more potential while produced through fermenter (Cerdá-Olmedo, 2001). Several strains
of Monascus are exploited for commercial production of red and yellow pigments (Fabre et
al., 1993).
SSF of Monascus using rice as substrate is well known in China and Japan. The red yeast,
Xanthophyllomyces dendrorhous (formally Phaffia rhodozyma) synthesizes astaxanthin and
zeaxanthin as its main carotenoids (Roy et al., 2008). Commercial production of carotenoids
using microorganism has been achieved in case of astaxanthin, by red yeast fermentation.
Rhodotorula also synthesize carotenoids and different studies are done with R. glutinis but
other important species are R.gracilis, R. rubra, and R. graminis (Simova et al., 2004; Tinoi
et al., 2005). Ascolor Biotech of Czech Republic is awarded patents of compounds from new
fungal strains that produce a red colorant which can be applied in the food industries. The
strain Penicillium oxalicum var. Armeniaca CCM 8242, obtained from soil, produces a
chromophore of the anthraquinone type. After evaluation, the red colorant Arpink Red was
recommended as 100 mg/kg in meat products are in non-alcoholic drinks, 200 mg/kg in
alcoholic drinks, 150 mg/kg in milk products including ice creams and 300 mg/kg in
confectionery items (Sardaryan et al., 2004). Wood hydrolyzates are potential substrates for
carotenoid production with X. dendrorhous (Yamane et al., 1997). Pine (Pinus pinaster)
wood meals are used in the production of carotenoids. Pine meals are treated with cellulases
from T. reesei (Celluclast) and cellobiase from A. niger (Novozym). The enzyme hydrolyzed
broth can be used for growth of X.dendrorhous, resulting in high growth rate (up to 0.07/h)
and high cell density (0.47 g/g of glucose, dry wt) and carotenoid yield up to 1.8 mg/l (Parajo
et al., 1997).
Extraction of Pigments
One way of colouring a food is to add a strongly coloured food (e.g., black currant) to the
food item (e.g., raspberry jam) that is to be coloured. This is the approach used in home-
cooking where spices may impart colour such as in case of turmeric and paprika. However,
for industrial production, this approach presents a series of problems as low concentration of
pigments in most foods means that a large amount would have to be added to give the desired
shade; unwanted flavour and insoluble matter (e.g., peel and seeds). Therefore, pigments are
extracted to overcome the problems of low concentration, flavour and insoluble material.
Lipid-soluble pigments like chlorophyll and carotenoids are usually extracted with organic
solvents, which are subsequently removed, yielding an oleoresin rich in pigments but also
containing other material such as triglycerides, sterols, wax and other lipid-soluble
compounds. Water-soluble pigments such as carminic acid and anthocyanins are in general
extracted with water or lower alcohols (Mortensen, 2006)
Methods for Pigment Extraction
Solvent extraction is the conventional method that is usually followed to extract colors
from plant materials. Anthocyanin and betalain pigments which are water soluble, are
extracted from the raw material with water and sometimes with aqueous methanol. For
carotenoids extraction, hexane is the solvent of choice and acetone is good solvent for
extraction of pigment from the plant material. After thorough extraction of the plant material,
the extract is concentrated and subjected to purification steps by using column
chromatography. Identification and quantification of the pigment is performed by
spectrophotometry or by high pressure liquid chromatography (HPLC). The advancements in
extraction of pigments from plant materials were necessary as the use of organic solvents is
harmful both for health as well as for environment (Naidu and Sowbhagya, 2012). Now a
days different advance techniques are followed in colour extraction as mentioned below:
 High Hydrostatic Pressure (HHP)

 Pulsed Electric Field (PEF)
 Sonication-assisted Extraction
 Gamma Irradiation
 Enzymatic Extraction
 Membrane Technology
1. High Hydrostatic Pressure (HHP) and Pulsed Electric Field (PEF)

HHP, PEF and sonication belong to environment friendly and energy efficient
technologies that enhance mass transfer processes within plant or animal cellular tissues.
These techniques increase the permeability of cytoplasmatic membranes which in turn
enhances extraction of valuable cell components. It also decreases the dielectric constant of
water under HHP combined with temperature leads to a decrease in the polarity of the media,
contributing to the higher yield of total phenolics and other antioxidants. PEF is reported to
enhance mass transfer rates by electroporation of plant cell membranes, improving tissue
softness and thus influencing the textural properties. PEF is reported to be an ideal method to
enhance juice production, increase the extraction of valuable components better than the
yields obtained by enzymatic maceration (Mason and Zhao, 1994; Naidu and Sowbhagya,
2012).
2. Sonication-Assisted Extraction
This method is used to enhance mass transfer phenomena by cavitation forces where
bubbles in the liquid/solid extraction can explosively collapse and generate localized pressure,
causing plant tissue rupture and improving the release of intracellular substances into the
solvent. It is widely explored for the extraction of secondary metabolites e.g., tea, mint,
chamomile and ginseng. A few studies on application of sonication to extraction of food
colours have been reported (Nayak et al., 2007). The optimal conditions for extraction of
anthocyanin from grapes with 50% ethanol as extraction solvent and conditions of
ultrasonication extraction were optimized (Corrales et al., 2007). Spigno et al. (2006)
highlighted the advantages of a mixture of ethanol/water for anthocyanin recovery from grape
skins. Sivakumar et al. (2009) have reported the ultrasound-assisted extraction of natural dye
from beet root in higher yield for industrial application.
3. Gamma Irradiation
Its pre-treatment to a plant material, increases cell wall permeabilization, resulting in
enhanced extraction of cell constituents in higher yield (Sowbhagya and Chitra, 2010). Nayak
et al. (2006) studied the effect of gamma irradiation as a pre-treatment prior to the solid–
liquid extraction on betanin extraction in comparison with control beetroot extraction.
4. Enzymatic Extraction
Now a days, Enzyme assisted extraction of plant materials (pigments, antioxidants,
flavours and phytochemicals) has been explored. Enzyme pre-treatment cannot be a complete
substitute for conventional solvent extraction but can result in increased yield of value added
cell components and a reduction in time of extraction and amount of solvent consumption.
Based on this approach, enzymes have been explored as a means to enhance the extraction of
carotenoids in marigold flowers (Rodriguez-Saona et al., 2001).
Enzymes are used to facilitate the extraction, increase pigment yield, improve quality and
stability of the result and colour preparation. For the commercial extraction of astaxanthin
from Phaffia rhodozyma, extracellular enzymes produced by Bacillus circulans are used.
These enzymes digest the yeast cell wall and allow easy extraction of the pigment by acetone
or ethanol. Endopolygalacturonase prepared from Aspergillus sp. is used for the extraction of
β-carotene from tissues and juices of various vegetables such as spinach, lettuce, squash,
green pepper, carrot, etc. Glycosidic and proteolytic enzymes such as β-galactosidase and
bromelain are used in extracting pigment. Similarly, treatment of pulped grape gives
satisfactory extraction of anthocyanin and fermentation time is reduced considerably.
Addition of invertase to pectinase treated beet juice increases betalins content by three fold
when the juice is purified and concentrated by membranes processing. Use of these enzymes
in extraction of anthocyanin from strawberries increases the pigment content upto 80%. The
maximum liquefaction of carrot can be achieved by cellulose plus pectinase treatment of the
carrot, which gives 25% enhancement of Carotenoids concentration in suspension (Sharma,
2014). Sampathu et al. (2006) have reported that, treating chilli powder/flakes with a multi
enzyme preparation and extracting using a solvent mixture results in chilli oleoresin with
enriched pungency and color.
5. Membrane Technology
This technology is fast and a emerging technique for the concentration and separation of
macro and micro molecules based on molecular size and shape in biotechnology and food
processing industries. Advantages of membrane processing includes, improved product
quality with higher yield; utilization of by-products; temperature and pH sensitive products
can easily be extracted without alteration; environmental friendly as no harmful chemicals are
being used and less energy is consume (Downham and Collins, 2001). In recent years,
membrane processes such as microfiltration, ultrafiltration and reverse osmosis have gained
importance for the concentration of natural colours. These existing membrane processes have
limitations of concentration polarization, membrane fouling and maximum achievable
concentration (only up to 25obrix).
Application of Nanotechnology to Colorants
Nanotechnology has been applied to natural colorants to convert fat soluble pigments into
water soluble formulations. Since application of fat soluble pigments to foods can cause
problems like non-uniform distribution and spots of colours in the processed foods, it is
desirable to convert fat soluble pigments to water soluble formulations. To use a liphophilic
natural pigment (β-carotene) in water-based foods, the colorant was entraped in a matrix of
Ca+ cross-linked alginic acid. Effects of different parameters e.g., solvent, alginic acid,
calcium chloride concentration on nanoparticle morphology were evaluated (Astete et al.,
2009). The nanoparticle stability was assessed by measuring aggregation against pH,
oxidation and particle precipitation as a function of time. The synthesized particle measured
120-180nm when formed with chloroform and 500-950nm when synthesized with ethyl
acetate. The particles were negatively charged -70 to -80mV and were stable at pH 3 to 7.
Addition of calcium increased nano particle density and improved β-carotene protection
against oxidation (Naidu and Sowbhagya, 2012).
Factors affecting the production of microbial pigments (Joshi et al., 2013; Kumar et al.,
2015)
1. Temperature: An Incubation temperature play a vital role in production process and it

is mainly depends on the type of microorganism involved For e.g., Growth of
Monascus sp.requires 25-28˚C for the production of pigment whereas Pseudomonas
requires 35-36˚C for its growth and pigment production.
2. pH: pH of the medium affects the growth of the microbe and type of pigment
production. Optimum pH for Monascus sp. and Rhodotorula sp. is 5.5-6.5 and 4.0-
4.5, respectively. Neutral to slight alkaline pH favours lycopene formation whereas
acidic pH favours β-Carotene synthesis.
3. Carbon source: The mycelial growth of pigment producing microorganism is affected
by the type of carbon sources, e.g., glucose, fructose, maltose, lactose, galactose, etc.
4. Type of Fermentation: The SSF yields three fold more pigment production than
submerged fermentation. As in case of Monascus purpureus, yields are superior in
solid cultures than submerged, though media composition, pH and agitation also
affect pigment production.
5. Minerals: Minerals also plays an important role in pigment production. Zn (2×10-3 M
and 3×10-3 M) stopped the growth in liquid medium whereas in solid medium
vigorous growth and pigmentation was observed.
6. Nitrogen source: Ammonium chloride is best for production of Monascus pigment

followed by ammonium nitrate and then glutamate. Potassium nitrate is the poorest
nitrogen source, while glutamate proved outstanding for the pigment production.
7. Moisture content: In SSF, the higher level of pigment production by Monascus ruber
occurs at the 70% initial moisture level in substrate i.e., rice.
8. Aeration rate: When the Monascus pigments are produced from SSF, the bed of rice
is continuously aerated by sparging with humidified air (95–97% RH). A forced
aeration rate of higher than 0.5L/min reduces the production of the pigments and
biomass as a consequence of water loss from the bed. Highest levels of pigments are
obtained at forced aeration rates of between 0.05 and 0.2 L/min.
STABILITY OF PIGMENTS
Pigments are no different from other compounds in that heat, light and oxygen are
detrimental to their activity. Actually pigments are rather unstable because they strongly
absorb light and are highly unsaturated molecules. The pH plays an important role in the
pigment production. However, there are large differences in stability. Vegetable carbon and
caramel are very stable pigments, withstanding heat, light and oxygen very well. Carminic
acid and carmine are also very stable pigments though not as stable as vegetable carbon and
caramel. At the other end of the stability scale, turmeric is rapidly bleached by light and
beetroot pigments turn brownish even under mild heating conditions. Lycopene is much more
unstable than β-carotene. Knowing the limitations of each pigment means that a specific
pigment can be avoided for certain applications, in which the conditions are unfavourable for
the pigment and that alternatives can be sought or that attempts can be made to increase the
stability of the pigments by chemical modifications or formulation (Mortensen, 2006).
Various natural pigments used in food with their stability pattern are discussed in Table 5.
Table 5. Different food based natural pigments with stability pattern (Joshi et al., 2003)
Name of the pigment with Water/fat Heat Light Oxygen pH

source soluble stability stability stability Stability
Anthocyanins (Fruits) Water H H H L
Betataines (Beet root) Water M H H H
Bixin (Seed coat of Bixa Fat M to L L H -
orellana)
Norbixin (Seed coat of Bixa Water M to L L H -
orellana)
Caramel (Heated sugar) Water H H H H
Carotenes (Leaves) Fat M to L L L H
Chlorophylls (Leaves) Water H H H L
Polyphenols (Tea leaf) Water H H H H
Xanthophylls (Fruits) Water M H H L
High: H; Moderate: M; Low: L.
Tolerance and Allergies to Biocolorants
The plant pigments are the most important precursors of several nutrients (e.g., β-
carotene is the precursor of vitamin A and many other carotenoids) and they have been
reported to be present in the diet. As interest in food allergy and intolerance has increased in
recent years, efforts to identify foods and food constituents that may cause reactions have also
increased. Thus, a variety of foods and food constituents have been identified which cause
reactions (Hefle et al., 1996). The consensus adopted by the Codex Alimentarius Commission
of the WHO in 1998 experts for investigating food-colorants; they consider eight foods or
food groups to be the major causes of food allergy (Codex, 1998). Natural colour additives
are justifiably not included among the foods and food groups identified by the Codex. Lucas
et al. (1998) critically evaluated the available information and demonstrated that reactions to
natural colour additives are rare. Studies of turmeric and carotenoid pigments administered in
mixtures with other food colourings failed to definitely identify reactions to either colour
additive or also found no reports of sensitivities to grape skin extract or grape colour extract
and hence concluded that the ingestion of natural colour additives presents a very low risk of
provoking adverse reactions (Rymbai et al., 2011).
LIMITATIONS OF BIOCOLORANTS
Bio-colorants inspite of having several potential benefits, natural dyes have some
limitations as well as follows (Rymbai et al., 2011):
 Tedious extraction procedures of colouring component from the raw material.

 Low colour value and longer time make the cost of dyeing with natural dyes
considerably higher than synthetic dyes.
 Some natural dyes are fugitive and require a mordant for enhancement of their
fastness properties while some metallic mordents are hazardous.
 Difficulty in the collection of plants and their particular species.
 Lack of standardization methods for extraction and dyeing.
 Instability of colorants in food products during processing and their sensitivity to
temperature, oxygen, light and pH.
 Colorants in food products decolourised or degraded during storage such as
Anthocyanin degradation and brown pigment formation cause colour loss in food
products. Curcumin is very prone to photo bleaching and beetroot colour has low
heat stability.
However, stability of these dyes or colours in food can be maintained by adding dextrin
additives extracted from tart cherries or maltodextrin extracted from Roselle as a stabiliser.
Increased glycosidic substitution and acylation of sugar residues with cinnamic acids will
reduce water activity and enhance stability. Anthocyanin pigments in dried forms can exhibit
remarkable stability.
APPLICATIONS OF BIOCOLOUR IN FOOD INDUSTRY

Biocolours have wide applications, as they play different roles in different food products
and some of their applications have been discussed below:
Food Preservatives
Most of the natural biocolorants possess antagonistic activity to certain bacteria, viruses
and fungi for protecting the food from microbial spoilage (Bridle and Timberlake, 1997).
Some are also active against protozoa (Leishmaniabrasiliensis) and insects
(Calliphoraerythrrocephala). Carotenoids are also to known to act as sun screen for
maintaining the quality of food by protecting them from intense light. Norton (1997) reported
that corn carotenoids inhibit the synthesis of aflatoxin produced by Aspergillus flavus and A.
parasiticus by 90% and 30%, respectively.
Quality Control Markers

Generally for maintenance of good manufacturing practices (GMP), level of anthocyanin
is used as an indicator to evaluate the quality of colored food (Boyles and Wrolstad, 1993).
Anthocyanin profiles have been used to determine the quality of fruit jams and it easily
detects that labeled black cherry jam which is prepared from common red cherries is real or
not. Besides, adulteration of blackberry jam with strawberries can also be detected efficiently
by the analysis of pelargonidin and cyanidin-3-glucoside content (Chattopadhyay et al.,
2008).
Nutritional Supplements
Bio-colorants possess chemical compounds produced by plant cells which are known as
the vegetal active principles. These are sources for obtaining drug substances (biologically
active) and many other natural compounds used in various industries such as food,
pharmaceuticals and cosmetics with important commercial value. Carotenoids are also used
as vitamin supplements, since β-carotene is the precursor of vitamin A (Haliwell, 1996).
Riboflavin is another example of natural food grade bio-colorant which is an essential vitamin
source and available in milk and in several leafy vegetables, meat, and fish (Counsell et al.,
1979; Hari et al., 1994). Yellow β-xanthins, in addition to their potential role as food colorant,
may be used as a means of introducing essential dietary amino acids into foodstuffs. Cotton
seed oil is not only a colorant but also enriched with essential amino and fatty acids. Gossypol
is a fat-soluble yellow pigment that occurs in bound and free forms in cotton seed meal but
the bound form of gossypol is particularly used to combine with free amino acids (Conkerton
et al., 1995). Cochineal colorant supplemented diet is recommended for hyperactive children
(Rymbai et al., 2011)
Therapeutic Properties
Biocolorants may also play an important role in human health as they contain some
biologically active compounds which possess a number of pharmacological properties such as
antioxidant, antimutagenic, anti-inflammatory and antiartheritic effect (Nagaraj et al., 2000;
Saleem et al., 2004). Carotenoids also act as biological antioxidants, protecting cells and
tissues from the damaging effects of free radicals and singlet oxygen and also as a good
source of anti-tumor agent (Zeb and Mehmood, 2004).
Lycopene, is particularly effective at quenching the destructive potential of singlet
oxygen (Di Mascio et al., 1989). Lutein, zeaxanthin and xanthophylls function as antioxidants
in the macular region of the human retina and they also act against aging, mascular
degeneration and senile cataracts (Landrum et al., 1997). Betacyanin also contain antioxidant
and radical scavenging properties. Since betanin exerts a good bioavailability, red beet
products may provide protection against certain stress related disorders. It has been
established that flavonoids present in different plant products show good antioxidant activity,
sometimes better than the commercially available antioxidants (Frankel, 1993). Allomelanins
(free of proteins) from plants are found to suppress growth of tumorigenic cells of mammals.
Grape seed extract is the commercial source of powerful antioxidants known as oligomeric
proanthocyanidins (OPCs), also generically called pycnogenol, a class of flavonoids.
Canthaxanthin also shows antioxidant property. Astaxanthin is another naturally occurring
xanthophyll with potent antioxidant properties (Haila et al., 1996). Lycopene prevents
oxidation of low-density lipoprotein (LDL) cholesterol and reduces the risk of developing
atherosclerosis and coronary heart disease. Other health benefits of biocolorants include
enhancement of immune system function, protection from sunburn and inhibition of the
development of certain types of cancers (Rymbai et al., 2011). Epidemiological studies
indicated that there is a correlation between the consumption of chlorophylls and decreased
risk of colon cancer (Fernandes et al., 2007).
Market Trend of Biocolour
The demand for food colour in global market in 2000 was 2400 MT which increased to
3000 MT by the year 2005 and further to increase to 8000 MT by the year 2010 and is
expected to increase to 15000 MT by the year 2015. The investment in natural food colour
market across the globe has touched to US $1 billion and is continuously growing as there is
demand for natural food colours against synthetic food colours. The market for natural food
colours is estimated to increase by approximately 10% annually. Many of the raw materials
for colours and flavours require growing conditions which are more favourable in countries
outside the EU. This makes EU a large importer of colours and flavours. Total imports of
natural colours, flavours and thickeners by the EU amounted to Euro 2,055 million or 475
thousand tonnes in 2008. Developing countries like India and China may play a major role in
supplying natural colours either in processed forms or as raw materials to the EU markets.
The potential market for astaxanthin pigment is apprx USD 200 million per year. Natural
pigments cost 5 to 1 times more than their synthetic pigments for e.g., β-carotene produced
by microalgae, costs approx USD 1000/kg against USD 500/kg produced by synthesis.
Despite their high cost, customers prefer β-carotene produced by fermentation. Therefore, in
the present scenenario customer want natural and healthy foodstuff and same in the case of
colours. Although, natural colours are on the rise but they are unlikely to be a total
replacement for synthetic dyes. It was estimated that to provide sufficient vegetable dyes to
dye cotton alone, about 462 million hectare would be needed ie, 31% of the world‗s current
agricultural land, which appear unlikely. Thus, natural dyes is likely to occupy a small niche
market, unless technology of horticultural practices and pigments extraction is redefined and
standardisation on modern scientific lines and with incorporation of advanced

biotechnological tool for better extraction and yield (Carvalho et al., 2007; EIBI, 2010;
Fiszman et al., 2012). There is a list of few companies involved in commercial production of
biocolour (Sharma, 2014):-
 Roche Products Pvt. Ltd., Australia (Carotenoids)

 Roche Vitamins fine Chemicals, Canada; BASF, Germany; Gist Brocades,
Netherlands (β-carotene)
 Synthetic Industrial chemicals Ltd., India (Xanthophylls)
 Bush Boake Allen Co, USA (Natural colours)
 Cyanotech Corporation and Aquasearch Inc, USA(Astaxanthin and Phycobiliproteins
from spirulina)
 Overseal Foods Ltd, UK (Natural carotenes, lutein and paprika)
 Burman Laboratories Pvt Ltd, India (Spirulina capsules)
 Warner-Jenkinson Europe Ltd. (UK)
 Quest International (Netherland)
 Kalsec Inc. (USA)
Regulatory Aspects Regarding Food Colour
Today all food colour additives are carefully regulated by federal authorities to ensure
that foods are safe to eat and accurately labeled. Food colorants are tested for biosafety before
its promotion and are controlled by various regulatory bodies around the world and regulation
varies with countries (Hallagan et al., 1995). As in US, FD and C (Food, Drug and Cosmetic)
numbers are given to synthetic food colorants approved by FDA that do not exist in nature,
while in EU, E numbers are used for all additives of food applications. Thus, the approved list
of food colors varies along the countries, it means each country has its own approved list,
including limit of maximum daily intake. Out of them, some other regulatory agencies are
there like PMDA (Pharmaceuticals and Medicinal Devices Agency) in Japan, SFDA (State
Food and Drug Administration) in China, CDSCO (The organization and function of the
Medicines Agency) in India and KFDA (Korea Food and Drug Administration) in South
Korea, etc. But most of the food grade biocolorants approved by FDA or EU are also
approved by other agencies. For example in India, Rule 26 of The Prevention of Food
Adulteration Act (PFA) permits 11 colors for food use for e.g., Lactoflavin, Caramel, Annato,
Saffron, Curcumin, etc. these colours are also approved by EU and FDA (Chattopadhyay et
al., 2008).
Under FDA regulations, a colorant added to a food product cannot be considered natural,
no matter what the source is. Unless the colorant is natural to the food product itself, for
example; strawberry juice or red beet color is used to make the ice cream a pink hue for
strawberry ice cream, it would not be considered as naturally colored because the colorant
from strawberry or beet are not a natural component of ice cream. FDA considered only few
colorants as food additives (FDA/IFIC, 1993). In 1958, additives were redefined and
classified with three categories as:
 Substances approved by the FDA or the USDA (United States Department of

Agriculture) during 1938 to 1958.
 GRAS substances do not require FDA evaluation.
 All other substances used in food are evaluated to fit with the recommendations of
FDA before commercialization. The natural colors under section 205.606 of FDA list
are only allowed in organic foods.
FDA uses the term indirect additives to group those additives which are used in the
coloration for animal feeds and in course the animals are used as human food. As some of the
natural colorants of non biological origin are decertified (Red Dye #2, amaranth). But the
accepted natural pigments or colors (from red beet, carrot, fruits, pepper, etc.) are grouped
under ‗exempt of certification‘ category. Further, legislation is often based on local,
traditional usage of coloring matter. Thus, lac, monascus, gardenia and spirulina are important
colorants in some parts of Asia but none of them are allowed in the EU or in the USA, where
there is no traditional use of the raw materials. In India, FSSAI is monitoring the regulation of
addition of colouring additive to food (Mortensen, 2006; Rymbaiet al., 2011; Sharma, 2014).
Table 6. Permitted natural and nature-identical colorants in the EU and the USA
(Mortensen, 2006)
S. No. EU Name (E- number) US name (USCFR number)

1 Anthocyanins (E 163) Grape color/skin extract (73.169, 73.170)
Not allowed Cottonseed flour (73.140)
2 Annatto, bixin, norbixin E 160b Annatto extract 73.30
3 Beetroot red (E 162) Dehydrated beets (73.40)
4 Cochineal, carminic acid, carmines (E Cochineal extract, Carmine (73.100)
120)
5 Copper complexes of chlorophyll(in)s Sodium copper chlorophyllin (73.125)
(E 141)
6 Caramel (E 150) Caramel (73.85)
7 Canthaxanthin (E 161g) Canthaxanthin (73.75)
8 Curcumin (E 100) Turmeric (73.600, 73.615)
9 Chlorophyll(in)s (E 140) Not allowed
10 Ethyl ester of β-apo- 8'-carotenoic acid
(E 160f)
11 Lycopene (E160d)
12 Lutein (E161b)
13 Mixed carotenes (E160a (i)) Carrot oil (73.300)
14 Paprika extract (E160c) Paprika (73.340, 73.345)
15 Riboflavin (E101) Riboflavin (73.450)
16 Vegetable carbon (E153) Not allowed
17 β-Carotene (E160a (ii)) β-Carotene (73.95)
18 β-Apo-8'-carotenal (E160e) β-Apo-8'-carotenal (73.90)
EU directive indicates which colors may be used for the coloring of foodstuffs with no
distinctions between synthetic and natural colors. These directives stipulate how the colors
can be produced and which demands of purity must be met. The label declaration of the color
has to mention the name of the recognized E-number. In the EU and the USA, the allowed
natural and nature-identical colorants shown in Table 6 (colorants only allowed in fish or
chicken feed for pigmenting flesh and/or eggs are not included).
CONCLUSION
A few years ago, some expressed doubts about the successful commercialization of
biocolours derived through fermentation due to high capital investment requirements for
fermentation facilities and the extensive and lengthy toxicity studies required by regulatory
bodies. Presently, some fermentative food grade pigments are on the market and the
successful marketing of algae-derived or vegetable-extracted pigments as a food colour,
reflects the presence and importance of niche markets in which consumers are willing to pay
a premium for all natural ingredients. Fermentative biocolorants production has a number of
advantages; cheaper production, easier extraction process, higher yields, no lack of raw
materials and no seasonal variations. Today, some fermented colors are already in production
as D. salina, B. trispora, spirulina and Monascus. Therefore, in the current scenario,
producers as well as consumers are increasingly aware of health and environmental issues and
they are willing to pay extra bucks for biologically origin colours. The need of hour is to
incorporate some strategic applications of biotechnology for the production of natural food
grade biocolorants. As technological limitation are the major bottlenecks for the commercial
exploitation of the source materials. Designing of a proper bioreactor would help to ease out
the situation. Commercial production of colorant by plant cell tissue culture could be
achieved only when a fully automated predictable process with a more advanced technology
is to be established by the joint effort of biotechnology and bioprocess engineering. Though
some scientists have isolated mutant varieties with acquired character of food ripening and
carotenoid accumulation but most of the regulatory genes are yet to be isolated. There is
scope to develop new microbial strains or transgenic plants and animals for over production
of coloring compound. Therefore, a giant leap forward in color production could be achieved
by combining genetic manipulation and fermentation. To conclude, there is need for proper
methods for extraction, documentation and characterization of dye yielding plants and
animals and microrganims for further development of new biocolour. But few things have to
be kept in mind before the launching of new bio-colorants in market, its acceptability,
regulatory approval and the size of the capital investment required to bring the product.
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Chapter 7
FOOD ALLERGENS: CHEMISTRY, DETECTION AND

FUTURE IMPLICATIONS ON HUMAN HEALTH
Priti Mudgil*
Department of Food Science and Technology, College of Food and Agriculture,
United Arab Emirates University, Al Ain, Abu Dhabi, UAE
ABSTRACT
Worldwide an estimated 22 −25 million people suffer from food allergies. Food
allergies are hypersensitive reactions of the human immune system with symptoms like
edema, redness, swelling, vomiting, asthma, difficulty in breathing, and anaphylaxis (life
threatening). Generally food allergies are caused by proteins known as allergens. Only
eight types of allergens are responsible for nearly 90% of food allergy cases and are
called as ―The Big 8.‖ These foods include eggs, fish, milk, peanut, shellfish, soy, tree
nuts, and wheat. Till now nearly 170 foods and more than 600 food allergens have been
identified and isolated. Still, only 206 of these are officially registered by the allergen
nomenclature subcommittee established by the International Union of Immunological
Societies (IUIS). Moreover, presence of multiple allergens in one food and cross
reactivity among them makes the allergy scenario worse and complicated. Under the
regulation of IUIS and codex alimentarius, till now 14 different food groups are required
for allergen labeling in the European Union and 5 in Japan. The increasing prevalence of
food allergies worldwide calls for some urgent solutions either in the form of regulations
or cure. The only way to control this increasing prevalence seems to be the development
of new regulations for food labelling, enlightening consumers about the risk and control
measures for food allergens via conducting awareness campaigns and following good
manufacturing practices in food factories. Moreover, detection, quantification and
policing food allergens remain problematic due to lack of standardized analytical
methods. Hence, new regulations and detection methods need to be developed.
Keywords: allergy, food allergens, hypersensitivity, immune system
*
Corresponding Author: Department of Food Science and Technology, College of Food and Agriculture, United
Arab Emirates University, Al Ain, Abu Dhabi, UAE, 15551, Email- priti.mudgil1@gmail.com; Tel: +971-
551016393.
156 Priti Mudgil
INTRODUCTION
Epidemical burden of allergies is on the verge of global explosion with adults and
children being most affected. Overall 30-4 % of world‘s population suffers from one or other
type of allergies. The global burden of common allergies like asthma, rhinitis and eczema has
been well-documented by researchers worldwide. However, no equivalent published data on
food allergies on a global platform exists. Food allergies (allergy against food proteins) due to
its higher prevalence have emerged as a shocking global allergic epidemic. With greater
socioeconomic impacts, food allergies significantly damage the quality of life. According to
recent statistics food allergies are riding high in terms of prevalence both in developed and
developing countries. Nearly, 220-250 million people suffer from food allergies globally with
high incidence of life threatening conditions among children (5-8%) as compared to adults (1-
2%). According to a study released in 2013 by the Center for Disease Control and Prevention,
food allergies among children increased approximately 50% between 1997 and 2011. With
the advent of modernization and sedentary lifestyle and increasing affluence of societies,
incidences of food allergies among infants have soared to approx 10% in some heavily
industrialized nations. Recent report concluded by CDC estimated that the number of food
allergy cases in American children has risen to 51% in 2011 as compared to 34% in 1999
(Pawankar et al., 2012; Prescott et al., 2013).
Food allergy can have a profound effect on the quality of life of not only the patient but
the whole family economically as well as emotionally (Pawankar et al., 2011). Recent
statistics released regarding the cost of food allergies indicated that the total cost (direct and
indirect) ranges from 40-50 billion USD (http://wwwworldallergyorg).
Normally an allergy is a hyper sensitive or over reactive immunogenic response of
body‘s immune system to an allergen (foreign substance). Generally, the immune system is
designed in a way to react only against harmful substances like pathogens and toxins to
protect the host body. However, some people have oversensitive immune system that can
elicit an immune response even against harmless substances and its severity could even vary
from mild discomfort to life threatening situations. Basically, allergens stimulate an immune
response via four routes i.e., inhalation, skin contact, ingestion through food and beverage and
injections (medication, insects stings). Most common allergies include eczema, hives, hay
fever, and asthma.
Types of most prevalent food allergies vary worldwide depending upon age, climatic and
geographical differences and chemical differences in allergens and have direct adverse
consequences on human health by inducing disease. Moreover, differences in eating and
culinary habits globally also leads to different prevalence in types of food allergies in
different regions. The burden of food allergies and their epidemiological characteristics are
different, For example 39% of food allergies among Scandinavian people arise from fish
consumption, while in USA milk, its products and nuts accounts for most cases, in France
46% population suffers from egg white allergy while 40% and 20% for peanuts and mustard.
In Holland 28% of food allergies were attributed to consumption of milk, 23% by broad
beans, 22% by coffee, 18% by tomatoes, 16% by egg white, 14% by chocolate, 13% by fish,
and 11% by oranges. While, In Asian countries majority of food allergies are caused by
distinctive food allergens like buckwheat, chestnuts, chickpeas, bird‘s nest, crustaceans and
shellfishes (Lee et al., 2008).
Food Allergens 157
Food allergens are normal proteins, present in all kinds of food products, meaning one
man' s meat is another man's poison, i.e., some people show an allergic reaction
(hypersensitivity) towards these proteins, resulting in gastrointestinal, respiratory,
dermatological and other problems (as e.g., anaphylaxis). Commonly, food allergens are
classified into three categories based on their origins i.e., plant, animal and seafood. The well-
known animal food allergens are cow milk, chicken egg and their products. Plant allergens
include cereals which contain gluten (wheat, rye, barley, oats, spelt) and products of these,
peanut, soybean and other legumes, tree nuts and nut products, and seeds (sesame, poppy,
cotton, mustard), while seafood allergens include fish, crustaceans, mollusks and shellfish.
Additionally cross reactions among different allergens make the situation more complex.
Due to complexity in allergens reactions and lack of direct and prophylactic treatments
for food allergies, the only way to prevent them is by developing deep understanding and
avoiding the contact with these food allergens as much as possible. Different strategies like
awareness among consumers, proper food labelling regarding the presence of possible food
allergens and its derivative upon use of related ingredients and implementation of allergen
control programs in form of hazard analysis and critical control point (HACCP) and their
detection can be used to avoid the risk of food borne allergies to a great extent. Past decade
has witnessed the development of more sophisticated tools for detection of these allergens
rather than depending upon conventional methods which takes months for detection.
However, the scope of food allergy and allergen is vast still attempts have been made in
this chapter to give an overview of the food allergens with main focus on mechanism,
detection, control and challenges ahead. First, an overview about the type, chemistry and
nature of different food allergens is given. Second, various detection methods, their limitation
and advancements in the field are compiled. Third, possible control measures are presented
and in last challenged ahead are given.
OVERVIEW, TYPES AND CHEMISTRY OF FOOD ALLERGENS

Presently a sturdy increase in the prevalence of food allergies can be noticed worldwide.
The prevalence of these allergies is more in developed countries as compared to developing
countries. According to an estimate approximately 35% population from highly developed
countries suffer from different forms of hypersensitive reactions, and their number is still
growing. According to INFOSAN Information (2006), the prevalence of food allergies is
estimated to be around 1%–3% in adults and 4%–6% in children, and more than 70 foods
have been reported as causing food allergies (FAO/WHO Bulletin, 2006). Mostly these food
allergies are caused by the presence of common allergen at the same time in many foods like
ingredients. Some common kind of food that are most commonly implicated in food allergy
cases are cereals containing gluten, crustaceans, eggs, fish, peanuts, soy, milk, and tree nuts.
Moreover, special attention is needed towards use of food additives (natural, synthetic or
biotechnologically produced) as they can also be responsible for causing such allergic
reactions. Many food additives like E102, E104, E110, E122, E124, E128, E129, E210, E211,
E212, E213, E214, E215, E216, E218, E219, E220, E231, etc. have revealed potential
capability to induce allergic reactions in sensitive patients (Arbes et al., 2005).
158 Priti Mudgil
Food allergens elicit their allergenicity by inducing an immunoglobulin E (IgE) response

upon their first interaction with host immune system and thereafter their subsequent exposure
can result into the clinical response. As allergenicity or potency of food allergens is
dependent mainly upon their abundance in food, presence of multiple linear IgE binding
epitopes, and resistance of the protein to digestion and processing. Deeper understanding into
their structure, biochemistry and molecular biology and factors that allow these food allergen
to be identified by immune system, survival in extreme food processing conditions and escape
through digestive enzymes of the gastrointestinal tract can help us to identify and develop
more stringent control measures. Generally impact of food allergens on an individual is
dependent on factors like genetic makeup of effected person, chemical structure and
physiological properties of allergen.
The understanding of allergen structures, their chemical nature and their pathogenicity is
very important in developing control strategies and detection methods. Therefore, over the
year different type of food allergens have been identified, purified, characterized and
categorized in different categories from different sources. These allergens have also been
characterized at a molecular level for better understanding towards mechanism of
immunopathogenesis of food allergy. Moreover, for their easy understanding and cataloguing,
International Union of Immunological Societies Subcommittee for Allergen Nomenclature
have started using a systematic terminology under different categories for pure allergens,
where the three first letters denote genus, then next single letter represent species and Roman
digit indicates the number for new allergen for example, allergen from hen egg white which
was previously known as ovomucoid and ovoalbumin are now represented as Gal d 1 and Gal
d 2 where gal stands for Gallus (Genus) and d for domesticus (species) (Chapman et al.,
2007).
Food allergens are classified by using different terminologies like depending on their
characteristics, stability, cross reactivity food allergens are classified into two types (Burks et
al., 2001).
Class 1: This class includes those food allergens, which are the major/primary water
soluble allergens made up of glycoproteins (MW 10-70kD) and generally cause allergy
through the gastrointestinal tract. These are highly stable to action of heat, acid, and proteases
(Table 1).
Class 2: This class comprises, food allergens that are cross-reactive with other proteins
mainly of plant origin making there pathogenesis and etiology more complex to understand
and control. These allergens are highly heat labile and difficult to isolate in pure forms,
therefore for their diagnostic procedure scientists had to resort more conventional methods for
their detection. Moreover these types of allergens can only cause hypersensitivity when taken
in raw form as food processing through heat treatment either denature or alter their
allergenicity by inducing changes in their epitopes for example: egg allergy (Table 1).
However depending on their origin, food allergens are classified into three main
categories i.e., Plant based food allergen, Animal originated food allergen including seafood
allergens and Food additives as allergens, their detailed description is as follows:
Food Allergens 159
Plant Based Food Allergens
Plants are the major source of foods for humans and comprises of thousands of different
proteins that can act as allergens. So far the allergens list of the International Union of
Immunological Societies (IUIS) allergen nomenclature subcommittee comprise of 130 plant-
derived food allergens (Hauser et al., 2008). Plant food allergens belong to protein families
and superfamilies‘ on the basis of their structure, biological function and their membership
among 8296 protein families of Pfam protein database. Most of plant food allergens belong to
31 protein families like prolamin, cupin, cysteine proteases, pathogenesis-related proteins,
profilins, lectins, oleosins etc. The prolamin, cupin, profilin, and PR-10 protein families
contain approximately 65% of all plant food allergens, while the remaining 27 allergen-
containing protein families refer to mostly the pathogenesis-related proteins (PRs)
(Breiteneder and Radauer, 2004; Radauer and Breiteneder, 2007).
Table 1. Examples of class I and class II allergens

(Hill et al., 1999; Ho et al., 2014)
Class I Class II
Cow’s milk:
Pathogen-related protein 2 group (glucanase)
Caseins (a, b, k), α-lactoalbumin, β-
Latex, avocado, banana, chestnut, fig
lactoglobulin, serum
Chicken and egg:
Pathogen-related protein 3 group (chitinase)
Albumin, Ovomucoid, ovalbumin,
Latex (Hev b6), avocado
ovotransferrin
Pathogen-related protein 5 (thaumatin-like) Cherry,
Peanut: Vicillin, conglutin, glycinin
apple, kiwi
Birch Bet v1 homologues (pathogen-related
Soybean: Glycinin, profilin, trypsin inhibitor proteins 10) Apple, cherry, apricot, peach, pear,
carrot, celery, parsley, hazelnut profiling
Birch Bet v2 homologues (celery-mugwort-spice
Shrimp and Fish:
syndrome)
Tropomyosin, Parvalbumins
Latex, celery, potato, pear, peanut, soybean
Fruits and vegetables: Lipid transfer proteins
(LTPs)
Prolamins
This family contains the largest number of food allergens. Major allergen include seed
storage proteins like LMW glutenin, α and γ gliadin (Wheat), cereal protease inhibitors like
(wheat, barley, maize and rice), 2 S albumins (walnut, mustard, brazil nut, peanut, sesame and
sunflower) and non-specific lipid transfer proteins (LTPs) (peach, apple, maize, apricot,
cherry). Allergens of this family are rich in sulphur and contain a conserved skeleton of 8
cysteine residues and are rich in glutamine and proline. All allergens among this family have
homologous 3D structures but low sequence similarities. Generally allergens from this family
are thermo-stable, resistant to digestion and stable to food processing conditions. These
allergens are soluble in alcohol-water mixture, low in molecular mass ranging from 7-65 kDa
with seed storage proteins having the highest molecular mass (31-65 kDa) while LTPs with
160 Priti Mudgil
lowest mass of 7-11 kDa. Allergies to these prolamins are not very frequent, Glutenin and
gliadin are responsible for physical properties of dough like elasticity and extensibility.
Generally gliadin in wheat are of two types i.e., low moleculat weight and high molecular
weight but only the former gliadin along with glutenin (α and γ) are implicated in majority of
IgE related food allergies to wheat Food allergens from this family are compiled in Table 2.
Cupins
This family is structurally and functionally diverse and has derived its name from a Latin
word cupa means barrel as majority of proteins in this superfamily have beta-barrel structures.
The proteins of this family are highly stable proteins and are classified into two types based
on the number of cupin domain iemonocupins and bicupins.
Table 2. Main plant food allergens of prolamins family
Prolamins Allergen Molecular

Source Characteristic Reference
categories Name mass (kDa)
Sec c 20
Rye
Hor V 21 15 They are rich in
Barley (Varjonen et al.,
Seed storage Tri a 19 sulfur, proline and
Wheat 1997; Varjonen et
protein (cereal α-gliadin 31-42 glutamine Only
Wheat al., 2000; Takizawa
prolamins) γ-gliadin 33-44 certain members
Wheat et al., 2001)
LMW- 65 are allergens
Wheat
glutenin
Pru p 1 Peach Thermostable
Mal d 3 Apple 9 monomeric
(Pastorello et al.,
Zea m 14 Maize 114 proteins,
Non-specific 2000; Scheurer et
Pruar 3 Apricot 9 Resistant to
lipid transfer al., 2001;
Pruav 3 Cherry 9 digestion, No
proteins Veličković and
Cor a 8 Hazelnut 9 glycosylation,
Jankulović, 2 14)
Aspa o 1 Asparagus 7 highly cross
Lac s 1 Lettuce reactive
Present mainly in
Hor v 1, 15 Barley
cereals,
Sec c 1 Rye
Cereal protease Thermostable, (Veličković and
Rag 1, 2, 5, Rice 12-16
inhibitors Stable to pro- Jankulović, 2014)
5b, 14, 14b, Maize
cesses such as
16, 17 barley
brewing
Jug r 1 Walnut 16
Allergen present in
Sin a 1 Yellow 14
raw
mustard
and roasted seeds
Bra j 1 Oriental 147 (Breiteneder and
shows increased
mustard Radauer, 2004;
activity,
2S albumins Ber e 1 Brazil nut 12 Hoffmann and
Severe allergen
Ara h 2 Peanut 175 Mills, 2009;
because of high
Ara h 6 Peanut 145 Hoffmann, 2011)
resistant to
Ara h 7 Peanut 158
proteolysis
Sesi 1 Sesame 9
SFA-8 Sunflower 12
Food Allergens 161
Monocupins (Germins)
Proteins with single domain are known as monocupin and are normally not involved in
allergy with and exception of Cit s 1 from orange (Ahrazem et al., 2006; Pöltl et al., 2007)
and a 28kDa protein from pepper (Leitner et al., 1998) Table 3.
Bicupins
Proteins with two cupin domains are known as bicupin and are further divided into two
types depending upon there sedimentation coefficient i.e., 7S vicilin-type globulins and 11S
legumin type globulins. These globular seed storage proteins are major source of protein with
high nutritional value and comprise 50% of total seed storage proteins in legumes and tree
nuts. Therefore, these 7S vicilin and 11S legumin globular seed storage proteins comprised
the main components of the human diets with those from soya being among the most widely
consumed. In contrast, globulins from other seeds, such as sunflower, sesame and poppy, are
eaten in much smaller amounts and this pattern of consumption also reflect in the number of
food allergy cases towards cupins (Mills et al., 2002; Hoffmann and Mills, 2009). Majority of
cupins allergens belong to either the 7 S vicilin-like or the 11 S legumin-like seed storage
globulin families and are shown in Table 3. Ara h 1, one of the main storage proteins is the
most characterized protein of allergenic vicilin from peanut, and is responsible for over 90%
cases peanut allergies. While legumin Ara h 3 is responsible for over 50% cases peanut
allergies Glycinin from soyabean is a major seed storage protein that makes up more than
20% of the soybean seed by dry weight and 35–40% of total soy protein.
Pathogenesis Related Proteins (PR Proteins)
Pathogenesis-Related Proteins are produced as a mechanism of plant protection against

environmental stress, pathogen infection, and antibiotic stimuli. PR-proteins constitute a
collection of 14 unrelated protein families like PR-2, PR-3, PR-4, PR-5, PR-9, PR-10 and PR-
14 containing enormous number of plant derived food allergens. These proteins are very
efficient in provoking immune response due to their smaller size, pH stability and resistant to
proteolysis. The first indication about possibility of food allergens in PR-1 family were
obtained from muskmelon allergen Cuc m 3 and is the only reported food allergen from PR-1
family (Asensio et al., 2004).
Proteins of PR-3 Family play important role against fungal and insect pathogens.
However, some evidences has suggested that class I chitinases from cherimoya, passion fruit,
kiwi, papaya, mango, and tomato resulted in food allergy and also have the capability of IgE
cross-reactivity with other allergens and have been implicated in the latex-fruit syndrome
(Kasprzewska, 2003; Karisola et al., 2005). Most of the allergenic activity of chitinases seems
to be due to the hevein-like domain.
162 Priti Mudgil
Table 3. Main plant food allergen of cupin superfamily

(Breiteneder and Radauer, 2004; Hause et al., 2008;
Hoffmann, 2011; Veličković and Jankulović, 2014)
Molecular
Type Allergen Name Source Characteristic
mass (kDa)
Cit s 1 Orange
Germins 28 kDa
28 kDa protein Bell Pepper
Ara h 1 Peanut 60 1Vicillins are homotrimeric
Len c 1 Lentil proteins of about 150 to 190 kDa
Pis s 1 Pea 2 Lack cysteine and disulfide bonds
Pis s 2 3 Differences in susceptibility to
Cor a 11 Hazelnut proteolytic, processing and
Vicillins Jug n 2 Black walnut glycosylation of the monomers
Jug r 2 English walnut
Sesi 3 Sesame 47
Ana o 1 Cashewnut
Pis v 3 Pistachio
β-conglycinin Soyabean
Ara h 3 Peanut 14 1 Allergenic legumins include the
Ara h 4 54 minor peanut allergens
Ber e 2 Brazil nut 2 These are hexameric proteins
Cor a 9 Hazelnut composed of two trimers
Sesi 6 Sesame
Sesi 7
Legumins
Ana o 2 Cahsewnut
Pis v 2 Pistachio
Glycinin Soybean 57
Coconut
AMP (almond Walnut
major protein) Almond
Proteins from PR-5 Family are generally abundant in fruits and are sub classified into
three categories depending upon their function i.e., (i) protein against pathogen infection, (ii)
protein against osmotic stress (osmotins), and (iii) protein against fungal pathogens. The
proteins from this family are generally resistant to proteolysis, processing and pH Molecular
mass is about 20kDa and generally consists of anti-parallel β-sheets that are further stabilized
by eight disulfide bonds.
Unlike other related families proteins from PR-10 family are comparatively heat-labile
and unstable towards digestion. These proteins are generally responsible for class II food
allergies where allergen Bet v 1 acting as sensitizing agent and leading to the induction of
cross-reactive IgE antibodies. Among the foods most commonly implicated with Bet v 1-
associated allergies are fruits of the Rosaceae (e.g., apple Mal d 1, pear Pyr c 1, peach Pru p
1, cherry Pruav 1, apricot Pruar 1, and strawberry Fra a 1), Fabaceae family (peanut Ara h 8,
mung bean Vig r 1), and Apiaceae vegetables (celery Api g 1, carrot Dau c 1), but some nuts
(hazelnut Cor a 1) (Table 4) also possess strong cross-reactivity towards the major birch
pollen allergen (Park et al., 2004; Breiteneder and Mills, 2005; Mogensen et al., 2007).
Food Allergens 163
Table 4. Main plant food allergen from plant defense system or pathogenesis
related proteins
Type Family Name Source Allergen

PR-1 Muskmelon Cuc m 3
Banana
PR-2 β-1,3-glucanases Potato β-1,3-glucanases
Tomato
Turnip Bra r 2
Chestnut Cas s 5
PR-3 Class I chitinases
Avocado Pers a 1
Wheat Tri a 18
PR-4 Hevein and Win like chitinases
Kiwi Act d 2
Apple Mal d 2
PR-5 Thaumatin like proteins
Cherry Pruav 2
Bell pepper Cap a 1
PR-9 Peroxidases Wheat Tri a Bd 36K
Celery Api g 1
Carrot Dau c 1
Gold kiwi Act c 6
Penaut Ara h 8
Soybean Gly m 4
Mungbean Vig r 1
Hazelnut Cor a 1
PR-10 Bet v 1-related proteins
Strawberry Fra a 1
Apple Mal d 1
Apricot Pruar 1
Cherry Pruav 1
Peach Pru p 1
Pear Pyr c 1
Raspberry Rub i 1
Peach Pru p 3
Apple Mal d 3
Apricot Pruar 3
PR-14 Nonspecific lipid transfer proteins
Hazelnut Cor a 8
Asparagus Aspa o 1
Lettuce Lac s 1
Prolifins
Profilins are a highly conserved allergen family of small size (12-15 kDa) actin binding
proteins molecules. The protein structure usually consists of seven stranded antiparallel β-
sheets enclosed on both sides by α-helices on one or both sides. The main role of profilins in
organisms is to maintain cell motility, cytokinesis, cell elongation and cytoplasmic streaming
(Ramachandran, et al., 2000). Because of their vast presence in almost all organisms and high
sequence similarities these profilins are highly cross reactive and are known as pan allergens.
Since they are capable of cross reaction with similar proteins from virtually all food proteins,
164 Priti Mudgil
exposure to them constitutes a risk factor for multiple pollen food allergies and class II food
allergy. In general, profilins are sensitive to heat denaturation and gastric digestion limiting
their allergic reaction to moderate oral symptoms arising from consumption of raw foods.
However, some profilins like celery profilin Api g 4 is slightly heat reaction and can elicit
allergy even in cooked foods. Till now several plant food-derived profilins characterized are
known to cross react with the first identified plant profilin i.e., grass and/or birch pollen Bet v
2 Furthermore, profilins are also involved in IgE mediated cross-reactivity between pollen
and exotic fruit, like lychee Lit c 1 and pineapple Ana c 1 All profilins that have been
characterized from different sources are compiled below in Table 5:
Table 5. Profilins as food allergen from different plant sources
Profilins
Source Allergen Source Allergen
Celery Api g 4 Apple Mal d 4
Carrot Dau c 4 Cherry Pruav 4
Pineapple Ana c 1 Almond Pru du 4
Muskmelon Cuc m 2 Peach Pru p 4
Peanut Ara h 5 Pear Pyr c 4
Soybean Gly m 3 Orange Cit s 2
Hazelnut Cor a 2 and 4 Litchi Lit c 1
Barley Hor v 12 Bell pepper Cap a 2
Rice Ori s 12 Tomato Lyc e 1
Strawberry Fra a 4 Banana Musxp 1
Other Plant Food Allergens Families
With the advancement happening in the field of allergens a number of novel and unique
food allergens are being identified. Many of these allergens are classified in the preexisting
families but a number of other allergens have been identified to less widespread allergen
families and are described in brief below (Table 6).
Patatin is described as a novel allergen from potato tuber and is denoted by Sola t1
(Seppälä et al., 1999; Astwood et al., 2000). Ptalins can cross-reacts with the major latex
allergen Hev b7 causing latex fruit syndrome.
Oleosins is a family of small size proteins (15-18 kDa) involved in the formation of oil
bodies and is classified as new allergen family. These are hydrophobic plant proteins found
only in association with small storage oil drops and contribute to stabilizing plant lipid
storage bodies. Recently, oleosins with allergenic activity were identified from legumes, nuts
and seeds for example Sesi 4 and Sesi 5 from sesame seeds (Leduc et al., 2006), Ara h 10 Ara
h 11from peanuts (Pons et al., 2002; Pons et al., 2005); Akkerdaas et al.,(2006) has identified
new oleosin allergens of 167 and 147 kDa from hazel nut.
Thioredoxins are small ubiquitous enzymes acting as general protein disulfide oxido-
reductases interacting with a broad range of proteins. Allergenic thioredoxins have been
identified in fungi as well as in plants i.e., Tri a 25 from wheat, Mala s 13 from
Food Allergens 165
Malasseziasympodialis and Zea m 25 from maize (Weichel et al., 2006; Limacher et al.,
2007).
Isovlavone reductases (IFR) However, these allergens are not major allergens, but can
be classified as pan allergens with members in pollen, fruits, and latex. These are a class of
oxido-reductases enzymes involved in plant secondary metabolism. Main allergens include
pyr c 5 from pear, lichee, and sharon fruit (Mills et al., 2003; Bolhaar et al., 2005).
Expansins These are a family of cell wall protein responsible for cell wall loosening and
have enigmatic effects of cell wall rheology. Recently these proteins are identified with
allergrnic properties (Cosgrove, 2015) For example: Act d 5 (Kiwellin, 28Kd) allergen from
kiwi fruits showed many fold similarities with the major grass pollen allergen Phl p 1, a
cysteine-rich glycoprotein representing one of the most important aeroallergens known to
date (Tamburrini et al., 2005; Uberti et al., 2015). All expansins consist of two active
domains; domain 1 is homologous to proteins in the glycoside hydrolase family 45 (GH45)
while expansin domain 2 is homologous to group-2 grass pollen allergens, and is responsible
for its biological function (Sampedro and Cosgrove, 2005).
Table 6. Newly identified allergen protein families
Family Name Source Allergen Family Name Source Allergen

Cystatins Kiwi Act d 4
Wheat Tri a 25 Kunitz-type Sola t 2
Thioredoxins
Maize Zea m 25 protease Potato Sola t 3
Inhibitors Sola t 4
Sesi 4
Pear Pyr c 5 Sesi 5
Isoflavone Sesame Hazelnut
Olive Ole e 12 Oleosins
reductases Peanut
Ash pollen Fra e 12 Ara h 10
Ara h 11
α-amylases Barley Hor v 17 Expansins Kiwi Soybean Act d 5 Gly m 2
Chlorophyll-
Glycoside
Date Ziz m 1 binding Celery Api g 3
hydrolases
Proteins
Seed specific
Lyc e 2
Patatin family Tomato Potato biotinylated Lentil Len c 2
Sola t 1
protein
Papain-like Pineapple Ana c 2 60s acidic
cysteine Kiwi Act d 1 ribosomal Almond Pru du 5
Proteases Soybean Gly m 1 Binding protein
Subtilisin-like
Muskmelon Cuc m 1
serine Proteases Kiwi Act d 3
Unidentified
Berberine bridge Pistacho Pis v 4
Celery Api g 5
enzymes
166 Priti Mudgil
Animal Originated Food Allergens
Though allergens of animal origin are not as diverse as plants still their share in total food
allergies is significantly high Similar to plant allergens animal allergens are also classified
into different families (Table 7) i.e., calcium binding EF hand proteins (parvalbumin),
tropomyosins and caseins being the major one while others like Lipocalins (β-lactoglobulin),
C-type lysozmye α-lactalbumin, Kazal-type protease inhibitors, serpin and transferrins, ATP:
guanidophosphotransferases, glycoside hydrolase family 22, immunoglobulins and serum
albumins contains only a few reported allergens (Hoffmann and Mills, 2009). The main
reason for the allergenicity of animal originated allergen is their homology to human proteins.
It has been reported that proteins with less than 50% homology are all allergen while proteins
with more than 60% homology are rarely involved in allergic reaction (Jenkins et al., 2007).
Hence, this low level of homology is responsible for allergenicity of these proteins and they
can be easily identified by the host immune system as foreign substances. Most of the
important food allergens are present in milk, egg, and seafood and hence their properties are
described further in this chapter.
Milk Allergens
Milk allergies are the first model of food allergy being the first food proteins
administered to infants and their incidences varies form 1-3% among children in developed
countries. Animal milk contains enormous number of proteins that can elicit immunogenic
response in sensitive individuals. Therefore milk from many species, their milk product and
even lactose are listed as allergens. Milk yields two main types of fractions upon acidification
or coagulation i.e., whey and curd both containing different types of proteins. Whey contains
essentially globular proteins like β-lactoglobulin, α-lactalbumin, and bovine serum albumin
while the curd fraction comprises the caseins. Milk Caseins are a heterologous group of
proteins containing fractions like αS1-casein, αS2-casein and β-casein and their major role
includes binding of calcium ions however, caseins constitute the major source of animal
allergens from their fractions, αs1-casein fraction and αs2-casein seem to be the most
important allergens, followed by β-casein. Moreover due to high level of sequence homology
between milks from different animals chances for cross reactive allergies are also very high.
For instance, proteins from cow's milk are very similar (≥9 %) to those from goats and sheep,
and can cause the same sorts of reaction in cow's milk-allergic subjects. Thus goat's or sheep's
milk cannot be used as a cow's milk substitute in allergic individuals. However, low sequence
identities (22–66%) are noticed for cow, mare, donkey and camel milk that explains the fact
that why these milks can be safely consumed by patients with cow milk allergy. Moreover,
casein fractions from goat or sheep milk have been recently reported to elicit immunogenic
responses similar to cow milk casein (Ah‐Leung et al., 2006).
The other important allergens in cows‘ milk are the whey proteins β-lactoglobulin, the
only lipocalin which acts as a food allergen, and α-lactalbumin, which like the egg allergen
lysozyme belongs to the glycoside hydrolase family 22 clan of the O-glycosyl hydrolase
superfamily. Lastly, one minor allergen identified in milk is the iron-binding protein,
Food Allergens 167
lactoferrinAllergen from milk, their molecular mass, function and cross-reactivity has been
described in Table 7.
Meat
Meat is an important food because of its high nutritional and functional properties. Meat
allergies are considered to be rare and there are conflicting reports about occurrence from
beef. Beef allergy is expected to cause some serious allergies children allergic to milk
because of cross reactivity as both foods contain bovine serum albumin, bovine gamma
globulin, and other proteins of high homology in significant quantities (Restani et al., 2009).
Laminin c-1 and collagen a-1 (VI) two IgE-binding proteins of 240 kDa and 140 kDa have
recently been reported from Bostauras (Takahashi et al., 2014).
Eggs
Eggs are well known for their allergic potential and represent one of the most frequent
allergies particularly in children with three times higher prevalence in children than in adults.
Both proportions of egg i.e., egg white and egg yolks are reported to have food allergens. Egg
white protein contains 23 different glycoproteins but the most abundant proteins in egg white
are ovomucoid, ovalbumin, ovotransferrin, and lysozyme C and represent 11, 54, 12, and
34% of total egg white proteins, respectively. These four are considered the major source of
allergens in egg white. Most of the allergens, their IUIC nomenclature, functionality,
molecular mass and other properties are cited in Table 8. Though egg white proteins represent
the major proportion of egg allergen, IgE-binding allergens have also been reported in the
yolk. Lysozymes are reported as minor hen‘s egg allergen and act as muramidase and
antimicrobial protein by hydrolyzing peptidoglycans of bacterial cell wall. Similarly, the
sulphur-rich iron-binding glycoprotein ovotransferrin has also been identified as minor
allergens in hen‘s egg (Takahashi et al., 2014). Many reports on cross reactions among
various avian eggs have been described for duck and goose egg with hen‘s egg allergy.
Allergy to bird meats is very rare but patients allergic to one bird‘s egg may be allergic to
others meat, including game birds. However, allergy to chicken meat is quite rare and allergy
to turkey is even less common.
Seafood Allergens
Seafood allergies are serious food allergies, especially in coastal regions and fish
processing communities where seafood is a common constituent of the diet. Besides peanuts
and tree nuts, fish and shellfish are among the most frequent causes of IgE-mediated allergic
reactions in adolescents and adults. The major allergens of fish are called as parvalbumins
while allergens of crustaceans such as shrimp, crab, and lobster are called as tropomyosin, a
muscle protein. The major allergens of seafood are described here in this chapter below and
Table 9.
Table 7. Allergens from milk proteins
Common name Allergen Protein Family Molecular mass Epitope Stability Function Cross reactivity
α-S1-Casein Bos d 8 Pfam 27-32 kDa AA 17-36, 39-48, 40% of total casein Goat (88%)
(Chatchatee et al., familyPF00363 69-78, 93-102, Rheomorphic fraction Sheep (85%)
2001) 109-120, 123-132, Proteolysis Sensitive Major source of Mare's milk (47%)
139-154, 159-174 Heat stable phosphate in milk Human milk
and 173-194 α-S2-Casein contains Precipitate upon calcium (32%)
cysteinesdisulphide- addition sequence
dimers Soluble in casein micellar homology
structure with other
caseins
α-S2-Casein Bos d 8 27-32 kDa AA 31-44, 43-56, 10% of total casein Goat (87%)
(Busse et al., 2002; 83-100, 93-108, fraction Sheep (89%)
Natale et al., 2004) 105-114, 117-128, Major source of sequence
143-158, 157-172, phosphate homology
165-188 and 191- Precipitate upon calcium
200 addition
Soluble in casein micellar
structure with other
caseins
β-casein Bos d 8 266 kDa AA 1-16, 45-54, 40% of total casein Goat and sheep
(Chatchatee et al., 83-92, 107-120 fraction of cow's milk (91%)
2001; Businco et and 135-144 with Less phosphorylated than Mare milk (58%)
al., 2000) weak binding to α-caseins
AA 57-66
Binds to calcium and Human milk
form nano clusters (56%)
Major source of sequence
phosphate and calcium homology
α-Lactalbumin Bos d 4, Glyco- 141-167 kDa AA1-16, 13-26, Stabilized by binding Evolved from a lysozyme Human (73%)
(Järvinen et al., Lactose hydrolyase 47-58 59-93, 93- to calcium Increase the rate of Sheep (97%)
2001; Chen et al., synthase B family 22 102, 99-108 and At 95°C denatures lactose formation (lactose Goat (95%)
2005; Redfield, protein Pfam PF00062 109-123 more slowly than β- synthase) sequence
2004) lactoglobulin No catalytic activity alone homology
Destabilized by Antimicrobiol activity
proteolytic enzymes (complete and peptide
at low pH and form form)
molten globule
Common name Allergen Protein Family Molecular mass Epitope Stability Function Cross reactivity
Immunoglobin Bos d 7 PfamPF00047 55 kDa Not known Not known Major role in humoral Not known
(Farrell et al., immunity
2004) Bind specifically to
enviromental agents like
viruses, bacteria, foods
and toxins
Bovine Serum Bos d 6 Serum albumin 664-133 kDa AA 524-542 Stable to heat and Present in low amount in Ovine serum
Albumin family isopropyl alcohol milk albumin (92%)
(Xu and Ding, Pfam PF00273 Stabilised by 17 Regulates osmotic sequence
2004) disulphide bridges pressure of blood homology
Resistant to Principal transporter of
proteolysis by pepsin fatty acids, hormones and
bilirubin
κ-Casein Bos d 8 Kappa Casein 19 kDa AA 15-24, 37-46, More structured than 10% of total casein Buffalo,
(Natale et al., family 55-80, 83-92 and α- and β-caseins Critical for casein micelle Sheep and goat
2004) Pfam PF00997 105-116 Heat resistant Due to stability (87-88%)
Disulphid bond Horse (45%)
Very sensitive to Human (35%)
Proteolysis sequence
homology
Lactoferrin Lactotransf Transferrin 67 kDa Not known Denature at 65°C for Glycoprotein with iron Not known
(van der Kraan et errin Pfam PF00405 apo- and 92°C for binding activity (Iron
al., 2005) iron loaded sequestration)
lactoferrin Antimicrobial (peptide
lactoferricin fraction 17 –
41 and 265-284)
Protect cells from free
radical damage
β-Lactoglobulin Bos d 5 Lipocalin 18 kDa AA 1-16, 31-48, Heat sensitive Binding with retinol Sheep (93%)
(Chen et al., 2005; Pfam PF00061 47-60, 67-78, 75- Extensive reduction Transport of hydrophobic Goat (94%)
Kontopidis et al., 86, 127-144 and in activity at 90°C molecules Horse (59%)
2004) 141-152 Human (44%)
Bold: strongest
Table 8. Allergens from Egg
Amino
Allergen Name Name Content (%) Mr (kDa) pI Carbohydrates Stability and biological Function
Acids
Egg White (Albumen)
Ovomucoid Gal d1 11 28 41 186 ~25% Serine-protease inhibitor
Ovalbumin Gal d2 54 43-45 45 385 ~3% Serpin family
Ovotransferrin Antimicrobial defense and iron-
Gal d3 12–13 76–78 6 686 260%
(= Conalbumin) binding protein
Lysozyme Gal d4 34–35 14 10 129 0% 1,4-β-N-Acetyl-muramidase C
Serum albumin Binding of water and Ca-, Na-, and
K- cations, fatty acids, hormones,
Gal d5 69-70 46-48 592 bilirubin, and drugs; regulation of
the colloidal osmotic blood
pressure
Ovomucin Glycosulfi protein, antiviral, and
15–35 1800–8000 45–50 ~18%–33%
antitumor properties
Egg Yolk
Lipovitellins 3-400
HDL 15
LDL 65
Phosvitins 10 ~175 210 Glycophosphoprotein
Livetins 10 45–150
α-Livetin Gal d5 70 43–57
Food Allergens 171
Parvalbumins
The major allergens identified so far in fish is the white muscle protein known as
parvalbumin, a protein which contains a calcium-binding domain known as an EF-hand
domain. Parvalbumin is resistant to heat, chemical denaturation, and proteolytic enzymes.
The parvalbumins are of two types known as α- and β-parvalbumins. Most of seafood
allergens are β-parvalbumins (Jenkins et al., 2007) with only one exception from α-
parvalbumin from a frog (Hilger et al., 2 2). β parvalbumins exist in two isoforms as β1 and
β2 and lead to high level of cross reactivity among fish allergen due to high homology
between sequences and structures. A number of parvalbumins have been identified in many
different fish species and are considered as pan allergens in fish among them 218 allergenic
isoforms of fish parvalbumins has been listed. Apart from parvalbumins a 50 kDa enzyme β-
enolase (Thu a 2), 4 kDaaldolase (Thu a 3) from yellowfin tuna and 472 kDa β-enolase (Sal
s 2) and 40 kDaaldolase A (Sal s 3) from Atlantic salmon have allergenic properties
(www.allergen.org).
Tropomyosin
Tropomyosins are a family of closely related proteins present in muscle and non-muscle
cells. Tropomyosin plays a key regulatory role in muscle contraction together with actin and
myosin. In non-muscle cells, tropomyosin is believed to play a role in the regulation of cell
morphology and motility. Tropomyosins are generally found in two invertebrate groups,
Crustacea and Mollusca and are generally referred to as shellfish. Tropomyosins are heat
stable cross-reactive food allergens present in crustaceans and mollusks. Invertebrate
tropomyosins are pan-allergens with significant sequence similarity identified in seafood and
insects such as storage and house dust mites and cockroaches. Consequently, tropomyosins
are also found as aeroallergens, which raise the possibility of sensitization by the respiratory
route. As a consequence of the lack of homology in the IgE epitopes, there is no reported
cross-reactivity between IgE from shellfish allergic individuals, and animal muscle
tropomyosins. As a result of their extensive homology, tropomyosins exhibit IgE cross-
reactivity between various crustacean and mollusk species. Tropomyosins were identified as
the major allergens of shrimp by several researcher and are documented in Table 9.
Food Additives as Allergens
Extensive use of food additives in food industry as antioxidants, flavorings and coloring,
preservatives, sweeteners, thickeners, and many others has raised the concern over their role
in food implicated allergic reactions. However, till now only some reports have suggested
their role in food allergies but still additional studies on other toxicological endpoints may be
required on a case-by-case basis. This chapter delineates the important food additives that can
react as possible food allergens. Although many additives have been reported to provoke
allergic symptoms of urticarial, angioedema and asthma, still their prevalence is nonexistent
because of poorly designed and properly conducted randomized trials. Some of the important
food additives implicated in allergic symptoms are compiled below in Table 10.
172 Priti Mudgil
Detection Methods for Allergens in Food
According to World Health Organization, food allergies are the fourth most important
public health problem and concern. Despite of years of research, till now there is no possible
cure exists other than the total averting of food containing allergens. Therefore in order to
ensure the safety of food from allergen point of view, regulatory authority for food safety
around the world have made it obligatory for labelling of food products with potential
ingredients that can be allergenic. These labelling regulations warn the consumers of the
presence of possible allergens in foodstuffs. However, in food processing sometime situations
lead to the unintentional presence of allergenic material in the final product, in which the
allergen is not an intended ingredient. Several practices and potential errors during production
may lead to the presence of unexpected allergen in a product. Therefore, food producers very
often use a so-called precaution labelling by mentioning on the packaging ―may contain
traces of‖ or ―produced in a factory handling‖ Still, in order to make foods safe for
consumption detection of these food allergens is advantageous for the producer for adequate
labeling of products as well as for consumer gaining information about presence or absence of
allergens in his/her food products.
For detection, identification and validation of allergens in food chain food specialists use
various methods that have been developed by researchers over a time. However detection
with these conventional methods is limited in terms of cost, labor, time taken (up to months to
validate and establish the limit), trace amount detection and quantitative determination.
Moreover interaction of allergens with food matrices and their cross reactivity with other
proteins made it more complex for detection. Therefore, major challenge today is the
detection and the quantification of trace amounts of allergens in miscellaneous food matrices,
which are able to provoke an allergic reaction, with the severity depending on the allergen
and the individual.
The only solution to the above problem can be the improvement in robustness of the
available analytical methods and development of new highly sensitive, fast, accurate, specific
and standard methods that can provide unambiguous identification and information about
food allergens to food catering industries and consumers. Some of these conventional and
new methods are hereby described in brief in this chapter.
Most of the method use markers whose presence tells about allergen rather than targeting
specific allergen proteins. Generally any component of food associated with allergen presence
can be targeted but most of the methods use either DNA of proteins as marker. Based on the
types of marker used detection methods are divided into two types i.e., protein based and
DNA based method. Protein-based methods usually involve immunological(antibody-based)
techniques where antibodies (Ab, raised in animals against pure allergens) or human IgE
DNA-based methods are based on the amplification of specific DNA fragments by means of
the polymerase chain reaction (PCR) in which specificity is achieved by the use of primers
that only facilitate amplification of DNA originating from the offending food. With the
advent of newer techniques more sensitive chip based and biosensor based methods has been
developed too.
Table 9. Major food allergens from seafoods
Source Allergen Family Molecular Mass Structure And Function Cross Reactivity References
Parvalbumin
Cod and Carp parvalbumin (Van do et al.,
Salmon Sal s 1 12-14 kDa
2005)
Thu o 101 Cod, Salmon and Carp parvalbumin (Swoboda et
Tuna 10-12 kDa
Thu o 102 al., 2002)
Epitopes are primarily
Alaska Cod parvalbumin (Van do et al.,
The c 1 12 kDa conformational
Pollock 2005)
Very much stronger in the
Cyp c 101 carp, cod, eel, perch and salmon (Untersmayr et
Carp 12 kDa presence of calcium
Cyp c 102 al., 2005)
EF hand Control flow of calcium
salmon, cod, sardine, tuna, Japanese eel, (Hamada et
Sco j 1, Sco a 1 calcium binding during muscle contraction
Mackerel 11 kDa horse mackerel, red sea bream, skipjack, al., 2003)
Sco s 1 proteins Quickly refold in the
bigeye tuna and Japanese flounder
Pfam PF00036 presence of calcium
Gad c 1 Fin fishparvalbumin (Van do et al.,
Heat stable
Gad m 1 Fishparvalbumin 2003)
Cod 125-51 kDa Calcium buffering
Gad m 2 Frog parvalbumin
Signal transduction
Gad m 3
Resistant to pepsin
Ran e 1 (α- Mackerel and Tuna β-parvalbumins (Higler et al.,
Parvalbumin) 2002)
Frog 10-12 kDa
Ran e 2 (β-
parvalbumin)
Tropomyocin
Shrimp (Miyazawa et
Squid Tod p 1 38 kDa
Simple helical structure al., 1996)
Tropomyosin Refold after denaturation Snail, white mussel, black mussel, oyster, (Chu et al.,
Hal rn 1 49 kDa
Abalone family Heat stable proteins and squid 2000; Lu et al.,
Hal d 1 38 kDa
Pfam PF00261 Control muscle 2004)
contraction Shrimp, lobster, fruit fly, blue mussel, crab (Leung et al.,
Crab Cha f 1 34 kDa
and Hom a 1 1998)
Arthropods: Crustacea and Insects (Leung and
Cra g 1 Gastropods: Abalone and whelk Chu, 2001)
Oyster Cra g 2 35-38 kDa Bivalves: mussel, pen shell, scallop, oyster
Cra g 103 and clam
Cephalopods: cuttlefish, squid and octopuss
Source Allergen Family Molecular Mass Structure And Function Cross Reactivity References
Hom a 1 329 kDa Lobster, crab, dust, mites, nematode, blue (DeWitt et al.,
Lobster
Pan s 1 34 kDa mussel and humans 2004)
Not known (Greenfield
Black tiger Pen m 1
34-38 kDa Possibly similar to other shrimp and Fowler,
shrimp
tropomyosin 2002)
Similar to others like oyster (Ayuso et al.,
Brown shrimp Pen a 1 34 - 38 kDa Heat stable
2002)
Proteolysis shows no
greasyback Lobster, crab, dust, mites, nematode, blue (DeWitt et al.,
Met e 1 34 - 38 kDa effect on allergenicity
shrimp mussel and humans 2004)
AA 153-160 and 50-66 are invertebrate tropomyosins (Nagpal et al.,
Indian prawn Pen i 1 or sa II 34 kDa
epitope Similar to Pen m 1 and Met e 1 1989)
clam, sea snail, octopus and shrimp and a (Asturias et al.,
Snail Hel as 1 36 kDa Similar to others
41 kDa protein from cuttlefish 2002)
Anisakis (Asturias et al.,
Ani s 3 Stable to heat or cooking Shrimp
simplex 2000)
moth, dust mite, cockroach, king prawn, (Yu et al.,
Black tiger
Pen m 2 40 kDa lobster, sand shrimp, lobster, crab, crawfish 2003)
shrimp
and mussel
less stable than
arginine kinases: Arthropods, moth, dust (Lin et al.,
tropomyosin
mite, cockroach, king prawn, lobster, and 1993)
Neptune rose Arginine kinase Catalyses the reversible
39 kD allergen 39 kDa mussel
shrimp production of high-energy
Pen m2: sand shrimps, lobster, crab,
phosphoarginine
crawfish
40 kDa Not known (France et al.,
Gulf shrimp 40 kDa
allergen 1997)
Denatured collagens Not known (Hansen et al.,
Major structural proteins 2004)
Cod Gelatine Not known 60 kDa of skin
Unlikely to be a clinically
important allergen
Food Allergens 175
Table 10. Main additives involved in allergic reactions
Additive Function Allergic Symptom References

Preservatives in Vascular headache
Nitrates and Nitrite (Simon, 2003)
meats Nitrosamines are carcinogens
Granulomatous panniculitis
low-calorie (Garriga et al.,
Aspartame Aspartame-provoked headaches
artificial sweetener 1991)
Urticaria
Chronic urticaria
Atopic dermatitis (Stevenson,
Tartrazine
Asthama 2013)
Hyperkinesis
Angioedema, headache, myalgia,
backache, neck pain, nausea,
Monsodium glutamate (Williams and
Flavor enhancer diaphoresis, tingling, flushing, and
(MSG) Woessner, 2009)
chest heaviness
Urticaria
Butylatedhydroxytoluene Antioxidant in
(BHT) and breakfast cereals (Goodman et al.,
Idiopathic urticaria
butylatedhydroxyanisole Maintain crispiness 1990)
(BHA) Reduces rancidity
(Hino et al.,
Sweetner
Erythritol 2000; Wilson
Food coloring anaphylaxis
Annatto, and Carmine and Bahna,
agent
2005)
Prevent browning
Anaphylaxis
of foods (Park et al.,
Sulfites Life threatening, asthmatic
Antimicrobial 2008)
reactions
agent
(Spurlock and
Spearmint flavoring Asthma
Dailey, 1990)
Table 11. Commonly used methods for the detection of food allergens (van Hengel,
2007; Kirsch et al., 2009; Alves et al., 2015b)
Methods Specificity Target LOD/LOQa

Immunological
Binding to human IgE or antibodies
ELISA Proteins 01–2 mg/kg
raised in animals
Dipstick Binding to antibodies raised in animals Proteins 1 mg/kg
Biosensor Binding to antibodies raised in animals Proteins 05–2 mg/kg
Allergenic
RAST/EAST Human IgE binding 1 mg/kg
proteins
Binding to human IgE or antibodies
Immunoblotting Proteins 25–5 mg/kg
raised in animals
Rocket immuno- Binding to human IgE or antibodies
Proteins 25–30 mg/kg
electrophoresis raised in animals
Molecular biological
PCR Primers DNA 1 ppm
PCR ELISA Primers DNA 10 ppm
Real-time PCR Primers and probe DNA 2–10 ppm
Others
Ab–Ag interaction through stripping
Nanoparticle based Monoclonal
analysis of enzymatically deposited 38 ng/ml
immunosensor antibodies
silver
176 Priti Mudgil
IMMUNOLOGICAL METHODS
Enzyme Linked Immune Sorbent Assay (ELISA)
ELISA is the most common immunological used for allergen detection because of its
fastness, high reliability and sensitivity. This method is a micro plate colorimetric method
where specific marker proteins are detecting by an enzyme linked antibody raised in
immunized animal against specific allergen. Quantification by ELISA method can be done by
measuring enzymatic activity of an enzyme linked protein specific second antibody which can
binds to the allergen primary antibody complex and upon addition of enzyme substrate
produces a colored product, for which the absorption is directly proportional or inversely
proportional to the quantity of allergen in the food sample. Recently a more advanced version
of ELISA i.e., ELISA-ICP-MS has been developed by combining it with inductively coupled
plasma MS (ICP-MS) to increase the sensitivity and the precision. In ELISA-ICP-MS the
secondary antibody is labeled with a stable isotope instead of an enzyme, and can be used for
quantification with a mass spectrometer (Careri et al., 2007; Careri et al., 2008; Koeberl et al.,
2014).
Dipstick Test
It is an alternative qualitative method to ELISA. This test is gaining interest in food

industry allergen detection as it is very inexpensive, simple, rapid and portable method that
does not require heavy instrumentation. The only limitation of its being qualitative method is
being resolved by researchers for example: recently an assay for egg was developed with high
specificity and extreme sensitiveness (002mg/kg).
Dot Immunoblotting
Dot immunoblotting is a simple, rapid, inexpensive semi quantitative (25 mg/kg) method
for allergen screening in foods where intensity of dot is propotionally related to the amount of
allergen. In this method sample are spiked in form of dot onto a nitrocellulose or poly-
vinylidenedifluoride (PVDF) membrane. After incubation with enzyme-labelled, protein
specific antibodiesdots can be visualized in form of a colored product after enzyme–substrate
interaction. In more advanced and sensitive form of dot immunoblotting radio actively
labelled antibodies and subsequently analysis is done by radiography (Blais and Phillippe,
2001).
Immuno-Electrophoresis (IE)
Immunoelectrophoresis is a combined method based on the principle of electrophoresis

and immune precipitation. Most commonly this method is used for identification and
characterization of proteins but over last many years the method has evolved to be use in a
Food Allergens 177
variety of other techniques like allergen detection. In allergen detection a modification of IE

known as Rocket immuno-electrophoresis is used. In this method antigens to be detected are
applied on a gel mixture containing specific antibodies. Antigens migrate through the gel
according to their electrophoretic mobility untilantigen–antibody complexes precipitate in the
gel. The height of the rockets is proportional to the amount of antigen applied. This method
has been utilized by many researchers for allergen detection but is not a method of choice due
to laborious gel preparation and immuno-staining procedures (Kirsch et al., 2009; Csako,
2012).
However these immunological methods discussed above are unable to differentiate
between similar proteins hence cannot be utilized for understanding and detection of cross
reactivity in food matrix which in turn can lead to false positive results. An exception to this
is SDS-PAGE followed by immunoblotting, which can provide information on the molecular
weight of proteins that are bound by the antibodies employed for the detection.
SDS-PAGE Immunoblotting
In SDS-PAGE immunoblotting, proteins are transferred to anitrocellulose or PVDF

membrane and protein-specific radio- or enzyme-labelled antibodiesare added after the
blotting. The allergens detected appear like protein bands on a 1D gel or like individualized
spots on a 2D gel SDS-PAGE immunoblot technique has been successfully utilized for
routine screening of low levels of potentially allergenic hazelnut and almond proteins in
chocolate with a sensitivity of 5 μg g of chocolate (Costa et al., 2015). However, similar to
other immunological procedure this method is also laborious and time consuming therefore
are not very well adapted for routine analyses.
Cell-Based Methods
These assays are basically used for the confirmation purpose and among these basophil
histamine release (BHR) assay and the β-hexosaminidaserelease assay are most common. The
working principle for these assays is the release of histamine and β-hexosaminidase
(mediators of allergy released by blood cells like ―basophils‖ and ―mast cells,‖ respectively).
The quantity of histamine or β-hexosaminidase is proportional to the concentration of the
specific allergen. These tests have been used for the detection of soybean allergens
(Yamanishi et al., 1995).
Another type of cell based method is BAT method, also called the ―flow-cytometric
allergen stimulation test‖ is more specific and sensitive method (82 μg mL) for allergen
detection This method targets the release of mediators like histamine, leukotriene C4,
interleukin-4, interleukin-13 and appearance of surface receptors (e.g., CD63, CD203c) on
basophils after exposure to allergens The quantification of allergen is performed by dye-
labelled antibodies which are detected by flow cytometry after binding to active receptors
(Ebo et al., 2006).
178 Priti Mudgil
MOLECULAR METHODS
PCR, Real-time PCR and PCR-ELISA
The PCR, a tool developed by Karry Mullis in 1985 has changed the area of molecular
detection completely within two and half decades. This tool can be used for indirect analysis
of allergen in food based on nucleic acids. Generally these methods target a small segment of
the gene responsible for allergen expression and amplification of this segment considered as
protein detection. DNA based detection tools have the advantage of being highly specific, less
laborious and sensitive and can even detect 10 mg/kg of allergen in foods. Advancement of
PCR method for allergen detection includes PCR coupled with ELISA and as real-time
PCRIn PCR-ELISA, the detection is gel-free since the amplified DNA fragments are
hybridized to a protein probe and detected by ELISA. Similarly, real-time PCR is also gel
free and less time consuming as compared to conventional PCR. This is based on florescence
dye like SYBR green which at each amplification results in fluorescence i.e., proportional to
the amount of the gene of interest in the food sample. The only disadvantage of these methods
is they are very expensive, require envy instrumentation.
Mass Spectrometry Based Method
Disadvantages related to immunological and molecular method in allergen detection has

always prompted researcher to develop new and alternative method which are fast, easy,
reliable and sensitive. One among such non-immunological methods are mass spectrometry
(MS) based methods that have been successfully investigated and developed to overcome the
drawbacks of ELISA. A mass spectrometer is composed of three different parts: ion source,
mass analyzer, and detector As ion source, matrix-assisted laser desorption/ionization
(MALDI) or electrospray ionization (ESI) are used most commonly. For mass analyzer, time-
of-flight (TOF) and ion trap (IT) are used. Other combination based hybrid MS systems like
ESI-qTOF, ESI-IT or MALDI-TOF has been further described. These hybrid MS systems can
be used to identify proteins and peptides with great sensitivity. The MS based method for
allergen identification is, however, not commonly applied for routine analysis but still they
have advantages of being easy, fast, multiple allergen testing, robust, stable and real time. In
summary, MS method overcomes the limitation of molecular and immunological methods.
Moreover, MS systems make it possible to generate information about amino acid sequences
of allergens as well as identifying cross reactivity and isoforms (Koeberl et al., 2014).
Biosensors
Biosensors and chip based technology are the most advanced yet uncommon technologies
that has not been explored fully for their potential in allergen detection. A biosensor is a self-
contained integrated device constituted by a biological recognition component (biochemical
receptor) and a signal transduction element connected to a data acquisition and processing
system. Biosensor based technique makes the allergen detection real time by allowing to
Food Allergens 179
measure specific molecular interaction of allergen in food matrix. The method generally use a
target molecule and depending upon the type of target used biosensors can be divided into
four types i.e., immunosensors (antibody based), Genosensors (DNA based), aptasensors
(aptamers; synthetic DNA or RNa strand with high affinity for allergen) (Alves et al., 2015b).
In the last decade a number of researchers has excavated the potential of biosensors for
allergen detection in food For instance, a gold based nanosensor bound to antibody has been
successfully utilized by for detection of peanut allergen Ara h 1(Alves et al., 2015a).
Similarly, an aptamer based biosensor was utilized by Tran et al., (2013), for Ara h 1 and Ara
h 2 detection in peanuts Bettazzi et al.,(2008), has investigated the presence of hazelnut major
allergens Cor a 104, Cor a 103 using genosensors. Many other examples and detailed
explanation for biosensor based allergen detection can be refereed from Alves et al.,(2015b).
FUTURE CHALLENGES
Generally food allergens are proteins hence one men food is other men poison and
absence of precise cure and medication make situation grimmer. Control of food allergens
poses more difficulties than other biological and chemical hazards in foods like chemicals and
pathogens as here the hazard is food itself that is required for most of the population for
nutrition. For food allergens common risk management approaches that aim to reduce overall
exposure to a hazard for all consumers cannot be applied as in case with chemical
contaminants and pathogens where they are considered as hazardous for the entire population.
The area of food allergy is an important public health concern globally and the only way to
stop is avoid eating foods that are allergic to an individual. Food allergy can only be
controlled by taking responsibility from both parties i.e., food producers by using specific
labelling and consumer by avoiding eating such foods that lead to potentially health
threatening allergic reactions. Different regulatory agencies worldwide publish new
information about food allergen and imply regulation about their proper manufacturing and
detailed labelling from time to time. In view of the increasing volume of international trade in
foods and food ingredients, international harmonization based on transparent risk assessment
and risk ranking processes will also be important. However as the process is complex, it is
important to remember that the goal is to prevent potentially life-threatening reactions by
providing allergic consumers with clear, complete, and accurate information about what is in
the foods they eat. Regulatory agencies work to protect the health of food allergic consumers
in a complex legal and scientific environment. Under regulatory standard for foods, all food
allergens that are intended to be present in a food (as ingredients or as components of
ingredients) must be identified on the label and preventive controls must be used to avoid the
presence of unintended (and undeclared) allergens.
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Chapter 8
RAPID METHODS FOR THE DETECTION

OF FOOD BORNE PATHOGENS
Divya Arora1,2, Nisha Sharma1,2, Vishal Sharma1,2, Vidushi Abrol1

and Sundeep Jaglan1,2,*
1
Quality Control, Quality Assurance and CMC Division, CSIR-Indian Institute of
Integrative Medicine, Jammu, India
2
AcSIR-Academy of Scientific and Innovative Research, India
ABSTRACT
Food borne pathogens cause severe infections, which result in human illness and
death in both developed and developing nations. Food borne pathogens can be found in
diverse foods, therefore, their detection and reckon in food is a requisite part of food
industry, food safety plans and public-health authorities. Conventional methods (culture-
based) used for detection of food borne pathogens are often time consuming and labor
intensive. With emerging technologies reliable, faster, sensitive, specific and convenient
methods come into place. Most of the rapid detection methods rely on nucleic acids and
antibody-based assays. The purpose of this chapter is to highlight the commonly
available rapid detection methods included are nucleic acids, biosensors, immunological,
flow cytometry and MALDI-TOF mass spectrometry based platform. The chapter will
cover an update on the current state of the knowledge including recent usage and
findings.
Keywords: foodborne pathogens, PCR, qPCR, ELISA, flow cytometry, biosensors, MALDI-
TOF-MS
*
Corresponding author: Email: sundeepjaglan@iiim.ac.in; Tel: +919419147422.
188 Divya Arora, Nisha Sharma, Vishal Sharma et al.
INTRODUCTION
Worldwide, there are abundant cases of deaths and food borne illness due to the
consumption of food contaminated with microbial pathogens or their toxins. According to
literature, there are 1,351 cases of deaths, 55,961 hospitalizations and 9.4 million episodes of
food borne illness per year in United States caused by 31 major pathogens (Scallan et al.,
2011). The scenario of casualties due to the contaminated food in developing countries is
more drastic. Microbial analysis is an important aspect in the food supply chain to ensure a
safe and pathogens free food. Therefore, there is a great need to implement systems such as
Hazard Analysis and Critical Control Points (HACCP) as a part of Good Manufacturing
Practices (GMP) and Sanitary and Phytosanitary measures (SPS) to control the quality of the
products produced for the presence of the microbial toxins and pathogens (Mandal et al.,
2011). HACCP is a systematic management system in which food safety is ensured by the
analysis and control of chemical, physical and biological hazards from raw material,
handling, production, distribution of the final product. The implementation of such preventive
systems has greatly improved food safety, but it will not be completely effective until better
techniques of analysis are developed. Traditional microbial detection methods like
microscopy, biochemical tests, phenotypic characteristics, Gram staining and growth on
different media (Croxatto et al., 2012), take several days for the final identification. Further,
minimally processed foods have a short shelf life, which limits the use of these traditional
methods. In recent years, many researchers have focused on the progress of rapid detection
methods for the food borne pathogens (Xu et al., 2013; Ryu et al., 2013; Wang et al., 2014;
Shukla et al., 2014; Lee et al., 2014; Leem et al., 2014; Chen et al., 2015; Xu et al., 2015).
These methods can be classified into various categories: nucleic acid based methods,
immunological methods, flow cytometry, biosensors based methods, matrix-assisted laser
desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) based detection of
food borne pathogens (Figure 1). The aim of this chapter is to review these rapid methods of
identification and detection of food borne pathogens.
Figure 1. Illustration of various rapid methods for the detection of food borne pathogens.
Rapid Methods for the Detection of Food Borne Pathogens 189
1. NUCLEIC ACID BASED METHODS

Detection of food borne pathogens mainly involves the combination of both conventional
as well as Polymerase Chain Reaction (PCR) based techniques. These methods include
enrichment (pre-enrichment and/or selective enrichment) followed by plating directly on to
selective agar and confirmation of presumptive bacterial colonies by biochemical tests,
staining and microscopic analysis. These methods are mainly used because of its cheapness,
detect only viable pathogens and yield isolates that can further be characterized and studied.
But there are many disadvantages as they are laborious, less efficient and relatively slow.
Advances in biotechnology have resulted in the development of rapid methods such as DNA
based assay: PCR, DNA hybridization and DNA microarray that make manipulation easy,
provide results in less time, and reduce cost. These methods are capable of detecting
microbial agents in food samples and also through precise genotyping of the contaminating
pathogen, the origin of contamination can be traced, which is the main basis of molecular
epidemiology (Adzitey et al., 2013).
Prior to final analysis of food sample it requires removal of any kind of inhibitors and in
some cases separation and concentrating the microorganisms (Kretzer et al., 2008). In context
of sample preparation, microbial lysis and efficient removal of inhibitors are necessary. Cell
lysis is done to release nucleic acid contents, various parameters have to be taken care of
during lysis, for example, the type and amount of organism to be lysed, the sample matrix,
etc. Multiple methods are available to lyse pathogens such as physical, chemical and
enzymatic, or combinations of these three methods. Common physical methods are
temperature based (freeze/thaw or heat), bead-beating, and sonication. In freeze/thaw lysis
method, the cellular suspension is transferred between freezing (dry ice-ethanol bath) and
warm conditions (37°C water bath). Heating at 95°C to 100°C is also used to lyse the cells.
The efficiency of heat lysis depends on the microorganism and is often combined with
chemical and enzymatic treatment. The heat treatment serves two functions, it breaks cells
and inactivates enzymatic activity. Enzymes such as Proteases are commonly used for
bacterial lysis. This enzyme is inactivated before adding sample to a PCR mix as proteases
will destroy DNA polymerase enzyme. High frequency sound waves are used in sonication to
create localized regions of low pressure that results in formation of micro bubbles that
implode, ultimately break and open cells. Bead-beating is used for breaking cells, in which
equal volume of silica or zirconium beads (0.1 mm diameter), are added to the sample and
mixed by vortexing for 3-5 minutes. Bead-beating generate foam that can be controlled by
common antifoaming agents. For chemical lysis of cells detergents such as Ionic detergents,
nonionic or zwitterionic detergents are of common usage. Most commonly Ionic detergents
such as sodium dodecyl sulfate (SDS) are used. Sodium dodecyl sulfate (SDS) is stronger
than nonionic or zwitterionic detergents and is used to disrupt the lipid bilayer. Detergents
disrupt the lipid bilayer surrounding cells. SDS has also the property of denaturing proteins,
such as DNAse and RNAse. As detergents cannot lyse cell walls, so samples are pre-treated
with enzymes (proteases or lysozyme) before the use of detergent. Other salts such as
Guanidinium thiocyanate and guanidinium chloride are also used to lyse cell membranes
(Burden, 2012).
Inhibitory compounds in the sample can lead to partial or complete inhibition of PCR.
Various inhibitory compounds are present in food and culture media that can inhibit PCR.
Food samples contain humic acid from soil. Animal samples contain protein and
aminoglycans such as hemoglobin, lactoferrin and heparin. Plant material contains
polysaccharides, ionic detergents such as phenol, ethanol, proteinase K, guanidinium, and
salts involve in extraction buffer, can also inhibit PCR. To control PCR inhibition by humic
acid, bovine serum albumin and hemoglobin are used. Another inhibitors were shown to
reduce single-stranded DNA binding protein from T4 bacteriophage (gp32), in addition
Tween® 20 or DMSO are also used. Bacterial enrichment before DNA extraction removes
PCR inhibitors and improves accurate detection (Xihong et al., 2014).
Bacterial strains can be rapidly identified without the need for isolating pure cultures.
Polymerase chain reaction (PCR) is a technique that is used for the amplification of target
DNA sample using temperature cycles (45–95.8 ⁰C and 30–40 cycles). Different components
used in PCR are synthetic oligonucleotide primers, (0.11–10 µM), a thermostable DNA
polymerase(0.6–1.25 U), DNA templates (5–25 ng), deoxyribonucleotide triphosphates
(dNTPs), magnesium (Mg2+) and buffer solutions have been used in different concentrations
to increase detection limits (Adzitey et al., 2013). PCR is the most sensitive and specific
method for the detection as well as quantification of small amount of DNA. This technique is
used to amplify genes that are specific to taxonomic groups of bacteria and also to detect
virulent genes present in food-borne bacteria. PCR can be used to detect even one molecule of
target DNA; this amplified product can be upscaled via in-vitro amplification methods to
detect pathogens indirectly that are present in extremely low concentration (Hill, 1996). PCR
based kits are commercially available for detection of specific food borne pathogens.
Different number of PCR based protocols have been developed for detection of food borne
pathogens detections such as single polymerase chain reaction(sPCR), multiplex polymerase
chain reaction (mPCR), quantitative or real time polymerase chain reaction (rtPCR),
isothermal amplification (Loop-mediated isothermal amplification and nucleic acid sequence-
based amplification) oligonucleotide DNA microarray, etc., (Law et al., 2015).
1.1. Single Polymerase Chain Reaction
In this reaction we use single set of primers that target specific genes to detect
microorganisms. Bacteria can be detected directly from the food sample with or without pre-
enrichment. However, direct detection of food borne pathogens by PCR assays in sample of
turbid nature can result in the detection of DNA in dead cells and give false negative results.
Before PCR detection, enrichment or the application of fluorescence in situ hybridization
(FISH) techniques have been suggested to curb this situation. Amplification of 16S rRNA
genes of bacteria using universal primers, helps in the identification of unknown or novel
bacterial species. Bacteria isolates picked directly from agar plates, can easily be confirmed
(Adzitey et al., 2013).
Single PCR has been used for detecting various species of Campylobacter recovered
from duck faeces and environmental waters that were contaminated with duck droppings
(Ahulreesh et al., 2010). 39 Campylobacter spp. from 110 duck meat samples were isolated
using the conventional bacteriological method in Iran and has been confirmed using this PCR
(Rahimi et al., 2011). One C. jejuni isolate from Philippine Mallard duck has been accurately
identified using PCR (Magistrado et al., 2001). Further this technique is used to amplify the
16S rRNA of Campylobacter spp., before sequencing for species identification (Adzitey and
Corry, 2011).
1.2. Multiplex Polymerase Chain Reaction
Amplification of more than one locus simultaneously is required for a rapid detection of
multiple microorganisms in a single reaction (Xihong et al., 2014). Basically it is a
methodology in which several specific primer sets are combined into a single PCR assay,
referred as Multiplex PCR (Chen and Lin, 2007). Apparently, primer designing is a key factor
in the development of a multiplex PCR assay. There should be some interaction between the
multiple primer sets, so that the primer concentrations have to be adjusted in order to generate
reliable yields of all the PCR products. The primer sets should be designed with a similar
annealing temperature, which provides a method to distinguish between amplicons following
thermal cycling. Kong et al. used mPCR method for the simultaneous detection of six
commonly encountered waterborne pathogens in a single tube. They targeted aerolysin (aero)
gene of Aeromonas hydrophila, invasion plasmid antigen B (ipaB) gene of Salmonella
typhimurium, attachment invasion locus (ail) gene of Yersinia enterocolitica, invasion
plasmid antigen H (ipaH) gene of Shigella flexneri, enterotoxin extracellular secretion protein
(epsM) gene of Vibrio cholerae, and 16S-23S rDNA (Vpara) gene of Vibrio
parahaemolyticus (Kong et al., 2001).
Food borne pathogens such as E coli, S aureus, Salmonella spp. and L monocytogenes
have been detected using mPCR assay in a single reaction. The mPCR used specific primers,
specific Stx2A primer of Escherichia coli O157:H7, specific Its primer of Salmonella spp.,
specific Cap8A-B primer of S. aureus, and specific Hly primer of Listeria monocytogenes.
DNA amplification of target pathogens, with these primers, produced products of 553, 312,
405, and 210bp, respectively (Park et al., 2006). Recently, a multiplex-PCR assay has been
established using fliCh7 and iap gene-specific primers for the simultaneous detection of
Escherichia coli O157:H7 and Listeria monocytogenes, alongwith, a modified method of
enrichment and harvesting leading to a highly sensitive and rapid single-reaction PCR
detection of both pathogens (Mukhopadhyay and Mukhopadhyay, 2007). In one mPCR assay
five Shiza toxin-producing E. coli genes i.e., stx1, stx2, eae, ehxA, and +93 uidA were
successfully detected and pathogenicity was assessed (Insook et al., 2014). Five pathogenic
Vibrio species i.e., Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio
alginolyticus and Vibrio mimicus were simultaneously detected in seafood by an innovative
assay (mPCR-HPLC) in which multiplex PCR was coupled with HPLC (Xu et al., 2015).
1.3. Quantitative PCR
Quantitative PCR (qPCR), also known as real-time PCR, enable one to monitor the PCR
product formation throughout the reaction. It offers simultaneous amplification, rapid and
sequence-specific-based detection of target genes and is highly applied in food microbiology
for detecting food borne pathogens (Xihong et al., 2014).
This assay involves Fluorescent dye such as SYBR green, this dye binds with the double-
stranded DNA as it is incorporated. Ultimately, these fluorescent amplicons are detected by
the fluorometer/photometer. Advantage of this dye over ethidium bromide is its 1,000-fold
more sensitivity. The amplicons are detected only when they reach the threshold range for
detection after ‗x‘ number of cycles. There is an inverse co-relation between cycle number
and the target copy number. The lesser the target sample is present in a sample, the more
cycles are required for the detection of amplicon. Real time PCR has the disadvantage of
false-positive results related with the production of nonspecific amplicons. Taqman PCR and
molecular beacons have reduced these false-positive results.
For the detection of food borne pathogens many commercially available qPCR kits have
also usage. The qPCR kits are reliable to detect food borne bacterial pathogens such as
Salmonella, E. coli, Listeria and Campylobacter; most of the kits are validated by
independent body (AOAC), U.S. regulatory body (FDA) or an ISO method for testing
relevant food. More details about the commercially available qPCR kits are presented in
Table 1.
1.3.1. Taqman PCR

Taq man probes mainly consist of fluorophore covalently attached to the 5' end of the
oligonucleotide probe and quencher at the 3' end. Different types of fluorophores are
available (e.g., 6-carboxyfluorescein, acronym: FAM, or tetrachlorofluorescein, acronym:
TET) and quenchers (e.g., tetramethylrhodamine, acronym: TAMRA). The quencher
molecule quenches the fluorescence emitted by the fluorophore via Fluorescence Resonance
Energy Transfer. Taq man probes are designed in such a way that they anneal with the DNA
region amplified by a specific set of primers. As the Taq polymerase synthesizes new strand,
due to 5‘ to 3‘ exo-nuclease activity the probe gets degraded. Degradation of probe releases
fluorophore, as before quencher was in close proximity to fluorophore, it was able to quench
the light emitted by the fluorophore but now due to release of fluorophore, distance between
fluorophore and quencher increases. Thus, fluorophore emits its light or fluorescence. Taq
man based real time PCR assays have been used for analysis and detection of foodborne
pathogens (Kimura et al., 1999; Lyon et al., 2001; Bej and Ward 2006; Rizvi et al., 2006;
Josefsen et al., 2007; Singh and Mustapha 2013).
1.3.2. Molecular Beacons

The internal probe consists of an internal nucleotide sequence complimentary to the
amplified product generated during PCR, flanked by the sequences that are complimentary to
each other, thus allowing 5‘ and 3‘ ends to anneal forming stem loop structure, fluorophore is
situated to one end and quencher at the other. 3‘end contains dideoxy nucleotide binding to
the nascent strand and degradation by exonuclease activity of DNA polymerase enzyme. The
molecular beacon hybridizes if there is any true amplicon, separates the fluorophore from
quencher and thus permits fluorophore to emit light upon excitation with UV radiation.
Development of recent qPCR assays eliminated the post- PCR step, by real-time
monitoring of the PCR product. Recently, the multiplex PCR approach has been implemented
in the qPCR using a set of TaqMan probes that were labelled with different fluorescent dyes.
Multiplex qPCR assay used the simultaneous detection of V. parahaemolyticus, V. cholerae
and V. vulnificus, using vmrA, zot and vuuA as target genes, respectively (Kim et al., 2012).
This procedure took approximately 12 hours, including the enrichment culture period.
Enrichment results a maximum sensitivity of 100 CFU/g food sample that meets the FDA
guidelines for acceptable level of these pathogens in food.
Table 1. Commercially available Quantitative PCR kits for the detection

of foodborne pathogens
Target Manufacturer’s/
Kit available and Manufacturer
Microorganism Supplier’s Web Address
Salmonella BAX® System Real-Time PCR assay, DuPont www.dupont.co.in
BAX® System PCR assay for screening, DuPont
BAX® System PCR assay for screening Salmonella
2, DuPont
AnDiaTec® Salmonella Real Time PCR, AnDiaTec www.andiatec.com
TaqMan™ Salmonella enterica detection, Applied www.lifetechnologies.com
Biosystems
MicroSeq® Salmonella spp. detection, Applied
Biosystems
ADIAFOOD® Salmonella, AES chemunex www.biomerieuxindia.in
Assurance GDS® for Salmonella, BioControl www.biocontrolsys.com
Systems
Foodproof Salmonella detection, BIOTECON www.bc-diagnostics.com
Diagnostics
GeneDisc® Salmonella spp., Pall Corporation www.pall.co.in
iQ-check® Salmonella II, Bio-Rad www.biorad.com
Listeria BAX® Listeria monocytogenes detection System, www.dupont.co.in
DuPont
TaqMan®Listeria monocyto genes Detection, www.lifetechnologies.com
Applied Biosystems
Foodproof Listeria Genus detection, BIOTECON www.bc-diagnostics.com
Diagnostics
Foodproof Listeria monocytogenes detection, www.bc-diagnostics.com
BIOTECON Diagnostics
GeneDisc® Listeria spp., Pall Corporation www.pall.co.in
ADIAFOOD® Listeria monocytogenes, AES www.biomerieuxindia.in
chemunex
Assurance GDS® Listeria monocytogenes, www.biocontrolsys.com
BioControl Systems
iQ-Check™ Listeria monocytogenes II Real Time www.biorad.com
PCR, Bio-Rad
iQ-Check™ Listeria spp. Real-Time PCR, Bio-Rad
Thermo Scientific™ SureTect™ Listeria Species www.thermofischer.com
PCR Assay, Thermofischer Scientific
Escherichia coli BAX® Real-Time PCR Assay E. coli O157:H7, www.dupont.co.in
DuPont
ADIAFOOD Detection System: E. coli O157, AES www.biomerieuxindia.in
chemunex
ADIAFOOD Detection System: E. coli O157:H7,
AES chemunex
Assurance GDS for E. coli O157:H7, BioControl www.biocontrolsys.com
Systems
Target Manufacturer’s/
Kit available and Manufacturer
Microorganism Supplier’s Web Address
FoodChek™ E. coli O157:H7, FoodChek Systems, www.foodcheksystems.com
Inc.
GeneDisc® E. coli, Pall Corporation www.pall.co.in
MicroSEQ® E. coli O157:H7 Detection Kit, www.lifetechnologies.com
Applied Biosystems
ThermoScientific SureTect Escherichia coli www.thermofischer.com
O157:H7 PCR Assay, Thermofischer Scientific
iQ-Check™ E.coli O157:H7 Real Time PCR, Bio- www.biorad.com
Rad
iQ-Check™ E.coli O157:H7, Bio-Rad
iQ-Check™ STE VirX and SerO Real-Time PCR,
Bio-Rad
Campylobacter BAX® System Real-Time PCR Assay for www.dupont.co.in
Campylobacter jejuni, coli, and lari, DuPont
ADIAFOOD Campylobacter JCL qPCR, AES www.biomerieuxindia.in
chemunex
ADIAFOOD Detection System: Campylobacter
Quantification, AES Chemunex
Campylobacter real-time PCR, Eurofins Genescan www.eurofinsus.com
iQ-Check™ Campylobacter Real-Time PCR, Bio- www.biorad.com
Rad
Taq man and a SYBR Green were also used for the identification and quantitative
detection of food borne pathogens such as S. aureus strains containing the enterotoxin gene
cluster regardless of their variants. Using optimized qPCR conditions, SYBR Green and
TaqMan qPCR assay, one can be able to quantitatively detect about 1 × 103 and 1 × 104 CFU
of the pathogen per millilitre raw milk (10 and 100 CFU equivalents of egc+ S. aureus per
reaction mixture) respectively.
1.4. Isothermal Amplification
Many novel methods have been developed to amplify nucleic acids under isothermal
conditions such as, loop mediated isothermal amplification (LAMP), nucleic acid sequence-
based amplification (NASBA) and oligonucleotide DNA microarray, etc. This approach has
simpler hardware requirements than PCR, as it works with a simple water bath setup and does
not require a thermal cycling system. Isothermal amplification techniques have better
tolerance to some inhibitory materials that affect the molecular amplification efficiency than
PCR (Xihong et al., 2014).
1.4.1. Loop-Mediated Isothermal Amplification (LAMP)

This method involves an rely on an auto cycling strand displacement DNA synthesis
performed by the Bst DNA polymerase large fragment and is different from PCR in that 4-6
primers are used to target 6-8 specific regions of the target gene. As the amplification is
performed under isothermal conditions between 59°C and 65°C, and the amplified products
are mixtures of many different sizes of stem loop DNAs with several inverted repeats of the
target sequence and cauliflower-like structures with multiple loops. In addition two loop
primers can be used to accelerate the reaction. This approach uses loop primers and yields
about 103-fold or higher levels of amplification product than conventional PCR in 60 min.
SYBR Green I dye is used to confirm the amplified products with naked eyes instead of
conventional gel electrophoresis analysis, this dye changes the color of the solution to green
in the presence of LAMP amplicons, otherwise, it remains orange for reactions with no
amplification.
StxA2 virulent gene in Escherichia coli O157: H7 cells, was first detected by this
technique (Maruyama et al., 2003). This technique was also used for identification and direct
detection of acidophilic thermophilic bacteria (ATB) contaminants in pure juices. This
method is able to detect 2.25 × 101 CFU/ml of ATB in juice samples within 2 h.
A large number of forms are available for this method such as reverse transcription
LAMP assay (Chen et al., 2011), in situ LAMP assay (Ye et al., 2011), multiplex LAMP
assay (Jasson et al., 2010) and real-time reverse transcription LAMP assay (Liu et al., 2009),
have been developed and employed for the detection of various foodborne pathogens, such as
Bacillus anthracis (Qiao et al., 2007), Vibrio parahaemolyticus (Nemoto et al., 2011),
Staphylococcus aureus (Yang et al., 2011), Salmonella (Zhao et al., 2010b), Pseudomonas
aeruginosa (Zhao et al., 2011), Escherichia coli O157 (Zhao et al., 2010a), and Listeria
monocytogenes (Wang et al., 2007).
1.4.2. Nucleic Acid Sequence-Based Amplification (NASBA)

NASBA is a technique of isothermal amplification, developed by Compton in 1991. In
this technique nucleic acid is amplified under isothermal conditions around 41⁰C. NASBA is
generally used for the RNA amplification, single strand RNA is converted into
complementary DNA (cDNA) with the help of reverse transcriptase enzyme during the
reaction. NASBA reaction requires two specific primers and three enzymes such as avian
myeloblastosis virus (AMV) reverse transcriptase, T7 RNA polymerase and RNaseH. After
completion of reaction, the amplicons are detected on agarose gel. The methods that are used
for amplicon detection (such as agarose gel electrophoresis or enzyme-linked gel assay), are
labor intensive and not cost-effective (Xihong et al., 2014). To overcome this problem real
time NASBA assay is used, in which fluorescently labelled probes (molecular beacons) detect
the single-stranded RNA amplicons. This method has been used for the detection of various
food borne pathogens such as Salmonella enterica, Staphylococcus aureus, Vibrio cholerae,
Campylobacter jejuni, and Campylobacter coli. This method is able to detect viable cells, but
it is necessary to treat samples with RNase-free DNase before NASBA assay, as RNase will
degrade mRNA from dead cells (Dwivedi and Jaykus, 2011).
1.5. Oligonucleotide DNA Micro-Array
Micro-array is mainly used for the study of gene expression, but recently this approach is
also used for the detection of food borne pathogens. Oligonucleotide probes of 25-28bp are
synthesized chemically and coated on glass slides or chips called micro array. Each oligo-
nucleotide probe is able to bind with specific sequence of target gene. The nucleic acid
sample mainly consists of fragments (DNA, mRNA or cDNA) that are denatured to generate
single strand and labeled with fluorescent dye followed by the binding of oligo-nulcleotide
with specific target sequence of array. Due to the formation of probe sample complex,
fluorescence signal is formed. The intensity of emitted light is directly proportional to the
concentration of labelled nucleic acid fragment of sample.
Micro-array has been used for the detection of pathogenic Shigella and E. coli serotypes.
Detection of various serotypes is very important as in E.coli, several sertotypes are found that
ranges from harmless strain of E. coli K-12 to deadly strain of E. coli O157:H7. This
approach was also used for the detection of various foodborne pathogens such as
Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, Clostridium
perfringens, Vibrio cholerae, Campylobacter jejuni, Shigella spp, Salmonella spp and
Bacillus cereus. With the advancement of technology, several companies provide ready to use
microarray based kits for foodborne pathogens detection, for example: StaphyChips® by
Affymetrix and Advanced Array Technology (ATT) by Eppendorf Array Technologies.
Table 2. Advantages and disadvantages of various molecular techniques used in the

detection of foodborne pathogens
Identification
Advantages Disadvantages References
method
Single PCR  Provides more sensitive,  Food components Adzitey et al.,
specific and accurate interfere with PCR 2013; Law et al.,
results provides misleading 2015; Mandal et
 Automated results al., 2011; Park,
 Rapid detection of  Optimization of PCR Aydin, 2014;
single genes condition required Zhang, 2013
 Provides reliable results  Do not distinguish
between non-viable and
viable cells
Multiplex PCR  Rapid detection of  Difficulty in Primer Adzitey et al.,
multiple pathogens are designing 2013; Law, 2015;
possible, thus reduces  Sometimes Primers Mandal et al.,
cost, time and sample interfere with each 2011; Park,
volumes other, thus some genes Aydin, 2014,
 Automated remain undetected Zhang, 2013
 Provides reliable results  Affected by PCR
inhibitors
 Do not distinguish
between viable and
non-viable cells
Real time PCR  Very high specificity  Expensive equipments Adzitey et al.,
sensitivity and and reagents are 2013; Law, 2015;
reproducibility required Mandal et al.,
 Post amplification  Requires trained 2011; Park,
processing is not personnel having high Aydin, 2014
required skills
 Less detection time  Difficulty in designing
 Real time monitoring of multiplex Real time
amplicon from PCR
pathogens  Very high cost
 Quantification is easy
Identification
Advantages Disadvantages References
method
Reverse  Only viable cells of  To handle unstable Adzitey et al.,
transcription PCR pathogens are detected RNA much skill is 2013
required
LAMP  Less time consuming  Designing of primer is Law 2015;
 Low cost very complicated Xihong et al.,
 Does not require  Unknown and 2014
thermal cycling system unsequenced target
cannot be detected
NASBA  Does not require  Requires only viable Law, 2015;
thermal cycling system results or Rnase-free Xihong et al.,
 Can detect viable cells DNase treatment before 2014
 Low cost NASBA assay should
 Specificity and be proper otherwise it
sensitivity will detect RNA from
non-viable cells and
provides misleading
results
Olignucleotide  High throughput  Very high cost Park, Aydin, 2014
DNA Micro array  Various pathogens can  Requires
be detected in a single oligonucleotide probes
microarray and fluorescent labeling
 Specific serotypes can of target gene.
be detected  Difficulty in
 Reduces time differentiation between
 Very high sensitivity viable and non-viable
and specificity cells
 Requires trained
personnel having high
skills.
Figure 2. Sandwich ELISA.

2. IMMUNOLOGICAL DETECTION METHODS

Immunological methods for the detection of food-borne pathogens are used to detect
antibodies by antigen or by antibody to detect an antigen of the pathogen. The major principle
is to detect the availability of organic molecule that can bind to specific domains present as
the target molecule due to its affinity. This technique is based on antigen-antibody binding
complex which can easily be detected and can be measured because the antibody will bind to
its specific antigen and based on its binding strength, it will be identified by its binding
sensitivity and specificity. There are various antibodies those have been designed in different
assay types for the detection of food-borne pathogens and microbial toxins. The various
Immunological based methods involve the use of polyclonal and monoclonal antibodies.
Many polyclonal antibodies which are derived from either rabbit or goat serum contain
antibodies with different origins with different specificities. Monoclonal antibodies are better
than polyclonal antibodies for the specific detection of a molecule and with the development
of monoclonal antibodies, immunological identification of microbial contamination has
become more sensitive, specific, reliable and reproducible (Leonard et al., 2003). Due to this,
many commercial immunological assays are available for the detection of huge number of
different microbes and their products include enzyme-linked immunosorbent assay,
immunomagnetic separation assay and lateral flow immunoassay, which are recently being
used for the detection of food-borne pathogens (Xihong et al., 2014). Generally,
immunological detection methods can be classified into two types i.e., Homogeneous
immunoassay and Heterogeneous immunoassay. Homogeneous immunoassay is also called
as marker free assay because in this assay, there is no requirement of separation of bounded
and unbounded antibody, the antigen and antibody complex (Ag-Ab Complex) is directly
visible and measurable. This process is very rapid as its incubation time is very less, for
example: Immunodiffusion, Agglutination reaction, Turbidimetry, etc. (Mandal et al., 2011).
In Heterogeneous immunoassay, the unbound antibodies need to be separated from the
bounded antibody. Its example is enzyme-linked immunosorbent assay (ELISA).
2.1. Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA is one of the most important and commonly used techniques for the detection of
food-borne pathogens. ELISA is mostly used for the detection of toxins present in foods like
Clostridium perfringens α, β, and ε toxin, staphylococcal enteroxins A, B, C, and E,
Escherichia coli enterotoxins and botulinum toxins (Aschfalk and Muller, 2002; Xihong et
al., 2014).
Sandwich ELISA is the most effective form of ELISA whereby it involves two
antibodies. The primary antibody which is immobilized coated on to a solid support of the
micro-titer plate wells. The target antigen like bacterial toxins and food-borne pathogens from
the sample, binds to the immobilized primary antibody and the remaining unbound antigens
are removed by washing. Then, an enzyme-conjugated secondary antibody is added and
coupled to an easily assayed enzyme. The complex consisting antigen sandwiched between
two antibodies, is formed (Figure 2) and it can be detected by adding a colorless substrate
which will be converted into a colored form in the presence of the enzyme (Zhang, 2013).
Enzymes commonly used are alkaline phosphatase, horseradish peroxidase (HRP) and beta-
galactosidase which can be easily detected using colorimetric substrates (Yeni et al., 2014).
Monoclonal antibodies along with sandwich ELISA were used for the detection of TDH-
related hemolysin (TRH) of pathogenic vibrio parahaemolyticus. Its detection limit was 103
cells of pathogenic Vibrio parahaemolyticus (Kumar et al., 2011).
Commercially available ELISA test kit (BIOLINE Salmonella ELISA Test) was also
used for the detection of Salmonella in food products. Detection limit of this test kit was 1
CFU/25 g sample with minimum 4 of the 20 food matrices tested (Bolton et al., 2000).
Recently, ELISA systems such as VITEK immunodiagnostic assay system (VIDAS-
BioMerieux) and Assurance Enzyme Immunoassay (EIA) (BioControl), are also introduced
as the high throughput and automated system for the detection of food borne pathogens
(Glynn et al., 2006). The VIDAS system performs entire ELISA procedure automatically and
utilizes enzyme-linked fluorescent immunoassay (ELFA) which is similar to ELISA, but it is
highly sensitive fluorescent immunoassay to analyze the results. This system generally
completes its assay in 45 min to 2 h majorly, depends on test kit. This VIDAS system uses the
reagent strip and a plastic tube which is also called as solid phase receptacle (SPR). The
liquid sample is placed in the reagent strip which contains all the required reagents which can
be used instantly. Automatically, the instrument will perform ELFA by transferring the
sample to the SPR which will be coated with antibodies in its interior wall in order to capture
the target pathogen or toxin followed by a series of wells that contain enzyme-conjugated
secondary antibodies and enzymes in a sequence. Further, the result will be analyzed and
interpreted (Fung, 2002; Vaz-Velho et al., 2000). Various studies applied VIDAS for the
detection of Salmonella in pork sample, fruits and vegetables (Gomez-Govea et al., 2012,
Vieira-Pinto et al., 2007). Escherichia coli O157:H7 in Minas Frescal cheese, fruits, and
vegetables (Carvalho et al., 2014; Gomez-Govea et al., 2012). Campylobacter spp in fruits
and vegetables Gomez-Govea et al., 2012) and staphylococcal enterotoxin in cheese, raw
milk (Cremonesi et al., 2007). Assurance Enzyme Immunoassay (EIA) is a commercially
available ELISA kit which allows automation and high-throughput testing for Escherichia
coli O157:H7, Listeria monocytogenes, Campylobacter and Salmonella (Fung, 2002),
Salmonella in alfalfa sprouts (Stewart et al., 2001), Salmonella in chicken meat (Taha et al.,
2010).
2.2. Immunomagnetic Separation Assay (IMS)
IMS uses super-paramagnetic particles which utilize immune-magnetic beads (IMBs) to

capture reagents which have been developed for microbial isolation and identification and are
coated with antibodies against the target organisms to selectively isolate the organisms from a
mixed population. Immuno-magnetic separation assay is analogues to selective culture
enrichment, which allows the growth of other bacteria is suppressed whereas the pathogen
which is of interest or target is allowed to grow. The separation of the cells is a process of two
fundamental steps; primarily, the target cells are mixed/suspended with immune-magnetic
particles for incubation of maximum 1 h and they are separated by an appropriate magnetic
separator. Secondly the magnetic complex then further washed several times to remove the
unwanted contaminants (Mandal et al., 2011). The most important factors for using IMS are
efficient magnetic handling, less time consumption and very slightly affect the target analytes.
Therefore, various bio-reactive molecules can be conjugated to the IMB surface for the
isolation, identification and immune-precipitation of bio-molecules like proteins, pathogens,
cells, etc. and also to improve the resolution of magnetic resonance imaging (MRI) (Stevens
and Jaykus, 2004).
Polystyrene beads that are coated with iron oxide and antibodies are the most effective
and common magnetic carriers from food products used for separation and concentration of
specific microorganism (Skjerve et al., 1990). IMBs have been used to capture Listeria
(Kaclikova et al., 2001), E. coli O157:H7 (Chapman and Ashton, 2003) and Salmonella
(Jordan et al., 2004). IMS coupled with PCR assays, has shown very promising results for the
detection of Salmonella enterica (Mercanoglu and Griffiths, 2005), Listeria monocytogenes
(Amagliani et al., 2006) and E. coli O157:H7 (Fu et al., 2005). Another techniques, involving
Ag-Ab binding mechanism that have also been developed and applied in detection of
foodborne pathogens and toxins, used IMS for the selective capture of target bacteria and
ATP bioluminescence for its quantification. Detection limit of about 20 CFU/100 ml was
achieved, which was below the action limits of 300 CFU/ml daily, or a 30 days moving
average of 126 CFU/100 ml set by United States Environmental Protection Agency (USEPA).
Therefore, the procedure took less than 60min to complete without an enrichment step. The
study indicated that the system combining IMS with ATP bioluminescence was effective and
expedient for detecting Escherichia coli in beach water (Lee and Deininger, 2004). Recently,
the increasing importance for the mass spectrometry for the identification and
characterization of protein toxins produced by food borne pathogens results the improved
sensitivity and specificity of mass-spectrometry based techniques specifically when combined
with affinity methods. Schlosser et al. reported a novel method based on the use of immune-
affinity capture and matrix-assisted laser desorption ionization time-of-flight mass
spectrometry for selective purification and detection of staphylococcal enterotoxin B
(Schlosser et al., 2007).
Figure 3. Lateral Flow Immunoassay (LFI) Test Strip.

2.3. Lateral Flow Immunoassay (LFI)
The technique lateral flow immunoassay was derived from latex agglutination assay
which was introduced in 1950s by Pilotz and Singer (Singer and Plotz, 1956). In this,
immunochromatographic strips and dipsticks have been introduced for rapid detection of food
borne pathogens. Lateral flow device is composed of four different portions: plastic backing
along with sample pad, conjugate pad, the absorbent pad and nitrocellulose membrane. In this
method, Lateral flow devices (LFD) are comprises of a simple dipstick which is made up of
porous membrane which contains the antibody coated with colored latex particles or colloidal
gold particles which specifically cross-link with antigens of the pathogen/target site to give
visible clumping for identification and detection of pure sample taken from foods. The
particles, on the base of the dipstick, are placed with direct contact of the enrichment medium
and give color. The color can be visualized approximately 2 min to 10 min after the addition
of sample. The movement of these conjugated cells/particles is taken place by capillary action
until it becomes immobilized capture antibodies (Ngom et al., 2010). The technique is
extremely easy to use, requires no washing or manipulation and extremely rapid to perform
(Aldus et al., 2003). There are a number of LFDs available in the market those have been
validated for detecting different food borne pathogens. Many companies are marketing lateral
flow assays for food borne pathogen, their examples are available on the websites of
Association of Analytical Communities (AOAC) and Food and Drug Administration (FDA)
(Jasson, Jacxsens, 2010).
Monodisperse latex, colloidal gold, carbon and fluorescent tags are being used as labels
for the detection of foodborne pathogens by lateral flow immunoassay (LFI) (Xihong et al.,
2014). Escherichia coli O157 and S. aureus were identified by immunochromatographic strip
labelled with colloidal gold particles. The detection limit for Escherichia coli O157 without
enrichment was 1.8 × 105 CFU/ml and along with enrichment was 1.8 CFU/mL (Jung et al.,
2005) and for S. aureus, 103 CFU/ml (Niu et al., 2014). A novel immunochromatographic
strip test which is also based on sandwich format with fluorescent microspheres as label to
detect Campylobacter jejuni and the detection limit was 106 CFU/mL (Xu et al., 2012).
Lateral flow immunoassay is also used for detection of other food borne bacterial pathogens
like Listeria spp. and Salmonella and this can also be used to detect toxins which may cause
foodborne diseases, for example brevetoxins and Staphylococcal enterotoxin B.
Immunochromatographic test strips are also available commercially for the detection of food
borne bacterial pathogens like Reveal test kits (Neogen), DuPont Lateral Flow System
(DuPont Qualicon) and VIP GOLD (BioControl Systems) for Listeria, Escherichia coli and
Salmonella (Rong-Hwa et al., 2010, Shukla et al., 2014).
3. FLOW CYTOMETRY BASED PATHOGENS DETECTION

Flow cytometry is a useful technique for the fast detection, identification and
susceptibility testing of bacteria. It is also useful for the study of various parameters of
eukaryotic cells as well as microbes, but still used infrequently in the clinical microbiology.
Need of the rapid susceptibility test is in demand now a day as the spread of multi resistant
bacteria has been increasing since the last decade (Nuding and Zabel, 2013). Control of yeasts
in breweries is well known by using flow cytometry, but the practice for monitoring bacteria
appears very less. Flow cytometry is labor saving, relatively easy and rapid method than
conventional methods of identification and analysis of pathogenic microorganisms in the
eatables like water, milk, wine, beer without the need of incubation step. It reduces analysis
time to a few minutes for the analysis of pathogens significantly in the raw materials or
products at different stages of production and contaminations on the surface of the food
production lines without sample pretreatment (Juzwa and Czaczyk, 2012).
Flow cytometry gives multiparametric data of a large number of individual cells within a
sample (Cronin and Wilkinson, 2010), as it implies microscopy and biochemical studies for
the analysis of the physical and chemical characteristics of cells when they move in a fluid
stream past optical or electronic sensors. Photodetectors detect the emitted light and
computers are used for its analysis. Multiple cellular parameters (structural and functional)
for example cell size and DNA content can be measured simultaneously by flow cytometry.
Sample amount required in flow cytometry is very small (Maukonen et al., 2003). Five
integrated systems constitute a flow cytometer. These are: light source- laser or mercury
lamp, optical filters for the detection at different wavelengths, flow chamber, light detectors
(photodiodes or photomultiplier tubes) for the detection and amplification of signals, data
processing unit. The cells in the sample intersect the source of light one at a time at the point
of interrogation and the scattered light and fluorescence is detected by the light detectors
(Paparella et al., 2012). Detection by flow cytometry is a three step process: separation of
microbes from food samples, labelling of the target cells, and study of the fluorescently
labelled target cells.
Semiconductor Quantum Dots (QDs) are also used to detect pathogens in bacterial cell
mixtures by using flow cytometric analysis. Semiconductor quantum dots (QDs) made up of
CdSe/ZnS core/shell bioconjugates, show greater fluorescence intensities and offer lower
detection thresholds and better precision in analyzing bacterial cell mixture composed of
pathogenic E.coli and harmless E.coli. QDs can make huge improvement in the field of flow
cytometry because of their higher absorption and lower emission capacity than the dyes and
ensure analysis of heterogeneous mixture of cells (prokaryotic pathogens and eukaryotic
cells) without complications (Hahn et al., 2008). Fluorescent-bacteriophage assay in
combination with flow cytometry was found to be a sensitive technique for the E. coli
O157:H7 detection in broth culture (Goodridge et al., 1999).
Acridine orange is used in direct epifluorescent filter technique (DEFT), but has some
disadvantages like the difficulty of the sample preparation and harsh conditions required for a
good staining. Flow cytometry is useful in the detailed understanding of spore forming Gram
positive bacteria like Bacillus cereus that is a potential hazard to the food and consumer
industries. It gives us information about the resistance mechanism of endospores, the effects
of various treatments on them, and recovery of damaged spores on storage. Offline FCM is
used in many cases for detection of Salmonella or Listeria spp. using fluorescein
isothiocynate labelled antibodies and ethidium bromide (Ueckert et al., 1995). It can perform
experiments impractical with any other technique and is capable of generating unique data
e.g., Analysis of expression of genes in a real time in a large number of respective cells. FCM
will be applied in more and more labs as a new generation of low cost flow cytometer (e.g.,
Acccuri‘s C6 Flow Cytometer System) is available commercially in the market now a day
(Cronin and Wilkinson, 2010). Flow cytometry has interesting analytical potential but it still
lacks standardization and validation for quality control analysis (Sohier et al., 2014). The
bacterial load estimation or specific identification of pathogens can be done precisely by

cancelling the background fluorescence and improving signal to noise. It provides results in
minutes compared to the ordinary determination of food safety that requires 48-56 hours
(Buzatu et al., 2014). The advantages that FCM offers over old techniques are: good
specificity, a detection limit of 102-103cfu/ml and requires only 30 minutes (Mandal et al.,
2011). Foodborne diseases are increasing rapidly both in the developing and developed
nations due to enhance in food production practices. To reduce the time required in exposing
microbial contamination and for food safety estimation the flow cytometry based detection
proves to be sensitive and reliable.
3.1. Fluorescent Labelling
Stains or probes that are added to cells to get a fluorescent signal, are called
Fluorochromes (Maukonen et al., 2003). Fluorescent labelling is the use of the fluorochromes
to distinguish between different microbial cell types (Paparella, Serio, 2012). The fluorescent
stains that can distinguish between the dead and living bacteria, are becoming highly
important (Maukonen et al., 2003). The fluorescent stains are the generic dyes that can be
used to detect the components of a cell, biological activities, and distinguish between the live
and the dead cells (Paparella, Serio, 2012). Physiological status of individual cells can be told
by using fluorogenic substrates that fluoresce when acted upon by an enzyme. Moreover, the
use of fluorescent compounds opens the door for their use in studying microbes on the
optically non-transparent surfaces (Maukonen et al., 2003).
Depending on the species involved, time for the identification of bacterial pathogens
varies by traditional methods which involve culturing and then biochemical testing. FISH
(fluorescence in situ hybridization) is utilized to save the time in identification of microbes as
it is a culture independent technique and employs oligonucleotides labelled with fluorophores
that are specific to a target DNA/RNA sequence. Detection of mastitis pathogens like
Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Enterococcus
faecalis, Enterococcus faecium, Escherichia coli, and Trueperella (Arcanobacterium)
pyogenes is done by FISH. FISH reliably identifies bacteria in number ≥ 1 6CFU/ml, but not
less than this number in mastitis milk samples (Gey et al., 2013).
Tape-FISH approach offers direct detection of Salmonella on the surface of the crop
produces prone to the contamination with the Salmonella spp. like tomatoes, spinach,
jalapeno peppers etc., Microbiological sampling tapes are used to remove microbes from the
surfaces and the Salmonella cells can be detected by performing FISH directly on the Tape.
103CFU/cm2 is the limit of direct detection via Fluorescence Microscopy and it can be carried
out within 1.5h (Bisha and Brehm-Stecher, 2009). FISHing offers a rapid, reliable and
promising tool for the detection of pathogenic bacteria in food (Rohde et al., 2015). Detection
of human pathogens in complex food matrices is also possible by FISH. For example,
Salmonella spp. and Listeria monocytogens causative agents for salmonellosis and listeriosis
respectively can be detected by FISH (Bisha, 2009; Churchill et al., 2006).
Figure 4. Illustration of main components of Biosensor and their classification on the basis of
bioreceptor and transducer.
4. BIOSENSORS IN FOODBORNE PATHOGENS DETECTION

The term biosensor is fusion of biology and sensing i.e., they are analytical devices which
transform the biological responses into an electrical signal. They have replaced the traditional
methods to detect food borne pathogens which are often time-consuming. A biosensor usually
comprises of two main components: a bioreceptor or biorecognition element (recognizes the
target analyte) which can be a tissue, organelle, antibody, microorganism, cell, enzyme,
biomimic, nucleic acid, etc. and a transducer (converts the recognition event into a
measurable electrical signal) which may be optical, thermometric, electrochemical, magnetic,
piezoelectric and micromechanical or combinations of one or more of the above techniques.
These biosensors can be classified on the basis of their bioreceptor and transducer type as
shown in Figure 4.
4.1. Biosensors on the Basis of Bioreceptors
A bioreceptor is the part of biosensor which interacts with the target analyte of interest
and results in its recognition (Velusamy et al., 2010). Bioreceptors can be classified in
different classes: Nucleic acid bioreceptors, Antibody bioreceptor, Enzyme bioreceptor and
Bacteriophage bioreceptor.
4.1.1. Nucleic Acid Bioreceptors

These include Deoxyribonucleic acid (DNA), Ribonucleic acid (RNA) and Peptide
nucleic acid (PNA) which act as bioreceptors for the biorecognition of pathogens like
Salmonella sp., Campylobacter jejuni, E. coli O157:H7, etc. A recent review by Liebana et al.
for the electrochemical genosensors, immunosensors and phagosensors detection of

Salmonella species is available in literature (Liebana et al., 2014). Sun et al. prepared
Au@Ag nanorods DNA biosensor based on the fluorescence detection of E. coli O157:H7
(Sun et al., 2015). Manzano et al. designed and characterized OLED based DNA biochip for
the detection of Campylobacter spp. in meat samples (Manzano et al., 2015). The PNA
(Peptide nucleic acid) is a synthetic oligo amide that binds firmly with the complimentary
oligonucleotide sequences. The drawback of PNA is its cost of synthesis, which is very high.
The purine rich PNA oligomers are weakly hydrophilic and tend to accumulate, which further
affects its use as a bioreceptor (Sharma et al., 2013).
4.1.2. Antibody Bioreceptors

These are universal bioreceptors employed in biosensors. The antibodies may be
monoclonal and polyclonal based on their selective synthesis and properties. These have the
ability to differentiate molecular structures, allow one to make antibodies that bind
specifically to any of the biomolecules or chemicals or microorganisms, etc. Karoonuthaisiri
et al. developed a method for the detection of Listeria monocytogenes using specific
monoclonal antibodies (Karoonuthaisiri et al., 2015).
4.1.3. Enzyme Bioreceptors

Enzymes as bioreceptors are highly sensitive, stable and not hazardous. Their
immobilization rises as a main aspect to evolve other biosensors with relevant properties like
good storage and operational stability, high selectivity, immense sensitivity, large
reproducibility and short response time. In most of the cases, beta-galactoxidase and
Horseradish Peroxidase (HRP) are used. The analysis of pathogenic bacteria such as
Escherichia coli, Campylobacter jejuni and listeria monocytogenes can be performed by
labelling of the antibodies with these enzymes. Creran et al. reported a viable strategy for the
low cost detection of bacteria, in which they use inkjet printing to co-pattern a nanoparticle
sensor, an enzyme and substrate on a paper based test strip for the rapid detection of bacteria.
A colorimetric response was shown on the paper substrate that allowed visual analysis of
bacteria without the need for the high-cost instrumentation (Creran et al., 2014).
4.1.4. Bacteriophage (Phage) Bioreceptors

Bacteriophages are viruses which are 20-200 nm in size, that bind to the specific
receptors on the bacterial surface in order to transfer their genetic information inside the
bacteria. Phages recognize the bacterial receptors by its tail spike proteins. Phages have the
following advantages: High specificity, reproducibility and ability to kill or lyse its host.
Additionally, they are harmless, economic, omnipresent, have longer shelf life and easily
produced (Sharma et al., 2013). Schmelcher et al. published the various applications of
bacteriophages for the detection of food borne pathogens (Schmelcher and Loessner, 2014).
Hiremath et al. used novel lytic phage based magnetoelastic biosensors for the detection of
Methicillin-resistant Staphylococcus aureus (MRSA) (Hiremath et al., 2015).
4.2. Biosensors on the Basis of Transducers
A transducer plays an important function in the identification and detection process of a

biosensor. Biosensors can also be manufactured on the basis of the transduction system they
have. The transduction methods may be of following classes: Electrical, optical and mass
based.
4.2.1. Electrical Biosensors

Electrical biosensors can directly convert the biological data to electrical signals, which
can be easily detected. Electrical biosensors are further categorized into: Amperometric
biosensors, Conductometric biosensors, Impedimetric biosensors and Potentiometric
biosensors. Amperometric biosensors are the universal electrical detection method which has
been widely exploited for the detection of pathogens. Fernandez et al. reported the rapid and
sensitive amperometric magneto immunosensor for the detection of Staphylococcus aureus.
In conductometric biosensors, the reactions result in the change in the ionic species
concentration, which further leads to a change in the current flow. Generally, a
conductometric biosensor formed of two metal electrodes separated by a definite distance and
then an AC voltage applied across the electrodes causes the electrical conductivity. During
detection, the ionic concentration changes and the difference in conductance between metal
electrodes are monitored (Arshak et al., 2009). Pal et al. developed a direct charge transfer
conductometric biosensor for the determination of Bacillus cereus in many food samples (Pal
et al., 2008). Impedance is defined as the resistance in an electric circuit to the flow of
alternating current, which corresponds to the real electrical resistance to a direct current.
Therefore, its principle is based on the changes in the conductance of the medium because of
the microbial metabolism of the inert substrates into electrically charged acidic-by-products
and ionic compounds. Thus, any microbial growth in a medium can be carefully measured by
its electrical conductance and impedance. Lillehoj et al. (2014) reported the rapid electrical
impedance detection method of bacterial pathogens by using immobilized antimicrobial
peptides. Potentiometric biosensors use ion selective electrodes for transduction of biological
reaction into an electrical signal. Zelada-Guillen et al., (2010) reported the label free detection
and identification of living bacteria in their article.
4.2.2. Optical Biosensors

Optical biosensors include fiber optics, surface plasmon resonance (SPR), waveguides
and others. The working principle of an optical biosensor involves monitoring change in
phase, amplitude, frequency or polarization of light. Fiber optic biosensors consist of light
source, immobilized biological recognition element (enzymes, microbes or antibodies),
optical transmission medium (wave guide, fiber, etc.) and optical probes (such as fluorescent
markers) for transduction and optical detection. The main accessories of fiber optic
biosensors are flow cell with pump and capillary tubing or flow injection system, cuvette
holder, optical filters and collimating lenses. These accessories are used for monitoring
reflectance, phosphorescence, luminescence, absorbance, fluorescence and transmission
(Narsaiah et al., 2012). This technique is broadly exploited for the detection of E. coli
O157:H7 (Liu et al., 2003), Salmonella sp. (Kramer and Lim, 2004), etc. Surface plasmon
resonance (SPR) based biosensors are based on the principle of optical illumination of a metal
surface and it can be used for biomolecular interaction detection. This is also a widely used
technique for the detection of Campylobacter jejuni (Wei et al., 2007), E. coli O157:H7
(Waswa et al., 2007), Staphylococcal enterotoxin B (Homola et al., 2002), Salmonella
typhimurium (Lan, Wang, 2008), listeria monocytogenes (Leonard et al., 2004), etc. Valadez
et al. (2009) reported an optical waveguide. The principle was to link a specific monoclonal
or polyclonal antibody with a target analyte to the core of the fiber optic waveguide and
finally detected by a photosensor. Zhu et al. (2005) performed the detection of water borne E.
coli O157 by integrating waveguide biosensor.
4.2.3. Mass Based Biosensors

Measurement of small changes in mass is a distinct configuration of transduction that has
been used in biosensors. Its principle is based on piezoelectric crystals. Binding of chemicals
results in the increase in mass which in turn transforms the oscillation frequency of the crystal
which can be monitored electrically and utilized in the detection of the additional crystal mass
(Sharma et al., 2013). Hiremath et al. (2015) detected Methicillin-resistant Staphylococcus
aureus (MRSA) by using lytic phage based magnetoelastic biosensors. Berkenpas et al.
(2006) performed the determination of E. coli O157:H7 with the help of the surface acoustic
wave sensors. Guo et al. (2012) developed a piezoelectric immunosensor for the detection of
pathogens by quartz crystal microbalance sensor.
5. MATRIX-ASSISTED LASER DESORPTION IONIZATION-TIME

OF FLIGHT MASS SPECTROMETRY (MALDI-TOF MS)
IN FOODBORNE PATHOGENS DETECTION
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-

TOF MS) has emerged as a very powerful technique for the routine identification of
microorganisms (Carbonnelle et al., 2011; Murray, 2012; Nori et al., 2013; Biswas and
Rolain, 2013). The advantages of MALDI-TOF MS are enhanced automation and high
throughput, reduced labour and cost, improved reproducibility, universal sample preparation
platform for bacteria, fungi and yeasts. The potential drawback of MALDI-TOF MS is the
high cost of instrument. However, considering the extremely less cost of consumables and the
ability to reduce labour costs, the total cost per sample may actually be reduced in the long
run in comparison with the other methods (Pavlovic et al., 2013). MALDI-TOF MS
instrument is used for the detection of bacteria, fungi, yeast, etc. in the food samples.
5.1. Bacterial Identification
MALDI-TOF MS technology allows accurate bacterial identification from 24 and 48

hours to a few minutes. The sample is picked with a sterile tip and smeared in a thin film on
to a MALDI target plate, overlaid with CHCA (α -Cyano-4-hydroxycinnamic acid) or DHB
(2, 5-Dihydroxy benzoic acid) and then introduced in the mass spectrometer. Spectra obtained
are compared with the reference database. An updated reference database is required to
improve the identification the identification performance of MALDI-TOF MS (De Carolis et
al., 2014). Improved database entries with multiple spectra of well characterized species will
result in identification rate close to 100% for Clostridia, mycobacteria, Neisseria,
Streptococci, Helicobacter pylori, Salmonella, Campylobacter (Wieser et al., 2012). Pignone
et al. (2006) identified cultured Mycobacterium species by using Matrix-assisted laser
desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Angelakis et al.
(2011) investigated the feasibility and accuracy of MALDI-TOF MS for identification of
bacteria at the species level in probiotic yoghurts and food. Detailed identification of
Escherichia coli O157:H7 was carried out by Mazzeo et al. (2006).
5.2. Yeast and Fungi Identification
Yeasts and fungi possess a thick cell wall, therefore, cell wall disruption needs an
additional extraction step in comparison to bacterial protocol. In this, colonies are inoculated
to 70% ethanol, the suspension is centrifuged resulting in a pellet, which is dried and
resuspended in 70% formic acid and acetonitrile. After centrifugation, one microliter of
supernatant is applied on the MALDI target plate, dried, covered with matrix and then
analyzed (Pignone et al., 2006). MALDI-TOF MS has the potential to distinguish within the
C. glabrata clade (C. bracarensis, C. glabrata and C. nivariensis) and among members of the
psilosis complex (C. metapsilosis, C. parapsilosis and C. orthopsilosis) whose identification
mainly depends on the molecular methods as biochemical ones do not permit the separation
of these species (Pinto et al., 2011). Fungi are difficult to identify due to their biological
complexity and because of the continuous classification. Filamentous fungi possess different
phenotypes depending on their portion of mycelium or conidia taken for the analysis, their
growth conditions, secondary metabolite production, making their protein extraction difficult
and low reproducibility of spectrum. However, some recent publications have shown the
applicability of the MALDI-TOF MS for the differentiation of fungi such as Penicillium,
Aspergillus, Fusarium, etc. (Santos et al., 2010).
CONCLUSION
Microorganisms are generally present everywhere in very low, medium or high
concentrations. Therefore, consumption of any type of food requires satisfaction of its
freshness, purity and hygienity. Easy and rapid detection of these microbes will facilitate
defensive measures to maintain healthy and fresh food. Molecular techniques involve the use
of two or more primers/methods resulted in the accurate detection of pathogens.
Immunological methods give best results in the absence of interfering agents in the sample
like DNA, non-targeted cells and proteins. Biosensors based methods are easy to operate,
inexpensive, fully automated and highly sensitive. MALDI-TOF-MS is also a quick, reliable
and flexible identification technique for the identification of food borne pathogens without
any false negative results. Finally, use of these rapid techniques could ensure to limit the
spread of food borne diseases resulted in healthy life.
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Chapter 9
ROLE OF ENZYMES IN FOOD INDUSTRIES
Gaurav Garg, Nirmala Sehrawat and Mukesh Yadav*

Department of Biotechnology, Maharishi Markandeshwar University, Mullana, Ambala,
Haryana, India
ABSTRACT
A vast array of functional proteins (enzymes) has already been commercialized for
different industrial processes. Many industries have accepted enzymes as an
indispensable part of their final products. In particular, food and pharmaceutical
industries are employing enzymes at large scale for different forms of processing and
packaging. Food enzymes are the most widely used enzymes at industrial level and
occupies the major share in enzyme market. Food enzymes from various sources are
commonly used in baking, beverages and brewing, dairy, nutraceuticals, dietary
supplements, fats, various oils and also in production of artificial sweeteners. This
chapter describes various enzymes which are used in food processing industries. The
specific roles of these food enzymes in food processing have also been discussed.
Keywords: enzymes, food processing, food industries, baking, confectionary, juice industry,
brewery, beverages, artificial sweeteners
INTRODUCTION
Enzymes are the functional proteins which catalyzes various chemical reactions.
Enzymes are the backbone of life. Cells cannot survive without these functional proteins.
Some fundamental and specific characteristics make enzymes as preferred molecules for
various industrial processes. These characteristics are (i) higher specificity and efficiency, (ii)
enzymes can be recovered after the product formation and (iii) enzymes can be produced
from rapidly growing microorganisms (Yadav et al., 2014). Enzymes are employed in
*
Corresponding author: Department of Biotechnology Maharishi Markandeshwar University Mullana-133207,
Ambala, Haryana, India, Email: mukeshyadav7@gmail.com; Tel: +91-7404206245.
220 Gaurav Garg, Nirmala Sehrawat and Mukesh Yadav
different industries involved in manufacturing and processing of food, feed, detergent,

textiles, laundry, tanning, as well as pharmaceuticals, cosmetics, and fine-chemicals (Miguel
et al., 2013). A large number of enzymes have already been commercialized for different
industrial processes. Industrial applications account for over 80% of the global market of
enzymes (van Oort, 2010). Food enzymes are the most widely used enzymes at industrial
level and represent the major share in enzyme market (Miguel et al., 2013). Food enzymes are
commonly used in baking, beverages and brewing, dairy, dietary supplements, as well as fats
and oils and also in synthesis of artificial sweeteners (Kirk et al., 2002; Xu et al., 2003;
Fernandes, 2010; Singh and Yadav, 2012). Development and advancement in process
technology has allowed the considerable increase in yield by fermentation, increased stability,
enhanced specificity and selectivity of enzymes (Poulsen et al., 2003; Miguel et al., 2013).
Microbial sources of enzymes are more advantageous than their equivalents from animal or
vegetable sources (Soares et al., 2012). The benefits, to use microbial enzymes, comprise
lower production costs, possibility of large-scale production in industrial fermentors, wide
range of physical and chemical characteristics, possibility of genetic manipulation, absence of
effects brought about by seasonality, rapid culture development and the use of non-
burdensome methods. The above characteristics make microbial enzymes suitable
biocatalysts for various industrial applications (Hasan et al., 2006). Non-enzymatic
processing and chemical based processing posses some drawbacks. Harsh and hazardous
processes involving high temperatures, pressures, acidity, or alkalinity need high capital
investment, and specially designed equipment and control systems (Information by
Novozymes®). High temperatures and/or high pressures needed to drive reactions lead to
high energy costs. Moreover, unwanted by-products may prove difficult or costly to dispose
of. High chemicals and energy consumption as well as harmful by-products have a negative
impact on the environment. In a number of cases, some or all of these drawbacks can be
virtually eliminated by using enzymes. Generally, there are two ways for the use of enzymes,
either to convert the raw material into the main product, or the enzymes are used as additives
to alter a functional characteristic of the product (Miguel et al., 2013). In the first situation,
the enzymatic process is undertaken in optimized and controlled conditions to enhance the
catalytic potential of the enzyme, whereas in the second situation it is more difficult to assure
optimal conditions and to control the enzymatic reaction (Illanes, 2008).
ENZYMES USED IN FOOD PROCESSING INDUSTRIES

Among the food enzymes, aspartase, laccase, amylase, glucose oxidase, pullulanase,
naringinase, inulinase, cellulase, pectinase and xylanase are commonly used in various food
processing technologies (Table 1). The specific roles of these enzymes have been described in
this chapter.
Role of Enzymes in Food Industries 221
Table 1. Different enzymes with potential applications in food industries
Enzyme Application in food industry Reference (s)

Asparaginase Reduce the formation of acrylamide in fried, Krishnapuraet al., 2015; Sinha
roasted or baked food products; et al., 2013
Pretreatment of potato slices and bread
dough before frying or baking
α-amylase Baking, brewing, preparation of digestive Couto and Sanromán, 2006;
aids, cakes and starch syrups; Decrease the Hopek et al., 2006; Pandey et
bread hardness; Improve bread quality; al., 2000; Kamel and Stauffer,
Partial replacement for the expensive malt 1993; Rameshkumar and
in brewing industries and also in flour and Sivasudha, 2011
baking industries; Manufacturing of high
molecular weight branched dextrins.
Aspartase Production of fumaric acid and aspartic Singh and Yadav, 2012;
acid; Fruit preservation Chibata et al., 1985; Xu et al.,
2003; Lean, 2004
Naringinase Debittering of grapefruit juices; Preparation Yadav et al., 2000; Busto et
of sugars (L-rhamnose) al., 2007; Puri et al., 2000;
2005; 2008
Laccase Improve strength of gluten structures in Brijwani et al., 2010;
dough and baked products. Increase volume,
improve crumb structure, and softness of
baked products; Use in juice processing and
wine industries
Inulinase Production of fructooligosaccharides Coussement, 1999; Singh and
(FOSs). FOSs are widely used as sugar Singh, 2010; Ricca et al.,
substitutes chiefly in dairy and bakery 2007; Singh and Gill, 2006
products. Production of high fructose syrup
for food industries.
Pullulunase Preparation of maltose syrup for food Singh et al., 2010a; 2010b;
industries Zoebelein and Böllert, 2001
Glucose oxidase Gluconic acid production, food Ramachandran et al., 2006
preservation, To remove residual oxygen
from fruit juices, beer, and wine and also
from dehydrated packaged foods
Cellulase Glucose feedstock from cellulose, Bio- Hardiman et al., 2010; Sadhu
ethanol, processing of animal feed, and Maiti 2013
clarification of fruit juices; improved texture
and quality of bakery products; improved
viscosity fruit purees; improved texture,
flavor, aroma, and volatile properties of
fruits and vegetables; controlled bitterness
of citrus fruits
Pectinase Degrade pectin present in cell wall, increase Li et al., 2012; Makky and
juice extraction, tea and coffee processing, Yusoff 2015; Tochi et al.,
animal feed, clarification of must for wine 2009; Chaudhri and Suneetha
2012; Hoondal et al., 2002
Xylanase Fruit-juice clarification; Improving rumen Li et al., 2012; Haros et al.,
digestion; Brewing industry, Improving 2002; Guy and Sarabjit, 2003
dough stability
Aspartase
Aspartase (L-aspartate ammonia lyase, EC 4.3.1.1) catalyzes the reversible deamination

of aspartate to produce fumarate and ammonia. The direction of reaction depends upon the
pH of the medium. In alkaline medium L-aspartic acid is produced from fumaric acid and
ammonia (Papierz et al., 2007). Aspartase has been considered as catabolic enzyme in the
metabolism of bacterial cells (Dougherty et al., 1972). It is an important enzyme of microbial
metabolic pathway, aspartase contributes significantly to Krebs cycle and therefore,
considered as important enzyme in metabolic pathway. Various amino acids, such as
asparagine, arginine, lysine, methionine, threonine and isoleucine are synthesized from
aspartic acid through fumarate and malate (Wang et al., 2006). Aspartase shows high catalytic
activity and is considered among the most substrate specific enzymes. The enzyme does not
show activity with D-aspartic acid, L-cysteic acid and crotonic acid (Virtanen and Ellfolk,
1955). Its native property to convert fumaric acid to aspartic acid makes it the enzyme of
interest in terms of food and pharmaceutical sciences. Different bacterial sources have been
reported for production of aspartase (Table 2). It is also reported from yeasts and molds.
Aspartase has also been reported from some plants (Virtanen and Ellfolk, 1955) and few
species of aquatic animals (Salvatore et al., 1965). Aspartase from higher organisms is not
well characterized because of poor yield and expensive time investment as compared to
microbial sources. Besides the production of L-aspartic acid for food based inductries,
aspartase has been examined for other possible applications.
Aspartase catalyzed the reversible reaction resulting in production of either fumaric acid
or aspartic acid depending on the pH conditions (Papierz et al., 2007) and substrate provided.
Both products are useful from industrial purpose. The industrial production of aspartase was
reported in 1960 (Kisumi et al., 1960). Aspartic acid is an essential part of artificial sweetener
aspartameTM which is approximately 180 times sweeter as compared to suger (Lean, 2004).
Artificial sweeteners are also used to prevent dental caries (Lean, 2004). L-phenylalanine has
also been produced by coupling the reaction of aspartase and transaminase (Xu et al., 2003).
Aspartase catalyzes the formation of L-aspartate from fumaric acid by amination reaction
while L-aspartate formed in this reaction is used by transaminase for the amination of
phenylpyruvic acetate (PPA) to L-phenylalanine. Aspartic acid has use in medicines, food
additives (Chibata et al., 1985). Fumaric acid, another product of aspartase catalysis, is also
used in food industry in fruit jellies and preserves. It controls acidity/alkalinity in food.
Laccase
Laccase (EC 1.10.3.2) are copper-containing enzymes that catalyzes the oxidation of
organic and inorganic substrates including polyphenols, amino phenols, methoxy phenols,
aromatic amines and ascorbate with concomitant four electron reduction of oxygen to water
(Galhaup et al., 2002; Madhavi and Lele, 2009). Laccases are widely distributed in higher
plants and fungi (Benfield et al., 1964; Messerschmidt and Huber, 1990; Brijwani et al.,
2010). Insects and bacteria have also been reported to produce laccase (Diamantidis et al.,
2000; Madhavi and Lele, 2009; Kunamneni et al., 2007). Laccases are distributed in broad
groups of fungi including Ascomycetes, Deuteromycetes, and Basidiomycetes and found
particularly abundant in some white rot fungi involved in lignin metabolism (Bourbonnais et
al., 1995; Leontievsky et al., 1997; Brijwani et al., 2010). The ability of laccase to oxidize
phenolic compounds and to reduce molecular oxygen to water has make it an enzyme of
interest for various scientific and industry based research groups.
Laccases have many biotechnological applications because of their oxidation ability
towards a broad range of phenolic and non-phenolic compounds (Imran et al., 2012;
Mohammadian et al., 2010; Claus, 2003). Applications of laccase in food industries have
been discussed by various authors (Imran et al., 2012; Madhavi and Lele, 2009) and mainly
these applications focuses on wine and juice based beverages industries and baking industries.
In food industry, wine stabilization is the main application of laccase (Duran and
Esposito, 2000; Rosana et al., 2002). Polyphenols have undesirable effects on wine
production and on its organoleptic characteristics. Removal of polyphenol is very necessary
from the wine (Rosana et al.,2002). Though, various treatments have been proposed for the
removal of phenolics which may result in discoloration, haze, and flavor changes but the
possibility of using enzymatic laccase as a specific and mild technology for stabilizing
beverages against discoloration and clouding provides an attractive alternative (Cantarelli et
al., 1989; Arora and Sharma, 2010). Further, the immobilized laccase can be used in
preparation of must, wine and in fruit juice stabilization (Minussi et al., 2002; Arora and
Sharma, 2010). The addition of laccase at the end of processing also improves the shelf life of
beer (Mathiasen, 1995).
Table 2. Various bacterial sources of aspartase

(Modified from Singh and Yadav, 2012)
Source Reference (s)

Aeromonas media Singh and Yadav, 2012
Alcaligenes metalcaligenes Vojtisek et al., 1986
Bacillus cereus Garza et al., 2000
Bacillus sp. Kawata et al., 1999
Bacillus brevis Kimura et al., 1983
Bacillus licheniformis
Bacillus aminogenes
Bacillus thermoaminophilus
Bacillus staerothermophilus Suzuki et al., 1980
Bacillus subtilis Iijima et al., 1977
Bacterium cadaveris Christman and Williams, 1952
Brevibacterium sp. Terasawa et al., 1985
Cytophaga sp. Kazuoka et al., 2003
E. coli Quastel and Woolf, 1926; Tokushige and
Mizuta, 1976
Enterobacter aerogenes Williams and Lartigue, 1967
Erwinia sp. Bagdasaryan et al., 2005
Escherichia alcalescens Malanikova et al., 1988
Hafnia alvei Wilkinsons and Williams, 1961
Propionibacterium freudenreichii subsp. Crow, 1987
Shermanii
Pseudomonas fluorescens Takagi et al., 1984
Pseudomonas putida Maalej et al., 1990
Laccase is also widely used to stabilize the fruit juices. Many fruit juices contain
naturally occurring phenolics and their oxidation products, which contribute to color and
taste. The interaction between proteins and polyphenols results in the formation of haze or
sediment in fruit juices. The color change, referred to as enzymatic darkening, increases due
to a higher concentration of polyphenols present in fruit juices (Ribeiro et al., 2010).
Therefore, clear fruit juices are typically stabilised to delay the onset of protein-polyphenol
haze formation (Siebert, 1999). Several authors have proposed the use of laccase for the
stabilization of fruit juices (Neifar et al., 2011; Sammartino et al., 1998; Giovanelli and
Ravasini, 1993; Stutz, 1993; Ritter et al., 1992; Cantarelli and Giovanelli, 1990; Maier et al.,
1990; Cantarelli, 1986), but results are contradictory. The phenolic content of juices has been
found to be greatly reduced after treatment with laccase along with an increase in color
stability (Ribeiro et al., 2010). Laccase treatment has also been found to be more effective for
color and flavor stability compared to conventional treatments, such as the addition of
ascorbic acid and sulphites (Minussi et al., 2002). Sammartino et al. (1998) have shown that
enzymatically treated juice was less stable than the one conventionally treated. Also,
Giovanelli and Ravasini (1993) and Gökmen et al. (1998) showed that laccase treatment
increased the susceptibility of browning during storage. Cantarelli (1986) described treatment
of black grape juice with mutant laccase from Polyporus versicolor and removal of 50% of
total polyphenols and higher stabilisation than the physical-chemical treatment. Effect of
laccase application on clarity stability of sour cherry juice has also been investigated (Artik et
al. 2004). High clarity was obtained by adding laccase. Moreover, the phenolic content was
decreased by around 70%. Neifar et al. (2011) reported use of a combined laccase
ultrafiltration process to control the haze formation and browning of the pomegranate juice.
The baking industry utilizes a wide range of enzymes to improve bread texture, volume,
flavor and freshness and also the machinability of dough (Imran et al., 2012; Brijwani et al.,
2010). Addition of laccase to dough results in oxidizing effect and therefore, improved
strength of gluten structures in dough and baked products. Machinability of dough was found
to be improved due to increased strength and stability along with reduced stickiness as a
result of laccase addition. Improved bread and dough quality was obtained when laccase was
used in processing of low quality flour (Minussi et al.,2002). The addition of laccase and
proteolytic to oat flour lead to a significant improvement to texture quality of oat bread, due
to increased loaf specific volume and lowering crumb hardness and chewiness (Renzetti et al.,
2010; Brijwani et al., 2010).
Asparaginase
L-Asparaginase (EC 3.5.1.1) is an amidohydrolase which catalyzes the breakdown of L-

asparagine to ammonia and L-aspartic acid. Asparaginase is widely reported from bacteria,
yeast, fungi and plant sources (Arif and Hussain, 2014). Its broad use in both food industry
and chemotherapy makes it an enzyme of interest for wide research. Though, asparaginase is
an important therapeutic enzyme but it is also of prospective use in food industry to reduce
the formation of acrylamide in fried, roasted or baked food products (Krishnapura et al.,
2015). Acrylamide (a carcinogen) has high concentration in heat-treated foods abundant in
carbohydrates (Tareke et al., 2 2). Acrylamide is formed by ―Maillard‖ reaction between
asparagine and reducing sugars which are abundantly present in such foods. This reaction is
prevented by adding asparaginase which converts asparagine into aspartic acid, making it
unavailable for Maillard reaction. The most common use of asparaginase is as a processing
aid in the processing of food (Kornbrust et al., 2009), which are marketed under the brand
names Acrylaway and PreventASe. Asparaginase is used in food industries to prevent
acrylamide formation in dough-based products, and starchy food, such as cookies and french
fries (WHO, 2007). Even small amount of asparaginase has been found sufficient to decrease
acrylamide formation (Boegl, 2006). Similarly, Anese et al. (2011) tested the effect of
asparagine in reducing acrylamide formation in biscuits. Intermediate concentrations of
asparaginase reduced the acrylamide formation without any significant change in colour or
taste of the final product.
A pretreatment of potato slices and bread dough with asparaginase before frying or
baking prevents acrylamide formation (Sinha et al., 2013; Pedreschi et al., 2008; Kuilman and
Wilms, 2007). Asparaginases from Aspergillus oryzae and A. niger are used in baking
industries to check the acrylamide formation (Morales et al., 2008). Recently, Ghonemy,
(2014) has discussed the applications of asparaginase in food industries. The ability of L-
asparaginases to selectively hydrolyze asparagines into L-aspartate and ammonia is a
potential way to reduce the amount of free L-asparagine in the starting materials of food
production, thus reducing the imminent risk of generating a potential carcinogenic and
neurotoxic compound called acrylamide that formed from L-asparagine and reducing sugars
in carbohydrate-containing foods (such as snacks and biscuits) when they are heated above
120oC (Friedman, 2003).
AMYLASE
Alpha amylases (E.C. 3.2.1.1) are starch degrading enzymes. The enzyme randomly
cleave the 1, 4-α- linkage between adjacent glucose units in linear amylase chains and
produce glucose, maltose and maltosetriose (Rameshkumar and Sivasudha, 2011). Amylases
constitutes important group of industrial enzymes, which alone covers approximately 25% of
the enzyme market. They have been in increasing demand for their use in many commercial
biotechnological processes including starch degradation, detergent, food products,
pharmaceutical, textile and paper industries (Mitchell and Lonsane, 1990; Sun et al., 2010;
Akpan et al., 1999; Oudjeriouat et al., 2003). Amylases may be found in yeasts, fungi,
bacteria and actinomycetes (Pandey et al., 2000; Rameshkumar and Sivasudha, 2011).
Among these sources, fungi and bacteria are major sources of amylase enzymes (Pandey et
al., 2000; Burhan et al., 2003). In particular bacterial amylase is most widely used at
industrial scale. α-amylases have potential application in a vast number of industrial processes
including food, textile, fermentation, detergent, paper and pharmaceutical industries. The
advances in biotechnology resulted in expansion of amylase application in more fields
including clinical, medicinal and analytical chemistry, as well in starch saccharification and in
the textile, food, brewing and distilling industries (Pandey et al., 2000; Kandraet al., 2003;
Gupta et al., 2003; de Souza and de Magalhães, 2010; Akcan, 2011). Due to the increasing
demand for these enzymes in various industries, there is massive interest in enzymes with
better properties suitable for industrial applications and their cost effective production
techniques (Sivaramakrishnan et al., 2006).
Bacterial and fungal amylases are widely used in different industries for various purposes
(Oliveira et al., 2007; Rameshkumar and Sivasudha, 2011). Amylases are also found in plants
and animals but they cannot be used at industrial level due to different limitations while
microbial amylase enzyme generally meets industrial demands (van der maarel et al., 2002;
Corrêa et al., 2011). Majority of amylases are metalloenzymes, which requires calcium ions
for their activity (Rameshkumar and Sivasudha, 2011). Calcium ion independent alpha
amylase from Bacillus KR-8104 has been reported by Sajedi et al. (2005). These enzymes
account for about 3 % of the world‘s enzyme production (van der maarel et al., 2002).
Bacillus is particularly endowed to produce thermostable α-amylase. Most of commercially
available enzymes are obtained from different species of Bacillus i.e., B. subtilis, B.
stearothermophilus, B. licheniformis, B. amyloliquefaciens and Bacillus subtilis (Burhan et
al., 2003; Nimkar et al., 2010; Akcan, 2011).
The most widespread applications of α-amylases are in the starch industry. Amylases are
generally used in starch hydrolysis (van der Maarel et al., 2002; Sundarram et al., 2014). In
the process of starch liquefaction, amylases are used to produce fructose and glucose syrups
(Bessleret al., 2003; Rameshkumar and Sivasudha, 2011). van der maarel et al. (2002) have
studied the properties and applications of starch-converting enzymes of the α-amylase family.
The enzymatic conversion of all starch includes: gelatinization, which involves the
dissolution of starch granules, thereby forming a viscous suspension; liquefaction, which
involves partial hydrolysis and loss in viscosity; and saccharification, involving the
production of glucose and maltose via further hydrolysis.
Amylases are extensively employed in processed-food industry such as baking, brewing,
preparation of digestive aids, cakes and starch syrups (Couto and Sanromán, 2006; Soares et
al., 2012). Amylases are routinely used in bread industry to decrease the bread hardness
(Hopek et al., 2006). These enzymes can be added to the dough of bread to degrade the starch
in the flour into smaller dextrins, which are subsequently fermented by the yeast. The addition
of α-amylase to the dough results in enhancing the rate of fermentation and the reduction of
the viscosity of dough, resulting in improvements in the volume and texture of the product.
Generally bacterial amylases are more thermostable as compared to amylases from other
sources, therefore, used in baker industries (Pandey et al., 2000). Fungal amylases are also
used for the same purposes. Powdered fungal (Aspergillus oryzae) alpha amylases are added
to baking flour. Its supplements or addition supports growth of yeast and results in dough
rising and improved bread quality (Kamel and Stauffer, 1993; Hopek et al., 2006). Bacterial
amylases was found to lowered the bread quality while the fungal amylases increased the
shelf life of bread and was not found to produce any detrimental effect (Hopek et al., 2006).
Amylases are used, as partial replacement for the expensive malt in brewing industries and
also in flour and baking industries (Rameshkumar and Sivasudha, 2011).
In the food industry amylolytic enzymes have a large scale of applications, such as the
production of glucose syrups, high fructose corn syrups, maltose syrup, reduction of viscosity
of sugar syrups, reduction of turbidity to produce clarified fruit juice for longer shelf-life,
solubilisation and saccharification of starch in the brewing industry (Pandey et al., 2000;
Nigam, 2013; Sundarram et al., 2014). The baking industry uses amylases to delay the staling
of bread and other baked products; the paper industry uses amylases for the reduction of
starch viscosity to achieve the appropriate coating of paper. Amylase enzyme is used in the
textile industry for warp sizing of textile fibers, and used as a digestive aid in the
pharmaceutical industry (Sivaramakrishnan et al., 2006).
GLUCOSE OXIDASE
Glucose oxidase [E.C. 1.1.3.4] is an enzyme that catalyzes the oxidation of beta-D-
glucose with the formation of D-gluconolactone. Glucose oxidase (GOx) is glycoprotein with
a carbohydrate mass percentage of 16–25% (Ramachandran et al., 2006). The enzyme
contains the prosthetic group flavin adenine dinucleotide (FAD) which enables the protein to
catalyze oxidation-reduction reactions (Kalisz, et al., 1997; Odebunmi and Owalude, 2007;
Soares et al., 2012). Different species of Penicillium and Aspergillus has been used for GOx
production. The enzyme (GOx) from Aspergillus niger and Penicillium amagasakiense has
been studied most extensively (Odebunmi and Owalude, 2007). P. adametzii has also been
investigated as an important fungal source of glucose oxidase (Mikhailova et al., 2007). GOx
is widely used at industrial scale for various applications including biofuel cell research for
implantable devices, sensor based applications and more important in food based industries.
The enzyme is used in the food industry for the removing of harmful oxygen. Oxygen level
during storage should be consequently minimal to avoid toxicity, the organism‘s death and
the consequent loss of the product‘s functionality (Soares et al., 2 12). Glucose oxidase is
used in the food industry for the removal of glucose from powdered eggs, for gluconic acid
production, and as a source of hydrogen peroxide in food preservation (Witt et al., 1998;
Odebunmi and Owalude, 2007). Glucose oxidase is also used extensively for the quantitative
determination of D-glucose in samples such as blood, food and fermentation products
(Wilson and Turner, 1992). It is also used in removing residual oxygen from fruit juices, beer,
and wine and also from dehydrated packaged foods (Ramachandran et al., 2006). Glucose
oxidase may be a biotechnological asset to increase stability of probiotic bacteria in yoghurt
without chemical additives. It may thus be a biotechnology alternative (Soares et al., 2012).
GOX is used to produce bread with improved texture and increased loaf volume
(Vemulapalli et al., 1998; Rasiah et al., 2005; Moorthi, 2009). Maillard non-enzymatic
browning is a result of reaction between the amino acid and reducing sugars. In the
production of dried egg powder this reaction results in undesirable browning. Glucose
oxidase (immobilized state) has been studied to remove glucose or sugars from egg before
spray drying (Moorthi, 2009; Sisak et al., 2006). The GOX mediated reaction consumes
oxygen therefore, GOX generally used as preservative, antioxidant and oxygen scavenger
component in food industries. GOX is also used for gluconic acid production for food based
industries (Ramachandran et al., 2006).
A simple, rapid, and sugar-selective glucose oxidase (GOx) based method to induce
gelation from glucose-containing samples has been described (Liu et al., 2012). GOx
selectively ―recognize‖ and oxidize glucose to generate gluconic acid, which acts to solubilize
calcium carbonate and release calcium ions. The release of calcium ions triggers gelation of
the calcium-responsive polysaccharide alginate to form a calcium-alginate hydrogel.
Rheological measurements confirmed that gel formation is triggered by glucose. This gel-
forming method can detect glucose in food/beverage products sweetened with glucose or
high-fructose corn syrups. These results indicated that the enzyme-induced gelation of
alginate may provide a simple means to test for sweeteners using components that are safe for
use on-site or in the home.
PULLULANASE
Pullulanase (EC 3.2.1.41) is an enzyme whose primary specificity is to hydrolyze the (1-
6)-α-D-glycosidic linkages in pullulan to yield maltotriose (Singh et al., 2010a; 2010b).
Pullulanases are widely distributed among animals, plants, fungi and bacteria (Singh et al.,
2 11; Domań-Pytka and Bardowski, 2004). It is an industrially important enzyme, which is
generally used in combination with other amylolytic enzymes (amylases) in the starch
processing industry for the production of sugar syrups (Singh et al., 2010a; 2010b). It is one
of the well known debranching enzyme. Pullulanase hydrolyzes the polysaccharide pullulan
and starch. Maltotriose syrup is produced by enzymatic hydrolysis of the polysaccharide
pullulan using the pullulanase (Singh et al., 2010b). This syrup possess many excellent
properties as low freezing point depression, mild sweetness, keeps in moisture, prevention of
retrogradation of starch in foodstuffs, less color formation compared with maltose syrups,
glucose syrups or sucrose, good heat stability, low solution viscosity, high fermentability and
favoring glassy states. These properties are useful in food and pharmaceutical industries
(Zoebelein and Böllert, 2001). High maltotriose syrup may be applied in the food industry for
the manufacturing of desserts, baking and brewing (Singh et al., 2011).
NARINGINASE
Naringin is a naturally occurring flavonoid in citrus fruits. It is present in oranges,
grapefruit, and lemon, these flavonoids may cause interference during the citrus fruit juice
processing and cause for the bitter taste (Konno et al., 1982). Naringinase (E.C.3.2.1.40) is an
enzyme which catalyzes the hydrolysis of naringin into prunin and then into naringenin,
which is non-bitter and tasteless. This enzyme has two different enzyme activities. One is α-
L-rhamonosidase (E.C.3.2.1.4 ) which acts on naringin to release prunin and α-L-rhamnose.
Second is β-D-glucosidase (E.C.3.2.1.21) which acts on prunin to release naringenin and β-D-
glucose (Puri et al., 2005; Puri and Kalra, 2005; Puri and Banerjee, 2000). Naringinase is
produced by many microorganisms but there are only few reports on the commercial
production of this enzyme (Mendoza-Cal et al., 2010; Puri and Banerjee, 2000). Naringinase
produced by several fungi (especially Aspergillus niger) is used for elimination of bitter
flavor from citrus fruit juice (Puri et al. 2005). The enzyme naringinase has gained wide
interest of researchers due to its potent applications in food industries. Rhamnosidase activity
of naringainase in combination with β-glucosidase and arabinosidase is considered suitable
for aroma enhancement in wine making. The immobilized enzyme has been used to develop a
continuous process for wine aroma enhancement (Caldini et al., 1975 and Gallego et al.,
2001).
The processing of citrus fruit juice has faced formidable problems in terms of bitterness
and delayed onset of bitterness. The bitterness affects its consumer acceptability. Two classes
of chemical compounds namely flavonoids and limonoids were found responsible for
bitterness in citrus juices. The use of α-L-rhamnosidases for debittering of grapefruit juices by
hydrolysis of naringin to prunin and L-rhamnose has been observed by many workers (Yadav
et al., 2000, Chen et al., 1990, Busto et al., 2007, Puri et al., 2008). Recently, Patil and Dhake
(2014) reported debittering of citrus fruit juice by partially purified naringinase of Penicillium
purpurogenum. Rhamnosidases play an important natural role in the modification of the

viscous property of gellan gum (Manzanares et al., 2001; Yanai and Sato, 2000; Hashimoto et
al., 2003). This enzyme has also attracted interest from biotech groups for its role in rhamnose
production (Puri and Banerjee, 2000).
INULINASE
Microbial inulinases are an important class of industrial enzymes, which hydrolyze inulin
to produce fructose and fructo-oligosaccharides (Singh and Lotey, 2 1 ). Inulin is a linear β-
(2→1) linked fructose polymer with a terminal glucose unit that occurs as a reserve
carbohydrate in many plants belonging to the Compositae, Liliaceae and Gramineae families
(Gupta and Kaur, 1997). This polymer is a well recognized source for the production of either
ultra-high fructose syrup or fructo-oligosaccharides (Gupta and Kaur, 1997; Kaur and Gupta,
2002). Inulinase production has been reported from various sources but higher inulinase
producing microorganisms are Penicillium, Aspergillus and Kluyveromyces (Singh and Lotey,
2010). Inulinases are classified as exo and endo acting on the basis of cleavage of β-2,1
linkage in inulin (Singh and Singh, 2 1 ). Exoinulinases (EC 3.2.1.8 ) cleave β-2,1 linkages
sequentially starting from the non-reducing end of inulin and split off terminal fructosyl units,
releasing fructose with a molecule of glucose, whereas endoinulinases (EC 3.2.1.7) act
randomly and hydrolyze internal linkages of inulin to yield fructooligosaccharides. Inulinases
often show a certain activity towards sucrose (Ricca et al., 2007; Singh and Singh, 2010).
However, sucrose hydrolytic enzymes are called invertases (EC 3.2.1.26) and have specific
characteristics. Because of the overlapping substrate specificity of invertases and inulinases,
their distinction is difficult and controversial. Generally, inulinases are distinguished from
invertases on the basis of I/S (inulinase activity/invertase activity) ratio (Ricca et al., 2007;
Vandamme and Derycke, 1983). If the I/S ratio is >10–2, the enzyme is referred to as an
inulinases, and if I/S ratio is <10–4, it is considered an invertase (Ettalibi and Baratti, 1987;
Singh and Singh, 2010). Fructose and fructo-oligosaccharides (FOSs) can be produced
through the hydrolysis of inulin. One of the approaches used for the production of FOSs is the
controlled hydrolysis of inulin by endoinulinases. Both fructose and fructo-oligosaccharides
are fast emerging as important ingredients in the food and pharmaceutical industry. Fructose
is sweeter than sucrose (1.2-2.0 times). Its GRAS status with lower cost and its technical
superiorities over sucrose has attracted many food and beverage industries (Singh et al.,
2008). Furthermore, fructose metabolism bypasses the known metabolic pathway of glucose
and therefore does not require insulin (Kaur and Gupta, 2002). Fructo-oligosaccharides are
prebiotic and their positive effect on human health has been widely known (Sangeetha et al.,
2005). Other important applications of inulinases are in the production of ethanol, gluconic
acid, sorbitol, pullulan and acetone-butanol (Singh and Gill, 2006). Production of fructose by
acid hydrolysis of inulin is not recommended because of undesirable colouring of inulin
hydrolysate, formation of difructose anhydride, which has practically no sweetening
properties (Vandamme and Derycke, 1983). Conventional fructose production from starch
needs at least three enzymatic steps and yields only 45% fructose. In contrast, the complete
hydrolysis of inulin by inulinase (EC 3.2.1.7, β-2,1-fructan fructanohydrolase) gives higher of
fructose (Gupta et al., 1994; Vranesic et al., 2002). Inulinase is found in filamentous fungi,
yeasts, bacteria and inulin storing tissues of plants. It has also received a great attention due to
the presence of relatively inexpensive and abundant substrate (inulin) for the production of
high fructose syrup.
CELLULASES
Cellulase causes the enzymatic hydrolysis of cellulose into soluble sugars by breaking the
β- 1, 4-glycosidic linkages (Rao and Mishra, 1989). It provides a key opportunity for
achieving the tremendous benefits of biomass utilization (Himmel et al., 1999). Cellulases
interactively promote the cellulose degradation (Rao and Mishra, 1989), to cope with the
problem of food and energy shortages expected in near future with explosive increase in
human population (Sakka et al., 2000). The cellulolytic organisms constitutively produce
three main types of enzymes, namely β-glucosidase (cellobiase or β-D-glucoside glucoyl
hydrolase; EC 3.2.1.21), exoglucanase (1,4-β-D-glucan cellobiohydrolyase; EC 3.2.1.91) and
endoglucanase (1,4-β-D-glucan glucanohydrolyase; EC 3.2.1.4) (Sakka et al., 2000; Tomme
et al., 1995). To hydrolyze and metabolize insoluble cellulose, the microorganisms must
secrete the cellulases that are either free or cell-surface-bound. Many bacteria can grow on
cellulose and many produce enzymes that catalyze the degradation of soluble derivatives of
cellulose or the amorphous regions of crystalline cellulose. However few bacteria synthesize
the complete enzyme system that can result in extensive hydrolysis of the crystalline material
found in nature. These bacteria should be called ―true cellulolytic‖ bacteria and those bacteria
that produce some endoglucanases and ß-glucosidases, but not the complete system, are
called ―pseudocellulolytic‖ (Coughlan and Mayer 2 13).
Fermentation is the process of conversion of complex substrates into simple compounds
by various microorganisms or by microbial products. Cellulase can be produced by either
submerged or solid state fermentation for their wide uses in industry. Over the years,
fermentation techniques have gained immense importance due to their economic and
environmental advantages (Sadhu and Maiti 2013). Both fungi and bacteria have been heavily
exploited for their abilities to produce a wide variety of cellulases (Table 3). These
microorganisms can be aerobic, anaerobic, mesophilic or thermophilic in nature. Fungi play a
significant role in the degradation of cellulose under aerobic conditions because of their
capability to produce copious amounts of cellulolytic enzymes. Among bacteria, the genera of
Clostridium, Cellulomonas, Thermomonospora, Bacillus and fungi Trichoderma, and
Aspergillus are the most extensively studied cellulose producer (Sun and Cheng, 2002;
Sukumaran et al., 2005). White rot fungus Phanerochaete chrysosporium, Soft-rot fungi,
Fusarium solani, Penicillum funiculosum, Talaromyces emersonii, Trichoderma koningii and
Trichoderma reesei. Aerobic cellulolytic bacteria which are having best-characterized
cellulase systems are as follows: Cellulomonas sp., Cellvibrio sp., Microbispora bispora and
Thermomonospora sp. Examples of anaerobic cellulolytic bacteria are as follows: Acetivibrio
cellulolyticus, Bacteroides cellulosolvens, Bacteriodes succinogenes, Clostridium
thermocellum, Ruminococcus albus and Ruminococcus flavefaciens (Saranraj et al. 2012).
Aspergillus and Trichoderma species are well known efficient producers of cellulases (Peij et
al., 1998). Several studies have been carried out to produce cellulolytic enzymes from
biowaste degradation process by many microorganisms including fungi such as Trichoderma,

Penicillium and Aspergillus species etc., (Mandels and Reese, 1985).
Cellulases are being used in animal feeds for improving the nutritional quality and
digestibility, in processing of fruit juice, in baking, in extraction of olive oil (Sadhu and Maiti
2013), but also for the significant role in bioconversion of agriculture wastes in to sugar and
bioethanol (Saranraj et al., 2012). In food industry, cellulases are used in extraction,
clarification and stabilization of fruit and vegetable juices. It also acts as an important
macerating enzyme for the extraction and clarification of fruit and vegetable juices to increase
the juics yield (Minussi et al., 2002; de Carvalho et al., 2008). The macerating enzymes
improves the cloud stability and texture and decrease viscosity of the nectars and purees from
tropical fruits such as mango, peach, papaya, plum, apricot, and pear (Sukumaran et al., 2005;
de Carvalho et al., 2008).
Table 3. Various microbial sources of cellulase

(Modified from Sadhu and Maiti 2013)
Source organism Reference (s)

Anoxybacillus flavithermus EHP2 Ibrahim et al., 2007
Anoxybacillus sp. 527 Liang et al., 2010
Bacillus sp.AC-1 Li et al., 2006
Bacillus sp. LFC15 Korpole et al., 2011
Bacillus sp Patel et al., 2005; Dey et al., 2002
Bacillus sp. NZ Nizamudeen and Bajaj, 2009
Bacillus licheniformis MVS1, Bacillus sp. Acharya and Chaudhary, 2012
MVS3
Cellulomonas cellulans MTCC 23, Cytophaga Mishra et al., 2007
hutchinsonii NCIM 2338
Clostridium thermocellum Zhuang et al., 2007
Streptomyces sp. BRC1, Streptomyces sp. Chellapandi and Himanshu, 2008
BRC2
Microbacterium sp. MTCC 10047 Sadhu et al., 2011
Bosea sp. MTCC 10045 Sadhu et al., 2012
Pseudomonas sp. Tewari et al., 1977; Ramasamy and
Varachtert, 1980
Pseudomonas fluorescens Yashikawa et al.,
1974
Bacillus pumilus EB3 Ariffin et al., 2006
Bacillus subtilis CEL PTK 1 Saraswati et al., 2012
Succinogens Groleu and Forsberg, 1981
Trichoderma reesei Muthuvelayudham and Viruthagiri, 2003
Chaetomium sp.NIOCC 36 Ravindran et al., 2010
Fomitopsis sp.RCK 2010 Deswal et al., 2011
T. aureoviride Zaldivar et al., 2001
Alternaria alternate Bailey and Poutaneu1988; Macris, 1984
Cellulase in combination with pectinase and hemicellulases improves the extraction of

olive oil. Olivex (a mixture of pectinase with cellulase and hemicellulase from Aspergillus
aculeatus), was the first enzyme used to improve the extraction of olive oil (Fantozzi et al.,
1977). Further, the use of macerating enzymes increased the antioxidants in extravirgin olive
oil and reduced the induction of rancidity (Galante et al., 1998).
Glucanases are added to improve the malting and fermentation of barley in beer
manufacturing and in wine industry, better maceration and color extraction is achieved by use
of exogenous hemicellulases and glucanases (Sukumaran et al., 2005; Galante et al., 1998;
Bamforth et al., 2009). These enzymes are added during mashing which hydrolyze glucan,
reduce the viscosity of wort, and improve the filterability (Bamforth et al., 2009; Canales et
al., 1988). The aroma of wines can be improved by using β-Glucosidases by modifying
glycosylated precursors. Macerating enzymes also improve pressability, settling, and juice
yields of grapes used for wine fermentation.
Cellulases are also used in carotenoid extraction in the production of food coloring
agents. Carotenoids are used as food colorants because these are natural products, null
toxicity and high versatility, with colour ranging from yellow to red (Cinar, 2005). Enzyme
preparations containing hemicellulase and pectinase in addition to cellulases are used to
improve the nutritive quality of forages. Improvements in feed digestibility and animal
performance are reported with the use of cellulases in feed processing (Dhiman et al., 2002;
Godfrey and West, 1996). Use of feed additive, Trichoderma cellulases helps in improving
the feed conversion ratio and increasing the digestibility of a cereal-based feed (Saranraj et
al., 2012). The cellulose enzymes remove the antinutritional factors present in the feed grains,
degrade certain feed constituents which cannot be digested by the animal, to improve the
nutritional value, the digestive enzymes such as proteases, amylases, and glucanases are
added. For instance, the dietary fiber consists of nonstarch polysaccharides such as
arabinoxylans, cellulose, and many other plant components including resistant dextrins,
inulin, lignin, waxes, chitins, pectins, β-glucan, and oligosaccharides (Ali et al., 1995).
PECTINASE
Pectinases comprises a heterogeneous group of enzymes that catalyze the breakdown of
pectin containing substrates. Pectinolytic enzymes are classified according to their mode of
action on the galacturonan part of the pectin molecule. Pectinases include depolymerizing and
demethoxylating enzymes. Depolymerizing enzymes are polygalacturonase (EC 3.2.1.15) and
polymethylgalacturonase, which cleaves the α-1, 4 glycosidic bonds, and pectate lyase (EC
4.2.2.2) and pectin lyase (EC 4.2.2.1 ), which catalyses a β-elimination reaction. De-
esterifying enzymes include pectin-esterase (EC 3.1.1.11), which catalyses the
demethoxylation of methylated pectin, producing methanol and pectin (Priest, 1984).
Pectin, another component of the plant cell wall that forms the major component of
middle lamella, is found between the primary cell walls of adjacent young plant cells. It is a
polysaccharide with a backbone of galacturonic acid residues, linked by α (1-4) linkage in
which 75% of the carboxyl groups of the galacturonate units are esterified with methyl
alcohol and others are combined with calcium or magnesium ions. They are commonly
substituted with L-rhamnose, arabinose, galactose and xylose side chains. These structural
polysaccharides are being degraded by enzymes pectinase. Pectinases attack and

depolymerize pectin by hydrolysis and transelimination as well as deesterification reactions
which hydrolze the ester bond between the carboxyl and methyl groups of pectin.
Pectinases are also produced from a wide variety of microbial sources such bacteria,
fungi, actinomycetes, yeast. Fungi include Aspergillus and Penicillium sp. (Botella et al.,
2007; Patil and Chaudhari, 2010). Aspergillus species such as Aspergillus alliaceus
(Sapunova et al., 1990), and A. niger (Fontana et al., 2005; Maciel et al., 2011) have been
reported for pectinase enzyme production. Pectinase production has been reported from
different species of Penicillium such as Penicillium chrysogenum (Banu et al. 2010), P.
italicum (Alana et al., 1990), P. roqueforti (Pericin et al., 1992), P. atrovenetum (Johnson et
al., 2012). Among the bacterial species, production of pectinases have been reported from
Bacillus sp. (Kapoor and Kuhad, 2002; Dey et al., 2011), Clostridium (Hla et al., 2005),
Erwinia sp. (Kothari and Baig, 2013), Tubercularia vulgaris (Fonseca and Said, 1995).
Pectinases play a crucial role to reduce the viscosity, increase the yield and clarification
of juice by liquefaction of pulps, remove off the peels (Chaudhri and Suneetha 2012; Makky
and Yusoff 2015) and in maceration of vegetables to produce various products like pastes and
purées (Demir et al., 2000; Tochi et al., 2009). In citrus juice processing, pectic enzymes
contribute in the removal of cloudiness and stabilization of juice (Braddock, 1981).
Pectinolytic enzymes were also applied in association with other cell wall degrading enzymes
such as cellulases and hemicellulases (Bhat, 2000). Croaka and Corredig (2006) reported the
changes occurring to orange juice cloud particles after addition of polygalacturonase and
pectin esterase. Soares et al., (2001) reported that yield of fruit and vegetable juice was
improved significantly by pectinase treatment. Singh and Gupta (2004) also reported the
effect of gelatin on the efficacy of pectinolytic enzyme from Aspergillus niger for
clarification of apple juice. In apple and grape, pectinases are used during pressing and
straining stages. Extraction by enzymatic maceration can increase yield by more than 90% as
compared to conventional mechanical juicing, besides improving the organoleptic, nutritional
properties and filteration efficiency (Rombouts et al., 1980).
The main functions of pectinolytic enzymes in the wine making process are to support the
extraction process, maximize juice yield, facilitate filtration and intensify the flavour and
colour (Chaudhri and Suneetha, 2012). Enzymatically treated wines showed more stability
with reduced filtration time in comparison to control wines (Jayani et al., 2005). Treatment of
macerated fruits with pectinolytic enzymes, before the addition of inoculum resulted in
improved characteristics of wine (Revilla and José, 2003; Praveen and Suneetha, 2014).
Clarification of must prior to the onset of alcoholic fermentation also improves the sensory
characteristics of white wine (Reddy and Reddy, 2009).
Vegetable oils of olive, sunflower, coconut, palm or canola are obtained by extraction
with organic solvents such as hexane, which is a potential carcinogen (Kashyap et al., 2001).
The use of pectolytic enzymes, in this case preferably alkaline, allows the extraction of
vegetable oils in an aqueous process by degradation of cell wall components (pectin). Now
days, the use of enzyme preparations containing cellulases, hemicellulases and pectinases has
begun for maximum extraction of oil. (Kashyap et al., 2001; Hoondal et al., 2002).
Pectinase treatment accelerates tea fermentation by breaking down the pectin which is
present in the cell walls of tea leaves and also destroys the foam forming property of instant
tea powders by destroying the pectins. The change in colour of tea during the fermentation
also results in the development of characteristic aroma (Praveen and Suneetha, 2014).
Pectinolytic microorganisms are also used in the fermentation of coffee to remove the
mucilaginous coat from the coffee beans. The robusta coffee mucilage layer is gelatinous and
viscous in nature, which is surrounded over the bean. Pectinases are added to remove the
pulpy bean layer consisting of pectic substances. Pectinolysis enabled reduction in
demucilisation time which was evident with reduction in pH and sugars released (Murthy and
Naidu, 2011).
The use of pectinases in production of ruminant feed decreases the feed viscosity and
increases the absorption of nutrients by ruminants, liberates nutrients by enzymatic action
which also reduces the amount of faeces (Hoondal et al., 2002; Praveen and Suneetha, 2014).
Ruminants diet was supplemented by cocktail of enzymes containing xylanases, pectinases
and cellulases. Supplementation of enzymes increases the digestibility of organic matter.
Improvements in animal performance due to the use of enzyme additives can be attributed
mainly to improvements in ruminal fiber digestion, resulting in increased digestible energy
intake (Ghorai et al., 2009). Feeds treatment with enzymes may improve digestibility of the
feed via a number of different mechanisms including direct hydrolysis, improvements in
palatability, changes in gut viscosity (Ghorai et al., 2009).
XYLANSES
Xylans are hemicelluloses and are the second most abundant polysaccharide in nature and
was surpassed only by cellulose in abundance (Collins et al., 2005; Whistler and Richards,
1970), comprising approximately one third of renewable carbon sources on earth (Prade,
1996). Xylan and cellulose are strongly associated, because a β-1, 4 linked xylan chain can
hydrogen bond to cellulose. Unlike cellulose, xylan is a complex heteropolysaccharide
(Gilbert and Hazlewwod, 1993). The backbone of xylans consist of approximately 2 β-1,4-
linked xylose units that are decorated with different side group depending on the origin of the
xylan. These compounds were present in the cell wall and in the middle lamella of plant cells.
Due to the complex structure of hemicelluloses, several different enzymes were needed
for their enzymatic degradation or modification. The two main glycosyl hydrolases
depolymerising the hemicellulose backbone are endo-xylanase and endomannanase
(Suurnakki et al., 1997). Since xylan is a complex component of the hemicelluloses in wood,
its complete hydrolysis requires the action of a complete enzyme system which is usually
composed of xylanase, xylosidase and debranching enzymes such as α-arabinofuranosidase,
α-glucuronidase, acetylxylan esterase, and hydroxycinnamic acid esterase that cleave side
chain residues from the xylan backbone. All these enzymes act cooperatively to convert xylan
to its constituents (Sunna and Antranikian, 1997). Microbial enzymes act co-operatively to
convert xylan to its constituent simple sugars: these enzymes include β- 1,4-endoxylanases
(xylanases ; EC 3.2.1.8), which cleave internal glycosidic bonds within the xylan backbone;
arabinofuranosidase (EC 3.2.1.55) which hydrolyses arabinose side-chains ; α-glucuronidase
which removes glucuronic acid side-chains from the xylose units; xylan esterases (EC
3.1.1.6)which release acetate groups; and finally xylosidase (EC 3.2.1.37), which hydrolyses
xylobiose to xylose (Wong et al.,1988).
Xylanases are widespread in nature. They occur both in prokaryotes and eukaryotes and
have been reported from marine and terrestrial bacteria, rumen bacteria, fungi, marine algae,
snails, crustaceans, insects and seeds of terrestrial plants (Dekker and Richards, 1976)
Xylanase enzyme have been investigated from a variety of microorganisms (Sanghi et al.,
2008; Collins et al., 2005). Xylanases are produced by diverse genera and species of
microorganisms (Bajaj and Singh, 2010; Nagar et al., 2010; Sanghi et al., 2008), and marine
algae, snails, crustaceans, insects and seeds of terrestrial plants (Liu et al., 1999; Sunna and
Antranikian, 1997). Fungi, yeasts, and some bacteria secrete extracellular xylanases, although
cell wall-bound and intracellular xylanolytic enzymes have also been reported (Dekker, 1985;
Dekker and Richards, 1976). The various important fungal xylanase producers are
Chaetomium cellulolyticum, Gloeophyllum trabeum, Trichoderma viride, Lentinus lepidius,
Tyromyces balsemeus, Heterobasidium annosum, Dactylomycescrustaceous, Fusarium
oxysporum, Penicillium jenthillenum, Phlebia radiata, Aspergillus niger, Poria placenta,
Coniophora puteana and Phanerochaete chrysosporium (Eriksson et al., 1990). Most of the
xylanase producing fungi belong to the Ascomycetes, Deuteromycetes, or Basidiomycetes
groups. Brown-rot fungi utilize hemicellulose and cellulose, leaving the lignin essentially
undigested while the white rot fungi cause the wood to become pale, eventually reducing it to
a fibrous, whitish mass. Among bacteria, Bacillus sp. were isolated from various habitats such
as soil, garbage dump, effluent treatment plant pulp and paper industry, urban solid waste
etc., (Kapilan and Arasaratnam, 2011; Giridhar and Chandra, 2010). Other xylanase
producing bacteria include Thermotoga thermarum, Streptomyces sp. (Sunna et al., 1996;
Keskar et al., 1989), Flavobacteriumsp. from forest soil (Bhatt et al., 1994).
Poultry diet contains some non-starch polysaccharides (NSP) such as cellulose, xylose,
arabinose, and galactonic acid that are not easily digested by poultry. The anti-nutritional
effect of these NSP is manifested by poor growth accomplished by depressed nutrient
utilization (Annison and Choct, 1991). Their adverse effects can be overcome by either
dietary supplementation or treating the diet with exogenous enzymes (Bedford, 1995).
Xylanases are used in poultry feed along with glucanases, pectinases, cellulases, proteases,
amylases, phytase, galactosidases and lipases. These enzymes break down arabinoxylans in
the ingredients of the feed reducing the viscosity of the raw material (Twomey et al., 2003).
The arabinoxylan found in the cell walls of grains has an anti-nutrient effect on poultry, they
might raise the viscosity of the ingested feed interfering with the mobility and absorption of
other components. Adeola and Bedford (2004) showed that supplementation of a high
viscosity wheat based diet with xylanase mitigated the growth performance reduction with an
accompanying decease in duodenal and ileal digesta viscosity and a subsequent increase in
nutrient use. Enzymes have been approved for use in poultry diets because they are natural
fermented products and, therefore, will not create a detrimental effect on the animal as well as
on consumers.
Xylanase has multiple applications in food industry; however much emphasis has been
paid for its use in bread production. Xylanases might be employed in bread making, together
with α-amylase, malting amylase, glucose oxidase and proteases. Due to the advancements in
baking industry and the ever-increasing demand for more natural products, enzymes have
gained much importance in baking industry. The xylanases broke down the hemicellulose in
wheat-flour helping in the redistribution of water and leaving the dough softer and easier to
knead. During the bread baking process, they delayed crumb formation allowing the dough to
grow. Xylanase increase the bread volumes, absorption of water and improve resistance to
fermentation (Camacho and Aguilar, 2003; Guy and Sarabjit, 2003). Amount of arabino-
xylooligosaccharides in bread would be beneficial to health. Many enzymes such as
proteases, xylanase and cellulases improve the strength of the gluten network and therefore,
improve the quality of bakery products (Gray and BeMiller, 2003). In biscuit making,
xylanase was recommended for making cream crackers lighter and improving the texture,
palatability and uniformity of the wafers.
Bread staling could be caused by progressive cross binding between protein and swollen
residues of starch granules, mediated by amylose molecules leached out during baking
(Martin et al., 1991). Inagaki et al.(1998) have reported that bread firming did occur, even
when the starch contained no amylose and they instead proposed that interaction between
swollen starch granules and the gluten matrix might occur during aging of the bread. The
simpler carbohydrates (monosaccharides and oligosaccharides) resulting from the enzyme
action could affect the water balance and may interfere with protein starch interaction during
bread storage, in the same way as specific dextrins have been described to interfere in the
amylopectin retrogradation (Haros et al., 2002).
Nowadays, xylanases in conjunction with cellulases, amylases and pectinases, leaded to
an improved yield of juice by liquefaction of fruit and vegetables, stabilization of the fruit
pulp, increased recovery of aromas such as essential oils, vitamins, mineral salts, edible dyes,
pigments etc., The mainly desirable properties of xylanases for use in the food industry were
high stability and optimum activity at an acid pH.
Xylanase and xylan are little used in the pharmaceutical industry. Sometimes, xylanases
are added in combination with a complex of enzymes (hemicellulases, proteases and others)
as a dietary supplement. Hydrolytic products of xylan such as β-D-xylopyranosyl residues can
be converted into combustible ethanol, solvents and artificial low-calorie sweeteners. The
first step is the delignification of hemicellulose material rich in xylan following by hydrolysis
by xylanases and hemicellulases to produce sugars such as β-D-xylopyranosyl units. Next, the
products are fermented by yeasts (Pichia stipitis and Candida shehatae) as outlined in Figure
2.3 in order to produce xylitol or ethanol (Screenath and Jeffries, 2000).
Xylitol was a polyalcohol with a sweetening power comparing to sucrose (Parajo et al.,
1998). The development of a more appropriate technology for xylitol production has
generated great hope of its wider use in the food, pharmaceutical and odontological
industries.
CONCLUSION
The use of enzymes in various industries is a well established environmental friendly
approach. These have been used in various food industries because they are natural fermented
products and, therefore, will not create a detrimental effect on the product as well as on
consumers. The replacement of chemicals with enzymes leads to decrease the burden of used
chemicals in the environment. Moreover, enzyme(s) increases the product yield and also
improves the product quality. Despite the developments made in this particular area but still
more development is required so that the cost of food production/processing can be reduced.
Enzymes properties such as thermostability, stability in acidic or alkaline environment, and
retaining the enzyme activity under severe reaction conditions such as in presence of other
metals and compounds must be improved to meet process prospects.
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Chapter 10
ROLE OF NANOTECHNOLOGY
IN FOOD PROCESSING
Sunita Dalal1, and Vidushi Malhan2

1
Department of Biotechnology, Kurukshetra University,
Kurukshetra, Haryana, India
2
Department of Microbiology, Panjab University, Chandigarh, India
ABSTRACT
The food processing industry is facing enormous challenges for developing and
implementing systems that can produce high quality, safe foods while also being
efficient, environmentally acceptable, and sustainable. To these challenges, innovation is
needed for harnessing new processes, products, and tools in the food industry.
Nanotechnology is gaining momentum and is used in a broad array of FDA-regulated
products, including medical products, foods and cosmetics. Desirable effects may be
achieved from altered chemical, biological, or magnetic properties, electrical or optical
activity, increased structural integrity, or other unique characteristics of materials in the
nanoscale range. Nanomaterials are an integrated part of conventional foods, because the
characteristic properties of many foods are based on nanometer sized components, like,
Nano emulsions and foams. The applications of nanotechnology include Nano particulate
Delivery Systems (Nano dispersions and Nano capsules), nanoencapsulation of bioactive
food compounds, Packaging (Nano laminates, Nano composites bottles, bins with silver
nanoparticles), Food Safety and Biosecurity (Nano sensors) etc. The ‗smart‘ packaging
containing Nano-sensors and antimicrobial activators may be used for detecting food
spoilage and releasing Nano-anti-microbes to extend food shelf life. The Nanotechnology
may be used to remove chemicals/pesticides from food. The edible nano wrapper to
envelope foods, prevents gas and moisture exchange. Nanobarcodes are used for safety
labeling and monitor distribution of food products. Ultimately, nanotechnology
application on foods supposes a great challenge but requires solving several aspects in the
near future. Once those barrier have been overcome, manufacturing of foods adapted to

Corresponding Author: Department of Biotechnology, Kurukshetra University, Kurukshetra-136119, E mail;
sdalal@kuk.ac.in; Tel: +91-9812001469.
254 Sunita Dalal and Vidushi Malhan
special necessities will be obtained. There would an improvement in antimicrobial

efficiency and it would lead to a better experience to consumer.
Keywords: nanoparticles, nanocomposites, nanotubes, food processing, food packaging
INTRODUCTION
Nanotechnology has emerged as one of the most innovative scientific fields in decades.
Materials in the nanoscale (10−9 metre) range are of approximately 1-100 nanometer (nm).
They can exhibit different chemical or physical properties, or biological effects because of
quantum size effects and large surface area to volume ratio, compared with their larger
counterparts. Hence, dimension-dependent properties or phenomena may be used for
functional effects such as increased bioavailability, decreased dosage, or increased potency of
a drug product, decreased toxicity of a drug product, better detection of pathogens, more
protective food packaging materials, or improved delivery of a functional ingredient or a
nutrient in food. It extends its potential from mechanics to medicine and able to create new
devices and techniques. ―Nanoparticle‖ is defined as the small object that acts as a whole unit
in terms of transport and properties. They are classified according to their characteristics, size
and structures. ―Nanomaterials‖ is a field that takes a materials science approach to
nanotechnology. Nanotechnology is catalyzing economic growth. Several nanotechnology
applications have evolved through the merge of novel materials with new device
architectures. Products routinely consumed include sunscreen composed of titanium
dioxide/zinc oxide nanoparticles, deep penetrating antiwrinkle cream etc. Applications of
nanotechnology in medicine and healthcare include diagnostic tools, methods for the early
detection and prevention of diseases, drugs and drug discovery, and prosthetics and implants.
Nanodevices, such as non-invasive or minimally invasive surgical tools, gene chips and
biochemical sensors have shown potential for the early detection and prevention of diseases.
Nanotechnology implants can monitor the body chemistry of patients and trigger drug release
at a specific location inside the body. Various drug delivery nanodevices are core-shell
nanoparticles, nanogels, liposomes, phospholipids-based vesicles, microemulsions, polymeric
micelles, polymersomes, dendrimers, nanotubes, and quantum dots. Real-time treatment
tracking is achieved by gold nanoparticles, quantum dots and magnetic nonoparticles (Sandhu
et al., 2002; Salem et al., 2003; Rosi et al., 2006; Weiss et al., 2006; Hellebust and Richards-
Kortum, 2012).
Over the counter products available are nanocoatings in sunglasses as antireflective,
scratch-resistant hydrophobic ultrathin polymer and nanocomposites in cars for scratch
resistance, light weight, and rust proof etc. So the nanotechnology market place is promising
and includes, the automotive, aerospace, electronics, energy, food, medical, and
pharmaceutical industries. Existing uses of nanotechnology typically focus on performance
improvement (Yu et al., 2002; Qiu and Kalita, 2006; Fogelstrom, et al., 2006).
Nanotechnology have potential applications in various aspects of food sciences viz.
agriculture (e.g., pesticide, fertilizer or vaccine delivery; animal and plant pathogen detection
and targeted genetic engineering), storage, quality monitoring, food processing, and food
packaging. Its application in the food industry ranges from intelligent packaging to creation of
on-demand interactive food that allows consumers to modify food, depending on the
Role of Nanotechnology in Food Processing 255
nutritional needs and tastes. Various nanoscale natural components in food include cell
membranes, hormones, enzymes, DNA, proteins, fats, carbohydrates, fatty acids and amino
acids etc. Researches in the field of food sciences have generated nanomaterial that will make
a difference not only in the taste of food but also in food safety and the health benefits. It can
be applied by two different approaches either ―bottom up‖ or ―top down.‖ in food processing
(Ravichandran, 2 1 ). The ―top down‖ approach involves a physical processing of the food
materials, such as dry-milling of wheat into fine flour generating a high water-binding
capacity. Similarly the antioxidant activity in green tea powder is improved by reducing size
of the powder to 1000 nm, the digestion and absorption also enhanced the activity of an
oxygen-eliminating enzyme (Shibata, 2 2). The ―bottom up‖ concept is inspired from self-
assembly and self-organization tendency of living organisms. More generally, molecular self-
assembly seeks to use concepts of supramolecular chemistry, and molecular recognition in
particular, to cause single-molecule components to automatically arrange themselves into
some useful conformation. For example, self-assembly structures through organization of
casein micelles or starch and the folding of globular proteins and protein aggregates creates
stable entities to form nanometer scale via self organization (Dickinson and Van Vliet, 2003).
Nanoparticals in food may appear in suspensions (mostly solid in liquids) or emulsions
(two liquid phases). These particles are known to have different structures and shapes, which
can be (homogeneous or heterogeneous) spherical, tubular, irregularly (non-spherical) shaped,
or can exist in fused aggregated or agglomerated forms. The delivery of bioactive compounds
for nutritional as well as development of functional food are possible through this technology.
The properties of the recent nanomaterial‘s like food colouring, delivery of substances in
targeted sites, and flavoring, nutritional additives provide functionally and antimicrobial
ingredients for food packaging offer many new opportunities for food industries. The
objective of this article is to provide a background on food nanotechnology and an up-to-date
account of known and possible futuristic applications of nanotechnology in the food
processing and packing industry.
OVERVIEW
Every living organism on earth exists because of the presence and interaction of various
nanostructures. Even food molecules such as carbohydrates, proteins, and fats are the result of
nanoscale-level augmentation of sugars, amino acids, and fatty acids. As it applies to the food
industry, nanotechnology involves using biological molecules such as sugars or proteins as
target-recognition groups for nanostructures that could be used, as biosensors on foods
(Charych et al., 1996). Such biosensors could serve as detectors of food pathogens and other
contaminants and as devices to track food products. Besides usefulness in encapsulation
systems for protection against environmental factors nanotechnology can be used in the
design of food ingredients by infusion of novel ingredients, flavors and antioxidants
(Imafidon and Spanier, 1994). The overall goal is to improve the functionality of such
ingredients with minimum concentration. This parameter leads to versatile application of
nanotechnology in food industry (Table 1).
Table 1. Applications of nanotechnology in the Food sector
Phases Applications Nanotechnology Functions

Production and Water purification Nanopore filters Removal of pathogens and
Processing contaminants.
Food machines/ Nanoceramic Large surface areas for catalytic
equipments/utensils devices, nanosize reactions, antimicrobial
silver/zinc oxide refrigerators, removal of toxins
coatings etc. released during cooking.
Preserving, Food items Nanoemulsions, Food stability against environmental
protecting and nanocapsules stress, ntimicrobials, taste-texture
product design enhancer.
Nanolaminates Coatings fruits, veggies, candies,
bakery items etc. to prevent
moisture, lipid, and gas losses.
Packing material Nanocomposites Reduces gas diffusion and prolongs
shelf life.
Nanosensors To detect food deterioration and to
monitor storage conditions.
Food Functional additives Nanofibers, Enhances shelf life, site-specific
enrichment delivery.
Nanocapsules Encapsulation of hydrophobic
nutraceuticals for enrichment of
non-fat or low-fat food products.
Food digestion Food engineering Nanodrops/ Phytosterol nanodrops inhibit
Nano-sized self- cholesterol transportation into the
assembled bloodstream and help in reducing
structural liquids cholesterol level.
(NSSA). NanoClusters increase wetness and
absorption of nutrients in the foods.
In Food industry, Nanotechnology could be viewed as an umbrella covering two major

aspects viz. ―Nano inside‖ and ―Nano outside.‖
―Nano inside‖ attributes various aspects of developing new food flavors, nutrients
delivery systems, supplements, processing and product development. Technologies like
nanodispersions, nanoemulsions and nanocapsules, to deliver nutrients; nanofibers as food
matrix and platform for bacterial cultures; nanoceuticals to enhance absorption of nutrients,
nanocochleates as effective tool for nutrient delivery to cells without affecting color and taste
of food products fall under this category. Vitamin sprays as nanodroplets for better absorption
of nutrients are also part of it. Nano capsules delivery systems play an important role in
processing sector and the functional property is maintained by encapsulating simple solutions,
colloids, emulsions, biopolymers and others into foods. Nano sized self assembled structural
lipids serves as a liquid carrier of healthy components that are insoluble in water and fats
called as nanodrops. They are used to inhibit transportation of cholesterol from the digestive
system into the bloodstream. Liposomes to deliver functional components such as
nutraceuticals, antimicrobials, and flavors to food (Were et al., 2003). Nanosized powders are
used for increasing absorption of nutrients. Supplementary aspect involves encapsulation
techniques where the needed probiotics, and other products are targeted into the human
system with the help of iron and zinc nano structured capsules. Thus, nanotechnology in food
supplement is very effective than common supplements because they react more effectively
with human cells due to their size.
―Nano outside‖ technology refers to various aspects of preventing spoilage of food and
enhancing shelf life of the product. Food packaging requires special physical, chemical, and
biological needs for protection, and tampering resistance. The food products are preserved by
maintaining an inert and low oxygen atmosphere preventing microbial growth and spoilage,
thus the material used should be impermeable to gases. The use of protective coatings and
suitable packaging by the food industry has become a topic of great interest because of their
potentiality for increasing the shelf life of many food products (Ahvenainen, 2003). By means
of the correct selection of materials and packaging technologies, it is possible to keep the
product quality and freshness during the time required for its commercialization and
consumption (Stewart et al., 2002). Nanocomposites are incorporated in the polymer matrix
of the substances due to their large surface area which favors the filler matrix interactions and
its performance. Nano reinforcement‘s acts as small, barriers to permeation of gases by
complicating the path of the material (Moore, 1999).
Nano clays are composite materials having complex metallic ores. They are naturally
obtained from volcanic ash (Montmorillonite) or polymer based clays (nylons, polyolefin,
PET, PA, epoxy resin, poly methane) are used for polymer matrix in food packaging.
Microbial growth leads to spoilage of food. Their growth is controlled by Silver nanoparticles
barriers. Zinc oxide‘s antibacterial nature increases with decreasing particle size, it can be
stimulated by visible light, and they are incorporated in number of polymers including
polypropylene. E. coli contamination can be controlled using Titanium dioxide as a coating in
packing material. It is also combined with silver to improve disinfection process. Chiston is a
biopolymer derived from chitin recently reported antimicrobial properties additional to
material for encapsulation. Antimicrobial packaging generates highly healthy and consumer
friendly products. Shelf life of the products could be checked and alarmed with the help of the
nanosensors. The food environment is continuously sensed for oxygen content, temperature,
pathogens and indicators are used for proper alarming. Some examples include gold nano
particle incorporated enzymes for microbe‘s detection, gas sensing related to condition of
food products, nanofibrils of perylene-based fluorophores indicates fish and meat spoilage by
detecting gaseous amines. Others include zinc oxide and titanium oxide nanocomposites for
the detection of volatile organic compounds. 5 nm thin nanoscale edible coatings, are
currently used on a wide variety of foods, including fruits, vegetables, meats, chocolate,
cheese, candies, bakery products, and French fries (Morillon et al., 2002; Cagri et al., 2004;
Rhim, 2004). These coatings or films could serve as moisture, lipid, and gas barriers.
Bioactive packaging materials need to be able to keep bioactive compounds, such as
prebiotics, probiotics, encapsulated vitamins or bioavailable flavonoids, in optimum condition
until they are released in a controllable manner into the food product (Lopez-Rubio et al.,
2006; Guerra et al., 2005; Brody, 2005).
Bioactive-packaging materials can help to control oxidation of foodstuffs and to prevent
the formation of off-flavors and undesirable textures of food. Bioactive compounds that are
encapsulated into the packaging itself are a promising approach because this would allow the
release of the active compounds in a controllable manner. Several already approved food
additives could be used for such as nanoencapsulation including carrageenan, chitosan,
gelatin, polylactic acid, polyglycolic acid, and alginate (Kumar, 2000; Agulla et al., 2003).
Nanobarcodes are used for tagging and also for security. The ―Smart sensors‖ are beneficial
to the consumers in terms of better quality identification and producers for rapid distribution
and authentication of the food products by detecting pathogens efficiently in real time and
with high sensitivity (Vo-Dinh et al., 2001; Mabeck and Malliaras, 2006). Nanotechnologies
are applied in food production machinery by coatings of machines and the use of nano-sieves,
to filter out bacteria.
Delivery Systems in Food Nanotechnology
Various methods to create nanoparticles in food technology include salting out,

spontaneous emulsification/diffusion, solvent evaporation, polymerization, and
nanoprecipitation. In addition, electro spraying has shown to be capable of producing uniform
particles of less than 100nm from polymer and biopolymer solutions. Nanotechnology
significantly contributed to the development of nanometric delivery systems, capable of
encapsulating functional ingredients, including simple solutions, association colloids,
emulsions, biopolymer matrices, and so on, of protecting them from undesired reactions, of
minimizing the impact on the organoleptic properties of the product, as well as of enhancing
their activity by promoting the mass transfer rates to sites of action (Chellaram et al., 2014).
Each type of delivery system has its own specific advantages and disadvantages for
encapsulation, protection, and delivery of functional ingredients, as well as cost, regulatory
status, ease of use, biodegradability, and biocompatibility. A number of potential delivery
systems based on nanotechnology are as follows.
Nano Dispersions and Nano Capsules
Functional ingredients vitamins, antimicrobials, antioxidants, flavorings, colorants, and

preservatives etc. exists in different molecular and physical forms. For many reasons of hard
surroundings and processing techniques they are rarely utilized directly in their pure form;
instead, they are often incorporated into some form of delivery system. Uricanu et al., (2004)
reported naturally available casein micelles (CM) nano-capsules deliver nutrients such as
calcium phosphate and protein to the neonate. A novel approach is to harness CM for Nano-
encapsulation and stabilization of hydrophobic nutraceutical substances for enrichment of
non-fat or low-fat food products. Such Nano-capsules may be incorporated in dairy products
without modifying their sensory properties.
Nano-Emulsions
Emulsions with droplet diameters of less than 100 to 500 nm with fascinating capability
of loading both hydrophobic and hydrophilic molecules, generated by high-pressure valve
homogenizers or microfluidizers are often referred to as ―nanoemulsions.‖ Functional food
components can be incorporated within the droplets, the interfacial region, or the continuous
phase (McClements, 2004). Functional food components could be encapsulated within the
inner water phase, the oil phase, or the outer water phase, thereby making it possible to
develop a single delivery system that contains multiple functional components This interfacial
engineering technology would utilize various food-grade ingredients like proteins,

polysaccharides, and phospholipids. This technology could be used to separate two aqueous
phase components that might adversely react with each other if they were present in the same
aqueous phase. Nano size emulsion-based ice cream with a lower fat content has been
developed by Nestle and Unilever (Renton, 2006). Nanoemulsions are shown to have many
advantages over conventional emulsions, such as optical clarity, high kinetic stability and
increased bioavailability. While nanoemulsions are considered to be one of the most
promising delivery systems for food applications, its use is still limited while its effects on
health and food safety investigated. Their stability is still an issue, being affected by various
factors.
Nanostructured Multilayer Emulsions
Use of multilayer emulsions can create novel delivery systems consisting of oil droplets
(the core) surrounded by nanometer thick layers (the shell) comprising different
polyelectrolytes. These layers are formed using a layer-by-layer (LbL) electrostatic deposition
method that involves sequential adsorption of polyelectrolytes onto the surfaces of oppositely
charged colloidal particles. Emulsions containing oil droplets surrounded by multilayer
interfaces have been found to have better stability against environmental stresses than
conventional oil-in-water emulsions with single-layer interfaces. In addition, it is possible to
develop smart delivery systems by engineering the properties of the nanostructured shell
around the droplets utilizing food-grade ingredients like proteins, polysaccharides, and
phospholipids. A functional component trapped within the core of a multilayer emulsion
delivery system could be released in response to a specific environmental trigger by designing
the response of the shell to the environment (Guzey and McClements 2006).
Nano Encapsulation
Nanoencapsulation is a favorable technique for masking unpleasant tastes, protection of

nutrients against undesirable condition of processing and storage and more application of
low-soluble compounds. It is defined as a technology to pack substances in miniature by
techniques such as nanocomposite, nanoemulsification, and nanoestructuration and provides
final product functionality. The protection and controlled release of various bioactive
compounds may be achieved using this technique for the production of functional foods with
enhanced functionality and stability. Nano encapsulation can significantly minimize the
amount of active compounds needed for formulation (Huang et al., 2009). Probiotics are
generally defined as live mixtures of bacterial species and can be incorporated in foods in the
form of yoghurts and yoghurt-type fermented milk, cheese, puddings and fruit based drinks.
Encapsulated forms of ingredients also achieve longer shelf life of the product.
Nanoencapsulation of bioactive compounds has versatile advantages for targeted site-specific
delivery and efficient absorption through cells.
Nano Fibers
Nanofibres with diameters from 10 to 1000 nm, make them ideal for serving as a
platform for bacterial cultures as well as structural matrix for artificial foods. Nanofibers have
exceptionally high surface areas and enjoy a high encapsulation efficiency that provides the
means to stabilize and maintain the quality of foods by stabilizing functional additives in
them. Increasing the surface area and the corresponding carrier dissolution rate enhance
material delivery. Food additives such as volatile or unstable flavors and antioxidants are
often susceptible to light, oxygen, and heat. Therefore, nanofibers that have normally a higher
thermal stability are used for the stabilization of functional additives (Kayaci and Uyar,
2012). Nanofibers of cellulose and cellulosic base present good potentials for use in food
systems. Cellulose in its natural form cannot be suitably used as delivery systems in food
because of its low solubility in water and large dimensions. However, cellulose can be
physically, chemically, or biochemically modified to change it for use in foods (Rezaei et al.,
2015). Caffeic acid encapsulated by Solid lipid nanoparticles to improve stability in oxidative
conditions and masking its bitter taste for food fortification (Fathi et al., 2013). In another
work, hesperetin was encapsulated by different biopolymers to increase its functionality in
functional foods and masking its bitter flavor. Gelatin is generally used as the gelling agent in
foods, pharmaceuticals, and cosmetics incorporated into cellulose acetate nanofibers.
Vitamins A and E have various biological actions and protective roles. Immobilized vitamins
A and E onto electrospun cellulose acetate nanofibers (247 to 265 nm) onto the nanofibers
resulted in the controlled release of vitamins over the test period (Taepaiboon et al., 2007).
Nanolaminates
A nanolaminate consists of two or more layers of material with nanometer dimensions

that are physically or chemically bonded to each other. One of the most powerful methods is
based on the layer-by-layer (LbL) deposition technique, in which the charged surfaces are
coated with interfacial films consisting of multiple nanolayers of different materials, Similar
to the preparation of multiple emulsions, electrostatic attraction causes polyelectrolytes and
other charged substances to be deposited onto oppositely charged surfaces. This LbL
technology allows precise control over the thickness and properties of the interfacial films,
which enables the creation of thin films (1 to 100 nm per layer). Since a variety of adsorbing
substances, like natural polyelectrolytes (proteins, polysaccharides), charged lipids
(phospholipids, surfactants), and colloidal particles (micelles, vesicles, droplets) could be
used to create the different layers. There gets enhanced possibility to incorporate variety of
active functional agents such as antimicrobials, antibrowning agents, antioxidants, enzymes,
flavors, and colors into the films. These functional agents are responsible for the enhanced
shelf life and quality of coated foods. Nanolaminates coatings over a wide variety of foods,
including fruits, vegetables, meats, chocolate, candies, bakery products, and French fries
could serve as moisture, lipid, and gas barriers (Park, 1999; Kotov, 2003; Decher and
Schlenoff 2003; Cha and Chinnan 2004; McClements et al., 2005).
Nanoceuticals
Nanoceuticals are a technology which encapsulates nutritional factors and speeds them
directly into the cells of the body. Worldwide commercial foods and food supplements
containing added nanoparticles are becoming available. A major growth area appears to be
the development of ‗nanoceuticals‘ and food supplements. The general approach is to develop
nano-size carriers or nano-sized materials, in order to improve the absorption and, hence,
potentially the bioavailability of added materials such as vitamins, phytochemicals, nutrients
or minerals. The materials can be incorporated into solid foods, delivered as liquids in drinks,
or even sprayed directly onto mucosal surfaces (Fulekar, 2010).
The number of food-related nano-products is increasing rapidly, and examples include:
 fruit drinks containing nanoparticles of carotenoids;

 synthetic lycopene;
 nano-sized micellar systems canola oil for vitamins, minerals or phytochemicals;
 nanocages or nanoclusters as delivery vehicles, for a sweet claimed chocolate drink
without added sugar or sweeteners;
 nano-based mineral supplements, Chinese Nanotea claimed to improve selenium
uptake;
 patented ‗nanodrop‘ delivery systems, to administer encapsulated vitamins,
transmucosally;
 large number of mineral supplements such as nano-silver or nano-gold are available.
To prevent the accumulation of cholesterol some of the nutraceuticals incorporated in the

carriers include lycopene, beta-carotenes and phytosterols.
When selecting a method to produce nanoparticles, food manufacturers should carefully
review the solvents required. Ultimately, more development efforts are needed to adapt these
methods to strictly use only food-approved processing aids and components.
Areas of Food Nanoscience
Scientists and industry stakeholders have identified potential uses of nanotechnology in

virtually every segment of the life from mechanics to cosmetics to food industry. Undeniably,
the most active area of food nanoscience research and development are food processing,
packaging and nutrient supplements (Figure 1).
Food Processing
Nanotechnology raises the opportunity of using molecules as starting material, so that

new interaction are achieved which in turn generates the required properties in food. The
paradigm shift resides in the fact the food formulation are based on the use of ―food matrix
precursors or structural elements.‖ New knowledge and techniques based on nanotechnology
generates the potential of the development of new textures and flavours, the possibility of the
design of low dense, low calorie foods but nutritionally enhanced. Those foods can be aimed
at a nutrition adapted to different lifestyle and consumer conditions (i.e., obesity cases).
Pray and Yaktine, 2009, recommended basis of foods formulation on two simultaneous
processes i.e.:
 Formation the structure: phase creation, reactions, biopolymer transformation.

 Stabilization of the system: vitrification, crystallization, network formation.
One advanced aspect of the use of nanotechnology is the possibility of acting straight
onto the food structure. Nanostructured foods aimed at producing better texturized, flavoured
and other properties. In particular it can be highlighted the possibility of producing cream-like
foods such as: ice-creams, spreads and low fat dressings. Leser et al., 2006, reported how
polar lipids (monoglycerides and phospholipids) can be used with this purpose. Some of these
polar lipids are capable of creating nanostructures that finally result in crystalline phases.
These structures can be used as a base for the development of spreads taking advantage on the
texture properties emerging from interaction between nanostructures clusters. The
manufacturing process depends on water relative quantity, stirring, and the system
temperature evolution. The contribution of particles to the stability of foams and emulsions is
another interesting aspect. Most of the foods are dispersions like emulsions or foams. Several
examples could be derived from bakery, confectionery, meat products, dressings and spreads.
Foam and emulsion stability can be improved in the presence of nanoparticles and
nanostructures.
Figure 1. Areas of Food Nanotechnology.

Most of the applications in processing technology are related with the use of
nanoparticles and nanocapsules, termed ‗Food Engineering‘. These particles enhance the
products functionality, and perform a protection function on active principle. Nanoparticles
avoid the degradation produced by the surrounding environment of the food or harsh
manufacturing process. Gökmen et al., 2011, reported successful nanoencapsulation of omega
3 fatty acid in bread baking. Besides the positive effect in taste masking, thermo-oxidation
was also reduced. This resulted in decline in the production of other degradation by-products
also (i.e., acrylamide). Other technological explorations like nanoparticles containing
essential oil were used to improve antimicrobial activity in juices (Donsì et al., 2011). In this
case, the addition of small amounts of nanoparticles containing terpenes to an orange and pear
juice delayed or avoided the microbial growth.
 Enhancement of the bioavailability is another breakthrough in food nanotechnology.

Nanoemulsions and nanoparticles suppose a delivery vehicle capable to override two
great problems of digestive system i.e.,
 Conservation in the food previous to the consumption and the stomach degradation.
 The absorption of the compounds by the organism.
Nanotechnology could improve the main absorption route of foods in the intestine. Direct
absorption of nanoparticles is controlled by size and surface chemistry of the particle.
Nanoparticles can be utilised by an active transport mechanism as well by a passive transport
(Acosta, 2009). Selective absorption can be conditioned by specific receptors in the cell
surface (enterocytes and M cells). The nanoparticles with a specific surface composition can
be absorbed by this way. Generally, the M cells present a better permeability. There is a great
interest in the development of lecithin nanoparticles since it could be an improvement in the
absorption of isoflavones by this route.
Cattania et al., 2010, studied in vivo of lipidic core nanocapsules. Nanocapsules delivered
the active principle once fixed by diffusion to gastrointestinal lumen. They act as active
principle reservoirs. There is a relation of particle size and bioavailability, as far the size
decreases, the bioavailability is improved. A size reduction under 500 nm produces a higher
absorption of the active principle and more particle uptake regardless of the system
composition (Acosta, 2009). So, it is possible to obtain more effect from a food additive with
the use of nanotechnology than with the use of the traditional approach.
Another interesting example of food engineering is regulating cholesterol through a
technology called nano-sized self-assembled structural liquids (NSSA). Minute compressed
micelles serve as a liquid carrier of healthy components called nanodrops. They are added to
food product and are able to pass through the digestive system untouched, and hence reach
directly to the absorption site, carrying phytosterols to the larger micelles of the body. These
phytosterols inhibit transportation of cholesterol from the digestive system into the
bloodstream and thereby help in reducing cholesterol level of body (Woodrow Wilson
International Center for Scholars, 2006a). Besides health parameters nanotechnology is used
in the diet industry to permit chocolate lovers to enjoy their chocolate without the burden of
excess sugar. NanoClustersTM are a nanosize powder that combines with foods to increase
wetness and absorption of nutrients in the foods. Cocoa is added to these clusters to enhance
the taste and benefit of this food (Woodrow Wilson International Center for Scholars, 2006b).
Food Equipment
Besides application of nanoparticles in food processing and packaging, they could play
crucial role in healthy cooking in households. Frying is common practice applied in kitchen
during which edible oil cooks food by transmitting heat to the objects being cooked.
Peroxides are formed during this cooking process. These peroxides are then transformed into
secondary substances, which result in generating higher viscosity and foul odor in cooking
oil. A catalytic device for refining frying oil is now available. This device is made of silver
infused ceramic pellets and is designed to capture the peroxides produced during the frying
process and turn them into stable compounds. The elimination of the secondary substances
formation prevents fatty and sticky residues forming on the surface of the cooked foods and
thus prevents the foul odors commonly released from used oil (Oil Fresh Corporation, 2005).
Refrigerators are now being designed with nano silver technology that makes use of the anti-
microbial properties of silver. Nanoparticles of silver are impregnated into the vegetable
compartment, meat compartment, and water-dispensing system. Researchers at the University
of California–Davis have verified that meat freshness and nutritional content can be
maintained for up to 15 days in these compartments (A to Z of Materials, 2006). Nano
Filtration systems work on less than 0.001 microns (10 angstroms) in size and rejects divalent
and multivalent ions. It has application in desalination, milk, whey and juice filtration,
demineralization, color removal, concentration of products, waste water treatment and water
purification (Tiwari et al., 2008).
PACKAGING
Food packaging is considered to be one of the earliest commercial applications of
nanotechnology in the food sector. About 500 Nano-packaging products are in commercial
use, and nanotechnology is expected to be used in the manufacture of 25% of all food
packaging within the next decade. The inclusion of nanoparticles in food contact materials
can be used to generate novel types of packaging materials and containers.
The major aims of innovative packaging solutions is the reduction of spoilage and real
time check system if any spoilage. So the packaging protocol includes nano antimicrobials
and nanosensors for pathogen for contaminant detection. Production, processing, and
shipment of food products could be made more secure through the use of nanosensors for
pathogen and contaminant detection. Silver, a well-known anti-microbial agent, is being
infused into storage containers to retard bacterial growth and enhance longevity storage of
foods. In a case study, the 24-hour growth of bacteria was reduced by over 98 percent because
of the silver nanoparticles (Woodrow Wilson International Center for Scholars, 2006c).
Nanomaterials are being developed with enhanced mechanical and thermal properties to
ensure better protection of foods from exterior mechanical, thermal, chemical, or
microbiological effects. Nanocomposites, for instance, are nanoparticles bonded in polymers
so that the materials have enhanced properties such a lighter weight and better recyclability,
as well as spoilage and flavor issues. Nanocomposite materials are currently being used in
beer bottles; allowing for a 6-month shelf life (A to Z of Nanotechnology, 2006). As milk
begins to spoil, various changes occur in its molecular properties. Researchers are hoping to
use these changes to react with nanoparticles embedded in the milk cartons, resulting in a
change of color of the carton. It would then be easy for retailers and consumers to tell if the
quality of the milk has diminished (A to Z of Nanotechnology, 2006). Nanoparticles of
pigments such as TiO2 become transparent but retain their UV absorption characteristics.
This suggests applications in transparent wraps, films or plastic containers where absorption
of UV radiation needs to be avoided. Layered composites containing nanoparticles (such as
nano-clays) are being used to generate long path lengths through materials to reduce gas
diffusion and prolong the shelf-life of products.
Applications for food contact materials (FCMs) using nanotechnology is as follows
(Guhan Nath, 2014):
 FCMs incorporating nanomaterials to improve packaging properties (flexibility, gas

barrier properties, temperature/moisture stability, light and flame resistant,
transparency, mechanical stability).
 ‗‗Active‘‘ FCMs incorporate nanoparticles with antimicrobial or oxygen scavenging
properties.
 ‗‗Intelligent‘‘ or ―smart‖ food packaging incorporating nanosensors for sensing and
signaling of microbial and biochemical changes, release of antimicrobials,
antioxidants, enzymes, flavours and nutraceuticals to extend shelf-life.
 Biodegradable polymer–nanomaterial composites by introduction of inorganic
particles, such as clay, into the biopolymeric matrix and can also be controlled with
surfactants that are used for the modification of layered silicate.
Nano Materials and Environmental Concern
Nanotechnologies promise many stimulating changes to enhance health, wealth and

quality of life, while simultaneously reducing the environmental stress. To save the
environment Bio degradable plastics came into effect but they lack mechanical strength and
are permeable to water and gases. These disadvantages of packing material are overcome by
nanotechnology by incorporating material made of natural or synthetic nanoparticles having
properties like bio-degradable, renewable resources having high mechanical strength.
Nanoparticles are obtained as the proteins, carbohydrates, lipids from animal and plant
materials, also metal oxides nanoparticles and carbon nanotubes are used. Highly porous in
nature Nanofibers, synthesized from collagen, cellulose, and zein obtained from corn. These
nano materials are added along with nanoclays and used for comfort packaging. They also
have additional novel properties like sensors, antibacterial action and as biocatalysts
(Hyder, 2003).
Although various nanomaterials seems to contribute in human health and preventing
pollution but the concern is that if changing the size of materials can lead to radical, albeit
useful properties, can we be sure how size will affect other properties and, in particular, the
potential toxicity of such materials? Nanomaterials, even when made of inert elements like
gold, become highly active at nanometer dimensions. So a new term Nanotoxicology evolved.
Its studies are intended to determine whether and to what extent these properties may pose a
threat to the environment and to human beings. Nanotoxicology is a sub-specialty of particle
toxicology. Nanoparticles have much larger surface area to unit mass ratios which in some
cases may lead to greater pro-inflammatory effects. In addition, some nanoparticles seem to
be able to translocate from their site of deposition to distant sites such as the blood and the
brain. Reactive oxidative stress (ROS) and free radical production is one of the primary
mechanisms of nanoparticle toxicity; it may result in oxidative stress, inflammation, and
consequent damage to proteins, membranes and DNA (Fenoglio et al., 2009; Bhatt and
Tripathi, 2011).
The Royal Society identifies the potential for nanoparticles to penetrate the skin, and
recommends that the use of nanoparticles in cosmetics be conditional upon a favorable
assessment by the relevant ―European Commission‖ safety advisory committee. Although the
products of nanotechnology intended for food consumption are likely to be classified as novel
products and require testing and clearance, there are concerns, particularly in the area of food
contact materials, that there could be inadvertent release and ingestion of nanoparticles of
uncertain toxicity. Such concerns need to be addressed because the ultimate success of
products based on nanotechnology will depend on consumer acceptance. While direct food
contact is evident, this application of nanotechnology is expected to have negligible
additional safety concerns in comparison with conventional techniques since carry-over to
food is expected to be negligible. The type of material (and wear-off as a result of the use) of
filters or coatings might require some attention, but this is not exclusively related to safety of
nanotechnologies (Bouwmeester et al., 2009). The Food and Drug Administration (FDA) has
provided its perspective on nanotechnology on its Web site (http://www.fda.gov/
nanotechnology/).One important fact to remember is that the FDA regulates products, not
technologies.
So the potential beneficial effects of nanotechnologies are generally well described, the
potential toxicological effects and negative impacts of nanoparticles have so far received little
attention. The main concern is that the conventional knowledge about health effects of
chemicals and materials, which is based on their chemical and physical properties, may not be
applicable when we are dealing with nanosize products. The size of these products alone may
alter their interaction with biological systems and affect toxicokinetic and toxicodynamic
applications. The toxicity of bulk sliver, for example, does not help predict the toxicity of
nanoparticles of that same material. Other issues, such as accumulation in the environment
and in the food chain, remain unknown.
CONCLUSION
Nanotechnology seems to transform the Food Industry in near future. Major focus of
nanotechnology is to enhance the product quality in terms of nutrition, and protection.
Promising results are available by incorporation of nanomaterials into food packaging leading
to reduce the use of valuable raw materials and the generation of waste. The development of
nanosensors to detect microorganisms and contaminants is a particularly highlight of this
nanotechnology. Challenges of the nanotechnology are technological and social. New
techniques and standardized test procedures are urgently needed to evaluate the impact of
nanoparticles on living cells, so that we can understand how the things are actually working at
the nanoscale. There are a social concern issues, regarding the accidental or deliberate
nanotoxicology in food, or food contact materials, which may provoke consumer concern.
The consumers should be able to exercise choice in the use of the products of nanotechnology
and that they have the information to assess the benefits and risks of such products. It is
widely expected that food products generated by application of nanotechnology at any phases
of development will be available increasingly to consumers worldwide in the coming years.
As developments in nanotechnology continue to emerge, its applicability to food industry will
increase potentially. But, technological and scientific considerations are not enough for the
development of nanotechnology. A great part of the possibilities and potential applications of
nanotechnology in the near future will depend upon public acceptance. Society must evaluate
critically and objectively the future use of nanotechnology.
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Chapter 11
SINGLE CELL PROTEIN:

MICROBIAL PRODUCTION AND ANALYSIS
Simran Preet Kaur

Department of Biotechnology, Sri Guru Gobind Singh College, Chandigarh, India
ABSTRACT
The worldwide food protein deficiency is becoming alarming day by day and with
increased population, pressure is exerted to produce affordable alternative protein sources
other than conventional plant or animal sources. Single cell protein (SCP) represents
microbial cells grown in mass culture and harvested for use as protein sources in foods or
animal feeds. They evolved as bioconversion processes which turned low-value by-
products, often wastes, into products with added nutritional and market value. Using
agricultural wastes and food processing leftovers in the production of single cell protein
as substrates would alleviate pollution. This chapter focuses on microbial single cell
proteins and will connect with its various aspects.
Keywords: microbial SCP, S. cerevisiae, bioconversion, non-conventional protein sources,

Spirulina
INTRODUCTION
Proteins are building blocks of all the living beings. Their role as enzymes in various
metabolic reactions of body, involvement in growth and development and occurrence as
histone proteins for nucleic acid structure make them a crucial metabolite. Most of the
developing countries of the world are facing a major problem of malnutrition. Due to rapid
growth in the population, deficiency of protein and nutrients are seen in human food and as

Corresponding author: Simran Preet Kaur. Department of Biotechnology, Sri Guru Gobind Singh College, Sec-26,
Chandigarh, 160019. E-mail: simmi44@gmail.com, tel: +91-9814277896.
272 Simran Preet Kaur
well as animal feed. The higher prices of protein sources derived from plants or animals have
generated a need for the search of more economical alternatives.
Single cell protein or microbial protein is the dried cell mass of algae, fungi, moulds and
bacteria grown in large scale to provide required supplements for human food and animal
feed. The first time, this term was used in the Massachusetts Institute by Carol Wilson. Using
the term ―SCP‖ was aimed to avoid microbial or bacterial words in nutritional field. They
evolved as bioconversion processes which turned low-value by-products, often wastes, into
products with added nutritional and market value (Schultz et al., 2006).
According to Pandey et al., (2000), application of agro-industrial residues in bioprocesses
such as cultivation of SCP on the one hand provides alternative substrates, and on the other
hand helps in solving pollution problems, which their disposal may otherwise cause.
The large-scale industrial production of microbial biomass has immense characteristic
advantage over plant or animal sources. The microorganisms possess high rate of
multiplication and high protein content (30-80% protein in terms of dry weight) with wide
amino acid spectrum, low fat content, higher protein carbohydrate ratio than forages. They
can utilize a large number of different low cost carbon sources including waste materials. The
microbial production installations occupy limited area, give high yield (except for algal
microbial production) and are independent of seasonal and climatic variations, therefore
easier to plan (Lee, 1996). Single cell proteins (SCP) have application in animal nutrient as
fattening calves, poultry, pigs and fish breeding. In food it is used as aroma carriers, vitamin
carriers, emulsifying aids and to improve the nutritive value of baked products in soups, ready
to serve meals and recipes. Also, SCP has been employed in paper processing, leather
processing and as foam stabilizers (Srividya et al., 2014). Although, employment of
microorganisms as a protein source for humans is controversial issue but for thousands of
years man has consumed either intentionally or unintentionally the microbial biomass along
with products such as alcoholic beverages, cheese, yoghurt and soya sauce, which is
responsible for their production.
MICROORGANISMS FOR SCP

Microorganisms are being extensively used for the production of a wide range of
proteins, enzymes, antibiotics, pharmaceuticals, food supplements and steroids. The choice of
microorganisms for SCP depends on the properties like: economical and easily available
substrates, non-pathogenicity, good nutritional values, easy use, non-toxic nature, ease of
downstream processing and cost-effective production. Various bacteria, mold, yeast and algae
have been employed for the production of single cell proteins. The desired microorganisms
are cultured on the medium under sterile condition. The bacteria include Brevibacterium,
Methylophilus methylitropous, Acromobacter delvaevate, Acinetobacter calcoacenticus,
Aeromonas hydrophilla, Bacillus megaterium, B. subtilis, Lactobacillus sp., Cellulomonas
sp., Methylomonas methylotrophus, Pseudomonas fluorescens, Rhodopseudomonas
capsulata, Flavobacterium sp., Thermomonospora fusca and others, some of the algae used
are Chlorella pyrenoidosa, C. sorokiana, Chondrus crispus, Scenedesmus acutus,
Porphyrium sp. and Sprulina maxima. The filamentous fungi that have been used include
Chaetomium celluloliticum, Fusarium graminearum, Aspergillus fumigates, A. niger,
Single Cell Protein: Microbial Production and Analysis 273
A.oryzae, Cephalosporium cichhorniae, Penicillium cyclopium, Rhizopus chinensis,

Scytalidium aciduphlium, Tricoderma viridae, T. alba and Paecilomyces varioti. Yeasts such
as Candida utilis (Torula yeast), C. lipolytica, C. tropicalis, C. novellas, C. intermedia and
Saccharomyces cerevisiae are among the various organisms that have been used for the
production of SCP (Bhalla et al., 2007).
COST EFFECTIVE PRODUCTION OF SCP

The most widespread and commonly used substrates for SCP production have been those
where the carbon and energy source is derived from carbohydrates. This is due to the fact that
their building blocks (mono and disaccharides) are natural microbial substrates, and that
carbohydrates are among widely distributed renewable resources.
Microorganisms have the ability to upgrade low protein organic material to high protein
food which has been exploited by the industries. Some of the cost effective substrates used for
SCP are enlisted in Table 1.
In many cases, these raw materials have been hydrolyzed by physical, chemical and
enzymatic methods before use (Jhojaosadati et al., 1999). Various hydrocarbon, nitrogenous
compounds, polysaccharides and agricultural wastes such as hemicelluloses and cellulose
waste from plants and fibrous proteins such as horn, feather, nail and hair from animals are
also abundant waste products used for the production of SCP (Pandey et al., 2000).
SCP production from cellulose and lignin substrates requires their breakdown into
assimilable forms, which can be rapidly taken up in solution by the growing organism. The
substrate must therefore be subjected to pretreatments in a number of steps which include
milling and chemical or enzymatic hydrolysis.
Table 1. Low cost substrates (wastes) used for single cell protein production
Substrate Microorganisms References

Orange peels A. niger and S. cerevisiae Azam et al., 2014
Pineapple skin S. cerevisiae MTCC 463 and C. Dhanasekaran et al., 2011
tropicalis MTCC184
Whey Kluyveromyces lactis, Moeini et al., 2004
Kluyveromyces marxianus and
Candida versatilis
Cucumber and orange fruit S. cerevisiae Mondal et al., 2012
wastes
Wheat bran Candida utilis Irfan et al., 2011
Waste of green gram and S. cerevisae Anbuselvi et al., 2014
Bengal gram husk
Rice husk Penicillium expansum Khan and Dahot, 2010
Molasses S. cerevisae Olbrich, 1973
Waste paper Scytalidium acidophilum Ivarson and Morita, 1982
Food industry waste S. cerevisiae and Kluyveromyces Aqqelopoulos et al., 2014
mixtures marxianus
Waste capsicum powder S. cerevisae Zhao et al., 2010
S. cerevisiae lacks the enzyme system which hydrolyzes the polysaccharides into
monosaccharide. Therefore, orange peels (Citrus aurantium, C. sinensis and C. paradisi)
were treated with HCl to obtain monosaccharides for the production of SCP by A. niger and
S. cerevisiae (Azam et al., 2014). Sulphuric and hydrochloric acids are the most commonly
used catalysts for hydrolysis of lignocellulosic residues. In contrast to these acids, phosphoric
acid can be more advantageous for hydrolysis. Phosphoric acid is less aggressive than other
acids which give solutions with higher concentrations of growth inhibitors of microorganisms
such as furfural or acetic acid (Romero et al., 2007). The higher amount of single cell protein
(1.64 g/L) with higher percentage of protein content (18.25%) was produced by Penicillium
expansum when grown on 0.6N H2SO4 pretreated rice husk (Khan and Dahot, 2010).
The pineapple skin waste was found to contain a good amount of reducing and non-
reducing sugars (10 and 13%, respectively), which is most favorable for the growth of
microorganisms. The pineapple waste can be used as an effective alternate carbon source for
SCP production using S. cerevisiae MTCC 463 and Candida tropicalis MTCC184
(Dhanasekaran et al., 2011).
Molasses is a by-product of the sugar manufacturing process. The concentrated sugar
solution obtained from the milling of sugar cane or sugar beet is cooled allowing the sugar to
crystalize. When no more sugar can be crystalized out of solution, the resulting liquid
(molasses), containing about 50% sucrose is eliminated. For every 100 Kg of plant, some 3.5
to 4.5 Kg of molasses may be obtained. Besides its high sugar content, molasses contains
minerals, organic compounds and vitamins which are valuable nutrients in fermentation
processes. Biomass production from molasses requires supplementation with a suitable
nitrogen source, as well as phosphorus. The traditional nitrogen sources used are ammonia or
ammonium salts, and phosphorus can be added in the form of salts. Baker‘s yeast was the first
microorganism to be produced in aerobic stirred fermentation on molasses (Olbrich, 1973).
An effective way of producing SCP (rich in essential amino acids) from soy molasses, a cost
effective substrate using Candida tropicalis was developed (Gao et al., 2011).
Whey is a yellow-green liquid by-product in cheese manufacturing, remaining after the
precipitation and removal of fat and casein from whole milk and represents about 85-90% of
the milk volume. The main composition of dried whey is 70% lactose, 9-14% protein, and 9%
ash. Several processes have been developed for the utilization of lactose in whey to produce
SCP. These processes are aimed for reducing the pollution load of dairy industry waste and
converting it to a marketable protein product (Waites et al., 2001). While most organisms do
not grow on lactose as a carbon source, strains of Kluyveromyces marxianus readily grow on
lactose (Ghaly and Kamal, 2004). A number of plants are operated using Kluyveromyces
lactis or K. marxianus (formerly K. fragilis) to produce protein, Protibel, which is used as a
nutritional supplement for both human and animal consumption (Waites et al., 2001).
Alkanes were considered as an attractive substrate for SCP production, particularly in the
former Soviet Union, where the structural deficit in feed protein was compensated by the
availability of oil. A large number of microorganisms are able to assimilate n-alkanes and 1-
alkenes in liquid culture, and these include yeasts like: Candida, Hansenula, Pichia,
Saccharomyces, Torulopsis and filamentous fungi like: Aspergillus, Fusarium, Mucor,
Penicillium, Rhizopus, Trichoderma (Riviere, 1977). Beux et al., (1995) compared the
cultivation of Lentinus edodes on cassava bagasse and sugarcane bagasse, individually or in
their mixture. Both the substrates were found suitable for mushroom production, but the best
results were obtained when a mixture of cassava bagasse (80%) and sugarcane bagasse (20%)
was used.
The A. terreus possessing a high protein value can be used as a better choice for SCP
production using cheap energy sources like Eichornia and Banana peel (Jaganmohan et al.,
2013). Evaluation studies on cucumber and orange peels were done for the production of
single cell protein using S. cerevisiae by submerged fermentation (Mondal et al., 2012).
A comparative study using S. cerevisiae on fruit wastes revealed that banana skin
generates highest amount of protein, followed by that of rind of pomegranate, apple waste,
mango waste and sweet orange peel respectively with 58.62%, 54.28%, 50.86%, 39.98% and
26.26% crude protein per 100 g of substrate used (Khan et al., 2010).
ANALYSIS OF SCP
The enormous complexity of the proteome and small amount of protein content present in
single cell poses difficulty in measurement of the proteins in single cells. Traditional bulk
protein analysis methods for single-cell level applications have met with varied degrees of
success. Proteomic measurements can be quite complex as there are various types of
measurements to be made such as protein abundance or expression levels, post-translational
modifications, protein translocation, interactions with other proteins, DNA, etc. and protein
activity. No single analytical method is found successful to measure all of these protein
parameters in a single cell hence, a consortium of biochemical and biophysical methods have
been employed.
The most established and user-friendly method for single cell protein analysis is flow
cytometry which allows measuring 10-15 fluorescent species per day. Its effectiveness
derives from the fact that while absolute protein amounts in a cell can be vanishingly small,
the localized protein concentrations can be larger and measurable if the cells are kept intact.
This improvement has been enabled by advancements in both instrumentation and availability
of highly specific antibodies (Wu and Singh, 2012). The use of multiparameter analysis using
multi-color flow cytometers to measure 10-15 key proteins in signaling pathways
simultaneously in single cells has been reported (Irish et al., 2004). The ability to perform
correlated measurements of multiple proteins in single cells has turned cytometry into a
powerful tool to semi-quantitatively analyze pathways underlying many diseases (Sachs et al.,
2005). While multi-parameter cytometry allowed high content screening (for example, of
multiple kinases) in cells, its throughput was still too limited to be useful for drug screening.
This limitation was overcome by developing a barcoding method where differently treated
cells are tagged with a combination of three dyes. Each dye, depending upon dilution, permits
up to 7 intensity levels and hence, combination of the 3 dyes allows processing of up to 343
samples from one pool of cells (Krutzik and Nolan, 2006). Flow cytometry has been most
commonly used for analysis of kinases and phosphatases; it is also useful for other types of
protein measurements such as glycosylation levels and cytokine production. Flow cytometry
can also be used for analysis of secreted proteins such as cytokines. This requires treating
cells with a vesicle formation inhibitor to trap synthesized cytokines in the Golgi, followed by
fixation and permeabilization to stain the trapped cytokines with fluorescent antibodies for
flow cytometric analysis (Karlsson et al., 2003).
Flow cytometry requires a large numbers of cells which makes it hard to analyze samples
that are limited in amount. To permit analysis of small number of cells (100-1000),
microfluidic platforms have been developed that integrate sample handling with flow
cytometry and sorting (Perroud et al., 2008). An integrated microfluidic device has been
developed which has reduced the amount of sample and reagent required and also provided a
means to perform two orthogonal modes of measurements, imaging and cytometry, in one
experiment (Srivastava et al., 2009).
The use of flow cytometry for single cells has a constraint of analyzing not more than 15
proteins simultaneously. A promising development has been an approach called mass
cytometry where the throughput of flow cytometry is combined with the ultra-high
dimensionality and sensitivity of mass spectrometry (Bandura et al., 2009). In mass
cytomtery, cells are stained with 20-30 antibodies conjugated to different metal isotope
containing polymers. The labeled cells are injected and nebulized and the metal tags are
quantified using mass spectrometry and can simultaneously profile various parameters,
including binding of antibodies, viability, DNA content, and relative cell size (Bendall et al.,
2011).
Surface immobilized antibody platforms can also be adapted to detect secreted proteins
like cytokines from single cells. The limitation of multiplexing more than three cytokines was
treated using high-throughput single-cell barcoded chip (Ma et al., 2011). Also, cytokines
released from single cells were detected by antibodies with covalently attached fluorescent
oligomers that can be amplified to increase detection sensitivity up to 200-fold (Choi et al.,
2011). Single cell mass spectrometry (MS) has the potential to provide label-free quantitative
analysis of the entire proteome of a single cell. The advantage of MS is that there is no need
for molecular labels, and femtomolar sensitivity is routinely achieved for pure proteins. Mass
spectrometry techniques used for single cell studies include electrospray MS, laser/
desorption/ionization (LDI-MS), and secondary ion MS (SIMS (Rubakhin and Sweedler,
2008). Using MS to study proteins in the single cells has its limitations. The biggest drawback
is the lack of sensitivity (low signal/noise) to detect low amount of proteins typically found in
single cells.
The use of electrophoresis or chromatography can greatly aid separations and
measurements of expressed proteins. Conventional scale separations such as HPLC or slab
gel electrophoresis are impractical for single cells because of their inability to process minute
amounts of proteins. Capillary electrophoresis and microfluidic chips are more promising
tools to separate and analyze proteome of a single cell (Hu et al., 2003). However, separation
based methods are not routinely used in analysis of single cells because they do not have the
peak capacity to resolve thousands of proteins in a cell and most of the detection methods
used do not have the ability to detect low-abundant proteins (Wu and Singh, 2012).
SPIRULINA AS SCP
Spirulina contains unusually high amounts of protein, between 55 and 70 percent by dry
weight, depending upon the source. It is a complete protein, containing all essential amino
acids, though with reduced amounts of methionine, cystine, and lysine, as compared to
standard proteins such as that from meat, eggs, or milk. It is, however, superior to all standard
plant protein, such as that from legumes (Phang et al., 2000). Spirulina is, like most
cyanobacteria, an obligate photoautotroph, i.e., it cannot grow in the dark on media
containing organic carbon compounds. It reduces carbon dioxide in the light and assimilates
mainly nitrates. The main assimilation product of spirulina photosynthesis is glycogen.
Spirulina is produced in at least 22 countries: Benin, Brazil, Burkina Faso, Chad, Chile,
China, Costa Rica, Côte d‘Ivoire, Cuba, Ecuador, France, India, Madagascar, Mexico,
Myanmar, Peru, Israel, Spain, Thailand, Togo, United States of America and Viet Nam.
Shimamatsu (2004) has reported that the total industrial production of Spirulina is about 3000
tonnes per year. Eight major environmental factors influencing the productivity of spirulina
are luminosity, temperature, inoculation size, stirring speed, dissolved solids, pH, water
quality and nutrients (Ciferri, 1983). The two influential characteristics of light for growth of
spirulina are average irradiance and specific light energy utilization rate (Zeng et al., 2001).
The effect of the rate of mixing on productivity of algal mass in relation to photon flux
density and algal concentration was quantitatively evaluated in cultures of Spirulina platensis
grown in a flat-plate photobioreactor. The higher the light intensity, the higher optimal culture
density, highest algal concentrations and productivity of biomass were obtained. But too high
a rate of mixing resulted in cell damage and reduced output rate (Hu and Richmond, 1996).
The culture of spirulina is practiced in different media, especially inorganic and
decomposed organic nutrients. Sanchez-Luna et al. (2004) found that the operation mode of
using urea intermittently would be preferable to reduce the production costs of Spirulina
platensis in large-scale facilities. In Bangladesh, spirulina is cultured in different agro-
industrial wastes such as sugar mill waste effluent, poultry industry waste, fertilizer factory
waste, urban waste and organic matter (Parvin, 2006). One of the main barriers to the small-
scale culture of spirulina is the cost and availability of inorganic nutrients.
Waste effluent from a fertilizer company in Nigeria was used for the production of
microalgal biomass and value-added biochemicals from Chlorella and Spirulina (Anaga and
Abu, 1996). Rice husk ash (RHA) and NaHCO3 were used as a source of carbon in Spirulina
culture (Akhter et al., 1996). The fermented wastewater of Thai rice noodle factories has the
potentiality as the source of nutrients for cultivation of Spirulina platensis (Vetayasuporn,
2004). Lagoon water can be used with some nutrient supplementation to grow Spirulina
platensis (Costa et al., 2004).
Clinical trials have shown that spirulina can serve as a supplementary cure for many
diseases. Spirulina capsules have proved effective in lowering blood lipid level, and in
decreasing white blood corpuscles after radiotherapy and chemotherapy (Ruan et al., 1988) as
well as improving immunological function. Phycocyanin of Spirulina platensis inhibits the
growth of human leukemia K562 cells when supplemented with diet (Liu et al., 2000). Its cell
wall consists of polysaccharide which has a digestibility of 86 percent, and could be easily
absorbed by the human body. Spirulina platensis powder is used as a health food tablet under
the brand name ―Linavina,‖ ―Lactogil‖ and ―Pirulamin‖ in Viet Nam. Spirulina platensis
contains γ-linolenic acid (GLA), which can help cure arthritis, heart disease, obesity and zinc
deficiency. Spirulina is well known to have very highly assimilable iron content and reported
to significantly reduce the blood glucose level. When the algal cells or filaments of spirulina
are transformed into powder it can provide the basis for a variety of food products, such as
soups, sauces, pasta, snack foods, instant drinks and other recipes (Henrikson, 1989).
LIMITATIONS OF SCP
SCP has high concentration of nucleic acids (6-10%) which elevates serum uric acid
levels and results in kidney stone formation. About 70 to 80% of the total nitrogen is
represented by amino acids while the rest occur in nucleic acids. This concentration of nucleic
acid is higher than other conventional protein and it is characteristics of all fast growing
organisms. High content of nucleic acids causes no problems to animals since uric acid is
converted to allantoin which is readily excreted in urine. The problem which occurs from the
consumption of protein with high nucleic acid concentration is the production of high
concentration of uric acid in the blood causing health disorders such as gout and kidney stone
(Nasseri et al., 2011). It has also been noted that the cell wall of the microorganisms may be
non digestible, there may be unacceptable color and flavors (especially in algae and yeast);
their cells should be killed before consumption. There may also be possible skin reactions
from consumption of foreign protein and gastrointestinal reactions may occur resulting in
nausea and vomiting. SCP from algae may not be suitable for human consumption because
they are rich in chlorophyll (except Spirulina), also it has low density i.e., 1-2 gm dry weight/
litre of substrate and there is lot of risk of contamination during growth. SCP from yeast and
fungi has high nucleic acid content, the filamentous fungi show slow growth rate than yeasts
and bacteria. Also, there is high contamination risk and some strains produce mycotoxins.
Therefore, they should be well screened before consumption. SCP from bacteria has also been
found to be associated with pitfalls which include: high ribonucleic acid content, high risk of
contamination during the production process and problematic recovering of the cells. Alkaline
extraction of microbial biomass at elevated temperature results in high protein yield with low
nucleic acid contaminations. However, this causes the production of potentially toxic
compounds like lysinoalanine which is reported to reduce digestion and induce kidney
changes in rats (Damodaran and Kinsella, 1983).
FUTURE PERSPECTIVES
SCP has a proven record as a source of protein which may be obtained with large
productivities in compact installations. The use of pesticides in foods of plant origin or the
appearance of carcinogens such as nitrosamines has already been reported in the past. SCP
products for animal and human nutrition are safer in this respect, since the components from
which they are produced are easily controlled, and their genetic background is well known.
The research on the production of microorganisms for animal and human consumption has
found various applications at the industrial levels. This research should also incorporate the
development of recombinant strains from nonconventional GRAS (Generally Regarded as a
Safe) yeasts and fungi. The potential of these techniques to improve the characteristics of
foods is considerable and holds a positive prospect.
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Chapter 12
BAKER’S AND BREWER’S YEAST: PRODUCTION,

APPLICATIONS AND GENETIC MANIPULATIONS
Priya Katyal, Parampal Sahota,

Gurvinder Singh Kocher and Jatinder Kaur
Department of Microbiology, Punjab Agricultural University,
Ludhiana, India
ABSTRACT
Saccharomyces cerevisiae is the best genetically characterized yeast. Its genome is
fully sequenced, procedure for its genetic manipulations are easily available and is
generally regarded as safe (GRAS) by Food and Drug Administration (FDA). With
regard to brewer‘s yeast (S. cerevisiae and S. carlsbergensis) a distinction is made
between top and bottom fermenting yeast. Wine yeast and baker‘s yeast are mostly
referred to as S. cerevisiae and S. ellipsoides, respectively. Modern industries require
very large amount of selected yeasts to obtain high quality reproducible product. As per a
latest report 0.4 million metric tonnes of yeast biomass is being produced worldwide
including .2 million tonnes of baker‘s yeast alone. Yeast biomass propagation of wine‘s,
distiller‘s and brewer‘s yeast is usually similar to baker‘s yeast propagation involving a
multistage fermentation that starts in a batch mode followed by fed-batch mode. The
emergence of advanced molecular biology techniques allowed a precise characterization
of different yeast and their genetic manipulations for improvement of strains as per
industrial requirements. Most of the genetically modified (GM) strains lack the resistance
marker and stability, but have been tested only upto pilot-scale. So far only two have
received approval for commercial use. The first is baker‘s yeast derepressed for maltase
and maltose permease for efficient dough-rising and second is brewer‘s yeast expressing
STA2 gene for glucoamylase. None of these GM yeast is being used commercially on a
large scale. Another GM yeast (ML01) is used to improve the taste and colour stability of
wine as well as to avoid the production of undesirable compounds (histamines). In
Canada, a GM yeast has been developed for reducing the presence of a ethylcarbamate (a

Corresponding Author: Department of Microbiology, Punjab Agricultural University, Ludhiana, Pb-141001, India,
Email- drpkatyal@pau.edu; Tel: +91-9888507787.
284 Priya Katyal, Parampal Sahota, Gurvinder Singh Kocher et al.
carcinogen - produced during fermentation). Public acceptance to GM strains is still

lacking. Efforts have to be made to increase awareness to advantages of recombinant
strains. Attempts are to be made to expand the knowledge and data obtained from
laboratory strains to industrial strains as genome of industrial strain are exposed to many
environmental stresses and their genome is more complex. Exploration of metabolic and
genetic control of gene expression for industrially relevant traits is the need of hour.
These complex traits which are under multigenic control can be studied using functional
genomic approach.
Keywords: Saccharomyces cerevisiae, baker‘s yeast, brewer‘s yeast, yeast biomass, alcohol,
genetic modification, applications
INTRODUCTION
Yeasts have been known to humans since ages, as they have been used in fermentation
processes like the production of alcoholic beverages and bread leavening. The industrial
production and commercial use of yeasts started at the end of the 19th century after their
identification and isolation by Pasteur. The word ―yeast‖ has been derived from the Sanskrit
word ‗yas‘ means ―to boil.‖ It has a remarkable ability to convert sugar or starch into alcohol
and carbon dioxide- the two main end-products required by brewing and baking industry.
Today, the knowledge of yeast biology and advance technology allows the industrial
production of yeast strains with specific properties to meet the requirements of baking and
brewing industry. Food grade yeasts are also used as sources of high nutritional value
proteins, enzymes and vitamins. These yeasts are having wide applications in the health food
industry as nutritional supplements, as food additives, conditioners and flavouring agents and
are essential component of different microbiological media and livestock feed. Yeasts are
included in starter cultures, for the production of specific types of fermented foods like
cheese, bread, sourdoughs, fermented meat and vegetable products, vinegar, etc. (Bekatorou
et al., 2006).
Popularity of Saccharomyces cerevisiae in basic and applied research is mainly due to its
classification as GRAS (generally regarded as safe) by the U.S. Food and Drug
Administration (FDA). S. cerevisiae has been used as a model organism to study various
biological processes such as aging (Bitterman et al., 2003), mRNA transport (Muller et al.,
2007), the cell cycle (Breeden, 2003), studying prions (Chernoff, 2004), basic and applied
virus research (Galao et al., 2007), ecotoxicology (Teixeira et al., 2007), as a tool for drug
research (Menacho-Marquez and Murguia, 2007) and for studying human diseases like cancer
(Spradling et al., 2006; Yuen et al., 2005; Sanchez and Waterston, 2006).
Metabolic Properties of Yeasts
Yeasts are facultative anaerobes. Under aerobic conditions, they convert sugars to CO2,
energy and biomass, whereas, during anaerobic conditions, as in alcoholic fermentation,
sugars are converted to intermediate by-products such as ethanol, glycerol and CO2.
Therefore, in yeast propagation, the supply of air is necessary for optimum biomass
Baker‘s and Brewer‘s Yeast 285
production. Glucose acts as a main source of carbon and energy which is metabolized to
pyruvate via Glycolysis. Further energy production from pyruvate: vary from respiration to
fermentation depending upon the concentration of glucose and oxygen. In Respiration,
pyruvate is decarboxylated in the mitochondrion to acetyl-CoA which is completely oxidized
via Krebs cycle to CO2, energy and intermediates to promote yeast growth. Whereas, glucose
is slowly utilized to produce the energy required just to keep the yeast cell alive during the
fermentation, in which the sugars are not completely oxidized to CO2 and ethanol. Similarly,
a high glucose concentrations leads to Catabolite Repression, during which gene expression
and synthesis of respiratory enzymes are repressed and yeast switch on to fermentative mode.
The tendency of yeast to grow via respiration when large amounts of oxygen are present is
known as the Pasteur Effect. Whereas, the tendency of yeast to grow via fermentation in the
presence of high levels of sugar is known as the Crabtree effect. On industrial scale yeast
biomass production, Crabtree effect may leads to incomplete fermentation, development of
off-flavours and undesirable by-products as well as loss of biomass yield and yeast vitality
(Feldman, 2005; Versetrepen et al., 2004; Ringbom et al., 1996). In baking industries, the
release of CO2 gas produces the leavening action in yeast products. The alcohol evaporates
completely during and immediately after baking. Dough raising ability of baker‘s yeast is
sensitive to temperature. Though yeast in dough is active between 0 and 55°C, but optimum
temperature for fermentation is 35°C and rate of growth is significantly reduced at
temperatures less than 20°C and over 40°C. In frozen dough, yeast can survive for a few
weeks at temperatures as low as -20°C, but it gradually loses its fermentation capacity.
Frozen doughs require yeast that retains its activity through extended frozen storage
conditions. Formation of ice crystals in the frozen dough will disrupt yeast cell walls and
reduces the amount of available water thereby increasing the solute concentration. Therefore,
yeast with a high osmotic tolerance is required for frozen dough.
Depending upon the type of process used and activity of selected yeast strain different
amount of yeast inoculums can be used. In traditional bread making, doughs are allowed to
ferment twice resulting in an increase in dough volume and development of characteristic
yeast flavours. But in advanced technologies involving ―No-time doughs‖ these are only
proofed once. The finished products of no-time dough are similar in quality to those of
traditional doughs, but increased levels of dough conditioners and yeast are required. During
bread making, fermentation by yeast generates a number of compounds including organic
acids, alcohols, aldehydes, esters, and ketones. Some of these compounds act as dough
conditioners and increase dough extensibility by relaxing the gluten depending upon the time
and pH. Addition of excess of salt for flavour, may sometimes limit yeast growth. Extra
nitrogen, as part of a dough improver, can be added to ensure optimum yeast growth and to
produce the necessary carbon dioxide to yield ―oven spring‖ i.e., sudden expansion of dough
by about one-third its original volume during the early baking stages. Table 1 compares the
leavening action of yeast and chemical leavening agent (baking powder).
Yeasts can metabolize various carbon substrates but mainly utilize sugars such as
glucose, sucrose and maltose. Sucrose is metabolized after hydrolysis into glucose and
fructose by the extracellular enzyme invertase. Maltose is transferred in the cell by maltose
permease, and metabolized after hydrolysis into two molecules of glucose by maltase. Some
yeasts can utilize a number of unconventional carbon sources, such as biopolymers, pentoses,
alcohols, polyols, hydrocarbons, fatty acids and organic acids. For example, lactose can be
utilized by yeasts that have the enzyme β-galactosidase. The yeasts of genera Kluyveromyces
and Candida can be efficiently grown in whey for yielding biomass. Biopolymers like starch,
cellulose, hemicellulose and pectin can be metabolized by some yeast directly, or after
hydrolysis by non-yeast enzymes. Some yeast species of Hansenula, Pichia, Candida and
Torulopsis are also able to grow on methanol as sole energy and carbon source. Finally,
elements like N, P, S, Fe, Cu, Zn and Mn are essential to all yeasts and are usually added to
the growth media. Most yeast are capable of assimilating directly ammonium ions and urea,
while very few species have the ability to utilize nitrates as nitrogen source. Phosphorus and
sulphur are usually assimilated in the form of inorganic phosphates and sulphates,
respectively.
Table 1. Leavening action of yeast and baking powder

(Reed and Nagodarithana, 1991)
Leavening Action Yeast Baking powder*

Leavener based on Flour 2.5% 6.0%
Leavener based on dough weight 1.47% 3.42%
CO2 evolved per g leavener 0.5g ** 0.15g ***
CO2 evolved per 100g dough 0.735g ** 0.513g ***
CO2 evolved per 100g dough 350 ml ** ***
*A double-acting baking powder containing 30% NaHCO3.
** CO2 evolution per hour.
*** Total CO2 evolution.
Baker’s Yeast
Increasing consumption of bread as a staple food rather than just a breakfast item and the
industry registering growth rate of around 7.5%, indicate good prospects for Baker‘s yeast in
the domestic market. Increasing number of nuclear families and working women in India
particularly in urban and semi urban areas and changing food consumption habits and pattern
of people, will drive the growth of Bakery industry and in turn the growth of Baker‘s yeast
demand. Baking is a millennia old process, and bakery products range in complexity from the
simple ingredients of a plain pastry to the numerous components of a cake. The term baking
applies not only to the production of bread, but to all food products in which flour is the basic
material and to which heat is applied directly by radiation from the walls and/or top and
bottom of an oven or heating appliance. More particularly, baking includes the production of
such items as bread, cake, pastry, biscuits, crackers, cookies, and pies where flour is the
essential and principal ingredient for the base product; baking also includes the toppings,
frostings, fillings, and so on that finish the baked product. Although many differences exist
between bakery products, they share two important issues of baking technology, baking
materials/ingredients and baking techniques (Lai and Lin, 2006).
Bakers yeast production depends on the ability of Saccharomyces cerevisiae to assimilate
inorganic nitrogen and grow via respiration instead of fermentation. Assimilating inorganic
nitrogen is important because it allows baker‘s yeast to be grown on molasses as an
economical source of sugar and other nutrients. Suitable baker‘s yeast strain should possess
high specific growth rate (m) and biomass yield (Yx/s), wide substrate utilization with high
affinity for substrate, simple nutritional requirements, ability to develop high cell density,
genetic stability, ability for genetic manipulations. Sugarcane or beet molasses is the primary
raw material for Baker‘s yeast production as it supplies the required sugar and energy with
needed nitrogen for the growth of yeast. It is relatively low in nitrogen and has to be
supplemented so that yeast can synthesize the protein and the other cell constituents they need
for growth. Because Saccharomyces cerevisiae can use inorganic nitrogen, a combination of
ammonia and ammonium salts are added to provide economical sources of nitrogen and pH
control. Growing via respiration is important because it is about eighteen times as efficient as
fermentation at converting sugar into yeast. The combination of Pasteur and Crabtree effects
in Saccharomyces cerevisiae is the reason commercial baker‘s yeast fermentations use high
aeration and incremental feeding to maintain high oxygen and low sugar levels throughout the
process. Reed and Nagodarithana (1991) gave the gross macromolecular composition of
bakers‘ yeast as shown in Table 2.
Table 2. Macromolecular composition of bakers’ yeast

(Reed and Nagodarithana, 1991)
Component Percentage
Moisture content 2-5
Protein 42-46
RNA and DNA 6-8
Minerals 7-8
Total Lipids 4-5
Carbohydrates 30-37
The European and Asian regions produced 51 million tons of bakery items, valued at US
$ 107 billion, in the year 2004-2005. As per the emerging global trend China is presently one
of the most promising markets for Baker‘s yeast, as its demand is continuously increasing
with the rise in population and changing demand of Bakery products. Commercial fresh
baker‘s yeast includes products in liquid, creamy or compressed forms and active dry yeast.
Compressed baker‘s yeast is the most commonly used product, consisting of only one yeast
species, S. cerevisiae. Special strains of S. cerevisiae can be used for the production of dry
yeast products, like active dry yeast (ADY) or instant dry yeast (IDY).
Instant dry yeast is produced from a faster-reacting yeast strain than that used for ADY.
The main difference between ADY and IDY is that ADY has to be dissolved in warm water
before usage, but IDY does not.
Unlike active dry yeast, inactive dry yeast is a product without leavening properties, used
for the conditioning of dough properties in baking or the development of characteristic
flavour. Baker‘s yeast market in developing countries is touching new highs with increasing
demand for processed foods and a consistent growth in Bakery items production. India‘s
bakery production in the year 2004-2005 registered a growth rate of around 20% producing
approximately 50 Lac tons of bakery items; valued at INR 69 billion. Of the total bakery
production, the bread production alone was estimated at around 27 Lac tons, indicating a
growth rate of 7.5%. India‘s estimated per capita consumption of bread is 2 Kgs per annum,
as compared to other European and developing countries of Asia it is far below the lowest. As
per Government of India trade statistics, the export for Baker‘s yeast in the fiscal year 2006-
2007(Apr-Jun), is 329.85 tons, valued at INR 28.44 million, with major export to Sri Lanka
followed by Saudi Arabia, Lebanon, Nepal, Mali, Egypt and Iran, while imports for the years
(2004-2007) stand cumulatively at a minimal 39.18 tons valued at INR 2.556 million.
Saf Yeast Co. Pvt. Ltd a Mumbai based unit with annual sales below US $1 million is
into production of baker‘s yeast. Blue Bird India Pvt. Ltd, Mumbai is marketing Dry yeast in
retail packing for home use. Baker‘s yeast is currently manufactured in the United States at 13
plants owned by 6 major companies producing two main types of baker‘s yeast, compressed
(cream) yeast and dry yeast. The total U. S. production of baker‘s yeast in 1989 was 223,5
megagrams (Mg) (245,000 tons). Of the total production, approximately 85 percent of the
yeast is compressed (cream) yeast, and the remaining 15 percent is dry yeast. Compressed
yeast is sold mainly to wholesale bakeries and dry yeast is sold mainly to consumers for home
baking needs. Compressed and dry yeasts are produced in a similar manner, but dry yeasts are
developed from a different yeast strain and are dried after processing.
It is propagated under controlled conditions in order to get consistent food quality
(Pattison and von Holy, 2 1). Baker‘s yeast production is one of the oldest technologies and
is considered as a ―ripened technology.‖ At the turn of the 2 th century, the baker‘s yeast
industry had developed from distilleries where high alcohol yield gave little yeast biomass to
full fledged baker‘s yeast manufacturing units. During the golden age of modern baker‘s yeast
manufacturing, from 1910 to 1940, much effort was put in to find the best and cheapest
growth media available (Gélinas, 2012). In addition, much interest was given to the control
of both infection and sugar concentration in such sugar-rich growth media to increase
biomass yields and reduce costs.
The objective of baker‘s yeast manufacturing is to harvest as fast as possible the highest
amount of living cell mass at the lowest cost. Nowadays, yeast biomass propagation of wine,
distiller‘s and brewer‘s yeasts are usually produced in baker‘s yeast plants. The procedure is
designed as a multistage-based fermentation, previously defined for the production of baker´s
yeast (G mez-Pastor et al., 2 11). So production of only baker‘s yeast is discussed here in
detail. First, fermentation tanks must be seeded with the strongest and purest microbial
starters, otherwise unwanted microorganisms will be harvested instead; both the gassing
power and the keeping properties of the concentrated yeast biomass will be reduced. Second,
such microorganisms must be fed under thoroughly controlled conditions to optimize yeast
biomass and gassing power, enough to raise dough, otherwise bakers will experience
variations in bread volume. Major steps of baker‘s yeast manufacturing, including
fermentation control, have been described in Gélinas (2006).
Raw Materials - Cane molasses and beet molasses are the principal carbon sources to
promote yeast growth. Molasses contains 45 to 55 weight percent fermentable sugars, in the
forms of sucrose, glucose, and fructose. The amount and type of cane and beet molasses used
depend on the availability, costs, and the presence of inhibitors and toxins. Usually, a blend
consisting of both cane and beet molasses is used in the fermentations. Once the molasses
mixture is blended, the pH is adjusted to between 4.5 and 5.0 because an alkaline mixture
promotes bacteria growth. Bacteria growth occurs under the same conditions as yeast growth,
making pH monitoring very important. The molasses mixture is clarified to remove any
sludge and is then sterilized with high-pressure steam. After sterilization, it is diluted with
water and held in holding tanks until it is needed for the fermentation process. A variety of
essential nutrients and vitamins is also required in yeast production. The nutrient and mineral
requirements include nitrogen, potassium, phosphate, magnesium, and calcium, with traces of
iron, zinc, copper, manganese, and molybdenum. Normally, nitrogen is supplied by adding
ammonium salts, aqueous ammonia, or anhydrous ammonia to the feedstock. Phosphates and
magnesium are added, in the form of phosphoric acid or phosphate salts and magnesium salts.
Vitamins are also required for yeast growth (biotin, inositol, pantothenic acid, and thiamine).
Fermentation – Baker‘s yeast begins as a pure culture of the desired strain, which is
inoculated from a small vial into a sterile flask of broth. From the flask it is transferred into a
larger vessel, then through several fermentation stages of increasing volume (Di Serio et al.,
2001). An efficient transformation of sugar in yeast protein requires that anaerobic
metabolites production such as ethanol and acetaldehyde (Bauer et al., 1999) is minimized,
i.e., that sugar metabolism is deviated to the oxidative pathway to achieving maximum ATP
energy yield and biomass formation (Van Hoek et al., 1998). Despite this, biomass formation
can be achieved by different metabolic pathways (Frick and Whitmann, 2005) and biomass
yield on substrate can reach values up to 50% in pure oxidative growth (Akinyemi et al.,
2005). To overcome the constraints on yeast biomass production two important variables are
of major importance: oxygen transfer rate and glucose concentration in the broth. In
heterogeneous gas-liquid reactions, e.g., in aerobic fermentation, the liquid phase controls
mass transfer processes due to the relative insolubility of gases. This limitation is minimized
by the use of bubble column reactors due to its good oxygen transfer with low cost operation
when compared to stirred tank reactors (Kantarcia et al., 2005). The minimum sugar
concentration in broth can be reached by the use of a fed-batch process. This process concept
is the current one in industrial scale and renders good biomass yields when appropriate
process control strategy is used.
Traditionally, in industrial production, molasses or another feedstock feeding follows a
strategy built on the basis of factory historic data and so it is peculiar to a determined strain
and other process conditions. Because of environmental policy, some industries are investing
in new strategies of process control to avoid emission of toxic pollutants (EPA, 1992). In
scientific literature, many articles can be found about baker‘s yeast production dealing with
yeast growth modeling and aiming at different goals such as productivity, yield and yeast
quality as well as new and robust online sensors (Rendez-Gil et al., 1999; Jones and Kompala,
1999; Soley, 2005). Evolution of fermentation control for baker‘s yeast production based on
patenting activity has been review by Gelinas (2014). The main objective of such
fermentation control was to improve yield and reduce the cost of commercial baker‘s yeast.
Moreover, fermentation products such as ethanol and, in particular, acetaldehyde are toxic.
This problem becomes especially relevant during cultivation at high biomass densities. Yeast
biomass production in a bubble column reactor based on a spreadsheet simulator suitable for
industrial application has also been investigated (Vieira et al., 2013). The larger-scale
fermentations take place in 25,000 to 50,000 gallon fermentors that are equipped for aeration,
cooling, incremental molasses feeding, pH control, and anti-foam addition. Each fermentation
step requires about a day, so that at the end of a week more than 500,000 pounds can be
produced from a single vial.
The initial stage of yeast growth takes place in the laboratory. A portion of the pure yeast
culture is mixed with molasses malt in a sterilized flask, and the yeast is allowed to grow for
2 to 4 days. The entire contents of this flask are used to inoculate the first fermentor in the
pure culture stage. Pure culture fermentations are batch fermentations, where the yeast is
allowed to grow for 13 to 24 hours. Typically, 1 to 2 fermentors are used in this stage of the
process. The pure culture fermentations are basically a continuation of the flask fermentation,
except that they have provisions for sterile aeration and aseptic transfer to the next stage.
Following the pure culture fermentations, the yeast mixture is transferred to an intermediate
fermentor that is either batch or fed-batch. The next fermentation stage is a stock
fermentation. The contents from the intermediate fermentor are pumped into the stock
fermentor, which is equipped for incremental feeding with good aeration. This stage is called
stock fermentation, because after fermentation is complete, the yeast is separated from the
bulk of the fermentor liquid by centrifuging, which produces a stock, or pitch, of yeast for the
next stage. The next stage, pitch fermentation, also produces a stock, or pitch, of yeast.
Aeration is vigorous, and molasses and other nutrients are fed incrementally. The liquor from
this fermentor is usually divided into several parts for pitching the final trade fermentations.
Alternately, the yeast may be separated by centrifuging and stored for several days before its
use in the final trade fermentations. The final trade fermentation has the highest degree of
aeration, and molasses and other nutrients are fed incrementally. Large air supplies are
required during the final trade fermentations, so these vessels are often started in a staggered
fashion to reduce the size of the air compressors. The duration of the final fermentation stages
ranges from 11 to 15 hours. After all of the required molasses has been fed into the fermentor,
the liquid is aerated for an additional 0.5 to 1.5 hours to permit further maturing of the yeast,
making it more stable for refrigerated storage. The amount of yeast growth in the main
fermentation stages described above increases with each stage. Yeast growth is typically 120
kilograms (270 pounds) in the intermediate fermentor, 420 kilograms (930 pounds) in the
stock fermentor, 2,500 kilograms (5,500 pounds) in the pitch fermentor, and 15,000 to
100,000 kilograms (33,000 to 220,000 pounds) in the trade fermentor. The sequence of the
main fermentation stages varies among manufacturers but a standard representation is shown
in Figure 1.
Processing - Once an optimum quantity of yeast has been grown, the yeast cells are
recovered from the final trade fermentor by centrifugal yeast separators. The centrifuged
yeast solids are further concentrated by a filter press or rotary vacuum filter. A filter press
forms a filter cake containing 27 to 32 percent solids. A rotary vacuum filter forms cakes
containing approximately 33 percent solids. This filter cake is then blended in mixers with
small amounts of water, emulsifiers, and cutting oils to form the end product. The final
packaging steps, as described below, vary depending on the type of yeast product. In
compressed yeast production (SCC 3-02-035-XX), emulsifiers are added to give the yeast a
white, creamy appearance and to inhibit water spotting of the yeast cakes. A small amount of
oil, usually soybean or cottonseed oil, is added to help extrude the yeast through nozzles to
form continuous ribbons of yeast cake. The ribbons are cut, and the yeast cakes are wrapped
and cooled to below 8°C (46°F), at which time they are ready for shipment in refrigerated
trucks.
Yeast broth from the fermentor at about 5 percent solids is concentrated in a centrifuge to
about 18 percent solids and washed with water. Cream yeast is simply this liquid yeast that is
cooled and delivered in bulk to the bakery. To make compressed (granular and cake) yeast,
cream yeast is passed through a filter, which removes water and increases the solids
concentration to about 30 percent. When a rotary vacuum filter is used, the cream is first
treated with salt, then sucked onto a thin layer of starch, rinsed with cold water to remove the
salt, and scraped off the starch. After filtering, small amounts of emulsifiers or oils are added
to assist in the extrusion and cutting of the yeast and to improve its appearance. Granular
yeast is then crumbled and packed in bags.
Figure 1. Schematic representation of fermentation steps involved in baker‘s yeast production

(Umacob, 2014).
Finished product - Fresh baker‘s yeast consists of approximately 30–33% of dry

materials, 6.5–9.3% of nitrogen, 40.6–58.0% of proteins, 35.0–45.0% of carbohydrates, 4.0–
6.0% of lipids, 5.0–7.5% of minerals and various amounts of vitamins, depending on its type
and growth conditions. The solids content can vary from about 27 to 33 percent, depending
on how it is filtered. The higher the yeast solids, the higher the activity. The protein level can
vary from about 40 to 60 percent and the carbohydrate level from 30 to 45 percent, depending
mostly on how fast the yeast is grown. Higher growth rates give higher protein, higher
activity, lower carbohydrate, and lower stability. Lower growth rates give lower protein,
lower activity, higher carbohydrate and higher stability.
Purity – Baker‘s yeast is grown under sanitary (not sterile) conditions to ensure that the
strain remains true to type and that no harmful organisms are present. Better sanitation
procedures, fewer fermentation stages, and lower fermentation pH give lower contamination
levels. All products should be free of Salmonella, Listeria, and E. coli, with low levels of
coliforms and other sanitation-indicator organisms. Lactic acid bacteria and ―wild‖ (non-
Saccharomyces cerevisiae) yeast are always present because these are common in the
environment and grow under the same conditions as baker‘s yeast. Normal levels of these
other microorganisms are not a problem because they occur naturally in flour, and the baker‘s
yeast cells greatly outnumber them.
Appearance - Compressed yeast can range in color from dark brown to nearly white, and
in texture from friable (easy to crumble) to gummy. Appearance is affected by yeast strain,
molasses source, fermentation conditions, processing aids, moisture level, and age. Low
fermentation pH gives higher purity but darker yeast. Emulsifiers give lighter color and
frozen storage gives mushier texture, but neither affects performance. High moisture makes
yeast darker but affects performance only to the extent that yeast solids are reduced. Storage
time, temperature, and oxygen make yeast darker and gummier and reduce its performance.
Old or damaged yeast frequently appears dark and gummy, but because there are so many
other factors involved, appearance alone is not a reliable indicator of yeast quality.
Applications
Baker‘s yeast has a long history of safe use in food (Tucker and Woods, 1995). Yeast cell
preparations have been commercialized as food supplements and medicines because of their
nutritional characteristics (Moyad 2 7, 2 8; Halasz and Lasztity, 1991). Baker‘s yeast, like
baking powder and baking soda, is used to leaven baked goods (breads, Danish pastries,
brioche, and croissants).
The principle use of Baker‘s yeast is as an essential bakery ingredient- for causing
fermentation in the dough used in making bakery items. This process helps making soft and
fluffy bakery items like variety of breads, bread rolls, pizza base, cracker biscuits, sweet
breads and burger buns etc. Within the past few years yeast extracts have become important
components in savoury flavours as well as in fermentation media. The growth of Baker‘s
yeast market is directly linked to the increasing trend of processed and fast food consumption,
especially bakery items.
Brewer’s Yeast
Pure brewer‘s yeast cultures are produced industrially to supply the brewing industry.
Lodder (1970) describes the cells of S. cerevisiae as being spheroidal, subglobose, ovoid,
ellipsoidal or cylindrical to elongate, occurring singly, in pairs, occasionally in short chains or
clusters. Cells may be grouped into three classes on the basis of size. A large type, 4.5-10.5 x
7.0-21.0 μm; a small-cell type falling within the range 2.5-7.0 x 4.5-11.0 μm and an
intermediate group with cells measuring 3.5-8.0 x 5.0-11.0 μm. Some yeast may form
filaments and brewer‘s yeast fit into any of these categories; however, they tend to be quite
large cells, a consequence of polyploidy (Boulton and Quain, 2001). The growth conditions
also influence cell size. Brewer‘s yeast during fermentation can accumulate high
concentrations of glycogen. Apart from the ‗structural‘ glycogen that is apparently associated
with the periplasm, the pool of glycogen that is used as a storage carbohydrate is located in
the cytoplasm. Yeast species are identified via an array of tests including fermentation and
assimilation of various carbon sources, growth on a selection of nitrogen sources and growth
at different temperatures and osmotic pressures. The number of tests required to achieve
identification hampers the use of such ‗keys‘ (Payne et al., 1998). Of the numerous
commercial approaches available for the identification of yeasts (Deak and Beuchat, 1996),
the bioMerieux API (Analytical Profile Index) series is the most widely used. The molecular
composition of dried wine yeast is given in Table 3 (Rosen, 1989).
Usually two Saccharomyces species are used: S. uvarum, formerly known as S.
carlsbergensis, which is used for the production of several types of beer with bottom
fermentation (lager yeasts), and S. cerevisiae, which conducts top fermentation (ale yeasts).
Due to recent reclassification both ale and lager yeast strains are considered S. cerevisiae
species.
Table 3. Component Percentage of dry weight Molecular composition

of dried wine yeast (Rosen, 1989)
Carbon 48.2
Hydrogen 6.5
Oxygen 33.8
Nitrogen 6.0
Phosphorus 1.0
Magnesium 0.1
Calcium 0.04
Potassium 2.1
Sulphur 0.01
Iron 0.005
Top-fermenting yeasts are used for the production of ales, porters, stouts, wheat beers,
etc., and bottom-fermenting yeasts are used for lager beers like Pilsners, Bocks, American
malt liquors, etc. (Goldammer, 2000). Top-fermenters are unable to ferment some types of
sugars, and the resulting beer is sweeter and ―fruitier‖. Inactive brewer‘s yeast preparations,
made from inactive yeast and other special ingredients, are produced commercially to be used
as nutrients to reinitiate or avoid sluggish and stuck fermentations.
Nutritional brewer‘s yeast - Commercial, nutritional brewer‘s yeast is inactive yeast
(dead yeast cells with no leavening power), remaining after the brewing process. Brewer‘s
yeast is produced by cultivation of S. cerevisiae on malted barley, separated after the wort
fermentation, debittered and dried. It is an excellent source of protein and it is used as a
nutrient supplement rich in B vitamins. Brewer‘s yeast products are usually found in the form
of powders, flakes or tablets, or in liquid form. Liquid yeast contains enzymatically digested
yeast for better digestion, absorption and utilization. Brewer‘s yeast should not be confused
with (brewer‘s type yeasts), which are pure yeasts usually grown on enriched cane or beet
molasses under controlled production conditions, and are not by-products of the brewing
process. Brewer‘s yeast is an excellent source of B vitamins, Ca, P, K, Mg, Cu, Fe, Zn, Mn
and Cr and has been studied extensively for its medicinal properties. It is often used for the
treatment of diabetes (regulation of insulin levels), loss of appetite, chronic acne, diarrhoea,
etc. (Sinai et al., 1974; McCarty, 1984; Bahijiri et al., 2000). It is also recommended as a
dietary supplement for healthy hair and nails. Nevertheless, according to some, brewer‘s yeast
is suspected of causing various problems, like chronic fatigue, memory disorders,
immunodeficiency, irritable bowel syndrome, allergies, etc., mainly due to the presence of
yeast antigens and high amounts of Cr and salicylates (Baldo and Baker, 1988; Smith, 1996).
Wine yeasts - A wide variety of pure yeast cultures, mainly Saccharomyces (S.
cerevisiae, S. bayanus, S. uvarum, S. oviformis, S. carlsbergensis, S. chevalieri, S. diastaticus,
S. fructuum, S. pasteurianus, S. sake, S. vini, etc.) are produced industrially for the use in
induced wine fermentations, according to the industrial demands for fermentation efficiency
and productivity. The suitable type of yeast is selected with respect to the geographical area,
climate, type of grapes and desirable organoleptic quality of the product (taste, aroma, colour,
tannin and glycerol content, etc.). Pure yeast cultures are also used to conduct specific types
of fermentations, like bottle fermentation of Champagne and sparkling wines, or to treat stuck
and sluggish fermentations. Seasonal wine production requires the development of highly
stable and Active dry yeast products. After biomass propagation by a process similar to that
of baker‘s yeast production, wine yeast cells are dehydrated to obtain ADY (Gonzalez et al.,
2005). Yeast cells are separated from media and are subjected to washing separations for
removal of nonyeast solids, because their presence can affect the proper rehydration process
of ADY. The separated product is in the form of a cream with 22% yeast solids, which is
further dehydrated to 30-35% solids and mixed with emulsifiers prior to its extrusion into
yeast strands. The yeast cake is extruded through a perforated plate, while particles are loaded
into the dryer and dehydrated to obtain a product with very low residual moisture (G mez-
Pastor et al., 2011). Finally, ADY is vacuum-packaged or placed in an inert atmosphere
before marketing. The drying temperature and rate can be critical for yeast resistance to
dehydration and rehydration (Laroche and Gervais, 2003; Simonin et al., 2007; Dupont et al.,
2010). Moreover, biomass propagation conditions have a major influence on yeast resistance
to dehydration-rehydration (Trofimova et al., 2010).
Distiller‘s yeasts - Distiller‘s yeasts are used for the industrial production of alcohol and
spirits (brandy, whiskey, rum, tequila, etc.). They are usually isolated from industrial
fermentations of fruit pulps and beet or sugar cane molasses. Their selection depends on the
desired product properties, including flavour, alcohol yield, productivity, and other
technological features. Generally, distiller‘s yeasts must exhibit low foam formation, high
stress-tolerance and high alcohol yields. They must also form controlled amounts of ethyl
esters, aldehydes, fatty acids and higher alcohols, which is an important prerequisite for the
production of fine quality distillation products. Distiller‘s yeasts must be able to ferment
various substrates, such as corn, barley, wheat, potato, etc. after hydrolysation in fermentable
sugars. They must be able to conduct fast fermentations with high productivities and low
production costs, and tolerate high temperatures, osmotic pressures and alcohol
concentrations (de Becze, 1964; Laube et al., 1987; Ibragimova et al., 1995).
Probiotic yeasts - Probiotics are live microbial feed supplements which beneficially affect
the host animal by improving its intestinal microbial balance, or by a wider definition
probiotics are microbial cell preparations or components of microbial cells that have a
beneficial effect on the health and well-being of the host (Salminen et al., 1999). Probiotic
properties of yeasts, like S. cerevisiae, have been reported and displayed as the ability to
survive through the gastrointestinal (GI) tract and interact antagonistically with GI pathogens
such as Escherichia coli, Shigella and Salmonella. Specifically, S. boulardii, a thermophylic,
non-pathogenic yeast, has been used for more than 50 years as a livestock feed probiotic
supplement as well as therapeutic agent for the treatment of a variety of gut disorders like
diarrhoea. This yeast is safe, it is resistant to antibiotics, achieves high cell numbers in the
intestine in short time, does not permanently colonize the intestine and is quickly cleared after
the cease of administration. Its probiotic effects are also enhanced by its ability to produce
polyamines, which are compounds that strongly affect cell growth and differentiation
(Lourens-Hattingh and Viljoen, 2001; Costalos et al., 2003). S. boulardii is widely used and is
available in various commercial formulations. Other yeasts allowed and commonly used in
animal feeds as probiotic additives are Candida pintolopesii, C. saitoana and S. cerevisiae
(Bovill et al., 2001; Leuschner et al., 2004).
Yeast extract - Yeast extract is the product of enzymatic digestion of the yeast cell
constituents by endogenous and exogenous yeast enzymes. It is rich in peptides, amino acids,
nucleotides and vitamins; therefore it is good for use as supplement in culture media. It is also
used in pharmaceuticals, as well as flavour and taste enhancer (replacing glutamates and
nucleotides) in many canned foods. Although brewer‘s yeasts contain residual beer flavour
compounds (mainly constituents of hops), they are commonly used for commercial food
grade yeast extract production, which is destined for use as supplement in both human and
animal foods, and as flavour enhancer (Chao and Joo, 2001).
Sourdough starters - Sourdough is a mixture of flour and water, containing yeasts and
lactic acid bacteria, used as starter culture to leaven bread. The use of sourdough has a
number of important advantages over baker‘s yeast, such as the development of characteristic
flavour (Hansen and Schieberle, 2005) and texture (Meignen et al., 2001), as well as
extension of preservation time through the in situ production of antimicrobial compounds
(e.g., bacteriocins) (Messens and De Vuyst, 2002). Therefore, sourdoughs are produced at
commercial level using various combinations of yeasts and bacteria, and are used for the
conditioning of dough, improvement of preservation time and the development of breads and
baking products with special organoleptic properties (Plessas et al., 2005).
The B-complex vitamins in brewer‘s yeast include B1 (thiamine), B2 (riboflavin), B3
(niacin), B5 (pantothenic acid), B6 (pyridoxine), B9 (folic acid), and H or B7 (biotin). These
vitamins help break down carbohydrates, fats, and proteins, which provide the body with
energy. They also support the nervous system, help maintain the muscles used for digestion,
and keep skin, hair, eyes, mouth, and liver healthy. Brewer‘s yeast does not contain vitamin
B12, an essential vitamin found in meat and dairy products. Vegetarians sometimes take
brewer‘s yeast mistakenly believing that it provides B12, which can be lacking in their diet.
Some studies suggest that chromium supplements may help people with diabetes control
blood sugar levels. People with diabetes either do not produce enough insulin - a hormone
needed to change sugar, starches and other food into energy or cannot use the insulin that
their bodies make. Chromium may lower blood sugar levels as well, improving glucose
tolerance and reducing the amount of insulin needed. A few studies suggest that brewer‘s
yeast may help lower LDL (―bad‖) cholesterol levels in the blood and raise HDL (―good‖)
cholesterol levels. Although some studies suggest that chromium may help reduce body fat,
the amount of fat lost is small compared to what can be lost with exercise and a well-balanced
diet. However, brewer‘s yeast is also used as a protein supplement and energy booster, so it
may help maintain a healthy weight. A study has found that brewer‘s yeast may improve acne
while another linked it to a reduced risk of a second skin cancer. And one large, preliminary
study found that taking a specific kind brewer‘s yeast product (EpiCor) may help prevent
colds and flu. Brewer‘s yeast is available in powder, flakes, tablet, and liquid forms.
Other approaches, which include testing the homology of DNA-DNA or ribosomal RNA
to DNA (Kurtzman and Blanz, 1998), are also finding application in determining
phylogenetic relationships. A more detailed phenotypic approach has been described (Duarte
et al., 1999) that exploits enzyme polymorphism between species. Electrophoretic
fingerprinting of glucose-6-phosphate dehydrogenase and three other enzymes enabled the
clear and reproducible differentiation of the different Saccharomyces species.
Genetic Manipulations
In traditional biotechnology (baking, brewing and wine making) yeast has tremendous
potential and hence a number of efficient transformation methods involving expression
vectors, reporter genes, immune-tags, and genetically selectable markers (Gueldener et al.,
2002; Janke et al., 2004; Sheff and Thorn, 2004) have been used for improvement of its
potential (Barnett, 2003; Gietz and Woods, 2001). Targeted manipulations within
chromosomes for homologus recombination have also been exploited (Klinner and Schafer,
2 4). The Baker‘s yeast was the first eukaryotic organism whose complete genomic
sequence was determined (Goffeau et al., 1996). Several databases such as the
Saccharomyces Genome Database (http://www.yeastgenome.org/) and the Comprehensive
Yeast Genome Database (http://mips.gsf.de/genre/proj/yeast/) contain an enormous amount of
information concerning its genetic make-up (Nevoigt, 2008).
S. cerevisiae has been used as a host organism for pharmaceutical protein production
(Porro et al., 2005). A number of its characteristic, including tolerance to low pH, high sugar
and ethanol concentrations, thereby lowering the risk of contamination, make it an important
organism in industrial biotechnology. This yeast is able to grow anaerobically and is fairly
resistant to inhibitors present in biomass hydrolysates. In contrast to classical methods of
strain improvement such as selection, mutagenesis, mating and hybridization (Attfield and
Bell, 2003; Panchal, 1990), Genetic engineering has conferred advantages such as the
directed modification of strains without unfavorable mutations and the introduction of foreign
genes with desirable traits. This part mainly focuses on the genetic engineering of yeast
strains including baker‘s, wine and brewer‘s yeast. Industrial Saccharomyces strains are
genetically diverse, prototroph, homothallic, and often polyploid, aneuploid, or even alloploid
(de-Winde, 2003).
Genetic Engineering in Baker’s Yeast
Engineering of baker‘s yeast has been extensively reviewed by Randez-Gil et al. (1999,
2003), Ostergaard et al. (2 ) and Dequin (2 1). Genetic improvement of baker‘s yeast is
mainly related to three parameters (i) efficient biomass production on cheap substrates (ii)
better dough-rising abilities and (iii) better product quality.
(i) Efficient biomass production on cheap substrates:- The most commonly used substrate
for yeast biomass production i.e., Molasses is a heterogenous mixture of sugars including
fructose, galactose, glucose, melibiose, raffinose and sucrose. The presence of glucose in
molasses prevents the simultaneous utilization of other sugars by the phenomenon known as
―glucose repression‖. This leads to lengthened process times during yeast propagation, dough
leavening and fermentation. Similarly, yeast cells must cope with various known stresses
during industrial propagation in molasses (Perez et al., 2005). A number of low-cost media
such as whey, starch, or lignocellulosics have also been considered as alternatives to molasses
for yeast propagation. But this requires extensive efforts to extend the substrate spectrum of S.
cerevisiae. As already explained another challenge for effective yeast biomass production is
―Crabtree effect‖ (Fiechter et al., 1981; Verduyn et al., 1984). Hence, optimal biomass yields
in industrial growth media can be obtained only if S. cerevisiae is grown in either well
controlled continuous or fed-batch fermentations.
Elbing et al. (2004) generated a yeast strain which is defective in the most important
hexose transporter genes (HXT1 to -7) and expresses a chimeric hexose transporter. This
fusion between HXT1 and HXT7 abolished the Crabtree effect in S. cerevisiae and resulted in
an exclusively respiratory phenotype to facilitate efficient yeast propagation. Other
approaches to prevent Crab tree effect in the presence of a high sugar concentration include
the expression of a water-forming NADH oxidase from Lactococcus lactis and an alternative
oxidase (AOX1) from Histoplamsa capsulatum (Heux et al., 2006a and 2006b; Vemuri et al.,
2007). The lower cytosolic NADH level in the strains reduced ethanol formation in batch
fermentation. In 1990s, there have been numerous attempts to improve yeast‘s tolerance
toward various stressors (Randez-Gil et al., 2003). Chen et al., (2006) worked on oxidative
stress tolerance by increasing the intracellular proline concentration, because supplemental
proline protected yeast cells from lethal levels of reactive oxygen species generated by
paraquat (Chen and Dickman, 2005). Moreover, deletion of PUT1-encoded proline
dehydrogenase conferred increased tolerance to hydrogen peroxide (H2O2) whereas PUT1
over-expression caused a strongly reduced intracellular proline concentration and a
hypersensitivity to oxidants such as H2O2 and paraquat (Chen et al., 2006). A high
intracellular proline has also been positively related to freeze tolerance (Terao et al., 2003),
desiccation (Takagi et al., 2000), and ethanol tolerance (Takagi et al., 2007) in yeast. Proline
may also serve as an osmoprotectant when present in the medium (Thomas et al., 1994).
(ii) Improvement of dough rising and product quality:-This has been targeted by
constructing the yeast with extended enzymatic capabilities, such as showing amylase,
hemicellulase, protease, and lipase activity. These enzymes alter the physicochemical
properties of the dough and thereby improving the flavour and shelf life of finished
product/bread (Randez-Gil et al., 1999 and 2003). Wheat flour, used in bread making,
contains starch, maltose, sucrose, glucose, fructose, and glucofructans. Maltose is also
released from starch during the baking process due to the activity of intrinsic amylases. A
major limiting factor for fermentation rate in baker‘s yeast is the carbon catabolite repression
of maltose-utilizing enzymes and the inactivation of maltase enzyme by glucose. This results
in a maltose consumption lag phase which can be avoided by de-repressing maltose-utilizing
enzymes. This has been achieved by replacing the native, glucose-controlled promoters of
maltase and maltose permease with constitutive ones. This engineered baker‘s yeast which
produces carbon dioxide more quickly than conventional baker‘s yeast was the first one to be
released in market for food use in the United Kingdom (Aldhous, 1990). Another strategy to
alleviate glucose repression of maltose-utilizing enzymes involves the deletion of proteins
Mig1p, which act as transcriptional regulator (Klein et al., 1998).
Additional desirable features for better dough performance include cryo-resistance in
frozen dough, osmotolerance in sweet frozen dough and freeze-thaw stress tolerance. Izawa et
al., (2004) have reported the most promising genetic modification for improving glycerol
accumulation by deleting FPS1, encoding a glycerol channel. It resulted in a roughly
threefold increase in intracellular glycerol accumulation. The engineered cells showed approx
80% survival after 7 days at -20°C as compare to 15% survival in the wild type. The
engineered cells also retained their high leavening ability even in frozen dough. Furthermore,
the car1arginase mutant accumulated higher levels of arginine or glutamate, which increased
leavening ability of frozen dough (Shima et al., 2003). Yeast strains over-expressing
heterologous aquaporin had been believed to provide new perspectives on the development of
freeze-resistant strains. Later studies, however, have shown that they have less potential for
use in frozen dough than originally thought (Tanghe et al., 2004). Another approach to
improve freezing tolerance has been the heterologous expression of antifreeze proteins. An
industrial yeast strain expressing the recombinant antifreeze peptide GS-5 from the polar fish
Myxocephalus aenaeus demonstrated improved viability during freezing; the number of
viable cells after 13 days at -20°C was 32-fold higher than that of the reference strain. There
was also a 30% increase in CO2 production after storage at -20°C for 40 days (Panadero et al.,
2005). Similarly, work has also been focused on the impact of unsaturated fatty acids on
freezing tolerance. The multicopy overexpression in S. cerevisiae of either FAD2-1 or FAD2-
3, encoding two different desaturases from sunflower, increased the dienoic fatty acid content
(Alper et al., 2006) and freezing tolerance. For example, about 90% of the FAD2-3-
overproducing cells survived for 35 days when frozen at -20°C (Rodriguez-Vargas et al.,
2007).
Wine Yeast
Approaches for genetic improvement of wine yeast are mainly focused on improved
fermentation performance, efficient use of nitrogen sources, ethanol tolerance, increased
glycerol production for body and fullness, the degradation of malic acid for acidity control,
improved processing efficiency, lesser contaminant rate, improved sensory qualities with
lesser content of deleterious compounds such as ethyl carbamate, biogenic amines, or even
ethanol and the increased amount of beneficial compounds like resveratrol (Pretorius and
Bauer, 2002). Integration of a linear cassette containing the Schizosaccharomyces pombe
malate permease gene (mae1) and the Oenococcus oeni malolactic gene (mleA) into the
URA3 locus led to the development of a genetically stable industrial yeast strain that can fully
decarboxylate 5.5 g/liter of malate. This malolactic yeast strain enjoys GRAS status from the
FDA and was the first genetically enhanced wine yeast to be commercialized (Husnik et al.,
2006). Increased glycerol production can lead to lower ethanol content in alcoholic beverages.
Cambon et al., (2006) combined (i) the overexpression of Glycerol 3-P dehydrogenase, the
rate-controlling step in glycerol biosynthesis, with (ii) the deletion of alcohol dehydrogenase
to decrease the ethanol content. But, a major drawback of glycerol overproduction is the
concomitant increase in other undesirable by-products such as acetoin and acetaldehyde.
Another approach for ethanol reduction in wine has been suggested by the Malherbe et al.,
(2003). It involves the expression of Aspergillus niger glucose oxidase in S. cerevisiae,
thereby, converting a portion of glucose into gluconic acid and reducing the amount of sugar
available for ethanol production. Elbing et al., (2004) developed a non-ethanol-producing
wine yeast strain by modifying hexose transporters. Moreover, introducing heterologous
enzymes to increase NADH oxidation can reduce ethanol formation in a wine yeast strain
(Heux et al., 2006 b).
Controlled and targeted modification of wine flavor is desirable to winemakers. Lilly et
al., (2006a), by altering the activities of the branched-chain amino acid transaminases altered
the formation of many aroma compounds, particularly higher alcohols. Another study by Lilly
et al., (2006b) examined the overexpression of four genes encoding ester-synthesizing and
ester-degrading enzymes in wine yeast. The genetic modifications resulted in specific ester
profiles and hence these approaches can be used to increase variability of wine flavor.
Approaches to modulate the formation of volatile sulfur compounds by wine yeast have
been reviewed by Swiegers and Pretorius (2007). Wine yeast transformants, expressing
foreign enzymes to have wide substrate utilization, have been reported to show several
positive effects on filterability, color and flavour of final product (van Rensburg et al., 2007).
Genetic engineering approaches have been applied to accelerate autolysis of S. cerevisiae in
order to enhance sparkling wine production (Tabera et al., 2006).
Brewer’s Yeast
In general, there are two types of brewer‘s yeast: (i) lager (bottom-fermenting) and (ii)
ale (top-fermenting) strains. The genome of the current lager strains may have resulted from
chromosomal loss, replacement, or rearrangement within the hybrid genetic lines. Indeed, the
complex genetics of lager brewer‘s yeast has complicated the engineering of those strains.
The identification of the entire genomic sequence of a commonly used lager brewer‘s yeast
strain represents a breakthrough in the molecular analysis of lager brewer‘s yeast (Kodama et
al., 2006). Brewer‘s yeast strain development principally aims to reduce time and cost of
production and improve product quality e.g., improved flocculation, reduced by-product
formation, ethanol reduction, glucanase expression for polysaccharide clarification, and
microbial spoilage prevention.
Good flocculation towards the end of primary fermentation is essential for a bright beer
with sufficient aroma and for the elaboration of sparkling wines. Late flocculation leads to
cloudy and hard to filter beer; and early flocculation leads to inefficient maturation. Yeast
flocculation is an asexual, calcium-dependent, reversible aggregation of cells into flocs that
involve interaction between a specific cell-surface lectin and cell-wall mannan (Stratford,
1992). This interaction is controlled by several dominant flocculation genes like FLO1, FLO5
and FLO8. Genetic transformation of wine yeast strain was achieved by transferring FLO1
gene, isolated from a flocculent S. cerevisiae strain (Bidard et al., 1995). Integration of the
ADH1-regulated FLO1 gene into the ADH1 locus led to a strong but early flocculation
phenotype (Watari et al., 1994). A major difficulty in the development of precise control of
flocculation is the lack of regulated promoters. However, a promoter (HSP30) that can be
induced by environmental factors prevalent towards the end of fermentation (high ethanol
concentration, depletion of sugars and nitrogen) has been successfully exploited under
laboratory conditions (Verstrepen et al., 2000).
Many attempts have been made to increase the glycerol yield during fermentation, which
is directly related to increased sweetness and decreased ethanol content of the beer. In S.
cerevisiae, glycerol is produced by reduction and subsequent dephosphorylation of
dihydroxyacetone phosphate, catalyzed by the glycerol- 3-phosphate dehydrogenase (GPDH)
and glycerol-3-phosphatase. Glycerol export and glycerol-3-phosphate dehydrogenase are
rate-limiting for glycerol production (Remize et al., 2001). Overexpression of GPD1 resulted
in a 2- to 3-fold increase in glycerol production with a consequent lower ethanol yield
(Nevoigt and Stahl, 1998; Remize et al., 1999). There was also a marked increase in the
production of acetaldehyde, diacetyl and acetoin (Nevoigt and Stahl, 1998).
Isoamyl acetate is an important aroma determinant for beer and wines. It is formed from
isoamyl alcohol - a by-product of leucine synthesis. Overexpression of LEU4 gene (coding α-
isopropylmalate synthase) (Hirata et al., 1992) and overexpression of the ATF1 gene (coding
alcohol acetyltransferase) (Fujii et al., 1994; Lilly et al., 2000) has been exploited for
increasing isoamyl acetate level, but with a simultaneous increase in the formation of esters
(ethyl acetate) which can be undesirable. Disruption of EST2 gene (coding for esterase)
resulted in a limited increase in isoamyl acetate production (Fukuda et al., 1998).
Spontaneous chemical reaction of ethanol and urea at elevated temperatures in acidic
media leads to the formation of ethylcarbamate- a potent carcinogen found in fermented foods
and beverages. Two approaches have been used to prevent its formation. First involve the
disruption of CAR1gene encoding arginase and thereby preventing the formation of urea from
arginine (Kitamoto et al., 1991). Second approach involves the expression of urease from
Lactobacillus fermentum to S. cerevisiae, with the aim of degrading urea. However, this
approach was ineffective, because S. cerevisiae lacks several proteins and cofactors required
for the proper folding of urease (Visser et al., 1999). To reduce the chances of contamination
by wild yeast, genes coding for production and immunity to Zymocins have been engineered
in brewer‘s yeast strains, but with limited success (Boone et al., 1990). Similarly, genes for
bacteriocins production have been inserted to cope up with bacteria (Schoeman et al., 1999).
Approaches to improve utilization of maltose and maltotriose by Saccharomyces yeast
strains are of interest in brewing because both of these are the major fermentable sugars in
brewer‘s wort. The MTTI gene encodes a maltotriose transporter, and its overexpression
resulted in a significantly increased maltotriose uptake in lager yeast (Day et al., 2002 a, b;
Dietvorst et al., 2005). Similarly dextrins, which make up about 25% of the carbohydrates in
wort, are usually non-fermentable. Dextrin utilization by brewer‘s yeast is necessary for the
production of low-calorie beer and dextrin-assimilating brewer‘s yeast is the second
genetically engineered strain to receive approval for commercial use from the British
Government (Akada, 2002). Efforts have been made to improve nitrogen uptake in brewer‘s
yeast. Proline, the most abundant amino acid in brewer‘s wort is virtually unassimilated
during brewing. Omura et al., (2 5) constructed a lager brewer‘s yeast with a modified
PUT4 gene to encode a highly specific proline permease. This modified strain efficiently
assimilated proline without negatively impacting beer quality. Accelerated lager beer
production is of great interest for the brewing industry, and there have been numerous
attempts to decrease diacetyl formation in brewer‘s yeast. During the maturation period,
diacetyl is enzymatically converted to acetoin and then to 2,3-butanediol, both of these have a
much higher taste threshold in beer than diacetyl or acetoin.
Expression of a bacterial acetolactate decarboxylase in yeast led to significant reduction
in the required maturation period. Interestingly, wine yeast possesses much higher 2,3-
butanediol dehydrogenase activity due to which diacetyl production is never a problem in
wine yeast. Sulfite is a key compound for the flavor stability of beer, so another target in
improving product quality is the increased production of sulfite, an antioxidant that forms
complexes with aldehydes that lead to ―cardboard flavor‖ in bottled beer as a result of
oxidative reactions. Donalies and Stahl (2002) reported the highest sulphite production in
laboratory yeast by the simultaneous overexpression of MET14 and SSU1, encoding
adenylylsulfate kinase and a sulfite pump, respectively. In breweries, which carry out high-
gravity brewing, highly concentrated wort is fermented (22 degrees Plato), it is worthwhile to
engineer yeast strains which better resist high osmolarity and high ethanol concentration.
Blieck et al., (2007) subjected a pool of UV-induced variants to consecutive rounds of
fermentation in very-high-gravity wort, and isolated variants of an industrial lager brewer‘s
yeast strain. Transcriptome analysis of these variants revealed a number of differentially
expressed genes and most of these variants have greater fermentation velocity with improved
final wort attenuation in highly concentrated wort.
Other Yeast Biomass-Derived Products
Industrial uses of yeast is not only limited to baking industry, rather there are several
other uses (Walker, 1998) like biomass (fodder yeast) and biomass extracted cell products
(yeast B-complex vitamins). Genetic engineering has been exploited here also to increase
iron-carrying yeast capacity by expression of heterologous ferritin (Chang et al., 2005), that is
bioavailable and have the potential to address the problem of iron deficiency.
Supplemental Starch-Degrading Enzymes
Efficient starch-degrading S. cerevisiae strains have immense potential in consolidated

bioprocessing for simultaneous production of enzymes for saccharification, hydrolysis, and
fermentation by a single microorganism. These strains can be of significant importance in
improving the economics of bioethanol production and can play a role in beer and whisky
production. Kondo et al., (2002) used Flo 1 protein for anchoring amylolytic enzymes on
yeast cell surface. An important enzyme i.e., Glucoamylase has been expressed either alone
(Sato et al., 2 2) or in combination with α-amylase (Eksteen et al., 2003) or pullulanase
(Janse et al., 1995). These heterologous genes encoding amylolytic enzymes can be from
amylolytic yeast Saccharomyces diastaticus, filamentous fungi (Aspergillus niger) and
several bacteria (Bacillus sp.). A hybrid glucoamylase-encoding gene has been constructed by
in-frame fusion of the S. diastaticus STA1 gene and the DNA fragment that encodes the
starch binding domain of A. niger glucoamylase (Latorre-Garcia et al., 2005).
Concerns Related to Use of GM Yeasts
The risk assessment procedure for genetically modified non-pathogenic microorganisms

used in fermentation is the same as that for GMOs from plant and animal origin (Sybesma et
al., 2006). As per European legislation the official approval of GMOs does not guarantee
successful commercialization. Another difficulty related to the use of genetically engineered
yeast in the food and beverage industry is the containment issue. The containment of
engineered yeast within the production unit cannot be guaranteed. All the engineered yeast
strains must pass safety evaluations and the product should follow labelling regulations. On
the international level, the Codex Alimentarius Commission has established principles for the
assessment of food-related safety issues of GMOs (http://www.who.int/foodsafety
/biotech/codex taskforce/en/) and the Cartagena Protocol on Bio-safety outlines principles for
the environmental impact of use of these genetically modified yeasts/microrganisms
(http://www.biodiv.org/biosafety/default.aspx). Presently, there is a very low acceptance of
GMOs in the food and beverage industry due to consumer acceptability concerns. Although
many advantageous industrial yeast strains have been engineered and two have obtained
approval from the British government for commercial use several years ago (Akada, 2002),
no genetically modified yeast has entered commercial use (Husnik et al., 2006). Various
complex cultural, social, ethical, environmental, and technical factors related to genetically
modified wine yeast has been reviewed by Pretorius and Bauer (2002). It has been suggested
that product labelling and improved education of the public on these matters can increase the
industrial use and acceptance of GM yeast products (Sybesma et al., 2006).
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Vieira, ED; Andrietta, MGS and Andrietta, SR. Yeast biomass production: a new approach in
glucose-limited feeding strategy. Brazilian Journal of Microbiology, 2013, 44(2): 551-58.
Visser, JJ; Coton, EPN; Bauer, F; Viljoen, M; Vuuren, HJJ van. Engineering an acid urease
for heterologous expression in Saccharomyces cerevisiae. Current Genetics, 1999,
35:321.
Walker, GM. Yeast physiology and biotechnology. 1998, J. Wiley and Sons, Chichester,
United Kingdom.
Watari, J; Nomura, M; Sahara, H; Koshino, S. Construction of flocculent brewer‘s yeast by
chromosomal integration of the yeast flocculation gene FLO1. Journal of the Institute of
Brewing, 1994, 100:73–77.
Yuen, KW; Montpetit, B; Hieter, P. The kinetochore and cancer: what‘s the connection?
Current Opinion in Cell Biology, 2005, 17:576–582.
Chapter 13
NUTRACEUTICALS:
A POTENT THERAPEUTIC AGENT
Sachin Gulati, Anita Yadav* and Neeraj Kumar

Department of Biotechnology, Kurukshetra University, Kurukshetra, Haryana, India
ABSTRACT
The concept of nutraceutical was stared from the survey in U.K., Germany and
France which concluded that diet is rated more highly by consumers than exercise or
hereditary factors for achieving good health. Various components of foods have clearly
established strong links with human health and it is learnt that their deficiencies provoke
diseases. Edible products possessing such fantastic healing capacity due to the presence
of more than 700 non nutrients functional food factor (FFFs). These are effective and
beneficial for health promotion, disease prevention and cure. The natural products
releasing such superb active components are known as nutraceutical foods. The explosive
growth, research developments, lack of standards, marketing zeal, quality assurance and
regulation will play a vital role in its success or failure. In this chapter we can try to
understand the different concept of nutraceuticals and their scope in the future.
Keywords: nutraceuticals, phytochemicals, nutrients, herbs and dietary supplements
INTRODUCTION
Epidemiological and clinical studies have demonstrated the relationship between diet and
health status. It is well known that populations consuming a large proportion of plant-based
foods, including fruits, vegetables, whole grains and cereals or those with a high intake of sea
foods, have a lower incidence of cardiovascular diseases and certain types of cancer (Lanou
and Barbara, 2011). Consumers are deeply concerned about how their health care is managed,
*
Corresponding author: Department of Biotechnology, Kurukshetra University, Kurukshetra, Haryana-136119,
India. *E-mail: ayadav@kuk.ac.in; Phone: 01744-238410 Extn. 2593; Mobile No. +91-9416291480; Fax No:
01744- 20277,21639,22565.
312 Sachin Gulati, Anita Yadav and Neeraj Kumar
administered and priced. They are frustrated with the expensive, high-tech, disease-treatment
approach predominant in modern medicine; the consumer is seeking complementary or
alternative beneficial products and the red tape of managed care makes nutraceuticals
particularly appealing.
Therefore, interest has been expressed in functional foods, nutraceuticals and dietary
supplements. Functional foods are defined as being similar in appearance to conventional
foods, are consumed as part of a usual diet, and are known to improve health status and
render physiological effects beyond basic nutritional function expected of conventional foods.
However, nutraceuticals are products produced from foods, but sold in the medicinal form of
capsule, tablet, powder or solution. They are not generally associated with food and have
demonstrated physiological benefits and/or provide protection against chronic diseases; these
are now referenced as ―natural health products‖ (Shahidi, 2007).
The term "nutraceutical" was coined from "nutrition" and "pharmaceutical" in 1989 by
Stephen DeFelice, MD, Founder and Chairman of the Foundation for Innovation in Medicine
(FIM), Cranford, NJ (Brower, 1998). According to DeFelice, nutraceutical can be defined as,
"a food (or part of a food) that provides medical or health benefits, including the prevention
and/or treatment of a disease" (Brower, 1998). However, the term nutraceutical as commonly
used in marketing has no regulatory definition. Nutraceuticals (often referred to as
phytochemicals or functional foods) are natural bioactive, chemical compounds that have
health promoting, disease preventing or medicinal properties.
Nutraceuticals are found in a mosaic of products emerging from the food industry, the
herbal, dietary supplement market, pharmaceutical industry, and the newly merged
pharmaceutical/agribusiness/nutrition conglomerates.
CATEGORIES OF NUTRACEUTICALS (DUREJA ET AL., 2003)

Nutraceuticals are non-specific biological therapies used to promote wellness, prevent
malignant processes and control symptoms. These can be grouped into the following four
broad categories:
1. Nutrients – substances with established nutritional functions, such as vitamins,

minerals, amino acids and fatty acids.
2. Herbals – herbs or botanical products as concentrates and extracts.
3. Dietary supplements – reagents derived from other sources (e.g., pyruvate,
chondroitin sulphate, steroid hormone precursors) serving specific functions, such as
sports nutrition, weight-loss supplements and meal replacements.
4. Phytochemicals – non-nutritive plant chemicals that have protective or disease
preventive properties. They are non-essential nutrients, meaning that they are not
required by the human body for sustaining life. It is well-known that plant produces
these chemicals to protect them but recent research demonstrates that they can also
protect humans against diseases. There are more than thousand known
phytochemicals i.e., lycopene in tomatoes, isoflavones in soy and flavanoids in fruits.
Nutraceuticals: A Potent Therapeutic Agent 313
Table 1. Common nutrients and their associated health benefits

(Dureja et al., 2003)
Nutrients Health benefits

Fat Soluble
Vitamins
Vitamin A Antioxidant, essential, for growth and development, maintains healthy vision, skin
and mucous membranes, may aid in the prevention and treatment of certain
cancers and in the treatment of certain skin disorders.
Vitamin D Essential for formation of bones and teeth, helps the body absorb and use calcium
Vitamin E Antioxidant, helps form blood cells, muscles, lung and nerve tissue, boosts the
immune system
Vitamin K Essential for blood clotting
Water Soluble
Vitamins
Vitamin C Antioxidant, necessary for healthy bones, gums, teeth and skin, helps in wound
healing, may prevent common cold and attenuate its symptoms
Vitamin B1 Helps to convert food in to energy, essential in neurologic functions
Vitamin B2 Helps in energy production and other chemical processes in the body, helps
maintain healthy eyes, skin and nerve function
Vitamin B3 Helps to convert food in to energy and maintain proper brain function
Vitamin B6 Helps to produce essential proteins and convert protein in to energy
Vitamin B12 Helps to produce the genetic material of cells, helps with formation
of red blood cells, maintenance of central nervous system and synthesize amino
acids and is involved in metabolism of fats, protein and carbohydrates
Folic acid Necessary to produce the genetic materials of cells, essential in first three months
of pregnancy for preventing birth defects, helps in red blood cell formation,
protects against heart disease
Pantothenic acid Aids in synthesis of cholesterol, steroids and fatty acids, crucial for intraneuronal
synthesis of acetylcholine
Minerals
Calcium Essential for building bones and teeth and maintaining bone strength, important in
nerve, muscle and glandular functions
Iron Helps in energy production, helps to carry and transfer oxygen to tissues
Magnesium Essential for healthy nerve and muscle function and bone formation, may help
prevent premenstrual syndrome (PMS)
Phosphorous Essential for building strong bones and teeth, helps in formation of genetic
material, energy production and storage
Trace elements
Chromium With insulin helps to convert carbohydrates and fats into energy coenzymes
Cobalt Essential component of vitamin B12, but ingested cobalt is metabolized in vivo to
form the B12
Copper Essential for hemoglobin and collagen production, healthy functioning of the
heart, energy production, absorption of iron from digestive tract
Iodine Essential for proper functioning of the thyroid
Selenium Antioxidant, essential for healthy functioning of the heart muscle
Zinc Essential for cell reproduction, normal growth and development in children,
wound healing, production of sperm and testosterone
Nutrients Health benefits

Vitamin like
compounds
Biotin Required for various metabolic functions
L- Carnitine Oxidation of fatty acids, promotion of certain organic acid excretion and
enhancement of the rate of oxidative phosphorylation
Choline Lipotropic agent used to treat fatty liver and disturbed fat metabolism
Vitamin F Involved in proper development of various membranes and synthesis of
prostaglandins, leukotrienes and various hydroxy fatty acids
Inositol Lipotropic agent necessary for amino acid transport and movement of
potassium and sodium
Taurine Aids in retinal photoreceptor activity, bile acid conjugation, white blood
cell antioxidant activity, CNS neuromodulation, platelet aggregation,
cardiac contractility, sperm motility, growth and insulin activity
1) Nutrients
These are the nutritional components in foods that an organism utilizes to survive and
grow. Macronutrients provide the bulk energy for an organism's metabolic system to function,
while micronutrients provide the necessary cofactors for metabolism to be carried out. Both
types of nutrients can be acquired from the environment (Whitney et al. 2005) (Table 1).
They are used to build and repair tissues, regulate body processes, and are converted to
and used for energy. Methods for nutrient intake are different for plants and animals. The
most commonly known nutrients are antioxidant, water and fat-soluble vitamins. Many
potential benefits have been attributed to antioxidant use in the form of dietary intake or
supplementation (Mobarhan, 1994; Agus et al., 1997; Rijk et al., 1997).
2) Herbals
Herbal is "a collection of descriptions of plants put together for medicinal purposes"
(Singer, 1923). Herbals are as old as human civilization and they have provided a complete
storehouse of remedies to cure acute and chronic diseases. The knowledge of herbals has
accumulated over thousands of years so that today we possess many effective means of
ensuring health care. Numerous nutraceuticals are present in medicinal herbs as key
components (Table 2).
3) Dietary Supplements
Dietary supplements have also been developed to manage a variety of diseases. For
instance, prepackaged, nutritionally balanced meals that meet the recommendations of
national health organizations influenzed multiple risk factors for patients with cardiovascular
disease and increased patient compliance with dietary restrictions (McCarron et al., 1997).
Ketogenic diets, comprised of foods high in fat and low in protein and carbohydrate content,
have been reported to improve seizure control. However, these diets are widely acknowledged
to be unpalatable, making sustained compliance with dietary restrictions difficult (Chapman
et al., 1997).
Table 2. Common herbal and phytochemical products (Dureja et al., 2003)
Compound Therapeutic activity
Aloe vera gel (Aloe vera L. N.L. Dilates capillaries, anti-inflammatory, emollient, wound
Burm.) healing properties
Chamomile (Matricaria recutita L.) Anti-inflammatory, spasmolytic, antimicrobial, wound healing
Echinacea (Echinacea purpurea L.) Immunostimulant, treatment of cold and flu symptoms
Eleuthera Adaptogen
(Eleutherococcus senticosus Rupr. and
Maxim., Maxim)
Ephedra (Ephedra sinica Stapf., Bronchodilator, vasoconstrictor, reduces bronchial edema,
Ephedra intermedia Schrank., Ephedra appetite suppressant
equisetina Bunge.)
Evening primrose oil (Oenothera Dietary supplement of linoleic acid, treatment of atopic eczema
biennis L.)
Feverfew (Tanacetum parthenium L.) Treatment of headache, fever and menstrual problems,
prophylactic to reduce frequency, severity and duration of
migraine headaches
Garlic (Allium sativum L.) Antibacterial, antifungal, antithrombotic, hypotensive,
fibrinolytic, antihyperlipidemic, antiinflammatory
Ginger (Zingiber officinale Rosc.) Carminative, antiemetic, cholagogue, positive inotropic,
treatment of dizziness
Ginseng (Panax ginseng, Panax Adaptogen
quinquefolius L.)
Ginkgo (Ginkgo biloba L.) Vasodilation, increased peripheral blood flow, treatment of
post-thrombotic syndrome, chronic cerebral vascular
insufficiency, short term memory loss, cognitive disorders
secondary to depression, dementia, tinnitus, vertigo
Goldenseal (Hydrastis canadensis L.) Antimicrobial, astringent, antihemorrhagic, treatment of
mucosal inflammation, dyspepsia, gastritis
Horehound (Marrubium Expectorant, antitussive, choleretic
vulgare L.)
Licorice (Glycyrrhiza glabra L., G. Expectorant, secretolytic, treatment of peptic ulcer
uralensis Fisch.)
Melissa (Melissa officinalis L.) Topical antibacterial and antiviral
Plantago seed (Plantago arenaria Cathartic
Waldst., Plantago arenaria Kit.
Plantago ovata)
Slippery elm (Ulmus rubra Muhl.) Mucilaginous demulcent, emollient and nutrient, used to sooth
irritated mucous membranes, ulcerations of the digestive tract
St. John‘s wort (Hypericum Anxiolytic, antiinflammatory, antidepressant, monoamine
perforatum L.) oxidase inhibitor
Valerian (Valeriana officinalis L.) Spasmolytic, mild sedative, sleep aid
Willow bark (Salix alba L., conditions, Antiinflammatory, analgesic, antipyretic, astringent, treatment
headache and gout S. daphnoides of rheumatic and arthritic
Villars, S. pentandra L.)
The Dietary Supplement Health and Education Act of 1994 (DSHEA) formally defined
"dietary supplement" using several criteria (Kalra, 2003). A dietary supplement (FDA, 1994):
 is a product (other than tobacco) that is intended to supplement the diet that bears or
contains one or more of the following dietary ingredients: a vitamin, a mineral, an
herb or other botanical, an amino acid, a dietary substance for use by man to
supplement the diet by increasing the total daily intake, or a concentrate, metabolite,
constituent, extract, or combinations of these ingredients.
 is intended for ingestion in pill, capsule, tablet, or liquid form.
 is not represented for use as a conventional food or as the sole item of a meal or diet.
 is labeled as a "dietary supplement."
 includes products such as an approved new drug, certified antibiotic, or licensed
biologic that was marketed as a dietary supplement or food before approval,
certification, or license (unless the Secretary of Health and Human Services waives
this provision).
Thus, nutraceuticals (as per the proposed definition) differ from dietary supplements in
the following aspects:
 Nutraceuticals must not only supplement the diet but should also aid in the
prevention and/or treatment of disease and/or disorder.
 Nutraceuticals are represented for use as a conventional food or as the sole item of
meal or diet.
4) Phytochemicals
Phytochemicals are naturally occurring biochemicals that give plants their color, flavor,
smell, and texture, which may help prevent diseases (Bland, 1996; Dulloo et al., 1999;
Brouns, 2002). They are biologically active natural products such as glucosinolates in
cruciferous vegetables, limonoids in citrus fruits, lignans in flaxseed, lycopene in tomatoes,
and catechins in tea. They all have specific actions and can be used variously for, e.g., as
antioxidants and have a positive effect on health (Burney, 1989; Whitman, 2001; Ramaa,
2006). Systematic classification on the basis of health benefits is given in Table 3.
CLASSIFICATION OF NUTRACEUTICALS
Regarding the promise of nutraceuticals, they should be considered in two ways:
 Potential nutraceuticals
 Established nutraceuticals
A potential nutraceutical is one that holds a promise of a particular health or medical

benefit; such a potential nutraceutical only becomes an established one after there are
sufficient clinical data to demonstrate such a benefit (Table 4). It is disappointing to note that
the overwhelming majority of nutraceutical products are in the 'potential' category, waiting to
become established (De Felice, 1995).
Table 3. Health benefit of nutraceutical (Umesh et al., 2012)
Diseases Nutraceuticals Reference

Cancer  Flavonoids which block the enzymes that produce Frydoonfar et al.,
estrogen- reduce of estrogen -induced cancers. 2003
 To prevent prostate/breast cancer a broad range of Limer et al., 2004
phyto-pharmaceuticals with a claimed hormonal activity,
called ―phyto -estrogens‖ is recommended.
 Soy foods source of isoflavones, curcumin from curry
and soya isoflavones possess cancer chemopreveventive Mandel et al., 2005
properties.
 Lycopene concentrates in the skin, testes, adrenal and Kucuk et al., 2002
prostate where it protects against cancer.
Gulcin et al., 2006
 Saponins (found in peas, soybeans, some herbs, spinach,
tomatoes, potatoes, alfalfa and clove) contain antitumor Aggarwal et al., 2003
and antimutagenic activitie.
 Curcumin (diferuloylmethane) which is a polyphenol of Thanopolou et al.,
turmeric possesses anticarcinogenic, antioxidative and 2006
anti-inflammatory properties.
 Top of Form Beet roots, cucumber fruits, spinach leaves,
and turmeric rhizomes, were reported to possess anti
tumor activity.
Obesity  Herbal stimulants, such as ephedrine, caffeine, Daly et al., 1993
mahuang-guarana, chitosan and green tea help in body
weight loss.
 Buckwheat seed proteins acting similar to natural fibers Si-quan, 2001
present in food.
 5-hydroxytryptophan and green tea extract may promote Bell, 2002
weight loss, while the former decreases appetite, the
later increases the energy expenditure.
 A blend of glucomannan, chitosan, fenugreek, G Woodgate, 2003
sylvestre, and vitamin C in the dietary supplement
significantly reduced body weight.
 Conjugated linoleic acid (CLA), capsaicin, Momordica Kasbia, 2005
Charantia (MC) possesses potential anti obese
properties.
Cardio-  Anti-oxidants, Dietary fibres, Omega-3 poly unsaturated German, 2000
vascular fatty acids, Vitamins, minerals for prevention and
Diseases treatment of CVD. Polyphenol (in grape) prevent and
control arterial diseases.
 Flavonoids (in onion, vegetables, grapes, red wine, Hollman et al., 1999
apples, and cherries) block the ACE and strengthen the
tiny capillaries that carry oxygen and essential nutrients
to all cells.
Diseases Nutraceuticals Reference

Diabetes  Ethyl esters of n-3 fatty acids may be beneficial in diabetic Sirtori, 2002
patients.
 Docosahexaenoic acid modulates insulin resistance and is Thomas et al.,
also vital for neurovisual development. 2006
 Lipoic acid, an antioxidant, for treatment of diabetic Coleman et al.,
neuropathy. 2001
 Dietary fibers from psyllium have been used for glucose Baljit, 2007.
control in diabetic patients and to reduce lipid levels in
hyperlipidemia.
Antiinflam-  Gamma linolenic acid (found in green leafy vegetables, Formica, 1995
matory nuts, vegetable oils i.e.-evening primrose oil, blackcurrant
Activities seed oil, hemp seed oil, and from spirulina, cyanobacteria) is
used for treating problems with Inflammation and auto-
immune diseases.
 Glucosamine and chondroitin sulfate are used against Alarcon et al.,
osteoarthritis and regulate gene expression and synthesis of 1994
NO and PGE2.
 Cat‘s claw has 17 alkaloids, along with glycosides, tannins, Balch et al., 2003
flavonoids, sterol fractions, and other compounds and work
as potent anti-inflammatory agent.
Parkinson  Vitamin E in food may be protective against Parkinson‘s Latif et al., 2007
Disease disease. Brower, 2005
 Creatine modifies Parkinson‘s disease features as measured
by a decline in the clinical signs.
Alzheimer‘s  β-Carotene, curcumin, lutein, lycopene, turmerin etc. may Glenville, 2006
Disease exert positive effects on specific diseases by neutralizing the
negative effects oxidative stress mitochondrial dysfunction,
and various forms of neural degeneration.
Allergy  Quercet (found in Onions, red wine and green tea) reduce Chidambara et al.,
the inflammation that results from hay fever, bursitis, gout, 2005
arthritis, and asthma.
Vision  Lutein (Helenium autumnale) known as helenien and Fisher, 2005
improving Zeaxanthin is used for the treatment of visual disorders
Agents found in mangoes, corn, sweet potatoes, carrots, squash,
tomatoes and dark, leafy greens such as kale, collards, egg
yolks and fruits, such as broccoli, green beans, green peas,
brussel sprouts, cabbage, kale, collard greens, spinach,
lettuce, kiwi and honeydew. Lutein and zeaxanthin are also
found in nettles, algae and the petals of many yellow
flowers. In green vegetables, fruits and egg yolk, lutein and
zeaxanthin exist in non-esterified forms.
Immune  Dietary nucleotides may improve growth and immunity; Vanderhoof et al.,
boosters optimize maturation, recovery and function of rapidly 1999
dividing tissue.
Osteoar-  Glucosamine (GLN) and chondroitin sulfate (CS) are widely Kalioraa, 2006
thritis (OA) used to alleviate symptoms of OA. These nutraceuticals
have both nutrient and pharmaceutical properties and seem
to regulate gene expression and synthesis of NO and PGE2,
providing a plausible explanation for their anti-
inflammatory activities.
Table 4. List of marketed nutraceutical products (Dureja et al., 2003)
Product Category Contents Manufacturer

Coral calcium Calcium Calcium and trace minerals Nature‘s answer,
supplement Hauppauge, NY, USA
Weight smartTM Nutritional Vitamins and trace elements Bayer corporation,
supplement Morristown, NL, USA
Omega woman Immune Antioxidants, vitamins and Wassen, Surrey, U.K.
supplement phytochemicals (e.g., Lycopene,
and resveratrol)
Appetite Appetite Caffeine, tyrosine and Natrol, Chatsworth, CA,
InterceptTM suppressant phenylalanine USA
ChaserTM Hangover Activated calcium carbonate Living essentials, Walled
supplement and vegetable carbon lake, MI, USA
Rox® Energy drink Taurine, caffeine and Rox America, Spartanburg,
glucuronolactone SA, USA
Mushroom Immune Mushrooms, polysaccharides Jarrow formulas, Los
optimizerTM supplement and Folic acid CA, Angeles, USA
BiovincaTM Neurotonic Vinpocetine Cyvex nutrition, Irvine,
CA, USA
Proplus® Nutritional Soy proteins Campbell soup company,
supplement Camden, NJ, USA
Snapple-a-dayTM Meal replacement Vitamins and minerals Snapple beverage group,
beverage White Plains, NY, USA
WelLife® Amino acid Granulated-L-glutamine Daesang America Inc.,
supplement Hackensach, NJ, USA
PNer plusTM Neuropathic pain Vitamin and other supplement NeuroHelp, San Antonio,
natural supplement Texas, USA
OlivenolTM Dietary Natural antioxidant, Cre Agri, Hayward, CA,
supplement hydroxytyrosol USA
Threptin® Diskettes Protein Proteins and vitamin B Raptakos, Brett and Co.
supplements Ltd., Mumbai, India
GRD® Nutritional Proteins, vitamins, minerals and Zydus Cadila Ltd.
supplement carbohydrates Ahmedabad, India
Proteinex® Protein Predigested proteins, vitamins, Pfizer Ltd., Mumbai,

supplement minerals and carbohydrates India
Calcirol D-3® Calcium Calcium and vitamins Cadilla healthcare

supplement limited, Ahmedabad,
India.
The food products used as nutraceutical are categorized as (Kokata et al., 2002):
Probiotic; prebiotic; dietary fiber; omega 3 fatty acid; antioxidant.
1) Probiotics
Probiotics are microorganisms that are believed to provide health benefits when
consumed Rijkers et al., 2011). Probiotics are live bacteria and yeasts that are good for your
health, especially your digestive system. We usually think of bacteria as something that
causes diseases. But your body is full of bacteria, both good and bad. Probiotics are often
called "good" or "helpful" bacteria because they help keep your gut healthy. Probiotics are
naturally found in your body. You can also find them in some foods and supplements. It's
only been since about the mid-1990s that people have wanted to know more about probiotics
and their health benefits. Doctors often suggest them to help with digestive problems and
because of their newfound fame, you can find them in everything from yogurt to chocolate.
2) Prebiotic
Prebiotics are the substances, which reach to colon in intact form i.e., without getting
depleted by the gastric pH and digestive acids. These prebiotics also selectively promote the
growth of colonel probiotic bacteria; hence they act as fertilizers for these bacteria. These are
collective term for non-digestive but a fermentable dietary carbohydrate that may selectively
stimulates growth of certain bacterial groups resident in the colon, such as Bifidobacteria,
Lactobacilli considered to be beneficial for the human host (Gibson and Roberfroid, 1995).
The prebiotic inulin, which is soluble dietary fibers and resistant to digestive enzyme
therefore reaches to large intestine or colon essentially intact, where it is fermented by
resistant bacteria, Lactobacilli (Peppelenbosch and Ferreira, 2009).
3) Dietary Fiber
Dietary fibers can act by changing the nature of the contents of the gastrointestinal tract
and by changing how other nutrients and chemicals are absorbed (Eastwood et al., 2005).
Some types of soluble fiber absorb water to become a gelatinous, viscous substance which is
fermented by bacteria in the digestive tract. Some types of insoluble fiber have bulking action
and are not fermented (Anderson et al., 2009). Lignin, a major dietary insoluble fiber source,
may alter the rate and metabolism of soluble fibers (United States Department of
Agriculture). Other types of insoluble fiber, notably resistant starch, are fully fermented
(Nugent, 2005).
4) Omega 3 Fatty Acid
Omega-3 fatty acids (also called ω-3 fatty acids or n-3 fatty acids (Omega-3 fatty
acids, 2012) are polyunsaturated fatty acids (PUFAs) with a double bond (C = C) at the third
carbon atom from the end of the carbon chain (Scorletti et al., 2013). The fatty acids have two
ends, the carboxylic acid (-COOH) end, which is considered the beginning of the chain, thus
"alpha", and the methyl (CH3) end, which is considered the "tail" of the chain, thus "omega."
The three types of omega-3 fatty acids involved in human physiology are α-linolenic acid
(ALA) (found in plant oils), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA)
(both commonly found in marine oils). Marine algae and phytoplankton are primary sources
of omega-3 fatty acids. Common sources of plant oils containing the omega 3 ALA fatty acid
include walnut, edible seeds, clary sage seed oil, algal oil, flaxseed oil, Sacha Inchi oil,
Echium oil, and hemp oil, while sources of animal omega-3 EPA and DHA fatty acids
include fish oils, egg oil, squid oils, and krill oil. Supplementation with omega-3 fatty acids
does not appear to affect the risk of death, cancer or heart disease (Rizos et al., 2012;
MacLean et al., 2006). Omega-3 fatty acids are important for normal metabolism (UNIH,
2014). Mammals have a limited ability to synthesize omega-3 fats.
5) Antioxidants
An antioxidant is a molecule that inhibits the oxidation of other molecules. Oxidation is

a chemical reaction involving the loss of electrons or an increase in oxidation state. Oxidation
reactions can produce free radicals. In turn, these radicals can start chain reactions. When the
chain reaction occurs in a cell, it can cause damage or death to the cell. Antioxidants
terminate these chain reactions by removing free radical intermediates, and inhibit other
oxidation reactions. They do this by being oxidized themselves, so antioxidants are often
reducing agents such as thiols, ascorbic acid, or polyphenols (Sies et al., 1997).
Antioxidants are widely used in dietary supplements and have been investigated for the
prevention of diseases such as cancer, coronary heart disease and even altitude sickness
(Baillie et al., 2009). Although initial studies suggested that antioxidant supplements might
promote health, later large clinical trials of antioxidant supplements including beta-carotene,
vitamin A, and vitamin E singly or in different combinations suggest that supplementation
has no effect on mortality or possibly increases it (Bjelakovic et al., 2013; Abner et al., 2011;
Bjelakovic et al., 2007). People who eat fruits and vegetables appear to have a lower risk of
heart disease, some neurological diseases (Stanner et al., 2004) and some cancers (WCRH,
2007). Since fruits and vegetables happen to be good sources of nutrients and
phytochemicals, this suggests that antioxidant compounds might lower risk against several
diseases (Table 5).
Table 5. Different Nutraceuticals and their food sources
Nutraceutical(s) Food Source

Catechins, theaflavins, thearubigins Teas (green and black)
Organosulfur compounds, quercetin, anthocyanins Onions
Conjugated linoleic acid (CLA), lactoferrin Dairy foods
Lignan, omega-3 (n-3) fatty acids Flaxseed
Allicin and related sulfur-containing compounds Garlic
Long-chain omega-3 (n-3) fatty acids Fatty fish (salmon, mackerel)
and fish oil
Anthocyanins, flavonoids, tannins Cranberries
Carotenes, quercetin, luteolin Peppers
Isoflavones and soy protein Soybeans
Carotenes, anthocyanins, lycopene Carrots
Lycopene Tomatoes
Sulfurophane and other glucosinolates Broccoli
Antioxidants have been investigated as possible treatments for neurodegenerative

diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis
(Di Matteo et al., 2003; Rao et al., 2002), and as a way to prevent noise-induced hearing loss
(Kopke et al., 2007). In general these trials have shown ambiguous effects on patient
outcomes (Crichton et al., 2013; Takeda et al., 2014; Harrison et al., 2012).
CLASSIFICATION BASED ON MECHANISM OF ACTION

Another means of classifying nutraceuticals is by their mechanism of action. This system
groups nutraceutical factors together, regardless of food source, based upon their proven or
purported physiological properties. Among the classes would be antioxidant, antibacterial,
antihypertensive, antihypercholesterolemic, antiaggregate, anti inflammatory,
anticarcinogenic, osteoprotective, and so on. Examples are presented in Table 6.
Table 6. Examples of nutraceuticals grouped by mechanisms of action

(Keservani et al., 2010)
Anticancer Positive Influence Antioxidant Anti inflame- Osteogenetic or

on Blood Lipid Activity matory Bone Protective
Profile
Capsaicin α-Glucan CLA Linolenic acid CLA
Genestein γ-Tocotrienol Ascorbic acid EPA Soy protein
Daidzein δ-Tocotrienol β-Carotene DHA Genestein
α-Tocotrienol MUFA Polyphenolics GLA (gammali- Daidzein
nolenic acid)
γ-Tocotrienol Quercetin. ω-3 Tocopherols Capsaicin Calc

CLA PUFAs Tocotrienols Ium
Casein
Lactobacillus Resveratrol Indole-3-carbonol Quercetin phosphopeptides
acidophilus Curcumin
FOS
Sphingolipids Tannins α-Tocopherol (fructooligosacc
Limonene β-Sitosterol Ellagic acid harides)
Diallyl sulfide Saponins Lycopene
Ajoene Guar Lutein Inulin
α-Tocopherol Pectin Glutathione
Enterolactone Hydroxytyrosol
Glycyrrhizin Luteolin
Equol Oleuropein
Curcumin Catechins
Ellagic acid Gingerol
Lutein Chlorogenic acid
Carnosol Tannins
L. bulgaricus
SCOPE OF THE MARKET

India is the most potential market for Nutraceuticals and Dietary Supplement products.
There has been an increasing awareness among the rising affluent middle class about health
and wellness. Nearly 400 million people in India belong to the middle class and have
disposable incomes which have made them capable of buying nutraceuticals and dietary
supplements. It is an inevitable fact that affluence is one of the causes of lifestyle diseases,
which nutraceuticals and dietary supplements often address. These factors will support the
double digit growth of the industry in the coming few years. The Indian nutraceuticals market
which has grown from $ one billion in 2008 to $1.8 billion in 2013, is likely to cross $ two
billion in 2014 and is expected to top $ four billion by 2018. According to experts,
nutraceuticals and functional foods would help mitigate malnutrition in India. Although, there
is a huge potential for the growth of this sector, yet its development is slow. The country has a
long way to go to en-cash much of its bio-agri wealth (Pharmabiz, 2014).
With the growing popularity of these supplemented foods, many multinationals are
investing in the nutraceuticals market in India. These include Monsanto, American Home
Products, DuPont, BioCorrex, Abbott Laboratories, GlaxoSmithKline Consumer Healthcare,
Johnson and Johnson, Nestle, Novartis, Yakult-Danone India, Herbalife etc. These players are
a major resource for nutraceuticals and related dietary supplements. Besides, India has many
local players such as Dabur India, Cadila Healthcare, British Biologicals, Himalaya Global
Holdings, Sami Labs, Sami Direct, Parry Nutraceuticals, Wockhardt etc.
The global demand for nutraceutical ingredients is forecast to rise 7.2 per cent annually to
$30 billion in 2017 according to report by Report linker. The best growth prospects will exist
in substances with clinically supported health benefits and broad applications in foods,
beverages, dietary supplements and adult and pediatric nutritional preparations.
The nutraceutical industry, which is perceived as consumer driven, must meet several
conditions to continue expansion. These conditions include
 a continued consumer emphasis on preventive health care and health maintenance;

 the Indian demographics of an aging population with both information access and
disposable income to pursue nutraceutical products;
 an increased acceptance and recommendation of nutraceutical-based products by the
medical establishment;
 products and marketing characterized by higher quality, more extensive scientific
documentation, and broader retail distribution; and affordability.
The primary consumers of nutraceuticals come from a range of backgrounds but often are
female, well educated, reasonably affluent, and middle aged (35 to 55 years [yr] (Childs,
1997). Product development tends to follow the scientific discoveries and health states that
preoccupy consumers. Scientific consensus recognized by a formal U.S. Food and Drug
Administration (FDA)-accepted health claim, such as the soy protein health claim for
cardiovascular health and the calcium osteoporosis claim, is believed to be a marketing
advantage; therefore, companies have targeted product development and reformulation to use
health claims and nutrient content claims on their food labels. This phenomenon emphasizes
the intimate and interconnected relationships among nutraceutical discovery and

documentation, product development, regulatory policy, and consumer behavior.
HEALTH BENEFIT OF NUTRACEUTICAL

Nutraceuticals are very beneficial for health. Various health benefits of nutraceuticals are
described in Table 7.
Table 7. List of some common chemical compounds isolated from plants used as
Nutraceuticals (Pandey et al., 2011)
S. No Chemical Properties
compounds/source
1 Allicin from Allium It is a powerful antifungal antibacterial. It has been shown to be an
sativum antioxidant and has been used to treat arteriosclerosis and serum
cholesterol.
2 Betaine (Trimethyl Reduces toxic buildup of
Glycine) from green leafy homocysteine.
vegetables and germinated
grains
3 Bromelain from Ananas It is pineapple protease enzyme used to prevent heart disease,
sp. reduce the effects of aging, improve the immune system, and to
reduce arthritis and inflammation.
4 Camphor from Used as an inhalant to treat
Cinnamomum camphora cold and flu.
5 Capsaicin or trans 8- Used for pain relief topically and as a digestive aid when taken
methyl- N-vanillyl-5 internally. It is also seen as a possible antioxidant for the body. It
nonenamide from can pose a risk of allergic reactions and the severe damage to the
Capsicum annum eyes or skin if used in higher doses.
6 Carnitine or L Carnitine Responsible for the transportation of long-chain fatty acids groups
from Asparagus into the mitochondria..
7 Ellagic Acid from this phytochemical fights
strawberries and cancer in humans.
raspberries
8 Ricinoleic acid from Contains ricinoleic acid the active ingredient. Castor oil is used
Castor both externally (multiple skin problems) and internally for
Oil or Ricinus communis constipation, upper respiratory problems, and liver and kidney
issues.
9 Chocolate (a mixture of Have positive effects on the heart and blood pressure due to the
cocoa obtained from flavonoids in chocolate. Chocolate also contains a
Theobroma cacao and neurotransmitter, serotonin, that acts as an antidepressant, and
vanilla from orchid) other
substances, such as theobromine and phenylethylamine. These
have
a stimulating effect.
10 Curcumin from Curcuma The colorant in turmeric a fraction of which has been shown by
longa studies done at the University of California in Los Angeles to clear
brain plaque
caused by Alzheimer's disease.
S. No Chemical Properties
compounds/source
11 Plant Glucosamine Chondroitin and glucosamine are part of normal cartilage and acts
as a cushion between the joints.
12 Glutathione (GSH) A tripeptide, which provides antioxidant properties thereby
protecting the cells against damage by free radicals.
13 Hesperitin Hesperitin is a GRAS ingredient that shows interest as a potential
anti-inflammtory.
14 Hydroxy Citric Acid Hydroxy Citric Acid found in Garcinia
15 Isoquercitin from Increases blood flow for varicose veins, and possible use for
mangoes and from Rheum arterial flow as well. Recent studies have shown possibilities in
nobile increased brain functions and it might be useful in the treatment of
(Enzyme Modified) progressive Alzheimer's disease.
16 Licorice or Glycirrhizic Licorice has been used as early as Roman and Greek times as a
acid decongestant, antiinflammatory, to treat stomach ulcers..
17 Lignan from rye, soybean Lignans are one of the two major classes of phytoestrogens.
and brocaoli Phytoestrogens are antioxidants and have been viewed as reducing
ill effects in the body as cellular destruction, aging, etc.
18 Lutein and Lutein Esters Extracted from marigold seeds, and also found in spinach,
from marigold rosemary and kale, it is a carotenoid which shows
healthful eye benefits.
19 Nattokinase Natto is Nattokinase is the enzyme produced by Bacillus natto used in the
fermented soybeans. fermentation.
20 Olive Oil from Olea Olive oil is high in monounsaturated fat and is a healthy oil in
europaea maintaining good cholesterol levels.
21 Omega 3 Fatty Acids from Among other positive effects (see rest of chart), omega 3 fatty
Linum usitatissimum acids have been associated with positive eye health.
22 Phloretin isolated from Obtained from the decomposition of phloridzin and used in the
apple leaves treatment of malaria as a quinine replacer. Studies have shown it
inhibits protein kinase C and effects the sodium/potassium transfer
across membranes.
23 Phytosterol obtained from Chemicals found naturally in foods that have the ability to lower
germinated corn cholesterol absorption in the digestive tract thereby lowering
overall cholesterol levels in the bloodstream.
24 Proanthocyanins from Help with urinary tract infections by inhibiting adhesion of
grapes microorganisms like e. coli to the urinary tract wall.
25 Resveratrol especially anti-inflammatory, inhibits COX-1 enzyme, blocks adhesion of
high in grape skin blood cells to vessel walls shown to reduce skin and breast cancer
26 Tall oil Derived phytosterols Has been seen to reduce arteriosclerosis, and
plasma cholesterol in rodents.
27 Zeaxanthin A carotenoid used as an antioxidant. Zeaxanthin is the coloring
agent in marigolds and is extracted from them. It is used for eye
health and some claim will retard the effect of 'aging eyesight' or
Age-Related Macular Degeneration (AMD).
THE FUTURE OF NUTRACEUTICALS

Increasing awareness levels about fitness and health, spurred by media coverage are
prompting the majority of people to lead healthier lifestyles, exercise more, and eat healthy.
The expanding nutraceutical market indicates that end users are seeking minimally processed
food with extra nutritional benefits and organoleptic value. In India, dietary supplements hold
the largest share, while the functional foods and beverages segment is relatively smaller.
Dietary supplement products are manufactured predominantly by pharmaceutical
manufacturers; pure play nutraceutical companies have also started to develop vitamin and
mineral supplements in this region. Fortified foods market is projected to grow at a rapid pace
with a compound annual growth rate, (CAGR) of 21.7 percent by 2018, as FMCG companies
have started entering the market with different product portfolio for functional food and
beverages. Vitamins and minerals hold 36 percent of the total Indian nutraceutical market,
followed by probiotic with a 9-percent share and omega-3 fatty acids with a share of 5
percent. This development, in turn, is propelling expansion in the nutraceutical markets
globally. The emerging nutraceuticals industry seems destined to occupy the landscape in the
new millennium. Its tremendous growth has implications for the food, pharmaceutical,
healthcare, and agricultural industries Many scientists believe that enzymes represent another
exciting frontier in nutraceuticals. Fermentation technology using microbes to create new
food products also represents potential. Global trends to healthy products cannot be reversed.
Companies taking the lead by investing strategically in science, product development,
marketing and consumer education will not go unrewarded.
CONCLUSION
The nutraceutical industry is growing at a rate far exceeding expansion in the food and
pharmaceutical industries. In tomorrow‘s market, the most successful nutraceutical players
are likely to be those companies in which functional product are just a part of a broad line of
goods satisfying both conventional and health value point. Future demand of nutraceutical
depends on consumer perception of the relationship between diet and disease. Although
nutraceuticals have significant promise in the promotion of human health and disease
prevention, health professional, nutritionists and regulatory toxicologist should strategically
work together to plan appropriate regulation to provide the ultimate health and therapeutic
benefit to mankind. Long-term clinical studies are required to scientifically validate the
nutraceuticals in various medical conditions. The interaction of nutraceuticals with food and
drugs is another area, which should be taken into consideration. The effect of different
processing methods on the biological availability and effectiveness of nutraceuticals remains
to be determined. As like drugs, there should be strict regulatory controls for nutraceuticals.
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Chapter 14
NUTRITIONAL GENOMICS:
NUTRIGENOMICS AND NUTRIGENETICS
Eshu Singhal Sinha, Radhika Mohan and Anu Mehta

Animal Biochemistry Division,
National Dairy Research Institute, Karnal, India
ABSTRACT
It has been a known fact for many years that the constituents of food have the
capability to improve health. The emerging science of nutritional genomics that aims at
using food to prevent and cure diseases has revolutionized the field of health and
nutrition. However, determining the cause or treatment of a disease cannot be conducted
based on the studies done in a small population due to variations in the genes at an
individual basis. Furthermore, due to variations in the genetic makeup; the use of
pharmaceuticals is not equally efficient in different individuals suffering from a similar
disease. Hence, understanding the influence of differences in genetic makeup on the
health and disease is very crucial. Accomplishing this complex task is one of the goals of
nutrigenetics, a branch of nutritional genomics. Another important aspect of this
emerging science pertains to the establishment of - how the diet affects the gene
expression and thus, the health of a person. The branch of nutritional genomics that
attempts to fulfill this aspect is known as nutrigenomics. Both of these sciences work in
parallel to accomplish the common goal of nutritional genomics i.e., improving health
through diet management and the use of healthy foods, also known as nutraceuticals.
Although nutritional genomics is at its beginning, it has been implicated in improving
skin health, discovering the genes involved in a disease and in the management of
diseases including phenylketonuria, obesity, cardiovascular diseases (CVD) etc. The
rapid progress achieved in the field has generated a hope that nutraceuticals will replace
pharmaceuticals in some, if not all, diseases in the near future.
Keywords: nutrition, genomics, nutrigenomics, nutrigenetics

Corresponding author: Biochemistry Department, Kurukshetra University, Kurukshetra 136119, Haryana, India.
Email-anubtk.oct@gmail.com; Tel-(+91)-9729702989.
332 Eshu Singhal Sinha, Radhika Mohan and Anu Mehta
INTRODUCTION
Nutritional genomics is the branch of science that attempts to study the relationship
between nutrition, genome and health (Figure 1) (Ziesel, 2011; https://en.
wikipedia.org/wiki/Nutritional_genomics). The effect of food on health occurs mainly due to
the regulation of gene expression by the molecular constituents of the diet. This is usually
mediated via modulating the activation or inhibition of transcription factors involved in
regulating gene expression. The completion of Human Genome Project has facilitated
nutritional genomic studies by providing a documented framework for single nucleotide
polymorphisms (SNPs) in candidate genes and determining their association with metabolic
imbalances (Subbiah, 2010). Based on the documented data for the involvement of a
particular gene in a metabolic disease, nutritional genomics can offer a preventive or curative
solution for that disease through modulation of diet. The science of nutritional genomics is
based on the following basic facts as provided by the Center of Excellence for Nutritional
Genomics at the University of California, Davis (http://www.nutrigenomics.ucdavis.edu).
1. The diet has some bioactive components that act on the genome leading to a change
in gene expression (Muller and Kersten, 2003).
2. Diet can be a risk factor for certain diseases (Muller and Kersten, 2003).
3. Some of the genes that are involved in the onset or progression of a disease can be
regulated by food constituents (Muller and Kersten, 2003).
4. An individual‘s genetic makeup can be responsible for his susceptibility or resistance
to a particular disease (Ziesel, 2007).
5. Based on the information about diet-regulated genes, an individual‘s genetic makeup,
and susceptibility to a disease, the modulation of the diet of a person can be used for
prevention, cure or management of a disease (Ferguson, 2014). Nutraceuticals can be
incorporated in the diet to serve this purpose. The word nutraceutical is a
combination of two words; ―nutrition‖ and ―pharmaceutical.‖ This term was coined
by Stephen L DeFelice in 1989 and refers to foods that provide health benefits to the
consumer.
Figure 1. Defining nutritional genomics.

Nutritional Genomics 333
Earlier, nutrition science emphasized more on elucidating the function of various

nutrients, nutritional deficiency diseases and their cure. With the introduction of high-
throughput -omics technologies, the science of nutrition opened an unparalleled scope and
opportunities to understand health disorders (Zhang et al., 2008). However, contrary to our
expectations the growth of this science is quite slow. The major focus of nutrition science is
to prevent chronic health disorders like CVDs, obesity etc. Therefore, using genetics and
genomics, we aim to understand the regulation of genes associated with the progression of
these diseases under the normal health conditions and how these genes expressed and their
regulation changes in the presence of a diet based bio-active components on an individual
basis. Hence, it requires the usage of high-throughput research tools. For instance, the
genome and transcriptome studies involve the use of microarray technology; proteome
analysis generally involves 2D-gel electrophoresis or Liquid chromatography-mass
spectrometry (LCMS). Metabolome studies are generally done by gas chromatography-mass
spectrometry (GCMS), liquid chromatography-nuclear magnetic resonance or LCMS. It is no
surprise that nutritional genomics involves the use of proteomics and metabolomics, since the
gene regulation can be well understood only if its products (proteins) and metabolism is
known at a depth. Further, the integrated use of these –omics technologies is helpful to
convert the data into meaningful knowledge through the use of bioinformatics (Zhang et al.,
2007). Following sites provide extensive reading resources for nutritional genomics:
 National Human Genome Research Institute (http://www.genome.gov).

 US Department of Energy Genome Programs (http://www.doegenomes.org).
 Center of Excellence for Nutritional Genomics at the University of California, Davis
(http://www.nutrigenomics.ucdavis.edu).
 Center for Human Nutrigenomics at Wageningen University, The Netherlands
(http://www.nutrigenomics.nl).
Nutritional genomics has been categorized under two areas (Ordovas and Mooser, 2004):
a. Nutrigenomics: It involves the study of the effect of diet (dietary constituents in

particular) on the genome.
b. Nutrigenetics: It covers the study of the influence dietary constituents on the
variation of an individual‘s genetic makeup on his susceptibility or resistance to a
disease.
Nutrigenomics
Nutrigenomics is the branch of nutritional science which deals with the study of the effect
of dietary nutrients on the genome, proteome (the sum total of all proteins), and metabolome
(the sum of all metabolites) at a global, population level (DeBusk et al., 2005; Mutch et al.,
2005). The change in gene expression leads to modulation of protein expression which is
finally translated into a change in metabolism of the nutrients in the body. Different bioactive
constituents of the diet exert differential influence on gene and protein expression which
results in altered metabolite production. Nutrigenomics usually involves understanding the
effect of a dietary constituent on thousands of genes or proteins simultaneously. It is a means

to establish a rationale between dietary intake and the induction/inhibition of a diseased
situation.
It aims to explain how food modulates the genome and how the dysregulation of gene
expression is translated to a disease. In short, nutrigenomics provides a means to study the
effect of diet on health via regulation of gene expression. Thus, the genome, transcriptome,
proteome and metabolome analyses are done by comparing healthy with diseased situation
(Figure 2). This also eases analyses of complex data by focusing mainly on the differences
between healthy versus diseased situation.
However, to study the effect of nutritional components on gene regulation, 2 approaches
are adopted for execution of experiments: direct and indirect. In the direct approach, a model
system is chosen for study and gene expression changes are recorded and analyzed with
nutritional variations. But, in the indirect approach, two subjects are taken into account,
healthy and diseased. And the gene expressions associated with various metabolic disorders
are analyzed in both the subjects and finally compared with the healthy subject, considering it
as a control for the experiment. The model systems used in either of the approach used may
vary from invitro to invivo. e.g., In vitro well characterized cell cultures, small animals
(mouse, rat, pig) etc. can be used for the studies. In other cases, nematodes like C. elegans are
increasingly used. Cell lines used for the studies provide a comprehensive way to understand
the effect of nutrient/s on gene expression, but the results obtained in these studies cannot be
applied as such, since they lack the natural complexity of the organ system. In cases, human
biopsies are also used, but due to medical or ethical issues researchers have found other ways
to make the studies easier and relevant too. Transgenic, knock-out and knock-in mice provide
an attractive animal based study approach. This is well elucidated by ApoE3L mouse, in
which human ApoE gene has been introduced and thus, has a more human – like lipid profile.
This model is preferably used for gene-diet interaction in context of metabolic syndromes
(Santo et al., 2004).
Figure 2. Techniques used to study nutrigenomics.

The implications of nutrigenomics to polygenic diseases, i.e., the diseases that involve
mutation or dysfunctioning of multiple genes, e.g., cancer, diabetes etc. is a difficult task.
However, most of the diseases that occur due to mutation or dysfunction of any gene are
monogenic in nature, i.e., these involve a single gene. Applying the modifications in dietary
intake to prevent monogenic diseases has been successful in some cases. For instance, due to
defective phenylalanine hydroxylase (PAH) enzyme in phenylketonuria patients; there is
impairment of the metabolism of phenylalanine to tyrosine. This leads to accumulation of
phenylalanine with a simultaneous decrease in tyrosine in the blood due to which there is a
risk of neurological damage (Folling, 1934). However, providing phenylalanine-restricted diet
to the people suffering from phenylketonuria along with increasing the intake of tyrosine rich
diet offers a nutritional solution to this disease (Gillies, 2003).
Although this science is complex and is at its beginning, it holds promise for providing
solution to prevent many diseases by defining health promoting foods and the development of
functional foods capable of disease management. It will establish scientific reasons to
rationalize the effect of the dietary constituents on health.
Role of Nutrients on Regulation of Gene Expression
Gene is the basic physical and functional unit of heredity. Primarily, the information
expressed by genes is located in the DNA sequence. Gene expression is a tightly regulated
process. This regulation is mediated by regulatory molecules which bind to the gene promoter
elements.
Figure 3. Ways by which diet nutrients regulate gene expression.
The binding of the intracellular regulatory molecules itself depends upon various
biomolecules/nutrients. Since the food is a source of various biomolecules, it is obvious that
nutrients play a role in gene expression. The dietary components can have direct or indirect
effect on the gene expression (Cousins, 1999), as shown in Figure 3. Vitamins derived
metabolites for instance; retinoic acid and calcitriol have a direct effect on gene expression.
The indirect effect of dietary components on gene expression is mediated through secondary
metabolites. e.g., butyrate, which is a short-chain fatty acid (SCFA), produced as a result of
fermentation by intestinal gut flora, is also a Histone Deacetylase Inhibitor (HDACi) and thus
known to influence expression of numerous genes in an epigenetic manner (Lea et al., 2010).
It also modulates cell signaling mechanisms (Shi et al., 2014). Here below, we explain the
prototype example for direct and indirect effects of nutrients on the expression of genes:
Direct: Vitamin D is itself not biologically active vitamin. In the liver and kidney, it is
converted into active 1, 25 (OH)2D3 by enzymes. This active form via cellular Vitamin D
receptor (VDR) binds as Retinoid X Receptor (RXR) heterodimer to Vitamin D response
elements present in the regulatory regions of the gene (Kerner et al., 1989; Ohyama et al.,
1996). This in turn allows the binding of various co-regulatory complexes which either turn
the gene- ON/OFF. Since, the active form of vitamin is directly involved in binding the
cellular receptors, thus it is directly involved in regulating the gene expression.
Indirect: Dietary fibre is the best prototypical example. Deciphering the effects of dietary
fibre is a difficult task as they are indirect and involve a number of other factors. Beneficial
health effects of high-fibre foods are well elucidated. Butyrate as mentioned earlier represents
one of the best examples to understand the indirect effect of nutrients. It is produced by the
fermentative action of microbes on the dietary fibre itself. It mediates its effects through G-
protein activation, which activates the down-stream signaling pathways (Wang et al., 2012).
This ultimately affects a number of gene expressions in an epigenetic manner.
Applications of Nutrigenomics
Nutrigenomics aims to study the genes whose expression changes by different nutrients
or different levels of nutrients are identified followed by study of the mechanism of their
action on gene regulation. This information has subsequently been exploited to understand the
development of diseases associated with that particular gene.
1. Nutrigenomics in anti-ageing: Free radicals are formed in the body due to exposure
to rays (particularly UV and γ rays), pollution, lack of healthy diet and most
importantly due to stress. Accumulation of excess free radicals in the cells leads to
ageing. Nutrigenomics provides a solution to prevent ageing of cells by incorporating
dietary supplements enriched with anti-oxidants and other combination of nutrients
to decrease the free radicals present in the cell either by scavenging the free radicals
or by increasing the elimination of free radicals from the cells (a blog by Leroy
Rebello at www.eternesseclinic.com).
2. Nutrigenomics in discovering the cause of diseases: Sitosterolemia is a disease
characterized by hyper-absorption and decreased excretion of dietary sterols leading
to hypercholesterolemia and accelerated atherosclerosis. In a nutrigenomic study
using a lipid metabolism altering drug, an unknown gene was discovered which
produces proteins involved in reverse transport of dietary sterols out of the intestinal
cells. Later on it was recognized as ABCG8 transporter gene. Hence, nutrigenomic
studies are very promising in discovering the genes associated with diseases as
evident from the establishment of mutation in ABCG8 gene as the cause for
sitosterolemia (Mutch et al., 2005).
3. Nutrigenomics in skin health: The use of nutragenomics has also been extrapolated to
skin health. The branch of nutragenomics or more accurately, the off-shoot of
nutrigenomics that deals with skin is known as dermagenetics. Although the topical
application of cosmetics and cosmeceuticals is considered beneficial for skin health,
skin tissue has also been demonstrated to derive benefits by oral administration of
nutraceuticals. In dermagenetics, the main focus is laid down on the genes involved
in the regulation of enzymes meant for the generation of inflammation, degradation
of pollutants and the breakdown of collagen (Subbiah, 2010).
4. Knowledge based functional foods: Nutrigenomics offers the knowledge for the
development of functional foods. Since nutrition can be employed in a natural way,
thus, nutrition based functional foods are easily accepted publicly and helps in
disease prevention among the masses (van der Greef et al., 2004).
Nutrigenetics
Brennan in 1977 introduced the term nutrigenetics, in his book entitled ―Nutrigenetics:
New Concepts for Relieving Hypoglycemia.” Nutrigenomics deals with a much broader scope
of interactions between nutrition and genome, whereas nutrigenetics provides an agenda for
personalized nutrition by understanding the genetics of the person. Nutrigenetics is the study
of influence of variations in individual genetic makeup on the response towards diet and the
susceptibility to diet-related diseases (Ordovas and Mooser, 2004). As observed in our daily
life, similar diet intake by two individuals experience different health outcomes. It is the
genetic makeup of an individual that is responsible for determining how he responds to the
dietary constituents. However, genome studies show that we are 99% genetically identical.
There is just 1% genetic variation among individuals which arise due to copy number
variation, indels, repeat sequences, SNPs or chromosomal aberrations. This 1% of genetic
variation, SNPs in particular, is responsible for the difference in the response to a dietary
constituent and leads to variations in health of individuals (Mutch et al., 2005).
Response to nutrition is multigenic, since digestion and assimilation of a particular kind
of nutrient involve a series of enzymes which are encoded by different genes. These enzymes
influence nutrient digestion, absorption, transport, biotransformation, uptake and elimination.
Permutations and combinations of factors responsible for genetic variations (copy number
variation, indels, repeat sequences, SNPs or chromosomal aberrations) associated with each
gene are huge. So, one can easily imagine how much variation in the biochemical action of a
single nutrient in different individuals exists. To have a good understanding of nutrigenetics,
knowing the candidate genes responsible for nutrient utilization becomes important. These
candidate genes are highly sensitive to diet and identified using a set of parameters in
particular.
Gene polymorphism encompasses qualitative and quantitative genetic variations.
Qualitative gene polymorphism includes changes associated with a gene in terms of sequence,
e.g., SNPs, small nucleotide indels and duplications. SNPs refer to alterations of single bases
in the DNA. These qualitative variations can affect regulatory regions or introns or exons of a
gene. Whereas, quantitative gene polymorphism includes large duplications or deletions that
affect the copy number of a gene and hence gene expression levels in a direct manner. In
nutrigenetics, the studies are conducted based on ‗Hypothesis-driven‘ approach. Here,
candidate genes involved in the metabolism of a nutrient in particular are taken into
consideration for studying the effect of a nutrient on different allelic forms of the gene. This
is well-exemplified by a diabetes mouse model db/db, from which leptin receptor encoding
gene was identified and then human homologue of this gene was tested in persons with
impaired glucose tolerance (Wauters et al., 2 1). However, now days, a new ‗holistic‘
approach has been introduced. Herein, the total genome is scanned for finding the risk-
conferring genes for a certain disorder. Gene polymorphism is initially found on a specific
chromosomal region and then attempts are made to detect whether a particular gene allele is
associated with a certain trait or not. Thus, this methodology not only helps to detect at least
one risk contributing gene per locus associated with a particular diseased trait, but also offers
the possibility to explain, inter-individual differences in risk at a given geographical location
(Cheverud et al., 2004). This in turn can offer individual based nutritional therapy.
Nutrigenetics in Translational Health
Tailoring food choices by measuring the unique genetic makeup requires an intervention
of nutrigenetics. Here below we have discussed applications of nutrigenetics in translating
health issues (Figure 4).
1. Metabolic diseases: Nutrigenetics identifies how the genotype of a particular

individual co-ordinates with response to various dietary nutrients. Monogenic
diseases, e.g., Phenylyketonuria (PKU), lactose or galactose intolerance due to lack
of the enzyme lactase, galactosemia due to lack of a liver enzyme to digest galactose
etc. can be modified by modifying dietary intake. In PKU, food containing the amino
acid phenylalanine, including high protein food such as fish, hen eggs, milk, cheese,
dried beans, nuts, and tofu must be avoided (Gillies, 2003). In case of defective
aldehyde dehydrogenase enzyme, alcohol must be avoided. Milk products, while, in
case of lactose patients should be avoided.
2. Cancer: Advanced genetic analysis has been able to explain the basis of complex
traits and the role of individual genotypes on the development of polygenic diet-
related diseases such as cancer. The genetic polymorphisms lead to, alteration of the
response to the dietary components by influencing absorption and metabolism.
Epigenetic events can induce changes in DNA methylation pattern and thus
influencing overall gene expression that can be modified in response to the food
components. The completion of the Human Genome Project has provided a vast
amount of data on inherited genetic variability. Studies have shown polymorphism in
folate metabolism was linked to risk of carcinogenesis. Genetic characteristics can
help clarify whether certain individuals may benefit from higher or lower intakes of
folate or nutrients relevant to folate metabolism (Ulrich, 2005). Polymorphisms in
5,10-methylene-tetrahydrofolate reductase (MTHFR), thymidylate synthase (TS),
methionine synthase, methionine synthase reductase, serine-hydroxymethyl-
transferase, cystathionine β synthase, methylene-tetrahydrofolate dehydrogenase, and
glutamate carboxypeptidase have been investigated in relation to the risk of
colorectal cancer or polyps. Evidences have supported that dietary factors can
interact with hormonal regulation and polymorphisms of gene encoding factors
involved in hormonal regulation are most strongly manifested in hormone dependent
tumors such as breast (Koh et al., 2003), prostate (Yuan and Ferguson, 2011),
ovarian and endometrial cancers (McCann et al., 2010).
3. CVDs: CVD is the primary diet-related chronic modern lifestyle disease

characterized as a group of multifactorial conditions associated with obesity,
atherosclerosis, hypertension, and thrombosis which are closely related to both
genetic factors and environmental influences. Polymorphic genes involved in energy
balance control provide the causes for the development of CVD. Genetic variation in
genes encoding for apolipoproteins, enzymes and hormones can alter individual
sensitivity to develop the CVDs. Studies have shown that some of these variants are
susceptible for dietary intervention, for example: individuals with the E4 allele in the
apolipoprotein E gene show high low-density lipoprotein-cholesterol (bad
cholesterol) levels (Loktionov, 2003).
4. Diabetes: The role of diet and lifestyle in the prevention and management of type 2
diabetes has been increasing at a rapid pace. Studies have found several
polymorphisms strongly associated with type 2 diabetes risk which may be modified
by diet e.g., a SNP in the transcription factor 7-like 2 protein gene has the strongest
association with type 2 diabetes (Florez, 2007). Several other genes have been
associated with type 2 diabetes risk include the sterol response element binding
protein, the peroxisome proliferator-activated receptors (PPAR), and the intestinal
fatty acid binding protein (Mutch et al., 2005). Effective diets to reduce type 2
diabetes risk in people with specific genotypes are still the subject of research.
5. Obesity: Obesity is a complex disease resulting from a chronic and long-term
positive energy balance in which both genetic and environmental factors are
involved. Dietary components not only fuel the body, but also participate in the
modulation of gene expression. Multiple polymorphic genes in central and peripheral
determinants of energy intake and expenditure have been researched recently. Food
intake control may be affected by polymorphisms in the genes encoding taste
receptors and a number of peripheral signaling peptides such as insulin, leptin,
ghrelin, cholecystokinin, and corresponding receptors (Loktionov, 2003). Studies
have demonstrated the expression pattern and nutritional regulation of several
obesity-related genes, as well as those that are differentially expressed by caloric
restriction e.g., the gene associated with fat mass and obesity (FTO) to be
significantly related to obesity. Certain lifestyles and diets reduce the incidence of
obesity even if individuals have the higher-risk genotype for the FTO gene or other
genes linked to obesity.
Networking in Nutrigenomics and Nutrigenetics
Since, nutrigenomics and nutrigenetics studies are based on enormous novel data sets,
thus data handling, acquisition and analyses are quite important to assure noteworthy use of
the data. In this view, the European Community has financed a network of excellence with
the name NuGO (www.nugo.org). This has been a noteworthy initiative to form a world wide
web of these two nutrition based branches of science.
Figure 4. Applications of nutrigenetics.
FUTURE PERSPECTIVES
Future scope of nutritional genomics lies in the application. The concept of nutraceuticals
is the best future scope that is replacing the conventional food products in the global food
market. The health benefits of these nutraceuticals can be either prevention or cure from a
disease. It includes nutrients, proteins, vitamins, minerals, cereals, soups, dietary supplements
and other healthy benefitting processed foods. Nutraceuticals are gaining importance and are
becoming part of a person‘s life worldwide. This is due to a worldwide increase in the diet-
related-diseases due to the consumption of non-healthy fast foods. Nutraceuticals are also
commonly called as functional foods as these can be used as a tool to cure or prevent
diseases. Thus, nutraceuticals deserve a special place in the field of nutritional genomics.
CONCLUSION
Food is the basic necessity of life and is known to provide health benefits to us. Despite
this, incidences of food-related diseases like CVDs, obesity, type 2 diabetes mellitus etc. are
increasing worldwide. This is due to decrease in physical activity and consumption of
unhealthy food as a result of changing lifestyles. Furthermore, some foods are believed to
provide protection from some diseases. For example, Azadirachta indica is beneficial in
diabetes. However, the clear molecular mechanism associated with food-mediated induction
or prevention of diseases is still ill defined. This problem is further aggravated by the
commonly observed incidences where two people of the same age group have the same food
habits and the lifestyle, but still one of thus acquires a disease but other does not. All these
observations clearly imply that the difference in the genetic makeup of two people is
responsible for this. Together, these facts lay the foundation for nutritional genomics.
Nutritional genomics believes in using food as an alternative for pharmaceuticals and hence,
the health promoting food is referred to as nutraceutical or functional food. The two branches
of nutritional genomics; nutragenetics and nutrigenomics have different approaches which,
when taken together hold strong promises to use nutraceuticals for prevention, cure or
efficient management of diseases. Notably, nutritional genomics have proven to be beneficial
in skin healing, anti-ageing, for discovering the genes associated with diseases like
sitosterolemia and in diseases like type-2 diabetes, phenylketonuria. Although this science is
at its beginning and is complex and ambitious, it offers a great solution of modulating diet for
being healthy.
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INDEX
additives, 4, 11, 17, 89, 110, 116, 124, 125, 128, 143,
# 146, 147, 150, 151, 157, 158, 171, 175, 184, 185,
186, 220, 222, 227, 234, 251, 255, 256, 257, 260,
2,3-butanediol, 300
284, 294
20th century, 26, 288
adenine, 227
adhesion, 34, 37, 40, 325
α adolescents, 167
adsorption, 31, 259
α-amylase, 32, 34, 165, 221, 225, 226, 235, 237, 239, adverse effects, 75, 76, 235
240, 242, 250, 301 aerospace, 254
aesthetic, 119
affluence, 156, 323
β aflatoxin, 144
agar, 189, 190
β-carotene, 120, 124, 125, 128, 129, 130, 134, 136, agglutination, 35, 201
137, 138, 140, 141, 142, 144, 145, 146, 149, 151 aggregation, 141, 299, 314
β-galactosidase, 21, 134, 140, 285 aging population, 323
aging process, 22
agriculture, 65, 96, 107, 108, 112, 231, 254, 280
A agrobacterium, 102, 103, 113, 129
agro-industrial wastes, 277
acetaldehyde, 5, 15, 289, 298, 299
AIDS, 39
acetic acid, 2, 3, 4, 15, 18, 47, 50, 83, 274
alanine, 239
acetoin, 2, 78, 298, 299, 300
albumin, 166, 167, 169, 170, 190
acetolactate decarboxylase, 300
alcohols, 2, 17, 21, 139, 285, 294, 298, 306
acetone, 134, 139, 140, 229
aldehydes, 17, 19, 77, 285, 294, 300
acetonitrile, 208
alfalfa, 123, 124, 199, 213, 215, 317
acetylcholine, 313
algae, 118, 122, 124, 128, 129, 132, 137, 148, 234,
acne, 293, 295, 306
272, 278, 318, 320
action of probiotics, 27, 30, 38
alkaline phosphatase, 199
active compound, 144, 257, 259
alkalinity, 220, 222
active dry yeast, 287
alkaloids, 318
active site, 243
alkanes, 274
active transport, 263
alkenes, 274
acute mountain sickness, 327
allantoin, 278
acylation, 143
allele, 338, 339
adaptation, 4, 31, 44, 79, 89
allergens, vii, 101, 112, 155, 156, 157, 158, 159,
adaptive immunity, 21
160, 161, 162, 163, 164, 165, 166, 167, 171, 172,
344 Index
173, 175, 177, 178, 179, 180, 181, 182, 183, 184, apoptosis, 303
185 appetite, 293, 315, 317
allergic reaction, 35, 41, 42, 101, 102, 123, 157, 164, appropriate technology, 236
166, 167, 171, 172, 175, 179, 324 aquaporin, 297, 309
allergic rhinitis, 44 arabinoxylo-oligosaccharides, 32, 44
allergy, 22, 143, 151, 155, 156, 157, 158, 161, 164, arginine, 174, 182, 222, 297, 300
166, 167, 177, 179, 180, 181, 182, 183, 184, 185, aromatic compounds, 62
186 arteriosclerosis, 324, 325
alloploid, 296 arthritis, 215, 277, 318, 324
altitude sickness, 321 ascorbic acid, 17, 224, 321
aluminium, 117 aseptic, 290
amines, 87, 117, 131, 222, 257, 298 asparaginase, 221, 224, 225, 237, 238, 243, 244, 246,
ammonia, 57, 127, 222, 224, 225, 241, 247, 274, 249
287, 289 asparagines, 225
ammonium, 123, 127, 142, 274, 286, 287, 289 aspartame, 175, 244
ammonium salts, 127, 274, 287, 289 aspartase, 220, 221, 222, 223, 237, 239, 240, 242,
amplitude, 206 243, 244, 249, 250, 251
AmulProLife Probiotic Dahi, 30 aspartate, 222, 225, 241, 242
amyotrophic lateral sclerosis, 322, 328 aspartic acid, 221, 222, 224, 239, 243, 246, 247, 250
anaerobic bacteria, 18 Aspergillus terreus, 241, 280
analgesic, 315 ASS, 242
anaphylactic reactions, 185 assay, 104, 150, 176, 177, 186, 189, 191, 192, 193,
anaphylactic shock, 123 194, 195, 197, 198, 199, 201, 202, 209, 211, 212,
anaphylaxis, 155, 157, 175, 182, 185 213, 214, 215, 216, 244
aneuploid, 296 assessment, 108, 110, 113, 115, 120, 179, 266, 267,
angioedema, 171 301, 342
angstroms, 264 assimilation, 37, 277, 292, 307, 337
aniline, 117 asthma, 22, 155, 156, 171, 184, 186, 318
annatto, 120, 121, 125, 134, 147, 150, 151, 175 astringent, 315
annealing, 191 ATF1, 299
anthocyanidins, 125, 126, 128 atherogenesis, 328
anthocyanin, 118, 126, 134, 135, 136, 140, 144, 151, atherosclerosis, 145, 336, 339
154 atomic force, 270
antibiotic, 26, 37, 91, 94, 101, 161, 316, 330 atopic dermatitis, 37, 43, 44, 186
antibiotic resistance, 26, 101 atopic eczema, 37, 315
antibody, 104, 172, 176, 177, 179, 183, 185, 187, ATP, 3, 166, 200, 212, 289
198, 201, 204, 207, 211, 212, 276 attachment, 191
anti-cancer, 47 authentication, 47, 258
anticancer drug, 306 authorities, 76, 82, 146, 187
antidepressant, 315, 324 authority, 172
anti-foam, 289 autoimmunity, 37
antifreeze gene, 100 autolysis, 298
antifreeze proteins, 297 avian, 167, 195
antigen, 177, 191, 198, 280
antimicrobial, 1, 4, 9, 12, 18, 23, 26, 27, 28, 30, 35,
36, 44, 75, 76, 77, 78, 80, 81, 82, 83, 88, 89, 91, B
92, 93, 94, 167, 169, 170, 175, 206, 213, 254,
bacillus, 26
255, 256, 257, 263, 265, 267, 268, 295, 315
Bacillus subtilis, 12, 57, 61, 223, 226, 231, 242, 245,
antioxidant, 17, 144, 145, 150, 152, 154, 175, 182,
248
227, 255, 300, 313, 314, 318, 319, 321, 322, 324,
bacterial cells, 31, 222
325, 327, 328, 329
bacterial colonies, 189
antipyretic, 315
bacterial fermentation, 40
antitumor, 170, 317
bacterial pathogens, 192, 201, 203, 206, 212, 213
Index 345
bacteriocins, vii, 3, 12, 18, 21, 35, 42, 75, 76, 77, 78, biomolecules, 205, 335
79, 80, 81, 82, 83, 85, 87, 88, 89, 90, 91, 92, 93, biopolymer(s), 256, 257, 258, 260, 262, 269, 285
94, 295, 300 biopreservation, 78, 80, 89, 90, 91, 94
bacteriophage, 190, 202, 211 biosafety, 146, 301
bacteriostatic, 76 biosensor(s), 172, 175, 178, 179, 187, 188, 204, 205,
bacterium, 98, 129, 136, 151, 239, 241, 244 206, 207, 209, 210, 211, 212, 213, 214, 215, 216,
bactibase, 81 217, 251, 255
baking, 219, 220, 221, 223, 224, 225, 226, 228, 231, biosynthesis, 79, 80, 94, 125, 138, 149, 150, 151,
235, 236, 243, 263, 284, 285, 286, 287, 288, 292, 246, 298
295, 297, 300, 303 biotechnological applications, 90, 223, 237, 244, 245
barriers, 257, 260, 277 biotin, 281, 289, 295, 314
basophils, 177 birth weight, 37, 40
beer, 49, 51, 55, 71, 73, 74, 127, 132, 202, 221, 223, bleaching, 122, 143, 250
227, 232, 245, 264, 292, 293, 295, 299, 300, 301 blood, 35, 37, 38, 67, 68, 130, 169, 170, 177, 227,
beet molasses, 287, 288, 293 266, 277, 278, 295, 313, 314, 315, 324, 325, 326,
beetles, 98 335
beneficial effect, 27, 36, 78, 266, 294 blood clot, 313
beneficial microbes, 25, 43 blood flow, 315, 325
beta-carotene, 101, 124, 261, 321, 327 bloodstream, 256, 263, 325
betacyanins (betalains), 121, 126, 154 Blue Bird, 288
betanines or betalains, 121, 126, 154 blueprint, 97
beverages, vii, 5, 6, 22, 47, 48, 49, 67, 70, 71, 72, 73, body fat, 295
74, 77, 90, 92, 121, 122, 123, 125, 126, 127, 130, body weight, 317
132, 219, 220, 223, 248, 272, 284, 298, 299, 308, bone form, 313
323, 326 bone mass, 43
bicarbonate, 279 bone mineral content, 43
bicolour, 118 boosters, 318
bifidiobacterium, 26 botrytis, 60
bile, 26, 34, 37, 40, 314 bowel, 36, 40, 41, 44, 45, 293
bilirubin, 169, 170 brain functions, 325
bioavailability, 1, 13, 21, 61, 145, 254, 259, 261, 263 branched-chain amino acid transaminases, 298
biocatalysts, 220, 265 breakdown, 21, 33, 224, 232, 273, 337
biochemical action, 337 breast cancer, 317, 325, 329, 341
biochemistry, 17, 158, 182, 186, 241, 248, 302 breathing, 155
biocompatibility, 258 breeding, 96, 98, 103, 248, 272
bioconversion, 21, 231, 271, 272 brevis, 5, 6, 12, 15, 16, 28, 35, 61, 85, 88, 223, 306
biodegradability, 258 bronchospasm, 185
biodiversity, 109 budding, 305
biofuel, 227 buffalo, 62, 70, 85, 93
bioinformatics, 333 building blocks, 271, 273
biological activities, 203 Burkina Faso, 277
biological processes, 284 bursitis, 318
biological responses, 204 buttermilk, 27, 30, 53, 62, 64, 72
biological samples, 210 butyrate, 13, 335, 336, 342
biological systems, 151, 266 by-products, 48, 62, 135, 140, 149, 206, 220, 263,
biologically active compounds, 144 271, 272, 284, 293, 298
bioluminescence, 200, 212
biomarkers, 36
biomass, 135, 136, 142, 230, 242, 248, 272, 277, C
278, 279, 280, 283, 284, 286, 288, 289, 294, 296,
cabbage, 14, 15, 60, 71, 121, 125, 126, 318
300, 305, 307, 309
cacao, 324
biomaterials, 269
caffeine, 317, 319, 327
bioMerieux API, 292
calcium carbonate, 227, 319
346 Index
caloric restriction, 339 cell surface, 78, 263, 301, 302, 308
campaigns, 155 cell-based methods, 177
campylobacter, 209 cellulase, 220, 221, 230, 231, 232, 237, 239, 242,
cancer, 25, 36, 40, 47, 61, 145, 268, 280, 284, 295, 243, 244, 245, 247, 248, 251
310, 311, 317, 321, 324, 325, 328, 329, 330, 335, cellulose, 88, 135, 140, 221, 230, 232, 234, 235, 240,
338, 341, 342 244, 245, 246, 247, 250, 251, 260, 265, 270, 273,
cancer cells, 280, 341 286
candida, 7, 12, 18, 59, 61, 88, 130, 133, 236, 273, central nervous system (CNS), 313, 314, 328
274, 279, 280, 286, 294 ceramic, 264
candidates, 39 certification, 121, 147, 151, 316
cane sugar, 63 ceruloplasmin, 245
capillary, 201, 206, 280 cheese, 5, 9, 10, 11, 22, 23, 59, 64, 69, 72, 76, 79,
capsule, 123, 312, 316 86, 87, 90, 91, 121, 125, 131, 132, 136, 199, 210,
caramel, 120, 127, 142, 146, 147 211, 257, 259, 272, 274, 279, 284, 338
carbohydrate(s), 2, 4, 8, 13, 33, 61, 81, 127, 135, chemical characteristics, 202, 220
224, 225, 227, 229, 236, 255, 265, 272, 273, 291, chemical interaction, 70
292, 295, 300, 313, 315, 319, 320 chemical preservatives, 76, 78, 87
carbon nanotubes, 265 chemical properties, 72
carbonization, 126 chemical reactions, 219
carboxyl, 232 chemiluminescence, 213
carboxylic acid, 320 chemoprevention, 329, 330
carcinogen, 224, 233, 250, 284, 299 chemotherapy, 224, 277
carcinogenesis, 338 chicken, 35, 125, 126, 148, 157, 167, 199, 213, 215
cardboard flavor, 300 chitin, 78, 257, 268
cardiovascular disease(s) (CVD), 25, 311, 315, 317, chitinase, 159
328, 331, 339 chitosan, 32, 34, 77, 78, 93, 94, 257, 268, 317
cardiovascular risk, 329 chloroform, 141
caries, 222 chlorophyll, 122, 127, 128, 131, 134, 139, 147, 150,
carotene, 101, 120, 124, 125, 128, 129, 130, 134, 165, 278
136, 137, 138, 140, 141, 142, 143, 144, 145, 146, cholera, 88, 100, 112
149, 151, 153, 321, 327 cholesterol, 37, 39, 47, 137, 145, 256, 261, 263, 295,
carotenoids, 115, 122, 124, 125, 127, 129, 130, 134, 313, 324, 325, 339
135, 136, 137, 138, 139, 140, 143, 144, 146, 149, choline, 342
150, 152, 153, 154, 232, 239, 261, 327, 330 chondroitin sulfate, 318
cartagena protocol, 301 chopping, 16
cartilage, 325 chromatography, 139, 154, 181, 216, 276, 333
casein, 8, 9, 166, 168, 169, 181, 255, 258, 270, 274 chromium, 295, 302, 306
catabolism, 11 chromosome, 305
catabolite repression, 285, 297 chronic diseases, 312, 314, 341
catalysis, 222 chronic fatigue, 293
catalytic activity, 168, 222 cirrhosis, 42
cattle, 67, 117, 126 classical methods, 296
CBS, 153 classification, 75, 80, 180, 182, 204, 208, 284, 316
CDC, 156 clinical trials, 38, 321
cDNA, 180, 182, 183, 185, 195 cloning, 134, 182, 183, 184, 185, 242, 304, 309
cell culture, 134, 151, 154, 334 clusters, 168, 262, 263, 292
cell cycle, 284 CMC, 187
cell death, 308 coal, 117
cell division, 151 coastal region, 167
cell line, 328 coatings, 131, 256, 257, 258, 260, 266, 267, 268, 269
cell membranes, 139, 189, 255 cobalamin, 21
cell signaling, 281, 336 cobalt, 313
cell size, 202, 276, 292 coccus, 2
Index 347
cocoa, 5, 324 coronary heart disease, 145, 321

codex alimentarius commission, 143, 301 correlation, 145, 153
coding, 299, 300 Costa Rica, 277
coenzyme, 37, 239 Côte d‘Ivoire, 277
coffee, 156, 221, 234 cotton, 96, 106, 107, 108, 144, 145, 149, 157
cognitive function, 327 coumarins, 121
colitis, 36, 38 country of origin, 110
collagen, 167, 185, 265, 313, 337 covering, 60, 66, 256
colon, 31, 33, 40, 145, 320, 328, 341 cracks, 63
colon cancer, 145, 328, 341 crop residue, 109
colonisation, 27, 30, 37 crystal structure, 183
colonization, 33, 36 crystalline, 63, 230, 246, 262
colorectal cancer, 40, 338, 342 crystallization, 250, 262
colour stability, 283 crystals, 207, 285
combined effect, 21 CSD, 90
commodity, 116, 242 Cuba, 277
communities, 1, 14, 47, 48, 73, 112, 167 cultivars, 240
community, 37, 48, 128 culture media, 190, 294
compatibility, 239 culture medium, 127
competition, 35, 88 cupins, 160, 161
complementary DNA, 195 curcumin, 122, 126, 317, 318, 326
complementary products, 330 curing process, 66
complex carbohydrates, 33 customers, 108, 145
complexity, 157, 208, 275, 286, 334 Cyanophyta, 137
compliance, 315 cycles, 85, 109, 190, 192
complications, 202 cycling, 191, 194, 197
composites, 253, 265 cyclodextrins, 32
compost, 248 cystathionine, 338
compressed yeast, 290 cysteine, 136, 159, 162, 165, 243
conditioning, 287, 295 cystine, 136, 276
conductance, 206 cytokines, 275, 276
conductivity, 206 cytokinesis, 163
configuration, 207 cytometry, 177, 187, 188, 201, 202, 210, 214, 215,
confinement, 65 275, 276, 279, 280
conjugation, 314 cytoplasm, 292
consensus, 80, 143, 323
conservation, 61, 66
constipation, 39, 40, 324 D
construction, 305
dairy industry, 215, 274
consumer education, 326
dairy products, 4, 22, 24, 26, 27, 30, 47, 65, 66, 72,
consumption habits, 286
76, 78, 92, 121, 122, 123, 125, 132, 258, 295, 306
containers, 5, 7, 31, 58, 66, 264, 265
data processing, 202
contaminant, 264, 298
data set, 339
contaminated food, 188
database, 81, 159, 207
contaminated soil, 100
DDT, 102
contamination, 87, 93, 111, 189, 198, 203, 211, 257,
deacetylation, 78
278, 291, 296, 300
deaths, 188
control measures, 155, 157, 158
decomposition, 240, 244, 325
controversial, 108, 229, 272
decongestant, 325
cooking, 48, 57, 58, 70, 123, 138, 174, 256, 264
defects, 313
cooling, 7, 8, 118, 289
deficit, 274
copper, 117, 147, 222, 289
degradation process, 231
coproduction, 251
dehydration, 294, 303, 306, 308, 309
348 Index
dementia, 315, 327 divergence, 15

demulcent, 315 diversification, 22
denaturation, 17, 164, 171, 173 diversity, 44, 47, 72, 74, 128, 135, 153, 181, 243
dendritic cell, 30 dizziness, 315
dental caries, 222 DNA polymerase, 189, 190, 192, 194
dephosphorylation, 299 DNase, 195, 197
depolymerization, 238 docosahexaenoic acid, 320
deposition, 259, 260, 266 dogs, 150
depression, 228, 315 DOI, 43, 90, 91
derivatives, 122, 131, 136, 230 dosage, 33, 127, 254, 302, 309
dermatitis, 36, 37, 43, 44, 175, 186 dot immunoblotting, 176
dermatology, 182 dough, 54, 59, 71, 86, 88, 93, 160, 221, 224, 225,
desiccation, 297, 309 226, 235, 247, 283, 285, 286, 287, 288, 292, 295,
desorption, 178, 188, 200, 207, 208, 213, 214, 215, 296, 297, 305, 306, 307
276 drainage, 64
destruction, 37, 47, 325 dressings, 130, 262
detection system, 181 drought, 48, 98, 100, 106
detergents, 189, 190, 239 drug delivery, 254, 269, 327
detoxification, 47, 48 drug discovery, 254
diabetes, 25, 36, 37, 39, 40, 43, 293, 295, 302, 318, drug release, 254
327, 330, 335, 337, 339, 340, 341 drugs, 27, 30, 118, 170, 254, 306, 326
diabetic neuropathy, 318 dry ice, 189
diabetic patients, 318 drying, 16, 21, 31, 34, 43, 48, 57, 66, 67, 70, 135,
diaphoresis, 175 227, 294
diarrhea, 30, 36, 37, 39, 41, 60, 330 DSHEA, 316
dielectric constant, 139 DSM, 302
dietary fiber(s), 33, 232, 319, 320, 327 dyeing, 118, 143, 153
dietary habits, 25, 37 dyes, 115, 116, 118, 120, 121, 143, 145, 149, 150,
dietary intake, 124, 314, 334, 335, 338 151, 192, 202, 203, 236, 275
dietary supplementation, 235 dyspepsia, 315
diffusion, 89, 256, 258, 263, 265
digestibility, 1, 34, 47, 49, 73, 231, 232, 234, 250,
277 E
digestion, 27, 31, 33, 61, 158, 159, 160, 162, 164,
ecology, 42
185, 221, 234, 255, 256, 278, 293, 294, 295, 337
ecosystem, 26, 109, 243
digestive enzymes, 158, 232
ecotoxicology, 284
dimensionality, 276
Ecuador, 277
dipstick test, 176
eczema, 37, 156, 315
directives, 117, 148
edema, 155, 315
disaster, 108
edible vaccines, 98, 100
discomfort, 156
education, 111, 301, 326
discrimination, 210, 213
effluent, 235, 249, 277, 279, 280
diseases, 25, 37, 39, 41, 44, 47, 83, 100, 184, 201,
eicosapentaenoic acid, 320
203, 208, 254, 275, 277, 284, 311, 312, 314, 316,
elaboration, 299
317, 318, 320, 321, 322, 323, 329, 331, 332, 333,
electric field, 76, 89, 149
335, 336, 337, 338, 340, 341
electrical conductivity, 206
disinfection, 257
electrical resistance, 206
disorder, 316, 338
electrodes, 206
dispersion, 63
electron(s), 3, 117, 222, 244, 321
displacement, 194
electrophoresis, 175, 176, 181, 195, 251, 276, 280,
disposable income, 323
333
distillation, 294
electroporation, 102, 139
distribution, 93, 96, 126, 141, 188, 253, 258, 323
ELISA method, 176
Index 349
elongation, 163, 184 eukaryotic, 83, 201, 202, 296, 304

emission, 202, 289 eukaryotic cell, 83, 201, 202
EMS, 92 Europe, 12, 14, 15, 96, 101, 116, 131, 146
emulsifiers, 290, 291, 294 European Commission, 121, 266
emulsions, 76, 253, 255, 256, 258, 259, 260, 262, European Community, 339
268, 269 European market, 99
encapsulation, 255, 256, 257, 258, 259, 260 European Union, 116, 121, 155
encephalitis, 213 evaporation, 258
encephalopathy, 36, 41, 42 evolution, 117, 238, 262, 286, 327
encoding, 297, 298, 299, 300, 301, 304, 308, 309, excitation, 117, 192
337, 338, 339 excretion, 134, 314, 336
entrapment, 43, 270 exons, 337
environmental conditions, 4, 25, 31, 34 exonuclease, 192
environmental degradation, 118 exopolysaccharides, 42
environmental effects, 111 expansins, 165, 181
environmental factors, 128, 255, 277, 299, 339 exploitation, 38, 148
environmental impact, 301 expression vectors, 295
environmental influences, 339 expulsion, 9
environmental issues, 148 extraction, 18, 32, 115, 122, 126, 132, 134, 135, 139,
environmental organizations, 108 140, 143, 145, 148, 149, 152, 153, 154, 190, 208,
environmental policy, 289 221, 231, 232, 233, 249, 278, 282
Environmental Protection Agency (EPA), 102, 108, extracts, 76, 93, 117, 120, 132, 135, 149, 292, 312
200, 289, 304, 320, 322 extrusion, 290, 294
environmental stress(es), 161, 256, 259, 265, 284
enzymatic activity, 176, 189
enzymatic extraction, 139, 140 F
enzyme immunoassay, 180
FAD, 227
Enzyme Linked Immune Sorbent Assay ELISA,
false negative, 190, 208
(ELISA), 42, 104, 175, 176, 178, 187, 197, 198,
false positive, 177
199, 209, 210, 212, 215
farmland, 96
enzyme-linked immunoassay, 209
fat soluble, 122, 141
EpiCor, 295
fatty acids, 11, 12, 13, 17, 35, 36, 44, 47, 48, 125,
epidemic, 156
126, 144, 169, 170, 255, 268, 285, 294, 298, 312,
epidemiology, 189, 328
313, 314, 317, 318, 320, 321, 324, 325, 326, 329,
epigenetics, 342
330
epilepsy, 327
federal authorities, 146
epithelial cells, 30, 41, 98
feedstock, 221, 289
epithelium, 27, 30
fermentation, vii, 1, 3, 4, 7, 8, 9, 11, 12, 13, 14, 15,
epitopes, 158, 171, 180, 181, 182
16, 17, 18, 19, 20, 21, 22, 23, 31, 33, 38, 39, 40,
equilibrium, 247
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
equipment, 14, 71, 220
60, 61, 62, 64, 65, 66, 67, 68, 70, 71, 72, 73, 76,
ESI, 178
78, 88, 94, 117, 120, 128, 130, 132, 133, 134,
essential fatty acids, 47
135, 136, 138, 140, 141, 145, 148, 149, 151, 220,
essential oil, 76, 77, 121, 236, 263, 268
225, 226, 227, 230, 232, 233, 235, 237, 238, 239,
EST2, 299, 304
240, 243, 245, 246, 247, 248, 250, 274, 275, 279,
estrogen, 317
280, 281, 283, 284, 285, 286, 288, 289, 291, 292,
ethanol, 2, 3, 7, 8, 15, 18, 19, 20, 32, 49, 50, 71, 131,
293, 296, 297, 298, 299, 300, 301, 302, 304, 305,
134, 135, 137, 140, 189, 190, 208, 221, 229, 236,
306, 307, 309, 325, 326, 335
248, 249, 251, 284, 289, 296, 297, 298, 299, 300,
fermentation technology, 71, 247
302, 305, 306, 307, 309
fermented beverages, 49
ethical issues, vii, 101, 334
fermented cereals, 47
ethyl acetate, 141, 299
fermented fish products, 64, 72
etiology, 158
350 Index
fermented foods, 1, 3, 4, 5, 11, 12, 13, 14, 20, 21, 22, food allergens, vii, 155, 156, 157, 158, 159, 160,
23, 24, 47, 48, 49, 53, 57, 60, 71, 72, 73, 74, 76, 161, 164, 166, 167, 171, 172, 173, 175, 179, 180,
82, 89, 284, 299 182, 183, 184, 186
fermented meat, 15, 16, 18, 23, 47, 66, 87, 284 Food and Drug Administration (FDA), 79, 102, 108,
fermented meat products, 16, 23, 66, 87 110, 121, 131, 146, 147, 150, 192, 193, 201, 253,
fermented milk products, 61, 62 266, 283, 284, 298, 316, 323, 328
fermented vegetables, 14, 15, 60, 88 food chain, 172, 266
ferritin, 301, 303 food fermentation, vii, 11, 20, 23, 47, 48, 73
fertility, 109 food grade yeasts, 302
fertilizers, 320 food habits, 341
fever, 156, 315, 318 food processing industry, 253
fiber(s), 31, 33, 226, 206, 212, 213, 215, 232, 234, food production, vii, 57, 90, 95, 96, 98, 100, 111,
317, 318, 319, 320, 327, 328 202, 203, 225, 236, 246, 258, 267, 305
fiber optics, 206 food products, 4, 13, 23, 25, 27, 74, 76, 82, 88, 92,
fibrinolytic, 315 110, 121, 125, 132, 143, 144, 157, 172, 199, 200,
field trials, 106 221, 224, 225, 243, 244, 253, 255, 256, 257, 258,
fights, 324 264, 267, 277, 286, 319, 326, 340
filters, 202, 206, 256, 266 food safety, vii, 49, 76, 93, 172, 187, 188, 203, 255,
filtration, 76, 233, 240, 241, 264 259
fingerprints, 280 food security, vii, 14, 49
fish oil, 321, 329 food spoilage, 83, 87, 253
fitness, 325 formula, 37, 44, 118
fixation, 215, 275 fouling, 141
flavonoids, 121, 126, 145, 153, 228, 247, 257, 318, fragments, 172, 178, 195
321, 324 free radicals, 145, 321, 325, 336
flexibility, 265 freezing, 83, 189, 228, 297, 308, 309
FLO1, 299, 302, 309, 310 freshwater, 64
FLO5, 299 frost, 96, 100
FLO8, 299 frozen dough, 285, 297, 305, 307
flocculation, 299, 302, 308, 310 fructo-oligosaccharides, 34, 229
flora, 6, 11, 12, 14, 17, 26, 39, 67, 70, 71, 87, 88, 93, fructose, 3, 32, 141, 221, 226, 227, 229, 241, 247,
335 285, 288, 296, 297
flour, 12, 52, 54, 55, 56, 93, 147, 154, 185, 221, 224, fruitier, 293
226, 235, 242, 255, 286, 291, 295, 297 functional food, 22, 25, 27, 38, 71, 255, 259, 260,
flow cytometry, 177, 187, 188, 201, 202, 203, 210, 311, 312, 323, 326, 329, 335, 337, 340, 341
214, 275, 276, 280 fungi, 70, 78, 88, 115, 118, 127, 131, 144, 164, 207,
flowers, 117, 118, 122, 125, 126, 140, 318 208, 222, 224, 225, 228, 229, 230, 233, 234, 242,
fluid, 66, 149, 202 245, 249, 250, 272, 274, 278, 301, 302
fluid extract, 149 fungus, 124, 132, 133, 138, 230, 240, 247, 280
fluorescence, 178, 190, 192, 196, 202, 203, 205, 206, fusion, 204, 296, 301
209
fluorescence in situ hybridization (FISH), 190, 203,
209 G
fluorochromes, 203
galacto-oligosaccharides, 34, 41
fluorophore, 192
gamma globulin, 167
fluorophores, 192, 203, 257
gamma irradiation, 139, 140
foams, 253, 262
gas diffusion, 256, 265
folate, 21, 338, 342
gastritis, 315
folic acid, 295
gastroenteritis, 37
food additive(s), 78, 79, 89, 110, 116, 122, 124, 125,
gastrointestinal tract, 26, 34, 42, 67, 79, 158, 320
128, 132, 146, 157, 171, 184, 185, 186, 222, 251,
GDP, 107
257, 263, 284
gel, 69, 177, 178, 195, 227, 276, 315, 333
gel formation, 227
Index 351
gelation, 227, 244 glycerol, 17, 250, 284, 293, 297, 298, 299, 304, 305,
gemma, 67 307
gene expression, 39, 43, 195, 246, 284, 285, 318, glycogen, 277, 292
331, 332, 333, 334, 335, 336, 337, 338, 339, 341 glycol, 39, 102
gene polymorphism, 337, 341, 342 glycolysis, 2, 18, 19
gene promoter, 335 glycopeptides, 22
gene regulation, 269, 333, 334, 336 glycoproteins, 158, 167
genes, 19, 80, 81, 97, 98, 101, 113, 128, 134, 148, glycoside, 165, 166, 328
149, 151, 185, 190, 191, 192, 193, 196, 202, 211, glycosylation, 160, 162, 275
295, 296, 298, 299, 300, 301, 303, 304, 305, 307, GM Regulations, 95, 108
309, 331, 332, 333, 334, 335, 336, 337, 339, 341 goat milk, 6, 7
genetic background, 278 gold nanoparticles, 211, 254, 269
genetic diversity, 135 good manufacturing practices, 77, 144, 155, 188
genetic engineering, 96, 97, 103, 108, 111, 112, 128, goose, 167, 214
134, 246, 254, 296, 303 gout, 278, 315, 318
genetic factors, 339 GOX, 227
genetic information, 110, 205 gracilis, 138
genetic manipulations, vi, 283, 287, 295, 302 granules, 44, 226, 236
genetically engineered crop, 103, 113 GRAS, 5, 26, 77, 78, 82, 89, 110, 124, 147, 229,
Genetically Modified (GM), vii, 23, 95, 96, 98, 99, 278, 283, 284, 298, 325
100, 101, 102, 104, 105, 106, 107, 108, 109, 110, grass, 122, 124, 164, 165
111, 112, 113, 114, 133, 209, 245, 248, 283, 301, gravity, 300, 302, 309
308, 310 green alga, 137, 149
Genetically Modified (GM) food, 96 groundnut oil, 54
genetically selectable markers, 295 groundwater, 100
genetics, 299, 302, 303, 307, 333, 337 growth dynamics, 305
genome, 41, 283, 299, 302, 309, 332, 333, 334, 337, growth factor, 61
338 growth rate, 138, 278, 286, 287, 291, 326
genomics, viii, 306, 309, 331, 332, 333, 340, 341, guardian, 269
342 guidelines, 108, 110, 193
genotype, 338, 339
genotyping, 189
genre, 296 H
genus, 2, 88, 131, 137, 158
habitats, 2, 235
germination, 33
hair, 273, 293, 295
germins, 161, 162
Hansenula, 12, 55, 274, 286
gestational diabetes, 39, 330
hardness, 221, 224, 226
ginger, 57, 58, 67, 68, 70
harmful effects, 89
ginseng, 140, 315
harmonization, 179
glucoamylase, 283, 301, 304, 305, 306, 308
harvesting, vii, 111, 191
glucose, 3, 6, 32, 78, 117, 136, 137, 138, 141, 154,
hay fever, 156, 318
220, 225, 226, 227, 228, 229, 235, 241, 243, 244,
hazards, 110, 113, 179, 188
245, 246, 247, 249, 251, 277, 285, 288, 289, 295,
haze, 223, 224, 248
296, 297, 298, 302, 304, 306, 309, 318, 326, 338,
headache, 175, 315
342
healing, 311, 313, 315, 341
glucose oxidase, 220, 227, 235, 243, 245, 246, 247,
Health and Human Services, 316
249, 251, 298, 306
health care, 311, 314, 323
glucose tolerance, 295, 302, 338, 342
health condition, 333
glucosidases, 230, 244
health effects, 119, 266, 336
glucoside, 144, 230
health promotion, 311
glucosinolates, 316, 321
health status, 38, 311, 312
glutamate, 11, 13, 120, 142, 175, 186, 297, 338
hearing loss, 322
glutamine, 13, 159, 160, 319
heart disease, 145, 277, 313, 321, 324
352 Index
Helicobacter pylori, 208

hemicellulose, 234, 235, 236, 286
I
hemoglobin, 190, 313
ideal, 87, 88, 131, 139, 251, 260
hemp, 318, 321
identification, 91, 149, 172, 176, 178, 182, 183, 184,
hepatic encephalopathy, 41, 42
185, 188, 190, 191, 194, 195, 198, 199, 200, 201,
hepatocytes, 342
203, 206, 207, 208, 209, 210, 214, 216, 242, 258,
heptane, 149
284, 292, 299, 302, 306
herbicide, 99, 109, 309
identity, 116
herbicide tolerance, 99
idiopathic, 39, 40
heredity, 335
illumination, 206
heterochromatin, 302
imbalances, 332
heterogeneity, 48, 280
immobilization, 205, 241, 249
heterologous ferritin, 301
immune function, 39
hexane, 126, 139, 233
immune response, 30, 37, 156, 161
hexose transporter, 296, 298
immune system, 35, 37, 41, 145, 155, 156, 158, 166,
High Hydrostatic Pressure (HHP), 87, 139, 149
313, 324
high protein food, 273, 338
immune-tags, 295
histamine, 177
immunity, 21, 35, 37, 44, 47, 79, 80, 91, 92, 169,
histone, 271, 341
300, 318
histone deacetylase, 341
immunization, 43
histone deacetylase inhibitor, 335, 341
immunocompromised, 36
history, 6, 48, 72, 83, 87, 292
immunodeficiency, 293
HIV, 30, 39
immuno-electrophoresis, 176, 177
HIV/AIDS, 39
immunoglobulin(s), 158, 166
hives, 156
immunomodulation, 26, 41
homeostasis, 37, 41
immunomodulatory, 23
homocysteine, 324
implants, 254
homothallic, 296
imports, 145, 288
hormone(s), 169, 170, 255, 295, 312, 338, 339
improvements, vii, 93, 226, 234
house dust, 171
impulses, 118
hue, 146
in situ hybridization, 190, 203, 209, 211, 216
human body, 277, 312
in vitro, 33, 37, 39, 41, 42, 44, 91, 137, 183, 184
human exposure, 38
in vivo, 31, 33, 42, 132, 137, 183, 263, 313
human health, vii, 22, 42, 43, 78, 108, 111, 118, 144,
incidence, 42, 156, 311, 339
150, 156, 229, 265, 311, 326
income, 106, 323
humidity, 16
incubation period, 136
humoral immunity, 169
incubation time, 86, 198
Hungary, 109
Indians, 60
hybrid, 96, 103, 178, 299, 301
indirect effect, 335, 336
hybridization, 189, 190, 203, 209, 211, 216, 279, 296
individual differences, 338
hydrocarbons, 77, 285
individuals, 49, 116, 166, 171, 302, 331, 337, 338,
hydrogen, 78, 124, 227, 234, 297
339
hydrogen peroxide, 78, 227, 297
inducer, 241
hydrolysis, 32, 77, 209, 226, 228, 229, 230, 233,
induction, 162, 232, 334, 340, 341
234, 236, 247, 248, 249, 250, 267, 273, 274, 285,
industrial environments, 213
301, 305
industrial processing, 43
hydroxyl, 136
industrial wastes, 247, 277
hypercholesterolemia, 38, 336
industrialization, 48
hyperlipidemia, 318
industrialized countries, 49
hypersensitivity, 149, 155, 157, 158, 182, 297
infants, 37, 40, 42, 44, 156, 166, 185, 303
hypertension, 339
infection, 39, 41, 161, 162, 288, 308
hypotensive, 315
infectious agents, 214
hypothesis, 329
inflammation, 266, 315, 318, 324, 337
Index 353
inflammatory bowel disease (IBD), 36, 41, 44 irradiation, 140, 153, 154
influenza, 37, 38, 43 irrigation, 212
influenza virus, 43 irritable bowel syndrome, 40, 41, 45, 293
infrastructure, 48 isoamyl acetate, 299, 304
ingestion, 89, 143, 156, 266, 316 isolation, 180, 199, 284
ingredients, vii, 15, 25, 31, 40, 47, 49, 52, 56, 59, 60, isoleucine, 222
61, 62, 70, 89, 96, 98, 104, 108, 118, 120, 129, isomaltulose/palatinose, 32
148, 157, 172, 179, 229, 235, 255, 258, 259, 286, isomerization, 32
293, 316, 323 isotope, 176, 240, 276
inheritance, 303 isozyme, 153
inhibition, 12, 27, 30, 37, 77, 78, 80, 87, 88, 89, 93, isozymes, 135, 238, 246
94, 145, 190, 246, 332, 334
inhibitor, 159, 170, 186, 275, 315
injections, 156 J
injuries, 83
JCC, 43
inoculation, 7, 8, 87, 90, 277
joints, 325
inoculum, 16, 57, 233
inositol, 289, 314
insect resistance, 98 K
insecticide, 99
insects, 102, 115, 118, 144, 156, 171, 235 kaempferol, 126
insertion, 305 Kefir, 5, 6, 7, 8, 23, 307
instant dry yeast, 287 ketones, 120, 285
insulin, 37, 229, 293, 295, 313, 314, 318, 339, 342 kidney, 278, 324, 336
insulin resistance, 318 kill, 75, 86, 99, 100, 109, 205
insulin sensitivity, 37 kinetochore, 310
integration, 22, 310 Kluyveromyces, 7, 229, 241, 249, 250, 273, 274, 285
integrity, 77, 78, 253 Koumiss, 8
interest groups, 108, 110 Krebs cycle, 222, 285
interference, 228
intermediaries, 118
intermolecular interactions, 268 L
international trade, 111, 179
intervention, 338, 339 labeling, 110, 111, 155, 172, 197, 253
intestinal flora, 26 labeling of GM foods, 111
intestinal tract, 39 Laccase, 220, 221, 222, 223, 224, 237, 238, 240,
intestine, 16, 25, 26, 33, 34, 35, 67, 68, 69, 263, 294, 241, 242, 244, 245, 248
320 lactase, 338
intoxication, 53 lactic acid, vii, 1, 2, 3, 4, 6, 7, 8, 9, 10, 15, 16, 17, 18,
introns, 337 19, 21, 22, 24, 26, 47, 50, 55, 59, 61, 62, 63, 67,
inulin, 32, 34, 35, 37, 43, 229, 232, 239, 243, 247, 70, 71, 76, 78, 81, 82, 83, 87, 91, 92, 93, 94, 120,
248, 249, 320, 322, 327 295, 308
inulinases, 229, 248, 250 lactic acid bacteria, vii, 1, 2, 10, 17, 18, 19, 21, 22,
inventors, 269 23, 24, 26, 50, 55, 59, 61, 67, 70, 71, 76, 78, 81,
invertebrates, 181 82, 87, 91, 92, 93, 94, 295, 308
investment, 128, 145, 148, 220, 222 lactobacillus, 2, 4, 5, 6, 9, 10, 11, 12, 15, 16, 19, 21,
investors, 112 26, 28, 29, 30, 34, 35, 37, 38, 39, 40, 41, 42, 43,
ionization, 76, 178, 181, 188, 200, 207, 208, 213, 44, 55, 56, 60, 61, 62, 82, 85, 88, 91, 92, 272,
214, 215, 276 300, 306, 322, 330
ions, 136, 166, 226, 227, 232, 264, 286, 309 lactoferrin, 77, 169, 190, 321
IPR, 73 lactose, 4, 6, 7, 8, 9, 21, 32, 36, 44, 62, 141, 166,
iron, 13, 27, 30, 54, 101, 117, 166, 167, 169, 170, 168, 248, 274, 285, 338
200, 256, 277, 289, 301, 303, 313 lactose intolerance, 21, 44
lactosucrose, 32
354 Index
lactulose, 32, 33, 35, 39

lamella, 232, 234
M
large intestine, 25, 26, 33, 34, 35, 68, 320
macrophages, 281
larvae, 98, 112
macular degeneration, 151
lateral flow immunoassay, 198, 200, 201
magnesium, 13, 122, 190, 232, 288, 309
lateral sclerosis, 322, 328
magnetic particles, 199
lecithin, 263
magnetic properties, 253
legend, 64
magnetic resonance, 200, 333
legislation, 124, 125, 147, 301
magnetic resonance imaging (MRI), 200
legume, 11, 12, 13, 21, 59, 73
Maillard reaction, 225
legumins, 162
malaria, 325
leptin, 337, 339
MALDI, 178, 187, 188, 207, 208, 209, 210, 213,
LEU4, 299, 305
214, 216, 281
leucine, 299
MALDI-TOF MS, 188, 207, 208, 214, 216
leukemia, 277, 280
malnutrition, 111, 271, 323
leukotrienes, 314
maltose, 32, 141, 221, 225, 226, 228, 283, 285, 297,
lignans, 316
300, 303
lignin, 222, 232, 235, 238, 273
mammalian cells, 269
lignocellulosic residues, 274
mammals, 145
linoleic acid, 21, 35, 315, 317, 321
management, 42, 43, 179, 188, 328, 329, 331, 332,
lipases, 235, 242
335, 339, 341
lipid metabolism, 329, 336
manganese, 289
lipids, 77, 81, 89, 121, 150, 256, 260, 262, 265, 269,
manipulation, 83, 97, 108, 134, 148, 189, 201, 220
291, 302, 308
mannitol, 15, 21, 121
lipolysis, 11
mapping, 304
liposomes, 254
marketplace, 109
liquid chromatography, 139, 154, 181, 216, 333
marriage, 52, 53, 55, 56, 67
liquid phase, 255, 289
masking, 21, 259, 260, 263
Listeria monocytogenes, 83, 87, 89, 90, 91, 92, 93,
mass spectrometry, 178, 181, 184, 187, 188, 200,
94, 191, 193, 195, 196, 199, 200, 205, 209, 210,
207, 208, 209, 210, 213, 214, 215, 276, 279, 333
212, 213, 214, 215, 216
mast cells, 177
liver, 295, 314, 324, 329, 336, 338
mastitis, 203, 211
liver disease, 329
materials science, 254
livestock, 96, 284, 294
matter, 80, 125, 136, 138, 146, 147, 234, 248, 277
locus, 191, 298, 299, 338
MBP, 180, 181
longevity, 26, 47, 264
measurement(s), 181, 227, 275, 276, 281, 309
Loop-Mediated Isothermal Amplification, 194, 210,
meat, 1, 3, 15, 16, 17, 18, 23, 24, 47, 49, 66, 67, 68,
213, 216, 217
69, 70, 72, 76, 77, 78, 83, 85, 87, 91, 92, 93, 94,
loss of appetite, 293
123, 131, 132, 137, 138, 144, 157, 167, 190, 199,
low-density lipoprotein (LDL), 37, 145, 170, 295,
205, 211, 212, 213, 214, 215, 257, 262, 264, 268,
339
276, 284, 295
lumen, 27, 30, 263, 267
media, 94, 139, 141, 188, 190, 211, 223, 249, 277,
luminescence, 206
284, 286, 288, 292, 294, 296, 299, 302, 304, 325
luminosity, 277
medical, 243, 253, 254, 312, 316, 323, 326, 334, 342
lung cancer, 330
medication, 156, 179
Luo, 305
medicine, 38, 153, 182, 185, 254, 307, 312
lutein, 122, 124, 125, 146, 150, 318
melanin, 127
lycopene, 124, 125, 141, 149, 150, 261, 312, 316,
mellitus, 340
318, 321
membership, 159
lysine, 11, 222, 276
membrane technology, 139, 140
lysis, 189
membranes, 80, 83, 135, 139, 140, 183, 189, 255,
lysozyme, 77, 166, 167, 168, 189
266, 267, 313, 314, 315, 325
memory, 293, 315
Index 355
memory loss, 315 moisture, 6, 9, 16, 31, 66, 142, 228, 253, 256, 257,
mental state, 36 260, 265, 269, 291, 294
mercury, 202 moisture content, 9, 31
meta-analysis, 40, 326, 327, 330 molasses, 117, 137, 273, 274, 279, 280, 281, 286,
metabolic, 2, 284, 304, 305, 307, 327, 338, 341 288, 289, 291, 293, 294, 296
metabolic disorder(s), 334 mold(s), 11, 14, 87, 137, 222, 272
metabolic pathways, 3, 134, 289 molecular beacons, 192, 195
metabolic syndrome, 334 molecular biology, 158, 283
metabolism, 2, 3, 11, 19, 20, 22, 23, 36, 40, 78, 154, molecular mass, 82, 159, 167, 184, 238
165, 206, 222, 229, 242, 289, 302, 304, 305, 309, molecular oxygen, 223
313, 314, 320, 321, 329, 333, 335, 336, 337, 338, molecular structure, 205
342 molecular weight, 76, 83, 160, 177, 221
metabolites, 4, 15, 30, 35, 70, 79, 140, 289, 333, 335 molecules, 3, 11, 13, 79, 83, 115, 140, 142, 163, 169,
metabolized, 285, 313 200, 219, 236, 255, 258, 261, 285, 321, 335, 341
metabolome, 333, 334 mollusks, 157, 171
metal ion(s), 136 molybdenum, 289
metal oxides, 265 momentum, 253
metalloenzymes, 226 monoclonal antibody, 183, 212
metals, 236 monomers, 32, 162
methanol, 32, 139, 232, 286 monosaccharide, 274
methodology, 191, 245, 249, 338 monosodium glutamate, 120
methyl group(s), 233 morphology, 35, 43, 141, 171
methylation, 338 mortality, 102, 321, 326, 327, 329
MHC, 244 mortality rate, 102
mice, 35, 39, 186, 334, 342 mosaic, 103, 312
microarray technology, 333 mosquitoes, 102
microbial cells, 118, 271, 294 mRNA, 195, 284, 307
microbial communities, 112 MTTI, 300
microbiota, 31, 33, 34, 35, 38, 39, 40, 42, 43, 44, 76, mucin, 33
79, 83, 328 mucosa, 26
micronutrients, 31, 314 mucous membrane(s), 313, 315
microorganism(s), vii, 2, 14, 16, 17, 20, 33, 38, 41, mucus, 33, 40
48, 49, 57, 59, 61, 62, 75, 76, 77, 78, 83, 85, 86, multigenic, 284, 337
87, 88, 89, 90, 93, 109, 115, 117, 118, 119, 120, multiplication, 136, 272
127, 128, 129, 130, 132, 134, 135, 136, 138, 141, mung bean, 154, 162
189, 190, 191, 200, 202, 204, 205, 207, 209, 213, muscle contraction, 171, 173
215, 216, 219, 228, 229, 230, 234, 235, 240,251, muscles, 295, 313
266, 272, 274, 278, 288, 291, 301, 319, 325 mustard oil, 70
microscopy, 188, 202, 270 mutagenesis, 296, 305
microspheres, 201, 216 mutant, 133, 134, 148, 224, 242, 244, 251, 297, 305
microstructure, 71 mutation(s), 134, 296, 309, 335, 336
microwave heating, 76 myalgia, 175
middle lamella, 232, 234 mycelium, 208
migraine headache, 315 mycobacteria, 208, 214
migration, 30 mycotoxins, 38, 217, 278
milk sugar, 6 myoglobin, 17
miniature, 259 myosin, 171
mitochondria, 77, 324
mixing, 7, 58, 59, 67, 111, 277, 280
model system, 334 N
modernization, 156
NADH, 297, 298, 305, 309
modules, 242
NADH oxidase, 297, 305
nano capsules, 258
356 Index
nano dispersions, 258 NMR, 184

nanoceuticals, 256, 261 novel materials, 254
nanoclusters, 261 nucleic acid, 78, 178, 187, 188, 189, 190, 194, 195,
nanocomposites, 254, 256, 257, 264 204, 271, 278, 279
nanodevices, 254 nucleic acid sequence-based amplification, 190, 194,
nanoencapsulation, 253, 257, 259, 263, 268 195
nanofibers, 256, 260 nucleotide sequence, 192
nanolaminate, 260 nucleotides, 294, 318
nanolayers, 260 nucleus, 97
nanomaterials, 265, 266 null, 232
nanometer, 253, 254, 255, 259, 260, 265 nutraceutical, 1, 258, 267, 311, 312, 316, 317, 319,
nanometer scale, 255 321, 322, 323, 324, 325, 326, 327, 328, 332, 341,
nanoparticles, 149, 211, 253, 254, 257, 258, 260, 342
261, 262, 263, 264, 265, 266, 267, 268, 269, 270 Nutrifit, 30
nanorods, 205, 215, 269 nutrigenetics, vi, 331, 333, 337, 338, 339, 340, 341,
nanostructures, 255, 262 342
nanotechnologies, 266, 267 nutrigenomics, vi, 331, 332, 333, 334, 335, 336, 337,
nanotechnology, viii, 141, 214, 253, 254, 255, 256, 339, 341, 342
258, 261, 262, 263, 264, 265, 266, 267, 268, 269, nutrition, 6, 14, 23, 38, 41, 42, 73, 179, 180, 181,
270 184, 243, 262, 266, 278, 312, 319, 329, 331, 332,
nanotubes, 254, 265 333, 337, 339, 341, 342
naringin, 228, 238, 243 nutritional genomics, vi, viii, 331, 332, 333, 340,
National Academy of Sciences (NAS), 194, 195, 341, 342
197, 330, 341 nylons, 257
National Health and Nutrition Examination Survey,
180
National Institutes of Health, 329 O
National Research Council, 23
obesity, 25, 35, 40, 43, 262, 277, 327, 328, 331, 333,
natural compound, 144
339, 340
natural food, 76, 117, 119, 124, 127, 132, 144, 145,
oleosins, 159, 164, 165, 183
148, 149, 152
oligomers, 205, 276
nausea, 30, 175, 278
oligosaccharide, 32, 34, 35, 37, 42
nematode, 174
olive oil, 231, 232
nervous system, 295, 313
omega-3, 268, 320, 321, 326, 329
Nestle Actiplus Probiotic Dahi, 30
omega-3 fatty acids, 268, 320, 326, 329
neurodegenerative diseases, 322, 329
opportunities, 89, 255, 333
neurological disease, 321
optical activity, 253
neuropathy, 318
optimization, 153, 154, 245, 247
neurotransmitter, 324
orchid, 324
neutral, 88, 121, 125
ores, 117, 257
next generation, 25
organ(s), 116, 334
NGOs, 108
organelle, 204
niacin, 14, 295
organic acids, 13, 18, 21, 39, 44, 66, 76, 77, 78, 135,
niche market, 129, 145, 148
279, 285
nicotine, 98
organic compounds, 118, 257, 274
NIR, 210
organic food, 147
nisin, 21, 29, 75, 76, 77, 78, 79, 80, 81, 82, 84, 85,
organic matter, 234, 277
86, 87, 88, 89, 90, 91, 93, 94
organic solvents, 122, 123, 139, 233
nitrates, 277, 286
organism, 26, 34, 96, 97, 103, 134, 137, 189, 227,
nitrite, 23, 66, 87, 131, 135
231, 255, 263, 273, 279, 284, 296, 314
nitrogen, 56, 59, 131, 142, 274, 278, 281, 285, 286,
oscillation, 207
288, 291, 292, 298, 299, 300
osmosis, 141
nitrosamines, 87, 278
osmotic pressure, 169, 292, 294
Index 357
osmotic stress, 162 pesticide, 254

osteoarthritis, 318 pests, 98, 102
osteoporosis, 25, 323 PET, 257
outreach, 25 petroleum, 117
oven spring, 285 pH monitoring, 288
overproduction, 298, 305, 307 phage, 26, 205, 207, 211
oxidation, 78, 123, 141, 145, 222, 223, 224, 227, pharmaceutical(s), 116, 117, 144, 219, 220, 222,
238, 246, 257, 263, 298, 309, 321 225, 226, 228, 229, 236, 254, 260, 272, 294, 296,
oxidation products, 78, 224 312, 317, 318, 326, 331, 332, 341
oxidative reaction, 300 pharmacology, 93
oxidative stress, 30, 37, 39, 266, 297, 303, 318, 329 phenol, 190
oxidative stress tolerance, 297 phenolic compounds, 21, 223
oxide nanoparticles, 254 phenotype(s), 208, 296, 299, 309
oyster(s), 65, 173, 174, 183, 213 phenylalanine, 222, 251, 319, 335, 338
phenylalanine hydroxylase (PAH), 335
phenylketonuria, 331, 335, 341
P phosphate(s), 2, 168, 246, 258, 286, 288, 295, 299,
307
PAA, 239
phospholipids, 254, 259, 260, 262
pantothenic acid, 289, 295
phosphorescence, 206
paradigm shift, 261
phosphorus, 274
parallel, 12, 39, 162, 331
phosphorylation, 27, 30, 314
partition, 77
photosynthesis, 277
parvalbumin(s), 159, 166, 167, 171, 173, 182, 185
phycobilin, 137
pasta, 88, 123, 277
phycocyanin, 131, 280
pasteurization, 11, 76, 83
physical activity, 340
patents, 138, 304
physical properties, 115, 160, 254, 266
pathogen derived resistance, 100
physicochemical properties, 297
pathogenesis, 158, 159, 163, 180, 341
physiology, 34, 35, 42, 310, 320
pathogens, vii, 18, 21, 26, 27, 30, 35, 37, 43, 44, 61,
phytoplankton, 320
75, 76, 83, 87, 90, 156, 161, 162, 179, 187, 188,
phytoremediation, 98
189, 190, 191, 192, 193, 194, 195, 196, 197, 198,
phytosterols, 261, 263, 325
199, 200, 201, 202, 203, 204, 205, 206, 207, 208,
Pichia, 236, 251, 274, 286
209, 210, 211, 212, 213, 215, 216, 217, 254, 255,
piezoelectric crystal, 207
256, 257,258, 294
pigmentation, 141
pathway(s), 2, 3, 18, 19, 21, 99, 134, 184, 222, 229,
pigs, 67, 272
275, 289, 336, 342
pitch fermentation, 290
PCT, 245
PKU, 338
pectinases, 88, 232, 233, 234, 235, 236, 239, 242,
placebo, 40, 41, 45, 182, 184, 327
243, 244
placenta, 235
pediocin, 29, 75, 76, 78, 80, 82, 83, 84, 85, 86, 87,
plaque, 324
89, 90, 91, 92, 94, 308
plasma membrane, 303, 308
pepsin, 9, 79, 169, 173
plasmid, 83, 102, 103, 191
peptic ulcer, 61, 315
plastics, 265
peptide(s), 3, 6, 9, 12, 17, 22, 75, 76, 78, 80, 81, 83,
platelet aggregation, 314
89, 91, 93, 168, 169, 178, 206, 213, 281, 294,
platform, 156, 187, 207, 256, 260, 281
297, 307, 339
Plato, 300
peripheral blood, 315
PM, 92, 94, 152, 153, 240, 270
peritonitis, 42
PMDA, 146
permeability, 37, 139, 263, 269
PMS, 313
permeation, 257
PNA, 204
permit, 208, 263, 276, 290
poison, 78, 157, 179
peroxide, 78, 227, 297
polar, 262, 269, 297
perylene, 257
358 Index
polarity, 139 probe, 175, 178, 192, 195

polarization, 141, 206 probiotic(s), vii, 5, 21, 22, 23, 24, 25, 26, 27, 28, 29,
policy, viii, 108, 111, 113, 289, 324 30, 31, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
pollen, 101, 102, 109, 112, 162, 164, 165, 180, 183 45, 208, 209, 227, 256, 257, 259, 294, 302, 306,
pollination, 96 308, 319, 320, 326, 329
pollutants, 289, 337 probiotic yeasts, 302
pollution, 100, 265, 271, 272, 274, 279, 336 process control, 289
polyamines, 294 producers, 81, 98, 134, 148, 172, 179, 230, 235, 258
polymer, 6, 94, 229, 254, 257, 258, 265, 308 product design, 256
polymer matrix, 257 production costs, 220, 277, 294
polymerase, 172, 181, 189, 190, 192, 194, 195, 211, programming, 97
213, 214, 215, 216, 306 pro-inflammatory, 266, 342
Polymerase Chain Reaction (PCR), 23, 42, 104, 105, prokaryotes, 234, 239
112, 172, 175, 178, 180, 181, 187, 189, 190, 191, prolamins, 159, 160
192, 193, 194, 196, 197, 200, 209, 210, 211, 212, proliferation, 87, 328
213, 214, 215, 216, 305, 306 prolifins, 163
polymerization, 258 proline, 159, 160, 297, 300, 303, 307, 309
polymers, 77, 88, 209, 257, 264, 276 promoter, 103, 299, 305, 335
polymorphism(s), 295, 332, 337, 338, 339, 341, 342 propagation, 107, 283, 284, 288, 294, 296, 307
polypeptide, 82, 270 prophylactic, 157, 315
polyphenols, 13, 222, 224, 321 proposition, 134
polyploid, 296 prostaglandins, 314
polyploidy, 292 prostate cancer, 342
polypropylene, 257 protease inhibitors, 159, 160, 166
polyps, 338 protective coating, 257
polysaccharide(s), 6, 88, 190, 227, 228, 232, 233, protective role, 260
234, 235, 237, 250, 259, 260, 273, 274, 277, 299, protein analysis, 275, 281
319 protein family, 180
polyunsaturated fat, 41, 320 protein kinase C, 325
polyunsaturated fatty acids, 320 protein structure, 163
polyvinyl alcohol, 268 proteinase, 190
ponds, 209 proteolysis, 11, 160, 161, 162, 169
potassium, 88, 288, 314, 325 proteolytic enzyme, 89, 134, 140, 168, 171, 247
potato, 32, 94, 100, 108, 159, 164, 180, 185, 221, proteome, 183, 275, 276, 333, 334
225, 238, 245, 294 proteomics, 112, 333
potential benefits, 110, 143, 314 protibel, 274
poultry, 21, 23, 77, 78, 123, 132, 213, 235, 272, 277, prototroph, 296
281 prototype, 336
poverty, 108 pruning, 281
prebiotics, vii, 25, 31, 32, 33, 34, 35, 36, 37, 38, 39, Pseudomonas aeruginosa, 129, 195, 217
40, 41, 42, 43, 44, 45, 257, 320 public awareness, 95
precipitation, 121, 141, 176, 200, 274 public health, 106, 172, 179
pregnancy, 313, 328 public interest, 108
premature infant, 303 pullulanase, 32, 220, 228, 249, 301, 305
premenstrual syndrome, 313 pulp, 4, 50, 235, 236, 241
preservation, 3, 14, 15, 18, 21, 31, 47, 48, 59, 64, 66, Pulse Based Fermented Foods, 57
68, 76, 81, 83, 89, 90, 91, 94, 151, 153, 221, 227, Pulsed Electric Field (PEF), 76, 89, 139, 149
245, 295 purchasing power, 49
preservative, 11, 21, 67, 75, 76, 77, 79, 82, 131, 137, purification, 139, 200, 237, 238, 240, 244, 245, 246,
227 247, 250, 251, 256, 264
primer designing, 191 purines, 61
prions, 284, 303 purity, 117, 148, 208, 291
private sector, 108 PVA, 268
Index 359
PVS, 184 reducing sugars, 59, 224, 225, 227, 274

pyridoxine, 136, 295 regions of the world, 9
pyrodextrins, 32 regression, 327
regulations, 95, 108, 118, 146, 155, 172, 301
regulatory agencies, 110, 128, 146, 179
Q regulatory bodies, 146, 148
regulatory controls, 326
quality assurance, 311
regulatory system, 106
quality control, 202
rehydration, 294, 308, 309
quality of life, 156, 265
reinforcement, 257
quantification, 23, 139, 155, 172, 176, 177, 183, 190,
relevance, 181, 309
200
reliability, 176
quantitative PCR, 191, 193
renewable resources, 251, 265, 273
quantum dot(s), 202, 211, 254
repair, 314
quartz, 207, 211
reparation, 57, 140
quercetin, 126, 321, 327
reporter genes, 295
query, 111
repression, 19, 244, 296, 297
quinones, 115, 127
reproduction, 313
requirement(s), vii, 6, 72, 79, 82, 128, 148, 194, 198,
R 283, 284, 287, 288, 342
residue(s), 4, 22, 32, 81, 99, 109, 143, 159, 232, 234,
race, 13 236, 243, 246, 248, 264, 272, 274, 281
radiation, 76, 119, 152, 192, 265, 281, 286 resins, 121
radicals, 145, 321, 325, 336 resistance, 26, 39, 95, 96, 97, 98, 100, 101, 102, 112,
radio, 176, 177 158, 202, 206, 235, 254, 257, 283, 294, 297, 303,
radiography, 176 308, 309, 318, 332, 333
radiotherapy, 277 resolution, 200
Raffinose, 32, 35, 296 resources, 30, 103, 251, 265, 273, 328, 333
rape seed, 145 respiration, 285, 286
raw materials, 4, 34, 67, 118, 145, 147, 148, 202, respiratory problems, 324
266, 273 response, 30, 37, 38, 124, 152, 156, 158, 161, 166,
reactive oxygen, 297 205, 245, 249, 259, 281, 336, 337, 338, 339, 341,
reactivity, 155, 158, 161, 162, 164, 167, 168, 171, 342
172, 177, 178, 181, 182, 183, 185, 308 response time, 205
reading, 333 restaurants, 67, 68
reagents, 196, 199, 312 restrictions, 315
real time, 178, 190, 192, 195, 202, 258, 264, 279 resveratrol, 298, 319
recall, 101, 102 retina, 145
receptacle, 199 retinol, 169
receptor(s), 82, 177, 178, 205, 263, 336, 337, 339, reverse osmosis, 141
342 reverse transcriptase, 181, 195
recognition, 91, 92, 116, 152, 178, 204, 206, 255, rheology, 165
267 rheumatoid arthritis, 215
recombinant DNA, 111, 134 rhinitis, 44, 156
recombination, 296 Rhizopus, 50, 88, 273, 274
recommendations, 147, 314 riboflavin, 14, 21, 59, 120, 128, 130, 295
reconciliation, 308 ribonucleic acid, 278
recovery, 117, 135, 140, 202, 236, 250, 302, 303, ribosomal RNA, 295
318 rice husk, 52, 274, 279
recurrence, 41 rings, 59
red blood cells, 313 risk assessment, 113, 179, 267, 301
red wine, 247, 317, 318 risk factors, 314, 329
redistribution, 235 risk management, 179
360 Index
RNA, 195, 197, 203, 204, 213, 287, 295, 306 self-organization, 255
rodents, 325 semiconductor, 211
room temperature, 7, 51, 53, 57, 68, 70 sensation, 116
root(s), 6, 32, 53, 118, 121, 133, 135, 140, 142, 151, senses, 116
317 sensing, 204, 257, 265
rotary vacuum filter, 290 sensitivity, 4, 37, 128, 143, 176, 177, 178, 192, 193,
rotavirus, 39, 41 196, 197, 198, 200, 205, 210, 258, 276, 339
routes, 156 sensitization, 171
Royal Society, 266, 268 sensor(s), 202, 205, 207, 209, 211, 227, 253, 254,
rubber, 150 257, 265, 269, 289
rules, 108, 112 sequencing, 191
rural areas, 48 serine, 165, 338
serotonin, 324
serum, 37, 40, 159, 166, 167, 169, 190, 198, 278,
S 302, 324
serum albumin, 166, 167, 169, 190
S. cerevisiae, 59
shear, 209
Saf Yeast, 288
sheep, 64, 68, 70, 166, 168, 179
salicylates, 293
shelf life, vii, 6, 9, 47, 57, 66, 76, 83, 89, 93, 96, 188,
salinity, 98, 116
205, 223, 226, 253, 256, 257, 259, 260, 264, 297
salmon, 85, 90, 92, 93, 94, 101, 124, 171, 173, 321
shellfish, 64, 93, 155, 157, 167, 171, 214
Salmonella, 30, 39, 88, 191, 192, 193, 195, 196, 199,
shock, 83, 123
200, 201, 202, 203, 204, 206, 208, 209, 211, 212,
shoot, 74, 336
213, 214, 215, 216, 217, 291, 294
shoots, 61
salt concentration, 9, 14
short term memory, 315
salt production, 64
short-chain fatty acid, 335
salts, 127, 135, 189, 190, 236, 274, 279, 287, 289
showing, 102, 119, 297
SAS, 152
shrimp, 65, 85, 94, 167, 171, 174, 180, 182, 184,
saturated fat, 125
186, 212
saturated fatty acids, 125
side chain, 232, 234
scaling, 128
side effects, 79
scarcity, 48, 111
signal transduction, 178
scavengers, 31, 239
signaling pathway, 275, 336, 342
sclerosis, 322, 328
signals, 202, 206
scope, 127, 148, 157, 311, 333, 337, 340
signs, 318
SDS-PAGE Immunoblotting, 177
silica, 189
seafood, 88, 90, 93, 157, 158, 166, 167, 171, 183,
silver, 117, 118, 127, 175, 253, 256, 257, 261, 264,
191, 212, 216
267
seasonality, 220
Sinai, 293, 308
secondary metabolism, 165
single cell protein (SCP), vi, viii, 271, 272, 273, 274,
secondary metabolites, 4, 79, 140, 335
275, 276, 278, 279, 280, 282
secrete, 230, 235
single nucleotide polymorphisms (SNPs), 332, 337,
secretion, 27, 30, 39, 134, 191, 246
339
sedative, 50, 315
skeleton, 137, 159
sedentary lifestyle, 156
skin cancer, 295
sediment, 152, 224
sludge, 247, 288
sedimentation, 161
small intestine, 33, 67
seed, 57, 97, 110, 144, 145, 149, 159, 161, 183, 184,
smoking, 16, 48, 65, 66, 67
315, 317, 318, 321
smuggling, 109
seedlings, 100
sodium, 66, 88, 93, 189, 279, 314, 325
seizure, 315
sodium dodecyl sulfate (SDS), 177, 189
selectivity, 13, 205, 220
solid phase, 199
selenium, 37, 261
solid waste, 235
self-assembly, 255
Index 361
solubility, 119, 260 sugarcane, 106, 274, 279

solution, 64, 66, 96, 132, 172, 195, 228, 273, 274, sulfate, 189, 318
312, 332, 335, 336, 341 sulfites, 127
solvents, 122, 123, 139, 149, 233, 236, 261 sulfur, 160, 298, 321
sonication-assisted extraction, 139 sulphur, 11, 136, 159, 167, 286, 308
soybeans, 12, 57, 58, 101, 108, 317, 325 Sun, 205, 215, 225, 230, 249, 341
soymilk, 76 supplementation, 31, 35, 37, 40, 43, 235, 274, 277,
specialists, 172 302, 314, 321, 327, 329
specific surface, 263 suppliers, 115, 123
specifications, 138 supply chain, 188
spectrophotometry, 139 surface area, 254, 256, 257, 260, 265
spectroscopy, 308 surface chemistry, 263
sperm, 313, 314 surface component, 91
spirulina, 131, 146, 147, 148, 271, 276, 277, 278, surfactants, 138, 260, 265
279, 280, 281, 282, 318 surplus, 137
spore, 11, 14, 86, 87, 202 survival, 31, 34, 43, 88, 93, 158, 297, 306
sprouting, 212, 215 susceptibility, 116, 162, 201, 214, 224, 332, 333, 337
Sri Lanka, 73, 288 suspensions, 255
SSA, 153, 256, 263 sustainability, 109
stabilization, 223, 224, 231, 233, 236, 239, 241, 248, Sweden, 29
258, 260 sweeteners, 1, 4, 171, 219, 220, 222, 227, 236, 261
stabilizers, 272 swelling, 155, 245
stakeholders, 261 Switzerland, 29, 109
standardization, 143, 202 symptoms, 155, 164, 171, 312, 313, 315, 318
staphylococci, 216 synbiotics, vii, 25, 34, 35, 36, 38, 40, 41, 42, 43, 44
starch granules, 44, 226, 236 syndrome, 36, 40, 41, 45, 159, 161, 164, 183, 293,
starch polysaccharides, 235, 237, 250 313, 315
statistics, 156, 287 synergistic effect, 89
sterile, 207, 272, 289, 290, 291 synthesis, 13, 62, 82, 99, 117, 118, 141, 144, 145,
steroids, 272, 313 194, 205, 210, 220, 237, 248, 285, 299, 313, 314,
sterols, 139, 336 318
stigma, 122
stimulation, 33, 138, 177, 307
stock, 49, 64, 290 T
stomach, 9, 60, 64, 68, 70, 102, 263, 325
T cell(s), 280
stomach ulcer, 325
tannins, 121, 318, 321
strain improvement, 296
tar, 117
stressors, 297
target, 21, 75, 76, 79, 80, 82, 91, 101, 103, 109, 178,
structural characteristics, 71, 182
179, 190, 191, 192, 194, 195, 197, 198, 199, 200,
structural gene, 83
201, 202, 203, 204, 207, 208, 255, 300
structural protein, 174
technological advances, 48
styles, 125
teeth, 313
subgroups, 80
terpenes, 263
submerged fermentation, 141, 237, 239, 245, 275
test procedure, 266
substitutes, 221
testing, 89, 108, 111, 178, 192, 199, 201, 203, 209,
substitution(s), 123, 143, 185, 305
214, 266, 295
substrate(s), 4, 33, 41, 47, 50, 51, 52, 88, 104, 117,
testing program, 209
138, 142, 154, 176, 198, 199, 203, 205, 206, 222,
testosterone, 313
229, 230, 232, 244, 246, 247, 271, 272, 273, 274,
textiles, 118, 220
275, 278, 285, 286, 289, 294, 296, 298, 308
texture, vii, 4, 6, 9, 11, 12, 13, 14, 16, 20, 62, 63, 67,
sucrose, 8, 32, 34, 117, 136, 228, 229, 236, 274, 285,
221, 224, 226, 227, 231, 236, 242, 256, 262, 291,
288, 296, 297
295, 316
sugar beet, 32, 274
Thailand, 12, 15, 65, 277
362 Index
therapeutic agents, 270 triggers, 227

therapeutic effect(s), 328 triglycerides, 17, 37, 139, 150
therapeutic use, 327 tropomyosin, 159, 167, 171, 173, 174, 180, 181, 182,
therapy, 185, 338 183
thermal properties, 264 trypsin, 79, 159, 186
thermal stability, 260 tryptophan, 243
thermostability, 236 tuff, 56
thiamine, 14, 59, 289, 295 tumor(s), 145, 317, 338
thin films, 260, 267, 268 type 2 diabetes, 37, 43, 302, 339, 340, 341
thioredoxins, 164, 165, 186 tyrosine, 319, 335
threonine, 222
thrombosis, 339
throws, 47 U
thyroid, 313
ulcer, 61, 315
tin, 66, 175
ulcerative colitis, 38
tinnitus, 315
ultrasound, 76, 140
tissue, 132, 134, 139, 148, 149, 204, 313, 318, 337
uniform, 13, 16, 118, 136, 141, 258
titanium, 117, 118, 254, 257
upper respiratory tract, 36, 42, 43
TLR, 239
urban, 235, 277, 286
tobacco, 100, 316
urban areas, 286
tofu, 338
urea, 277, 281, 286, 299, 305
Togo, 277
urease, 300, 310
torulopsis, 7, 12, 61, 274, 286
uric acid levels, 278
total cholesterol, 37
urinary tract infection, 325
total energy, 3
urine, 278
total product, 288
urticaria, 175, 182
toxicity, 48, 95, 115, 121, 128, 148, 227, 232, 254,
USDA, 96, 102, 108, 114, 147, 330
265, 266, 309
UV radiation, 192, 265
toxicology, 182, 265
toxin, 30, 39, 99, 100, 109, 112, 191, 198, 199, 209,
210, 211, 216, 302 V
trace elements, 319
trade agreement, 111 vaccine, 112, 254
traits, 98, 284, 296, 338 vacuum, 94, 290, 294
transaminases, 298 validation, 112, 172, 202
transcription, 195, 197, 213, 302, 303, 332, 339 valuation, 147
transcription factors, 332 valve, 258
transcriptional regulator, 297 vapor, 269
transducer, 204, 206 variables, 16, 289
transduction, 173, 178, 206, 207 variations, 4, 5, 36, 148, 272, 288, 331, 334, 337
transfection, 269 varicose veins, 325
transformation, 103, 113, 247, 262, 289, 295, 299, varieties, 4, 5, 9, 10, 11, 48, 59, 63, 64, 67, 148
304 vector, 102
transgene, 103, 104, 112 vegetable oil, 17, 122, 124, 233, 318
translocation, 91, 275 versatility, 232
transmission, 66, 100, 206 vertigo, 315
transparency, 48, 265 vesicle, 275
transplantation, 44, 100 vessels, 290
transport, 27, 30, 64, 78, 254, 263, 284, 303, 304, vicillins, 162
307, 314, 336, 337 Vietnam, 65, 106
transportation, 256, 263, 324 viral gastroenteritis, 37
trial, 39, 40, 41, 42, 44, 45, 96, 101, 327 virology, 304
tricarboxylic acid, 19 viruses, 36, 78, 97, 144, 169, 205
Index 363
viscosity, 221, 226, 228, 231, 232, 233, 234, 235, workers, 228
237, 264 working women, 286
vision, 313 World Health Organization (WHO), 61, 62, 71, 77,
vitamin A, 14, 101, 132, 143, 144, 270, 321, 327 143, 157, 172, 181, 225, 251
vitamin B1, 21, 295, 313 worldwide, 1, 11, 48, 62, 73, 101, 106, 115, 155,
vitamin B12, 295, 313 156, 157, 179, 267, 271, 283, 340
vitamin C, 313, 317, 326, 328 wound healing, 313, 315
vitamin D, 313, 336, 341, 342 WTO, 110, 112
vitamin E, 270, 321, 327
vitamins, 1, 13, 21, 35, 42, 47, 48, 59, 61, 116, 127,
236, 257, 258, 260, 261, 274, 284, 288, 291, 293, X
294, 295, 301, 312, 314, 319, 340
xanthones, 121
volatile organic compounds, 257
xanthophyll, 124, 145, 152
xylans, 234
W xylitol, 236, 246
waste water, 240, 264, 270, 277, 281

water purification, 264 Y
water quality, 277
yakult, 28, 30, 323
water vapor, 269
yeast(s), viii, 11, 12, 18, 19, 20, 23, 50, 51, 57, 61,
wavelengths, 116, 202
70, 71, 74, 120, 127, 134, 135, 136, 138, 140,
wealth, 265, 323
150, 207, 208, 224, 226, 233, 244, 272, 273, 274,
web, 81, 339
278, 279, 280, 283, 284, 285, 286, 287, 288, 289,
weight loss, 317, 330
290, 291, 292, 293, 294, 295, 296, 297, 298, 299,
well-being, 22, 294
300, 301, 302, 303, 304, 305, 306, 307, 308, 309,
wellness, 312, 323
310
whey, 9, 32, 34, 37, 43, 44, 63, 64, 69, 71, 153, 166,
yeast extract, 292, 295, 303
239, 264, 273, 274, 279, 280, 281, 286, 296
yoghurt, 4, 5, 27, 30, 62, 86, 117, 227, 259, 272
white blood cells, 38
yolk, 132, 167, 180, 318
white blood corpuscle, 277
wholesale, 288
wild type, 297 Z
Wilson, Woodrow, 263, 264, 270
wine, 5, 18, 19, 22, 23, 24, 50, 71, 117, 124, 132, zinc, 13, 254, 256, 257, 277, 289
137, 202, 221, 223, 227, 228, 232, 233, 247, 283, zinc oxide, 254, 256, 257
288, 292, 293, 295, 296, 298, 299, 300, 301, 302, zirconium, 189
303, 305, 306, 307, 308, 317, 318, 328 zymocins, 300
Wisconsin, 101, 113
wood, 138, 152, 234, 235, 241, 248

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BIOTECHNOLOGY IN AGRICULTURE, INDUSTRY AND MEDICINE

FRONTIERS IN FOOD BIOTECHNOLOGY

Additional books in this series can be found on Nova‘s website

Additional e-books in this series can be found on Nova‘s website

FRONTIERS IN FOOD BIOTECHNOLOGY

NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

ISBN  (eBook)

Published by Nova Science Publishers, Inc. † New York

Chapter 11 Single Cell Protein: Microbial Production and Analysis 271

The application of biotechnology in food sciences has subsequently increased the

Dr. Chetan Sharma

Dr. Anil K. Sharma

Prof. K.R. Aneja

LACTIC ACID BACTERIA (LAB)

Namita Rokana1, and Harsh Panwar2

Keywords: lactic acid bacteria (LAB), fermented foods, homofermentation,

Table 1. Characteristic of homo and heterofermentative lactic acid bacteria

Characterization Homofermentative LAB Heterofermentative LAB

Adapted from Modler et al., 1990.

MICROBIOLOGY OF DIFFERENT TYPES OF FERMENTED PRODUCTS

Table 2. Types of fermented foods and their main constituent

Raw material Products Starter culture

Figure 2. Schematic presentation of the procedure of yogurt preparation.

1.2. Fermented Milk Beverages

acidophilus, L. helveticus, L. delbreuckii subsp. bulgaricus, L. rhamnosus, L. gasseri, L.

Figure 3. Flow diagram of kefir preparation.

Figure 4. A general diagram of procedure of koumis preparation.

Figure 5. Schematic diagram of cheese preparation and ripening.

Table 3. Different types of cheese varieties and associated microflora

Category Cheese Starter cultures Nonstarter lactic Other

2. Cereal and Legume Based Fermented Foods

Table 4. Worldwide popular traditional cereal/legume based fermented foods

Products Substrates Starter culture Country

Figure 6. A general processing of cereals and legumes during fermentation.

Figure 7. Biochemical Changes in Cereal-based Fermented Foods.

Preservation of meat by fermentation is done in different parts of world. Several types of

Table 5. Worldwide popular fermented vegetables

Product Vegetable/Fruit Other ingredients Place

Table 6. Basic characteristics of three major types of fermented meats

Types of fermented sausage Characteristics

Figure 9. Flow diagram of biochemical changes during meat processing.

Figure 10. Flow diagram of wine manufacture.

Figure 11. Carbohydrate metabolism and ethanol production by yeast.

1. Improvement of Nutrition Value

3. Preservation and Enhancement of Safety

Tanous, C; Kieronczyk, A; Helinck, S; Chambellon, E; Yvon, M. Glutamate dehydrogenase

PROBIOTICS, PREBIOTICS AND SYNBIOTICS:

Akshay Joshi, Vikram B. Lanjekar, Prashant K. Dhakephalkar

Keywords: bacteria, health, gut, dairy, functional food, disease, diet

Probiotic Microbes, Their Selection Criterion and Mode of Action

The information related to modes of action and mechanism of inhibition by probiotics is

Figure 1. Selection Criteria for Probiotic Microorganisms.

Table 1. List of probiotic bacteria, their antimicrobial compounds and commercially

Table 2. Different modes of action of probiotics

Action Probiotics Pathogens Mechanism of inhibition Reference

Table 3. Probiotic dairy products available in Indian market

Product Name Product Type Company Probiotic

 Protection against oxygen

Type Source Composition Method of manufacture

Selection of the Suitable Prebiotic

 Resistance to gastric acid, enzymatic digestion, and intestinal absorption

Substances Used as Prebiotics

The substances used as prebiotics should be ‗selective‘ or ‗specific‘ towards ecological

ISBN (eBook)