Chilled foods microbiology 157 understood, e.g. vacuum packed cod( Gram and Huss 1996). In general, a greater understanding is needed of the relationship between specific spoilage microorganisms in particular foods and the deterioration in sensory quality 7.4.2 Food-borne pathogens ith many human pathogens, the greater the number of cells consumed, the greater the chance of microbial invasion, as the larger number of cells may be able to evade/swamp the bodys defence mechanism. Higher numbers may also result in a shorter incubation period before the onset of disease. Consequently ontrol, and preferably inhibition, of growth in foods is essential. However, with ome invasive pathogens(e.g. viruses, Campylobacter), the infectious dose is ow and growth in the food may not be necessary. Other pathogenic microorganisms may produce a toxin in the food which results in disease Preformed toxins are usually produced at high cells densities and so usuall growth has occurred. If the toxin is heat stable, it may remain although all microorganisms have been eliminated from the food. Consequently it is important to control growth at all stages of the chill chain 7.5 Factors affecting the microfora of chilled foods 7.5.1 Initial microflora with healthy animal and plant tissues, microbial contamination is absent or at a low level except for the exterior surfaces. For example, fresh muscle from healthy animals is usually microbiologically sterile, and aseptically drawn milk from healthy cows contains only a few microorganisms(mainly streptococci and micrococci)derived from the teat canal. Similarly, the interior of healthy damaged vegetables does not contain microorganisms although the exterior ay be contaminated with a wide range of microorganisms of soil origin During slaughter or harvesting, subsequent processing and packaging, these raw materials become contaminated from a wide range of sites. Typically, these sites nclude water, air, dust, soil, hides/fleece/feathers, animals, people, equipment and other food materials. Consequently, a large range of microorganisms can be isolated from foods. Those which are able to grow may potentially give rise to microbial spoilage or public health issues. The hygienic practices of all food operations, from slaughter/harvesting through retail sale to consumer use, will affect the level of microbial contamination of products. In general, the lower the initial level of contamination, the greater the time until microbial spoilage is evident 7.5.2 Food type The intrinsic properties(e.g. pH, water activity, acidity, natural antimicrobials) of different foods vary greatly. Such factors affect the ability of microorganisms
understood, e.g. vacuum packed cod (Gram and Huss 1996). In general, a greater understanding is needed of the relationship between specific spoilage microorganisms in particular foods and the deterioration in sensory quality. 7.4.2 Food-borne pathogens With many human pathogens, the greater the number of cells consumed, the greater the chance of microbial invasion, as the larger number of cells may be able to evade/swamp the body’s defence mechanism. Higher numbers may also result in a shorter incubation period before the onset of disease. Consequently, control, and preferably inhibition, of growth in foods is essential. However, with some invasive pathogens (e.g. viruses, Campylobacter), the infectious dose is low and growth in the food may not be necessary. Other pathogenic microorganisms may produce a toxin in the food which results in disease. Preformed toxins are usually produced at high cells densities and so usually growth has occurred. If the toxin is heat stable, it may remain although all microorganisms have been eliminated from the food. Consequently it is important to control growth at all stages of the chill chain. 7.5 Factors affecting the microflora of chilled foods 7.5.1 Initial microflora With healthy animal and plant tissues, microbial contamination is absent or at a low level except for the exterior surfaces. For example, fresh muscle from healthy animals is usually microbiologically sterile, and aseptically drawn milk from healthy cows contains only a few microorganisms (mainly streptococci and micrococci) derived from the teat canal. Similarly, the interior of healthy undamaged vegetables does not contain microorganisms although the exterior may be contaminated with a wide range of microorganisms of soil origin. During slaughter or harvesting, subsequent processing and packaging, these raw materials become contaminated from a wide range of sites. Typically, these sites include water, air, dust, soil, hides/fleece/feathers, animals, people, equipment and other food materials. Consequently, a large range of microorganisms can be isolated from foods. Those which are able to grow may potentially give rise to microbial spoilage or public health issues. The hygienic practices of all food operations, from slaughter/harvesting through retail sale to consumer use, will affect the level of microbial contamination of products. In general, the lower the initial level of contamination, the greater the time until microbial spoilage is evident. 7.5.2 Food type The intrinsic properties (e.g. pH, water activity, acidity, natural antimicrobials) of different foods vary greatly. Such factors affect the ability of microorganisms Chilled foods microbiology 157
158 Chilled foods to grow and the rate of growth and will be discussed in more detail in subsequent sections of this chapter. With different food types, the nutritional status varies although foods are generally not nutritionally limiting for microorganisms Foods rich in nutrients(e.g. meat, milk, fish) permit faster growth than those with a lower nutritional status (e.g. vegetables) and so are more prone to spoilage. Slaughter and harvesting practices may affect the intrinsic properties of a food. For example, poor practices in the husbandry and slaughter of pigs may lead to pork being classified as DFD(dark, firm, dry) or PSE(pale, so exudative), both of which are more prone to spoilage thannormal pork. With DFD meat, the pH is higher, so permitting faster growth, whilst nutrient leakage and protein denaturation from Pse meat also allow more rapid microbial proliferation Even within a single food ingredient or product, variations in the ph, aw and redox potential may occur and so affect the nature and rate of microbial multiplication. The situation may be further complicated in multi-component foods where migration of nutrients and gradients of pH, aw and preservatives ay occur. In addition, microorganisms unable to grow on one ingredient may come into contact with a more favourable environment and so permit growth 7.5.3 Processing Chill storage The time of storage will affect microbial numbers. Generally, microbial numbers increase with time in chilled foods at neutral pH values, low salt concentrations and the absence of preservatives. However, low pH values or high salt concentrations in foods may cause microbial stasis, injury or even death. At chill temperatures however, the rate of death is often reduced and so the microorganism may survive for longer periods compared with higher (e.g ambient) temperatures. In many cases a combination of processing and preservation factors may be used to achieve a safe, high quality product with an acceptable shelf-life. Such combination treatments have been reviewed by Gould (1996) The ability of individual microorganisms to grow and their rates of growth are affected by temperature. As discussed previously, some microorganisms (mainly psychrotrophs) are better adapted to growth at chill temperatures Therefore during chill storage not only will the total number of microorganisms hange, but also the composition of the microflora will alter. For example, with freshly drawn milk, the microflora is dominated by Gram-positive cocci and ods, which may spoil the product by souring if stored at warm temperatures. At hill temperatures, these microorganisms are largely unable to grow and the microflora rapidly becomes dominated by psychrotrophic Gram-negative rod- shaped bacteria(most commonly Pseudomonas spp )(Neill, 1974). A similar change in the microflora composition has also been reported for other chill- (Huis in't Veld, 1996)
to grow and the rate of growth and will be discussed in more detail in subsequent sections of this chapter. With different food types, the nutritional status varies although foods are generally not nutritionally limiting for microorganisms. Foods rich in nutrients (e.g. meat, milk, fish) permit faster growth than those with a lower nutritional status (e.g. vegetables) and so are more prone to spoilage. Slaughter and harvesting practices may affect the intrinsic properties of a food. For example, poor practices in the husbandry and slaughter of pigs may lead to pork being classified as DFD (dark, firm, dry) or PSE (pale, soft, exudative), both of which are more prone to spoilage than ‘normal’ pork. With DFD meat, the pH is higher, so permitting faster growth, whilst nutrient leakage and protein denaturation from PSE meat also allow more rapid microbial proliferation. Even within a single food ingredient or product, variations in the pH, aw and redox potential may occur and so affect the nature and rate of microbial multiplication. The situation may be further complicated in multi-component foods where migration of nutrients and gradients of pH, aw and preservatives may occur. In addition, microorganisms unable to grow on one ingredient may come into contact with a more favourable environment and so permit growth. 7.5.3 Processing Chill storage The time of storage will affect microbial numbers. Generally, microbial numbers increase with time in chilled foods at neutral pH values, low salt concentrations and the absence of preservatives. However, low pH values or high salt concentrations in foods may cause microbial stasis, injury or even death. At chill temperatures however, the rate of death is often reduced and so the microorganism may survive for longer periods compared with higher (e.g. ambient) temperatures. In many cases a combination of processing and preservation factors may be used to achieve a safe, high quality product with an acceptable shelf-life. Such combination treatments have been reviewed by Gould (1996). The ability of individual microorganisms to grow and their rates of growth are affected by temperature. As discussed previously, some microorganisms (mainly psychrotrophs) are better adapted to growth at chill temperatures. Therefore during chill storage not only will the total number of microorganisms change, but also the composition of the microflora will alter. For example, with freshly drawn milk, the microflora is dominated by Gram-positive cocci and rods, which may spoil the product by souring if stored at warm temperatures. At chill temperatures, these microorganisms are largely unable to grow and the microflora rapidly becomes dominated by psychrotrophic Gram-negative rodshaped bacteria (most commonly Pseudomonas spp.) (Neill, 1974). A similar change in the microflora composition has also been reported for other chillstored foods (Huis in’t Veld, 1996). 158 Chilled foods
Chilled foods microbiology 159 Heating As part of their manufacture, many chilled foods undergo a heating process. This will reduce microbial numbers, generally resulting in a pasteurised rather than a sterilised product, otherwise chill storage would be unnecessary. Food pasteurisation treatments have been reviewed by Gaze(1992). The degree of heat applied will affect the types of microorganisms able to survive. In general the Gram-negative rod-shaped bacteria, which proliferate in chilled foods, are sensitive to heat and are readily eliminated. Although these bacteria may be isolated from, and even spoil, heated foods, their presence is usually attributable to post-heating contamination Some Gram-positive bacteria are tolerant to mild heat and classified as thermoduric(e.g. some Lactobacillus, Streptococcus and Micrococcus species Jay 1978). However pasteurisation processes are designed to destroy all vegetative cells. Other bacteria however, produce heat-resistant bodies, called spores, which may survive. The genera of concern are Bacillus and Clostridium species, which include both pathogenic and spoilage strains. Whilst these bacteria are generally out-competed in chilled foods by the Gram-negative rod- aped bacteria, Bacillus and Clostridium species may grow relatively unhindered in heated foods subsequently stored chilled Acidification Several types of chilled foods are naturally acidic(e.g. fruit juices)or acidified using either a fermentation process(e.g. yoghurt) or by the direct addition of acids(e.g. coleslaw). As with temperature, microorganisms have pH limits for growth. The pH optima for most pathogenic bacteria is usually in the range 6.8- 7.4(Jay 1978), which is similar to the human body ph in which they are adapted to grow. Typical minimum pH values for growth are shown in Table 7.2. The minimum pH for the major spoilage bacteria of meat, poultry and dairy products is approximately 5.0 whereas other microbial types, in particular yeasts and moulds, may grow at pH values of 3.0 or less. Consequently, mildly acidified products may be spoiled by acid-tolerant bacteria (lactic acid bacteria and some Enterobacteriaceae) whilst more acid products are spoiled by yeasts and moulds Both pH and temperature interact, and the minimum pH for growth at optimal temperatures may be significantly less than that at chill temperatures( George et al. 1988). At pH values below the minimum for growth, some microorganisms will die rapidly in the food, whilst others may persist for the life of the product. Of particular concern in acid foods is the pathogen E. coli O157: H7, which more acid tolerant than other pathogens. It may grow at pH values of 4.0 or below and survive for considerable periods at lower pH values(Conner and Kotrola 1995, Deng et al. 1999) In addition to the pH, the acid type used affects the microbial stability of the foods. The organic acids (lactic, acetic, citric and malic) are more antimicrobial than the inorganic acids(hydrochloric, sulphuric). Care is needed with published literature, as the minimum pH values reported often have used inorganic acids Therefore the minimum pH for growth in foods is often higher than that quoted
Heating As part of their manufacture, many chilled foods undergo a heating process. This will reduce microbial numbers, generally resulting in a pasteurised rather than a sterilised product, otherwise chill storage would be unnecessary. Food pasteurisation treatments have been reviewed by Gaze (1992). The degree of heat applied will affect the types of microorganisms able to survive. In general, the Gram-negative rod-shaped bacteria, which proliferate in chilled foods, are sensitive to heat and are readily eliminated. Although these bacteria may be isolated from, and even spoil, heated foods, their presence is usually attributable to post-heating contamination. Some Gram-positive bacteria are tolerant to mild heat and classified as thermoduric (e.g. some Lactobacillus, Streptococcus and Micrococcus species, Jay 1978). However pasteurisation processes are designed to destroy all vegetative cells. Other bacteria however, produce heat-resistant bodies, called spores, which may survive. The genera of concern are Bacillus and Clostridium species, which include both pathogenic and spoilage strains. Whilst these bacteria are generally out-competed in chilled foods by the Gram-negative rodshaped bacteria, Bacillus and Clostridium species may grow relatively unhindered in heated foods subsequently stored chilled. Acidification Several types of chilled foods are naturally acidic (e.g. fruit juices) or acidified using either a fermentation process (e.g. yoghurt) or by the direct addition of acids (e.g. coleslaw). As with temperature, microorganisms have pH limits for growth. The pH optima for most pathogenic bacteria is usually in the range 6.8– 7.4 (Jay 1978), which is similar to the human body pH in which they are adapted to grow. Typical minimum pH values for growth are shown in Table 7.2. The minimum pH for the major spoilage bacteria of meat, poultry and dairy products is approximately 5.0 whereas other microbial types, in particular yeasts and moulds, may grow at pH values of 3.0 or less. Consequently, mildly acidified products may be spoiled by acid-tolerant bacteria (lactic acid bacteria and some Enterobacteriaceae) whilst more acid products are spoiled by yeasts and moulds. Both pH and temperature interact, and the minimum pH for growth at optimal temperatures may be significantly less than that at chill temperatures (George et al. 1988). At pH values below the minimum for growth, some microorganisms will die rapidly in the food, whilst others may persist for the life of the product. Of particular concern in acid foods is the pathogen E. coli O157: H7, which is more acid tolerant than other pathogens. It may grow at pH values of 4.0 or below and survive for considerable periods at lower pH values (Conner and Kotrola 1995, Deng et al. 1999). In addition to the pH, the acid type used affects the microbial stability of the foods. The organic acids (lactic, acetic, citric and malic) are more antimicrobial than the inorganic acids (hydrochloric, sulphuric). Care is needed with published literature, as the minimum pH values reported often have used inorganic acids. Therefore the minimum pH for growth in foods is often higher than that quoted, Chilled foods microbiology 159
160 Chilled foods Table 7.2 Typical minimum pH and aw values for growth of microorganisms(Anon 1991b, Gould 1989, Mitscherlich and Marth 1984, ACMSF 1995) Microorganism Minimum pH Minimum aw Bacillus cereus Campylobacter jejun 306 0.98 Clostridium botulinum(non-proteolytic) Clostridium botulinum(proteolytic) Clostridium perfringens Escherichia coli Escherichia coli o157: H7 3.84.2 Lactobacillus species Many yeasts and mould 0.8-0.6 Yersinia enterocolitica 4.6 Minimum ph with toxin production as organic acids are present. Within the organic acids, the order of decreasing antimicrobial efficiency is usually acetic, lactic, citric then malic acid. With the organic acids, the undissociated form of the acid is effective against microorganisms and the degree of dissociation is dependent on the ph of the food. Organic acids and their use in food systems have been discussed by Kabara nd eklund (1991). The pH and acid composition does not remain constant during the life of some foods. Changes in pH will affect the types of microorganisms able to grow nd their growth rates. With some foods, fermentation results in a pH decreases during storage whilst in others an increase can be noted. For example, during maturation of mould-ripened cheeses, the pH value of cheese near the surfaces ncreases owing to proteolytic activity of the mould, and this has been related to he ability of Listeria monocytogenes to grow in these products, but not in the unripened cheeses(Terplan et al. 1987) Reduced a The aw is a measure of the amount of water available in a food which may be used for microbial growth. As the aw of a food is reduced, the number of microorganisms able to grow and their rate of growth is also reduced(Sperber 1983)(Table 7.2 ). The aw of a food may be reduced either by the removal of water (i.e. drying)or by the addition of solutes(e.g salt or sugar). In response to diet and health issues, many jam and sauce products have reduced their sugar ontent. Thus the intrinsic preservation system (i.e. low aw)of the product has been compromised and some microorganisms, mainly yeasts, may now grow These products generally recommend refrigeration after opening to prevent microbial growth. The aw of a product may interact with other preservation factors, including temperature, to maintain the safety of chilled foods(Glass and
as organic acids are present. Within the organic acids, the order of decreasing antimicrobial efficiency is usually acetic, lactic, citric then malic acid. With the organic acids, the undissociated form of the acid is effective against microorganisms and the degree of dissociation is dependent on the pH of the food. Organic acids and their use in food systems have been discussed by Kabara and Eklund (1991). The pH and acid composition does not remain constant during the life of some foods. Changes in pH will affect the types of microorganisms able to grow and their growth rates. With some foods, fermentation results in a pH decreases during storage whilst in others an increase can be noted. For example, during maturation of mould-ripened cheeses, the pH value of cheese near the surfaces increases owing to proteolytic activity of the mould, and this has been related to the ability of Listeria monocytogenes to grow in these products, but not in the unripened cheeses (Terplan et al. 1987). Reduced aw The aw is a measure of the amount of water available in a food which may be used for microbial growth. As the aw of a food is reduced, the number of microorganisms able to grow and their rate of growth is also reduced (Sperber 1983) (Table 7.2). The aw of a food may be reduced either by the removal of water (i.e. drying) or by the addition of solutes (e.g. salt or sugar). In response to diet and health issues, many jam and sauce products have reduced their sugar content. Thus the intrinsic preservation system (i.e. low aw) of the product has been compromised and some microorganisms, mainly yeasts, may now grow. These products generally recommend refrigeration after opening to prevent microbial growth. The aw of a product may interact with other preservation factors, including temperature, to maintain the safety of chilled foods (Glass and Table 7.2 Typical minimum pH and aw values for growth of microorganisms (Anon. 1991b, Gould 1989, Mitscherlich and Marth 1984, ACMSF 1995) Microorganism Minimum pH Minimum aw Bacillus cereus 4.9 0.91 Campylobacter jejuni 5.3 0.985 Clostridium botulinum (non-proteolytic) 5.0 0.96 Clostridium botulinum (proteolytic) 4.6 0.93 Clostridium perfringens 5.0 0.93 Escherichia coli 4.4 0.95 Escherichia coli O157:H7 3.8–4.2 0.97 Lactobacillus species 3–3.5 0.95 Pseudomonas species 5.0 0.95 Salmonella species 4.0 0.95 Staphylococcus aureus 4.0 (4.6)a 0.86 Many yeasts and moulds 2.0 0.8–0.6 Yersinia enterocolitica 4.6 0.95 a Minimum pH with toxin production. 160 Chilled foods
Chilled foods microbiology 161 Doyle 1991). In general, yeasts and moulds are more tolerant than bacteria of low aw values in foods (Jay 1978). As bacterial growth is largely inhibited yeasts and moulds may then grow and cause the spoilage defects in such products Preservatives In order to maintain their microbial stability, many chilled products contain natural or added preservatives, e.g. salt, nitrite, benzoate, sorbate. The presence of these compounds affects the type and rate of product spoilage that may occur Their applications and mechanisms of action have been reviewed in Russell and redominate on chilled fresh meat. The addition of curing sa. c sodin o Gould(1991). As discussed previously, Pseudomonas species tend chloride and potassium nitrite)to pork meat to form bacon, largely inhibits the growth of these microorganisms and spoilage is caused by other microbial groups(e.g. micrococci, staphylococci, lactic acid bacteria)( Gardner 1983, Borch et al. 1996). Similarly, the British sausage is largely a fresh meat product, but is preserved by the addition of sulphite. This prevents the growth of the Pseudomonas species, and microbial spoilage will be caused by sulphite resistant Brochothrix thermosphacta or yeasts( Gardner 1983) The number and type of microorganisms able to grow in preservative- containing chilled foods depend on the food type, preservative type, pH of the food, preservative concentration, time of storage and other preservation mechanisms in the food. Overall, yeasts and moulds tend to be more resistant to preservatives compared with bacteria and so may dominate the final spoilage microflora. Recent trends in food processing have tended to reduce or eliminate the use of preservatives. Care is needed with such an approach, as even small hanges may compromise the product safety and microbiological stability Storage atmosphere The use of modified atmospheres, including vacuum packaging, for the storage of chilled foods is increasing. Often these are chosen to maintain sensory characteristics of a product, but many will also inhibit or retard the development of the normal spoilage microflora. Pseudomonas species, the major spoilage group in chilled proteinaceous foods, require the presence of oxygen to grow Therefore, the use of vacuum packaging or modified atmospheres excluding oxygen will prevent the growth of this microbial group. Whilst other microorganisms can grow in the absence of oxygen, they generally grow more lowly and so the time to microbial spoilage is increased. The microflora of vacuum-packed meats is usually dominated by lactic acid or Brochothrix thermosphacta(Borch et al. 1996). In some cases, Entero iaceae or coliforms may cause the spoilage of vacuum-packed and modified- atmosphere-packed(MAP) foods(Gill and Molin 1991) Most commercial MAP gas mixtures for chilled food usually contain a combination of carbon dioxide, nitrogen and oxygen. The inhibition of bacteria becomes more pronounced as the amount of carbon dioxide increases. The
Doyle 1991). In general, yeasts and moulds are more tolerant than bacteria of low aw values in foods (Jay 1978). As bacterial growth is largely inhibited, yeasts and moulds may then grow and cause the spoilage defects in such products. Preservatives In order to maintain their microbial stability, many chilled products contain natural or added preservatives, e.g. salt, nitrite, benzoate, sorbate. The presence of these compounds affects the type and rate of product spoilage that may occur. Their applications and mechanisms of action have been reviewed in Russell and Gould (1991). As discussed previously, Pseudomonas species tend to predominate on chilled fresh meat. The addition of curing salts (i.e. sodium chloride and potassium nitrite) to pork meat to form bacon, largely inhibits the growth of these microorganisms and spoilage is caused by other microbial groups (e.g. micrococci, staphylococci, lactic acid bacteria) (Gardner 1983, Borch et al. 1996). Similarly, the British sausage is largely a fresh meat product, but is preserved by the addition of sulphite. This prevents the growth of the Pseudomonas species, and microbial spoilage will be caused by sulphiteresistant Brochothrix thermosphacta or yeasts (Gardner 1983). The number and type of microorganisms able to grow in preservativecontaining chilled foods depend on the food type, preservative type, pH of the food, preservative concentration, time of storage and other preservation mechanisms in the food. Overall, yeasts and moulds tend to be more resistant to preservatives compared with bacteria and so may dominate the final spoilage microflora. Recent trends in food processing have tended to reduce or eliminate the use of preservatives. Care is needed with such an approach, as even small changes may compromise the product safety and microbiological stability. Storage atmosphere The use of modified atmospheres, including vacuum packaging, for the storage of chilled foods is increasing. Often these are chosen to maintain sensory characteristics of a product, but many will also inhibit or retard the development of the ‘normal’ spoilage microflora. Pseudomonas species, the major spoilage group in chilled proteinaceous foods, require the presence of oxygen to grow. Therefore, the use of vacuum packaging or modified atmospheres excluding oxygen will prevent the growth of this microbial group. Whilst other microorganisms can grow in the absence of oxygen, they generally grow more slowly and so the time to microbial spoilage is increased. The spoilage microflora of vacuum-packed meats is usually dominated by lactic acid bacteria or Brochothrix thermosphacta (Borch et al. 1996). In some cases, Enterobacteriaceae or coliforms may cause the spoilage of vacuum-packed and modifiedatmosphere-packed (MAP) foods (Gill and Molin 1991). Most commercial MAP gas mixtures for chilled food usually contain a combination of carbon dioxide, nitrogen and oxygen. The inhibition of bacteria becomes more pronounced as the amount of carbon dioxide increases. The Chilled foods microbiology 161