Food Microbiology

Food Microbiology
Sven-Olof Enfors KTH - Biotechnology Stockholm 2007
Content Chapt 1 Introduction...................................................................................1 Chapt 2. The ecological basis of food spoilage ...........................................5 2.1 The microflora ........................................................................5 2.2 The physico-chemical properties .............................................8 2.3 Chemical reactions ................................................................15 Chapt 3. Spoilage of different types of food .............................................22 Chapt 4. Food borne pathogens.................................................................38 4.1 Microbial food intoxications .................................................39 4.2 Food borne microbial infection .............................................44 Chapt 5. Food preservation.......................................................................51 5.1 Heat sterilisation and pasteurisation ......................................51 5.2 Irradiation ................................................................................. 5.3 High-pressure preservation........................................................ 5.4 Freezing .................................................................................... 5.5 Chemical preservatives.............................................................. Section 2-5 are not yet available. Please refer to the lecture notes Chapt 6 Fermented foods .........................................................................64
1 Chap 1 Introduction
Living organisms are usually classified as animals, plants, algae, protozoa, bacteria, archae or viruses. All viruses, archae, bacteria and protozoa plus the unicellular algae and some fungi, so called micro-fungi, are collectively called microorganisms. The microfungi can be further divided into yeast and molds, a classification that is based on the cell morphology. Based on DNA analysis, the group previously called bacteria is further divided into eubacteria and archae and today the word bacteria is usually used as synonym to eubacteria. Most microorganisms that we encounter in the normal spoilage of food belong to the eubacteria, here called bacteria, yeasts and molds. When it comes to food born diseases, also viruses, some protozoa and archae, i.e. the blue-green algae, are involved. A full species name is composed of two parts: the genus name plus the specification defining the species within that genus. sometimes these genera are grouped into families. This is illustrated in Table 1.1. Note that the genus name is spelt with leading capital letter, while the species name is spelled with lower case letters: Eschericia coli, Penicillium chrysogenum. The family, genus, and species names should always be written with italic letters. It is common in food microbiology literature that the full species name is not used since many species within the same genus are discussed. Then, Bacillus sp. means one not defined Bacillus species and Salmonella spp. means several not defined Salmonella species.
Table 1.1. Examples of family names, genus names and species names Species Escherichia coli Salmonella typhimurium Salmonella enterica Bacillacae Bacillus Bacillus subtilis Bacillus cereus Bacillus anthracis Bergeys Manual of Determinative Bacteriology divides bacteria into 35 groups. Groups, families, and genera which are most relevant in food microbiology are listed in Table 2.1. Family Enterobacteriacae Genus Escherichia Salmonella
In bacterial classification, the cell morphology, the relation to oxygen, and the Gram staining reaction are important parameters. Most common morphological types are rods, cocci (spheric cells), and vibrioforms (short bent rods). The Gram reaction gives information about the cell envelope. Gram negative cells have an outer membrane outside the cell wall that prevent the staining. Obligate aerobes require molecular oxygen for their energy metabolism (aerobic respiration). Anaerobes have an alternative energy metabolism that does not need oxygen. It may either be anaerobic respiration (with e.g. nitrate as electron acceptor) or fermentation. Oxygen is often toxic
Introduction
for anaerobic cells. Facultative anaerobic cells use oxygen and aerobic metabolism if oxygen is available but switch to anaerobic metabolism in absence of oxygen. Microaerophilic cells require low concentrations of oxygen, while normal air contact is inhibitory. Lactic acid bacteria (e.g. Lactobacillus and Lactococcus) have an obligately anaerobic metabolism but are still resistant to oxygen.
Table 2.1. Some of the bacterial groups (according to Bergeys Manual of Determinative Bacteriology) which are commonly encountered in food microbiology. Group nrp 2 4 5 Description Gram-neg., aerobic, mobile, vibrioformed Gran-neg., aerobic rods or cocci Gram-neg., facultatively anaerobic rods Food related organisms Campylobacter Pseudomonas, Shewanella, Legionella Family Enterobacteriacae (e.g. Escherichia, Enterobacter, Salmonella, Shigella, Yersinia, Erwinia) Vibrio Staphylococcus, Streptococcus, Lactococcus, Enterococcus, Micrococcus, Leuconostoc Bacillus Clostridium Lactobacillus Brochothrix Listeria
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Gram-pos. cocci
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Gram-pos endospore formers: aerobic or facultatively anaerobic: obligate anaerobes:
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Gram-pos, non-sporulating rods
There is a number of common groups of microorganisms that is not well defined but often used. Some food related examples are: Gram-negative psychrotrophic rods: This includes the genera Pseudomonas, Achromobacter, Alcaligenes, Acinetobacter, and Flavobacterium. Lactic acid bacteria (LAB) includes the food related genera Lactobacillus, Lactococcus, Pediococcus och Leuconostoc. Coliform bacteria is not synonymous to E. coli but includes Escherichia coli and Enterobacter.
Introduction
A special problem with the microbial taxonomy is that the names often are date dependent due to repeated re-classification of species. One example is the lactic acid bacteria which previously were called Streptococcus lactis, Streptococcus cremoris a.o. These so called lactic streptococci are now referred to a new genus and galled Lactococcus lactis, Lactococcus cremoris etc. Other previous Streptococcus spp. wich are associated with the intestines are now called Enterococcus, while yet another group of the previous Streptococcus genus remain as Streptococcus. When it comes to pathogenic organisms a further classification problem is that only some strains of a certain species may be pathogenic while other strains are harmless. An example is Escherichia coli to which species the feared EHEC (enterohaemorrhagic E. coli) belong. In such cases immunological or DNA analyses are required for proper classification.
Streptococcus is a genus with species of very different impact for humans. Some of todays Lactococcus and Enterococcus were previously classified as Streptococcus. They were then referred to as the lactic group and the enteric group of the streptococci, respectively. A classification of the old streptococci according to current nomenclature is: 1. Lactococci (Lactococcus lacits, L. cremoris a.o.). These organisms are often used for fermentation of food. 2. Enterococci (Enterococcus faecalis, E. faecium a.o.) are in most cases not pathogenic, but certain strains have been reported to cause serious infections. Such contradictions are due to the limitation in the current nomenclature which is based on phenotypic properties. These organisms are common in the intestinal flora. The presence of enterococci in food is not considered to be a health risk per se, but it is used as an indication of bad hygiene and that constitutes a risk, since other organisms of faecal origin like Salmonella may be present For this reason enterococci are called as indicator bacteria. 3. Hemolytic streptococci. There are two types of hemolytic streptococci, and these organisms remain in the genus Streptococcus: -hemolytic and -hemolyic. the hemolytic streptococci are named the viridans group and they are common on mucous membranes in the mouth and respiratory tract and on the teeth. The -hemolytic streptococci are named the pyogenes group and among them there are serious pathogens involved in several diseases and wound infections. -hemolytic organisms produce a greenish discolorisation zone around the colonies on blood agar while -hemolytic cells produce a clear zone.
Lactic acid fermentation is to a large extent also employed for production of food, namely some of the fermented foods: cheese, yoghurt, fermented sausages, and fermented vegetables like sauerkraut, pickles, olives a.o. However, this fermentation is also involved in food spoilage. Then the type of
Introduction
lactic acid fermentation may be important for the taste development. Some lactic acid bacteria mainly produce lactic acid which while others also produce other products.
The lactic acid bacteria are grouped according to their type of lactic acid fermentation. Homofermentative lactic acid bacteria produce mainly lactic acid from the sugar, and no CO2. To this category belong all all all some Streptococcus Lactococcus Pediococcus Lactobacillus
Heterofermentative lactic acid bacteria produce, besides lactic acid, also acetic acid, ethanol, CO2 and formic acid. Some can also convert citric acid (in milk) to diacetyl. In this group are all most Leuconostoc Lactobacillus
Chapter 2 2.1 The microflora
The ecological basis of food spoilage
Food consists to a large extent of cells from plants or animals (meat, fish, fruits, vegetables) and biological material with this origin (milk, juice, fat, starch etc). When discussing the shelf life of food it must be done from an ecological view. All biological material in Nature is degraded to simple molecular components, eventually down to inorganic components. This is called mineralization and it is a integrated part of the carbon and nitrogen cycles in Nature (Fig 2.1) which is a prerequisite for life on Earth. If the process is interrupted all nutrients would eventually be bound in dead biological material. The circumstance that we select some part of this biological material for food purpose does not change the natural destination of the food for degradation. However, it means that our interest in a long shelf-life of food is in conflict with the natural processes.
CO2 + N2 Light Fig 2.1. Microorganisms, especially bacteria and fungi, account for the main recirculation of carbon and nitrogen to the atmosphere from where it is adsorbed for generation of plants which constitute the original source of food.
Animals
Plants
Organic materia
Archae Bacteria Fungi Algae Protozoa
Dead organisms The degradation of biological material is mainly catalysed by microorgansims, which together carry an enormously diversified metabolic capacity. This is illustrated in fig 2.2 which summarises the main paths of the biological energy metabolism. All energy is generated, with exception of photosynthesis, by oxidation (combustion) of reduced substances (energy sources). Higher organisms like animals and also some microorganisms make this by oxidation of reduced carbon compounds, e.g. sugars. These compounds are oxidised in many steps in which oxidised co-enzymes (e.g. NAD+) constitute the oxidant, which then become reduced (e.g. NADH). These co-enzymes must be re-oxidised and eventually molecular oxygen in the air is used as the ultimate oxidant for this in the respiration. The reduced compound or energy source is called electron donor and the ultimate oxidant (oxygen) is called electron acceptor in this
2. The ecological basis of food spoilage
energy metabolism. The electron donor in this case ends up as carbon dioxide while the electron acceptor oxygen is reduced to water. this respiration process is also coupled to phosphorylation of ADP to ATP.
Re-oxidation of co-enzymes
S 2N2 H2O + NAD Ethanol Electron donors (energy source)
Cred
NH3
S 2-
Fe 2+
H2
ATP
NAD+
NADH
O2 NO3 SO42Electron acceptors Pyruvate
NADH
ADP
Fermentation Respiration
CO2
NO3
SO4-
Fe 3+
H2O
Fig 2.2. Summary of different types of energy metabolism. Common principle is that energy is derived by oxidation in several steps of a reduced compound (C, N, S, Fe, H2 a.o.) by means of co-enzymes, here represented by NAD+. Re-oxidation of the reduced co-enzyme can be achieved with respiration, in which molecular oxygen, nitrate or nitrite, and sulphate are common oxidants (electron acceptors). An alternative to respiration is fermentation, in which a partially oxidised carbon compound from the metabolic path (e.g. pyruvate) is used as electron acceptor for re-oxidation of the co-enzyme and then becomes reduced, in this case to ethanol.
When oxygen is used as electron acceptor the process is called aerobic respiration, while the use of alternative electron acceptors like nitrate, nitrite, sulphate etc. is called anaerobic respiration. Many facultatively anaerobic bacteria use oxygen if it is available but can switch to anaerobic respiration (mainly nitrate respiration) or fermentative metabolism in absence of molecular oxygen. Of these respiration types, it is mainly the aerobic respiration and nitrate respiration that take place in food. Some microorganisms can use other reduced compounds than carbon compounds as energy source. Some examples are ammonia and nitrite which are oxidised by nitrifying bacteria, and sulphide, ferrous iron, and hydrogen gas. These reactions are very important in the environment but seem to play a little role in the handling of food. One alternative energy metabolism that is common in microorganisms growing in food is fermentation, in which a reduced intermediate is used as electron acceptor in the re-oxidation of reduced co-enzymes. There is a number of different fermentative metabolic pathways , named according to the dominating
products, like ethanol fermentation, lactic acid fermentation, mixed-acid fermentation etc. Some of these reactions are detrimental for the food while others are utilised in processing of food. The main fermentative pathways and their role in food microbiology are further discussed in the section on degradation of carbohydrates. To increase the shelf-life of food means that the progress of the natural degradation path must be prevented or delayed. However, food spoilage is not exclusively a matter of microbial degradation. Other spoilage reactions are dehydration, oxidation of fat, and endogenous metabolism (over-maturation of fruits and vegetables), but microbial metabolism is the most important type of reaction that reduces the quality of food during storage. The common microbial food spoilage usually does not make the food unsafe or even reduce its nutritional value, but it makes the product unpalatable. The negative perception of food which is severely contaminated by microorganisms is an important defence mechanisms for us, since the risk associated with eating food increases considerably if it is spoilt by microbial metabolism. This is due to the risk that some organisms among the spoilage flora may be pathogens. It is impossible to give a simple and yet comprehensive description of the microbial spoilage of food since this is a very diversified process. What is said in this booklet must be seen as typical and common cases, to avoid the use of very large lists of microbial names. When, for instance, it is stated below that the activities of Pseudomonas spp. limits the shelf-life of refrigerated fresh meat and fish, it means that most investigations - but not all- show that Pseudomonas species dominate the spoilage flora but there are usually a number of other species involved, usually in the group "psychrotrophc, Gramnegative rods". Another problem is that it is not always sure that the dominating microflora is responsible for the main spoilage reactions. An example is that it may require 10 times more Achromobacter cells than Shewanella cells to make fresh fish unacceptable in taste. Another example is the lactic acid bacteria of the homo-fermentative type which have a relatively low impact on the spoilage due to the domination of lactic acid in the metabolic products. Most food raw material have a primary flora of microorganisms which origins from the production environment. During the continuing processing of the raw material and additional contamination (or secondary) flora infects the food. It may come from the air, especially from dust in the air, from process water, process equipment, or from humans that handle the food. During the subsequent storage of the product the different species develop differently depending on the environment. The primary plus initial contamination flora
usually is in the order of 103 cells/cm2 of solid foodstuff if the quality is very good (see table 2.1). Depending on the conditions for growth some of these species will grow exponentially (see Fig 2.3) up to concentrations above 107/ cm2 (or per gram). The finally dominating microflora may origin from the primary or the contamination microflora. When the number of cells exceed 107 to 108 cells/cm2 (or per gram) the product usually develops bad smell and the microflora is then called the spoilage flora. It is the nutritional (for microorganisms) properties of the food and the environment (temperature, water activity, pH etc.) that determine which species will dominate the spoilage flora, their metabolic products and how fast this spoilage process will proceed. In the sections below the environmental parameters will be discussed and in Chapter 2.2 the most important chemical reactions of food spoilage are presented.
Table 2.1 Typical size of different food microfloras at good production hygiene Product Internal tissues of healthy animals Plant surfaces Fish skin Egg shell Milk Meat Fish fillet Spoilage flora on most food types Microbial concentration
0
Primary flora 103 cells/ cm2 Contamination flora 103 cells / ml Contamination flora 103 cells / cm2 107 - 108 cells / cm2 or gram
2.2 The physico-chemical properties The possibility of the food to serve as a substrate for microbial growth depends on a number of physical and chemical properties: - Temperature - Water activity (aw) - pH and buffer capacity - Oxygen concentration and transfer - Mechanical barriers - Metabolisable energy sources - Metabolisable nitrogen sources - Chemical inhibitors
Temperature. The temperature influences of course the rate of growth, and thereby the shelf-life of the product. But it has also an impact on selection of species in the microflora. This is probably the explanation why reduction of temperature in the refrigeration range (0-8C) has such a dramatic influence on the growth rate, as demonstrated by experimental data Fig 2.3. The organisms growing at 20C have an initial generation time of about 4.8 h, while the generation time at 0C is about 25 h, and represents psycrotrophic organisms.
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Log 9 (cfu) 7 5 3 1
0
20C
10C 8C
4C 0 C
t(d)
150
Fig 2.3. Influence of temperature on the total bacterial count (colony forming units, cfu) on fresh meat. The dotted line indicate the typical level of spoilage. Note that the growth initially is exponential.
Microorganisms are usually classified in four groups according to their relationship to temperature. Fig 2.4 illustrates this. In general the mesophiles have the highest maximum growth rate and an optimum temperature in the range of 30-40 C .
Relative growth rate
Mesophiles Psychrotrophes Psychrophiles Thermophiles
10
20
30
40
50
60 C
Fig 2.4. Schematic illustration of the temperature dependence of the growth rate of different classes of microorganisms. There are no general and exact limits for the temperature ranges.
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The psychrophiles have the lowest maximum growth rate, but can grow quite fast at refrigerator temperature. Thermophiles have an optimum above 40C and some can grow even above 100C. The psychrotrophic organisms constitute an important group in food microbiology. They grow well in the 2035 C range like the mesophiles but they can also grow relatively fast at refrigerator temperature.
The growth rate of microorganisms is expressed either with the generation time (tg, h) or with the specific growth rate constant (, h-1). The generation time is the time needed to double the amount of cells. The specific growth rate expresses the rate of cell formation per cell. The correlation between these parameters can be derived from a mass balance of the cell number: dN = N dt where N is the number of cells, (h-1) is the specific growth rate and t (h) is time. Integration with N0 cells at t = 0 and Nt cells at time t, gives:
ln(N t ) = t ln(N 0 )
After one generation time, tg is the cell number 2N0. Insertion of this in the equation above gives:: ln(2N 0 ) ! = t g ln(N 0 ) from which the correlation between generation time and specific growth rate is obtained: ! ln(2) 0.69 = tg !
Water activity (aw) This is one of the main parameters that determines how fast and by which type of organisms the food is spoilt. The water activity of food can be determined as the water vapour pressure (pH2O) in a closed vessel in aw = pH2 O pH2 O* which the product is enclosed in relation to the water vapour pressure of pure water (pH2O*): For a water solution with low molecular weight compounds (e.g. salt or sugar) the water activity is approximately:
aw =
where
nw nw + ns
2. The ecological basis of food spoilage nw = number of moles water ns = number of moles of dissolved molecules Some common food components that reduce the water activity are: - Ions (e.g. salts) - Dissolved molecules (e.g. sugars) - Hydrophilic colloids (e.g. starch) - Ice
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The water activity is a measure of the availability of the water for the microorganisms. It is not only the water concentration that determines the water activity but also the capacity of the material to bind water. This is illustrated in Fig 4.2 which shows sorption isoterms for some materials with different water binding capacity. Cellulose get a relatively high water activity and starch a lower water activity at the same water concentration. Water concentration (%) 30 20 10
Cellulose Fruit
Rel Relative rate reaktionshastighet
Starch Meat
Lipid oxidation
Lipolysis Proteolysis
0 0 0.9
Molds Yeast s
Bacter iar
0.3
0.6
Water activity
0. 2
0.
0.
Water activity 4 6
0. 8
Fig 2.5. Sorption isoterms for different materials show that aw is not the same as water concentration
Fig 2.6. Schematic view of how the aw influences the rate of enzyme reactions and microbial growth.
Most biochemical reaction rates decline with declining water activity. However, the sensitivity to reduced water activity varies, as illustrated in Fig 2.6. Among microorganisms, molds and yeasts are generally more resistant to low aw and many enzymes keep their activity at even lower water activity. But there are many exceptions to this rule. Three types of microorganisms prefer reduced water activity. These are osmophilic (sugar preferring) yeasts, xerophilic (drought preferring) fungi, and halophilic (salt preferring) bacteria. These organisms not only grow faster than most other organisms at lower water activity, but they also prefer a reduced water activity. See further in Table 2.2.
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Table 2.2 Examples of typical minimum water activity for growth of some microorganisms and corresponding aw in food. Organism Min aw Food Food aw Milk, fish, meat 0.99 Pseudomonas 0.97 E. coli 0.96 Sausage, 7% salt 0.96 Clostridium 0.95 Brochothrix thermosphacta 0.94 Bacillus 0.93 Ham, 12% salt 0.93 Lactobacillus 0.93 Streptococcus Lactococcus 0.93 Micrococcus Jam, 50% socker 0.91 Hard cheese, bread Herring, 20% salt 0.87 Staphylococcus 0.86 Yeasts in general 0.85 Molds in general 0.80 Halophilic bacteria 0.75 Grains w.10% water 0.7 Xerophilic molds 0.65 Osmophilic yeasts 0.60 Dried fruits, 15% 0.6 water Dry milk, soups etc < 0.5 Dry bread Halophilic = salt preferring; xerophilic = drought preferring; osmophilic = preferring high osmotic pressure (of sugar).
The water activity of food has a large impact on the rate of spoilage but also on the type of spoilage since it exerts a selection pressure on the microflora. Many of the common food spoiling microorganisms are very sensitive to reduced water activity and the growth rate of these declines rapidly when the water activity drops below the optimum, which is close to 1 for Pseudomonas and Enterobacteriacae. Many conclusions can be drawn from Table 2.2. Pseudomonas, which dominate the spoilage of refrigerated fresh meat and fish does not create problems in sausages and salted herrings or if meat and fish is dried. Such products get a spoilage flora of more low-aw resistant organisms like lactic acid bacteria, molds and yeasts. The table also explains why molds are the main problem during storage of cheese and bread, and why dried products like flour, grains, dry milk are not attacked by microorganisms at all, provided they are stored in a dry environment so they do not absorb water. It is also obvious that the toxin producing Staphylococcus, which are commonly present on human hands, constitute a threat at "smrgsbord" and other buffets.
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Note that the figures in Table 2.2 are collected from different sources. The actual minimum aw for and organism depends on other parameters like pH, temperature, and nutritional conditions. Thus, such data are only approximate and indicative of relative sensitivities. pH is another parameter of large impact for the shelf-life of food. The pH influences both the growth rate and the type of organisms that will dominate during storage. Most food products have pH below 7 (Table 2.3) and most food spoiling bacteria require a relatively neutral pH, with the large exception lactic acid bacteria (Table 2.3). The lactic acid bacteria grow well down to a pH in the range 4-5. In Nature there are many examples of bacteria that can grow at very low and very high pH values, but these organisms are not relevant in food microbiology. Comparing these tables give one reason why fruits and many vegetables mainly are degraded by molds and sometimes yeasts.
Table 2.3. Typical pH-values of common food products Shrimps 7 Cabbage 5.5 fish 6.7 Potatoes 5.5 Corn 6-7 Tomatoes 4.2 Milk 6.5 Orange juicee 4 Melon 6.5 Yoghurt 3.5 Butter 6.2 Apples 3 meat 5.1-6.4 Lemon 2 Cheese 5.9 Oysters 5-6
Table 2.4. Generalised picture of pH ranges for microbial growth pH range pH optimum Most food spoilage bacteria 4-9 71 Molds 2 - 11 51 Yeasts 2.5 - 7 4-5
Oxygen availability and the diffusion rate of oxygen are important parameters that influence the type of metabolism. The rate of growth may be slower in anaerobic than in aerobic environments but on the other hand is the anaerobic metabolism associated with much more detrimental products for the shelf-life. An exception to this is the lactic acid bacteria which have anaerobic metabolism but usually produce less ill-smelling compounds than most other anaerobic organisms. Anaerobic conditions are a prerequisite for growth of the dangerous pathogen Clostridium botulinum, and therefore special precautions must be taken when storing some types of food under anaerobic conditions.
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The mechanical structure may be important for the shelf-life of food. On whole meat bacteria grow only on the contaminated surface, where they dwell on the exudate, i.e. the glucose and amino acid rich liquid that leaks from damaged cells and blood vessels. If the meat is minced this surface and exudate increase enormously which leads to much higher microbial activity and growth in the inner anaerobic parts of the minced meat. Fruits and vegetables are protected from microorganisms by the outer shell or skin and by the gelatine-like pectins which cements adjoining plant cells together. Outside the skin/shell the water activity is low and there is a lack of nutrients for growth of the contaminating microflora. But if the product is mechanically damaged or if the organism can produce pectinases the nutrients become available and the spoilage rate increases. It is mainly molds that produce pectinases, and this, together with the often low pH of these products, explains why this type of food often is spoilt by molds. Yeasts that also grow well at low pH often come as a second infection after the initial mold attack. Erwinia is one of few bacterial genera with pectinase producing species that attack plant material. Antimicrobial substances. Many food raw materials, especially vegetables and other food with plant origin, contain antimicrobial compounds which hamper the microbial growth. Some examples are listed in Table 2.5. Many microorganisms produce antimicrobial substances (antibiotics) and in food there is often growth of lactic acid bacteria some of which produce known antibiotics (Table 2.6). Nisin is a polypeptide antibiotic naturally produced in fresh (unpasteurised) milk by Lactococcus lactis which belong to the normal flora transmitted during milking. Other antibiotics, like acidocin B and reuterin are mainly produced in processed milk if it is inoculated with the producing organism.
Table 2.5. Some examples of naturally occurring antimicrobial substances. Food Inibitor Horseradish Allyl isothiocyanate Onion and garlic Allicin and diallylthiosulphinic acid Tomato Tomatin Radish Raphanin Lingonberry Bensoic acid Oregano Eteric oils Table 2.6. Antibiotic substances produced by lactic acid bacteria Antibiotic Organism Nisin (in milk) Lactococcus lactis Salvaricin Lactococcus. salvaricus Acidocin B (fermented milk) Lactobacillus acidophilus Reuterin (fermented milk) Lactobacillus reuterii
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Some definitions of antimicrobial compounds Antibiotics Microbial product with an antimicrobial (bactericide/ fungicide or bacteristatic/fungistatic) activity and which have low toxicity to humans. If the latter is not added to the definition most mycotoxins would also be classified as antibiotics. Microbial cultures, mainly lactic acid bacteria, which are consumed for stabilisation of the intestinal microflora of humans or animals. They are believed to act by establishing on the intestinal mucouse membrane and prevent, possibly by production of antibiotics, the growth of other disturbing organisms. Components (oligosaccharides) in the food that are not digested in the intestines but are assumed to promote the beneficial microflora. Bacterial proteins or peptides with bactericidal effect mainly on related species and strains.
Probiotics
Prebiotics
Bacteriocines
bactericide = bacteria killing; fungicide = fungi killing; bacteri/fungi-static = inhibiting growth of bacteria/fungi.
2. 3 The chemical reactions The most important chemical reactions involving food components during microbial spoilage of food are: - Degradation of N- compounds - Degradation of fat - Degradation of carbohydrates - Pectin hydrolysis Degradation of nitrogen compounds The dominating and usually the first reaction is oxidative deamination of amino acids: amino acid + O2
deaminase
NH3 + organic acid
This reaction is assumed to be the dominating spoilage reaction in refrigerated fresh meat and fish. The amino acid is then used as energy source by splitting off the amino group with an oxidative deaminase, which leaves the organic acid that enters the energy metabolism.
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Proteolysis. One would think that proteolysis would be a common spoilage reaction. However, most microorganisms do not secrete proteases and those who do usually do not produce them until there is a lack of nitrogen source. In later stages of spoilage, however, proteases and peptidases may degrade the protein: Proteins
proteinase
peptides
peptidase
amino acids
Many peptides have strong taste, bitter or sweet, and this sometimes contributes to the spoilage. These reactions are also important for the development of characteristic tastes of many fermented products (Chapter 6). Putrification is a set of anaerobic reactions with amino acids which results in a mixture of amines (e.g. cadaverine, putrescine, histamine), organic acids, and strong-smelling sulfur compounds like mercaptans and hydrogen sulphide:
Anaerobic metabolism
amino acids
Amines Organic acids S-compounds Indol
Many of these compounds have terrible odour. Cadaverine, putrescine, and histamine are formed by decarboxylation of lysine, ornithine, and histidine, respectively (Fig 2.6) While cadaverine and putrescine in food probably have no health impacts, only spoil the food due to the odour, histidine causes intoxication problem since it may induce a serious anaphylactic shock. This is often associated with microbial activity in histidine rich fish products (e.g. tuna fish). Putrification is typical for microbial degradation of meat and other protein rich foods at higher temperature (> 15C). Bacillus and Clostridium species may then grow fast and rapidly make the food toxic, but under refrigeration conditions these organisms are usually not active and under these conditions the oxidative deamination spoils the food before the putrification becomes dominating.
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Fig 2.6. Histamine, cadaverine and other amines are formed by decarboxylation of amino acids.
Reduction of trimethylamine oxide (TMAO). Marine animals may contain high concentrations of trimethylamine oxide, which is believed to have a function in protecting proteins from denaturation at low temperatures, high pressure and high osmolarity. Certain microorganisms, like Pseudomonas and Shewanella, can utilise TMAO as electron acceptor in anaerobic respiration: CH3 H3 C - N = O CH3 TMAO TMAOreductase CH3 H3 C - N CH3 TMA
This results in formation of trimetylamin (TMA) which gives a typical "fishy" smelling. TMA can also be formed by enzymatic hydrolysis of lecithin. Degradation of fat When fat is degraded it becomes rancid and this rancidification depends on many different reactions which are not all well known in detail. One attempt of classification is shown in Fig 2.7. The hydrolytic rancidification results in free fatty acids (FFA) and glycerol. Our organoleptic tolerance of free fatty acids depend on the type of the fatty acids, especially the carbon chain length. Up to 15% FFA is said to be acceptable in beef, which has long fatty acids, while only up to 2% is acceptable in olive oil. If very short FFA are formed, e.g.
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butyric acid from butter, only traces of the acids can be accepted. The hydrolysis can be spontaneous but then at a very low rate, while it may proceed fast if lipolytic enzymes from the foodstuff or from the contaminating microflora are present.
Fig 2.7. Different types of rancidification reactions.
The oxidative rancidification requires presence of oxygen. Autooxidative rancidification is catalysed by metal ions and is accelerated by light. In this process peroxide radicals (ROO*) are produced and they react with other fatty acids to form instable hydroperoxides (R-OOH) which later on decompose to aldehydes and ketones which give the rancid taste (Fig 2).
Fig 2.8. Autooxidation of a fatty acid (RH) results in aldehydes and ketones. The chain reaction is initiated by a radical (R*) which is produced from the fatty acid under catalysis of Fe2+ and other metal ions and light. The radical reacts with molecular oxygen to form a peroxide radical (ROO*). Antioxidants in food are used to scavange the peroxide radical that otherwise

continuous the chain reaction by reacting with another fatty acid to produce a new radical (R*) and a hydroperoxide (R-OOH). The hydroperoxide is instable and decomposes to ketones or aldehydes.
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-oxidation is the common metabolic route for degradation of fatty acids and each cycle results in generation of one acety-oA and a new fatty acid with 2 C shorter C-chain (Fig 2.9). Some microorganisms have a side reaction in the last step of the -oxidation cycle, by which very aromatic methyl ketones are formed and may contribute to bad taste (rancidity) of the food.
Fig 2.9. Methyl ketones may be formed as by-products in the -oxidation of fatty acids.
Lipoxydaser are common enzymes in plant and animal tissues and they are also produced by some molds. The enzyme oxidises unsaturated fatty acids with cis-cis 1-4 pentadien configuration to hydroperoxides which decompose spontaneously to ill-tasting aldehydes and ketones. This configuration occurs in linolic and linolenic acids in plants and in arachidonic acids in animal tissues. To prevent this type of rancidification during some vegetables , e.g. frozen spinach and peas, are heat treated to inactivate the plant enzyme. These aldehydes and ketones are not always unwanted products in food. They are also important ingredients in certain types of cheeses (see Chapter 6, Fermented foods). Degradation of carbohydrates Microorgansims growing on food mainly use various sugars as carbon- and energy source. Under aerobic conditions the energy source is combusted to carbon dioxide and water but under oxygen limiting or anaerobic conditions
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many species switch to fermentative metabolism which results in various fermentation products (see Fig 2.2). The most common fermentative pathways are listed in Table 2.7.
Table 2.7. Common fermentation types Fermentation type Alcohol fermentation Homofermentative lactic acid fermentation Heterofermentative lactic acid fermentation Propionic acid fermentation Butyric acid fermentation Mixed-acid fermentation 2,3-butanediol fermentation Products Ethanol, CO2 Lactic acid Lactic acid, Acetic acid, Ethanol,CO2 Propionic acid, Acetic acid, CO2 Butyric acid, Acetic acid, CO2, H2 Lactic acid, Acetic acid, CO2, H2, Ethanol CO2, Ethanol, Butanediol, Formic acid
Of these fermentation types, it is the butyric acid, mixed acid and butanediol fermentations that are most detrimental for the food taste. The mixed-acid and butanediol fermentations are typical for organisms in the Enterobacteriacae family. Butyric acid fermentation is common among saccharolytic Clostridium. Lactic acids is mainly produced by lactic acid bacteria but it proceeds also under aerobic conditions since these bacteria are relatively indifferent towards oxygen although they always use the fermentative metabolism. A more detailed picture of the different fermentation pathways from glucose via the common intermediate pyruvate is shown in Fig 2.10.
Glucose
Lactic acid fermentation

NAD
+
ATP
NAD
Ethanol fermentation Acetaldehyde

NAD
+
NADH
Lactate
Pyruvate
Ethanol
Oxaloacetate
ATP ATP NAD
+
AcetylCoA
Formate
AcetylCoA +
CO2
ATP
+H2
NAD
Acetate Acetoin
+
Ethanol
H2
CO 2
ATP NAD
+
AcetacetylCoA
Acetate
Succinate
Mixed acid fermentation
NAD
Butyrate
Acetone
+
Propionate Propionic acid fermentation
Butandiol Butandiol fermentation

NAD
+
NAD
Butanol
2-propanol
Butyric acid fermentation
Fig 2.10 Summary of the six main fermentative pathways. The main end product are emphasised by frames. Sites of co-enzyme generation and ATP formation are indicated.
21
Pectin hydrolysis Pectins are carbohydrate polymers mainly composed of partially methylated poly--(1,4)-D-galacturonic acid. They are present in all fruits and vegetables where they function as a glue between the plant cells and gives mechanical rigidity. During ripening of fruits and berries indigenous pectinases are synthesised or activated and start hydrolysing the pectins which makes the structure soft. Also mechanical damages on fruits and vegetables activate the pectinases and this opens for microbial attack. However, also some microorganisms produce and secrete pectinases. Many molds have this capacity and among bacteria plant pathogens in the genus Erwinia also produce pectinases which serve as tools for the microbial invasion resulting in soft rot. Slime production Microbial spoilage of meat and fish sometimes results in a slimy surface layer, composed of microbial polysaccharides. Such polysaccharide slime can also appear as a result of microbial growth on vegetables, wine and vinager. A special case of slime formation is the so called ropiness of bread which is caused by B. subtilis which may survive the baking as spores and then germinate and grow if the water activity is high and the temperature kept too high after the baking. Usually the slime formation on cold-stored fresh meat comes after the meat has become unacceptable due to smelling. Some species of lactic acid bacteria produce polysaccharides and this sometimes utilised in various fermented milk products to give a higher viscosity (yohurt, Swedish lngmjlk). However, the viscosity of yoghurt is mainly caused by protein precipitation due to low pH.
22 Chapter 3. Spoilage of different types of food From a microbiological viewpoint it is convenient to classify different types of food according to the conditions they provide for microbial growth which gives an indication of the food shelf-life. One such classification is shown in Table 1.3.
Food properties Water-rich Protein-rich Relatively neutral pH Water-rich Protein-poor Relatively sour Water-poor Exampel Meat Fish Milk Cooked food Fruits Vegetables Root-fruits Grains Flour Bread See Chapter 6 Protection None
Low pH Inhibitors Mechanical structure Low aw Often low aw + low pH Microbial competitors Microbial inhibitors Low aw Low pH Low pH, low aw, inhibitors No microflora Small initial microflora Often in combination with chemical preservatives
Fermented food
Preserved food
Salted Pickled Smoked Sterilised Pasteurised
3.1 Water and protein rich foods Fresh meat, fish and milk belong to this category. They have a water activity close to 1, contain lots of energy sources and other nutrients for microbial growth, are relatively pH neutral and contain no or little microbial inhibitors. If not treated by preservation methods these food stuffs are spoilt by microbial activity in a couple of days or shorter at room temperature. Therefore these products are always stored at refrigerator temperatures to reduce the rate of microbial growth. At a first look one would expect that eggs should belong to this category, but for obvious reasons Nature has build a sophisticated system that keeps the egg
3.
Spoilage of different types of food
23
protected from microbial attach for several weeks at room temperature. This is described in Fig 3.11. Meat At the moment of slaughter, the breathing and aerobic respiration cease abruptly but the cells in the body tissues continue their metabolism for several hours and these reactions are important for the later microbial development. During the post mortem metabolism glucose is metabolised through the glycolysis, but due to lack of oxygen, lactic acid is produced from the pyruvate. Glycolysis generates two ATP molecules per glucose molecule, which is much less than in the aerobic respiration but still enough to prevent the formation actomyosin complex in the muscle (See Fig 3.1). However, the formation of lactic acid reduces the tissue pH from neutral towards pH 5.5-6. Eventually the low pH inhibits the glycolysis and the ATP generation ceases which results in formation of actomyosin from the components actin and myosin which are kept dissociated by ATP. Formation of actomyosin results in muscle contraction and it is observed as rigor mortis.
Fig 3.1. The post mortem glycolysis generated protons and ATP. The ATP forces the equilibrium between actin + myosin and the actomyosin towards the dissociated state. When pH has dropped too much the ATP generation through glycolysis ceases and the equilibrium shifts towards formation of the actomyosin complex, which results in muscle contraction, i.e. rigor mortis. After some time (Table 3.2) the actomyosin complex is hydrolysed by proteases (cathepsins and calpains).
The time course of this most mortem metabolism and the final pH depends on the animal species (Table 3.1). The final pH is considered important for the shelf-life. This pH is not only dependant on the animal species but also on the condition of the animal before slaughtering. An animal that has been stressed has a lower blood glucose level and the post mortem metabolism can then cease due to glucose limitation rather than pH inhibition and the result is a meat with higher pH. Since the dominating spoilage flora on refrigerated fresh meat and fish is Pseudomonas (and other Gram negative psychrotrophic rods)
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and these organisms quite sensitive to pH below about 5.5-6, the final pH of the meat is considered important for the shelf-life.
Table 3.1. Typical pH of meat from different animals and lenth of rigor mortis.
Animal type Cow Swine Chicken Fish
Rigor mortis 10-20 h 4-8 h 2-4 h min-h (longer on ice)
final pH 6 - 5.5 6 6.4 - 6 6.8 - 6.4
The meat contains many nutrients for the microorganisms (Table 3.2) which only grow on the exudated from damaged tissue. Furthermore, it is only on the surface of meat the cells grow, unless the meat has been mechanically perforated or minced. Therefore, the microbial count is expressed as cells/ cm2 or cfu/ cm2, where cfu means colony forming units on agar plates.
Table 3.2 .Example of microbial nutriens in meat exudate Component Concentration g/Kg Lactic acid 9 Creatine 5 Inosine 3 Carnosine 3 Amino acids 3 Glucose-6P 1 Nucleotides 1 Glucose 0.5
Meat is usually stored at refrigerator temperature which gives a shelf life around one week, however longer for beef, but this shelf life depends strongly on other factors like the hygiene during slaughter and handling of the meat. It is often assumed that also a low pH after rigor mortis is important. Under these conditions the microflora at spoilage is dominated by Gram negative psychrotrophic rods of the genera Pseudomonas, Achromobacter, Alcaligenes, Acinetobacter och Flavobacterium. These organisms are often obligate aerobes. Many investigations report Pseudomonas, and especially P. fragi as common spoilage flora on fresh cold-stored meat. there are also reports that state that this type of microflora on meat is universal and not dependent on which animal the meat comes from. The flora is always dominated by bacteria, only small amounts of yeasts and molds are developoing under these conditions. During storage, the bacteria initially grow exponentially, sometimes after a lag phase that can be due to shift of domination microflora. The cell concentration increases from about 103 cells/cm2 on a meat of highest
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hygienic quality towards 107 - 108 cells /cm2. Then the spoilage becomes apparent through bad odour, and sometimes discolorisation and slime formation. Typical growth curves on refrigerated pork and chicken are shown in Fig 3.2 It is apparent that the shelf life of such products depends on the growth rate, which is mainly determined by the temperature, and the initial amount of cells that can grow, which is strongly related to the hygiene during and after slaughter.
log N/cm 8 7 6 5 4 3 2 1 0 2 4 Tid (d) Days 6 8 10
2
slime odour chicken

kyckling
slem odr
pork grisktt
Fig 3.2. Example of microbial growth "total aerobic count" during storage of fresh pork and chicken meat at refrigerator temperature.
According to one hypothesis, the shelf-life of fresh meet depends on the availability of glucose at the surface. As long as glucose is available, this is the main energy source for the bacteria, but when it is exhausted, other organic compounds, e.g. amino acids provide the energy. When aminoacids are used as energy source, ammonia is split off by oxidative deamination and produces bad odour. This is supported by the data shown in Fig 3.3 which shows how the glucose gradually is exhausted at the surface when the microflora approaches the spoilage stage. It can also be an explanation to why meat from stressed animals has a lower shelf-life, since short intensive stress before the slaughter may reduce the blood glucose concentration. .
400 400
N*10-7= 2.7
Glucose (g/g)
6.3 32 110 280

20 0
Fig 3.3. Glucose concentration gradients and microflora development during cold storing of fresh meat. At N=32*107 cm-2 the meat was classified as spoilt and this coincides with glucose exhaustion at the surface.
Distance from surface (mm)
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Carbon dioxide and vacuum packages Vacuum packaging of meat, both fresh and cured meat, increases dramatically the shelf-life. It was originally believed that the main mechanisms of vacuum packaging is that oxygen is removed and that this hampered the main spoilage flora. However, storing meat under nitrogen atmosphere does not improve the shelf-life. Fig 3.4 shows that the microflora develops slower, but the fermentative metabolism that dominates under anaerobic conditions produces more off-flavour, unless it is a lactic acid microflora that dominates. The figure also shows that storing the meat under CO2 atmosphere significantly reduces the rate of microbial growth. When the CO2 packed meat was opened subjected to air, the microbial growth rate immediately increased.
logN / cm2 9 8 7 6 5 4 3 0 Luft air
Luft air
Kvve N2
air Luft
CO2
CO
82 16 Tid (dagar) 24 32
Fig 3.4. Influence of the gas atmosphere on the growth rate of microorganisms on refrigerated fresh pork meat. Some of the CO2 stored samples were opened and further exposed to air, as indicated in the CO2plot.
When the composition of the microflora was investigated under these conditions it became clear that the atmosphere exerts a selecting pressure, see table 3.3. In air the dominating microflora usually is Pseuomonas. These organisms are obligate aerobes or use anaerobic respiration in absence of oxygen. In nitrogen atmosphere different species from the Enterobacteriacae family dominate. These organisms possess a strong fermentation capacity with ill-tasting products from the mixed-acid fermentation or 1,3 butandiol fermentation pathways. The CO2 not only reduces the rate of growth on the meat, but it also exerts a selective pressure that favours growth of
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Lactobacillus, which with their lactic acid fermentation have less impact on the spoilage than the Pseudomonas .
Table 3.3. Dominating spoilage flora on cold stored pork in different atmospheres.
O2 % 20 80 80 10 N2 % 80 100 20 20 90 100 CO2 % Pseudomonas Enterobacteriacae Aeromonas Brochothrix Lactobacillus
+ + + + + + + +
The selective pressure of CO2 is explained by the different inhibitory effect this gas has on various microorganisms. Pseudomonas belongs to the most CO2 sensitive bacteria while lactic acid bacteria are very resistant to this gas. Most molds are very sensitive while yeasts are very resistant to CO2.
Fig 3.5 Relative sensitivity of microorganisms to inhibition of growth by carbon dioxide.
When fresh meat is vacuum packed after slaughter, which is often the case for meat that is to be stored for tendering, CO2 is released from the tissues during the first day and since the vacuum package plastic film has a low gas permeability and the gas headspace is removed by the vacuum, the partial pressure of CO2 raises rapidly and exert a CO2 protecting function. Also the shelf-life promoting effect of vacuum packing of cured meat products is similar but in that case it is the metabolic activity of the microflora that produces the CO2. The strong protecting effect of CO2 on meat and meat products have
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The selective pressure of the gas atmosphere, can be explained by the relationship of the dominating types of microorganisms to oxygen and CO2, as summarised in Table 3.4
Table 3.4 Some characteristics of the organisms that dominate the spoilage flora on cold-stored meat in different atmospheres. Organism Properties Pseudomonas Aerobic Very CO2-sensitive Enterobacteriaceae Aeromonas Brochothrix thermosphacta Facultative Intermediate CO2-sensitivity Facultative Intermediate CO2-sensitivity relatively CO2 resistant Facultative Resistant to low aw Very CO2-resistant Indifferent to oxygen
Lactobacillus
The inhibitory effect of CO2 seems to be synergistic with low temperature in storage of meat as shown in Fig 3.6. This may partly be due to the increasing solubility of CO2 at declining temperature. Even if CO2 dissolves in water and partly is hydratized and dissociates to bicarbonate, it is the gaseous CO2 molecule that has the inhibitory effect. This also means that the effect is strongly pH dependent and declines with increasing pH.
C
Fig 3.6. Time needed to reach 10 cells cm on pork meat stored at different temperatures in air or in CO2.
6 -2
3.
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The antimicrobial effect of CO2 on many spoilage organisms has been utilised also for direct applications in which food is stored under so called controlled atmosphere in which carbon dioxide is the growth inhibiting compound and oxygen often is present to avoid anaerobic metabolism or discolorisation. Vacuum packing of food is applied also for other reasons than to provide microbial inhibition via CO2. One common reason for vacuum packing is to prevent oxidative rancidification or other oxidising reaction with molecular oxygen (e.g. peanuts), or to prevent evaporation of flavour compounds (e.g. coffe). When cheese is packed in vacuum tight plastic films it is likely that a mold inhibiting CO2 atmosphere develops, but on the other hand, molds are obligately aerobic so the lack of oxygen is also a mold-protecting mechanism. Fish. The post mortem metabolism is important also in the fish. An important reaction is the degradation of ATP which results in a transient accumulation of inosine monophosphate (IMP). this compound is considered to contribute to the sensoric appreciation of "fresh fish" taste. IMP is also utilised as a flavour improving additive in the food industry, in analogy with the meat flavour enhancing effect of glutamine.
Fig 3.7. During the post mortem metabolism in the fish tissue inosine monophosphate (IMP) is transiently accumulated.
This metabolism has been utilised to develop a "fish-freshness" biosensor (Fig 3.8). Since the absolute level of the IMP varies much between fish sorts and even between individuals, it is not sufficient to analyse only the concentration of IMP . Instead the ratio IMP/(IMP + inosin + hypoxanthine) is used as a fish-freshness index. The enzymatic biosensor measures the oxygen consumption catalysed by xanthine oxidase. If only xanthine oxidase is present in the analysis, the oxygen consumption represents the concentration of hypoxanthine. If also the nucleotide phosphorylase is present, the oxygen consumption represents the concentration of hypoxanthine + inosine. By
3.
30
including also the 5'-nucleotidase the oxygen consumption also includes the IMP.
Fig 3.8. Principle of a "fish-freshness" biosensor based on analysis of the degradation of IMP degradation. The oxygen consumption catalysed by xanthine oxidase is analyses with or without the enzymes nucleotide phosphorylase and 5'-nucleotidase and a index that represents the concentration of IMP In relation to the sum of the metabolites is calculated.
The microbial spoilage of refrigerated fresh fish has large similarities with that of fresh meat. Pseudomonas is often dominating in the spoilage flora (Fig 3.9). A similar organism, Shewanella putrifaciens (previously called Pseudomonas putrifaciens or Alteromonas putrifaciens) is another spoilage organism specifically associated with marine fishes. It has the capacity to produce both hydrogen sulfide from cysteine and trimetylamine (TMA) by anaerobic respiration with TMAO as electron acceptor. Due to this capacity to produce bad odour the fish may be spoilt at 10 times lower total microflora if Shewanella putrifaciens dominates.
Fig 3.9. Distribution of spoilage organisms on refrigerated fresh fish. Aeromonas is mainly associated with freshwater fishes and Shewanella with marine fishes.
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Milk is a very good substrate for microbial growth. However, it is protected by several antimicrobial mechanisms that favour the development of lactic acid bacteria if the temperature is not too low. The lactoperoxidase system is one of these antimicrobial systems in milk (Fig 3.10). Milk contains the enzyme lactoperoxidase and small concentrations of its substrate thiocyanate. The milk is contaminated with lactic acid bacteria during the milking. These bacteria are catalase negative and therefore the hydrogen peroxide, which always is produced as a by-product in the metabolism, is not removed by catalase as in other microbial systems. Instead The lactoperoxidase oxidises the thiocyanate with the hydrogen peroxide to hypothiocyanate. This compound is strongly oxidising and reacts with sulfhydryl groups in transport proteins in the bacterial membrane, especially in Gram negative bacteria, while the lactic acid bacteria are relatively resistant. The lactoperoxidase system has been reported to have an antimicrobial function also in tears and other body-fluids. O2 oxidase catalase H2 O2 H2 O thiocyanate SCN OSCN hypothiocyanate
LP
HO-S-protein HS-protein
Fig 3.10. The lactoperoxidase system. The milk contains thiocyanate. H2O2 is produced in small quantities by all aerobic cells and usually degraded by catalase. However, lactic acid bacteria, which are first contaminating milk during the milking process, do not have catalase. The lactoperoxidase use the hydrogen peroxide to oxidise thiocyanate to the strongly oxidising hypothiocyanate which oxidises transport proteins in bacterial membranes. Especially Gram negative bacteria are sensitive to the hypothiocyanate.
When the milk leaves the udder it becomes infected by about 100 so callled udder cocci per milliliter. During the further handling in tha cow house the milk is infected with several types of microorganisms as shown in Table 3.5
Table 3.5 The initial milk contamination microflora Infection Source Feces E. coli Enterococcus Micrococcus Bacillus spores Air Mold spores Yeasts Lactococcus Lactobacillus Milking equipment Gram-negative rods
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If the milk is stored at room temperature the "lactic streptococci", i.e. the Lactococcus will first dominate the microflora and protect it from most of the other microorganisms with lactic acid. Eventually Lactobacillus, which can grow at lower pH will dominate. This fermented milk similar to yoghurt was previously produced on the farms (Swedish filbunke). If the milk is stored further proteolytic molds will finally raise the pH and it will be further destroyed by putrification by Clostridium and Bacillus. These reactions do not take place in a refrigerated milk. When the milk is cooled after milking and stored refrigerated on the farm, psychrotrophic gram negative rods (Pseudomonas and similar) will dominate. These bacteria will not make it sour as does the lactic acid bacteria. If stored too long the milk is spoilt by ammonia, peptides and free fatty acids. This psychrotrophic microflora, which itself is very heat sensitive, is known to produce comparatively heat resistant proteases and lipases which may create problems in the later storage. When the milk reaches the dairy it is pasteurised (see chapter 5) which efficiently eliminates the psychrotrophic Pseudomonas flora and most other bacteria. However, some of the more heat resistant organisms, mainly Lactobacillus and Micrococcus will survive, and the bacterial endospores from Bacillus will not be influenced at all by the pasteurisation. After the pasteurisation the milk is re-infected with the dairy equipment microflora. This may restore the psychrotrophic Pseudomonas flora or at bad hygiene even the Enterobacteriacae flora. The final spoilage of the refrigerated milk therefore differs depending on the contamination flora. Members of the Enterobacteriacae family may spoil the milk with fermentation, Bacillus spores my germinate and spoil by proteolysis. This is especially common in fatty products like cream. Also proteolysis and lipolysis by enzymes from the early Pseudomonas flora may contribute to the final spoilage of milk. However, the old days souring by lactic acid bacteria is not the common fate of refrigerated pasteurised milk. The egg is infected on the surface when the hen lays the egg. this flora is dominated by Pseudomonas, Staphylococcus, Micrococcus and fecal bacteria. It is not uncommon that the hen is infected with Salmonella and during the 1990ths many reports on Salmonella infected egg yolks appeared in England. The surface microflora is usually not infecting the egg due to a number of defence mechanisms, which are illustrated in Fig 3.11. When this protection fails and the egg becomes invaded by bacteria it is Pseudomonas fluorescens that dominates (80%). These infections can be detected by illumination of the egg with UV-light.
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Albumin: viscous, high pH (pH9,5), riboflavin + pyridoxin complexing

No protect ion
outer mucin layer 1-10m pores in shell inner keratin
Con-albumin: Fe2+ complexing Avidin: Biotin complexing Lysozyme: kills G+ bacteria
membrane
Fig 3.11. The egg is protected against bacterial infections an multiple ways: The shell and the two membranes gives is the first and mechanical hinder. The high pH in the egg white is non-optimal for many bacteria. Lysozyme ruptures cell walls of many bacteria. Albumin, conalbumin and avidin make several nutrient unavailable by strong complex formations.
3.2 Fruits and vegetables Fruits and vegetables do have a high water activity but they develop another spoilage scenario than meat, fish and milk. Many of these products are protected mechanically by the pectins which constitute a "glue" between the cells and gives rigidity. When fruits and berries ripen, endogeneous pectinases start to hydrolyse the pectin and this also makes the products more susceptible to microbial attacks. Another common protection the low pH of some of these products. This group of foods also has a much lower concentration of free amino acids and other nutrients than meat, fish, and milk. For these reasons it is usually not the Pseudomonas and other spoilage bacteria mentioned above which dominates the spoilage. Instead it is often pectinase producing organisms, which mostly means molds, that initiate the spoilage of fruits and vegetables. In the later phase, when the pectinolytic organisms have opened up the defence structure, also yeasts participate in the spoilage. One of few bacteria involved in spoilage of vegetables is the plant pathogen Erwinia carotovora. This organism has been subject to studies of the corum sensing phenomenon which plays a central role in the ecology of many organisms. In this case the corum sensing is based on accumulation of Nacylated homoserine lactones (AHL) which accumulates around the cells (Fig 3.12). When the concentration of AHL is high enough this compound induces the pectinase synthesis. The strategic advantage of not producing the pectinase constitutively is obvious, since the plants have their defence systems which generated antimicrobial chemicals when the plant is attacked. Only by delaying the pectinase synthesis until the number of bacteria is large
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enough, can the hydrolysis of the pectins be fast and efficient enough. Once the pectinases have damage the structure of the fruit/vegetable, other organisms follow and contribute to the soft rot. Due to the often low pH, molds and yeasts, rather than bacteria are common in the spoilage of these products. plant cell pectinolytic bacteria
AHL AHL AHL AHL AHL
Fig 3.12. Erwinia carotovora utilises corum sensing to invade plants. They start by hydrolysing the protecting pectin layer with extracellular pectinases. When the plant recognises a microbial attack it defends itself by producing antimicrobial ( ) compounds. Instead of initiating this defence response at low concentration of Erwinia cells, they first accumulate acylated homoserine lactones (AHL) and when the concentration is high enough this is a signal for induction of the pectinase ( ) production. By delaying the attack until there are many cells that can produce much pectinase, Erwinia gains increased virulence.
It is estimated that only about 20% of the fruits and vegetables are spoilt by microorganisms. The endogenous metabolism of the products, which leads to over-maturation plays a major role for the spoilage. Furthermore, drying also contributes to the spoilage. To reduce and better control the endogenous metabolism, fruits and to some extent also vegetables are stored in modified atmospheres (Controlled Atmosphere, CA-storage). Common principles are to increase the CO2-concentration, which also ha a microbial inhibition effect, and to reduce the oxygen concentration by adding nitrogen gas. Many fruits produce ethylene gas, which acts as a maturation hormone, and for some products absorption of the ethylene is included in the CA storage. Addition of ethylene or cessation of the absorption is then used to initiate the ripening. Table 3.6 gives an example of a modified atmosphere for fruits. the exact composition is optimised for each product.
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Table 3.6. Example of modified atmosphere for storage of fruits O2 : 0-5% CO2 : 2 - 10 % N2 : 90-95 % Relative humidity: 90-95%
3.3 Cereals Grains on the field usually has a primary flora of 103 - 106 bacteria g-1. Lactic acid bacteria, coliform bacteria and Bacillus spores dominate. A weather dependent flora of fungal spores is also present. At humid conditions the mold spore count can be 105 g-1. Different species of Aspergillus and Penicillium usually dominate. If the grains are soaked in water the bacterial flora will dominate, the regulations set a maximum water concentration of 13% for storage of grains and then no significant microbial activity is expected due to the low water activity. If the water content exceed 15% mold growth begins. Even if the grains are kept dry enough according to the regulations, local humid zones may appear in the silos, e.g. due to water condensation on walls. Under these conditions mold growth and mycotoxin formation may appear. During the milling of the grain most of the microflora follows the hull but some is of course transferred to the flour. Typical microbial counts are 102103 bacteria plus about 100 mold spores per gram sifted flour and about 10 times more in course flour. At correct dry storage of the flour there is no microbial activity, but as soon as water is added a vigorous growth starts. The surface of the bread becomes sterilised in the oven and a dry hard bread surface protects the bread against mold growth. If the bread is cut before packing the surfaces are usually infected and if the bread is kept to moist in a plastic bag mold growth will spoil it. The inner part of a bread is usually heated to 95-99C which means it is essentially sterile with respect to vegetative cells and mold spores. There is however a rare bakery problem called ropiness, which is caused by polysaccharide formation by Bacillus subtilis. The organism has then survived the baking in spore form and if the temperature is kept at 30-45 C too long and the bread has not become dry enough during the baking the B. subtilis spores germinate and grow very fast and produces the polysaccharides. During storage of the bread, spoilage is entirely caused by molds which have contaminated the bread after the baking. To reduce the rate of mold growth propionic acid or propionates are often used a preservatives in industrial baking. Dry bread (knckebrd) is not subject to any microbial spoilage, provided it is stored dry. Under such conditions the very slow spoilage is eventually caused by rancidification.
3. 3.4 Preserved foods
36
The spoilage of preserved food depends on the type of preservation. In general, if the preservation prevents microbial spoilage, the ultimate fate is usually spoilage by rancidification, which usually is a very slow process. Dried products. In the drying process the water activity is reduced to so low levels that no microorganisms are active. If the storage conditions are not dry enough, mold formation may occur, but otherwise the shelf-life is limited by rancidification processes, which depend very much on the fat composition of the product. During a spray-drying process the food is exposed temperatures that kile the most sensitive bacteria, but endospores, mold spores and more heat resistant vegetative bacteria as Enterococcus, Lactococcus, Micrococcus, and Lactobacillus may survive. When such products (e.g. dry milk, soups, sauces, etc.) are reconstituted with water they are usually very susceptible to fast microbial spoilage and considerable risks for food poisoning. Cured meat products are usually protected by the low water activity created by salt additions. If the products are fermented (salami and other fermented sausages) they are also protected by the lactic acid and the competitive effects of the lactic acid bacteria. These products are often further protected with nitrite. A common bacterium in vacuum packed cured meat products, is Brochothrix thermosphacta. This organism is similar to Lactobacillus (CO2 resistant and tolerant against low aw) which usually dominates vacuum packed meat products, but it is a severe spoilage organism since it produces stinking metabolites. Also the low-aw tolerant Micrococcus and Lactobacillus are common in these products. Salted fish and fish preserves are also protected mainly by the low water activity and chemical preservatives. In salted fish products mainly halophilic strains of Pediococcus, Micrococcus, and yeasts grow and they do this at a very low rate with slow spoiling. Usually these products are to be cold stored. and the main shelf-lime limitation is usually rancidification of the fat.
Table 3.7. Summary of common spoilage floras on different types of food Fresh meat Fresh fish Vac/CO2-pack Salted/Smoked Fish preserves Pseudomonas Pseudomonas Lactobacillus Lactic acid bacteria Lactic acid bact Shewanella Aeromonas Brochotrix Micrococcus putrefaciens Enterobacteriacae thermosphacta Yeasts Aeromonas Brochotrix Micrococcus (freshwater) thermosphacta Enterococcus Molds (not in vacuum)
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Table 3.8.Propertie of common food related organisms Organsim Properties Gramneg. rods: - Psychrotropic - Pseudomonas - Aerobic - Sensitive to low aw - Sensitive to low pH - CO2-sensitive - Shewanella - H2S-producer putrefaciens Lactic acid bacteria: -Lactobacillus - O2-indifferent -Lactococcus - Resistant to low aw -Pediococcus - Resistant to low pH Enterococcus - CO2-resistant B. thermpsphacta Grampositive cocci: - Facultative -Micrococcus - Resistent to low aw -Staphylococcus - Resistant to low pH - Heat resistant - Lipo-/proteolytic Spore formers: - Extremt vrmeresistenta -Bacillus - Mesofila -Clostridium - Starkt fermentativa Enterobacteriaceae: -E.coli -Enterobacter m.fl. -Erwinia Molds - Strongly fermentative - Pektinase-active - Aerobic - CO2-sensitive - Pektinase-active - Resistant to low aw - Resistant to low pH - Lipo-/proteolytic - Facultative - CO2-resistant - Resistant to low aw - Resistant to low pH
Products Refrigerated fresh: -Meat -Fish -Milk Fish Fish preserves Vacuum packed Fermented food Smoked/salted/dried meat/fish Pickles Fish preserves Vacuum packed Fermented food Smoked/salted/dried meat/fish Pickles ade livsmedel Heat sterilised food Reconstituted dried food. Pre-cooked Milk: B.cereus Milk Pre-cooked food Vegetables Vegetables Fruit Dried food
Yeasts
Vegetables Fruit Low-pH preserves Sweett products
38
Chapter 4.
Foodborne pathogens
Most cases of so called food poisoning are caused by microorganisms. Only a few per cent of the food poisoning cases are reported to be caused by toxic raw materials like toxic mushrooms or plants or contamination by toxic impurities like heavy metals. The remaining cases of food poisoning can be divided into microbial food intoxication, when microorganisms have produced toxins in the food and microbial food borne infections, when pathogenic microorganisms in the food are ingested and infect the human body. Intoxications and infections caused by microorganisms in food and water account for a large number of fatal cases and large economic loss in the society. Food borne pathogens causes millions of death cases every year, especially in poor countries and it is especially children that are the victims. It is difficult to estimate the true statistics behind the food borne diseases, since most cases are never confirmed by clinical analysis. This is especially true for "mild" but common diseases like Bacillus cereus intoxication and Clostridium perfringens infections, since they are usually confirmed by analyses only in large outbreaks. On the other hand, statistics on the severe Clostridium botulinum intoxication is probably reflecting the true cases, at least in the industrial world.
Fig 4.1 Number of cases with food borne diseases reported to the Swedish Institute for Infectious Disease Control according to the law for report on certain diseases (Average number per year during 1997-2005).
4. Foodborne pathogens
39
Only some of the microbial food poisoning diseases are reported to authorities according to law. See Fig 4.1. Other sources of statistics that also include organisms that are not covered by obligatory reporting gives a similar picture, namely that Campylobacter, Salmonella and Norovirus (earlier called calicivirus) are among the most frequent causes of food borne illness, but it also shows that Clostridium perfringens and Staphylococcus often occur in the outbreaks (Fig 4.2).
Cases Outbreaks
Fig 4.2 Statistics of food borne diseases in Sweden for a 5 year period. Calicivirus = Norovirus .Source: Vr Fda, nr 5, 1999.
4.1 Microbial food intoxications Staphylococcus aureus. The probably most common microbial food intoxication is caused by certain strains of Staphylococcus aureus. This organism is also known as a common pathogen causing infections in wounds and blood, but these infections are not considered to be transferred via food. S. aureus produces a series of toxins and other virulence factors (Table 4.1) but it is mainly the enterotoxins that cause food poisoning after ingestion of food on which S. aureus has grown and produced the enterotoxins.
Table 4.1 Some virulence factors of S. aureus Toxins Membrane damaging toxins (several) Epidermolytic toxin Toxic shock syndrom toxin Pyrogenic exotoxin Enterotoxin ( 6 serotypes) Exoenzymes Coagulase Staphylokinase Proteases Phospholipase Lipase Hyaluronidase
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S. aureus is a common inhabitant on animals and humans where it grows on mucose membranes, for instance in the nose, even on healthy individuals, and it is frequently found in pus and wounds. The common source of food contamination is therefore human hands. This organism is very resistant to low water activity (Table 2.2) which means that they can grow on salted and relatively dry products. It does not grow under refrigerator conditions, and without growth no toxin is produced. It has low competitive power compared to many other bacteria, like lactic acid bacteria and Pseudomonas. For this reason, a small amount of S. aureus is usually accepted in food ( e.g. 102 - 103 g-1) before it is classified as not acceptable (Swedish: otjnligt) which means the product must be withdrawn from the market. The intoxications are associated with a large number of foods, often food that has been cooked which eliminates competing microorganisms and food that is handled by human hands: Chicken, ham, salads, pizza, kebab, sauses, paseries etc. The enterotoxins are very heat stable and contaminated food may therefore still be poisonous after re-heating when all vegetative cells have been killed. The disease caused when eating S. aureus enterotoxis is characterised by a violent nausea with vomiting, diarrhoea and convulsions. It is one of the few cases when the eating of infected food results in an almost immediate illness, within one or a couple of hours. The patient usually recovers in 1-2 days and the disease is not associated with further complications. Bacillus cereus. This organism is a facultatively anaerobic endospore former that is ubiquitously present in Nature. Therefore vegetables are usually contaminated with this organism. It is also frequently present in milk, probably since the dusty air in the barn contaminates the milk and the subsequent pasteurisation has no effect on the endospores, while most competing organism are killed. B. cereus produces three enterotoxins which cause relatively mild diarrhoeal illness with an incubation time of 6-24 hours, and an emetic toxin, cereluid. The haemolysins are inactivated in the stomach and this type of disease is actually an infection where the toxin is produced locally by B. cereus growing in the intestine. The cereluid is a heat stable protein and this disease is considered to be a true intoxication. It has a shorter incubation time, 0.5-6 hours, and is especially associated with rice dishes. B. cereus is assumed to be a very common agent of food poisoning, but both diseases are usually proceeding fast with little complications and therefore isolated outbreaks are normally not identified and the statistics becomes unsure. B. cereus is not so competitive but after heating of a product the spores may become the dominating organisms and if the food after that is kept too long in the temperature range 15-45C the spores may germinate and grow and produce
41
the toxin. Like most Bacillus this organism is typical mesofilic with respect to temperature, but certain strains are reported to be psychrotrophic and may grow down to about 4 C. Clostridium botulinum. The most well-known and feared microbial intoxication is botulism, which is caused by one of several toxins of Cl. botulinum. This organism is an obligately anaerobic spore forming bacterium that is very common in soil and water. The toxin is classified according to serotype A-F, where type A, B, E, and F are toxic to humans. Cl. botulinum type E is commonly found on fishes and this toxin is relatively heat labile, destroyed by boiling, while type A is more heat resistant. The endospores make also heat treated food potentially dangerous since surviving spores may grow out. The botulin toxin is a very toxic protein that is produced during growth of the vegetative cells in food. Cl. botulinum does not grow at temperatures below 4C, at pH below 4.5, or in presence of oxygen. The toxin acts as a neurotoxin paralysing the central nervous system. It is one of the most potent toxins known with a lethal dose of about 10-6 g. After an incubation time of 18-36 hours, the illness sometimes starts with nausea and is followed by the effects on the CNS caused by blocking of the acetyl choline release at the nerve synapses: double-seeing, difficulties to swallow and finally paralysing of the breathing. At this stage the mortality is high. In US statistics during 1950 - 1970 the number of fatal cases was almost as high as the number of reported cases. After that an anti-toxin became available but mortality is still considerable. Fortunately, the number of cases is low, in Sweden the average is less than one/year. The few cases of botulism in Sweden are associated with home preserved (marinaded or smoked) fish and home preserved meat. The precautions that must be taken to avoid botulism in association with food preservation are low pH (often vinegar), high salt concentration and storage below 4C. In commercial preservation nitrate also plays an important role. This is further described in Chapter 6. The so called infant botulism has another mechanism. It is caused by a Cl. botulinum infection of the intestines where the spores germinate, grow and produce the toxin. This disease is only associated with babies under one year age who have not obtained the normal competitive intestinal microflora, and the infection origin has exclusively been honey which often (10%) contains spores of Cl. botulinum. For this reason authorities recommend not to give honey to babies.
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Mycotoxins. Intoxications by fungal toxins, mycotoxins, are not found in the statistics on food borne diseases. The reason is that these diseases, contrary to the other diseases discussed here, do not cause acute symptoms. Most reports on mycotoxins describe their effects as cancerogenic or liver or kidney damaging, with symptoms emerging long time after consumption of the food. An exception is patulin, which is associated with intestinal illness but it is also a suspected carcinogen. There are hundreds of mycotoxins described in the literature. Biochemically they are typical secondary metabolites produced by moulds. It means they are mainly produced late in or after the growth phase. Most mycotoxins are resistant to temperatures used in cooking. Fig 4.3 shows the chemical structure of some mycotoxins. For some of the mycotoxins (e.g. aflatoxins, ochratoxins and patulin, there are regulatory concentration limits for food, based on TDI values (TDI=tolerable daily intake). TDI-values are usually in the range below 1 mg/Kg body weight.
Aflatoxin B1 Fig 4.3 Examples of mycotoxins
Ochratoxin A
Table 4.2 lists some well-known mycotoxins, producing organisms and food they are typically associated with. The table demonstrates two characteristics of mycotoxins: several species, even from different genus, may produce the same mycotoxin and one mycotoxigenic organism may produce several mycotoxins. There are several variants of chemically related aflatoxins. Aflatoxin B1 is the most commonly observed and most toxic of the aflatoxins and it is a strongly potent carcinogen. In animal experiments daily intake of less that 100 ng/kg body weight causes liver tumours. Aflatoxin M1 is found in milk and it is a degradation product of aflatoxin E.
43
Table 4.2 Examples of mycotoxins, mycotoxigenic molds, and associated food Toxin Aflatoxins Ochratoxin A Patulin Penicillinic acid Zearalenon Roquefortin Organism Aspergillus flavus Asp. parasiticus Asp. ochraceus Penicillium viridicatum Pc. exapnsum Pc roqueforti Pc. cyclopium Pc viridicatum Fusarium graminearum Pc. roqueforti Associated food Nuts, figs, corn Grains, coffee, wine, beans Fruits, berrys Peas Grains Bread Infertility Effect Liver cancer Kidney/liver damage, teratogenic Diarrhoea
zone
1 2 3 4
g aflatoxin/ kg bread Bread 1 Bread 2 Bread 3

>> 15 000 600 100 n.d 150-300 n.d n.d 40-80 20 n.d
Fig 4.4 Analysis of aflatoxin distribution in three breads that were inoculated with A. flavus and incubated until a colony was formed (Vr Fda, 31, 390-399, 1979).
The extremely high toxicity of aflatoxin and the fact that mould colonies often grow on bread raises the question about how far the toxin reaches from the fungal colony. In an investigation 3 breads were inoculated at the surface with an aflatoxin producing strain of Aspergillus flavus, as indicated in Fig 4.4. Samples were taken from 4 zones at different distances from the colony and analysed for aflatoxin B1, B2, G1, and G2. The table in Fig 4.4 shows the sum of the aflatoxin concentrations in the zones after one week. The permitted level in bread was 5 g/Kg. While aflatoxins are mainly associated with nuts and figs, ochratoxins are generally found in food and especially in food that is consumed in large amounts, like cereals. Ochratoxin is also spread via meat from animals fed on grains. It has been shown to cause damages on liver and kidney and it is also teratogenic. The TDI is 14 ng/Kg body weight but due to expected but not
44
proved cancerogenic effects the TDI value used by some authorities is considerably lower. Patulin was first studied as a potential antibioticum but is now classified as a mycotoxin. The source in nature is fruits and berries, and it is frequently found at very low concentrations in commercial fruit juices and jam. Several strains of P. roqueforti isolated from commercial blue cheeses have been shown in the laboratory to produce mycotoxins, among them PR toxin and roquefortine. This organism is also a common contaminant in many foods and it is a predominant organism in silage where it is said to have a positive effect on the acceptance by cattle. Roquefortine C has been reported to have a neurotoxic effect and it is an inhibitor of Gram positive bacteria. The uncertainty of the real effects of consumption of mycotoxins with food has resulted in the general recommendation to avoid mold infected food. Algal toxins. Planktonic algae called dinoflagellates are responsible for different types of shellfish poisoning: Paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and others. The PSP is observed as respiratory paralysis within 0.5 - 2 hours after consumption of the toxic food and it may get severe consequences if not treated. The DSP causes diarrhea within 0.5 - 3 hours and lasts for 2-3 days with no after effects. These types of poisoning are associated with filter-feeding molluscs, like mussels, clams, scallops and oysters. Cyanobacteria, previously called blue-green algae, are involved in so called algal blooms, some of which may make the water toxic. Nodularia spumigena is one of the most common toxic cyanobacteria in algal blooms in the Baltic sea. These intoxications are normally not associated with food or drinking water, however. 4.2 Food borne infections Food borne diseases caused by microbial infection of the consumer is much more frequent than the intoxications caused by ingestion of microbial toxins produced in the food. Two of the most frequent diseases in the statistics (Fig 4.1) namely Campylobacter and Salmonella are infections caused by eating contaminated food. The pathogens may then grow in the intestines and cause the disease. In general, this type of disease has an incubation time of one to several days, which often results in difficulties to identify the food that was
45
causing the problem. The incubation times reported for infections varies much with the status of the individual and with the infecting dose. Also reported minimal infectious doses are very unsure figures and depend on the condition of the person. Mostly elderly people and children are much more sensitive that grown-up and healthy individual. In Table 4.3 common infections are grouped according to the probable source of contamination. Bacteria with fecal origin may enter the food from water or raw material that has been in contact with feces, which is the natural environment for these organisms. Alternatively, the food has got this infection directly from feces contaminated hands of someone handling the food. Most of the food pathogens of fecal origin belong to the family Enterobacteriaceae, which includes among others the genera Salmonella, Shigella, Yersinia and Escherichia belong to the
Table 4.3 Classification of common food pathogens based on their probable source Fecal origin Water origin Soil origin Campylobacter Listeria monocytogenes Clostridium perfringens Salmonella Aeromonas hydrophila Bacillus cereus (diarrhoeal) Shigella Vibrio parahaemolyticus Yersinia enterocolitica Pathogenic E. coli
Campylobacter is a common inhabitant of intestines of many types of warm blooded animals without causing any symptoms in the animal. It is mainly the species C. jejuni that causes the food borne infections. It is a Gram negative bacterium and it is environmentally sensitive: it grows only in the range between 25 and 42 C, is microaerophilic, and very sensitive to drying, freezing and disinfectants. The food contamination source is often chicken, unpasteurised milk or water. Flies are also suspected to transmit the bacteria from feces to food. Several large outbreaks have been caused from municipal water, but mostly it is chickens that are associated with Campylobacter infections. The chicken (and also other types of meat) becomes contaminated from its feces during the slaughter and since the infectious dose is very small ( 500 cells) infected meat can cause disease even if it has been stored so that no further growth has been possible. The only way to avoid this disease is to apply good hygiene in the food preparation and to heat the food enough to kill the cells, which requires 65 C through all parts of the meat. The infection gives diarrhoea and other typical gastroenteritis symptoms for about 2-5 days but sometimes reactive arthritis prolongs and complicates the disease. Salmonella. There are more than 2000 different serotypes of Salmonella and some cause relatively mild diseases while other strains cause severe illness. S.
46
typhi and S. paratyphi cause the most dangerous infections. Salmonella is frequently found in poultry and swine. It is environmentally very resistant which explains why they are widely spread in Nature even if they grow mainly in animals. The organisms are usually distributed via meat that is contaminated with feces during slaughter. Infected animal feed is another carrier of Salmonella. It is also common in spices and vegetables, probably through contamination with infected water or soil.. The disease breaks out after 12-48 hours and lasts for a couple of days, with some exceptions when there are complications with reactive arthritis or septicemia with subsequent infection of organ systems. Humans may also become carriers of Salmonella without showing any symptoms. The infective dose varies much but as little as 15-20 cells has been reported. According to FDA the number of cases of salmonellosis is 2-4 millions/year in the US and the frequency is rapidly increasing. Especially S. enteritides is rapidly spreading in US and Europe. Shigella. Contrary to Salmonella these organisms are very host specific and grow only in the intestines of humans and apes. The food borne infections are mostly caused by bad personal hygiene but also by vegetables that have been contaminated with water containing human feces. Infected humans may recover and still be "healthy carrier" of the organisms. This, together with the very low infectious dose (10 cells), also makes shigellosis (bacillus dysenteri) directly transferable between individuals. Shigella multiply intracellulary in the epitheleal cells which results in tissue destruction. Some strains produce shiga toxin which is similar to the toxin produced by EHEC. This protein, when produced by the bacteria in the infected human host cell, inhibits the protein synthesis and results in cell death with severe hemorrhage in the patient. Yersinia enterocolitica. There are three pathogenic Yersinia species. Y. pestis, Y. pseudotuberculosis and Y. enterocolitica. The latter is associated with food borne infections, mainly from pork, since swine is a common reservoir of this organism. Also dogs and cats are frequent carriers of Y. enterocolitica which grows not only in the intestinal tract but also in mucous membranes in the mouth and throat. This is one of few psychrotrophic pathogens which can grow at high rate in the refrigerator, even down to 0C. Most cases are associated with pork and vacuum packed meat products but also water and un-pasteurised milk have been involved in out-breaks. Vacuum packed meat products have often been heat treated, which removes most vegetative cells inclusive the quite heat sensitive Y. enterocolitica, and if the product then is re-infected and stored for long time in the refrigerator the product may cause infection. The disease breaks out 3-7 days after the infection and it lasts for 1-3 weeks. The disease is relatively rare in the statistics which partly may be due to the
47
difficulties to isolate the bacteria. It is also assumed that only certain strains of Y. enterocolitica are pathogenic. Pathogenic E. coli. There are four enteropathogenic groups of E. coli. They are classified according to serotype. The nomenclature is not strict, but a common classification is: EHEC ETEC EPEC EIEC enterohemorrhagic E. coli enterotoxigenic E. coli enteropathogenic E. coli enteroinvasive E. coli
EHEC (enterohemorrhagic E. coli ) produces shiga-like toxins, also called verotoxins. The most well-known EHEC are characterised and analysed as the serotype O157:H7, but these shiga-like toxins are produced also by other E. coli serotypes. Alternative names of EHEC are STEC ( shiga-toxin producing E. coli ) or VTEC (verotoxin producing E. coli). The natural reservoir of EHEC is probably the intestines of cows, who are not themselves showing any symptoms, and then the distribution occurs via fecal contamination. EHEC infections have been associated with hamburgers, un-pasteurised milk, water, and alfalfa sprouts. In some cases it has been assumed that humans keeping indoors in a cow-house can be infected directly from this environment. The very low infectious dose,10 cells, means that the bacteria do not need to grow on food to make it infective. EHEC has unusually high resistance to low pH, and the cells can survive extended periods in sour products like juice, yoghurt, and fermented sausages, products that usually have been considered as safe in this respect. The two shiga-like toxins are coded by genes (stx1 and stx2) which are located on lambda phages and integrated as inactive prophage genes in the bacterial genome. Only after induction, which can be by agents resulting in the SOS response or by iron limitation, does the prophage enter the lytic phase which induces the toxin production. The toxin kills the cells in the intestines and causes bloody diarrhoea. In severe cases, especially in children, the infection is spread to the kidney which may be permanently destroyed. ETEC, or enterotoxigenic E. coli causes a relatively mild gastroenteritis with watery diarrhea, often called travelers' diarrhea. These infections are also common among children in poor countries. Large infective doses (> 108 cells) are required and the incubation time is about 1 day. ETEC is not common in countries with good sanitary standards but when the water is contaminated with human feces there is a risk for ETEC infections in food.
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EPEC are strains of E. coli that cause the infantile diarrhea in newborn babies. It is not assumed to be food associated. EIEC invade the epithelial cells of the intestine, resulting in a mild form of dysentery. It is not known if this is a food associated infection. Water and soil are reservoirs for several pathogenic bacteria that may contaminate food: Listeria monocytogenes, Clostridium perfringens, Bacillus cereus, Aeromonas hydrophila, and Vibrio parahaemolyticus. Listeria monocytogenes is widely spread in nature both in water, soil, plants, and in animal intestines. It grows often in biofilms which are common in food manufacturing facilities. L. monocytogenes is therefore very common in food. Also humans are often carrying this organism in its intestinal flora without any symptoms. Many strains are pathogenic to some extent. Listeriosis is not primarily a gastroenterit but it is rather manifested as septicemia, meningitis, and cervicial infections in pregnant women which may result in spontaneous abortion. Sometimes the symptoms are preceded by gastrointestinal symptoms like nausea, vomiting, and diarrhea. The bacteria invade the human phagocytic cells and propagate intracellulary. In this way the infection is spread to organs with the blood. The organism is one of the psychrotrophic pathogens which can grow to dangerous concentrations also in a refrigerator. It is frequently found in vacuum packed smoked or marinaded fish and in soft cheeses made on un-pasteurized milk. Cooking at 70C kills the bacteria. Food associated with Listeria outbreaks are often such food where the organism gets the chance to grow during production and then is consumed without further heating, e.g. in soft cheeses, marinaded meat, and smoked fish. The infective dose is unknown. The reason why the number of identified disease cases in not larger while Listeria is often present in food is probably that only some strains are pathogenic and the pathogenecity factors are not known enough to be the goal for analysis. Clostridium perfringens is a common spore forming soil bacterium which means that vegetables often are contaminated. It can also grow in the intestines of humans and animals without causing any symptoms. Many strains of Cl. perfringens produce enterotoxins. There are a number of facts that together makes this one of the most frequent diseases associated with large-scale cooking, especially with soups and casserols: 1) Being a common soil bacterium it is often added to food as spores in contaminating vegetables, 2) The spores not only survives cooking at 100C but even become activated to germinate, while most competing bacteria are killed. 3) The boiling also removes the oxygen which otherwise prevent growth of this obligately
49
anaerobic bacterium. 4) In rich media and optimal temperature it can grow extremely fast (8 min doubling time at 45C). If the cooling from this temperature down to below 15C is not fast enough, or if the food is kept warm at too low temperature (should be 60C), conditions for growth of Cl. perfringens are excellent. The common form of perfringens poisoning is characterized by intense abdominal cramps and diarrhea that come within 8-24 hours after consumption of foods with large numbers of cells and lasts for about 24 hours. The infective dose is very large , over 108 cells. Bacillus cereus. This organism produces the toxin cereluid that act as food intoxication causing vomiting. But many strains of B. cereus also produce one or several of three enterotoxins: haemolysin BL, non-haemolytic enterotoxin, and cytokine K. These proteins do not survive the passage through the stomach, and therefore its is considered that the bacteria also can establish themselves in the intestines and produce the enterotoxins there. This diarrhoeal disease is often associated with meat and vegetable dishes and sauces. Also the spores may germinate and grow in the intestines, so contaminated food may cause disease even after heating. This is assumed to be a very common source of mild illness, that is seldom investigated clinically, and therefore the statistics is uncertain. Vibrio parahaemolyticus is widespread in marine environments and brackish waters all over the world, especially in areas with warm climate. It is associated with infections from fish, shellfish, shrimps and other seafood, especially raw food that has not been heat treated. The organism attaches itself to the small intestine and secretes a toxin. The illness comes after about 24 hours and lasts for a couple of days. All the common food poisoning symptoms may be involved: diarrhea, abdominal cramps, nausea, vomiting, headache, and fever. Aeromonas hydrophila. This organism has only recently been recognised as a food pathogen and there is not much information available on this. However, in several cases it has been isolated from stools of patients with gastroenteritis without any other sign of infection. A. hydrophila is a common bacterium in soil and water, even in drinking water pipes. It grows well down to 5C and it grows in vacuum packed food. Shrimps, ham, sausages are examples of food that has been associated with these infections. Virus. There are several virus infections spread with food and water. Viral gastroenteritis is usually a mild illness characterized by nausea, vomiting, diarrhea, and fever. The infectious dose is not known but is presumed to be low. These infections are either spread via contaminated water or food or
50
through direct contacts between people. Norovirus is one of these viruses that cause short but intensive gastroenteritis especially in children. It has previously been named Calicivirus. The virus is present in the feces of infected persons. It is assumed that only 10 virus particles is enough for an infection and this may explain why this disease also is very contagious and not only distributed via food and water.
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Chapter 5. Heat sterilisation and pasteurisation Sterilisation is based on either of two principles: All living cells, including spores, are physically removed from the medium or they are killed. Each principle involves a number of methods. Heating is the most coming method for killing microorganisms in food. If this is made with the goal to kill even endospores temperature in the range of 120 C or higher must be used and this can result in real sterilisation, i.e. also the endospores are killed. If the goal is to eliminate the majority of vegetative cells, temperatures in the range of 70-90C are used and this is called pasteurisation, and it has no inactivating effect on endospores. Also ionising irradiation (Co60 or electron beams) can be used for sterilisation, and if the doses used only kills the more sensitive cells and leaves the endospores, also this is called pasteurisation. Killing of microorganisms can also be made with chemical agents, and this is usually referred to as disinfection, and applied mainly to surfaces 5.1 Mechanisms of heat inactivation of microorganisms. Microorganisms may be classified in two groups with respect to heat sensitivity: 1) Bacterial endospores and 2) Vegetative cells and spores of other types, e.g. fungal spores. Endospore formation is mainly found in the genera Bacillus and Clostridium, but also Sporosarcina, Desulfotomaculum, Sporolactobacillus and Thermoactinomyces may form endospores. Endospores are extremely resistant to heat, UV and ionising radiation, drying and chemical agents. It takes heat treatment in the range of 100C and higher to inactivate these spores. Note that other spores, e.g. fungal spores, may be quite resistant to drying but they are only slightly more resistant to heating than are the corresponding vegetative cell. To inactivate these spores and vegetative cells in general, heat treatment in the range of 50 to 90 C (pasteurisation) may be efficient and it does not provide complete sterility since it has no effect on the endospores. Fig 5.1 shows the main structures of a bacterial endospore. The exact mechanisms behind the extraordinary resistance of bacterial endospores are not known, though some information is available from mutants lacking different components in the spore: The spore has three distinctive structures: The core, containing the DNA, a few key enzymes and 2-10% dipicolinic acid (DPA) in complex with Ca2+ and the DNA. The core also contains some basic proteins that are quickly hydrolysed and serve as amino acid source during the germination. The water content of the core is low, which together with DPA is
5. Sterilization and pasteurization
52
assumed to contribute to the large thermal stability of the spore. The basic proteins contribute to the high UV radiation resistance. The surrounding cortex contains negatively charged peptidoglucans and the water in the cortex is freely exchangeable with surrounding water. The difference in water concentration between the core and the cortex makes the spore refractile and gives it a light appearance in a phase contrast microscope, while vegetative cells appear dark. The cortex is surrounded by a spore coat of proteins that confer the chemical resistance to the spore. The size of a spore is somewhat smaller than the vegetative cell, as indicated in Fig 5.2
Core: DNA, Ca-DPA, few ribosomes, key enzymes, no water Cortex: Negatively charged peptidoglucanes Coat: chemically resistant proteins
Fig 5.1 Structure of a bacterial endospore.
Transformation of a spore to a vegetative cell involves a number of reactions (Fig 5.2). The activation is a reversible reaction, which is poorly understood. Activation may be needed to make a spore competent for the next stage, germination.
activation germination
dormant spore lysis
activated spore
germinated spore outgrowth vegetative cell growth
sporulation
Fig 5.2 The endospore germination-sporulation cycle. The dormant spore may need activation before the initiation of germination can take place. Activation is a reversibel raction and does not change the resistance or appearance of the spore. At the initiation of germination all resistance properties disappear and the spore then grows out to a vegatative cell which divides a number of times until harsh environmental conditions induce sporulation. The spore is eventually liberated by cell lysis.
53
Agents that cause activation are, for instance, sub-lethal heat treatment, high pressure and extreme pH. Spores that are difficult to activate are called super dormant spores. It is difficult to differentiate between super dormant spores and dead spores, since it is only when the spore has been provoked to germinate that it has been proven that it was not a dead spore. The activation reaction does not result in any visible change of the spore structure or composition nor any observable metabolic reaction. An activated spore may be initiated to germinate by several chemicals like amino acids, nucleotides etc. The initiation of germination is seen as a swelling of the spore and it is associated with migration of the Ca2+ ions from the DPA complex in the core to the cortex where they neutralise the electronegatively expanded cortex, which shrinks and in this process water enters the core which swells. The germination of one spore takes only a couple of minutes. In a phase contrast microscope the appearance of the spore is changed from a bright reflecting structure of the ungerminated spore to a dark colour, like that of the vegetative cell, of the germinated spore. Germination of a whole spore population can also be observed as a reduction of the absorbance in a spectrophotometer. The germination process of a whole population of spores may be completed as fast as within 15 minutes, but it may also take much longer time. The initiation of the germination is characterised by a complete loss of all the resistance factors of the spore. Electron microscopy reveals that the germination is associated with an expansion of the core and a thinning of the surrounding cortex (Fig 5.2). During this phase a sequence of metabolic reactions and synthesis of enzymes is initiated. The last phase of the germination is called outgrowth. During this phase, which takes about one generation time, all the normal metabolic reactions are restored and the spore is gradually converted to a vegetative cell. Heat inactivation of the endospore is believed to be a matter DNA damage, but also heat denaturation of essential proteins in the core may be involved. Heat inactivation of vegetative cells involves quite different reactions, and it is mainly a matter of disorganisation of the cell membrane. This is indicated by several phenomena observed in the heat surviving fraction of a population, as for instance the increased osmosensitivity and increased leakage of cell components. Also DNA damages and denaturation of proteins may be observed during heat killing of vegetative cells. The thermal resistance of vegetative cells is also influenced by the level of its heat shock proteins, which participate in the protection against thermal denaturation of proteins. Since the heat shock proteins may be induced by e.g. thermal (sub-lethal) chock and other stress agents, the thermal stability of a vegetative cell depends not only on the
54
environment but also on its history. Also endospore stability depends on environmental factors like the composition of the medium during the sporulation. Metal ions like Ca2+ and Mn2+ are often required for the endospore to aquire full heat resistance. 5.2 Kinetics of heat inactivation of cells Heat inactivation of spores as well as vegetative cells can be described with the same mathematical model. Therefore the same methods of calculation may be employed for sterilisation and pasteurisation. The rate of heat inactivation of a population is proportional to the number of cells, N. If N represents the number of organisms in the total volume of medium to be sterilised, the rate of inactivation becomes:
dN = "kN dt
-1
(1)
where k (min ) is the specific heat inactivation constant, also called the death rate constant and t (min) is the time. Integration from time zero with the initial number of cells (No)! gives
"N% ln$ ' = (kt # N0 &
(2)
which can be rearranged to

!
ln( N ) = ln(N 0 ) " kt N = N 0e"kt
(3) (4)
or to
!
for calculation of the number of surviving cells after a given time. Eq. 3 is illustrated in Fig 5.3. Experimentally determined inactivation curves often show ! deviations from this model. Some examples of this are shown and explained in Fig 5.3. Note that this first order kinetic model does not permit calculation of the time when the number of cells reaches zero, which is the time it takes to sterilise a sample! However, when N is below one cell (N < 1) the sample is in practice sterile. An interpretation of this is that when N < 1 (i.e. ln(N) <0) there is a certain statistic probability that the sample is sterile. This will be used for calculation of the sterilisation time in Section 5.3
55
The inactivation constant, k, is a characteristic of the cell but it depends also on many environmental parameters. The higher the temperature is the larger is the k-value. The heat inactivation constant depends on temperature like most rate constants of chemical reactions. This is usually described with the Arrhenius equation:
k = Ae"#E /RT
(5)
where A (min-1) is a constant which gives the order of magnitude of the inactivation reaction, E ( J mole-1 ) is the activation energy which describes ! the temperature dependence of the inactivation reaction, R ( 8.31 J mole-1 K-1 ) is the universal gas constant, and T (K) is the temperature.
Fig 5.3. Heat inactivation curves. The left hand figure shows two inactivation curves with different death rate constants. The right hand figure shows some deviations from the model: 1. This form may be caused by super-dormant spores, which are activated by the first heat treatment and do not germinate unless they get this treatment; 2. This may be observed in samples that contain aggregates of cells, since analysis is usually made by viable count that gives number of colony forming units rather than number of cells. The viable count does then not decline until the last cell in an aggregate is killed. This curve form can also be caused by an experimental artefact, if heat transfer is not fast enough. 3. Non-uniform heat resistance in the population, e.g. when the sample contains species with different thermal sensitivity. This is the expected curve for a mixed microflora.
Eq. 5 can be only used to calculate the effect of a temperature change on the rate of heat inactivation within a limited temperature range, where the inactivation is caused by the same reaction. The constants A and the activation energy, E, can be obtained from the logarithmic form of the Arrhenius equation:
ln(k) = ln(A) " #E 1 R T
(6)
56
which shows that a plot of the logarithm for the thermal death rate, k, against the reciprocal absolute temperature will have the slope E/R and an intercept with the ln(k) axis corresponding to ln(A). See Fig 5.4.
1
spores !E 280 kJ/mole
k (1/min)
0.1
Thiamine !E 92 kJ/mole
112 C 0.01 2.1 2.2 2.3 2.4 2.5 2.6 2.7
96 C 2.8 2.9
Fig 5.4 Arrhenius plots of inactivation of B. stearothermophilus spores and thiamine. Note that a temperature increase has a larger effect on the spore inactivation rate than on the vitamin inactivation rate.
1/T
The heat treatment does not only cause cell death but also increased rates of other chemical reactions, which may be beneficial or detrimental to the product. Examples are inactivation of vitamins and other nutrients, lipid oxidation, and so called Maillard reactions. The latter is a group of reactions involving reducing sugars and amino groups and it is an important class of reaction in food processing, including both wanted and unwanted reactions, depending on the situation. The principle reaction is shown in Fig 5.5.
high T, low aw, high pH
Fig 5.5. A Maillard reaction is a reaction between reducing sugar and an amino acid favoured by high temperature, low aw and high pH.
57
Depending on which sugars and amino acids that are involved, the products may be have different taste, be toxic, and cause colourisation of the product. A large group of such mellanoidines are important for the organoleptic properties of food. Also chemical reactions like vitamin inactivation and Maillard reactions can be modelled with first order kinetics. In analogy with eq. 2 :
"C% ln$ ' = ( kC t # C0 &
(7)
where C and Co are the time dependent and the initial concentration of the ! compound, respectively, and kc is the inactivation rate constant. The temperature dependence of these reactions also follows the Arrhenius equation (eq. 5) and can be characterised with the activation energy. Table 5.1 lists some examples of activation energies for inactivation of endospores and for some other chemical reactions. There is a tendency that the activation energy for cell killing is higher than the activation energy of most of the chemical reactions. This can be utilised to minimise the chemical reactions during sterilisation by applying continuous sterilisation.
Table 5.1 Examples of E values for heat inactivation of spores and some chemical reactions. Inactivation of E (kJ mol-1 ) B. subtilis spores 318 B. stearothermophilus spores 283 Cl. botulinum spores 343 Folic acid 70 d-panthotenyl alcohol 88 Cyanocobalamin 97 Thiamine HCl 92 Maillard reactions 125
5.3 Calculation of sterilisation time According to the model for heat inactivation, eq. 3 and Fig 5.3, it is not possible to calculate the time needed to reach zero concentration of viable cells. Yet, when the cell number, N, in eq. 3 is below 1, the medium is sterile. If eq. 3 is used to calculate the time needed to reach e.g. 10-3 cells, it means that there is a probability that one batch of 1000 sterilised batches will be infected. The time
58
needed to reach this probability of sterility does not only depend on the death rate constant, k, but also on the initial number of organisms, No, as is obvious from Fig 5.3. Thus, a sterility criterion, (nabla) has to be defined:
#N & " = ln% 0 ( %N ( $ f'
(8)
where Nf is the final number of organisms. Eq. 2 can now be used to estimate ! the sterilisation time, F (min), needed to satisfy the sterility criterion " :
F= " k
(9)
This sterilisation time depends also on the temperature applied since k is a function of temperature. The sterilisation time (FT ) required to satisfy the same ! sterility criterion at another temperature (T K) than the reference temperature (Tref K) at which the sterilisation time Fref once has been assessed, can be obtained from eq. 9 written for the two sterilisation temperatures:
" = Fref k ref = FT kT
(10)
which, after substitution of k according to the Arrhenius model (eq 4), gives the wanted sterilisation time at a different temperature T K:
!
FT = Fref e
"#E $ 1 1' & " ) R & Tref T) % (
(11)
5.3.1 Batch sterilisation If the death rate constant, k, is known and constant during the sterilisation, eq. 9 ! gives the answer for the sterilisation time required to satisfy the sterility criterion. However, in batch sterilisations the temperature is first increasing, then constant, and finally declining (See Fig 5.6). Since the thermal death rate constant, k, depends on the temperature, this effect must be included in the calculation. In the beginning of the sterilisation, when the temperature is low, the rate of sterilisation is low. We must then introduce a time dependent relative sterilisation dose (t)= ln(No/N(t)), during the sterilisation. The sterility criterion is then satisfied when (t) = . Combining eq. 9 with eq. 5 gives an expression that shows how the sterilisation dose depends on time:
#$E
"(t) = t k(T) = t Ae RT
(12)
59
Since the temperature varies with time during batch sterilisation, the total sterilisation dose is obtained as the integral of eq. 12
t
"(t) = A % (e#$E / RT )dt

0
(13)
The batch sterilisation has reached the criterion on sterility when t = . This can be calculated without knowledge of the constant A in equation 11. ! According to eq 4 and eq 8, the sterility criterion can be written
" = Fref Ae
#$E /RTref
(14)
Division of both sides of eq 13 by eq 14 gives the ratio between (t) and :

!
t
)dt "(t) = 0 #$E / RTref " Fref e

#$E / RT
% (e
(15)
Fig 5.6 shows an example of a batch sterilisation temperature profile and the sterility according to eq.15. In this example the heating phase is relatively short ! and it contributes only with some 20 per cent of the total sterilisation dose. Since cooling from the highest temperature first is very efficient, the contribution to sterilisation from the cooling phase is very small. Note also that the exponential dependence of the sterilisation rate on the temperature means that good temperature control at the holding phase is important for the precision of the sterilisation.
Fig 5.5. Simulation of the progress of the sterilisation (t /) and inactivation of a temperature sensible compound (C/Co) during a batch sterilisation. The sterility was calculated according to eq.15 and the concentration of compound according to eq. 7 and eq. 5. Parameters: =20, A= 1035.8 sec-1 and 109 sec-1 for sterilisation and chemical reaction, respectively. E= 282 kJ mole-1 and 92 kJ mole-1, respectively. R= 8.31 J mol-1 K-1 .
60
5.3.2 Continuous HTST sterilisation During the low temperature part of the batch sterilisation, the rate of sterilisation is relatively low, but other chemical reactions, like vitamin inactivation, lipid oxidation and Maillard reactions may take place at a considerable rate, which is sometimes detrimental for the food quality. An interesting property of the sterilisation reaction is that it has a relatively high activation energy, as pointed out in Table 5.1. The HTST sterilisation (High Temperature Short Time) utilises the different sensitivity to temperature of the two reactions "sterilisation" and "chemical reaction", which was expressed as different values of the activation energy. This is exemplified in Fig 5.7, which shows how the rate constant for spore inactivation increases much faster with a temperature increase than does the rate constant of thiamine inactivation, because the former has a higher activation energy (slope of the curve). Therefore, the vitamin inactivation will be reduced if an increased sterilisation temperature is combined with a reduced sterilisation time to give identical sterility criterion, . This effect of increased sterilisation temperature is demonstrated in Fig 5.6.
30 1
C / Co
20
F (min) C / Co
10
0 115
120
125
130 T
135
140
0 145
Fig 5.7 Effect of sterilisation temperature on a chemical reaction in a continuous sterilisation. C/Co is the fraction of non-reacted chemical compound. F is the sterilisation time according to eq. 9. C/Co was obtained from eq. 7 and the temperature dependence of the two reaction rate constants, k, was obtained from the Arrhenius equation (eq. 5). Parameters as for the batch sterilisation, Fig 5.6.
5.4 The D-value and the Z-value The theory of heat sterilisation was developed in the food industry during the first part of the 20th century. It was then common to use logarithms with the base of 10 and much literature on sterilisation, and especially constants on heat sensitivity and temperature dependency, are still based on this nomenclature, which uses a D-value and a Z-value to describe the inactivation rate and the temperature sensitivity of the inactivation rate, respectively.
61
The rate of heat inactivation according to eq. 2 can be written on a 10log basis as
10
"N% 1 log$ ' = ( t D # N0 &
(16)
where 1/D is the slope of the curve when the number of surviving cells (N) is ! plotted against time during heat exposure (Fig 5.8). The decimal reduction time (D, min), is the time needed to reduce the number of cells to one tenth of the previous value.
Fig 5.8 Inactivation curve plotted on 10log basis showing the definition of the D-value.
Solving eq. 2 for t = D and N = No/10 gives the correlation between the inactivation constant k and the D-value:
D= ln(10) k
(17)
D-values for inactivation of spores as well as for inactivation of vegetative cells are available in the literature and, if not found, can be determined ! experimentally by plotting the data as in Fig 5.3 or 5.8. Table 6.2 shows some examples of D-values. For endospores D-values are often standardised to minutes at 121 C but for vegetative cells lower temperatures, e.g. 60C, are often used. These D-values (and k) depend much on the environment in which the heating is performed. As a general rule one may say that the heat resistance increases
62
with reduced water activity but it decreases when the organism is subjected to other extreme conditions like extreme pH, toxic compounds etc. The effect of the water activity means that it may be very difficult to heat sterilise media with suspended solids like starch, in which spores may stay relatively dry. Sterilisation of dry materials like glass and other equipment requires much higher temperature and/or prolonged heating. While water solutions mostly are sterilised by some 15 minutes at 120C (steam sterilisation) the corresponding sterilisation of dry materials (dry heat sterilisation) may require about 4-6 hours at 160C or 1.5 h at 170C to give similar effect. Data given in this chapter refers to sterilisation in water solutions.
Table 5.2 D-values (min) for heat inactivation of microorganisms D121 D60 D50 Organism Endospores (general) 0.1-4 Cl .botulinum 0.2 Cl. thermosaccharolyticum 10-20 Micrococcus spp. 5-20 Streptococcus spp. 5-20 Fungal spores 5-20 Virus 1-10 Mesophilic bacteria 1 Psychrotrophic bacteria 1-5 Psychrophilic bacteria <1
The temperature influence on the D-value is expressed by the Z-value, (C) which is the temperature increase that is needed to reduce the D-value by a factor of 10. The definition is exemplified in Fig 5.9
Fig 5.9 The temperature dependence of the D-value and the definition of the Z-value.
63
The mathematical expression of the temperature dependence shown in Fig 5.9 is

10
1 log(D) = " T+10 log(DT = 0 ) Z
(18)
where log (DT=0) is just a constant that represents the extrapolation to temperature zero. This constant has no physical meaning, since the model is ! relevant only for a limited temperature range where the nature of the heat inactivation reactions are identical. In analogy with the calculation of sterilisation time at another temperature than the reference temperature (eq. 11), it is possible to show that this calculation can be done based on the Z-value:
FT = Fref 10
(Tref "T )/Z
(19)
The Z-value for endospores is usually in about 10C while it is lower, about 5C for inactivation of vegetative cells (pasteurisation).
!
64 Chapter 6. Fermented foods All food raw materials are contaminated by microorganisms, which take part in the mineralisation of organic materials in Nature. Therefore, Man had early to learn to live with microbially infected food. The microbial reactions mostly resulted in spoilage of the food. However, Man learnt to handle some foods in ways that extended their shelf-life. These preservation methods were mainly based on drying or fermentation. Food fermentations are still used to produce so called fermented food, but today preservation is not the main objective of the fermentation, but it is rather the specific taste and texture that is the goal of the fermentation. Food fermentation is applied to a all main types of food, as meat (sausage), milk (cheese and yoghurt), grains (beer and bread), fruit juice (wine) and vegetables (sauerkraut and pickles). In Africa and Eastern Asia many other types of food fermentation are applied. For a European, the most well-known of these products is soy sauce, which is produced by fermentation of soy, sometimes supplemented with rice. Table 6.1 lists the main types of fermented food in the Western world together with the main biochemical reactions employed in these fermentations. In most food fermentation the basis of fermentation control is inoculation and adjustment of the oxygen concentration and the water activity: 1) Inoculation with a microflora. In traditional fermentations the inoculum was a contamination from earlier production via the equipment or addition of some product that had already been fermented. Some processes still rely on the spontaneous natural microflora. This method is now gradually replaced by the use of pure starter cultures, as the production becomes more industrial, since inoculation increases the control and reproducibility of the process. 2) Adjustment of the oxygen concentration. Ethanol fermentations are inhibited by oxygen and therefore require un-aerated conditions. Lactic acid bacteria are independent on the oxygen concentration, but since anaerobic metabolism of competing organisms is slower than aerobic metabolism, also lactic acid fermentation is favoured by anaerobic conditions. Acetic acid fermentations require oxygen. Also moulds, which are important producers of hydrolytic enzymes in some food fermentation, are obligately aerobic organisms. 3) Reduction of water activity. Several food fermentation processes are controlled by reduction of the water activity by addition of salt. This is the case in fermentation of meat, fish, vegetables and soy sauce (the lactic acid stage). The background to this is that lactic acid bacteria, which are active in these fermentations, are relatively resistant to reduced water activity and therefore are favoured in this environment. In sausage fermentation the salt is mixed with the minced meat and in the other cases the raw material is placed in a salt brine.
6. Fermented foods
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Table 6.1 Fermented foods, their raw materials and main biochemical reactions Raw material Meat Fish Milk Products Sausages Sour herring Cheese Main type of reaction Lactic fermentation Enzymatic hydrolysis and lactic fermentation Enzymatic hydrolysis and lactic fermentation and (sometimes mold) fermentation Lactic (thermophilic) fermentation Lactic (mesophilic) fermentation Lactic fermentation1) Lactic fermentation Lactic fermentation Ethanol fermentation Enzymatic hydrolysis and ethanol fermentation Soy sauce Fruits Wine Cider Vinegar Cocoa Coffee Olives Enzymatic hydrolysis by moulds, lactic and ethanol fermentation Ethanol (and malo-lactic) fermentation Ethanol fermentation Ethanol and acetic acid fermentation Ethanol and acetic acid fermentation Microbial pectin hydrolysis Lactic fermentation
Yoghurt Fermented milk Butter Vegetables Cereals Sauerkraut Pickles Bread Beer
1) In some countries the cream is fermented before the churning of butter to provide diacetyl as aroma compound.
6.1 Beer brewing Production of beer by ethanol fermentation of grains dates back to at least 4000 BC, when it was applied in Egypt. In the ancient beer production lactic acid fermentation probably played a role, and certain beer types are still produced with mixed cultures of yeast and lactic acid bacteria. Hops was introduced as an aroma compound and preservative during the 15th century. Around 1840 the lager type of beer was introduced in Bavaria, characterised by slow fermentation at low temperature (below 10 C) and maturation before bottleing.
6. Fermented foods
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The beer brewing process is outlined in Fig 6.1. It contains a large number of biochemical reactions. The raw materials of beer are malt, sometimes supplied with other grains called adjunct, hops and water. Yeast, either Saccharomyces cerevisiae or Saccharomyces uvarum, is added as a biocatalyst and sometimes also additional enzymes of microbial origin are added to improve the enzymatic reactions.
Fig 6.1 Summary of the beer production process.
Malting. The first stage of beer production is the malting of barley. The barley should be of low nitrogen type, as opposite to the fodder barley. The grains are first soaked in water in a steeping process during about two days to raise the water content to 45%, which initiates sprouting of the grains. The grain content of giberellic acid is important for the resulting germination. This germination involves respiration, and the grains must be aerated to provide oxygen and remove the carbon dioxide. Since the reaction is exothermic cooling must also be provided and the grains are mechanically turned to provide homogeneous conditions. During the malting process many of the barley enzymes are activated and start to
6. Fermented foods
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hydrolyse the grain: Hemicellulases, proteinases, - and - amylases. Roots also develop from the grain during the germination, which may take about 4-6 days to be completed. The germination and the emerging enzymatic reactions are interrupted by the kilning, in which the temperature is gradually raised to 65-85 C. During the kilning, the high temperature results in Maillard reactions between reducing sugars and amino-groups, that colour the malt, darker the higher the temperature is used. This is the main way of controlling the beer colour. Maillard reaction products also contribute to the taste of the malt and the beer. During the kilning the water content is reduced so the malt can be stored for later use. Thus, malt can be considered as a package of hydrolytic enzymes, notably - and -amylases, packed with the enzyme substrates, mainly starch. The last stage of the malting is the removal of the rootlets which, like most other by-products from the beer production, are used as fodder. Malt is not always produced by the brewer, but often obtained from specialised malting companies.
Table 6.2 Composition of barley grains before and after malting Compound % in barley % in malt Starch 64 59 Sugar 2.5 9 -glucans 9 7 Cellulose 5 5 Amino acids and peptides 0.5 1.5
Mashing. The malt is milled, coarsely to facilitate the later separation of the husk. The milled malt is mixed with hot water to extract starch and enzymes from the grains in the mashing process at about 65 C. Some brewers supply additional starchy materials, adjuncts, that are cheaper than malt, like maize, wheat or rice. Even sugar may be used. This also reduces the protein concentration of the wort, which may be an advantage if the malt is too protein rich, since proteins may cause problems with precipitations in the beer. On the other hand, the use of starchy adjuncts requires higher enzyme activity in the malt. Starch is composed of amylose, that is a straight chain of -1,4-linked polyglucose, and amylopectin which besides -1,4 bonds also contains branching points with -1,6 bindings (Fig 6.2). During starch hydrolysis -amylase randomly hydrolyses -1,4 bonds between the glucose units in the starch, which results in smaller poly-glucose molecules called dextrins. Thus, hydrolysis by -
6. Fermented foods
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amylase gradually reduces the mean molecular weight and the viscosity of the starch solution but little fermentable sugar is produced in this reaction.
Fig 6.2. Hydrolysis of amylopectin to dextrins, maltotriose and maltose by amylase and -amylase. Both enzymes hydrolyse at the -1,4 site leaving the branching -1,6 sites in low molecular weight dextrins. Oligosaccharides larger than maltotriose are not fermented by the yeast.
The -amylase hydrolyses -1,4 bindings two glucose units from the nonreducing terminal of amylopectin, amylose or dextrin to produce the disaccharide maltose, which is the main fermentable sugar in the wort (Table 6.4). Thus, the longer the mashing continues the higher becomes the concentration of fermentable sugar. However, these enzymes can not hydrolyse the branching points (-1,6 bonds) of the amylopectin and therefore small branched dextrins are left. These dextrins are not fermentable and they remain in the beer and contribute to sweetness and viscosity of the product. Additional enzymes like proteases or -glucanases, may also be added to improve the proteolysis or the -glucan hydrolysis. Pullulanase, a debranching enzyme that hydrolyses -1,6 bonds in the amylopectin, may also be used to increase the concentration of fermentable sugar from the starch.
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Table 6.3 Temperature and pH optima of the main malt enzymes Enzyme pH Temperature -amylase 5.7 70 -amylase -glucanase proteinase 5.5 5.1 4-5 60 57 40-50
These enzymes have different temperature optima (Table 6.3). During the mashing different temperature programmes can therefore be used to control the hydrolysis of the macromolecules. The proteolysis should furnish the wort with amino acids for the growth of the yeast during the fermentation but it should also degrade proteins that would otherwise precipitate in the beer. Likewise, the glucanolysis is important to reduce later precipitations and it yields oligosaccharides. The main reaction during mashing is the degradation of starch to fermentable sugars and non-fermentable dextrins. A typical composition of the wort is shown in Table 6.4
Table 6. 4. Components of starch hydrolysis in wort. Product % of total starch Maltose 51 Maltotriose 12 Glucose 9 Fermentable Fructose 2 Sucrose 2 Maltotetrose 3 Non-fermentable Dextrins 21
The enzymatic hydrolysis is interrupted by boiling of the wort for 1-2 hours. pH has then dropped from 5.8 to 5.4. Before this, the husks and precipitated proteins are removed from the wort and hops are added. It is the dried non-fertilized female flower of Humulus lupulus that is used. Today also pelleted hops and even hops extract is used by the brewer. During the subsequent wort boiling, aromatic compounds are extracted from the hops, some unwanted aroma compounds are evaporated, all enzymatic activity ceases and the wort becomes essentially sterile. Hops contain two main types of flavour compounds: humulones (the so called alpha acids) and lupulones (called beta acids).
Fig 6.3 Basic structure of the humulones of hops.
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The molecules isomerise during the wort boiling which makes them more water soluble and more bitter. Negatively charged tannins are also extracted from the hops and they form precipitate with proteins. After the wort boiling the hops residuals are separated off together with the precipitated proteins and used as fodder. The so clarified wort is cooled and inoculated with yeast. Fermentation. The fermentation process is performed in a batch according to either of two principles. In top fermentation Saccharomyces cerevisiae is used. This yeast flotates to the top when the fermentation has ceased due to lack of fermentable sugar. The bottom fermentation processes utilise Saccharomyces uvarum (carlsbergensis) which sediments to the bottom after the fermentation. Bottom fermentation is typical for lager beer and pilsner and it is performed at low temperature: 5-10 C for about one to two weeks, until all visible fermentation has ceased. Top fermentations is applied to produce the beers of ale, stout and porter type and this fermentation is made at higher temperature, around 20C, which results in more ester production.
N*10-6/mL
60
E (%)
5
EtAc (mg/L)
50
N E
EtAc
50
100
150
Time (hrs) Fig 6.4 Progress of a lager beer fermentation at 10C. N = yeast cell number; E = ethanol concentration; EtAc = concentration of ethyl acetate.
During the fermentation, the yeast biomass concentration increases about four times (Fig 4.4). Cells separated from the beer after fermentation are partly used to inoculate next batch and partly used as fodder. To permit growth of the yeast during the conditions in the wort, oxygen must be available for synthesis of cell membrane constituents. Therefore the wort is saturated with oxygen from air before inoculation. This oxygen is quickly consumed by the cells and then the process is strictly anaerobic. From this time in the process much effort is focused on keeping the beer free from oxygen since the shelf-life is strongly reduced by
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oxidations in the beer. All fermentable carbohydrates (Table 6.4) are converted during the fermentation to biomass carbon dioxide, ethanol and other organic compounds that contribute to the taste. Since the yield coefficient for ethanol from maltose is about 0.5 g/g, the final alcohol concentration can be predicted from the concentration of wort used to start the fermentation. However, it depends also on the extent of the starch hydrolysis to fermentable sugars. To make a low-caloric beer there is only one way: reduce the wort concentration, since most of the energy of the sugar is preserved in the ethanol. Depending on the extent of starch hydrolysis, the low caloric beer can either be a low alcohol beer with a normal alcohol to dextrin ratio or a low dextrin beer with normal alcohol content. Ethanol is a major contributer to the taste of beer, but minor quantities of organic acids, higher alcohols, esters and other aroma compounds are also produced and make important contributions to the taste of the beer. However, also less pleasant compounds are produced and for this reason a post-fermentation process is included. One of these unwanted compounds is diacetyl. It is not produced directly by the yeast cells, but -acetolactate is secreted by the cells during the later phase of the primary fermentation (see the ethyl acetate curve in Fig 6.4) and then spontaneously decarboxylated to diacetyl. The main fermentation results in a "green" beer which must be matured in a postfermentation process at 0 - 10 C before use. Lager beer is generally matured for a longer period, 2 weeks to 2 months at a temperature close to 0 C, while ale is stored at higher temperature for a much shorter period of time. Many less characterised reactions takes place during the post- fermentation. One of the products from the main fermentation, -acetolactic acid, spontaneously decarboxylates to diacetyl, which is considered unpleasant in beer. However, during the late stage of the fermentation, and further during the post fermentation, this diacetyl is resorbed by the remaining yeast cells, and the concentration of remaining diacetyl is sometimes used as a measure of the post-fermentation progress. A problem in this process is that it is the decarboxylation of the acetolactate that is the rate limiting step. New technology has been developed to achieve the postfermentation by means of an accelerated decarboxylation induced by continuous heat treatment in a heat exchanger followed by diacetyl removal by immobilised yeast in a packed bed column. In this way, the post fermentation reactions can be accomplished with about 2 hours residence time during which almost all diacetyl is resorbed by the cells. After the post-fermentation the beer is clarified by centrifugation or filtration. To reduce effects of microbial infections, the beer is often pasteurised or sometimes sterile filtered. It is mainly other yeasts and lactic acid bacteria that can interfere with beer during storage, due to the low pH, the alcohol content and the high partial pressure of carbon dioxide. As long as these infections can be avoided the
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shelf life of some 3-6 months is mainly limited by oxidation reactions. To reduce these reactions ascorbic acid is commonly added as an anti-oxidant in beer. 6.2 Fermented milk products Lactic acid bacteria is a group of species that are characterised by fermentation of sugar to lactic acid. The group is divided into two categories, homofermentative and heterofermentative lactic acid bacteria, depending on whether the metabolism yields mainly lactic acid (homofermentative) or also considerable amounts of acetic acid, ethanol and carbon dioxide is formed (heterofermentative). This classification is not strict, since cultivation conditions may influence the product pattern. Table 6.5 lists typical representatives of lactic acid bacteria in these groups.
Table 6.5 Lactic acid bacteria classification according to the product pattern Homofermentative Heterofermentative Lactococcus spp . (all) Leuconostoc spp .(all) Pediococcus spp. (all) Lactobacillus spp. (some) Lactobacillus spp . (some)
The lactic acid bacteria play an important role in fermentation of food. Table 6.1 shows that they are involved in fermentation of milk, meat, fish and vegetables. In these cases the lactic acid fermentation plays an important role to stabilise the product against microbial spoilage. The mechanism of this food preservation effect is not at all generally known. It is well known, however, that many lactic acid bacteria, when grown in mixed culture in the laboratory, are very competitive. This competitiveness has been ascribed a number of factors like production of inhibitors and resistance against low pH and low water activity (aw) as depicted in Table 6.6.
Table 6.6 Competition advantages associated with lactic acid bacteria Antagonistic products Lactic acid Acetic acid Hydrogen peroxide Antibiotics, e.g. nisin and reuterin Properties of the bacteria Ubiquitous on food raw materials (inoculum) Oxygen indifferent Relatively fast growing Tolerant to carbon dioxide Tolerant to low pH Tolerant to low aw
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6.2.1 Fermented milk and yoghurt. Fermentation of milk with lactic acid bacteria is probably the oldest method to preserve milk. It is widely used all over the world, probably because it has been the safest way to consume milk. Milk that is not quickly fermented with lactic acid bacteria soon becomes infected with a number of potentially pathogenic bacteria. Only lately has it become possible to store non-fermented milk safely for several days in refrigerators. Milk is fermented with lactic acid bacteria in many different ways in different countries. Here only two types of fermentation will be considered: a mesophilic fermentation employing a mixture of Lactococcus spp. and yogurt, that is a thermophilic fermentation employing Lactobacillus spp. as well. These two types are summarised in Table 6.7 and Fig 6.4.
Note that the Lactococcus genus in older literature is called Streptococcus. Only the so called lactic streptococci are re-named Lactococcus. Streptococcus of the enteric, viridans and pyogenes types are still classified in the Streptococcus genus.
The mesophilic fermentation employs two types of Lactococcus spp.; the acidifiers Lactococcus lactis and Lactococcus cremoris, which are homofermentative and have the task to quickly reduce pH and produce lactic acid, and the heterofermentative aroma bacteria Lactococcus diacetilactis and Leuconostoc cremoris, which are slow fermenters but produce diacetyl, which is a desired aroma contributor in dairy products. The species mentioned in Table 6.7 are used by Swedish dairies, but many variants of this concept may be utilised. The American fermented buttermilk, Swedish filmjlk, Danish ymer and Finnish villi belong to this category . Villi is, however, also inoculated with a surface growing mould, Geotrichium candidum, that contributes to the flavour and the surface crust. Lactobacillus spp. are generally slower to initiate the lactic acid fermentation, but they are more resistant to low pH. Reduction of pH inhibits the glycolysis in all starter organisms but Lactococcus spp stop the fermentation at about pH 4.5, while the Lactobacillus fermentation continues to pH 3.9. Thus, pH in the Lactococcus fermented milk is higher than in yogurt.
Table 6.7 Examples of two starter cultures for fermentation of milk Mesophilic (20C) Thermophilic (44C) "Filmjlk" Yogurt Lactococcus lactis Lactococcus cremoris Lactococcus diacetilactis Leuconostoc cremoris Lactococcus thermophilus Lactobacillus bulgaricus
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Another difference between the two types of fermented milk is the consumption of lactose. The starter culture is inoculated to a concentration of about 106-107 cells/ml which grow to about 108-109 cells/ml. For this purpose lactose is used as the energy source. The organisms of the yogurt starter culture do hydrolyse lactose to glucose and galactose, but only glucose is consumed leaving the galactose. Since the total biomass produced is similar or even higher in yoghurt, the result is that yoghurt has lower concentration of lactose than the common mesophilically fermented milk (Fig 6.5).This may be of significance in many parts of the world, since adults generally do not accept too much lactose. The so called lactose intolerance among adults, expressed as abdominal pains and diarrhoea because of inability to hydrolyse the lactose in the intestines, is unevenly distributed over the world. Generally, North Europeans and the white population in America have a large tolerance to lactose while Asians and Africans generally have very low lactose tolerance. Many alternative species of lactic acid bacteria are used for fermentation of milk, sometimes with the claim to give a more healthy product. The basis of these properties would be that the cells colonise the intestine. Examples of such starter organisms are Lactobacillus acidophilus, which grow very slowly compared to other starter bacteria, and Bifidobacterium spp., which is frequently isolated from the gastrointestinal tract. Other fermented milk types, like kefir and koumiss contain yeast species, e.g. Candida spp and Saccharomyces spp , which contribute to the flavour by production of alcohols and esters in very small quantities.
Fig 6.5 Schematic presentation of the lactose consumption in a fast thermophilic yoghurt fermentation and mesophilic 'filmjlk' fermentation with a Lactococcus based starter culture.
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6.2.2 Cheese. Like beer production, manufacturing of cheese is a combination of enzymatic and microbial processes and the origin of the product dates back to prehistoric times. The main steps of hard cheese production is outlined in Table 6.8. However, the variety of cheeses available on the market is reflected by a large number of process variations. Only some common features and examples from two main types of hard cheeses and the mould fermented cheeses will be treated. The milk selected for cheese production is pasteurised (with some exceptions) at for instance 72C for 15 seconds. It is extremely important that it is antibiotic free, since the starter cultures used are very sensitive to antibiotics. Especially penicillin, which is often used to treat mastitis, may accidentally be present in the milk. Lactic acid bacteria are extremely sensitive to penicillin. Antibiotics in the milk may delay the lactic fermentation and it gives the opportunity for Clostridium spores to germinate. Especially Cl. tyrobutyricum is a problem and a spore concentration below 10 spores per 100 ml milk is required. Clostridial growth in cheese may cause excessive gas production, butyric acid off-flavour and even health hazards. Thus, special quick-test kits have been developed to analyse the presence of antibiotics in the milk before cheese production.
Table 6.8 Main stages of cheese production Action Main purpose Pasteurisation Inactivate pathogenic and competing organisms Fermentation Reduce pH. Produce lactic acid Produce cells for later function Hydrolyse and precipitate casein to a curd Formation of the cheese
Addition of rennet Cutting and pressing of curd, whey separation Storing
Maturation of the cheese
Cow's milk contains about 87% water. The main ingredients of the dry matter are shown in Table 6.9. Cheese is composed mainly of the caseins, except for part of the -casein that is removed by enzymatic hydrolysis, the fat and part of the salts.
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Table 6.9. Main ingredients of cow's milk Component Concentration (%) Water 87 Lactose 5 Fat 3.8 Protein 3.4 Caseins 2.8 1.7 0.6 0.1 0.4 Whey proteins 0.6 albumins globulins Salts 0.9 CalciumCitrates-
The milk is inoculated with starter cultures that have much concordance with those used to produce fermented milk. Two main types may be distinguished for hard cheese production: The Emmentaler and Gruyre type of cheese is based on thermophilic Lactobacillus and Propionibacterium mixture while the Cheddar and Gouda type is based on a mesophilic Lactococcus mixture (Table 6.10). the purpose of the fermentation is to initiate the casein precipitation by reduction of pH and to provide cells which are entrapped in the precipitated curd to take part of the later maturation process. Also the soft cheeses like Camembert, Brie, Roquefort, Stilton and Gorgonzola are started with Lactococcus mixtures but they are also inoculated with a mould species before the maturation and the action of these organisms takes place during the maturation(Table 6.11). Since moulds are obligate aerobes, they grow only on the surface, unless the cheese is perforated by holes.
Table 6.10 Examples of starter cultures for cheese production Cheddar / Gouda Emmentaler / Gruyre Lactococcus cremoris Lactococcus. thermophilus Lactococcus lactis Lactobacillus helveticus Lactococcus diacetylactis Lactobacillus lactis Leuconostoc spp. Lactobacillus bulgaricus Propionibacterium shermanii
The declining pH during the fermentation contributes to precipitation of casein at its isoelectric point 4.6, as in the case of milk fermentation. However, proteases
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are also added to the milk during cheese manufacturing, and these enzymes contribute to an efficient precipitation of the main part of the casein. The major protease preparation is calf-rennet, which is an enzyme extract from young calves. The proteases of rennet are mainly chymosin (rennin) and pepsin. When the calf grows older the proportion of pepsin increases, which makes the extract less useful for cheese production, since pepsin hydrolysis is too extensive which reduces the curd yield. A relative lack of calf rennet has provoked the development of microbial proteases for cheese production. Such microbial rennet is in extensive use in some countries. Calf chymosin has been cloned to a yeast, Kluyveromyces sp., to produce chymosin in bioreactors. The process has been scaled up and introduced on the market.
Table 6.11 Mould species used for maturation cheeses Cheese type Example Mold specie (example) White moulded cheeses Camembert Penicillium camemberti Brie Blue-vein cheeses Roquefort Gorgonzola Stilton Penicillium roqueforti
The casein is present in milk as colloidal micelles of very complex structure as illustrated in Fig 6.6. The micelles with a diameter around 100 nm, are composed of submicelles. The inner part of the submicelle is composed of - and - caseins which interact by their hydrophobic parts and via Ca2+ ions also between their hydrophilic parts. The stabilisation of the submicelle is achieved by a surface layer of -casein which is divided into a very hydrophilic part, turned outside, and a hydrophobic part turned inwards the submicelle. The main action of the rennet enzymes is a selective cleavage in the region between the hydrophobic and hydrophilic parts of -casein. This removes the hydrophilic surface layer from the micelle which, by hydrophobic interactions, start to aggregate and precipitate as the cheese curd. Thus, the hydrophilic parts of the -casein make up the whey proteins together with the globulins and the albumins. The whey also contains the lactic acid and most of the remaining lactose. It is important that the cells and some of the rennin enzymes and a little lactose are entrapped in the curd, since they form the basis for the maturation process.
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Fig 6.6 Schematic illustration of the composition of a casein submicelle in milk. The casein molecules have characteristic hydrophobic (dotted) and hydrophilic (white) regions. - casein forms chains which are interlinked by hydrophobic interaction. -casein binds to the hydrophobic areas of this chain and Ca2+ ions stabilises the complex by ionic bindings between the hydrophilic parts. Finally, the submicelle increases its hydrophilicity of the surface by attracting -casein units which bind their hydrophobic ends inwards against the hydrophobic sites. Chymosin and pepsin act by specific hydrolysis in the region between hydrophilic and hydrophobic parts of -casein, thus exposing a hydrophobic surface. The degraded micelles start to interact by hydrophobic binding and precipitate as a cheese curd.
The precipitated curd is cut in pieces, separated from the whey, washed and pressed etc., according to different procedures for the different types of cheeses. During the subsequent storing for some months, a large number of biochemical reactions takes place to give the product its special texture and taste. First the starter culture cells resume a slow growth, since the pH, that had declined to stop the glycolysis during the initial fermentation, is increased after removal of most of the lactic acid with the whey. During this stage heterofermentative lactic acid bacteria or propionic bacteria produce gas that is entrapped in the cheese to give the characteristic holes. Heterofermentative lactic acid metabolism also results in diacetyl formation which is important for the flavour, as is the lactic acid and in some cases propionic acid and other microbial products of the primary metabolism. Eventually the microorganisms die and lyse, thus releasing proteases and lipases. These enzymes, together with the traces of the rennet proteases and the low activity extracellulary cellbound proteases of the lactic acid bacteria induce a very slow proteolysis and lipolysis that produce peptides and fatty acids to contribute to the flavour. Furthermore, in mould inoculated cheeses, extracellular proteases and lipases gradually diffuse from the mycelium to slowly soften and mature the cheese. Moulds also produce lipoxydases, enzymes that catalyse oxidative degradation of fatty acids which results in methylketones. Among these
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degradation product are 2-heptanone and 2-nonanone considered to be especially important for the cheese flavour. 6.3 Fermented meat products In Europe, fermented meat is mainly found in some types of sausage, like the salami and many other of the hard, often smoked sausages. Meat is normally contaminated by an aerobic psychrotrophic flora dominated by Pseudomonas spp. that normally spoil the meat by growth on the surface. When meat is minced this flora is mixed into the product and it is furnished by a surplus of nutrients from the damaged meat cells. Thus, minced meat is extremely sensitive to microbial spoilage. However, since long time ago, Man learnt that if the minced meat was salted and stuffed in a gut it did not develop the unpleasant odour but stabilised and could be used as food for very long time. This is still the basic procedure in production of fermented sausages. There are several mechanisms that stabilise the meat in a fermented sausage: Addition of salt reduces the water activity to prevent the Pseudomonas spp. to develop. These organisms are especially sensitive to reduced water activity, while lactic acid bacteria are especially tolerant in this respect. It is also essential that lactic acid bacteria are present in the mixture so that lactic acid is quickly produced and pH declines. This prevents organisms of the Enterobacteriaceae family, Clostridium spp. and Bacillus spp., which are normally present at low concentrations, to develop. Traditional formulations often included garlic or spices with antimicrobial compounds that further increased the stability. To increase the safety with respect to the dangerous Clostridium botulinum , also nitrite is added nowadays, since the undissociated acid, HNO2, is known to be very efficient in preventing endospore germination. Actually, the name botulinum comes from Latin botulus = sausage, since botulism was formerly often associated with infected sausages. The package of the sausage, originally guts from animals but nowadays often synthetic materials, also protects the meat from infection during the storage. It is quite common that the surface becomes covered with certain species of mould during the storage. This growth of moulds is even utilised for the processing in certain case, like the production of Salami. In the traditional procedures the inoculum was obtained either automatically from the not too clean vessels used to mince and mix the meat. Some formulations also contain milk or other sources of lactic acid bacteria. It is also common to mix old product into the fresh unfermented mixture to inoculate the meat. Today it has been common practice to use starter cultures to make the process more safe and reproducible. Lactobacillus plantarum, Pediococcus spp , Lactococcus spp. and in some cases even Micrococcus spp are used as starter cultures for fermentation
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of sausages. After the fermentation the sausages are often smoked, which further contributes to the preservation of the product. 6.4 Fermented vegetables Vegetables are not fermented to a large extent in Europe. Some common products are sauerkraut, that is fermented cabbage, pickles, that is a mixture of fermented vegetables and fermenter cucumber. The history of fermented vegetables is, however, probably as old as that of the other fermented foods. It is documented that large scale fermentation was applied to furnish the workers with food during construction of the Great Chinese Wall during the third century BC. The microbiology of fermented vegetables is not so well documented as that of beer, wine or cheese production. It is also just recently that starter cultures has been adopted. The common method is still to place the vegetables in a 3-6% salt brine and wait till the natural flora starts the fermentation. This takes some time since the lactic acid bacteria are present only at very low concentrations. Meanwhile, the main flora that may be Enterobacteriaceae members and Bacillus spp. are retarded by the salt and they gradually die. Typically, this fermentation starts with Leuconostoc mesenteroides and is followed by Lactobacillus brevis, that can ferment pentoses and Lactobacillus plantarum that is the main acid producer. Also Pediococcus cerevisiae is commonly found in fermented vegetables. After the main fermentation a slow post-fermentation by yeasts is common During the fermentation, that may take a couple of weeks, the low concentration of fermentable sugars is further reduced, which is part of the stabilisation of the product against other microorganisms. Lactic acid is also produced at concentrations determined by the available sugar concentration. 1-2% lactic acid is achievable. The product obtains special characteristics not only by the taste of the acid produced, but also by the effect of the low pH that makes the vegetable crispy. Another important function of the fermentation is that it may inactivate some of the plant enzymes, like pectinases, that otherwise would hydrolyse pectins to soften the product.

Food Microbiology

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Food Microbiology

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Food Microbiology

Sven-Olof Enfors KTH - Biotechnology Stockholm 2007

Gram-pos endospore formers: aerobic or facultatively anaerobic: obligate anaerobes:

Gram-pos, non-sporulating rods

Chapter 2 2.1 The microflora

The ecological basis of food spoilage

Archae Bacteria Fungi Algae Protozoa

2. The ecological basis of food spoilage

O2 NO3 SO42Electron acceptors Pyruvate

2. The ecological basis of food spoilage

2. The ecological basis of food spoilage

2. The ecological basis of food spoilage

Relative growth rate

Mesophiles Psychrotrophes Psychrophiles Thermophiles

2. The ecological basis of food spoilage

Rel Relative rate reaktionshastighet

2. The ecological basis of food spoilage

2. The ecological basis of food spoilage

2. The ecological basis of food spoilage

2. The ecological basis of food spoilage

NH3 + organic acid

2. The ecological basis of food spoilage

Amines Organic acids S-compounds Indol

2. The ecological basis of food spoilage

2. The ecological basis of food spoilage

Fig 2.7. Different types of rancidification reactions.

2. The ecological basis of food spoilage

2. The ecological basis of food spoilage

Lactic acid fermentation

Ethanol fermentation Acetaldehyde

Mixed acid fermentation

Propionate Propionic acid fermentation

Butandiol Butandiol fermentation

Butyric acid fermentation

2. The ecological basis of food spoilage

Salted Pickled Smoked Sterilised Pasteurised

Spoilage of different types of food

Spoilage of different types of food

Animal type Cow Swine Chicken Fish

Rigor mortis 10-20 h 4-8 h 2-4 h min-h (longer on ice)

final pH 6 - 5.5 6 6.4 - 6 6.8 - 6.4

Spoilage of different types of food

slime odour chicken

6.3 32 110 280

Distance from surface (mm)

Spoilage of different types of food

Spoilage of different types of food

Fig 3.5 Relative sensitivity of microorganisms to inhibition of growth by carbon dioxide.

Spoilage of different types of food

Spoilage of different types of food

Spoilage of different types of food

Spoilage of different types of food

Spoilage of different types of food

Spoilage of different types of food

Albumin: viscous, high pH (pH9,5), riboflavin + pyridoxin complexing

outer mucin layer 1-10m pores in shell inner keratin

Con-albumin: Fe2+ complexing Avidin: Biotin complexing Lysozyme: kills G+ bacteria

Spoilage of different types of food

AHL AHL AHL AHL AHL

Spoilage of different types of food

3. 3.4 Preserved foods

Spoilage of different types of food

Spoilage of different types of food