Microb Ecol (1986) 12:15-30
MICROBIAL ECOLOGY 1996 Springer-Veflag
Ecology of Food Microorganisms G. Hobbs Torr~ Research Station, PO Box 31, 135 Abbey Road, Aberdeen AB9 8DG, UK Abstract. The behavior of microorganisms in foods is governed by the constraints applied to the microflora by a variety of envffonmental and ecological factors. These include water activity, pH, Eh, chemical composition, the presence o f natural or added antimicrobial compounds, and storage temperature, as well as processing factors such as the application of heat and physical manipulation. Control of these factors will govern whether the food spoils or not, whether any microbial health hazard arises, and whether desired microbial processes are successful or not. While much is known about the effects of individual environmental factors, the effects due to their interactions are less understood. The two main problems now facing the food microbiologist are optimization of environmental parameters and the selection of strains with specific properties. A better understanding of the mechanisms of acf~on and interactions between the various environmental factors, coupled with the application o f modern techniques to produce strains with particular properties, will lead to optimum use of food supplies and improvements in quality. There is also potential for the development o f new and novel foods.
Introduction The preservation of animal and plant tissues for use as food essentially consists of manipulating the environmental factors ~n the food so that the normal degradation processes brought about by microorganisms are slowed down or prevented altogether. The success or otherwise o f preservation processes depends on how the microbial population responds to the constraints applied by handling and processing. It will depend on how successfully the procedures prevent undesirable organisms from gainirtg access and how successfully they inhibit microbial growth or permit the preferential growth o f desirable organisms. Post-slaughter or post-harvest foods immediately have a particular microflora, the actual composition of which will depend on the nature of the food and the slaughter or harvesting conditions. ~n healthy plants and animals the tissues are essentially sterile and microorganisms are present primarily on the outer surfaces, and in the case of animals, in the intestinal tract. Subsequent handling, processing, artd storage wil~ cause changes in the micro~tora in response to various ecological pressures. Ttte precise nature o f these pressures
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teria to reducing temperatures below a m b i e n t and thus provide a measure o f spoilage [11]. Although the relationship is empirical, it does relate bacterial growth and temperature sufficiently well to be useful. Where spoilage is mainly bacterial, the spoilage rate at different temperatures can be predicted from knowledge o f spoilage at 0~ Evidence is beginning to accrue now to show that the relationship can be used to predict shelf life o f chilled foods. Lower temperatures therefore reduce microbial growth until it eventually ceases. Freezing and subsequent thawing have the additional effect o f killing a proportion o f the microflora and damaging others. T h e reduction in n u m b e r s is relatively small, however, i.e., less than 1 log cycle, and it is doubtful if it has a noticeable effect on the spoilage o f thawed frozen foods. At higher temperatures inactivation reactions b e c o m e significant. Proteins, including enzymes, nucleic acids, and other cell c o m p o n e n t s are all sensitive to high temperatures, and there is a wide variation in the response o f different organisms. Some extreme psychrophilic organisms will not survive long at 20~ and above, whereas extreme thermophiles will survive and even grow up to 100~ For the majority o f organisms, however, temperatures f r o m 65~ upwards are lethal. The death rate o f microorganisms is logarithmic; exposure time and initial n u m b e r s are therefore i m p o r t a n t considerations. The m o s t highly resistant forms o f microorganisms are bacterial spores. While they do not grow at high temperatures, they survive t h e m very well. C o m m e r c i a l sterilization o f foods requires t r e a t m e n t with steam u n d e r pressure, and laboratory sterilization procedures c o m m o n l y require a temperature o f 12 I~ for 20 rain.
Water Activity The water activity o f a food is the ratio o f the water v a p o r pressure o f the food to that o f pure water at the same temperature, and is a measure o f the water available for microbial growth. It is therefore related to the a m o u n t o f solutes present in the food as well as the a m o u n t o f water. As water activity falls, the growth rates o f microorganisms are reduced until growth ceases. Below this value, which varies for different organisms, there will be a slow decline in the numbers o f organisms surviving. Most bacteria will not grow below an aw = 0.90, whereas some yeasts and molds will grow as low as aw = 0.60. T o relate these values to solute concentration, a value o f aw = 0.995 corresponds to a salt concentration o f 0.87% or a sugar concentration o f 0.92%, whereas a~ = 0.90 corresponds to 16.2% and 14% respectively, and aw = 0.753 is equivalent to a saturated sodium chloride solution. The o p t i m u m range o f water activity for most microorganisms is aw = 0.95--0.99.
Acidity and Alkalinity Each organism has a range o f p H within which growth is possible, and it will have an o p t i m u m within this range. Most natural e n v i r o n m e n t s have a p H between 5 and 9, and the majority o f microorganisms have o p t i m a within this range. Very few organisms can grow below p H 2 or above p H 10. T h e range
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over which an individual organism can grow is usually not more than 3 to 4 units the majority of bacteria having an optimum pH around neutrality and molds and yeasts at slightly acid values (pH 5-6). At 91-I values outside a particular organism's range a proportion of the population will die, and prolonged storage under these conditions will result in a further decline.
Oxidation-Reduction Potential The range of redox potentials (E~,) found in nature is +0.82 to - 0 . 4 2 volts, and it can be poised at different levels with various chemical agents. In foods, the Eh will depend on the level of oxidation or reduction of the various components and on the composition of the atmosphere surrounding the food. In most cases the Eh will not be uniform throughout a particular product; for instance, in contact with air the outside of a piece of meat or fish will be highly aerobic with a high positive Ej, whereas the inside will be anaerobic with a negative E~. Obligate aerobic organisms require oxygen or a highly oxidized environment for growth, whereas obligate anaerobic organisms require highly reduced conditions and the absence of oxygen. Between these extremes are large numbers of facultative organisms that can grow under most conditions, though they have varying optimum Eh conditions. In general, aerobic metabolism is more energy efficient and usually results in more growth. Anaerobic metabolism, however, although less efficient, usually results in a greater production of odorous and flavorous compounds which are relevant to the acceptability of foods.
Nutritional Status and Preservatives Raw plant and animal tissues generally provide an excellent nutrient medium for the growth of a wide range of microorganisms. Without the application of preservation procedures they would be rapidly decomposed by microorganisms and this is the normal course of events in nature with dead plants and animals. S o m e tissues tend to favor one group of organisms because o f specific components. An example of this is the presence of organic acids artd low pH values in many fruits. Many bacteria are inhibited by these conditions, and degradation tends to be brought about by molds and yeasts. A number of preservatives are permitted for general use in foods because they have been used in traditional preservation techniques for a very long time. Salt, various sugars, acetic acid, and smoke constituents are examples. In the main, however, the addition of chemical inhibitors to foods is strictly controlled in most countries. The most commonly used include organic acids and their esters, generally with chain lengths less than Cm, some antibiotics, and inorganic salts such as sulphite and nitrite. In addition to reducing p H and water activity, chemical preservatives generally have some specific antimicrobial effects frequently against a wide range of organisms but sometimes against a particular ~roup.
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Interactions Between Environmental Factors There is a substantial a m o u n t o f i n f o r m a t i o n available on the individual effects o f the various e n v i r o n m e n t a l factors on growth o f microorganisms and thus on food preservation. It is relatively recently, however, that the full extent o f interactions between these parameters has been realized. T h e interactions between water activity, pH, and t e m p e r a t u r e are probably the best known. I f two o f these are o p t i m u m , growth will occur at extremes o f the third o v e r a range characteristic for the particular organism. This range is appreciably narrower, however, when either or both o f the others are not optimal. A n o t h e r example is the increased sensitivity o f bacteria to heat in the presence o f curing salts and with reduced pH. Also, m a n y bacteria are m o r e sensitive to the inhibitory effects o f nitrite in the presence o f increased concentrations o f s o d i u m chloride. An extreme example is the difference in sensitivity o f bacteria to "wet heat" and " d r y heat." The former, where the water activity is close to 1.00, is m u c h more effective than the latter at killing bacteria. In only a few cases are the mechanisms o f action o f e n v i r o n m e n t a l factors understood; in still fewer cases is the m e c h a n i s m o f interactions between various factors understood. T h e evidence available to explain these m e c h a n i s m s was recently reviewed [6], and the conclusion is that as far as resistance o f bacterial spores to heat and the effects o f lowering water activity and p H on growth are concerned, the cell mechanisms operate to maintain homeostasis with respect to water inside the cells. W h e n the cell is no longer able to do this growth ceases, or in the case o f bacterial spores, heat resistance is lost. Such a c o m m o n mechanism could well explain why when two or m o r e factors are acting together there are synergistic effects.
Preservation of Foods
lnitial Microbial Flora There are extensive reports in the literature describing the i m m e d i a t e postslaughter or post-harvest flora o f a wide range o f foods [7, 8, 12]. As might be expected, the initial flora is very varied, not only between different foods but between foods from different geographical locations. It will depend, a m o n g other things, on the e n v i r o n m e n t a l conditions o f the living animals or plants, the conditions prevailing during slaughter or harvest, and the nature o f the animals and plants themselves. In the case o f meat and fish, the tissues o f n o r m a l healthy animals contain few, if any, microorganisms and the flora is present on the outer surfaces and in the intestinal tract. A major difference between fish and other animals normally eaten as food is that fish are cold blooded whereas the others are for the most part warm blooded. T h e intestinal flora o f w a r m - b l o o d e d animals will therefore have a high proportion o f mesophilic organisms, whereas that o f fish will bepend m u c h m o r e on the e n v i r o n m e n t . Geographical factors will therefore have a bigger influence on the flora o f fish than on that o f m e a t animals. Fish
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from colder climates will have an essentially psychrotrophic flora, whereas fish from tropical countries will have a higher proportion o f mesophiles. Contaminants arising from soil or the processing environment in tropical countries will also tend to be mesophilic as opposed to those in colder climates. In meat, fish, and birds the initial microflora is primarily bacterial, with small numbers of yeasts and molds. The commonest groups of bacteria found are the gram-negative Pseudomonas, Alteromonas, Moraxella, Acinetobacter, Flavobacterium, Cytophaga, Vibrio, Aeromonas, and some Enterobacteriaceae and the gram-positive Micrococcus, coryneform, Lactobacillus, Streptococcus, Bacillus, and Brochothrix thermosphacta. They originate from the outer surfaces and intestines of the animal and from the processing environment. In raw milk the flora is again primarily bacterial; it originates from the interior of the udder, the outside surfaces of the animal, and the milk-handling equipment. The flora is predominantly the gram-positive groups Streptococcus, Micrococcus and Staphylococcus with some gram-negative Pseudomonas and AIcaligenes strains. Where a disease such as mastiffs exists in the animals, of course there are large numbers of pathogenic members o f these or other groups. Vegetables acquire most of their flora from the soil in which they are grown and from the processing area. There is evidence that some healthy, intact vegetable tissues can contain bacteria but in relatively small numbers. The main groups found on freshly harvested vegetables are the gram-positive coryneforms, lactic acid bacteria, Bacillus and hlicrococcus, and the gram-negative Pseudomonas and Enterobacteriaceae. Molds and yeasts are usually present in significant numbers. The flora of vegetables, however, will depend very much on climatic conditions and crop husbandry as well as on the harvesting methods. Fruits provide a rather different situation, the pH of most fruits being somewhat acid. Therefore the normal bacterial flora in the orchard do not survive on fruit and the flora is made up of yeasts, molds, and some lactic acid bacteria. Again the tissues of undamaged healthy fruits are sterile. Cereals and nuts are generally contaminated by soil organisms, and penetration of microorganisms into the tissues only occurs with physical damage or disease. Because of their low water activity molds and yeasts are more significant than bacteria in the initial flora. The main genera of molds in these initial flora are Alternaria, Fusarium, Helminthosporium, and Cladosporium. Manufactured foods will carry an initial flora derived from the raw materials and processing areas.
Heat Processing Heat processing, irradiation, and some chemical treatments are the only pro.Cessing parameters that function primarily by killing microorganisms. SterilIzation, by definition, should kill all microorganisms, and any microbiological spoilage is a result of post-process contamination or inadequate processing. Pasteurization, however, selectively kills the more sensitive microorganisms. Heating at substerilization temperatures will kill many organisms and injure
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others; a few will survive unharmed. Injured organisms may not be able to grow in the food, depending on the extent of the injury and the efficiency of repair mechanisms. Microorganisms vary considerably in their resistance to heat, but bacterial spores are far more resistant than vegetative cells. They can survive several minutes at 120~ or several hours at 100~ whereas vegetative cells are killed generally after a few minutes at 70-80"C. Among the vegetative organisms, gram-positive bacteria tend to be more heat resistant, and frequently strains of Micrococcus or the coryneform group survive pasteurization processes. Where foods are to be consumed without further treatment, a carefully controlled process is usually designed to ensure the killing of pathogenic bacteria. Pasteurization of milk is the prime example of such a process. Others, such as blanching of vegetables, are primarily designed to inactivate tissue enzymes. Various cooking systems applied to shellfish are primarily done to facilitate removal of the meats from the shell, and pasteurization of wines and beers is designed specifically to prevent spoilage. Because pasteurization does not kill all organisms, other controlling factors are usually necessary for further storage. Fortunately, the organisms best able to grow at low temperatures, such as the gram-negative spoilage organisms, are most sensitive to heat. Refrigeration of pasteurized products is therefore highly effective. Pasteurization of products with a low pH and water activity will frequently give a shelf stable product. Cured meats and fish products are of this type, and with refrigeration they have a shelf life of several months. Foods with a pH less than 4.5 do not require steam heating to render them microbiologically stable; the combined effect of low pH and heating is sufficient. Similarly, a combination of low pH, alcohol, and pasteurization is sufficient to render wines stable.
Refrigerated Storage The effects of freezing on microorganisms have already been described: storage of foods in the frozen state results in a gradual decrease in numbers. As with other factors, freezing and cold storage tend to kill a greater proportion of the gram-negative flora, the gram-positive organisms such as micrococci, and coryneforms being resistant. At chill temperatures, 0-5~ some microorganisms can still actively grow, and storage of foods at these temperatures has a marked selective effect on the microbial flora of foods. With raw flesh foods such as meat, poultry, fisfi, and shellfish, stored in the presence of air, a gram-negative spoilage flora develops almost irrespective of the initial flora. Bacteria belonging to the gram-negative groups Pseudomonas, Alteromonas, Moraxella and Acinetobacter become numerically preponderant. Storage temperature is the main reason for this along with the fact that most microbial spoilage occurs at the surface of such products. The generation time of these gram-negative bacteria at 0~ is of the order of 10-12 hours, whereas the gram-positive bacteria grow very slowly, if at all, below 5~ Other gramnegative bacteria such as the Enterobacteriaceae have a m i n i m u m growth temperature in the region of 6-80C, and although yeasts and molds are capable of
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growth at chili temperatures, their generation times are much longer than the gram-negative bacteria, and they start off in relatively small numbers on these products. The present state of the taxonomy of the gram-negative spoilage bacteria does not permit their characterization to species level or beyond. There are indications that if this were possible art even greater degree o f selection could be seen. Not all the gram-negative flora produce the spoilage changes, and the fact that there are sensory and chemical differences in the spoilage of different chilled foods suggests that there are differences in the spoilage flora. In general, the Pseudomonas-Alterornonas group is the more active spoiler, this having been shown by inoculating individual strains on to sterile meat and fish samples. Some Pseudomonas strains produce odors of ammonia or sulphide, some produce strong fruity odors due to esters of lower fatty acids, and Alteromonas strains produce strong ammoniacal and putrid odors. Moraxe[la strains, on ~the other hand, produce no significant odor changes, though they clearly grow quite well. The addition of other environmental constraints to chilled foods results in different spoilage characteristics in the food, reflecting selection of a different microflora. Restriction of oxygen, as when meat and fish are vacuum packed, results in the selection of psychrotrophic organisms capable of growth in microaerophilic conditions. In general, the gram-negative spoilage flora described above grows poorly under such conditions, and a facultative gram-positive flora might be expected to develop. Since they grow slower than the gramnegative ttora, the spoilage processes might also be expected to be slower. Both these things do in fact happen with meat products. The spoilage flora that develop is primarily lactic acid bacteria, coryneforms, and micrococci, and frequently Brochothrix therrnosphacta is the main spoilage organism. In some products there is competition between lactobaciUi and Brochothrix thermosphaeta; the former can inhibit the latter. However, in cooked and cured meats, Brochothrix frequently predominates. The development of the gram-positive fora in meat products is helped by the fact that fresh meat is normally between pH 5.5 and 6.2 during chill slorage, and this is near to the lower limit for growth of the gram-negative flora. In marine fish the same considerations do not apply. First, the pH of fresh fish does not fall as low and, secor~d, marine fish will support the anaerobic growth of at least some of the gram-negative spoilage bacteria. These organisms can in fact grow anaerobically by using trimethylamine oxide as an alternative terminal electron acceptor to oxygen [41. Trimethylamine oxide is present in appreciable amounts in marine fish but nat in fresh water fish or mammalian tissues. The oxide is reduced to trimethylamine which accumulates in the fish and has in fact proved to be a useful spoilage indicator. As a result of this, marine fish stored under reduced oxygen conditions will still develop a predominantly gram-negative flora. The rate of growth is somewhat slower, hence there is still some small shelf-life extension with vacuum packed fish. It has been known for many years that carbon dioxide in high enough concentrations can inhibit bacterial growth, and indeed this is one of the factors acting on the flora of vacuum-packed foods. Metabolism of the active flora in sUch packs rapidly uses any remaining oxygen and leaves an elevated carbon
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dioxide concentration. Controlled atmospheres, with increased levels of carbon dioxide, have been used for many years for bulk storage of fruits. More recently there has been considerable interest in packaging fresh meats and fish in individual packs with increased levels of carbon dioxide. In large container storage the composition of the atmosphere is controlled throughout storage. In individual retail packs this is not practical and the gas mixture is fixed at packaging but will change during storage. To distinguish the two storage conditions, the latter is best referred to as modified atmosphere packing or storage. This form of storage gives some extension of shelf life, particularly in products where the spoilage is primarily microbial, and presents an attractive and practical consumer pack. Bacteria vary in their sensitivity to carbon dioxide, but again it is the gram-negative spoilage groups that are most sensitive and the gram-positive lactic acid bacteria, micrococci, and coryneforms that are more resistant. The spoilage patterns in modified atmosphere-packed meats and fish are similar to those in vacuum-packed products. The gram-negative flora are still able to grow to some extent in marine fish but not in meat products.
Cured and Fermented Products A wide range of food products exists which are prepared by curing and fermenting. Preservation using these 2 processes is traditional and in different parts of the world a vast range of different products has been developed. The effective parameters are primarily reduction of water activity, lowering of pH, and in some, the addition of chemical preservatives. Manipulation of these 3 parameters in different ways gives rise to the wide range of products. Reduction of water activity by drying alone is used to preserve a range of products including meat, fish, fruit, vegetables, grains, coffee, milk, and eggs. When carried out properly there are no microbiological problems. These arise usually as a result of rehydration, and mold spoilage is the usual result. Removal of water by concentration of liquid foods is also widely used, for instance, with meat extracts, tomato pastes, fruit juices, and milk. Usually additional processing such as heat treatment, the addition of chemical preservatives, further reduction of water activity with various solutes, or freezing is necessary to render these products stable. The use of salt to reduce water activity is common to most cured and fermented products. It is usually used along with acids, to reduce pH, and chemical preservatives such as nitrites; sugar is also added to some products to help reduce the water activity and to add flavor to the product. Many cured and fermented products are also smoked. This introduces a mild heat treatment, some loss of water, and the addition of chemical preservatives from the smoke as further constraints on the microbial population. The gram-negative spoilage flora of fresh meat and fish products is more sensitive to most of the constraining parameters applied to cured and fermented products, and these develop in only a few very mildly cured foods. Whether the gram-positive bacteria, molds, or yeasts will develop depends on the nature of the cure. At the extreme of heavily cured products, the water activity is very low and only obligate halophilic organisms are able to grow. An example of
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this is heavily salt-cured fish where the salt concentration is > 20% and often saturated. Two common spoilage problems with these products are a "pink'" condition, resulting from the growth of pink pigmented bacteria of the genera Halococcus and Halobacterium, and a condition known as " d u n , " resulting from the growth of brown molds of the genera Sporendonema and Oospora. Both groups of organisms are strict aerobes and obligate halophiles and do not grow below 5~ Curing brines often include nitrite and nitrate as well as chloride; in addition to reducing water activity these also have some preservative effect, particularly the nitrite ion. Another reason for using nitrite, particularly in meat products, is to retain the expected and desired color of the product. In a raw, cured meat product such as bacon, there is no fermentation element and the curing salts are sufficient to inhibit the gram-negative spoilage flora. Various gram-positive organisms, usually Micrococcus and Leuconostoc species, will grow and cause spoilage. In some products halophilic Vibrio species are present, and these can account for a significant amount of spoilage. In dried and smoked bacon the additional constraints result in virtually no bacterial spoilage. Some molds are still able to grow, but being strict aerobes this is restricted to surface growth. Where large cuts of meat, such as whole sides of bacon, are concerned there is a very different situation deep in the tissues. Penetration of curing salts may be slow, and smoking will have little effect. Here facultative or strict anaerobic bacteria can grow. Spoilage is often caused by bacteria belonging to the Vibrio, Alcaligenes, Micrococcus, Proteus, and Clostridium genera. As with meats, cured fish products are extremely varied and may or may not be smoked. Lightly cured and smoked fish products require further measures such as refrigeration for microbial stability and safety. Again with the milder cures it is still possible to get a gram-negative spoilage flora developing. This occurs in products such as the kippered herring and finnan haddock where the salt concentration can be as low as 1.5%. The smoke deposits are light on these products and the process only raises the fish to 30~ during smoking. At the other end of the scale is the heavily cured "'red'herring which has a salt concentration of 10-12% and during smoking the fish temperature is raised to 60"C. These products are virtually sterile when prepared. Intermediate between these extremes there is a range of smoked and cured fish products; the spoilage flora developing on these is essentially micrococci, yeasts, or molds. Most other cured products involve some degree of fermentation. The conditions are adjusted so that varying degrees of fermentation are allowed to OCCur depending on the desired nature of the product. The primary environmental constraints are reduced water activity and pH, and these are achieved in different ways and to different degrees. At one end of the scale are the traditional European and Scandinavian cured fish where there is little degradation of the tissues, the gram-negative spoilage flora is inhibited, and gramPositive micrococci and lactic acid bacteria are allowed to grow to the extent of contributing to the characteristic flavors of these products. Brining solutions consist of salt and acetic acid, sometimes with sugar added. The final products have salt concentrations in the region of 8-9% and pH values of about 4.5 or less. Chemical preservatives, for example, benzoate or sorbate may also be
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added in some cases. At the other end o f the scale are the fish sauces and pastes of South East Asia. Proteolytic enzymes, either from the fish itself or added, actively decompose the tissues and a considerable amount of microbial fermentation also occurs. Salt levels are normally high, and oxygen is usually excluded during the process. The main active bacteria in these processes therefore belong to the genera Lactobacillus, Pediococcus, Leuconostoc, and Bacillus. The pH rapidly falls to below 4.0 in the first stages of fermentation, and the salt concentration is usually >20% in the final product. The products are generally stable, but sauces are often pasteurized as well to give a long shelf life. There are many variations on this process. In some, carbohydrate is added to encourage a lactic fermentation; in others, carbohydrate previously fermented with yeasts is added. Other variations are introduced by stopping the degradation processes at various levels so that a wide range of products from fully liquefied to those with the flesh more or less intact are produced with a large number of regional variations, giving different products. Fermented meat products consist mainly of the many varieties of sausages. Their manufacture involves reduction of water activity by salting and drying, reduction of pH, addition of chemical preservatives such as nitrate and nitrite, and smoking which adds more chemical preservatives and gives a mild heat treatment. Normally various spices are added, some of which also have antimicrobial effects. There are basically 2 types of fermented sausage, i.e., dried products which have an aging time of several months and a semi-dried product where the aging time is 1-3 weeks. The dried sausages have a water content below 40% (i.e., water activity below 0.93), are heavily spiced, and are not usually smoked. The semi-dried products have a water content of 40-60% (i.e., water activity 0.930.98) and are frequently smoked. They are not microbiologically stable and should be stored refrigerated. The microbial flora of both types of sausage is gram-positive and microaerophilic, the normal gram-negative spoilage flora of fresh meats being suppressed. In modern processing, starter cultures are used to ensure an efficient lactic fermentation of the desired type. Starter cultures are combinations of strains belonging to the genera Lactobacillus, Pediococcus, and Micrococcus. Vegetables are also preserved by fermentation and acidification. In North America and Western Europe the commonest vegetable prepared in this way is cabbage, though there are several others. Cabbage is usually chopped and dry salted. The salt draws out plant juices containing carbohydrates and other nutrients to form a brine. Large numbers of soil bacteria and organisms from the processing plant are present, and a series of changes occur in the flora ending with a predominant lactic fermentation. In the initial stages, gram-negative organisms tend to predominate, and since the fermentation vats are covered to restrict the access of oxygen, these are facultative organisms such as coliforms. The temperature of fermentation is usually controlled at 20-25~ Once the lactic acid bacteria begin to grow, the pH drops to 4.6-4.9. The predominant organism in the early stages of lactic fermentation are Leuconostoc spp. and these are followed by more acid-tolerant lactobacilli and pediococci. After a few weeks the process is complete and the final product has a pH of 3.5-3.8.
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The finished sauerkraut is marketed raw, pasteurized, or canned. Spoilage can occur if the initial lactic fermentation fails and the coliform organisms cause spoilage; this usually happens because of uneven or inadequate salting. If oxygen is allowed in the process, oxidative yeasts can grow, using the lactic acid and causing a rise in pH. Less acid-tolerant organisms are then able to grow and spoil the product. A wide range of fermented milk products is available throughout the world, including fermented milks such as yoghurt and a wide range o f cheeses. Fermentation is brought about by lactic acid bacteria, sometimes along with yeasts. Commercially, starter cultures are used, and the characteristic properties o f the different products result largely from the use of appropriate microorganisms and manipulation of the fermentation conditions. Microbial stability in these products is due to low pH along with refrigerated storage and sometimes pasteurization. In a similar way a vast range o f alcoholic beverages is produced, essentially by fermentation of carbohydrate substrates with yeasts which primarily produce ethyl alcohol. The pH of fruit juices used for wines is naturally low, but acid is frequently added to ensure a low pH and favor the yeasts. The characteristic sensory properties of the individual products are achieved by selection of substrate and individual yeast strain and manipulation o f the processing conditions. Microbial stability depends primarily on low pH, alcohol concentration, chemical additives (particularly sulphur dioxide), and sometimes pasteurization. In commercial production, selection of the appropriate fermentation flora is ensured by pasteurization of the raw material and inoculation with the appropriate starter culture and then adjusting the fermentation conditions to favor these particular organisms.
Chemical Preservatives The distinction between chemical preservatives, curing salts, and fermentation processes is one of convenience. The various salts, organic acids, alcohols, and antibiotics present in these processes are still chemical preservaties. There remain a number of other chemicals that are deliberately added to foods to inhibit or kill microorganisms. The amount and type of additives permitted in foods are carefully controlled by government agencies and vary in different countries. Organic acids and their esters are commonly added. It is the undissociated molecule that is responsible for the antimicrobial activity (as opposed to any effects due to their ability to reduce the pH). Because the pK values of organic acids are low (pH 3-5), they are therefore more active in high acid foods. They are also more active against molds though in high enough concentration they inhibit a wide range of organisms. Acetic acid is one of the most widely used, and the presence of 1-2% of undissociated acid in meat, fish, and vegetables will usually inhibit or kill most microorganisms. Only Acetobacter spp. and certain lactic acid bacteria are appreciably resistant, and Acetobacter spp. are in fact used to manufacture Vinegars. Benzoic acid is primarily active against molds and yeasts; many
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spoilage bacteria will not be inhibited at the levels permitted. Similarly, propionic acid is highly effective against molds and is useful against some bacteria. Sorbic acid has a wider spectrum of activity. It acts against catalase-positive organisms including molds, yeasts, and bacteria but is ineffective against catalase-negative bacteria. It is therefore most useful for inhibiting aerobic growth in fermented or high acid foods. An exception to the general statement about low pH and activity is p-hydroxybenzoic acid and its esters; they have a relatively high pK value and are effective against yeasts and molds up to neutral pH values. The activity of organic acids and their esters is particularly affected by other environmental parameters; pH has been discussed. Others, such as water activity, Eh, and temperature, have marked effects, and synergism is common between organic acids, their esters, and these parameters, as well as between different preservatives. Carbon dioxide has already been mentioned as a preservative; other gases are also effective in some circumstances. Sulphur dioxide is widely used, often applied as sulphite, bisulphite, and metabisulphite salts. Its antimicrobial activity is largely related to the unbound nonionized molecular form and hence is most active in high acid foods. Its commonest use is with soft fruits, fruit juices, wines, and sausages, and it is most effective against molds, yeasts, and gram-negative bacteria. Antibiotics were, for a time, thought to be very useful for inhibiting food spoilage bacteria; however, for a variety of reasons, most are now prohibited as food additives. Two are still found useful and have no therapeutic applications: natamycin and nisin. Natamycin is produced by Streptomyces natalensis and is primarily active against fungi. It is used, for instance, to inhibit molds in sausages and as a treatment for cheese rinds, against to prevent mold growth. Nisin is produced by Streptococcus lactis and, therefore, occurs naturally in soured milks and farmhouse cheese. It has a relatively narrow range of activity, affecting only gram-positive bacteria. It is most stable at acid pH values and is of little use for preventing spoilage of fresh meat and fish. It could, however, have specific applications in a variety of foods to inhibit grampositive food poisoning bacteria, though this requires further investigation.
Microbial Health Hazards Food poisoning microorganisms behave in the same ways as other organisms to changing environmental factors. In many cases they are less resistant than spoilage organisms, and processes designed to prevent spoilage will automatically prevent their growth. In designing or changing processes it is important, however, to take the properties of food poisoning organisms into consideration. With heat processed foods the main health hazards arise with spore-forming bacteria, particularly Clostridium botulinum, Clostridium perfringens, and Bacillus cereus. The proteolytic strains of C. botulinum are the most heat-resistant pathogens, though some strains of B. cereus are equally so. Sterilization procedures are governed by the higher heat resistance of putrefactive spore formers which cause spoilage. Post-process recontamination, however, can arise with
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a range of food poisoning bacteria, not necessarily spore formers. Since the process has eliminated the normal microflora, if such contamination does occur, t.he contaminating organism grows rapidly with no competitive flora. Pasteurtzatton processes were originally introduced to eliminate pathogens, e.g., Mycobacterium tuberculosis from milk. Other preservation methods--refrigerated storage being the commonest--have to be applied, however, since the process does not kill all organisms. Many other pathogenic bacteria have heat resistances comparable to spoilage bacteria, yeasts, and molds; these include Brucella spp., Salmonella spp., and Staphylococcus aureus. Mild heat processing will not inactivate all preformed microbial toxins, the prime example being staphylococcal toxins; these are relatively beat resistant, and if they have been produced before heat processing, can survive the process. Refrigerated storage will control most food poisoning organisms provided the temperature is properly controlled. Salmonella, Shigella, and Staphylococcus spp. have a minimum growth temperature of 6-8"C. With C. perfringens and many E. coil strains the minimum is about 12~ and these should present rio problems in refrigerated foods. The nonproteolytic strains o f C. botulinum, however, will grow in temperatures as low as 3.5~ and Yersinca enterocolitica will grow slowly at 0~ These organisms can give rise to problems in refrigerated foods, especially if other treatments such as mild heat treatment and smoke curing have inhibited or killed the normal gram-negative spoilage flora. Growth of the psychrotrophic C. botufinum strains can be a particular problem in such foods, since they do not produce spoilage odors and flavors. Cured and fermented foods rely on low water activity and pH primarily. Salmonella spp. and Staphylococcus aureus are controlled below pH 4.fi, proteolytie strains of C. botulinum below pH 4.5, and nonproteolytic strains below pH 5.0. These figures do not take account of synergism with other factors when inhibition can be achieved at somewhat higher values. Similarly, low water activity inhibits most food poisoning bacteria as well as spoilage bacteria. In the range of fresh foods, at aw = 0.98 or higher, C. per.fringens and C. botulinum will grow, and problems arise with these spore formers in heatprocessed foods. Salmonella spp. and most Enterobacteriaceae will compete well with spoilage organisms in these foods at temperatures above about 10~ Staphylococcus aureus and toxin-producing molds do not compete well in this type of food primarily because of their slower growth rates. Most food-borne pathogens are inhibited in the range ofaw = 0.98-0.93; however, S. aureus will grow down to a,~ --- 0.85. Bacteriological media with 10-12% added salt are frequently used to selectively isolate this organism. For most food-poisoning bacteria the inhibitory levels of most environmental factors and chemical additives are known so that safe parameters can usually be specified. Achieving these parameters in food products is, however, not always an easy matter.
Conclusions Fresh animal and plant tissues that are suitable for human food are equally, if not more suitable as food for microorganisms. At the point of slaughter or
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harvest, raw foods have a relatively large and varied microbial population. Preservation o f foods for h u m a n use is therefore primarily a question o f m a nipulating e n v i r o n m e n t a l a n d processing factors to kill m i c r o o r g a n i s m s or reduce their growth, and in some cases to encourage the growth o f specific desired organisms. Definitions o f desirable and undesirable when applied to acceptability o f foods are subjective. Spoiled foods in one c o m m u n i t y can be a culinary delicacy in another. G i v e n a knowledge o f the desired properties o f the food, a knowledge o f the effects o f ecological pressures and processing parameters on the growth and m e t a b o l i s m o f m i c r o o r g a n i s m s should p e r m i t specification o f optimal processing a n d storage conditions. While there is a considerable b o d y o f knowledge concerning the effects o f the various parameters individually, i n f o r m a t i o n on their interactions is only beginning to accumulate. Indeed, techniques for efficient investigation o f m u l t i - p a r a m e t e r situations are only just b e c o m i n g available. O p t i m i z a t i o n o f control and processing p a r a m eters is one o f the m a i n investigatory fields facing the f o o d microbiologist in the near future. A n o t h e r field recently opened up is the use o f genetically engineered strains in food processing. Traditionally, selection o f particular features in a m i c r o o r g a n i s m has been based on r a n d o m m u t a t i o n and selection o f mutants. The possibility o f designing strains for specific purposes n o w exists. Even where optimal processing and storage conditions are known, there is still r o o m for i m p r o v e m e n t o f control procedures; m o s t food poisoning outbreaks occur when something goes wrong. References 1. Baldham CD (1856) Prose halieutics, John W Parker & Son, London 2. Cutting CL (1955) Fish saving. A history of fish processing from ancient to modern times. Leonard Hill (Books) Ltd, London 3. Deraniyagala PEP (1933) Cured marine products of Ceylon. Celon J Sci (C) 5:49 4. Easter MC, Gibson DM, Ward FB (1982) A conductance method for the assay and study of bacterial trimethylamine oxide reduction. J Appl Bacteriol 52:357-365 5. Erichsen I (1983) Fermented fish and meat products: the present position and future possibilities. In: Roberts TA, Skinner FA (eds) Food microbiology SAB Symposium Series No 11, Academic Press, London, pp 271-286 6. Gould GW, Brown MH, Fletcher BC (1983) Mechanisms of action of food preservation procedures. In: Roberts TA, Skinner FA (eds) Food microbiology SAB Symposium Series No 11, Academic Press, London, pp 67-84 7. International Commission on MicrobiologicalSpecifications for Foods (1980) Microbial ecology of foods. Vol. I. Factors affectinglife and death of microorganisms. Academic Press, New York 8. International Commission on MicrobiologicalSpecifications for Foods (1980) Microbial ecology of foods. Vol II. Food commodities. Academic Press, New York 9. Pedersen CS (1979) Microbiology of food fermentations. AVI Publishing Co, Westport, Connecticut 10. Radcliffe W (1921) Fishing from earliest times. John Murray Ltd, London. I 1. Ratkowsky DA, Olley J, McMeekin TA, Ball A (1982) Relationship between temperature and growth rate of bacterial cultures. J Bacteriol. 149(1):1-5 12. Roberts TA, Skinner FA (eds) (1983) Food microbiology, advances and prospects. SAB Symposium Series No 11, Academic Press, London 13. Wee-Ching W Yen (1908) The importance of fishing and its allied industry, the manufacture of salt. Address before the International Fishing Congress, Washington DC, USA 14. Wilkinson Sir JG (1878) The manners and customs of ancient Egyptians. Revised by Birch S. John Murray, Lid, London
MICROBIAL ECOLOGY 1996 Springer-Veflag
Ecology of Food Microorganisms G. Hobbs Torr~ Research Station, PO Box 31, 135 Abbey Road, Aberdeen AB9 8DG, UK Abstract. The behavior of microorganisms in foods is governed by the constraints applied to the microflora by a variety of envffonmental and ecological factors. These include water activity, pH, Eh, chemical composition, the presence o f natural or added antimicrobial compounds, and storage temperature, as well as processing factors such as the application of heat and physical manipulation. Control of these factors will govern whether the food spoils or not, whether any microbial health hazard arises, and whether desired microbial processes are successful or not. While much is known about the effects of individual environmental factors, the effects due to their interactions are less understood. The two main problems now facing the food microbiologist are optimization of environmental parameters and the selection of strains with specific properties. A better understanding of the mechanisms of acf~on and interactions between the various environmental factors, coupled with the application o f modern techniques to produce strains with particular properties, will lead to optimum use of food supplies and improvements in quality. There is also potential for the development o f new and novel foods.
Introduction The preservation of animal and plant tissues for use as food essentially consists of manipulating the environmental factors ~n the food so that the normal degradation processes brought about by microorganisms are slowed down or prevented altogether. The success or otherwise o f preservation processes depends on how the microbial population responds to the constraints applied by handling and processing. It will depend on how successfully the procedures prevent undesirable organisms from gainirtg access and how successfully they inhibit microbial growth or permit the preferential growth o f desirable organisms. Post-slaughter or post-harvest foods immediately have a particular microflora, the actual composition of which will depend on the nature of the food and the slaughter or harvesting conditions. ~n healthy plants and animals the tissues are essentially sterile and microorganisms are present primarily on the outer surfaces, and in the case of animals, in the intestinal tract. Subsequent handling, processing, artd storage wil~ cause changes in the micro~tora in response to various ecological pressures. Ttte precise nature o f these pressures
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G. Hobbs
teria to reducing temperatures below a m b i e n t and thus provide a measure o f spoilage [11]. Although the relationship is empirical, it does relate bacterial growth and temperature sufficiently well to be useful. Where spoilage is mainly bacterial, the spoilage rate at different temperatures can be predicted from knowledge o f spoilage at 0~ Evidence is beginning to accrue now to show that the relationship can be used to predict shelf life o f chilled foods. Lower temperatures therefore reduce microbial growth until it eventually ceases. Freezing and subsequent thawing have the additional effect o f killing a proportion o f the microflora and damaging others. T h e reduction in n u m b e r s is relatively small, however, i.e., less than 1 log cycle, and it is doubtful if it has a noticeable effect on the spoilage o f thawed frozen foods. At higher temperatures inactivation reactions b e c o m e significant. Proteins, including enzymes, nucleic acids, and other cell c o m p o n e n t s are all sensitive to high temperatures, and there is a wide variation in the response o f different organisms. Some extreme psychrophilic organisms will not survive long at 20~ and above, whereas extreme thermophiles will survive and even grow up to 100~ For the majority o f organisms, however, temperatures f r o m 65~ upwards are lethal. The death rate o f microorganisms is logarithmic; exposure time and initial n u m b e r s are therefore i m p o r t a n t considerations. The m o s t highly resistant forms o f microorganisms are bacterial spores. While they do not grow at high temperatures, they survive t h e m very well. C o m m e r c i a l sterilization o f foods requires t r e a t m e n t with steam u n d e r pressure, and laboratory sterilization procedures c o m m o n l y require a temperature o f 12 I~ for 20 rain.
Water Activity The water activity o f a food is the ratio o f the water v a p o r pressure o f the food to that o f pure water at the same temperature, and is a measure o f the water available for microbial growth. It is therefore related to the a m o u n t o f solutes present in the food as well as the a m o u n t o f water. As water activity falls, the growth rates o f microorganisms are reduced until growth ceases. Below this value, which varies for different organisms, there will be a slow decline in the numbers o f organisms surviving. Most bacteria will not grow below an aw = 0.90, whereas some yeasts and molds will grow as low as aw = 0.60. T o relate these values to solute concentration, a value o f aw = 0.995 corresponds to a salt concentration o f 0.87% or a sugar concentration o f 0.92%, whereas a~ = 0.90 corresponds to 16.2% and 14% respectively, and aw = 0.753 is equivalent to a saturated sodium chloride solution. The o p t i m u m range o f water activity for most microorganisms is aw = 0.95--0.99.
Acidity and Alkalinity Each organism has a range o f p H within which growth is possible, and it will have an o p t i m u m within this range. Most natural e n v i r o n m e n t s have a p H between 5 and 9, and the majority o f microorganisms have o p t i m a within this range. Very few organisms can grow below p H 2 or above p H 10. T h e range
Ecologyof Food Microorganisms
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over which an individual organism can grow is usually not more than 3 to 4 units the majority of bacteria having an optimum pH around neutrality and molds and yeasts at slightly acid values (pH 5-6). At 91-I values outside a particular organism's range a proportion of the population will die, and prolonged storage under these conditions will result in a further decline.
Oxidation-Reduction Potential The range of redox potentials (E~,) found in nature is +0.82 to - 0 . 4 2 volts, and it can be poised at different levels with various chemical agents. In foods, the Eh will depend on the level of oxidation or reduction of the various components and on the composition of the atmosphere surrounding the food. In most cases the Eh will not be uniform throughout a particular product; for instance, in contact with air the outside of a piece of meat or fish will be highly aerobic with a high positive Ej, whereas the inside will be anaerobic with a negative E~. Obligate aerobic organisms require oxygen or a highly oxidized environment for growth, whereas obligate anaerobic organisms require highly reduced conditions and the absence of oxygen. Between these extremes are large numbers of facultative organisms that can grow under most conditions, though they have varying optimum Eh conditions. In general, aerobic metabolism is more energy efficient and usually results in more growth. Anaerobic metabolism, however, although less efficient, usually results in a greater production of odorous and flavorous compounds which are relevant to the acceptability of foods.
Nutritional Status and Preservatives Raw plant and animal tissues generally provide an excellent nutrient medium for the growth of a wide range of microorganisms. Without the application of preservation procedures they would be rapidly decomposed by microorganisms and this is the normal course of events in nature with dead plants and animals. S o m e tissues tend to favor one group of organisms because o f specific components. An example of this is the presence of organic acids artd low pH values in many fruits. Many bacteria are inhibited by these conditions, and degradation tends to be brought about by molds and yeasts. A number of preservatives are permitted for general use in foods because they have been used in traditional preservation techniques for a very long time. Salt, various sugars, acetic acid, and smoke constituents are examples. In the main, however, the addition of chemical inhibitors to foods is strictly controlled in most countries. The most commonly used include organic acids and their esters, generally with chain lengths less than Cm, some antibiotics, and inorganic salts such as sulphite and nitrite. In addition to reducing p H and water activity, chemical preservatives generally have some specific antimicrobial effects frequently against a wide range of organisms but sometimes against a particular ~roup.
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G. Hobbs
Interactions Between Environmental Factors There is a substantial a m o u n t o f i n f o r m a t i o n available on the individual effects o f the various e n v i r o n m e n t a l factors on growth o f microorganisms and thus on food preservation. It is relatively recently, however, that the full extent o f interactions between these parameters has been realized. T h e interactions between water activity, pH, and t e m p e r a t u r e are probably the best known. I f two o f these are o p t i m u m , growth will occur at extremes o f the third o v e r a range characteristic for the particular organism. This range is appreciably narrower, however, when either or both o f the others are not optimal. A n o t h e r example is the increased sensitivity o f bacteria to heat in the presence o f curing salts and with reduced pH. Also, m a n y bacteria are m o r e sensitive to the inhibitory effects o f nitrite in the presence o f increased concentrations o f s o d i u m chloride. An extreme example is the difference in sensitivity o f bacteria to "wet heat" and " d r y heat." The former, where the water activity is close to 1.00, is m u c h more effective than the latter at killing bacteria. In only a few cases are the mechanisms o f action o f e n v i r o n m e n t a l factors understood; in still fewer cases is the m e c h a n i s m o f interactions between various factors understood. T h e evidence available to explain these m e c h a n i s m s was recently reviewed [6], and the conclusion is that as far as resistance o f bacterial spores to heat and the effects o f lowering water activity and p H on growth are concerned, the cell mechanisms operate to maintain homeostasis with respect to water inside the cells. W h e n the cell is no longer able to do this growth ceases, or in the case o f bacterial spores, heat resistance is lost. Such a c o m m o n mechanism could well explain why when two or m o r e factors are acting together there are synergistic effects.
Preservation of Foods
lnitial Microbial Flora There are extensive reports in the literature describing the i m m e d i a t e postslaughter or post-harvest flora o f a wide range o f foods [7, 8, 12]. As might be expected, the initial flora is very varied, not only between different foods but between foods from different geographical locations. It will depend, a m o n g other things, on the e n v i r o n m e n t a l conditions o f the living animals or plants, the conditions prevailing during slaughter or harvest, and the nature o f the animals and plants themselves. In the case o f meat and fish, the tissues o f n o r m a l healthy animals contain few, if any, microorganisms and the flora is present on the outer surfaces and in the intestinal tract. A major difference between fish and other animals normally eaten as food is that fish are cold blooded whereas the others are for the most part warm blooded. T h e intestinal flora o f w a r m - b l o o d e d animals will therefore have a high proportion o f mesophilic organisms, whereas that o f fish will bepend m u c h m o r e on the e n v i r o n m e n t . Geographical factors will therefore have a bigger influence on the flora o f fish than on that o f m e a t animals. Fish
Ecologyof Food Microorganisms
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from colder climates will have an essentially psychrotrophic flora, whereas fish from tropical countries will have a higher proportion o f mesophiles. Contaminants arising from soil or the processing environment in tropical countries will also tend to be mesophilic as opposed to those in colder climates. In meat, fish, and birds the initial microflora is primarily bacterial, with small numbers of yeasts and molds. The commonest groups of bacteria found are the gram-negative Pseudomonas, Alteromonas, Moraxella, Acinetobacter, Flavobacterium, Cytophaga, Vibrio, Aeromonas, and some Enterobacteriaceae and the gram-positive Micrococcus, coryneform, Lactobacillus, Streptococcus, Bacillus, and Brochothrix thermosphacta. They originate from the outer surfaces and intestines of the animal and from the processing environment. In raw milk the flora is again primarily bacterial; it originates from the interior of the udder, the outside surfaces of the animal, and the milk-handling equipment. The flora is predominantly the gram-positive groups Streptococcus, Micrococcus and Staphylococcus with some gram-negative Pseudomonas and AIcaligenes strains. Where a disease such as mastiffs exists in the animals, of course there are large numbers of pathogenic members o f these or other groups. Vegetables acquire most of their flora from the soil in which they are grown and from the processing area. There is evidence that some healthy, intact vegetable tissues can contain bacteria but in relatively small numbers. The main groups found on freshly harvested vegetables are the gram-positive coryneforms, lactic acid bacteria, Bacillus and hlicrococcus, and the gram-negative Pseudomonas and Enterobacteriaceae. Molds and yeasts are usually present in significant numbers. The flora of vegetables, however, will depend very much on climatic conditions and crop husbandry as well as on the harvesting methods. Fruits provide a rather different situation, the pH of most fruits being somewhat acid. Therefore the normal bacterial flora in the orchard do not survive on fruit and the flora is made up of yeasts, molds, and some lactic acid bacteria. Again the tissues of undamaged healthy fruits are sterile. Cereals and nuts are generally contaminated by soil organisms, and penetration of microorganisms into the tissues only occurs with physical damage or disease. Because of their low water activity molds and yeasts are more significant than bacteria in the initial flora. The main genera of molds in these initial flora are Alternaria, Fusarium, Helminthosporium, and Cladosporium. Manufactured foods will carry an initial flora derived from the raw materials and processing areas.
Heat Processing Heat processing, irradiation, and some chemical treatments are the only pro.Cessing parameters that function primarily by killing microorganisms. SterilIzation, by definition, should kill all microorganisms, and any microbiological spoilage is a result of post-process contamination or inadequate processing. Pasteurization, however, selectively kills the more sensitive microorganisms. Heating at substerilization temperatures will kill many organisms and injure
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G. Hobbs
others; a few will survive unharmed. Injured organisms may not be able to grow in the food, depending on the extent of the injury and the efficiency of repair mechanisms. Microorganisms vary considerably in their resistance to heat, but bacterial spores are far more resistant than vegetative cells. They can survive several minutes at 120~ or several hours at 100~ whereas vegetative cells are killed generally after a few minutes at 70-80"C. Among the vegetative organisms, gram-positive bacteria tend to be more heat resistant, and frequently strains of Micrococcus or the coryneform group survive pasteurization processes. Where foods are to be consumed without further treatment, a carefully controlled process is usually designed to ensure the killing of pathogenic bacteria. Pasteurization of milk is the prime example of such a process. Others, such as blanching of vegetables, are primarily designed to inactivate tissue enzymes. Various cooking systems applied to shellfish are primarily done to facilitate removal of the meats from the shell, and pasteurization of wines and beers is designed specifically to prevent spoilage. Because pasteurization does not kill all organisms, other controlling factors are usually necessary for further storage. Fortunately, the organisms best able to grow at low temperatures, such as the gram-negative spoilage organisms, are most sensitive to heat. Refrigeration of pasteurized products is therefore highly effective. Pasteurization of products with a low pH and water activity will frequently give a shelf stable product. Cured meats and fish products are of this type, and with refrigeration they have a shelf life of several months. Foods with a pH less than 4.5 do not require steam heating to render them microbiologically stable; the combined effect of low pH and heating is sufficient. Similarly, a combination of low pH, alcohol, and pasteurization is sufficient to render wines stable.
Refrigerated Storage The effects of freezing on microorganisms have already been described: storage of foods in the frozen state results in a gradual decrease in numbers. As with other factors, freezing and cold storage tend to kill a greater proportion of the gram-negative flora, the gram-positive organisms such as micrococci, and coryneforms being resistant. At chill temperatures, 0-5~ some microorganisms can still actively grow, and storage of foods at these temperatures has a marked selective effect on the microbial flora of foods. With raw flesh foods such as meat, poultry, fisfi, and shellfish, stored in the presence of air, a gram-negative spoilage flora develops almost irrespective of the initial flora. Bacteria belonging to the gram-negative groups Pseudomonas, Alteromonas, Moraxella and Acinetobacter become numerically preponderant. Storage temperature is the main reason for this along with the fact that most microbial spoilage occurs at the surface of such products. The generation time of these gram-negative bacteria at 0~ is of the order of 10-12 hours, whereas the gram-positive bacteria grow very slowly, if at all, below 5~ Other gramnegative bacteria such as the Enterobacteriaceae have a m i n i m u m growth temperature in the region of 6-80C, and although yeasts and molds are capable of
Ecology of Food Microorganisms
23
growth at chili temperatures, their generation times are much longer than the gram-negative bacteria, and they start off in relatively small numbers on these products. The present state of the taxonomy of the gram-negative spoilage bacteria does not permit their characterization to species level or beyond. There are indications that if this were possible art even greater degree o f selection could be seen. Not all the gram-negative flora produce the spoilage changes, and the fact that there are sensory and chemical differences in the spoilage of different chilled foods suggests that there are differences in the spoilage flora. In general, the Pseudomonas-Alterornonas group is the more active spoiler, this having been shown by inoculating individual strains on to sterile meat and fish samples. Some Pseudomonas strains produce odors of ammonia or sulphide, some produce strong fruity odors due to esters of lower fatty acids, and Alteromonas strains produce strong ammoniacal and putrid odors. Moraxe[la strains, on ~the other hand, produce no significant odor changes, though they clearly grow quite well. The addition of other environmental constraints to chilled foods results in different spoilage characteristics in the food, reflecting selection of a different microflora. Restriction of oxygen, as when meat and fish are vacuum packed, results in the selection of psychrotrophic organisms capable of growth in microaerophilic conditions. In general, the gram-negative spoilage flora described above grows poorly under such conditions, and a facultative gram-positive flora might be expected to develop. Since they grow slower than the gramnegative ttora, the spoilage processes might also be expected to be slower. Both these things do in fact happen with meat products. The spoilage flora that develop is primarily lactic acid bacteria, coryneforms, and micrococci, and frequently Brochothrix therrnosphacta is the main spoilage organism. In some products there is competition between lactobaciUi and Brochothrix thermosphaeta; the former can inhibit the latter. However, in cooked and cured meats, Brochothrix frequently predominates. The development of the gram-positive fora in meat products is helped by the fact that fresh meat is normally between pH 5.5 and 6.2 during chill slorage, and this is near to the lower limit for growth of the gram-negative flora. In marine fish the same considerations do not apply. First, the pH of fresh fish does not fall as low and, secor~d, marine fish will support the anaerobic growth of at least some of the gram-negative spoilage bacteria. These organisms can in fact grow anaerobically by using trimethylamine oxide as an alternative terminal electron acceptor to oxygen [41. Trimethylamine oxide is present in appreciable amounts in marine fish but nat in fresh water fish or mammalian tissues. The oxide is reduced to trimethylamine which accumulates in the fish and has in fact proved to be a useful spoilage indicator. As a result of this, marine fish stored under reduced oxygen conditions will still develop a predominantly gram-negative flora. The rate of growth is somewhat slower, hence there is still some small shelf-life extension with vacuum packed fish. It has been known for many years that carbon dioxide in high enough concentrations can inhibit bacterial growth, and indeed this is one of the factors acting on the flora of vacuum-packed foods. Metabolism of the active flora in sUch packs rapidly uses any remaining oxygen and leaves an elevated carbon
24
G. Hobbs
dioxide concentration. Controlled atmospheres, with increased levels of carbon dioxide, have been used for many years for bulk storage of fruits. More recently there has been considerable interest in packaging fresh meats and fish in individual packs with increased levels of carbon dioxide. In large container storage the composition of the atmosphere is controlled throughout storage. In individual retail packs this is not practical and the gas mixture is fixed at packaging but will change during storage. To distinguish the two storage conditions, the latter is best referred to as modified atmosphere packing or storage. This form of storage gives some extension of shelf life, particularly in products where the spoilage is primarily microbial, and presents an attractive and practical consumer pack. Bacteria vary in their sensitivity to carbon dioxide, but again it is the gram-negative spoilage groups that are most sensitive and the gram-positive lactic acid bacteria, micrococci, and coryneforms that are more resistant. The spoilage patterns in modified atmosphere-packed meats and fish are similar to those in vacuum-packed products. The gram-negative flora are still able to grow to some extent in marine fish but not in meat products.
Cured and Fermented Products A wide range of food products exists which are prepared by curing and fermenting. Preservation using these 2 processes is traditional and in different parts of the world a vast range of different products has been developed. The effective parameters are primarily reduction of water activity, lowering of pH, and in some, the addition of chemical preservatives. Manipulation of these 3 parameters in different ways gives rise to the wide range of products. Reduction of water activity by drying alone is used to preserve a range of products including meat, fish, fruit, vegetables, grains, coffee, milk, and eggs. When carried out properly there are no microbiological problems. These arise usually as a result of rehydration, and mold spoilage is the usual result. Removal of water by concentration of liquid foods is also widely used, for instance, with meat extracts, tomato pastes, fruit juices, and milk. Usually additional processing such as heat treatment, the addition of chemical preservatives, further reduction of water activity with various solutes, or freezing is necessary to render these products stable. The use of salt to reduce water activity is common to most cured and fermented products. It is usually used along with acids, to reduce pH, and chemical preservatives such as nitrites; sugar is also added to some products to help reduce the water activity and to add flavor to the product. Many cured and fermented products are also smoked. This introduces a mild heat treatment, some loss of water, and the addition of chemical preservatives from the smoke as further constraints on the microbial population. The gram-negative spoilage flora of fresh meat and fish products is more sensitive to most of the constraining parameters applied to cured and fermented products, and these develop in only a few very mildly cured foods. Whether the gram-positive bacteria, molds, or yeasts will develop depends on the nature of the cure. At the extreme of heavily cured products, the water activity is very low and only obligate halophilic organisms are able to grow. An example of
Ecology of Food Microorganisms
25
this is heavily salt-cured fish where the salt concentration is > 20% and often saturated. Two common spoilage problems with these products are a "pink'" condition, resulting from the growth of pink pigmented bacteria of the genera Halococcus and Halobacterium, and a condition known as " d u n , " resulting from the growth of brown molds of the genera Sporendonema and Oospora. Both groups of organisms are strict aerobes and obligate halophiles and do not grow below 5~ Curing brines often include nitrite and nitrate as well as chloride; in addition to reducing water activity these also have some preservative effect, particularly the nitrite ion. Another reason for using nitrite, particularly in meat products, is to retain the expected and desired color of the product. In a raw, cured meat product such as bacon, there is no fermentation element and the curing salts are sufficient to inhibit the gram-negative spoilage flora. Various gram-positive organisms, usually Micrococcus and Leuconostoc species, will grow and cause spoilage. In some products halophilic Vibrio species are present, and these can account for a significant amount of spoilage. In dried and smoked bacon the additional constraints result in virtually no bacterial spoilage. Some molds are still able to grow, but being strict aerobes this is restricted to surface growth. Where large cuts of meat, such as whole sides of bacon, are concerned there is a very different situation deep in the tissues. Penetration of curing salts may be slow, and smoking will have little effect. Here facultative or strict anaerobic bacteria can grow. Spoilage is often caused by bacteria belonging to the Vibrio, Alcaligenes, Micrococcus, Proteus, and Clostridium genera. As with meats, cured fish products are extremely varied and may or may not be smoked. Lightly cured and smoked fish products require further measures such as refrigeration for microbial stability and safety. Again with the milder cures it is still possible to get a gram-negative spoilage flora developing. This occurs in products such as the kippered herring and finnan haddock where the salt concentration can be as low as 1.5%. The smoke deposits are light on these products and the process only raises the fish to 30~ during smoking. At the other end of the scale is the heavily cured "'red'herring which has a salt concentration of 10-12% and during smoking the fish temperature is raised to 60"C. These products are virtually sterile when prepared. Intermediate between these extremes there is a range of smoked and cured fish products; the spoilage flora developing on these is essentially micrococci, yeasts, or molds. Most other cured products involve some degree of fermentation. The conditions are adjusted so that varying degrees of fermentation are allowed to OCCur depending on the desired nature of the product. The primary environmental constraints are reduced water activity and pH, and these are achieved in different ways and to different degrees. At one end of the scale are the traditional European and Scandinavian cured fish where there is little degradation of the tissues, the gram-negative spoilage flora is inhibited, and gramPositive micrococci and lactic acid bacteria are allowed to grow to the extent of contributing to the characteristic flavors of these products. Brining solutions consist of salt and acetic acid, sometimes with sugar added. The final products have salt concentrations in the region of 8-9% and pH values of about 4.5 or less. Chemical preservatives, for example, benzoate or sorbate may also be
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added in some cases. At the other end o f the scale are the fish sauces and pastes of South East Asia. Proteolytic enzymes, either from the fish itself or added, actively decompose the tissues and a considerable amount of microbial fermentation also occurs. Salt levels are normally high, and oxygen is usually excluded during the process. The main active bacteria in these processes therefore belong to the genera Lactobacillus, Pediococcus, Leuconostoc, and Bacillus. The pH rapidly falls to below 4.0 in the first stages of fermentation, and the salt concentration is usually >20% in the final product. The products are generally stable, but sauces are often pasteurized as well to give a long shelf life. There are many variations on this process. In some, carbohydrate is added to encourage a lactic fermentation; in others, carbohydrate previously fermented with yeasts is added. Other variations are introduced by stopping the degradation processes at various levels so that a wide range of products from fully liquefied to those with the flesh more or less intact are produced with a large number of regional variations, giving different products. Fermented meat products consist mainly of the many varieties of sausages. Their manufacture involves reduction of water activity by salting and drying, reduction of pH, addition of chemical preservatives such as nitrate and nitrite, and smoking which adds more chemical preservatives and gives a mild heat treatment. Normally various spices are added, some of which also have antimicrobial effects. There are basically 2 types of fermented sausage, i.e., dried products which have an aging time of several months and a semi-dried product where the aging time is 1-3 weeks. The dried sausages have a water content below 40% (i.e., water activity below 0.93), are heavily spiced, and are not usually smoked. The semi-dried products have a water content of 40-60% (i.e., water activity 0.930.98) and are frequently smoked. They are not microbiologically stable and should be stored refrigerated. The microbial flora of both types of sausage is gram-positive and microaerophilic, the normal gram-negative spoilage flora of fresh meats being suppressed. In modern processing, starter cultures are used to ensure an efficient lactic fermentation of the desired type. Starter cultures are combinations of strains belonging to the genera Lactobacillus, Pediococcus, and Micrococcus. Vegetables are also preserved by fermentation and acidification. In North America and Western Europe the commonest vegetable prepared in this way is cabbage, though there are several others. Cabbage is usually chopped and dry salted. The salt draws out plant juices containing carbohydrates and other nutrients to form a brine. Large numbers of soil bacteria and organisms from the processing plant are present, and a series of changes occur in the flora ending with a predominant lactic fermentation. In the initial stages, gram-negative organisms tend to predominate, and since the fermentation vats are covered to restrict the access of oxygen, these are facultative organisms such as coliforms. The temperature of fermentation is usually controlled at 20-25~ Once the lactic acid bacteria begin to grow, the pH drops to 4.6-4.9. The predominant organism in the early stages of lactic fermentation are Leuconostoc spp. and these are followed by more acid-tolerant lactobacilli and pediococci. After a few weeks the process is complete and the final product has a pH of 3.5-3.8.
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The finished sauerkraut is marketed raw, pasteurized, or canned. Spoilage can occur if the initial lactic fermentation fails and the coliform organisms cause spoilage; this usually happens because of uneven or inadequate salting. If oxygen is allowed in the process, oxidative yeasts can grow, using the lactic acid and causing a rise in pH. Less acid-tolerant organisms are then able to grow and spoil the product. A wide range of fermented milk products is available throughout the world, including fermented milks such as yoghurt and a wide range o f cheeses. Fermentation is brought about by lactic acid bacteria, sometimes along with yeasts. Commercially, starter cultures are used, and the characteristic properties o f the different products result largely from the use of appropriate microorganisms and manipulation of the fermentation conditions. Microbial stability in these products is due to low pH along with refrigerated storage and sometimes pasteurization. In a similar way a vast range o f alcoholic beverages is produced, essentially by fermentation of carbohydrate substrates with yeasts which primarily produce ethyl alcohol. The pH of fruit juices used for wines is naturally low, but acid is frequently added to ensure a low pH and favor the yeasts. The characteristic sensory properties of the individual products are achieved by selection of substrate and individual yeast strain and manipulation o f the processing conditions. Microbial stability depends primarily on low pH, alcohol concentration, chemical additives (particularly sulphur dioxide), and sometimes pasteurization. In commercial production, selection of the appropriate fermentation flora is ensured by pasteurization of the raw material and inoculation with the appropriate starter culture and then adjusting the fermentation conditions to favor these particular organisms.
Chemical Preservatives The distinction between chemical preservatives, curing salts, and fermentation processes is one of convenience. The various salts, organic acids, alcohols, and antibiotics present in these processes are still chemical preservaties. There remain a number of other chemicals that are deliberately added to foods to inhibit or kill microorganisms. The amount and type of additives permitted in foods are carefully controlled by government agencies and vary in different countries. Organic acids and their esters are commonly added. It is the undissociated molecule that is responsible for the antimicrobial activity (as opposed to any effects due to their ability to reduce the pH). Because the pK values of organic acids are low (pH 3-5), they are therefore more active in high acid foods. They are also more active against molds though in high enough concentration they inhibit a wide range of organisms. Acetic acid is one of the most widely used, and the presence of 1-2% of undissociated acid in meat, fish, and vegetables will usually inhibit or kill most microorganisms. Only Acetobacter spp. and certain lactic acid bacteria are appreciably resistant, and Acetobacter spp. are in fact used to manufacture Vinegars. Benzoic acid is primarily active against molds and yeasts; many
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spoilage bacteria will not be inhibited at the levels permitted. Similarly, propionic acid is highly effective against molds and is useful against some bacteria. Sorbic acid has a wider spectrum of activity. It acts against catalase-positive organisms including molds, yeasts, and bacteria but is ineffective against catalase-negative bacteria. It is therefore most useful for inhibiting aerobic growth in fermented or high acid foods. An exception to the general statement about low pH and activity is p-hydroxybenzoic acid and its esters; they have a relatively high pK value and are effective against yeasts and molds up to neutral pH values. The activity of organic acids and their esters is particularly affected by other environmental parameters; pH has been discussed. Others, such as water activity, Eh, and temperature, have marked effects, and synergism is common between organic acids, their esters, and these parameters, as well as between different preservatives. Carbon dioxide has already been mentioned as a preservative; other gases are also effective in some circumstances. Sulphur dioxide is widely used, often applied as sulphite, bisulphite, and metabisulphite salts. Its antimicrobial activity is largely related to the unbound nonionized molecular form and hence is most active in high acid foods. Its commonest use is with soft fruits, fruit juices, wines, and sausages, and it is most effective against molds, yeasts, and gram-negative bacteria. Antibiotics were, for a time, thought to be very useful for inhibiting food spoilage bacteria; however, for a variety of reasons, most are now prohibited as food additives. Two are still found useful and have no therapeutic applications: natamycin and nisin. Natamycin is produced by Streptomyces natalensis and is primarily active against fungi. It is used, for instance, to inhibit molds in sausages and as a treatment for cheese rinds, against to prevent mold growth. Nisin is produced by Streptococcus lactis and, therefore, occurs naturally in soured milks and farmhouse cheese. It has a relatively narrow range of activity, affecting only gram-positive bacteria. It is most stable at acid pH values and is of little use for preventing spoilage of fresh meat and fish. It could, however, have specific applications in a variety of foods to inhibit grampositive food poisoning bacteria, though this requires further investigation.
Microbial Health Hazards Food poisoning microorganisms behave in the same ways as other organisms to changing environmental factors. In many cases they are less resistant than spoilage organisms, and processes designed to prevent spoilage will automatically prevent their growth. In designing or changing processes it is important, however, to take the properties of food poisoning organisms into consideration. With heat processed foods the main health hazards arise with spore-forming bacteria, particularly Clostridium botulinum, Clostridium perfringens, and Bacillus cereus. The proteolytic strains of C. botulinum are the most heat-resistant pathogens, though some strains of B. cereus are equally so. Sterilization procedures are governed by the higher heat resistance of putrefactive spore formers which cause spoilage. Post-process recontamination, however, can arise with
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a range of food poisoning bacteria, not necessarily spore formers. Since the process has eliminated the normal microflora, if such contamination does occur, t.he contaminating organism grows rapidly with no competitive flora. Pasteurtzatton processes were originally introduced to eliminate pathogens, e.g., Mycobacterium tuberculosis from milk. Other preservation methods--refrigerated storage being the commonest--have to be applied, however, since the process does not kill all organisms. Many other pathogenic bacteria have heat resistances comparable to spoilage bacteria, yeasts, and molds; these include Brucella spp., Salmonella spp., and Staphylococcus aureus. Mild heat processing will not inactivate all preformed microbial toxins, the prime example being staphylococcal toxins; these are relatively beat resistant, and if they have been produced before heat processing, can survive the process. Refrigerated storage will control most food poisoning organisms provided the temperature is properly controlled. Salmonella, Shigella, and Staphylococcus spp. have a minimum growth temperature of 6-8"C. With C. perfringens and many E. coil strains the minimum is about 12~ and these should present rio problems in refrigerated foods. The nonproteolytic strains o f C. botulinum, however, will grow in temperatures as low as 3.5~ and Yersinca enterocolitica will grow slowly at 0~ These organisms can give rise to problems in refrigerated foods, especially if other treatments such as mild heat treatment and smoke curing have inhibited or killed the normal gram-negative spoilage flora. Growth of the psychrotrophic C. botufinum strains can be a particular problem in such foods, since they do not produce spoilage odors and flavors. Cured and fermented foods rely on low water activity and pH primarily. Salmonella spp. and Staphylococcus aureus are controlled below pH 4.fi, proteolytie strains of C. botulinum below pH 4.5, and nonproteolytic strains below pH 5.0. These figures do not take account of synergism with other factors when inhibition can be achieved at somewhat higher values. Similarly, low water activity inhibits most food poisoning bacteria as well as spoilage bacteria. In the range of fresh foods, at aw = 0.98 or higher, C. per.fringens and C. botulinum will grow, and problems arise with these spore formers in heatprocessed foods. Salmonella spp. and most Enterobacteriaceae will compete well with spoilage organisms in these foods at temperatures above about 10~ Staphylococcus aureus and toxin-producing molds do not compete well in this type of food primarily because of their slower growth rates. Most food-borne pathogens are inhibited in the range ofaw = 0.98-0.93; however, S. aureus will grow down to a,~ --- 0.85. Bacteriological media with 10-12% added salt are frequently used to selectively isolate this organism. For most food-poisoning bacteria the inhibitory levels of most environmental factors and chemical additives are known so that safe parameters can usually be specified. Achieving these parameters in food products is, however, not always an easy matter.
Conclusions Fresh animal and plant tissues that are suitable for human food are equally, if not more suitable as food for microorganisms. At the point of slaughter or
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harvest, raw foods have a relatively large and varied microbial population. Preservation o f foods for h u m a n use is therefore primarily a question o f m a nipulating e n v i r o n m e n t a l a n d processing factors to kill m i c r o o r g a n i s m s or reduce their growth, and in some cases to encourage the growth o f specific desired organisms. Definitions o f desirable and undesirable when applied to acceptability o f foods are subjective. Spoiled foods in one c o m m u n i t y can be a culinary delicacy in another. G i v e n a knowledge o f the desired properties o f the food, a knowledge o f the effects o f ecological pressures and processing parameters on the growth and m e t a b o l i s m o f m i c r o o r g a n i s m s should p e r m i t specification o f optimal processing a n d storage conditions. While there is a considerable b o d y o f knowledge concerning the effects o f the various parameters individually, i n f o r m a t i o n on their interactions is only beginning to accumulate. Indeed, techniques for efficient investigation o f m u l t i - p a r a m e t e r situations are only just b e c o m i n g available. O p t i m i z a t i o n o f control and processing p a r a m eters is one o f the m a i n investigatory fields facing the f o o d microbiologist in the near future. A n o t h e r field recently opened up is the use o f genetically engineered strains in food processing. Traditionally, selection o f particular features in a m i c r o o r g a n i s m has been based on r a n d o m m u t a t i o n and selection o f mutants. The possibility o f designing strains for specific purposes n o w exists. Even where optimal processing and storage conditions are known, there is still r o o m for i m p r o v e m e n t o f control procedures; m o s t food poisoning outbreaks occur when something goes wrong. References 1. Baldham CD (1856) Prose halieutics, John W Parker & Son, London 2. Cutting CL (1955) Fish saving. A history of fish processing from ancient to modern times. Leonard Hill (Books) Ltd, London 3. Deraniyagala PEP (1933) Cured marine products of Ceylon. Celon J Sci (C) 5:49 4. Easter MC, Gibson DM, Ward FB (1982) A conductance method for the assay and study of bacterial trimethylamine oxide reduction. J Appl Bacteriol 52:357-365 5. Erichsen I (1983) Fermented fish and meat products: the present position and future possibilities. In: Roberts TA, Skinner FA (eds) Food microbiology SAB Symposium Series No 11, Academic Press, London, pp 271-286 6. Gould GW, Brown MH, Fletcher BC (1983) Mechanisms of action of food preservation procedures. In: Roberts TA, Skinner FA (eds) Food microbiology SAB Symposium Series No 11, Academic Press, London, pp 67-84 7. International Commission on MicrobiologicalSpecifications for Foods (1980) Microbial ecology of foods. Vol. I. Factors affectinglife and death of microorganisms. Academic Press, New York 8. International Commission on MicrobiologicalSpecifications for Foods (1980) Microbial ecology of foods. Vol II. Food commodities. Academic Press, New York 9. Pedersen CS (1979) Microbiology of food fermentations. AVI Publishing Co, Westport, Connecticut 10. Radcliffe W (1921) Fishing from earliest times. John Murray Ltd, London. I 1. Ratkowsky DA, Olley J, McMeekin TA, Ball A (1982) Relationship between temperature and growth rate of bacterial cultures. J Bacteriol. 149(1):1-5 12. Roberts TA, Skinner FA (eds) (1983) Food microbiology, advances and prospects. SAB Symposium Series No 11, Academic Press, London 13. Wee-Ching W Yen (1908) The importance of fishing and its allied industry, the manufacture of salt. Address before the International Fishing Congress, Washington DC, USA 14. Wilkinson Sir JG (1878) The manners and customs of ancient Egyptians. Revised by Birch S. John Murray, Lid, London
