С. Дж. Уокер и Г. Бете, Campden and Chorleywood Food Research Association
On the huge constantly developing market of chilled food products, a very wide range of them is presented. Traditionally, among them were meat, poultry, fish and dairy products, but in recent years, products have become more diverse and more complex . With the release of new products, the number of ingredients has grown, many of which come from different parts of the world and relatively little can be known about their microbiological state. The number and types of microorganisms that can be isolated from chilled products are quite different. When storing refrigerated products, their microflora is not static and depends on many factors, mainly on the time and temperature of storage. Damage and preservation of refrigerated products are complex phenomena, including physical, chemical, biochemical and biological changes. Often they are interrelated, and some changes affect the speed of others. This chapter will only address microbiology issues related to chilled foods.
Progress in the production and transportation of chilled and frozen products made it possible to move quickly to other countries and sometimes to other continents. Therefore, if a microbiological problem occurs, it can spread widely, which will further increase the value of the microbiological state of the cooled products. Strengthening control, both domestically and internationally, will allow for faster identification, identification, and resolution of microbiological problems.
What you need cooling?
Cooling reduces the rate of decline in food quality. This concerns not only their chemical and biochemical changes, but also the activity of microorganisms. The effect of temperature on the growth of microorganisms is shown in Fig. 7.1. When the storage temperature decreases, the lag-phase before the beginning of the growth of microorganisms (the time until an obvious increase in their number) increases, and the growth rate decreases. In addition, when approaching a minimum temperature, the maximum possible size of a population of microorganisms often decreases. At the cellular level, the effect of temperature on the growth of microorganisms is a complex process that captures the structure of cell membranes, substrate uptake, respiration, and other types of enzyme activity (see  for more on this).
The range of temperatures in which microorganisms can grow is unusually wide. In , it was reported that a number of microorganisms, mainly yeasts, are capable of growing at temperatures below 0 ° C, and yeast isolated from oysters grew at -34 ° C. Therefore, to completely prevent the growth of all microorganisms, only cooling is not enough; it is necessary to use low temperatures, which reduce the rate and level of growth of microorganisms.
Microbiologists have repeatedly attempted to characterize microorganisms based on their ability to grow at different temperatures. The most widely used specific growth temperature (minimum, optimum and maximum). For
Fig. 7.1. Effect of temperature on the growth of microorganisms
chilled foods. The main concern is the minimum growth temperature (minimum growth temperature, MGT), the lowest temperature at which a certain microorganism can grow. If the MGT microorganism is above 10 ° C, then this microorganism will not grow during refrigerated storage. MGT values have been published for some microorganisms, but they must be approached with caution. If the duration of the study, on the basis of which the value is given, was too small, or the intervals between sampling were too long, then the value obtained will be incorrect. For example, although it was reported for Listeria monocytogenes that the value of MGT is -0,4 ° C, the lag-phase before the start of growth was greater than 15 days . If the study had ended before this time, the resulting MGT would have been higher. MGT is influenced by other factors, including pH, salt, preservatives and prior heat treatment. The true MGT value can be determined only with the optimal values of the other factors.
When microorganisms are found at temperatures below MGT, they can die out, but quite often the microorganisms survive, and if the temperature subsequently rises, their growth will resume.  noted that at temperatures slightly below MGT, the viability of Salmonella was worse than at lower temperatures. Storage of products at temperatures below the minimum for growth should not be considered lethal for microorganisms, since in many cases their growth will resume with a subsequent increase in temperature.
The optimal growth temperature is the temperature at which the biochemical processes that control the growth of a particular microorganism work most efficiently. At this temperature, the lag-phase before growth is minimal, and the growth rate is maximum. When the temperature rises above the optimal growth rate of the microorganism will decrease until the maximum growth temperature is reached. Usually the maximum growth temperature is only a few ° C above the optimum. In some specific microorganisms isolated from hot springs, the maximum growth temperature may exceed 90 ° C . At temperatures slightly above the maximum for growth, cell damage begins to occur. If the temperature is then reduced, growth may resume, although it may take some time to repair the cell. At higher temperatures, the inactivation of one or more vital enzymes in the microorganism becomes irreversible, and lethal cell damage occurs. At the same time microorganisms will not be able to recover and resume growth with a decrease in temperature. The concepts of cell damage and cell death are discussed in more detail in .
Based on the relative position of the cardinal temperatures, the microorganisms can be divided into four groups, namely, psychrophilic, psychrotrophic, mesophilic, and thermophilic (Table 7.1). For refrigerated products, psychrophilic and psychrotrophic microorganisms are most important. In the past, these terms were used as synonyms, leading to considerable confusion. It is now accepted that the term psychrophilic should be used only for microorganisms having a low (i.e. less than 20 ° C) maximum growth temperature .
|Temperature, ° C||psychrophilic||Psihrotrofnye||Mesophilic||Thermophilic|
|Minimum||<0-5||<0-5||(5 to) * 10||(30 to) * 40|
|Optimal||12 - 18||20 - 30 (35) *||30 – 40||55 – 65|
|Maximum||20||35 (40 - 42) *||45||(70) * and> 80|
* Numbers in parentheses sometimes written for microorganisms belonging to a particular classification.
True psychrophilic organisms in food microbiology are rare and usually limited to certain microorganisms obtained from the deep-sea fish. Basic spoilage microorganisms chilled foods inherently psihrotrofny.
Effect of microbial growth
Under appropriate conditions, most microorganisms grow or multiply. Bacteria multiply by division into two parts, that is, each cell divides and forms two daughter cells, and therefore the population of bacteria grows exponentially. Under ideal conditions, some bacteria can grow and divide every 20 min, so that one cell per 8 h can produce 16 million cells. Under unfavorable conditions, for example, when stored chilled, the generation time (doubling time) increases. For example, if you increase this time to 2 h via 8 h, the population size will be equal to only 16 cells. Even under ideal conditions, growth does not continue unhindered, but is limited by a number of factors — depletion of nutrient reserves, accumulation of toxic by-products, changes in experimental conditions, or lack of space.
With the growth of bacteria in food, they consume nutrients from them and form metabolic by-products - gases or acids. In addition, they can produce a number of enzymes that destroy the cellular structure or its components (for example, lipases and proteases). If only a few spoilage microorganisms are present, the effects of their growth may be implicit. If, however, microorganisms have multiplied, the formation of gases, acids, the appearance of foreign smell and taste, or the destruction of the structure of the food product can lead to its deterioration. In addition, the presence of microorganisms can manifest as a visible colony, mucus formation or turbidity of liquids. Some enzymes formed by harmful bacteria can remain active even after the destruction of the bacteria themselves in the food product as a result of heat treatment.
The relationship between the number of microorganisms and the spoilage of products is complex and depends on the number, type and activity of the microorganisms present, the type of products, its properties and external conditions. In some cases, this relationship is obvious, as in the case described in  (cod in a sealed vacuum pack). In general, a greater understanding of the relationship between certain microorganisms causing spoilage of certain products and deterioration of organoleptic characteristics is necessary.
Pathogenic microorganisms in foods
In the case of many microorganisms pathogenic for humans, the greater the number of cells absorbed by a person, the greater the likelihood of infection, since a large number of cells may be able to suppress the protective mechanisms of the body. Higher amounts may also lead to a shorter incubation period before the onset of the disease, and therefore it is necessary to limit or better stop the growth of microorganisms in food. In the case of some pathogenic microorganisms (for example, Campylobacter viruses), the dose that causes the infection is small and their growth in the food product may not be necessary for infection. Other pathogens can form toxins in food products leading to diseases. At high densities of microbial cells, toxins are usually preformed, and cell growth occurs. If the toxin is heat-resistant, it can persist even when all microorganisms are removed from the product, therefore it is important to control growth at all stages of the cold chain.
Factors affecting the microflora of chilled products
In healthy animal and plant tissues, infection with microorganisms (with the exception of external surfaces) is absent or is at a low level. For example, fresh muscle tissue of healthy animals is usually microbiologically sterile, and aseptically obtained milk of healthy cows contains only a few microorganisms (these are mainly streptococci and micrococci) from the nipple canal. Similarly, the inner part of healthy intact vegetables does not contain microorganisms, although outside the vegetables can be contaminated with many microorganisms from the soil. When slaughtering animals or harvesting, as well as subsequent processing and packaging, these raw materials are polluted from many sources - water, air, dust, soil, hides and feathers, from animals, people, equipment and other materials. Therefore, many types of microorganisms can be isolated from food products. Those that are capable of growth can lead to microbial deterioration of the product or harm to the health of consumers. Sanitary and hygienic measures taken during all operations with food products (from animal slaughter and harvesting to retail) affect the level of microbial contamination of products. Usually, the lower the initial level of contamination, the longer the microbial deterioration does not become apparent.
type of product
Different food products properties (for example, water activity, acidity, the presence of natural antimicrobial substances) may be different. These factors affect the growth ability of microorganisms and their growth rate and will be discussed in detail in the following sections of this chapter. Depending on the type of food product, the nutritional conditions of microorganisms vary, although the availability of nutrients in foods is usually not a limiting factor for the growth of microorganisms. Foods rich in nutrients (for example, meat, milk, fish) create conditions for faster growth and nutrition of microorganisms than foods poor in nutrients (for example, vegetables), and therefore they are more susceptible to spoilage. Methods of slaughtering animals and harvesting can affect the properties of food. For example, poor farming and slaughtering methods can lead to the fact that pork will be classified as DFD (“dark, solid, dry”) or PSE (“pale, soft, watery”). In both cases, it is more susceptible to spoilage than “normal” pork. Meats characterized as DFD have a higher pH, which allows faster growth of microorganisms. Nutrient loss and protein denaturation of meat, characterized as PSE, also make it possible to more quickly replicate microorganisms.
Even for a single food ingredient or product, deviations in pH, aw and redox potential can occur, which can affect the nature and speed of reproduction of microorganisms. The situation may be even more complicated in multi-component food products, where nutrient migration, pH, aw and preservative gradients may occur. In addition, microorganisms that are unable to grow on one ingredient can come into contact with a more favorable environment, which will make their growth possible.
Treatment. Cold storage
The storage time affects the amount of microorganisms, and usually their amount in cooled products at neutral pH, low salt concentrations and the absence of preservatives increases with time. However, low pH or high salt concentrations in foods can cause microorganism stagnation, damage, and even death. At low temperatures, the death rate often decreases, and therefore the microorganism can live longer than at a higher temperature (for example, at ambient temperature). In many cases, to obtain a safe (harmless) high quality product with an acceptable shelf life during storage, a combination of processing and preservative factors can be used. An overview of the types of such combined processing is given in .
The ability of individual microorganisms to grow and their growth rate is influenced by temperature. As noted above, some microorganisms (mostly psychrotrophic) are better adapted to grow at low temperatures. Therefore, during refrigerated storage, not only the total number of microorganisms will change, but also the composition of the microflora. For example, gram-positive cocci and sticks predominate in the microflora of freshly milk, which can lead to souring, and thus spoil the product if it is kept warm. At low temperatures, these microorganisms are largely incapable of growth, and psychrotrophic gram-negative rod-shaped bacteria (most often Pseudomonas species)  begin to predominate in the microflora. Similar changes in the composition of the microflora have also been described in other products stored at low temperatures .
The pre-heat treatment component of the production process of many refrigerated products. This reduces the number of microorganisms, which usually results in a pasteurized rather than sterilized product — otherwise refrigeration would be unnecessary. An overview of the various pasteurization methods is given in . The degree of heating will determine what types of microorganisms can withstand treatment. Heat-sensitive and easily destroyed mainly gram-negative rod-shaped bacteria that multiply in chilled foods. Although these bacteria can be isolated from heated foods and even spoil the heated food, their presence is usually attributed to infection after heating.
Some gram-positive bacteria tolerate moderate heat and are classified as heat resistant (for example, some species of Lactobacillus, Streptococcus and Micrococcus) . However, pasteurization processes are designed to destroy all microbial vegetative cells. Other bacteria, however, form heat-resistant spores that can withstand heat. Of concern is the bacillus of the genus Bacillus and varieties of clostridia, which include both pathogenic strains and food spoilage. If bacteria are mostly supplanted in chilled foods by gram-negative rod-shaped bacteria, Bacillus and Clostridium spp. In cooked foods that are then kept chilled can grow relatively unhindered.
Some types of chilled foods (for example, fruit juices) contain natural acid or they are made acidic by fermentation (for example, yogurt) or by the direct addition of acids (for example, cabbage lettuce filled with mayonnaise). As in the case of temperature, microorganisms have a pH limit for growth. The most favorable pH range for most pathogenic bacteria is usually 6,8-7,4 , which corresponds to the pH of the human body, to the growth in which these bacteria are adapted. Typical minimum pH values for microbial growth are given in table. 7.2. The minimum pH for the main bacteria that cause spoilage of meat, poultry and dairy products is about 5,0. At the same time, other types of microorganisms (especially yeasts and molds) can grow at pH values of 3,0 or below. Therefore, slightly acidic products can be spoiled.
|Microorganism||The minimum pH||Minimum aw|
|Clostridium botulinum (neproteoliticheskiy)||5,0||0,96|
|Clostridium botulinum (протеолитический)||4,6||0,93|
|Escherichia coli 0157:H7||3,8 – 4,2||0,97|
|Lactobacillus species||3 – 3,5||0,95|
|Staphylococcus aureus (S. aureus)||4,0 (4,6) *||0,86|
|Yeast and mold||<2,0||0,8 – 0,6|
* The minimum pH for the allocation of toxins.
acid-tolerant bacteria (lactic acid bacteria and some enterobacteria), and more acidic products - yeast and mold. The effects of temperature and pH are interrelated, and the minimum pH for growth at the optimum temperature can be significantly lower than at low temperatures . At pH values below the minimum for growth, some microorganisms in products die quickly, while others may continue to exist during the life of the product. Of particular concern in acidic foods is pathogenic E. coli E. coli 0157: H7, which is more acid tolerant than other pathogens. It can grow at pH values of 4,0 or lower and withstand lower pH [22,29] for a long time.
In addition to pH, the type of acid used affects the resistance of food to microorganisms. Organic acids (lactic, acetic, citric and malic) have a greater antibacterial effect than inorganic (hydrochloric, sulfuric). Care should be taken in the literature, as published minimum pH values are often related to the use of inorganic acids. Therefore, the minimum pH for microbial growth in food products is sometimes higher than that indicated in the literature, since organic acids are present in food products.
The antibacterial action of organic acids usually decreases in the following order: acetic, lactic (a-hydroxypropionic), citric, and then malic acid. Organic acids in undissociated form are effective against microorganisms, and the degree of dissociation depends on the pH of the products. Organic acids and their use in food systems are discussed in .
During the life of some products, their acid composition and pH do not remain constant. Changes in pH affect the types of growthable microorganisms and their growth rate. In some products, fermentation leads to a decrease in pH during storage, while in others an increase in acidity may be observed. For example, when ripening cheese with mold, the pH of the cheese at the surface increases due to the proteolytic activity of the mold, which is associated with the ability of Listeria monocytogenes to grow in such products, but not in the unripe cheese .
Low water activity
Aw water activity is a measure of the amount of water that is in a food product and can be used by microorganisms for their growth. By reducing the aw of a food product, the number of microorganisms capable of growth and their growth rate also decrease  (see Table 7.2). The aw water activity in food can be reduced by removing moisture (that is, drying) or by adding solutes (such as salt or sugar). Due to the great attention paid to diet and healthy nutrition, many types of jam and mashed potatoes reduce the sugar content. At the same time, the internal defense system, that is, the low aw of the product, is weakened, and some microorganisms (mainly yeasts) are able to grow. To prevent the growth of microorganisms, these products are usually recommended to be stored refrigerated after opening. The effect of water activity on maintaining the preservation of chilled products is associated with other preservative factors, including temperature . Yeast and mold are more resistant to low aw water activity in food than bacteria . Since the growth of bacteria is generally suppressed, yeasts and molds can grow and cause food spoilage.
To maintain microbiological stability, many refrigerated products contain natural or added preservatives (for example, salts, nitrites, benzoates, sorbates). The presence of these compounds affects the appearance and rate of possible damage to the product. Their use and mechanisms of action are considered in the work.
. As noted above, species of Pseudomonas often dominate in chilled fresh meat. Adding preservative salts (i.e. sodium chloride and potassium nitrate) to pork to produce bacon basically inhibits the growth of these microorganisms, and spoilage is caused by their other groups (for example, micrococci, staphylococci, lactic acid bacteria) [16, 39]. English sausage is mainly a product of fresh meat preserved by the addition of sulfites, which prevents the growth of microorganisms of the Pseudomonas species, and microbial spoilage is caused by Brochothrix thermosphacta sulfite-resistant or yeast .
The number and type of microorganisms capable of growing in chilled products containing preservatives depends on the type of product, the type of preservative, the pH of the product, the concentration of preservative, storage time and other preservative mechanisms in the product. Yeast and molds are often more resistant to preservatives than bacteria, and therefore can at the final stage prevail in the spoilage microflora. Recently, the tendency to reduce or eliminate the use of preservatives has increased, but this approach requires caution, since even small changes can lead to damage to the product and disruption of its microbiological stability.
Gas storage media
The use of controlled gaseous media, including vacuum packaging, for the storage of refrigerated products is expanding. Often they are chosen to maintain the organoleptic properties of the product, but many environments suppress or inhibit the development of normal microflora, causing spoilage. The genus Pseudomonas, the main group of microorganisms that cause spoilage of chilled protein products, needs to grow in the presence of oxygen, and therefore the use of vacuum packaging or CGS without oxygen prevents the growth of this group of microorganisms. Although other microorganisms can grow without oxygen, they usually grow more slowly, and therefore the occurrence of microbial spoilage is delayed. In the microflora that causes spoilage of meat in vacuum packaging, lactic acid bacteria or Brochothrix thermosphacta  usually prevail. In some cases, bacteria of the Enterobacteriaceae family or coliforms  can cause spoilage of products in vacuum packaging or packaging in the CSG.
Most industrial gas mixtures for packaging refrigerated products in a CSG usually contain a combination of carbon dioxide, nitrogen and oxygen. Inhibition of bacteria becomes more pronounced with increasing amounts of carbon dioxide. The effect of carbon dioxide on microbial growth was discussed in . Later, other gases (including neutral) and high levels of oxygen  were used to extend the shelf life of refrigerated products.
In order to maximize the potential benefits of the CSG and vacuum packaging, good temperature control is necessary. If a temperature violation occurs, the speed of damage can be close to the speed of damage without CGS. It has been suggested that CGS suppresses the microflora that usually cause food spoilage, but the growth of some anaerobic or facultatively anaerobic (capable of anaerobic development) pathogenic microorganisms (for example, Clostridium species, Listeria monocytogenes, Yersinia enterocolitica species, Salmonellala and Aeroneoneonocolitica species, Salmonellala species, and Aeroneoneonotroneslae Arononemonella species, Aeteroneonlosa Aeroneonaslaeratone, a species of Yersinia enterocolitica, Salmonellala, and Aeroneononelosis. will change. Therefore, the products can have a satisfactory appearance, but contain microorganisms that cause food poisoning. Of particular concern is the growth potential of the psychotrophic sticks of Clostridium botulinum botulinum . Molds usually require oxygen for their growth, so it is unlikely that they will cause problems in products subjected to vacuum packaging or packaging in the CGS (excluding oxygen). However, many yeasts can grow in or without oxygen, although aerobic growth is usually more efficient, and therefore faster.
Combinations of different factors preservatives
In many refrigerated products, microbiological stability is provided by a combination of several preservative factors described above that can effectively inhibit the growth of microorganisms . Care must be taken in the production, sale and retail of such products, since the inappropriate regulation of one factor can make it possible for microorganisms to grow rapidly. In addition, the use of two or more systems in their combination for certain types of microorganisms may be beneficial . For example, pasteurization in vacuum packaging includes vacuum packaging of products, followed by relatively moderate heat treatment (pasteurization). Heat treatment kills vegetative forms of microorganisms, but does not destroy spore-forming bacteria. During subsequent refrigerated storage (up to 30 days) in vacuum-packed anaerobic spore-forming bacteria, including CL botulinum sticks, can grow in the absence of other microorganisms. To prevent this, the product should be stored at a temperature below the minimum growth temperature CL. botulinum, and the composition of the product must be chosen to inhibit the growth of microorganisms or more efficient heat treatment  should be applied.