USE OF BIOTECHNOLOGY TECHNIQUE LIKE PCR & RFLP IN

FOOD INDUSTRY
USE OF BIOTECHNOLOGY TECHNIQUE LIKE PCR & RFLP
IN FOOD INDUSTRY

B.K.K.K.Jinadasa,

Department of Food Science & Technology,

University of Sri Jayawardanapura,

July-2009
Contents 
Introduction: ...…………………………………………………………………………………………..1

Polymerase Chain Reaction: .....................................................................................................1

Restriction Fragment Length Polymorphism:........................................................................... 2

Uses of Biotechnology: ................................................................................................................... 3

Agricultural Biotechnology: ....................................................................................................... 4

Biotechnology use in food industry: ......................................................................................... 5

Issues and relevant to food industry:............................................................................................ 8

Conclusion:....................................................................................................................................... 8

References:....................................................................................................................................... 9

 
1.0 Introduction:

According to the Codex statements in March 2000, biotechnology is “any technological

application that uses biological systems, living organisms, or derivatives thereof, to make or
modify products or processes for specific use”. As the some other definition "Any
technological application that uses biological systems, dead organisms, or derivatives
thereof, to make or modify products or processes for specific use." A broad definition of
biotechnology is "The application of indigenous and/or scientific knowledge to the
management of (parts of) microorganisms, or of cells and tissues of higher organisms, so that
these supply goods and services of use to the food industry and its consumers. Biotechnology
provides powerful tools for the sustainable development of agricultural, fisheries and forestry
and food industry. There are different techniques and applications.

Biotechnology has included wide range f diverse technologies and they may be applied in
many of the different field in food and agricultural sectors. Biotechnology includes
technology such as gene modification and transfer, use of the molecular markers,
development of recombinant vaccines and DNA based methods like PCR for diseases
characterization and diagnostics, in vitro vegetative propagation of plants, embryo
transferring technique and other reproductive technologies in animals like fish, DNA typing
and cloning of plants and animals. Tissue culture has produced plants that are increasing crop
yields by providing farmers with healthier planting material. Rice has been genetically
engineered to contain pro-vitamin A (beta carotene) and iron which could improve the health
of many low income communities. As well as biotechnology includes a range of technologies
used to process the raw food materials produced by crops, fishery and livestock sectors.
Biotechnology in the food processing sector targets the selection and improvement of
microorganisms with the objectives of improving process control, yield and efficiency as
well as quality safety and consistency of bioprocesses products. Microbes are generic terms
for the group of living organisms which are microscopic in size. It includes bacteria, yeast
and moulds.

1.1. Polymerase Chain Reaction:

In 1983 Kary B. Mullis was driving through California on a moonlight night. He was
pondering how to use DNA polymerase with oligonucleotide primers in order to identify a
given nucleotide at a given position in a complex DNA molecule, such as the human
genome. During this drive he invented or discovered the elegant method of making unlimited
DNA copies from a single copy of DNA, and called the method: "Polymerase Chain
Reaction" (PCR). A couple of months later he conducted the first successful experiment. Ten
years after his drive in California, he was awarded the Nobel Prize in Stockholm for his
brilliant discovery (Carr, 1993).

PCR was first published in 1985 with Klenow polymerase used as the elongation enzyme.
Due to the heat instability of the Klenow polymerase, new enzyme had to be added for every
new cycle, and the maximum limit of the product length was 400 bp. In 1988 the first report
using DNA polymerase from Thermophilus aquaticus (Taq-polymerase) was published. This
polymerase greatly enhanced the value of PCR, and the introduction of the automatic
programmable heating block in the same report also took the tedious need for three different
water baths out of the procedure. Currently the PCR technique is utilized in most molecular
biology laboratories as a routine tool which is suitable for performing a great number of
different experiments. The method is frequently chosen for conducting experiments, such as
cloning, making mutations, sequencing, detecting, typing, etc. The polymerase chain reaction
(PCR) is a relatively simple technique that amplifies a DNA template to produce specific
DNA fragments in vitro. Traditional methods of cloning a DNA sequence into a vector and
replicating it in a living cell often require days or weeks of work, but amplification of DNA
sequences by PCR requires only hours. While most biochemical analyses, including nucleic
acid detection with radioisotopes, require the input of significant amounts of biological
material, the PCR process requires very little. Thus, PCR can achieve more sensitive
detection and higher levels of amplification of specific sequences in less time than previously
used methods. These features make the technique extremely useful, not only in basic
research, but also in commercial uses, including genetic identity testing, forensics, industrial
quality control and in vitro diagnostics. Basic PCR has become commonplace in many
molecular biology labs where it is used to amplify DNA fragments and detect DNA or RNA
sequences within a cell or environment. However, PCR has evolved far beyond simple
amplification and detection, and many extensions of the original PCR method have been
described.

The PCR process was originally developed to amplify short segments of a longer DNA
molecule. A typical amplification reaction includes the target DNA, a thermo stable DNA
polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction
buffer and magnesium. Once assembled, the reaction is placed in a thermal cycler, an
instrument that subjects the reaction to a series of different temperatures for varying amounts
of time. This series of temperature and time adjustments is referred to as one cycle of
amplification. Each PCR cycle theoretically doubles the amount of targeted sequence
(amplicon) in the reaction. Ten cycles theoretically multiply the amplicon by a factor of
about one thousand; 20 cycles, by a factor of more than a million in a matter of hours.

Each cycle of PCR includes steps for template denaturation, primer annealing and primer
extension. The initial step denatures the target DNA by heating it to 94°C or higher for 15
seconds to 2 minutes. In the denaturation process, the two intertwined strands of DNA
separate from one another, producing the necessary single-stranded DNA template for
replication by the thermo stable DNA polymerase. In the next step of a cycle, the temperature
is reduced to approximately 40–60°C. At this temperature, the oligonucleotide primers can
form stable associations (anneal) with the denatured target DNA and serve as primers for the
DNA polymerase. This step lasts approximately 15–60 seconds. Finally, the synthesis of new
DNA begins as the reaction temperature is raised to the optimum for the DNA polymerase.
For most thermo stable DNA polymerases, this temperature is in the range of 70–74°C. The
extension step lasts approximately 1–2 minutes. The next cycle begins with a return to 94°C
for denaturation.

Each step of the cycle should be optimized for each template and primer pair combination. If
the temperature during the annealing and extension steps are similar, these two steps can be
combined into a single step in which both primer annealing and extension take place. After
20–40 cycles, the amplified product may then be analyzed for size, quantity, sequence, etc.,
or used in further experimental procedures.
1.2. Restriction Fragment Length Polymorphism:

RFLP (pronounced as a rif-lip) mentioned in the name are sections of a DNA sequence
which have been cut by a restriction enzymes. Lengths of the fragments are polymorphic
because certain differences in DNA strands such as base substitutions, additions, deletions
and other sequence rearrangements. Researchers have developed a method of comparing
DNA sample based on this differential cleavage of DNA by restriction enzymes. First step of
perform RFLP of extracted DNA from the sample. The DNA must then cut with one or more
restriction enzymes. Each resulting fragments is placed into the agarose gel. The
electrophoresis separates the restriction fragments by size. The shorter fragments are travel
more quickly than larger ones. The final step is visualizing the DNA. When DNA running
with Ethilium Bromide, are facilitates to see the bands fluorescence under UV light.

Biotechnology facilitates in agricultural fields to produce hybrid seeds, bio fertilizers, bio
pesticides, plant extract, plant genetic engineering and plant tissue culture fields also. At the
food industrial field biotechnology use in industrial enzymes, bio fuels, polymers, fermented
products, microbial strains, biocatalysts and oligonucleotides etc.

2.0 Uses of Biotechnology:

2.1 Agricultural Biotechnology:

World population is increasing very fast; concern about that the quality of food, bio
agricultural technique should developed to fulfil that needs. Normally agricultural farmers
wish to take more return than investment and also increase productivity by using these
techniques.

The challenge of producing more food grains to feed the ever increasing world population.
Plant breeding has been enhanced considerably by in vitro development of improved verities
which are better adapted to a specific environment. Using the techniques of modern
biotechnology, one or two genes (Smartstax from Monsanto will use 8, starting in 2010) may
be transferred to a highly developed crop variety to impart a new character that would
increase its yield. However, while increases in crop yield are the most obvious applications
of modern biotechnology in agriculture, it is also the most difficult one. Current genetic
engineering techniques work best for effects that are controlled by a single gene. Many of the
genetic characteristics associated with yield (e.g., enhanced growth) are controlled by a large
number of genes, each of which has a minimal effect on the overall yield. There is, therefore,
much scientific work to be done in this area. The application of tissue cultural has some
advantages like rapid reproduction and multiplication, availability of seed materials
throughout the year. Tissue cultural technique very popular technique, because do not need
very high tech instruments, and knowledge. As an example tissue cultural technique use to
take banana seed in Sri Lanka.

As well as biotechnology help to reduce the use of agrochemicals. It will help to enhance the
productivity, reduction in toxic elements in final product and environment also.
Biotechnology use many ways to increase yield; for example by increasing flowering
capacity and increasing photosynthesis. The plant cloning can help reduce the work in
harvesting period. It can use to take more uniform character in plant like growing speed,
ripening at same time. As well as scientist have been able to produce insect resistant plant
verities such as BT corn.

Food shortages would not exist in many countries if the problems of post harvest losses could
be solved. In the future genetic engineer may be used to remove plant components that cause
early deterioration of the harvest. Recently the genetic engineer had used to take box type
water lemon, it is reduced the storage space when transport. Other example is reduce the
presence of normal tomato enzymes involved in the softening of ripe tomato fruit has been
patented and would be found very useful for enhance the shelf life of crops varies verities.

When we consider livestock and poultry fields; several growth enhancing novel genes have
been introducing into pigs. By using this several qualities enhance the meat; example meat is
lean and tenderer. Many modifications to milk have been done by gene manipulation
technology; as an example Newzealand developed GM cows that produce milk with
increased levels of casein protein. Use of that protein increases the efficiency of cheese
production. Other application of genetic modification in animal production in the early stages
of research and development include improvement of disease resistance, increased birth rates
of sheep, altered sex ration in poultry, increased egg production in poultry by creating two
 active ovaries, and improved feed conversion in the pig. When we consider aquaculture field,
enhanced the growth of Atlantic salmon containing a growth hormone gene from Chinook
salmon is likely to be first GM animal on the food market. These fish grow 3-5 times faster
than their non transgenic variety. At least eight other farmed fish species have been
genetically modified for growth enhancement. One of above species has introduced in Sri
Lanka few years ago call GIFT Tilapia (genetically improved farm Tilapia).

2.2 Biotechnology use in food industry:

Biotechnology in food processing sector targets the selection and improvement of

microorganisms with the objectives of improving process control, yields and efficiency as
well as the quality, safety and consistency of bio-processed products.

Fermentation is one of the oldest forms of food preservation method which is conversion of
organic substance by microorganisms or enzyme of microbial, plant or animal origin. Bread,
cheese, wine, yogurt, sausages, soy sauce, cider, cocoa, coffee, kefir, miso, salami, sauerkraut
are the typical example for that. Food fermentation is important to food safety, nutritional
value and food security. Even though fermentation technology has been used many years, the
technology is very simple as well as not developed comparing with time. Some bacteria used
in food fermentation produce compounds that kill other food poisoning and spoilage bacteria.

Microorganisms have been essential in food industry as a source of many food additives and
processing aids. Many food additives are substances nature has provide microbial origin, such
as xanthan gum, guar gum. Many of amino acid supplements, flavours, flavour enhancers,
vitamins added to breakfast cereals are produced by microbial fermentation. The enzymes
produced by microbial fermentations play essential roles as processing aids in the food
industry. Before the bio technique, the enzymes like rennin had are extracted from stomach of
calves, lambs and bay goats, but it now produced by microorganisms that were given the gene
for this enzymes.

Modern biotechnology can be used to slow down the process of spoilage so that fruit can
ripen longer on the plant and then be transported to the consumer with a still reasonable shelf
life. This alters the taste, texture and appearance of the fruit. More importantly, it could
expand the market for farmers in developing countries due to the reduction in spoilage.
However, there is sometimes a lack of understanding by researchers in developed countries
about the actual needs of prospective beneficiaries in developing countries. For example,
engineering soybeans to resist spoilage makes them less suitable for producing tempe which
is a significant source of protein that depends on fermentation. The use of modified soybeans
results in a lumpy texture that is less palatable and less convenient when cooking.

The first genetically modified food product was a tomato which was transformed to delay its
ripening. Researchers in Indonesia, Malaysia, Thailand, Philippines and Vietnam are
currently working on delayed-ripening papaya in collaboration with the University of
Nottingham and Zeneca.

Biotechnology in cheese production; enzymes produced by micro-organisms provide an

alternative to animal rennet – a cheese coagulant - and an alternative supply for cheese
makers. This also eliminates possible public concerns with animal-derived material, although
there are currently no plans to develop synthetic milk, thus making this argument less
compelling. Enzymes offer an animal-friendly alternative to animal rennet. While providing
comparable quality, they are theoretically also less expensive.

About 85 million tons of wheat flour is used every year to bake bread. By adding an enzyme
called maltogenic amylase to the flour, bread stays fresher longer. Assuming that 10-15% of
bread is thrown away as stale, if it could be made to stay fresh another 5–7 days then perhaps
2 million tons of flour per year would be saved. Other enzymes can cause bread to expand to
make a lighter loaf, or alter the loaf in a range of ways.

When we consider health factors, plant scientist have able to make healthier cooking oils
which is decreased in total amount of saturated fatty acids in certain vegetable oils. Other vice
normal hydrogenation process of oil form trans-fatty acids. But using biotechnology heat
stability of oil has improved. Produce high protein potato verity using amaranth gene transfer,
canola oil with high amount of vitamin A, increase the ellagic acid like cancer protective
agent in strawberry, are the other example for health and nutrional and health benefits. Most
of food allergens also can remove from food by using biotechnology.
Biotechnology helps to improve product quality, especially in raw materials quality. Increase
the shelf life of fresh fruits and vegetables using this technique, improving the crispness of
carrots, peppers and celery, certain seedless verities of grapes and melons, improving the
flavour of tomato, lettuce, pepper, peas and potatoes and creating caffeine free coffee and tea.
Recently the Japanese scientists have identified the gene which is produce enzymes that make
s cry when we slice onion. By using biotechnology, they are able to make the tearless onion.
The normal potato with high water content absorbed the high amount of oil when frying
process, it is not healthier. Now scientist able to produce high starch potato verity, which are
absorbed less oils as well as less cost to handle.

Biotechnology is helping many ways to enhance food safety. It is providing many tools to
detect microorganisms and the toxins they produce. Monoclonal antibody tests, biosensors,
PCR, and DNA probes are being developed to determine the presence of harmful bacteria
such as Listeria and Clostridium, E. coli 0157:H7. Biotechnology based detection method
have been developed now to detect toxin like aflatoxin. Unhygienic processing and handling
help to add food borne pathogens into the food, it will critical for ensuring the safety to
consumers. Traditional detection method takes need 3-4 days, but biotechnological method
need maximum 36 hrs and it also specific, sensitive and faster than normal conventional
method. As well as PCR based method are also used for the detection of ingredients in food
product such as soy in meat products, whet in non whet products or allergens in diverse food.

3.0. Issues and relevant to food industry:

Application of biotechnology to food processing in developing countries is in issues for a

long time. A Socio economic and cultural factor is one of issues. Traditional food
fermentation processes are low input with minimal investment requirement. These product
and process normally uncontrolled, unhygienic and inefficient as well as product variable
quality, short shelf life. But fermented food play major role in food security and nutrition in
developing countries.
Infrastructural and logistic factors are the next important one. Physical infrastructural
requirement need for the manufacture, distributor and storage of generally these technologies.
Other issues are the consumer education, intellectual property rights should be considering.

4.0. Conclusion:

DNA-based methods are increasingly used for the detection of foreign food constituents, such
as microbial pathogens, or the presence of genetically modified crop material. Furthermore,
the detection of plant and animal species as well as allergens, certain ingredients or
contaminants in the final food products has been shown to be feasible with DNA-based
methods. The methods are in general fast, very specific and provide a sensitive tool for the
detection of specific food constituents. The choice of the analytical method applied is
however, mainly dependent on the food concerned (availability of specific PCR primers) and
on the history of processing involved during food production (degradation or even removal of
DNA). However, it has to be considered that PCR-based assays detect only the presence of
DNA from a living entity. However, this is not always the compound of concern. PCR-based
assays do not detect for example the allergen or mycotoxin itself. Therefore, the PCR-based
result cannot be tied to actual allergenic exposure or an actual contamination of the sample
with mycotoxins. Nevertheless, PCR-based detection methods provide an excellent alternative
to more traditional methodologies in the quality and safety assurance of food.

References:

U.S. Department of State International Information Programs, “Frequently Asked Questions

about biotechnology”, USIS online available from:
http://usinfor.state.gov/ei/economic_issues/biotechnology/biotech_faq.html.

Bio-2008: biotechnology industry organization, Washington DC, www.bio.org

Molecular biology training manuals-2007: Anfaco - Cecopesca, Spain.

Executive summary of food biotechnology in Asia, 2008: Asian food information centre
(AFIC), www.afic.org.
Modern food biotechnology, human health and development an evidence based study, 2005:
World health organization, www.who.int.foodsafety