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MICROBES IN FOOD AND DAIRY PRODUCTION

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Bisen PS. Microbes in Practice, New Delhi, IK International , 2014 pp 1089-1157.

21

MICROBES IN FOOD AND DAIRY PRODUCTION

21.1 INTRODUCTION

The production of safe food, largely free from pathogenic and spoilage organisms, is

vitally important with the rapid changes in the pattern of food distribution now taking place in

highly industrialized countries. Cities are so large that the inhabitants are becoming increasingly

dependent on regular supplies of reasonably priced food, pre-packaged and available all the year

round. Such foodstuffs, marketed over a wide area, must be microbiologically safe. Only

companies with large capital resources and wide networking can afford the necessary amount of

research and quality control to produce food to these high standards. Further, in order to keep the

large food factories running economically, constant supply of animals, vegetables and fruits are

needed, of standard size and quality. This in turn has led to intensive farming practices with

extremely high standards of hygiene and scientific principles of nutrition with rigid

microbiological control at all stages. The same pattern will be repeated with fish, crustaceans,

and mollusks. Rising world population continue to pressure us to search and to make new foods,

either entirely microbiological in origin, or bland starch and protein, suitably modified in flavor

and nutrient status by the action of micro-organisms.

Food microbiologists are concerned with the level of spoilage organism in the raw

materials, often of animal and plant origin, the standard of hygiene during processing, the

efficiency of preservation and storage methods, as well as the incidence of organisms responsible

for food poisoning.

21.2 THE NATURAL MICRO FLORA

The natural micro flora of food or beverage can consist of three main components. i.e.,

those associated with the raw material, those acquired during processing, and those surviving

preservation and storage. They can be further subdivided into harmless organisms, producing

either desirable or undesirable flavor changes in the food, and pathogens forming dangerous

enterotoxins.

Micro-organisms spoiling the aroma, taste, color, or texture of the product may be

moulds, yeasts, or bacteria. The precise flora that is present at any stage will depend on the

nutrient status of a food, its temperature, pH, water content, etc., as well as on the nature of the

organisms themselves. Thus, many moulds flourish below pH 3.8 in the presence of air, even at

low levels of available water. Aerobic yeasts are similarly acid- tolerant and can spoil pickles,

dry wines, and ciders. Some yeast has special properties, thus can be found in many habitats with

Debaryomyces sp. are aerobic, salt-tolerant, and can utilize nitrite as a sole source of nitrogen;

commonly found on the cut surfaces of cured meats, low water activity, such as sea water, from

which it was initially isolated, cheese, wine, beer, fruit and soil as well as in high-sugar

products.. Fermenting yeasts can grow anaerobically at low pH and are thus associated with

spoiled fruit juices; osmophilic yeasts are contaminants of sugar; highly sugared or salted

products. Similarly, bacteria can be tolerant of heat (thermophilic), cold (psychrophilic), low pH

(acid-tolerant), salt (halophilic) etc. Even foods without very marked characteristics carry an

association of bacteria, e.g., fresh meat and fish have Pseudomonas and Achromobacter species

that give way to a micrococci/lactobacilli association when the flesh cured. Often the natural

flora is succeeded by a factory flora, as in cheese and cider making. Many foods and beverage

are stable only because of the early development of particular bacteria reduce the pH by

producing lactic acid and thus inhibit the development of food pathogens. Again, foods are often

rendered unpalatable by spoilage organisms before pathogens have had time to develop in

sufficient numbers.

Food pathogens include salmonellae, Clostridium botulinum, C. Perfringens, and some of

the staphylococci and streptococci. All non-spore forming pathogens are killed by pasteurization

of the food, but preformed enterotoxins are not destroyed by the temperature used in

pasteurization. Where the raw material contains pathogenic organisms, there is a danger of re-

infection of the pasteurized product. Staphylococci are more salt-tolerant than many spoilage

organisms, and this can give them an advantage in some preserved foods. Faecal streptococci

(Streptococcus faecalis and other group D streptococci) and coli forms, including Escherichia

coli, are common in foodstuffs; but in this situation are not reliable indicators of faecal

contamination, though they may be so in water supplies. These organisms grow readily on food

residues in badly maintained processing lines. Thus their numbers in food are generally

considered giving an indication of the degree of factory hygiene. They are normally absent from

liquids pasteurized and processed in a closed system. Massive numbers (106 to 107/g) in other

foods would be regarded as a potential hazard to health, since, in such circumstances, they have

been suspected of causing food poisoning. The general food poisoning bacteria grow optimally at

37º C and little or no multiplication occurs under refrigeration, a technique widely used to

restrict bacterial multiplication.

21.3 METHODS OF FOOD PRESERVATION

Any product, whether solid or liquid, can be sterilized if heated long enough and/or

treated with a suitable concentration of germicide. Usually the treatment with substrates of

neutral pH is so drastic that accompanying changes in the flavour, texture, and colour render the

product unacceptable. Techniques are designed to inhibit the multiplication of the spoilage flora

for such type of product prior to consumption of the food. The treatments available are

numerous, the choice depending not only on the nature of micro flora, but also on the chemical

and enzymatic constitution of the product (Figure 21.1).

21.3.1 Chemical Inhibition

The use of herbs and spices in the middle Ages moderate or mask the effects of food spoilage is

well known. Essential oils e.g. clove has been shown to have only mild anti-bacterial properties,

so that they are now used only as flavoring agents. The details of the methods of ancient and

modern food preservation are shown in Figure 21.1.

21.3.2 pH

The range of pH over which an organism grows is defined by three cardinal points: the minimum

pH, below which the organism cannot grow, the maximum pH, above which the organism cannot

grow, and the optimum pH, at which the organism grows the best. Microorganisms which grow

at an optimum pH well below neutrality (7.0) are called acidophiles. Those which grow best at

neutral pH are called neutrophiles and those that grow best under alkaline conditions are called

alkalophiles. In general, bacteria grow faster in the pH range of 6.0-8.0, yeasts 4.5-6.5 and

filamentous fungi 3.5-6.8, with the exception of lactobacilli and acetic acid bacteria with optima

between pH 5.0 and 6.0 (Table 21.1).

Lowering the pH is an obvious method of controlling micro-organisms by chemical means.

Lactic acid is preferred in many products, not only for its lack of distinctive flavor, but also

because of its very flat preservation. This allows considerable pH change without unpleasant

increase in acid taste. Both addition of acid and encouraging lactic acid bacteria are used in

practice. Growth of food pathogens is rare below pH 5.5; lactic acid bacteria grow readily down

to pH 3.8 but only very slowly down to pH 3.0. Several mould and yeast will continue to grow at

pH 2.3. Lowering the pH also improves the anti-microbial action of preservatives which are

effective in the form of the undissociated molecule. Acetic acid has a similar function where its

flavor is acceptable or traditional. The approximate pH ranges of some common food

commodities are shown in Table 21.2.

21.3.3 Water Activity ( aW

) Water is often the major constituent in foods. Even relatively ‘dry’ foods like bread and

cheese usually contain more than 35% water. The state of water in a food can be most usefully

described in terms of water activity.

Water activity of a food is the ratio between the vapour pressure of the food, when in a

completely undisturbed balance with the surrounding air, and the vapour pressure of pure water

under identical conditions. Water activity, in practice, is measured as Equilibrium Relative

Humidity (ERH) and is given by the formula:

Water Activity (aW

) = ERH / 100 Water activity is an important property that can be used to predict food safety, stability and

quality. The various applications of water activity includes; maintaining the chemical stability of

foods, minimizing non enzymatic browning reactions and spontaneous autocatalytic lipid

oxidation reactions, prolonging the desired activity of enzymes and vitamins in foods, optimizing

the physical properties of foods such as texture.

Water activity scale extends from 0 (bone dry) to 1.00 (pure water). But most foods have

a water activity in the range of 0.2 for very dry foods to 0.99 for moist fresh foods. Based on

regulations, if a food has a water activity value of 0.85 or below, it is generally considered as

non-hazardous. This is because below a water activity of 0.91, most bacteria including the

pathogens such as Clostridium botulinum cannot grow. But an exception is Staphylococcus

aureus which can be inhibited by water activity value of 0.91 under anaerobic conditions but

under aerobic conditions, it requires a minimum water activity value of 0.86. Most molds and

yeasts can grow at a minimum water activity value of 0.80. Thus a dry food like bread is

generally spoiled by molds and not bacteria. In general, the water activity requirement of

microorganisms decreases in the following order: Bacteria > Yeast > Mold. Below 0.60, no

microbiological growth is possible. Thus the dried foods like milk powder, cookies, biscuits etc

are more shelf stable and safe as compared to moist or semi-moist foods.

Factors that affect water activity requirements of microorganisms include the following-

kind of solute added, nutritive value of culture medium, temperature, oxygen supply, pH,

inhibitors. Each microorganism has a minimal water activity for growth as shown in Table 21.3.

Water activity of some foods and susceptibility to spoilage by microorganisms is shown in Table

21.4.

Water acts as an essential solvent that is needed for most biochemical reactions by the

microorganisms. Water activity of the foods can be reduced by several methods: by the addition

of solutes or hydrophilic colloids, cooking, drying and dehydration: (e.g., egg powder, pasta), or

by concentration (e.g. condensed milk) which restrict microbial growth so as to make the food

microbiologically stable and safe. A wide variety of foods are preserved by restricting their

water activity. These include:

21.3.3.1 DRIED OR LOW MOISTURE FOODS

These contain less than 25% moisture and have a final water activity between 0.0 and 0.60. e.g.,

dried egg powder, milk powder, crackers, and cereals. These products are stored at room

temperature without any secondary method of preservation. These are shelf stable and do not

spoil as long as moisture content is kept low.

21.3.3.2 INTERMEDIATE MOISTURE FOODS

These foods contain between 15% and 50% moisture content and have a water activity between

0.60 and 0.85. These foods normally require added protection by secondary methods such as

pasteurization, pH control, refrigeration, preservatives, but they can also be stored at room

temperature. These include dried fruits, cakes, pastries, fruit cake, jams, syrups and some

fermented sausages. These products are usually spoiled by surface mold growth.

21.3.3.3 CHEMICAL PRESERVATIVES

Organisms can also be inhibited specifically by the addition of small quantities of a chemical

preservative. The ideal properties of an antimicrobial food preservative are summarized below:

1. It is preferable that the preservative should kill rather than inhibit the microorganisms.

Having killed them it should itself decompose into innocuous products.

2. A bacteriostatic preservative would be equally satisfactory if it is destroyed only during

final cooking, but if it is to be used in conjunction with thermal control processes it

would need to have adequate heat resistance.

3. The range of specificity should correspond with the range of micro-organisms able to

develop in the food, i.e., it must inhibit both food poisoning and spoilage organisms.

4. Any preservative used as a supplement to thermal processing should give similar

protection against Clostridium botulinum as that given by the standard thermal processing

alone,i.e., a reduction in viable spores by a factor of 1012.

5. A preservative should not be inactivated or removed by chemical reaction with the food,

by some specific inhibitor in the food, or by-products of microbial metabolism.

6. The preservative should not stimulate the development of resistant strains, and should be

avoided totally if the same is also used therapeutically or as an additive to animal feeds.

7. There should be a chemical/analytical method for analyzing the effective portion of the

preservative.

No known antibacterial food preservative has all these properties, and there is a dearth of

suitable compounds active in the pH range 5.0 to 7.0, a range which is important especially for

wet foodstuffs of high nutritive value such as fish meat, and milk.

Food preservatives can be divided into two groups on the basis of their mode of action. In

the first, which includes acids, esters, and phenols, the compound is absorbed by the solid

components of the bacterial cell and, if of high lipid solubility, is concentrated on the cell

membrane and various cell structures. The metabolic effects of salicylic acid, for example, are

related to its influence on ATP formation in the mitochondria. The action of such preservatives

become increasingly less effective with an increase in the lipid and solid content of the food to

which it is added. The antimicrobial effect of the second group, quinines and nitrofurans,

depends primarily on their ability to penetrate cell membranes. Quinones are able to penetrate

any type of cell, whereas nitrofurans have a selective action on bacteria which they can

penetrate, but are unable to penetrate yeasts. Once inside the cell, excessive amounts of

coenzyme are required to produce the corresponding hydroquinones and aminofuran compounds,

ultimately disrupting electron transport in the cell. The role of preservatives in the prevention of

spore germination is also important. No known preservative will prevent germination of Bacillus

cereus spores, but at minimum inhibitory concentrations nisin, subtilin, diethyl pyrocarbonate,

and sodium nitrite prevent growth immediately after germination. Spores that shed their spore

wall are prevented from elongating into vegetative cells by sodium benzoate, whereas tylosin,

sodium sorbate, sodium metabisulphite, and sodium chloride allow some increase in length but

prevent cross wall formation. At greater concentrations, all preservatives prevent any

development after germination. Nisin is used to prevent gas formation by clostridia in cheese, but

attempts to use it in other foodstuffs have not been entirely successful; part of its activity is lost

during heating or curing and on storage it is eventually lost completely so that any bacterial

spores present can then develop.

Knowledge of a range of organisms sensitive to a preservative is most important. Thus,

growth of yeasts and moulds follows suppression of bacteria by the use of tetracyclines.

Attempts to use tetracyclines for delaying spoilage in poultry carcasses led to the selective

growth of salmonellae, which may be relatively resistant to antibiotics.

Chemically, it is easier to conserve acid foodstuff and beverages. Sulphurous acid

(sulphur dioxide) controls the acid-tolerant bacteria of ciders and wines, but moulds and

fermenting yeasts, common to such environments, are highly resistant. Sorbic acid is used in

cheese wrapping to inhibit surface mould growth. It has some activity against yeasts and in

France it is permitted in sweet white wines, together with sulphur dioxide, which is antibacterial

and an antioxidant. Benzoic acid, used as a preservative in soft drinks, is effective against many

types of yeast, but Saccharomyces acidifacienscan metabolizes it to some extent in the presence

of sugar. Diethyl pyrocarbonate is active against yeasts, particularly in the presence of alcohol,

so that it would be more effective in wines than in freshly pressed fruit juices. All substances

suggested for use as antimicrobial food preservatives must be submitted to a statutory two year

evaluation programme that includes feeding to rat and dogs, before being permitted in foods and

beverages.

Chemical inhibition of growth can also be obtained with gases. Thus with the original

Boehi process non-sterile apple juice was impregnated with eight atmospheres pressures of

carbon dioxide and stored at ambient temperature. The process had to be modified, since

contaminating lactic acid bacteria (acid-tolerant and microaerophilic) grew and spoiled the

product. The juice is now concentrated to a third or fourth of its original volume, saturated with

the same percentage of carbon dioxide (0.8% wt) and held at 20C. There is a cheaper process in

France for the sterile storage of flash-pasteurized juice at ambient temperature under a blanket of

nitrogen gas. Stringent inspection is required since traces of oxygen would encourage the growth

of any mould and yeast contaminants; gas production by the latter would burst the tanks.

Smoking meat, fish, cheese etc. (normally in conjunction with salting), is an ancient

method of food preservation brought up to date. Essentially it is a vapour absorption process, the

solid particles playing only a minor role. The active components of the vapour include volatile

fatty acids which have both a bactericidal and a residual mild bacteriostatic effect. Usually

micrococci and staphylococci are inhibited, leaving a flora consisting mainly of lactic acid

bacteria.

21.3.3.4 DEHYDRATION AND USE OF CONCENTRATED SOLUTIONS

A wide range of foods is preserved by dehydration or the use of concentrated solutions of salt

or sugar. The lower the moisture content of a product, the less liable it is to support microbial

growth. A similar effect is obtained the higher the osmotic pressure of food or beverage, whether

this is due to added salt or sugar. Hence, the terms xerophile, halophile, and osmophile are used

to describe organisms likely to be found in extremes of such environments.

Moisture requirements of micro-organisms for survival or growth are expressed as water activity

(aw), i.e. equal to one-hundredth part of the corresponding relative humidity. Water activity can

also be related to osmotic pressure and absolute temperature.

Each species of micro-organism has its own characteristic optima and range of aw values..

Bacteria normally exist only between 0.995 to0.990 aw, although staphylococci can exist down to

0.86 aw, and a few halophiles down to as low as 0.75 (saturated sodium chloride solution). Yeasts

can withstand drier conditions than bacteria; ‘osmophilic’ species such as Zygosaccharomyces

barkeri (S. rouxii var. polymorphus) can exist at aw as low as those tolerated by most moulds.

However, some moulds can exist at lower aw values than any other micro-organisms, some

species tolerating an aw as low as 0.62. Reducing the available water below the optimum serves

merely to increase the lag phase and decrease the rate of growth; at aw values 0.65 to 0.62 mould

spoilage would be unlikely to become serious in less than one-and-a-half to two years.

It is possible to compare the susceptibility of all types of foods and beverages to microbial

growth by using the concept of available water. Thus the aw in frozen foods is given by the ratio

of the vapours pressure of ice to that of water at a temperature under consideration. At -50C, -

100C, and -150C the corresponding aw values are 0.9526, 0.9074, and 0.8642, so that the inability

of bacteria to grow on frozen food below -50C could be due to the limitations of unsuitable aw.

The ability of specific moulds to grow below this temperature is due to their tolerance both of

low temperature and low aw values. Some specific effects are also due to the toxic effect of ions;

for an osmophilic yeast these are in the order of toxicity K+ < Na+ < Mg++ < Ca++ Li+; < Cl- <

SO4--. It must not be forgotten that enzyme reactions can still continue even in a dry substrate at

0.35 aw albeit very slowly indeed and irrespective of whether water is a reactant (hydrolytic

enzymes) or not oxidizing enzymes).

21.3.4 Temperature

21.3.4.1 HIGH TEMPERATURE

In practice the terms ‘sterilization’ and ‘pasteurization’ are used to differentiate different

levels of heat treatment. The second usually implies that only some of the spoilage organisms or

food pathogens are destroyed. With both processes, it is first necessary to know the amount of

heat required to inactivate an organism or, if spore-forming, its spores. This will vary with the

chemical nature of the food e.g., its pH, water activity (aw), and the presence of inhibitors.

Secondly, heat penetration studies must be carried out on the food in its actual container during

the heating process. The rate of penetration is modified by the volume and shape of the

container, and the viscosity of the contents, presence of solid particles, etc. Thirdly, since the

death/time curve approaches zero only at infinity, it is never possible to kill every organism in

every container in every batch produced. Hence, some statistical limit must be agreed initially,

e.g., a reduction in the number of viable spores of Clostridium botulinum by a factor of 1012 in

the canning of certain foods, such as wet food at neutral pH.

Cases of botulism have been reported in the past, mainly in the USA, following the

consumption of vegetables, mushrooms, etc., canned or bottled in the home. The heat treatment

given was sufficient to destroy the spoilage that would normally keep Clostridium botulinum in

check, but left spores of the latter undamaged. If the subsequent storage temperature were

sufficiently high the spores grew and produced toxin, without altering the physical appearance of

the food. These infections have now disappeared where governmentally sponsored and tested

recipes are used in conjunction with domestic pressure cookers. With acid foods and beverages a

relatively mild heat treatment will give satisfactory results, since Clostridium botulinum will not

develop at low pH.

The amount of heat required to inactivate bacterial spores (units of lethal heat) is usually

calculated in terms of F values, or the number of minutes at 1210C. This assumes instantaneous

heating and cooling but, as this is impossible in practice, the equivalent heating effects occurring

during the heating and cooling processes must also be calculated from standard sets of tables as

graphs. The values would be inconveniently small if used for calculating heat requirements in

pasteurization, which is carried out below 100 0C. Instead, Pasteurization Unit (P.U.) is used; for

pickles the reference temperature is usually 82 0C, while 60 0C has been proposed for brewing. It

is often difficult to determine the effectiveness of sterilization or pasteurization, except by

incubating large numbers of samples. With clear liquids, membrane filtration of the contents of a

specified number of containers is a routine measure, while chemical determination of the

remaining amount of the enzyme phosphatase is a suitable test for milk and beer. For milk, there

is also the short Methylene Blue test carried out 24 hours after heating, to determine the amount

of post-pasteurization infection.

Normally, packaged goods other than acid or cured products are processed to at least the

Clostridium botulinum spores standard but difficulties are sometimes experienced with other

heat-resistant organisms, e.g., the spores of thermophilic bacteria. The problem is greater with

very viscous products of neutral pH, like packaged rice pudding, whose flavor and appearance

would be spoiled by the excessive heating needed to kill extreme thermophiles. Whether the

contents of the can remain, sound will depend on the minimum growth temperature of the

organisms remaining after heating and the temperature at which cans are stored. Canners now

have rigid standards for the numbers of thermophiles they will accept in sugar, starch, and other

additives. Staphylococcal food poisoning from consumption of commercially packaged

vegetables has now been eliminated following recognition that the source of the infection was

the operatives handling the newly processed cans. The cans are sterile when leaving the cooker,

but 2 to 3 % of cans with good commercial seams will leak temporarily during cooling. Hence,

infection liquid on the outside of the same could be drawn into the cans while it was cooling. The

amount of water entering is 300 x 10-4 per mL for large leaks and 8 to 30 x 10-4 per mL for

small. The packaging industries have adopted following measures to solve above problems (1)

rigid control of cooling water chlorination, (2) no mechanical damage to the can seam while it is

in motion, (3) keeping the wet runways between cooler and dryer to a minimum (surface count

on the runway must be < 500 organisms/4 sq. inch), (4) keeping any subsequent runways dry, (5)

rigid sterilization programme for the equipment, (6) no manual handling of wet cans. Typhoid

outbreaks, such as the one in Aberdeen in 1964, associated with imported packaged meats have

usually originated from the use of non-chlorinated river water, contaminated with sewage, for

cooling the cans after heating.

Considerable developments are now taking place in sterile canning. The product is flash-

heated in a heat exchanger to the required temperature, then filled into sterilized cans that are

sealed for the required time before cooling. Basically, the same process is used for the U.H.T.

process for milk (1380C for a few seconds) and the flash-pasteurization of carbonated beer or

cider under pressure prior to bottling

21.3.4.2 LOW TEMPERATURE

Food and beverages may be chilled to between 0 and 50C to delay spoilage while

awaiting sale or consumption, or to -180C for extended storage. Three main groups of organisms

are important in this respect in deep frozen foods i. food poisoning bacteria, ii. psychrophiles,

and iii. moulds. Psychrophiles are defined as organisms that grow well on solid media at O0C,

forming colonies visible to the naked eye in one week. It is important to note that this is not their

optimum temperature. Most psychrophilic bacteria are strains of the genus Pseudomonas, with

some from the genera Flavobacterium, Achromobacter, Alcaligenes, Escherichia, and

Aerobacter. They are very rare amongst Gram-positive bacteria. Psychrophilic yeasts usually

belong to the genera Candida and Rhodotorula. Vegetative forms of moulds are more sensitive to

cold than are spores. Spores of certain species of the genera Monilia, Chaetostylum,

Cladosporium, Aspergillus, Fusarium, Mucor, Thamnidium, and Botrytis, are particularly

resistant. Some of these are able to grow slowly at low levels of water activity and prevention of

growth therefore requires the addition of CO2 or exclusion of air from the package.

It is difficult to determine accurately the minimum temperatures at which organisms will

grow. The freezing of the medium alters the ability of the organism to grow, but the addition of

antifreezes of low osmotic pressure (e.g., glycerol) or super-cooling without crystallization are

beginning to show that some organisms can be induced to grow at -50 to -70C. Without these

techniques, minimum growth temperatures are normally determined by extrapolation of the

growth rate or the reciprocal of the lag period. The lag phase before growth occurs may be so

long that the results of the experiment depend more on its duration than on the experimental

conditions. With these provisos it is generally accepted that (1) the lowest temperature for

bacterial growth is -10°C, (2) growth of Staphylococcus aureus and Salmonella sp. has not been

reported at +6.7°C (Enterotoxin production by staphylococci is unknown below + 18°C), (3)

Clostridium botulinum (types A, B, C, and D) do not grow or produce toxin below +10°C, and

(4) Clostridium botulinum type E can grow and produce toxin at +3.30C after prolonged

incubation, but there is no recorded case of botulism arising from this type of activity at low

temperatures.

Frozen foods are not sterile and most spoilage is due to improper handling prior to

freezing. Freezing is not generally bactericidal unless it is carried out slowly and the produce

subsequently stored at a comparatively high temperature (0 to -100C). Fluctuating storage

temperatures within this range also reduce the bacterial load ; food pathogens are found to die

more rapidly between these limits (maximal at -20C) than they do below -170C. Hence the

commercial freezing temperature cannot be guaranteed to destroy pathogens, particularly with

modern freezing techniques using rapid freezing at -35°C followed by storage at 18°C. The

cryogenic technique of extremely rapid freezing, by spraying with liquId nitrogen, has hardly

any effect on bacterial numbers at all. Hence prevention of food poisoning from frozen foods

depends on the use of good quality foodstuffs, on maintaining impeccable hygienic standards

during processing and on the avoidance of contamination and multiplication of micro-organisms

in the thawed product. All vegetables and some meat and fish products are heat treated before

freezing. The cooling period between the two processes should be as short as possible to avoid

bacterial growth. Products containing uncooked meat, especially imported frozen boned meat

(readily contaminated during the boning procedure) are particularly troublesome. Salmonellae,

usually from infected animals, are liable to be present and even with some heat pre-treatment

there is a danger of cross-infection between raw and cooked meat. Similarly egg products are

likely to contain salmonellae and must be pasteurized at 65°C for 2·5 minutes and not re-infected

afterwards.

After a frozen food is thawed it does not then spoil any more rapidly than it would have

done had it not been frozen. But microbial control must be exercised during thawing; this is done

either in cold rooms or by using dielectric heating, the latter being too rapid for bacterial growth

to occur. Normally even if unheated frozen vegetables are thawed and stored above the

minimum growth temperature, the food is damaged by obvious taints from the harmless spoilage

flora rather than by clostridia. The latter organisms appear to be inhibited by these psychrophiles.

Similarly, food being de-frosted reaches the minimum temperature for growth of the

psychrophilic microflora before that of the toxigenic staphylococci. Danger would arise in

products containing fairly high concentrations of salt, since staphylococci may then grow faster

than less salt-tolerant spoilage bacteria. The ability of Salmonella typhimurium to grow in the

presence of a competing microflora appears to depend on the nature of the food. No frozen food

should contain salmonellae, irrespective of whether it will be cooked before being eaten or not.

This is perhaps an ideal situation, practicable only in pasteurized products and frozen vegetables.

Faecal indicators and enterococci rarely grow below 5°C but their presence in substantial

numbers in a food thawed above this temperature could falsely indicate insanitary processing

conditions.

Two industrial processes based on both freezing and heating have been developed. The

first is the dielectric defrosting of blocks of frozen meat, fish, and crustacea, which takes 20 to 30

minutes compared with 25 to 30 hours needed for cold room defrosting. The average temperature

after dielectric defrosting is still just below 0°C, consequently there is very little change in the

bacterial count. The second is freeze-drying, when a frozen food is dried by microwave heating

under vacuum at a temperature just below the incipient melting point. These products, however,

need the same care in their pre-freezing and re-constitution phases as do other frozen products

21.3.4.3 MISCELLANEOUS METHODS Clear liquids can be clarified and then filtered free of all micro-organisms. By this process

organisms are removed from fluids and may be separated from their soluble products of

metabolism, e.g. exotoxins. Filters are made from many materials including unglazed porcelain,

diatomaceous earth, asbestos fibers, sintered glass and synthetic membranes of cellulose nitrate

(collodion) and cellulose acetate. All types are available in a range of grades of different pore

size of particle which is to be removed. Membrane filters are now made in a wide range of pore

size including many much smaller than the smallest bacterium. Many millions of gallons of

wines, beers, ciders, clear fruit juices, soft drinks, sugar syrups, etc., are sterilized annually in

this manner. In contrast to these well-tried method, efforts have been made over the past several

years to use atomic or ionizing radiation for food preservation. Unfortunately off-odours and

taints tend to form at the dose levels needed to sterilize foods.

21.4 SPECIFIC FOODS AND BEVERAGES

The common microbiological spoilage defects that occur in different foods with some examples

are shown in Table 21.5 and are being discussed below in brief for different food.

21.4.1 Milk

Milk is synthesized by cells within the mammary gland and is virtually sterile when

secreted into the alveoli of the udder. Beyond this stage of milk production, bacterial

contamination can generally occur from three main sources; within the udder, outside the udder,

and from the surface of equipment used for milk handling and storage. Cow health, environment,

milking procedures and equipment sanitation can influence the level of microbial contamination

of raw milk. Equally important is the milk holding temperature and length of time milk is stored

before testing and processing that allow bacterial contaminants to multiply. All these factors will

influence the total bacteria count (SPC) and the types of bacteria present in raw bulk tank milk.

Milk drawn aseptically from the udder shows a predominance of staphylococci (the great

majority coagulase-negative) and diphtheroids (mainly heat sensitive corynebacteria), both

groups being part of the cow's normal skin flora. Some udders harbour mastitis streptococci,

coagulase-positive staphylococci have become the commonest cause of mastitis. However,

thermoduric organisms are uniformly absent from milk collected aseptically. Under usual

milking conditions further contamination occurs from milking utensils, the dust of the dairy, the

udder of the animals, and from the milking operatives. Of these the most important source is the

milking utensils which, if unsterilized, become coated with large numbers of bacteria and

contribute enormous numbers of organisms to the milk.

Not unnaturally, single farm samples show wide variations both qualitatively and

quantitatively, ranging from those in which udder organisms are predominant to those with a

denser and more complex flora. Counts of thermoduric bacteria (micrococci, corynebacteria,

aerobic spore-forming bacilli, and thermophilic strains of Streptococcus faecalis) above 102-10

3/

mL are indicative of heavy infection from contaminated equipment. Bulk samples, particularly

from non-refrigerated tanks, carry heavy contamination and give evidence of bacterial growth,

particularly of Streptococcus lactis and Streptococcus kefir. Holding the raw milk unchilled (10

to 20°C) allows an active growth of the two last-named species, coagulase-negative

staphylococci and Gram-negative rods (Alcaligenes viscolactis and fluorescent and non-

fluorescent pseudomonads). Finally, the flora is likely to be dominated by Streptococcus lactis,

resulting in souring owing to fermentation of lactose. Lower temperatures favour the growth of

Gram-negative rods which turn the milk alkaline and eventually putrid by their proteolytic

activity.

Under laboratory conditions, milk pasteurized by being held at 63°C for three minutes

succumbs to deterioration by Bacillus cereus (including Bacillus mycoides). In commercial

practice, whether flash (High Temperature Short Time - HTST) or in-bottle pasteurization

methods are used, Bacillus cereus again overgrows the slower growing coryneforms, but is itself

overtaken by Gram-negative rods derived from recontamination. These in turn are outgrown by

the milk-souring organisms, Streptococcus lactis and Streptococcus cremoris. Bacillus cereus

causes not only rapid sweet curdling but also ‘bitty' cream and 'ropiness'. 'Sterile' milk has long

been produced commerciaIly by heating the bottled milk above l00°C. It is now replaced by

Ultra-High Temperature treated (UHT) milk, filled aseptically into sterile bottles and Tetrapaks.

21.4.2 Butter

Butter is made from separated cream, which is usually soured, since butter made from soured

cream keeps better than that made from sweet. The souring of the cream is brought about by

organisms naturally present in the milk, or the milk may first be pasteurized and specific starters

then added. Organisms concerned in the making of butter include Streptococcus lactis and

Streptococcus cremoris, which ferment lactose and sour the cream, and two other capsulated

streptococcal-like organisms, Leuconostoc citrovorum and L. dextranicum. The latter organisms

attack citric acid, a by-product of lactose fermentation, to produce diacetyl which imparts a

buttery aroma to the cream. Many other identified strains are also present and add their particular

flavours to the butter.

The temperature at which the cream is soured is very important. Temperatures around

20°C are low enough to prevent growth of thermophilic spoilage organisms, which have

survived pasteurization, and yet are sufficiently high to allow growth of the desirable

streptococci. In butter-making the soured cream is churned. This causes the fat globules to

aggregate to form butter, leaving the buttermilk which can be drained off.

Bacteria are restricted to the moisture droplets throughout the butter. Washing of butter to

replace these droplets of buttermilk by water deprives bacteria of nutrients and limits their

growth, thus improving the keeping quality of the butter. 'Working' of butter to break the

moisture into smaller droplets also improves the keeping quality by limiting the available

nutrients within a droplet. This does not apply to moulds, which can penetrate the surrounding

wall of fat and move to other droplets.

21.4.3 Cheese Cheese is one of the most important milk products. The widely used Cheddar cheese has

a close texture, firm mellow body, and a mild nutty flavour. The raw milk is heated to 68°C for

15 seconds to destroy most vegetative organisms, including coagulase-positive staphylococci

(which have increased with the wider use of milking machines) as well as the udder-derived

coagulase-negative strains. It is important that the milk be pasteurized without delay to ensure

that any strains of Staphylococcus aureus which may be present would not have had time to

produce enterotoxins. The pasteurized milk is not sterile and will contain thermoduric organisms.

A starter is required to produce lactic acid in situ in the curd; this can be either Streptococcus

lactis, Streptococcus cremoris, or a mixture of these, with or without Leuconostoc cremoris, etc.

Great care must be taken not to build up specific bacteriophages in cheese rooms, particularly

where a single strain of bacterium is always employed. Single strains are used when waxy,

bland-flavoured cheese is favoured, whereas multi-strains commonly produce the more mellow,

nutty flavour. Antibiotics used to control mastitis cause problems in cheese making and

penicillinase-producing strains of coagulase-negative staphylococci have been used both to

remove the antibiotic and because their lipolytic action can contribute to the cheese flavour. The

starter rapidly gives a low redox potential, produces protein degradation product and a pH of 5·0,

all conditions essential to the development of the lactobacilli and unfavourable to other bacteria;

clostridia are inhibited by the antibiotic nisin produced by some strains of streptococci. If the

streptococci fail, so also do the lactobacilli and the cheese develops such faults as taints and gas

produced by coliforms and clostridia, and faulty colour by a variety of organisms. Toxigenic

staphylococci would also find the conditions favourable.

Renneting with calf rennet follows immediately after the starter which also produces the

optimum pH for casein decomposition and clotting. The coagulum is cut into small pieces to

release the whey, the degree of syneresis being dependent on the temperature and the rate of acid

production by the bacteria. Finally the curd is consolidated into blocks under light pressure until

they become plastic, when they are broken into pieces, sprinkled with salt and pressed into

moulds. The shaped blocks are first dipped in molten paraffin wax to exclude air and thus to

prevent external mould growth and weight loss, and then ripened at 13°C for quick process

cheese or 5.5°C for the old slow-ripening type. The optimum period of maturing varies from 2 to

14 months, according to the previous treatment and type of cheese required. During cheese

ripening the numbers of lipolytic organisms are low; the lactic streptococci used as starters are

soon inhibited while the numbers of lactobacilli increase progressively during the later stages of

ripening of all cheeses, especially the hard varieties. The main flora consists of

homofermentative types (Lactobacillus casei and Lactobacillus plantarum); Lactobacillus brevis

and Lactobacillus. lactis occur much less frequently. The total number of lactobacilli and

streptococci, both alive and dead, is extremely large and probably accounts for as much as 0·1%

of the weight of the cheese. In Camembert cheese, Streptococcus lactis is dominant during the

first phase of growth, Penicillium and Oidium in the second and L. casei in the third phase. Blue

cheeses are made from open textured cheese through the action of varieties of the mould

Penicillium roqueforti. The drying cheese coat supports the growth of moulds, yeasts (Torulopsis

sp.), streptococci, and lactobacilli. The microflora of the coat of Stilton cheese is characteristic of

the factory producing it. Clammy rind on Dutch cheese is caused by the growth of corynebacteria

that develop only at pH 5·3 and is favoured by the prior development of yeasts (such as

Debaryomyces, Candida, Trichosporon sp.) that decompose lactic acid and form ammonia from

proteins, thus altering the pH in favour of the corynebacteria. On the other hand, surface ripened

cheeses such as Liederkrantz, Trappist, Tilsiter, etc., that have a brown surface smear of aerobic

bacteria, have antibiotic activity due to Brevibacterium linens.

21.4.4 Fermented milks Many different strains of lactobacilli, either alone or in combination with lactose

fermenting yeasts and streptococci, are used to produce characteristic fermented beverages. In

Europe and the Middle East yoghurt is made from milk thickened by heating or the addition of

dried milk solids and fermented with Lactobacillus bulgaricus and Streptococcus thermophilus.

It has custard like consistency and is often flavoured with ground fruit or nuts. Kefir is a fizzy

beverage made from fermented mares' milk using 'grains' that contain yeasts, streptococci,

lactobacilli, and micrococci. The grains are strained off after their action is complete and added

to the next batch of milk. Koumiss, also made from mares' milk, is a greyish-white liquid with

uncurdled casein, often bottled and allowed to carbonate by the action of the micro-organisms. It

normally contains 1 - 2.5% v/v alcohol. Acidophilus milk has been a fashionable drink for

several decades since it was thought that Lactobacillus acidophilus would survive in the gut and

replace so-called harmful bacteria. However, it is doubtful whether this organism would survive

without a high carbohydrate diet rich in growth factors.

The most common usage for bacteria in food preparation is with dairy fermentations. Yogurt and

cheeses have been made for centuries using bacteria. The ancients may not have known exactly

what kind of bacteria that was needed or if what were needed was, indeed, bacteria. All they

knew was that the previous batch was required to make a new one. Many people lack the ability

to break down and absorb lactose, the sugar molecule in milk. As a result, it enters the gut,

producing acid and gas, causing pain and diarrhea. Fermented milk products metabolize lactose

into lactic acid, which is more tolerable for many people. It is found in two isomer form D and L

(Figure 22.4). The most common fermented milk product is yogurt. The lactobacilli used in the

making of many yogurts, however, may not be the same type as found within the common flora

of humans as there are many different strains (Probiotics). Some of the bacteria used in the diary

industry are listed in Table 21.6.

Lactic acid bacteria are a heterogenous family of mainly low G+C Gram positive

anaerobic, non sporulating and acid tolerant bacteria. They can ferment various nutrients through

a homo or heterofermentative route into primarily lactic acid, but also into by products such as

acetic acid. Formic acid, ethanol and carbon dioxide. They contribute to rapid acidification of

food products, but also to flavous, texture and nutrition. The genome sequencing of food and

health related LAB has been booming. Around forty LAB genomes have been fully sequenced,

including the genera Lactobacilli, Lactococci, Streptococci, Pediococcus, Oenococcus and

Leuconotoc (Figure 21.2).

Acidophilus milk is made with Lactobacillus acidophilus. Butter is made from pasteurized

cream, to which a lactic acid starter has been added. The starter contains, for example,

Streptococcus cremoris or S. lactis, but requires Lactobacillus diacetylactis to give it its

characteristic flavor and odor. Cheese is often made with Streptococcus and Lactobacillus

bacteria. Fermentation lowers the pH, thus helping in the initial coagulation of the milk protein,

as well as giving characteristic flavors (Figure 21.3).

In such Swiss cheeses, the typical flavor is the result of the use of Propionibacterium.

Cheese can be classified within two groups -- ripened and unripened. Unripened cheeses consist

of cottage cheese, cream cheese, and Mozzarella, for example. These are soft cheeses and are

made by the lactic acid fermentation of milk. Many different bacteria are used to produce the

various cheeses, but Lactococcus lactis and Leuconostoc cremoris are used most often. Soft

cheeses can take one to five months to ripen; hard cheeses, three months to a year or more; and

very hard cheeses, like Parmesan, can take twelve to eighteen months. The blue veins found in

cheeses, are caused by growth of Penicillium roqueforti, which is deliberately added to cheese.

Originally, it was found as a natural contaminant of the areas where it was made. The holes in

Swiss cheese are the result of Propionibacterium shermanii. The surfaces of Camembert and

Brie are innoculated with Penicillium camembertii, which then develops in a skin on the surface.

Limburger is soaked in brine to encourage the growth of Brevibacterium linens (it should come

as no surprise that this is the same bacteria isolated from smelly feet!). Kefir includes many

different microbes, including yeasts, lactobacilli, lactococci, and leuconostocs. Depending on

geographical locations, the precise types of microbes will vary. Yogurt usually requires the

addition of Lactobacillus bulgaricus, Lactococcus thermophilus, and/or Streptococcus

thermophilus to the milk. Lactic acid bacteria comprise of an ecologically diverse group of

microorganisms with the formation of lactic acid as the primary metabolite of sugar metabolism.

These bacteria utilize sugars by either homo- or heterofermentative pathways (Figure 21.4).

21.5 CONTROL OF FOOD SUPPLIES

The increase in urban populations and improvements in methods of food preservation

have led to large-scale transport of basic foods from the producer to the consumer areas. This has

inevitably increased the risk of infection of many people from a common food source. This risk

can be considerably reduced by suitable precautions.

21.5.1 Milk supplies

Consumer-milk is usually transported by bulk-collection services; it is therefore most essential

that adequate measures of control are observed since contaminated milk from one source may be

mixed with a large volume of clean milk. The first requirement is good animal husbandry and

dairy technique to produce a clean product of high quality. As an additional safeguard most

milks are heat-treated to kill pathogenic bacteria which may be present and at the same time to

reduce the number of contaminants thereby improving keeping quality.

21.5.1.1 METHODS OF HEAT TREATMENT OF MILK

Heat treatment is the most widely used method of preservation of milk. Three methods are

currently used.

Pasteurization This method of partial destruction of the microbial population by heat was first

introduced by Pasteur to kill contaminating organisms which interfered with the fermentation

processes in the manufacture of wine. Its application to milk was first used in Denmark to

safeguard pigs against infection from bovine pathogens, but its widest industrial application

today is in treating milk for human consumption. By holding the milk for a defined time at a

standard temperature, as, for instance, 15 seconds at 161°F (71.7°C) in the High Tempera ture

Short Time process, most non-sporing organisms, including all nonsporing pathogens, are killed.

This renders the milk safe for drinking and extends its keeping quality.

Boiling Greater numbers of micro-organisms are killed when milk is held at a temperature not

less than 100°C for a period, which ensures that it will comply with the turbidity test, and the

bottles are sealed immediately afterwards. This process imparts a caramelized flavour to the milk

and homogenization occludes a visible cream line. Milk subjected to this process is often called

Sterilized but total sterility is not achieved and it will not keep indefinitely at normal

temperatures.

Ultra heat treated By this method, the milk is exposed to a temperature of not less than 270°F

(I32.2°C) for at least one second. Usually this treatment renders it sterile and therefore gives it

excellent keeping qualities. There is no cream line, the cream being dispersed throughout the

milk as in homogenized milk. From 10 to 20 per cent of vitamins are destroyed and a slight

flavour is imparted, but this becomes less upon storage. Because of the excellent keeping quality

of the milk it is likely to become very popular since less frequent deliveries to the consumer will

be possible and export to other countries is facilitated.

21.5.1.2 STATUTORY STANDARDS FOR MILK

The one official test to which an untreated milk must comply is the Methylene Blue

Test. A standard solution of methylene blue is added to a sample of the milk on the day after it

left the farm and the sample is then incubated at 37°C. Satisfactory milk will not reduce the dye

to the colourless leuco-form within 30 minutes. Methylene blue is a redox potential indicator and

is reduced by the microbial activity in badly contaminated samples.

Pasteurized milk is required to pass two official tests prescribed by the Ministry of

Health. A Methylene Blue Test similar to the one described for untreated milk is performed on

the milk 24 hours after it has been pasteurized. This determines the bacterial cleanliness of the

milk at the time when it would normally reach the consumer, and measures contamination which

may arise from improperly cleaned bottles subsequent to processing. The other test, the

Phosphatase Test, is a quantitative method of detecting the amount of the natural enzyme,

phosphatase, normally present in milk. In pasteurized milk, most of this enzyme is destroyed and

only a minimal amount is detectable. This test checks that the milk has been adequately

pasteurized.

Sterilized' milk must conform to the Turbidity Test. Proteins are denatured by boiling, but

if the milk has received inadequate heating, soluble proteins remain and may be detected by the

turbidity test. The milk is first saturated with ammonium sulphate and filtered; a tube of the clear

filtrate is then plunged into boiling water and kept in it for 5 minutes. The formation of a

turbidity, due to the heat denaturation of proteins, indicates that the milk had been insufficiently

heated. Ultra Heated Milk is tested by a Colony Count. A standard loopful of the milk is cultured

in 5 mL of yeastral milk agar at 37°C for 48 hours. Not more than 10 colonies are permitted if

the milk is to pass the test. Neither the phosphatase nor the turbidity test can be applied to Ultra

Heated Milk since phosphatase and undenatured proteins remain after the ultra heat treatment.

21.6 PROBIOTICS

Probiotics are defined as foods containing live microorganisms, which actively enhance

health of consumers by improving the balance of microflora in the gut when ingested live in

sufficient numbers (FAO/WHO 2001). Functional food is defined as food product that contain

one or more functional compounds based on scientific evidence having certain physiological

function and health benefit, and has been proved to be safe (Figure 21.5). The recommended

doses of probiotics is 108–1011 CFU per serving with one to several servings per day.

These days’ consumers are concerned about the synthetic chemicals used as

preservatives in food, and there is resulting trend towards less processed food. The untreated

foods can harbour dangerous pathogens which are able to multiply under refrigeration and

without oxygen. A solution to this dilemma is the use of antimicrobial metabolites of

fermentative microorganisms. Many antimicrobial chemicals have been in use for a long time

without any known adverse effects. Many of the organic compounds which have stirred interest

are antimicrobial metabolites of bacteria used to produce, or associated with fermented foods. In

fermentation, the raw materials are converted by microorganisms (bacteria, yeast and molds) to

products that have acceptable qualities of food. In common fermented products such as yogurt,

lactic acid is produced by the starter culture bacteria to prevent the growth of undesirable

microorganisms. Food fermentations have a great economic value and it has been accepted that

these products contribute in improving human health. LAB has contributed in the increased

volume of fermented foods worldwide especially in foods containing probiotics or health

promoting bacteria.

The recent advances in biotechnology have significantly increased the production of high

quality, nutritious and tasteful foods that remain fresh for long time and are completely safe and

less reliant on artificial additives. The potential application of bacteriocins as consumer friendly

biopreservatives either in the form of protective cultures or as additives is significant. Besides

being less potentially toxic or carcinogenic than current antimicrobial agents, lactic acid bacteria

and their by products have shown to be more effective and flexible in several applications.

Evidence is accumulating that confirms that probiotics can benefit the host by improving

intestinal well being (Table 21.7). In order to have functional probiotic strains with predictable

and measurable beneficial effects, strict attention to strain selection is required. Different species

of Lactobacillus listed in 21.8 has been widely used as probiotics.

The basic morphological characteristics are the rod-shaped usually non motile non spore

forming. It produces lactic acid lowering final pH of the medium to 2.2 - 3.4, resistant to acids

and is microaerophillic. They grow well in milk. The identification of genus is based on usual

morphological and biochemical tests such as Gram reaction, motility, presence of flagella,

oxidase test, catalase reaction, presence of extracellular gelatinase and nitrate reduction. The

acceptable manufacturing process of probiotics has been defined in Figure 21.6.

21.6.1 Nisin

Nisin is bacteriocins which are ribosomally synthesized closely related antimicrobial peptides or

proteins produced by strains of Lactococcus lactis subsp. lactis showing a narrow or broad antimicrobial

activity spectrum against Gram positive bacteria sensitive to proteolysis enzymes. They are heat stabile

and are active in a wide pH range (maximum stability at pH 3).

Nisin preparation consists of nisin and sodium chloride with an activity of not less than

900 units per mg. Nisin may be produced in a sterilized medium of non-fat milk solids or of a

non-milk-based fermentation source, such as yeast extract and carbohydrate solids. Nisin can be

recovered from the fermentation medium by various methods, such as injecting sterile,

compressed air (froth concentration); membrane filtration; acidification; salting out; and spray-

drying. Bacteriocins act by destabilization and permeabilization of the cytoplasmic membrane

through the formation of transitory poration complexes or ionic channels that cause the reduction

or dissipation of the proton motive force (PMF) (Figure 21.7).

It is a polycyclic antibacterial peptide with 34 amino acid residues used as a food

preservative. It contains the uncommon amino acids lanthionine (Lan), methyllanthionine

(MeLan), didehydroalanine (Dha) and didehydroaminobutyric acid (Dhb). These unusual amino

acids are introduced by post-translational modification of the precursor peptide. In these

reactions a ribosomally synthesized 57-mer is converted to the final peptide. The unsaturated

amino acids originate from serine and threonine, and the enzyme-catalysed addition of cysteine

residues to the didehydro amino acids result in the multiple thioether bridges.

21.6.1.1 HISTORY OF NISIN

• Produced by group N Streptococcus Inhibitory Substance, end – IN (revised into

Lactococcus lactis (Schleifer et al., 1985)

• 1928 – 1947: research continued on nisin

• 1953 – Commercial production started on nisin

• 1969 – joint expert FAO/WHO approved nisin as bio-preservative

• 1989 – nisin obtained GRAS status by US FDA

• Nisin is adopted by BPOM as one of food preservatives

• 30% (6 of 20) of human milk samples contains nisin-producing bacteria suggests that

humans may have a long history of consuming nisin-producing bacteria. Nisin-producing

L. lactis may protect mothers (mastitis) and infants (toxication) from pathogenic skin

flora, such as Staphylococcus aureus (Beasley and Saris, 2004).

Nisin is produced by fermentation using the bacterium Lactococcus lactis. Commercially, it is

obtained from the culturing of Lactoccus lactis on natural substrates, such as milk or dextrose,

and is not chemically synthesized. It is used in processed cheese, meats, beverages, etc. during

production to extend shelf life by suppressing Gram-positive spoilage and pathogenic bacteria

(Table 21.9). While most bacteriocins generally inhibit only closely related species, Nisin is a

rare example of a "broad-spectrum" bacteriocin effective against many Gram-positive organisms,

including lactic acid bacteria (commonly associated with spoilage), Listeria monocytogenes (a

known pathogen), etc. However, when coupled with the chelator EDTA, Nisin has also been

known to inhibit Gram-negative bacteria, as well. Nisin is soluble in water and can be effective

at levels nearing the parts per billion ranges. In foods, it is common to use Nisin at levels ranging

from ~1-25ppm, depending on the food type and regulatory approval. Due to its naturally

selective spectrum of activity, it is also employed as a selective agent in microbiological media

for the isolation of gram-negative bacteria, yeast, and moulds. Subtilin and Epidermin are related

to Nisin. All are members of a class of molecules known as lantibiotics. Following two methods

illustrate the production of Yogurt (Figure 21.8).

21.7 MEAT

The microflora of freshly dressed meat from healthy animals is derived only slightly

from the flesh (mainly the lymph nodes) but mainly from dirt on the animal, its faeces, the

personnel and instruments in the abattoir, and the air flora of the chill rooms (Figure 21.9).

Animals must not be fatigued or distressed before slaughter, otherwise rigor mortis sets in early

and the meat putrefies rapidly. The high muscle glycogen content of a rested animal ensures the

continued presence of some lactic acid, which is unfavourable to spoilage bacteria in the meat.

Efficient stunning suspends the heart action. Otherwise contaminants from the knife or from the

cut area circulate around the body during the bleeding process. Strains of Serratia,

Achromobacter and Pseudomonas, which are resistant to the bactericidal action of fresh

mammalian blood, are particularly important in this respect. Immobilization of pigs with carbon

dioxide prior to bleeding is helpful for both these reasons; its use could well be extended to

cattle and sheep. During evisceration it is essential to remove the intestines from the carcass

cutting area immediately; this reduces the amount of contamination by bacteria from the gut.

When the carcasses are hanging in the chill room (2° to 3°C for 2 to 3 days) before cutting up,

the microflora can include bacteria, moulds, and yeasts. Most of the bacteria are mesophiles and

tend to die out during chilled storage; only a few are associated with any specific defect or

spoilage of stored meat. Moulds and yeasts may then form between 1 to 10% of the flora. Growth

on the uncut surfaces which are covered with a layer of fat and connective tissue is very limited.

During chilled storage any of the following changes may take place, depending

on the original microbial 'load':

1. Psychrophilic bacteria grow readily on moist surfaces of the meat, thus producing

individual colonies that coalesce, especially if there is condensation, forming slime and

off-odours. Pseudomonas sp. are virtually the sole slime formers when the meat is stored

at l00C, while at 15°C approximately equal amounts of Micrococcus and Pseudomonas

occur.

2. Putrefactive spoilage can occur in the deep tissue of large thick pieces. Clostridium navy;

and other putrefactive anaerobes have been implicated with bone taint, while salt-tolerant

bacteria, able to grow between 0 to 3°C in bone marrow, are responsible for ham souring.

3. The mould Cladosporium herbarum can grow as black spots on chilled meat during

transhipment. Carcasses of home-killed meat, held for long periods in chilled rooms may

become infected with Cladosporium, Rhizopus, Mucor, and Thamnidium species but

these moulds rarely develop with the shorter storage periods and damper conditions now

in vogue.

4. Rancidity can be caused by lipolytic bacteria and yeasts.

After a chilling period the carcasses are transported, cut and re-chilled. Each cut re-

distributes the organisms and adds to the total number of bacteria, so that minced meat, which is

cut most of all, has the highest count. The microfloras of chilled carcasses and cut meat are

mainly aerobic surface contaminants with very few anaerobic spore-formers. Good quality meat

has an initial load of ca 103-10

4 bacteria/cm

2 and should not become slimy before 14 days at 0°C.

When slime does occur the count is usually 3 to l0 x 107

bacteria/cm2, mainly Pseudomonas and

Achromobacter species.

When meat has lost its freshness it becomes offensive and is not likely to be eaten. The

consumer, however, may be unable to judge whether apparently fresh meat has come from a

diseased animal and he is protected against this risk by inspection of animals slaughtered for

food. Qualified inspectors examine the animal both before and after death and only satisfactory

meat is released for human consumption. Premises where meat is sold or prepared are regularly

inspected. The prevention of cross-infection from meat is the complete eradication of certain

diseases from the domesticated animal population within a country.

21.8 BACON

Originally bacon is prepared by packing split pig carcasses in dry salt for a period, giving a

brown tough product. It is now customary to inject the meat with brine containing sodium

chloride, potassium nitrate, sodium nitrite, polyphosphate, etc., followed by steeping in a similar

solution for several days. The brine tanks contain large numbers of bacteria (109/mL), mainly

salt-tolerant micrococci and some lactobacilli, part of whose function is the reduction of nitrate

to nitrite. The sliced bacon is vacuum-packed in oxygen-impermeable plastic wrapping; the

exclusion of air prevents the attractive pink colour of nitrosomyoglobin from fading. The

bacterial flora of micrococci (105

to 106/g) is fairly stable at low temperatures, but it changes

rapidly at higher storage temperatures. Slices with a normal salt content (5 to 7 per cent w/v in

the aqueous phase) spoil after ca 15 days at 20°C with the scented sour smell derived mainly

from lactic acid bacteria. At 30°C the slices putrefy from the proteolytic action of the mesophilic

coagulase-negative staphylococci. Increasing the salt content in the bacon to 8 to 12 % reduces

the number of lactic acid and proteolytic bacteria, thereby increasing the storage life at 30°C.

Sliced bacon can support the growth of Staphylococcus aureus if the storage temperature is 30°C

and the numbers of competing organisms are low. Packs of sliced bacon should therefore be kept

cool during sale and in the home. The same is also true of cooked or smoked hams.

21.9 FISH The flesh of healthy fish is largely free of organisms, but their external surfaces carry an

appreciable bacterial flora (skin 102

to 107/ cm2, gill tissue and intestines 103 to 108 /g). The

aerobic bacteria of marine fish are largely psychrophiles, species of Pseudomonas,

Achromobacter, coryneforms, Flavobacterium, micrococci, Vibrio, and possibly Alcaligenes.

The percentage composition of the flora differs in fish from different parts of the world, with

larger numbers of mesophiles on fish from warmer seas. The intestines usually contain some

strictly anaerobic bacteria, similar to those found in deposits on the sea bottom. In certain parts

of the world there has been a progressive increase in the incidence of botulism caused by fish

contaminated with Clostridium botulinum Type E, an organism able to grow at low temperatures.

Intestines of fish caught in sewage polluted areas commonly contain typical coliform and food

poisoning bacteria. Fish caught by deep-sea trawlers is eviscerated and packed in ice, that caught

inshore may be so treated after landing. Evisceration contaminates the flesh with intestinal

organisms and a low storage temperature is essential to prevent these from multiplying with

consequent spoilage of the fish. As on meat, Pseudomonas species are the most active spoilage

organisms at 0°C.

Cooling usually ceases once the fish reach the market leading to an increase in the flora

already present. Further contamination is likely from fish boxes, filleting knives and boards, etc.

The nature and amount of the microflora developing depends upon the temperature during transit

and sale. Many fish are brined and/or smoked, with numerous permutations of treatment on both

unsplit and split fish. The overall effect of brining is to reduce the Gram-negative bacteria and to

increase Gram-positive types such as micrococci and coryneforms. Cold smoking, while

reducing the total bacterial load considerably, has little effect on the composition of the flora.

Moulds never occur on fresh fish but they are a problem on smoked fish, the spores being

derived from the sawdust used in smoke production. In contrast, hot-smoked fish (i.e., processed

at temperatures of 65-75°C for 30 minutes or longer) are usually sterile unless Clostridium

botulinum was present originally and the salt concentration is below 3 per cent. Probably one of

the safest fish products, as far as food poisoning organisms are concerned, is fish sticks. For

these, frozen rectangular blocks of cod and haddock fillets are cut or sawn to size, dipped in

batter, fried at 205 to 260°C for 2 minutes, packed and re-frozen. The bacterial counts remain at

a very low level since the interior of the sticks remain frozen throughout.

21.10 FERMENTED EASTERN FOODS AND BEVERAGES Fermentations of bland starch (cereal products) and protein (particularly fish) with

moulds, yeasts, and bacteria have been practised for centuries in the Orient to add flavour,

improve nutrient quality, or prevent spoilage. Some of these fermented foods and beverages are

an important source of minerals and vitamins in the diet of large populations. Table 21.10 lists a

few of the many preparations known. Fermented drinks, prepared from millet or other local

cereals, are also an important source of vitamins in the diet of many African populations.

21.10.1 Lactic pickles

Pickling is the process of preserving food by anaerobic fermentation in brine or vinegar.

The resulting food is called a pickle. This procedure gives the food a salty or sour taste. In South

Asia, edible oils are used as the pickling medium with vinegar. They are made from certain

individual varieties of vegetables and fruits that are chopped into small pieces and cooked in

edible oils like sesame oil or brine with many different Indian spices like asafoetida, red chili

powder, turmeric, fenugreek, and plenty of salt. Some regions also specialize in pickling meats,

mushroom and fish. Vegetables can also be combined in pickles to make mixed vegetable pickle.

Some varieties of fruits and vegetables are small enough to be used whole. When both salt

concentration and temperature are low, Leuconostoc mesenteroides dominates, producing a mix

of acids, alcohol, and aroma compounds. At higher temperatures Lactobacillus plantarum

dominates, which produces primarily lactic acid. Many pickles start with Leuconostoc, and

change to Lactobacillus with higher acidity. Another distinguishing characteristic is a pH less

than 4.6, which is sufficient to kill most bacteria. Pickling can preserve perishable foods for

months. Antimicrobial herbs and spices, such as mustard seed, garlic, cinnamon or cloves, are

often added. If the food contains sufficient moisture, pickling brine may be produced simply by

adding dry salt. For example, German sauerkraut and Korean kimchi are produced by salting the

vegetables to draw out excess water. Natural fermentation at room temperature before sunlight,

by lactic acid bacteria, produces the required acidity. Other pickles are made by placing

vegetables in vinegar. Unlike the canning process, pickling (which includes fermentation) does

not require that the food be completely sterile before it is sealed. The acidity or salinity of the

solution, the temperature of fermentation, and the exclusion of oxygen determine which

microorganisms dominate, and determine the flavor of the end product. Pickles produced in

traditional method contain Lactobacillus, produced by fermentation in brine (salt and water).

Pickles produced using vinegar does not contain Lactobacillus. Lactobacillus makes traditional

pickles probiotic.

While vinegar is commonly used in the Western world in the preparation of pickles,

many vegetables are preserved by fermentation with lactic acid bacteria. Thus in the preparation

of Sauerkraut, the solid, round-headed cabbages are washed to remove the undesirable Gram-

negative bacteria on the outer leaves (Pseudomonas, Flavobacterium, and Achromobacter spp.)

and then sliced and mixed with brine. Sugar, mainly sucrose, nutrients, and mineral salts are

leached into the liquid which is then fermented by a succession of three bacterial species, derived

from the innermost leaves. The heterofermentative Leuconostoc mesenteroides, and

Lactobacillus brevis produce lactic and acetic acids, carbon dioxide, alcohol, mannitol, and

dextran while the homofermentative Lactobacillus plantarum produces lactic acid only. The

optimum temperatures and salt concentrations are 13-18°C and 1.8 – 2.25 % salt. At the upper

ends of these limits, the homofermentative species Streptococcus faecalis and Pediococcus

cerevisiae tend to replace the less salt-tolerant Leuconostoc mesenteroitks. At 3 % salt the

product is tough and can develop a pink coloration with the growth of carotenoid-producing

yeasts. At too low a salt concentration the product is unpleasantly soft due to the growth of

bacteria capable of digesting pectin and cellulose. Pure culture inoculations have never produced

the succession of species that gives the best flavoured product. Olives and small cucumbers are

also brined and preserved by the action of lactic acid bacteria.

21.11 COCOA AND COFFEE

Fermentation is also used to prepare certain products for processing, as with cocoa and

coffee beans, where removal of unwanted outer layers is facilitated by the action of micro-

organisms. Coffee and chocolate require Erwinia dissolvens, Leuconostoc, and Lactobacillus

species plus the yeasts of the genus Saccharomyces to remove the tough outer coats. The

microbes do not affect the taste of coffee but are necessary to confer the characteristic taste to

cocoa and chocolate. The bacteria S. napoli and S. eastbourne often use chocolate as a vector. It

is thought that the chocolate provides protection for the bacterium as it passes through the acidic

environment of the stomach. This was observed when higher incidents of illness were reported in

children.

21.12 EGGS

The contents of some 90% of freshly laid hens' eggs are sterile; their shells and the interior of

the remainder contain Gram-positive bacteria of ovarian origin. However, the predominant

species inside rotten eggs are Gram negative rods and extra-genital in origin, derived from the

nesting material and not the faeces. The predominant species are Alcaligenes faecalis,

Aeromonas liquefaciens, Proteus vulgaris, non-proteolytic strains of Cloaca sp., Citrobacter sp.,

and Pseudomonas fluorescens. These organisms possess combinations of proteolytic and

lecithinase activity, pigment and hydrogen sulphide production. The progress of bacterial rotting

follows a definite pattern. First there is contamination and penetration of the shell, followed by

growth in the cell membranes. The extent of growth is controlled by the antimicrobial action of

the conalbumin in the egg albumen. Eventually the yolk makes contact with the shell membrane

and the organisms are able to utilize the glucose of the albumen and infect the complete contents

of the egg.

Salmonellae can proliferate in eggs without producing any visible symptoms other than a

faint turbidity in the albumen. Salmonella typhimurium, S. anatum, and S. enteritidis are

frequently encountered even in eggs from clinically normal birds and the importance of duck

eggs as a source of human salmonellae is well established. Humans can become infected by

salmonellae not only from fresh whole eggs but also from frozen, liquid, or spray-dried products

made from lower grade eggs. Because of the lack of obvious spoilage symptoms, salmonella

infections are not apparent and the contents of such eggs are added to the main bulk for

processing. This is in addition to any contamination from the hands of the process workers or

from the containers into which the eggs are poured.

The control of infection from egg sources relies on reduction of the incidence of infection

in the flock, reduction of contamination of the shells by faeces, special attention to the sites

where eggs are laid, the sale of only clean, unwashed eggs, and the education of kitchen staff to

encourage adequate cooking of food containing eggs or its products.

21.13 BREAD

In the conventional bread making process, a dough of flour, water, salt, etc., is allowed to

ferment with a culture of the yeast Saccharomyces cerevisiae, originally overnight, now for 3

hours. The dough is worked, allowed to rise, then reworked, placed into tins, allowed to rise for a

further 50 minutes (final proof) and baked. This is now being replaced by the high-energy system

(Chorleywood Bread Process) in which the dough is worked mechanically. The amount of yeast

is increased by 50% and the addition of a fast-acting oxidizing agent and fat are essential parts of

the process. Production time is decreased by some 60 %, much less space at controlled humidity

is required in the bakery, the loaf has a lower staling rate, and the yield of bread is increased 4-5

per cent. Yeasts produced by controlled hybridization are now available and are particularly

suitable for modern methods of bread making.

Both compressed yeast and bakery products are subject to contamination. Bacteria

contaminating compressed yeasts are mainly lactobacilli, normally considered harmless;

only heavy infections of the slime-forming Leuconostoc mesenteroides are liable to affect

the baking properties of the yeast. Nowadays contamination with wild yeasts is rare, but

before rigid sanitation programmes were instituted in yeast factories the presence of such

aerobic yeasts as Candida krusei, C. mycoderma, C. tropicalis, Trichosporon cutaneum, T.

candida, and Rhodotorula mudlaginosa was not uncommon. The widespread use of

moisture-impervious wrappings has made control of moulds such as Aspergillus and

Penicillium species and Oidium (Oospora) lactis of special importance, since they can form

unsightly blotches on the surface of the yeast block beneath the wrapping. Acid calcium

phosphate and acetic acid are commonly used as preservatives in bakery products to prevent

the development of bacterial ropiness. The addition of propionates in bread, sorbic acid and

its salts in flour, and both these in backed products are being allowed to use for the

prevention of mould growth. Reducing the available water content (aw ) to as low a level as

possible without spoiling texture and appearance helps to delay mould growth but, in the

absence of fungicides, cannot prevent it. Strict cleanliness within the bakery is absolutely

essential to ensure that any waste flour is quickly removed, otherwise it becomes mouldy and

spores are discharged into the atmosphere.

Sour dough bread is made with a mixture of Lactobacillus brevis (provides aroma),

L. plantarum (provides crumb elasticity), and yeast (for raising power). Increasing the

holding temperature decreases the percentage of yeast in the mix, until at 35°C it is

practically yeast-free.

21.14 BEER AND LAGER

An alcoholic beverage is a drink that contains ethanol, commonly known

as alcohol (although in chemistry the definition of “alcohol” includes many other compounds).

Beer has been a part of human culture for 8,000 years. In Germany, Ireland, the United

Kingdom, and many other European countries, drinking beer (and other alcoholic beverages) in a

local bar or pub is a cultural tradition. Non-alcoholic beverages are drinks that usually contain

alcohol, such as beer and wine, but contain less than 0.5% alcohol by volume. This category

includes low-alcohol beer, non-alcoholic wine, and apple cider.

Wines are made from a variety of fruits, such as grapes, peaches, plums or apricots. The

most common wines are produced from grapes. The soil in which the grapes are grown and the

weather conditions in the growing season determine the quality and taste of the grapes which in

turn affects the taste and quality of wines. When ripe, the grapes are crushed and fermented in

large vats to produce wine. Beer is also made by the process of fermentation. A liquid mix,

called wort, is prepared by combining yeast and malted cereal, such as corn, rye, wheat or barely.

Fermentation of this liquid mix produces alcohol and carbon dioxide. The process of

fermentation is stopped before it is completed to limit the alcohol content. The alcohol so

produced is called beer (Figure 21.10). It contains 4 to 8 per cent of alcohol.

Beer is made by fermenting a hop-flavoured extract of barley malt with a top-fermentation strain

of Saccharomyces cerevisiae. The bottom-fermenting yeast, S. carlsbergensis, is required for

lager. Barley is converted into malt by allowing the soaked grain to germinate under controlled

conditions of temperature and humidity, leading to the formation of α-and β-amylases, and the

breakdown of the protein, hordein, to amino acids. The process is stopped by raising the

temperature and removing the rootlet and plumules. The exact treatment depends on the type of

malt required. In some countries, the protein is fully converted into amino acids, lager malts still

contain appreciable quantities of protein, for beer manufacture. Stout malts are dried at 105°C to

give a dark-coloured extract; malts from distilleries and vinegar breweries receive no final

kilning and still contain limit-dextrinase.

An extract or wort is now prepared from the ground malt, the exact conditions of extraction again

being varied according to the type of beer. In some countries starch is hydrolysed by the action of the

amylases to glucose, maltose, malto-triose and -tetrose, and oligosaccharides; glucose and fructose are

also present in the extract. Worts from lager also need extensive proteolytic action during mashing.

Sometimes flaked barley or maize, or gelatinized unmalted cereal grits are added to supplement the starch

supply (Table 21.11 and Table 21.12). When extraction is complete the wort is boiled under pressure with

hops, the tannins from which co-precipitate with any remaining proteins. A proportion of the humulone

and lupulone complexes are converted into their corresponding isomers during boiling, ultimately giving

bitterness to the beer and conferring some resistance to Gram-positive bacteria. The cooled and filtered

wort is 'pitched' with yeast and allowed to ferment. Distillery and vinegar brewers' worts are not boiled

(so that further saccharification due to the enzyme limit-dextrinase can take place during fermentation),

neither are they hopped. Bacterial infection is often restricted by inoculating part of the wort with

Lactobacillus delbrueckii and, when acidified sufficiently, boiling this before returning it to the remainder

of the wort ready for yeasting; sulphuric acid may be used as an alternative method of reducing the pH.

Again, some power-alcohol distilleries sterilize the wort chemically with 1,000 ppm ammonium fluoride

and use a fluoride-adapted yeast.

The two types of brewing yeasts differ not only as to species but also in other properties.

Saccharomyces cerevisiae has round cells, most of the crop rises to the surface at the end of the

fermentation and, characteristically, when supplied with raffinose ferments only one third of the

molecule, leaving melibiose. Saccharomyces carlsbergensis is cylindrical, forms a deposit and can

ferment the whole of raffinose. There are strains of both yeasts with intermediate morphology and

brewing properties; the latter can be modified of course by the brewing system employed. Beer wort is

yeasted with between 2 and 6 g moist yeast/L, depending on its original specific gravity. After 12-18

hours at 15°C a rapid fermentation ensues, the temperature is raised by 3 to 7°C and the pH falls from 5·2

to 4.1. The thick yeast head that collects after 21 days is skimmed off, fermentation slackens and the beer,

still containing some sugar, is ready for clarification and natural conditioning in tanks before sale. Some

modern breweries use a non-flocculent yeast that ferments very rapidly; the fermentation is terminated at

the desired sugar content by centrifugation. Traditionally, part of the yeast crop (often a balanced

collection of strains) was collected and used for the next fermentation. Nowadays pure cultures are being

used increasingly and are replaced after 12 to 20 brews, depending on the standard of hygiene. Lager has

always been made with a pure culture, the crop being collected from the bottom of the tank after the main

fermentation, subsidiary crops in the pre-fermentation and conditioning tanks being discarded. The

pitching temperature, 5 to 90C, is lower than with beer; fermentation at 120C is more protracted (7 to 14

days), and storage at 20C may take 6-40 weeks before the lager is considered sufficiently conditioned and

stabilized for sale. Distillery worts of high specific gravity are pitched at 210C with a heavy inoculum of a

yeast specially bred for alcohol tolerance; fermentation is rapid, the temperature rising to 25-300C during

the process. Either a fresh culture is used for each fermentation or the previous crop is acid-washed and

re-used.

21.15 CIDER AND WINE

Successful brewing is dependent not only on the biochemical properties of a yeast strain but also

on the physical properties of the mass culture. In contrast, cider and wine were made by controlling a

natural flora of yeasts, moulds, and bacteria, so that only organisms with desirable properties were

allowed to grow. The microbiology of cider is being discussed here, that of wine is similar in many

respects. In the traditional cider-making countries, the raw material consists of special varieties of apples,

high in tannin and low in acidity. Now a days these are supplemented with dessert and culinary apples

graded as unsuitable for the fresh fruit market; other countries use these latter sorts as their sole source of

raw material. The fruit as it arrives at the factory carries both an internal and an external microflora. Some

of the common moulds are Candida pulcherrima, Kloeckera apiculata, Torulopsis jamata, Aspergillus

niger, Botritis cinerea, Penicillium sp. together with the 'black yeast' Aureobasidium pullulans and

occasionally rhodotorulae, Candida, and Torulopsis spp. Saccharomyces spp. and bacteria are rare in

sound fruit but mould-damaged specimens carry large populations, not only of mould spores but also of

acetic acid bacteria and fermenting yeasts. Both the moulds and acetic bacteria produce sulphite binding

compounds that are of great significance at later stages of processing. Hence sound, preferably washed

fruit, is essential. The apples are milled to a pulp and pressed hydraulically in cloth envelopes between

wooden drainage racks. Formerly the racks and cloths were washed very infrequently, consequently they

soon supported a dense population of Saccharomyces species; these were the yeasts mainly responsible

for the subsequent fermentation. At first Candida spp. and other poorly fermenting yeasts are most

numerous but they are rapidly overgrown by Kloeckera sp. after a few degrees drop in specific gravity.

The latter then co-exist and in turn are overgrown by Saccharomyces sp. while in the fully fermented dry

ciders film yeasts and acetic bacteria become more important. The whole process is improved by

treatment of the freshly pressed juice with sulphur dioxide, the actual amount being varied according to

the pH. Below pH 3.3, 100 ppm is sufficient, while 150 ppm is desirable between 3.3 and 3.8; to be

effective more S02 is required above 3.8 than is allowed by law (200 ppm), but most factories process a

mixture of apples whose juice pH is generally 3.5-3.6. It is vitally important that the concentration of

sulphite binding compounds be kept low in the juice, otherwise no free sulphur dioxide remains in the

solution and this is the only effective moiety (sometimes referred to as undissociated sulphurous acid).

After treatment only Saccharomyces spp. remain, so that in effect sulphiting selects a pure culture from

the original microflora. However, with higher standards of cleanliness in the press rooms, the juices have

a flora similar to that of the fruit, i.e., virtually free of fermenting yeasts. Hence, the addition of pure

cultures of bottom-fermenting wine yeasts, following sulphiting, is becoming almost universal in cider

factories. It also means that both the time needed for fermentation and the flavour of the cider become

more predictable, since there is no longer any need to rely on what were virtually factory contaminants.

A second fermentation of malic acid to lactic acid and carbon dioxide, due to lactic acid bacteria, nearly

always occurs during either the yeast fermentation or storage of the cider. The organisms that can bring

about this change include both homo-and heterofermentative lactic rods and cocci, including the

organisms responsible for producing polysaccharide slime or ropiness. The bacterium found most

frequently is Lactobacillus pastorianus var. quinicus. If citric acid is present, as in perry but not cider,

diacetyl, lactic, and acetic acids are also formed and the flavour is thereby spoiled. The source of these

bacteria is still in some doubt since they appear to be absent from many sulphited juices. Ciders stored in

contact with air, especially those held in small wooden containers, soon show surface growths of

Acetobacter xylinum and the film yeasts Pichia membranaefaciens and Candida mycoderma. The

problem is now rare in large factories where juice sulphiting and very large storage tanks are normal

practice.

Basically, wines are made by a similar process but many other fermentation treatments are

possible, due to the much wider range of climatic conditions in which grapes are grown. Thus grapes can

be 'raisinified' by being left on the vine or by being dried in the sun; wines made from their high gravity

juices are fermented with osmophilic yeasts such as Saccharomyces rouxii. Sauterne is made from grapes

naturally infected with the mould Botrytis cinerea that dehydrates the grapes, metabolizes some

of the acid and produces glycerin and the mild antibiotic botryticin. Consequently the subsequent

fermentation of the very sugary juice ceases prematurely, leaving a soft, sweet, luscious-

flavoured wine. Climate also has an effect on the type of yeasts found on the grapes. The greater

the ambient temperature, the greater the concentration of alcohol-tolerant Saccharomyces yeasts

and sporing apiculate yeasts (Hanseniaspora valbyensis) and the fewer there are of their non-

sporing counterparts (e.g., Kloeckera magna and K. apiculata). Finally a number of

Saccharomyces species have been isolated from both cider and wine. These are S. acidifaciens,

S. carlsbergensis, S. cerevisiae, S. delbrueckii, S. elegans, S. florentinus, S. fructuum, S.

oviformis, S. rosei, S. rouxii, and S. uvarum. In addition, there are other Saccharomyces spp.

apparently characteristic of each beverage. Brettanomyces sp. have been isolated from cider and

wine, in the Bordeaux area and in dry wines made within a 40-mile radius of Cape Town. There

is one wine for which no parallel exists in cider-making, namely, sherry. The yeasts responsible

for the characteristic fermentation of alcohols and organic acids, sometimes called S. beticus, S.

cheresiensis, etc., develop as a veil on the surface of tlte dry wine when it is exposed to air.

Unlike C. mycoderma and other spoilage film yeasts, the sherry yeasts form acetaldehyde and

other characteristic flavours from the alcohol; the remaining process is a complicated blending

system (the solera) also used for the sun-baked wine, Madeira.

21.16 FOOD AS A SOURCE OF INFECTION AND DISEASE

The two most common types of food-borne illness are intoxication and infection. A food

borne infection is caused by the ingestion of food containing pathogenic microorganisms (i.e

bacteria, virus or parasite) which must multiply within the gastrointestinal tract, producing

widespread inflammation. The food-borne illness may be due to Campylobacter jejuni,

Clostridium botulinum, Clostridium perfringens, Cyclospora cayetanensis, Escherichia coli,

(0157:H7), Shigella, Salmonella, and Staphylococcus aureus. Campylobacter jejuni can be

transmitted to humans via unpasteurized milk, contaminated water, and raw or undercooked

meats, poultry, and shellfish. C. botulinum, which causes botulism, is the most deadly of all food

pathogens. Toxins, not bacteria, cause illness. Bacteria that produce toxins include S. aureus and

C. botulinum. Symptoms may include nausea, vomiting, diarrhea, abdominal pain or distress, and

fever. Pathogens may be introduced by flies or, most important of all, by the handling of foods

by human carriers of food poisoning organisms. Most risk is entailed with foods prepared for

consumption the following day, the organisms multiplying if the food is kept at a temperature

conducive to growth.

21.16.1 Food Poisoning

A special group of diseases contracted by the ingestion of food are designated 'food

poisoning'. Outbreaks of these are usually explosive in character and may be related to ingestion

of a specific meal or common food source. Food poisoning may be due to poisons derived from

plant or animal sources, or to chemicals added inadvertently or as preservatives at too high

concentrations, or to the presence of harmful micro-organisms or toxins produced by them. The

symptoms produced by different poisons may be similar and the final diagnosis thus depends

upon laboratory tests. The time from the consumption of the poisonous food to the onset of

symptoms varies from 10 minutes to 2 hours with some forms of chemical poisoning, to around

6 hours with bacterial toxins, 12 to 72 hours when poisoning is due to living bacteria and 2-3

days when poisonous toadstools are the cause.

21.16.1.1 BACTERIA CAUSING FOOD POISONING

The types of foods involved in food poisoning are those favouring growth of the causative

organisms, so that large numbers of bacteria or their products are present in the food at the time

it is ingested. Outbreaks of food poisoning frequently involve a large proportion of the people

who have eaten a particular food. Two groups of agents are recognized: pathogenic bacteria

which can infect the alimentary canal and produce symptoms of gastro-enteritis, and bacteria (or

their toxins) which do not infect the gut but produce various toxic symptoms when ingested with

food.

21.16.1.1.1 Infective Food Poisoning

Salmonellosis

Salmonellosis is a form of food infection that may result when foods containing Salmonella

bacteria are consumed. In this form of food poisoning the infective agent is able to establish

itself in the gut, multiply rapidly and produce symptoms of disease over a number of days, often

for about a week or longer. This form of poisoning is mainly· limited to species of Salmonella.

When infection is established, endotoxins, resulting from the breakdown of dead bacterial cells,

cause irritation to the gut linings resulting in symptoms of acute gastroenteritis. The Salmonella

group includes many hundreds of types of serologically related organisms which are found in

almost every species of animal, including birds and reptiles. In Great Britain meat, milk,

synthetic cream, and eggs (particularly duck eggs) are the foods most commonly contaminated

with salmonellae. Meats from cattle and pigs have been found to be a frequent source of

infection, particularly when made up into processed foods such as brawns, sausages or pies,

which may be incompletely cooked or subsequently contaminated during handling. Ideally

salmonella should be absent from food but if adequately cooked, these pathogens are destroyed

and there is no risk of food poisoning, since there are no residual toxins to produce food

poisoning symptoms. Symptoms of salmonella food poisoning vary in severity but include acute

gastro-enteritis accompanied by severe headaches followed by nausea, vomiting, abdominal

pain, and diarrhoea. Fever frequently occurs. These symptoms usually arise between 12 and 72

hours after the meal has been consumed, by which time the organisms are established in the gut.

Symptoms of toxic food poisoning may arise earlier if sufficiently large numbers of organisms

are ingested . In favourable cases the symptoms subside within a week, but in a small percentage

of patients organisms invade the tissues from the gut and may cause death. Even when symptoms

disappear, the patient may continue to excrete the pathogens and thereby remain a potential

source of infection for susceptible individuals and of contamination of food. If this continues for

a long period after recovery, the patients are termed carriers. It is most important to ensure that

such people are not employed in food handling.

Cholera

Cholera is a severe water-borne diarrheal disease caused by Vibrio cholerae, a gram-

negative curved bacillus bacterium, transmitted almost exclusively via contaminated water, food,

etc. V. cholerae and V. parahaemolyticus are pathogens of humans. Both produce diarrhea, but in

ways that are entirely different. V. parahaemolyticus primarily affects the colon while V.

cholerae is noninvasive and affects the small intestine by secreting an enterotoxin.

V. cholerae produces cholera toxin, the model for enterotoxins, whose action on the mucosal

epithelium is responsible for the characteristic diarrhea of the disease cholera. The clinical

description of cholera begins with sudden onset of massive diarrhea. This results from the

activity of the cholera enterotoxin which activates the adenylate cyclase enzyme in the intestinal

cells, converting them into pumps which extract water and electrolytes from blood and tissues

and pump it into the lumen of the intestine. This loss of fluid leads to dehydration, anuria,

acidosis and shock. The watery diarrhea is speckled with flakes of mucus and epithelial cells and

contains enormous numbers of vibrios. The loss of potassium ions may result in cardiac

complications and circulatory failure. The immediate treatment of the disease is the oral

rehydration therapy with NaCl plus glucose to estimate water uptake by the intestine. Antibiotics

such as tetracyclin, trimethoprim-sulfamethoxazole or ciprofloxacin are commonly used for

treatment.

Camphylobacteriosis and Helicobacteriosis

Campylobacteriosis is caused by consuming food or water contaminated with

Campylobacter jejuni. C. jejuni generally inhabits intestinal tracts of healthy animals (especially

chickens) and in untreated surface water. Raw and inadequately cooked foods and non-

chlorinated water are the main sources of human infection. The organism grows best in a reduced

oxygen environment, is easily killed by heat. Diarrhea, nausea, abdominal cramps, muscle pain,

headache and fever are common symptoms. Onset usually occurs two to five days after eating

contaminated food. Duration is two to seven days, but can be weeks with such complications as

urinary tract infections and reactive arthritis.

Infection of gastric epithelial cells with Helicobacter pylori induces strong pro

inflammatory responses by activating nuclear transcription factors NF-KB and AP-1. Several

reports indicate that multiple bacterial factors and cellular molecules are involved in this

signaling.

Listeriosis

Listeriosis is a dangerous infection caused by eating food contaminated with bacteria

called Listeria monocytogenes, small Gram-positive rods and non-spore forming. The L.

monocytogene infections are common in wild animals, domesticated animals, and in soil and

water. Raw milk or products made from raw milk may carry these bacteria. The bacteria most

often cause a gastrointestinal illness. In some cases, it may lead to septicemia or meningitis. The

bacteria may cross the placenta and infect the developing baby. Infections in late pregnancy may

lead to stillbirth or death of the infant within a few hours of birth. About half of infants infected

at or near term will die.

21.16.1.1.2 Bacterial Cells or their Products as cause of Food Poisoning

In this form of food poisoning no infection of the gut occurs but bacterial cells or

exotoxins of bacterial origin produce symptoms of food poisoning when ingested in foods.

Since these toxins are absorbed by the gut wall they are called enterotoxins. They are preformed

in the food by growth of the pathogenic organism and although the vegetative cells may be

killed by heating the food prior to eating, many of the toxins, being moderately resistant to heat,

remain and cause symptoms of food poisoning when ingested. The most important bacterial

toxic food poisonings are caused by the toxins of specific strains of Staphylococcus aureus and

Clostridium botulinum, the latter frequently causing a fatal form of food poisoning known as

botulism. Strains of Clostridium perfringens also cause toxic food poisoning and certain non-

pathogenic bacteria, such as Proteus sp., not generally considered to be toxigenic, produce acute

gastro-enteritis when consumed in sufficiently large numbers.

Staphylococcal Food poisoning

Most outbreaks of this type of food poisoning are associated with milk and its products (e.g.,

cream fillings in cakes and milk deserts such as custards) and prepared meat (e.g., pies or

brawns). Staphylococcal food poisoning is due to enterotoxin B and the symptoms are

characterised by severe vomiting, diarrhoea, abdominal pain and cramps. Recovery usually

occurs within 6 to 24 hours. These foods favour the rapid multiplication of staphylococci (and

consequent toxin production) in warm weather if they are not refrigerated. In non-pasteurized

milk the organisms may come from an infection of the cow's udder, but since staphylococci are

frequently found on human skin and in the naso-pharynx, there are many opportunities for

contamination of the food during preparation. Fortunately only a few types of staphylococci

(generally phage types of group III) are able to produce the enterotoxin which is formed in

significant amounts only when the bacterial count reaches 105

to 106

per gram of food. Symptoms

of staphylococcal food poisoning may arise after only 1 hour but more usually after about 12

hours from ingestion of the toxin, and include violent vomiting, diarrhoea, and prostration but

there is no fever and recovery is rapid, often within 24 hours. This type of poisoning is only

occasionally fatal.

Botulism

Clostridium botulinum is a spore-bearing anaerobe occurring in soil. It may be ingested

with food but does not multiply in the body. Inadequate sterilization of foods originally

contaminated from the soil and stored under anaerobic conditions conducive to the growth of this

organism are responsible for the majority of outbreaks. There are 7 types (type A, B, C, D, E, F,

and G) of these neurotoxins recognised on the basis of specificity. The neurotoxin of C.

botulinum is a protein and a dose as low as 0.01 mg is said to be fatal to human beings. A and B

producing strains often found on fruits and vegetables and honey. Symptoms appear 12-36 hours

after ingestion and include nausea and vomiting (B and E) visual impairment: blurred, ptosis,

dilated pupils, loss of mouth and throat function (A and B) dry mouth, throat, tongue, sore throat

fatigue and loss of coordination respiratory impairment abdominal pain and either diarrhea or

constipation in general, cranial nerve first affected and then descend. Sixty to seventy percent

cases of botulism die. Neurotoxin (BoTox) is water soluble, produced as a single polypeptide

150,000 MW (progenitor), cleaved by a protease to form two polypeptides (H and L chains)

which then become S-S bonded. All serotypes block the exocytotic release of acetylcholine from

synaptic vesicles at peripheral motor nerve terminals. No case of botulism is known to have

arisen as a result of eating fresh foods. The preformed toxin, when ingested, is absorbed through

the mucosa of the stomach and upper part of the intestine. The symptoms differ from all other

forms of food poisoning in that the toxin acts directly upon the central nervous system. Nausea

and vomiting usually first occur within 24 hours and are followed several days later by paralysis

of specific muscles due to damage of the nerve centers controlling them. Thus double vision

results from paralysis of the eye muscles, difficulty in swallowing follows paralysis of the throat

muscles. If paralysis of the muscles of the respiratory tract occurs, death from respiratory failure

results.

Non-specific bacterial causes of food poisoning

Occasionally outbreaks of food poisoning have been reported in which the foods involved were

heavily contaminated with species of Proteus, Escherichia, Streptococcus, or Bacillus. It is

generally thought that large numbers of these organisms have an irritating effect on the gastro-

intestinal mucosa and set up symptoms of toxic food poisoning. This emphasizes the fact that

gross contamination of food with organisms generally considered to be non-pathogenic, followed

by storage at temperatures conducive to bacterial propagation, presents a potential hazard to the

consumer.

21.16.1.2 POISONOUS FUNGI

Even edible species of fungi are liable to prove indigestible and to cause minor illness if

eaten in excess and individuals may be allergic to particular species. A few agarics, however, are

very poisonous. The most deadly species in Britain is the death cap, Amanita phalloides. The

consumption of even a small piece of a fruit-body of this species may be fatal and at best will

cause a long and serious illness. Attempts have been made to prepare a serum against this species

but this is effective only if it is known at once that the death cap has been eaten. More often the

victim is unaware that he has eaten a poisonous variety and the symptoms develop after only one

or two days by which time treatment is useless. The best safeguard is the ability to recognize this

and a few other poisonous species. The toxins of A. phalloides, which cause degeneration of liver

and kidneys, and progressive paralysis of the central nervous system, are not destroyed by

cooking, but those of some other species are inactivated by heat, so that fruit-bodies which are

dangerous when eaten raw are safe after adequate cooking. For instance, the Ascomycete,

Gyromitra esculenta produces a heat labile toxin, helvolic acid, which acts on red blood cells.

The sclerotia of the Pyrenomycete Claviceps purpurea the ergot of rye, are of medicinal value,

owing to the presence of an alkaloid which is extracted and used to stop excessive bleeding,

particularly in childbirth. They are also poisonous; rye grass infected with ergot can cause

abortion in sheep and cattle, and flour prepared from grain admixed with the ‘ergots’ causes

serious disease (formerly known as St. Anthony's Fire) in persons consuming it. Another

Pyrenomycete, Gibberella zeae, infects the ears of cereals and renders the grain poisonous to

man, pigs, and dogs but not to sheep or cattle. The darnel grass fungus, a non-sporing systemic

endophyte of Lolium temulentum, is said to render the seeds of this grass poisonous to stock, but

this has not been proved and it has been suggested that in the instances reported contamination

with ergot had occurred.

Aflatoxins are toxic metabolites produced by certain fungi in/on foods and feeds. They

are probably the best known and most intensively researched mycotoxins in the world.

Aflatoxins have been associated with various diseases, such as aflatoxicosis, in livestock,

domestic animals and humans throughout the world. The occurence of aflatoxins is influenced by

certain environmental factors; hence the extent of contamination will vary with geographic

location, agricultural and agronomic practices, and the susceptibility of commodities to fungal

invasion during preharvest, storage, and/or processing periods. Aflatoxins have received greater

attention than any other mycotoxins because of their demonstrated potent carcinogenic effect in

susceptible laboratory animals and their acute toxicological effects in humans. As it is realized

that absolute safety is never achieved, many countries have attempted to limit exposure to

aflatoxins by imposing regulatory limits on commodities intended for use as food and feed.

21.17 LABORATORY DIAGNOSIS OF BACTERIAL FOOD POISONING

Confirmation of clinical diagnosis rests either on laboratory isolation of the causal

organism, principally from faeces, or demonstration of the toxin either in the patient or in the

incriminated food, if some is still available. Since in staphylococcal food poisoning the causal

organism is rarely present in the faeces of patients, isolation and subsequent bacteriophage

typing is attempted from food, vomit, etc. Often the staphylococci have been killed by

subsequent heating of the food and then confirmation of this type of food poisoning is only

presumptive. Whilst many animal tests to detect staphylococcal enterotoxin in food and culture

filtrates have been explored, the only reliable method is to feed small amounts of culture filtrates

to human volunteers in an attempt to reproduce food poisoning symptoms. The organism

causing botulism is spore-bearing and its spores are highly heat-resistant. It may survive in

cooked food, from which it may be isolated. As a further test, the toxin, obtained as a sterile

filtrate from a suspension of the food or gut contents, is injected intraperitoneally into mice.

Ifmice, protected against botulism by the injection of specific antiserum survive whilst

nonprotected ones die, proof of the presence of botulism toxin is established.

21.17.1 Tools and methods to detect Microbes in Food

Many of these molecular tools have been accepted and implemented in standard protocols

for detection and quantification of the most important pathogenic bacteria, such as Salmonella sp

and Listeria monocytogenes. Despite of rapid diffusion of molecular tools in microbiology

laboratories, there are still many drawbacks and obstacles concerning specificity, reproducibility

and reliability of nucleic acids and antibody based technologies for microbial detection. That led,

to some extent, to an underestimation of those methods as a permanent alternative to

conventional culture-based detection techniques that sill represent the “golden standard” for

microbial diagnostic. The complexity of food and water matrices and the cross reaction of some

molecular probes to target sites of innocuous bacteria closely related to pathogenic ones are the

main enthralling challenges which researchers are still arguing with. In order to maintain food

safety standards, conventional microbiological methods are still being used to detect bacteria and

other organisms in food (Table 21.13). However, these techniques are not ideal, as often it can

be many days before results are known-which may be of particular economic importance for

those foods with a short shelf-life. The introduction of newer technology, such as nucleic acid

probe and related amplification technology in other fields, has transformed the detection of many

organisms. The Polymerase Chain Reaction (PCR) allows nucleic acid probes, with their

inherent specificity, to be used to detect organisms present in very low numbers within a short

period of time. However, at present, in food microbiology, there are technical problems with

using the PCR, as certain components in food interfere with the reaction.

21.17.2 Commercial food safety diagnostics

The implementation of Food Safety should be seen as an ongoing process, which is influenced

by environmental, socio-economical, political and cultural factors. Food safety issues need to be

addressed on a continuous basis, from a regional, national, European and global point of view.

New, flexible tools are required for evaluating and managing new food safety challenges (Figure

21.11). To guarantee the safety of foodstuffs producers have therefore shifted their focus towards

the use of food safety management tools, most important HACCP (Hazard Analysis Critical

Control Point), and the consequent application of hygienic measures, based on Good

Manufacturing/Hygienic practice (GMP/GHP). Food safety management tools use input of

scientific information to identify critical contamination points in the food chain and the

production process, and design measures to control them. However, the lack of reliable data is

often limiting the usefulness of this approach and therefore data collection is one of the priorities

for future food safety strategies. In the absence of relevant data, other strategies might still have

to be used to control food hazards. The Food and Drug Administration (FDA) has been involved

in the regulation of in vitro diagnostic devices (IVDs or laboratory tests) since the introduction of

the Medical Device Amendments of 1976. IVDs developed as kits or systems intended for use in

multiple laboratories require review by the FDA before being marketed to ensure appropriate

performance and labeling.

So, what is there in existence to manage quality and safety, and how do they relate to

each other? Below are listed the most well known methods to manage quality and/or safety, and

these will be briefly discussed individually and then how they integrate with each other. The

food safety rules and their relationship are explained in Figure 21.11 and Figure 21.12.

Good Hygienic Practices / Good Manufacturing Practices

The terms GHP and GMP refers to measures and requirements which any establishment

should meet to produce safe food. These requirements are prerequisites to other and more

specific approaches such as HACCP, and are often now called prerequisite programmes. In

recent years the term Standard Sanitary Operating Procedures (SSOP) has also been used in the

US to encompass basically the same issues, i.e. best practices.

Hazard Analysis Critical Control Point (HACCP)

Hazard Analysis Critical Control Point (HACCP) is a systematic approach which

identifies, evaluates, and controls hazards which are significant for food safety (CAC, 1997).

HACCP ensures food safety through an approach that builds upon foundations provided by good

manufacturing practice. It identifies the points in the food production process that require

constant control and monitoring to make sure the process stays within identified limits. Statistical

Process Control systems are relevant to this operation. HACCP is legislated in many countries,

including the USA and the European Union. The combination of GHP/GMP and HACCP is

particularly beneficial in that the efficient application of GHP/GMP allows HACCP to focus on

the true critical determinants of safety.

Quality Control

It is an important subset of any quality assurance system and is an active process that

monitors and, if necessary, modifies the production system so as to consistently achieve the

required quality. It can be argued that QC is used as part of the HACCP system, in terms of

monitoring the critical control points in the HACCP plan. However, traditional QC is much

broader than purely this focus on critical control points for safety systems.

Quality Assurance / Quality Management

This can be defined as all the activities and functions concerned with the attainment of

quality in a company. In a total system, this would include the technical, managerial and

environmental aspects. The best known of the quality assurance standards is ISO 9000 and for

environmental management, ISO 14000. The term quality management is often used

interchangeably with quality assurance. In the seafood industry, the term quality management

has been used to focus mostly on the management of the technical aspects of quality in a

company, for instance, the Canadian Quality Management Programme which is based on

HACCP but covers other technical issues such as labelling.

The International Organization for Standardization (ISO) in Geneva is a worldwide

federation of national standards bodies from more than 140 countries.ISO's work results in

international agreements which are published as International Standards. The vast majority of

ISO standards are highly specific to a particular product, material, or process. However, two

standards, ISO 9000 and ISO 14000, mentioned above, are known as generic management

system standards. Over half a million ISO 9000 certificates have been awarded in 161 countries

and economies around the world and in 2001 alone over 100 000 certificates were awarded, 43%

of which were the new ISO 9001:2000 certificate. Historically, the ISO 9000 series of standards

of relevance to the seafood industry included: ISO 9001 Quality systems - Model for quality

assurance in design/ development, production, installation and servicing ISO 9002 Quality

systems - Model for quality assurance in production and installation. More recently, the new ISO

9001:2000 certificate is the only ISO 9000 standard against whose requirements a quality system

can be certified by an external agency and replaces the old ISO 9001, 9002 and 9003 with one

standard.

It is important to note that the ISO 9000 standards relate to quality management with

customer satisfaction as the end point, and that they do not specifically refer to technical

processes only. ISO 9000 gives an assurance to a customer that the company has developed

procedures (and adheres to them) for all aspects of the company's business.

ISO 14000 is primarily concerned with environmental management. Introduced much

later than the ISO 9000 series, there are now over 35 000 ISO 14000 certificates awarded in 112

countries or economies of the world. During 2001, nearly 14 000 certificates were awarded,

around 40% of the total awarded since the introduction of the standard.

In most countries, implementation of ISO 9000 quality management systems or ISO

14000 environmental systems are voluntary.

Quality Systems

This term covers organizational structure, responsibilities, procedures, processes and the

resources needed to implement comprehensive quality management (Jouve et al., 1998). They

are intended to cover all quality elements. Within the framework of a quality system, the

prerequisite programme and HACCP provides the approach to food safety.

Total Quality Management (TQM)

TQM is an organization's management approach, centred on quality and based on the

participation of all its members and aimed at long-term success through customer satisfaction

and benefits to the members of the organization and to society (Jouve et al., 1998). Thus TQM

represents the organizations' "cultural" approach and together with the quality systems provides

the philosophy, culture and discipline necessary to commit everybody in the organization to

achieve all the managerial objectives related to quality.

21.17.3 Business outlook for food diagnostics

Consumer demands safe food today. The commercial food industry's ability to identify

bacterial pathogens and unsafe residues has resulted in an almost five fold increase in food

recalls by major manufacturers since 1988. New technology allows Government regulatory

agencies to identify a bacterial pathogen and trace it back to its source more rapidly (Figure

21.13). The key to this new technology is the availability of rapid food safety diagnostics.

These rapid food safety diagnostics provide a quick, "positive or negative" answer before

the food product enters the distribution system. Tests take up to 30 hours to complete because of

the requirement for a bacterial growth enrichment period. This growth enrichment period is

necessary to increase the total number of bacteria so they can be detected using current

technology. A negative answer means that the product does not contain that particular bacteria or

toxin and no further testing is required. A positive answer means that further testing is needed at

a reference laboratory using standard laboratory methods to confirm the actual presence of the

bacteria or toxin. For example, rapid screening for Salmonella typhimurium requires

approximately 24 hours to complete.

21.17.4 Next generation diagnostics industry

The most promising breakthroughs of the development of on line or on site, sensitive,

low-cost, rapid methods for routine use are expected to be made in the area of sensor technology.

Many prototypes for food diagnostic application in the food and drink industry are currently

being developed. They have high potential for automation and allow the construction of simple

and portable equipment for fast analysis. These properties will open up many applications within

quality and process control, control of fermentation processes, quality and safety control of raw

materials, and for HACCP monitoring.

A new method must have high sensitivity, high specificity, high precision (repeatability),

at the same time rapid, robust and cheap. There is currently no method that will fulfil all

requirements. Antibody based methods as represented by the ELISA assay and DNA based

methods as the polymerase chain reaction are the most widely used technologies in food

diagnostics today. Also immunomagnetic separation (IMS) is exploited in a number of

commercially available kits and will become an even more important technology in the future.

While some microarray based systems are commercially available, most of the biosensors and

microarray based applications are still on the level of prototypes. For other promising methods

with future potential, but with less momentum for commercial development specifically in food

diagnostics, as flow cytometry, bacteriophage technology or adenylate kinase, only a basic

description of the method is given together with brief background information on the current

stage of its development. Sensor Technology covers a wide area of diverse techniques, including

opto-chemical sensors and biosensors. Biosensors are a subgroup of chemical sensors where the

analytical devices are composed of a biological recognition element such as enzymes, antibodies,

receptors, proteins, oligonucleotides, or even a whole cell coupled to a chemical or physical

transducer. A transducer measures the changes that occur when the sensor couples to its analyte.

The sensitivity of the system is determined by the type of tranducers employed. Biosensors can

be used for the detection of very different analytes such as pathogens, pesticides and toxins.

Biosensors can be grouped according to their 15 biological recognition element into

immunosensors using antibodies and hybrid sensors using DNA or RNA probes. There have

been many sensors developed for the detection of food borne pathogens with the goal to

overcome problems associated with traditional microbiological detection techniques such as

being time and labour intensive (Baeumner, 2003). In fact, biosensor advancements have greatly

improved our ability to detect minute quantities of analytes as research into biosensors has

mainly focused on detection platforms with very low detection limits (Rider et al., 2003). It has

been estimated that 38% of reported pathogen biosensors in the past 20 years were designed for

the food industry. However, only a limited amount of methods are combined and currently

exploited for their use in food diagnostics. As recognition elements, bioaffinity based receptors

that use the selective interaction between ligand and receptor, antibody or nucleic acid are most

widely used. As transducers, electrochemical and optical systems have gained practical

importance. As nanobiotechnology progresses, sensors to detect pathogens or their constituents

become smaller and more sensitive. Owing to the nature of these nanoscale sensors, the sample

size from which the detection is being made is typically a microliter or smaller. Therefore, the

challenge for scientists developing detection methods for pathogens in foods is in the sample

preparation. Although the sample preparation requirements will vary from one food product to

another, research into this step is required to bridge the emerging field of nanosensors with the

food industry. Thus, while the organism with the largest number of diagnosed cases may

fluctuate from year to year, the food industry will always be looking for detection systems that

will help identify all pathogens of concern in its food products (Rider et al., 2003).

Optical biosensors have been developed for rapid detection of contaminants in foods,

including pathogens, and several have evolved into commercial prototype systems. The analyte

in the food interacts with the bioactive molecule, usually an antibody. Antibodies can be

immobilized directly on the fibre, either on the blunt end or along the sides of a fibre tip. The

binding of antibody and analyte is detected as a change in an optical signal measured through the

fibre-optic assembly. The light from a laser travels to the fibre tip and penetrates into the area

outside the tip. A fluorescently labelled complex binds to the antibodies on the tip. The

fluorescent signal then radiates in all directions, and some of it travels back up the fibre tip to the

detector. Detection of molecules in solution can be made either by direct binding to the biosensor

coating molecules, or by competition binding with soluble capture molecules added together

with the sample. Future applications might include protein quality and the detection of allergens

genetically modified proteins, BSE prions, pathogens and biocide residues.

21.18 EXPERIMENTS

21.18.1 Bacterial Count

The bacterial count in milk is the most reliable well accepted indication of sanitary quality.

Although, the human pathogens may not be in a high count it may indicate a diseased udder,

unsanitary handling of milk. In general, a high count means that there is a possibility of disease

transmission. On the other hand, it is necessary to avoid the wrong interpretation of low plate

counts, since it is possible to have pathogens such as brucellosis and tuberculosis with

permissible number of bacterial count. Following methods are normally used for microbiological

examination of milk.

21.18.1.1 DIRECT MICROSCOPIC COUNT OF ORGANISMS

This method is especially useful to determine the milk quality in much shorter time than

is possible with a standard plate count. In addition to being much faster than the SPC, the direct

microscopic count has two other distinct advantages. First of all, it will reveal the presence of

bacteria that do not form colonies on an agar plate at 350C; thermopiles, psychrophiles, and dead

bacteria would fall in this category. Secondly, the presence of excessive number of leukocytes

and pus-forming streptococci on a slide will be evidence that the animal that produced the milk

has an udder infection (mastitis).

Direct Microscopic count is accomplished by staining a measured amount of milk that

has been spread over an area of one square centimeter on a slide. The slide is examined under oil

and all of the organisms in an entire microscopic field are counted. Several fields are counted to

get average field counts in order to maintain accuracy. It is necessary to measure the field area in

order to translate the readings into organisms per mL.

High quality milk will have very few organisms per field, necessitating the examination

of many fields. A slide made of poor-quality milk, on the other hand, will reveal large numbers

of bacteria per field, thus requiring the examination of fewer fields. In view of all these

advantages, it is apparent that the direct microscopic count has real value in milk testing. It is

widely used for testing raw milk in creamery receiving station and for diagnosing the types of

contamination and growth in pasteurized milk products. Milk that has been separated, blended,

homogenized, and pasteurized will lack leukocytes and normal flora.

Shake the milk sample for nearly 50-60 times so as to break the large bacterial clumps,

transfer 0.01 mL of the milk to one square on the slide using micropipette. Allow the slide to air

and then place it over a beaker of boiling water for 5 minutes dry to steam fix it. Flood the slide

with xylol to remove fat globules (Figure 21.14). Remove the xylol from the slide by flooding

the slide with 95% ethyl alcohol. Gently immerse the slide into a beaker of distilled water to

remove alcohol under running water. The milk film will wash off. Stain the smear with

methylene blue for 15 seconds and dip the slide again in water to remove the excess stain.

Decolorize the smear to pale blue with 95% alcohol and dip in water to stop decolorization.

Allow the slide to completely air dry before examination.

Least Count of Microscopic Measurement

The relationship of the field to a milliliter is the microscopic factor (MF). Stage

micrometer is to be used to calculate the microscopic field. πr² formula is used for measuring the

area. Suppose 0.01 mL milk is used in the smear and the area of the slide is 1cm². MF can easily

be calculated. Measurement will require to focus the stage micrometer on the microscope under

oil immersion keeping in mind that each space is equal to 0.01 mm. Field area is calculated in

square millimeters employing πr² formula where π is equal to 3.14. Square millimeters are

converted to square centimeter by dividing with 100. Calculate the number of fields in one

square centimeter by dividing one square centimeter by the area of the field in square

centimeters. To get the part of a milliliter that is represented in a single field (MF), multiply the

number of fields by 100. The value should be around 500,000. Therefore, a single field

represents 1/500,000 of each ml of milk.

It is observed that the number per milliliter is higher than a standard plate count (SPC)

but a clum count is closed to the SPC. Both methods viz. individual cells and clumps are

normally employed. The details of the methods is depicted in following Figure 21.15.

21.18.1.2 LITMUS MILK TEST

Milk is a complex nutritional source that contains proteins (mainly casein) in an aqueous

solution of lactose and minerals. Bacterial enzymes alter the media and may bring about various

changes. Litmus is added to the medium to detect pH changes that may occur as a result of these

enzymatic reactions. Litmus is blue at above pH 8.3 while below a pH of 4.5 it is red. By

producing acid from the fermentation of lactose present in milk a bacterium may also cause the

milk to curdle or clot in the bottom of the tube. Litmus acts as an electron acceptor thus

becoming reduced by bacterial metabolism. This reaction is sometime observed as a white color

in the medium. Litmus milk test is used to distinguish between different types of bacteria. The

lactose (milk sugar), litmus (pH indicator), and casein (milk protein) contained within the

medium can all be metabolized by different types of bacteria. The test itself tells whether the

bacterium can ferment lactose, reduce litmus, form clots, form gas, or start peptonization (Figure

21.16).

21.18.1.3 QUALITATIVE SCREENING OF MILK QUALITY (METHYLENE BLUE

TEST)

Milk having enough amount of microbial population will quickly consume dissolved

oxygen resulting to reduce the oxidation reduction potential. Methylene blue loses its color when

it is reduced. It is also known as reductase test. In this test, 1 mL of 1:25000 diluted aqueous

methylene blue solution is added in freshly collected 10 mL milk sample. The tube is sealed with

a rubber stopper and slowly inverted 3-4 times to mix well, incubated at 350C

in a water bath and examined after every 30 minutes for up to 6 hours. The time taken for

methylene blue to become colourless is the methylene blue reduction time (MBRT). The shorter

the MBRT, the poorer the quality of milk. An MBR of 30 minutes is considered as the good

quality of milk. Raw milk primarily contains Streptococcus lactis, Escherichia coli which are

strong reducer. The test is used for screening the milk at milk collection center. However, the test

will not identify the psychrophiles and thermophiles which are in large number in milk (Figure

21.17).

21.18.1.4 STANDARD PLATE COUNT: A QUANTITATIVE TEST

This determines the total number of bacteria in a specified amount of milk, generally a

millilitre (mL). This is used for grading of milk. Under aseptic conditions, one mL of milk is

added to 99 mL of distilled water or buffer. One ml and one-tenth mL samples are then

transferred to sterile petri dishes and prepare 1:100 and 1: 1000 dilutions of the milk,

respectively. Other dilutions may also be prepared successively (Figure 21.18).

A growth medium like plate count agar or tryptone glucose yeast extract agar is then

added, and the milk samples are mixed with the medium. The dishes are incubated at 37°C for

24-48 hours. The plates are then placed on a counting device like a Quebec colony counter and

the number of bacterial colonies is recorded. The colony count falling between 30 and 300 is

selected and multiplied by the reciprocal of the dilution factor to obtain the bacterial count per ml

of milk. If 248 colonies appeared on the 1: 100 plate and 16 colonies on, the 1:1000 plate, 248

would be selected and multiplied by 100, giving 24,800 total bacteria per ml of milk sample.

The standard plate count (SPC), also referred to as the aerobic plate count or the total

viable count, is one of the most common tests applied to indicate the microbiological quality of

food. The significance of SPCs, however, varies markedly according to the type of food product

and the processing it has received. When SPC testing is applied on a regular basis it can be a

useful means of observing trends by comparing SPC results over time.

Three levels of SPC are listed below based on food type and the processing/handling the food.

Level 1 applies to ready to eat foods in which all components of the food have been cooked in

the manufacturing process/preparation of the final food product and, as such, microbial counts

should be low.

Level 2 applies to ready to eat foods which contain some components that have been cooked and

then further handled (stored, sliced or mixed) prior to preparation of the final food or where no

cooking process has been used.

Level 3 SPCs not applicable. This applies to foods such as fresh fruits and vegetables (including

salad vegetables), fermented foods and foods incorporating these (such as sandwiches and filled

rolls). It would be expected that these foods would have an inherent high plate count because of

the normal microbial flora present.

Note: An examination of the microbiological quality of dairy and food product should not be

based only on SPCs. The significance of high (unsatisfactory) SPCs cannot truly be made

without identifying the microorganisms that predominate or without other microbiological

testing for pathogenic and nonpathogenic organisms.

21.19 FOOD MICROBIOLOGY

The detection of pathogenic bacteria is key to the prevention and identification of

problems related to health and safety. Legislation is particularly tough in areas such as the food

industry, where failure to detect an infection may have terrible consequences. Throughout the

world, food production, preparation and distribution have become increasingly complex, and raw

materials are often sourced globally. Changes in food processing techniques, food distribution

and the emergence of new food pathogens have changed the epidemiology of food-borne

diseases. Food-borne microorganisms are continuously changing due to their inherent ability to

evolve and their amazing capacity to adapt to different forms of stress. New primary production

technologies and food manufacturing practices are introduced all the time; food consumption

patterns and the demographic structure of many countries continue to change.

Approximately 1/3rd of all food manufactured in world is lost due to spoilage. On the basis of

the shelf life, food can be classified as non-perishable foods (pasta), semiperishable foods

(bread) and perishable foods (eggs). The conditions for spoilage relates to water, pH, physical

structure, oxygen and temperature. Various intrinsic factors are also responsible like

composition, pH, presence and availability of water, oxidation-reduction potential, altered by

cooking, physical structure and presence of antimicrobial substances. Microbial content of foods

(microbial load) can be evaluated by determine bacterial titer.

The intestinal tract of warm-blooded animals contains a variety of bacterial species.

When an animal is butchered, these bacteria may be spread from the gut to the edible portions of

the carcass. While such bacteria generally are not harmful to humans (beef is most often

contaminated with E. coli, a normal inhabitant of the human intestine), some types of bacteria

(such as Salmonella, found frequently in chickens) can cause food poisoning. It is, therefore,

essential that meats be thoroughly cooked prior to eating. The non-pathogenic varieties of

bacteria in foods may cause chemical changes which alter the flavour and texture of food

(Figure 21.19). Because of this, meats are refrigerated or frozen to retard the growth of bacteria.

Since some bacteria can grow over a wide range of temperatures (from less than 10° C to greater

than 45° C for E. coli), refrigeration is adequate for only short term storage of meats.

21.19.1 Microbial Spoilage of Canned Food

Canned foods may spoil either due to biological or chemical reasons. Biological spoilage

of canned food occurs due to the action of various microorganisms. Spore forming bacteria, e.g.,

Clostridium, Bacillus represent the most important group of canned food spoiling

microorganisms because of their heat resistant nature (thermophilic nature). In addition, there are

other microorganisms which are not heat resistant (mesophilic) but enter through the leakage of

the container during cooling and spoil the food. In this way, we can divide biological spoilage of

canned into following two categories.

21.19.1.1 BIOLOGICAL SPOILAGE BY THERMOPHILIC BACTERIA

Canned foods under processing results in spoilage by thermophilic bacteria, the bacteria that

grow best at temperature of 50°C or higher. Five types of this spoilage can be recognized.

Flat sour spoilage In canned foods, production of acid and no gas is referred to as flat sour

spoilage because the food becomes sour, but the can shows no evidence of food spoilage because

no gas is produced, i.e., the can remains flat. Thus, the spoilage cannot be detected unless the can

is opened. The spoilage is caused by Bacillus spp. such as B. coagulans and B.

stearothermophilus resulting in sour, abnormal odour, sometimes cloudy liquor in food content

of the can.

Thermophilic anaerobic (TA) spoilage Clostridium thermosaccharolyticum, an obligate

thermophile, causes spoilage. The can swell and may burst due to production of CO2 and H2. The

food becomes fermented sour, cheesy, and develops butyric odour.

Sulfide spoilage Clostridium nigricans is involved in this spoilage. It produces H2S gas which is

absorbed by the food product. The latter becomes usually blackened and gives “rotten egg”

odour.

Putrefactive anaerobic spoilage Clostridium sporogenes causes spoilage through putrefaction.

The can swells and may burst. Putrefaction may result from partial digestion of the food. The

latter develops typical “putrid” odour.

Aerobic sporeformer’s spoilage Bacillus spp., the aerobic bacteria, causes spoilage. If the

canned food is cured meat, swelling of the can is observed.

21.19.1.2 BIOLOGICAL SPOILAGE BY MESOPHILIC MICROORGANISMS

Bacillus spp., Clostridium spp., Yeast, and other Moulds which are mesophilic

(an organism growing best at moderate temperature range of 25 to 400C) are mainly responsible

for this type of canned food spoilage. As stated earlier, these organisms enter through the leakage

of the container during cooling. Clostridium butyricum and C. pasteurianum result in butyric

acid type of fermentation in acidic (tomato juice, fruits, fruit juices, etc.) or medium acidic (corn,

peas, spinach, etc.) food with swelling of the container due to the production of CO2 and H2.

Bacillus subtilis and B. mesenteroides have been reported as spoiling canned sea-foods, meats,

etc. Other mesophilic bacteria which have been reported in cans are Bacillus polymixa, B.

macerans, Streptococcus sp., Pseudomonas, Proteus, etc. Yeasts and moulds have also been

found present in canned foods. Yeasts result in CO2 production and swelling of the cans.

21.19.1.3 MICROBIAL SPOILAGE OF REFRIGERATED MEAT

Lipases are class of enzymes, which catalyze the hydrolysis of long chain triglycerides.

These are glycerol ester hydrolases that also catalyses esterification, interesterification,

acidolysis and aminolysis in addition to the hydrolytic activity on triglycerides. Beside these

activities, there is two more class of enzymes, mostly secreted by microorganisms and these are

the oxygenases and the reductases which also bring about changes in lipids.

21.19.1.4 BACTERIAL COUNTS

SPC (Standard Plae Count), milipore filter and multiple tube tests is normally used much in the

same manner that they are used on water and milk to determine the total counts and the presence

of coliforms. However, to get the organisms in suspension the food blender is used for blending

meat, dry fruits, frozen foods etc as described in Figure 21.20. Fruit juice and other beverages

are used in the manner as we do for milk and water.

Canned Food

Microbial spoilage of commercially canned food and heat processed is confined to heat

resistant endospore. Canning normally involves heat exposure for long period at temperature

that is adequate to kill spores of most bacteria especially to the processing of low acid foods in

which Clostridium botulinum can thrive to produce botulism food poisoning.

Spoilage occurs when the heat processing fails to meet accepted standards. This is

because of several reasons (1) usually in case of home canning (2) carelessness in handling the

raw materials before canning, resulting in an unacceptably high level of contamination that

ordinary heat processing may be inadequate to control (3) malfunctioning of equipment resulting

to undetected underprocessing (4) defective containers that permit the entrance of organisms

after the heat process. There are three types of food spoilage caused by heat resistant bacteria (a)

Flat Sour – pertains to spoilage in which acids are formed with no gas production; results sour

food in cans that have flat ends (b) T.A. Spoilage - caused by thermophlic anaerobes which

produce acid and gas (CO2 and H2 but not H2S) in low acid foods (c) Stinker Spoilage – Cans

swell to various degrees, sometime bursting due to the spore formers that produce H2S and

blackening of the can and contents. Blackening is due to the reaction of H2S with the iron in the

can to form iron sulfide. To perform the experiments on microbial spoilage follow the Figure

21.21 with the inoculation of cans with following 5 cultures viz. Bacillus stearothermophilus,

Bacillus coagulans, Clostridium sporogenes, Clostridium thermosaccharolyticum and Escerichia

coli sepeartely. Incubation temperature for Clostridium thermosaccharolyticum and Bacillus

stearothermophilus be 550C, Clostridium sporogenes and Bacillus coagulans at 370C and 300C

for Escerichia coli. If cans begin to swell during incubation they should be placed in refrigerator.

After incubation place the cans in hood to open with the help of punch type can opener. Hold an

inverted plastic funnel over the can in case the can is swollen during perforation to minimize the

effect of any explosive release of contents. Remove about 10 mL of the liquid through the

opening, pour it into a small plastic beaker, cover with parafilm and later use for making stained

slides for gram staining and endospore staining and examine under brightfield oil immersion.

Refrigerated Meat

The wide spread use of refrigeration to store and preserve food stuffs, provide great diversity of

nutrient rich habitat for psychrophilic and psychrotolerant food spoilage microorganisms. The

lipases produced by certain organisms are significant to the food industry in that they improve

the traditional chemical processes of food manufacture. However, certain microorganisms like

Arthobacter sp., Pseudomonas fragi, Pseudomonas fluorescence and Serratia marcesans which

produce cold active lipases, were isolated from refrigerated milk, meat products and other

spoiled food samples.

Dressed poultry are highly susceptible to spoilage by many microorganisms. The flesh

and other parts like the liver, has hardly any microorganisms in any living animals; therefore,

most of the contamination comes during the slaughtering processes like bleeding, defeathering,

removal of viscera, washing and subsequent handling. In order to prevent spoilage by these

microorganisms, meat is refrigerated usually at -10 to -15°C. However, during selling these are

stored at -2 to -5°C over the counter. Sometimes it has been observed that the temperature of the

counter reaches 0 to 2°C and the material is held in this temperature for more than 8 h. Such sort

of practice leads to increase in aerobic microbial count especially like those of the psychrophiles.

These microorganisms bring about biochemical changes, which can be termed as spoilage, as the

material is not accepted for human consumption. Some of the spoilage causing microorganisms

gets inactivated during refrigeration; others survive the low temperature. The meat is also

subjected to changes by its own enzymes. Such autolytic changes include proteolysis and

lipolysis. The excessive autolysis is called as souring. The spoilage activities of microorganisms

include the hydrolysis of fats and sometime subsequent oxidation of the fatty acids liberated,

leading to loss of flavor and production of off odors due to certain aldehyde and acids. This may

even change the normal colour of the poultry meat to shades of green, brown or gray as a result

of the production of oxidized compound e.g., peroxides. It is well known that the chicken meat

flavor is primarily due to arachidonic acid which is, like all meat flavors, is fat soluble present as

a glyceride and gets liberated during processing. However, this arachidonic acid is β-oxidized by

microbes leading to the formation of different N-hydroperoxyeicosatetraenoic acids with

hydroperoxide substitution at C5, C8, C11, C12 and C15. Figure 21.22 illustrates the overall

procedure to perform the experiment for bacterial count of refrigerated meat.

Count the colonies after incubation for two weeks in the back of the refrigerator where

the temperature will remain between 00C to 50C on all the plates and calculate the number of

psychrophiles and psychotrophs per gram of meat. Microorganism that grow between 50 and 00C

are classified as being either psychrophilic or psychotrophic. The difference between the two

groups is that psychrophiles seldom grow at temperature above 250C. While the optimum growth

temperature range from for psychrophiles is 15-180C, psychotrophs have an optimum growth

temperatue range of 250C-300C. It is the psychrotrophic microorganisms that cause most meat

spoilage during refrigeration. Select a colony from the plates and preparea gram stain, endospore

stain and flagella staining etc and observe under oil immersion with the aid of bright field

microscope.

Demonstration of Fermentation

Wines are available commercially in two forms viz. white and red wines are fermented at 130C

and 240C, respectively after peeling off the skin. The only difference between the two is that the

distillers use red grapes with the skin left on during the initial stage of the fermentation process.

Fermentation can be demonstrated by using grape juice in the following way (Figure 21.23)

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Important Links

http://www.cnawater.com/food-a-dairy-microbiology-mainmenu-44 http://foodscience.psu.edu/directory/research-areas/food-microbiology http://www.ndri.res.in/ndri/Design/dairymicrobiology.html http://foodsci.wisc.edu/ http://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm2006949.htm http://www.dairyscience.info/cheese-starters/49-cheese-starters.html http://www.titanmedia.in/dairy-microbiology.php http://osp.mans.edu.eg/fsmp/leader.asp https://www.boundless.com/microbiology/industrial-microbiology/the-microbiology-of-food/microbes-and-dairy-products/ http://www.foodnavigator.com/Science-Nutrition/From-dairy-microbiology-to-canning-practice-an-overview-of-CSIRO

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