Food microbiology by WC Frazier.pdf

# Food as a Substrate for Microorganisms The interactions between microorganisms, plants, and animals are natural and constant. The ecological role of microorganisms and their importance in all the geochemical cycles in nature is well documented. Since the human food supply consists basically of pl...

# Food as a Substrate for Microorganisms The interactions between microorganisms, plants, and animals are natural and constant. The ecological role of microorganisms and their importance in all the geochemical cycles in nature is well documented. Since the human food supply consists basically of plants and animals or products derived from them, it is understandable that our food supply can contain microorganisms in interaction with the food. In most cases, microorganisms use our food supply as a source of nutrients for their own growth. This, of course, can result in deterioration of the food. By increasing their numbers, utilizing nutrients, producing enzymatic changes, and contributing off-flavors by means of breakdown of a product or synthesis of new compounds they can "spoil" a food. This is a normal consequence of the action of microorganisms, since one of their functions in nature is to convert reduced forms of carbon, nitrogen, and sulfur in dead plants and animals to the oxidized form required by plants, which in turn are consumed by animals. So by simply "doing their thing" in nature they frequently can render our food supply unfit for consumption. To prevent this we minimize the contact between microorganisms and our foods (prevent contamination) and also eliminate microorganisms from our foods, or at least adjust conditions of storage to prevent their growth (preservation). When the microorganisms involved are pathogenic, their association with our food supply is critical from a public health point of view. Many of our foods will support the growth of pathogenic microorganisms or at least serve as a vector of them. Here again, we attempt to prevent their entrance and growth in our foods or eliminate them by processing. Interactions between microorganisms and our foods are sometimes beneficial, as exemplified by the many cultured products consumed and enjoyed. What are the governing factors in these interactions? Why is this interaction beneficial at some times and not at others? Why do some foods support the growth of microorganisms more readily than others? Why are some foods very stable in regard to microbial deterioration? Food is the substrate, and so the characteristics of a food are an important consideration. The type of microorganisms present and the environmental conditions also are important. However, the food or substrate dictates what can or cannot grow. By understanding the characteristics of the food or substrate one can make predictions about the microbial flora that may develop. A knowledge of the factors that favor or inhibit the growth of microorganisms is essential to an understanding of the principles of food spoilage and preservation. The chief compositional factors of a food that influence microbial activity are hydrogen-ion concentration, moisture, oxidation-reduction (O-R) potential, nutrients, and the presence of inhibitory substances or barriers. ## Hydrogen-Ion Concentration (pH) Every microorganism has a minimal, a maximal, and an optimal pH for growth. Microbial cells are significantly affected by the pH of food because they apparently have no mechanism for adjusting their internal pH. In general, yeasts and molds are more acid-tolerant than bacteria. The inherent pH of foods varies, although most foods are neutral or acidic. Foods with low pH values (below 4.5) usually are not readily spoiled by bacteria and are more susceptible to spoilage by yeasts and molds. A food with an inherently low pH would therefore tend to be more stable microbiologically than a neutral food. The excellent keeping quality of the following foods is related to their restrictive pH: fruits, soft drinks, fermented milks, sauerkraut, and pickles. Some foods have a low pH because of inherent acidity; others, e.g., the fermented products, have a low pH because of developed acidity from the accumulation of lactic acid during fermentation. Molds can grow over a wider range of pH values that can most yeasts and bacteria, and many molds grow at acidities too great for yeasts and bacteria. Most fermentative yeasts are favored by a pH of about 4.0 to 4.5, as in fruit juices, and film yeasts grow well on acid foods such as sauerkraut and pickles. On the other hand, most yeasts do not grow well in alkaline substrates and must be adapted to such media. Most bacteria are favored by a pH near neutrality, although some, such as the acid formers, are favored by moderate acidity, and other, e.g., the actively proteolytic bacteria, can grow in media with a high (alkaline) pH, as found in the white of a stored egg. The buffers in a food, i.e., the compounds that resist changes in pH, are important not only for their buffering capacity but also for their ability to be especially effective within a certain pH range. Buffers permit an acid (or alkaline) fermentation to go on longer with a greater yield of products and organisms than would otherwise be possible. Vegetable juices have low buffering power, permitting an appreciable decrease in pH with the production of only small amounts of acid by the lactic acid bacteria during the early part of sauerkraut and pickle fermentations. This enables the lactic to suppress the undesirable pectin-hydrolyzing and proteolytic competing organisms. Low buffering power makes for a more rapidly appearing succession of microorganisms during a fermentation than does high buffering power. Milk, on the other hand, is fairly high in protein (a good buffer) and therefore permits considerable growth and acid production by lactic acid bacteria in the manufacture of fermented milks before growth of the starter culture is finally suppressed. The pH of a product can be readily determined with a pH meter, but this value alone may not be sufficient for predicting microbial responses. It is also desirable, for example, to know the acid responsible for a given pH, because some acids, particularly the organic acids, are more inhibitory than others. The inhibitory properties of many of the organic acids, including acetic, benzoic, citric, lactic, proprionic, and sorbic acids, make them widely used as acidulants or preservatives in foods. Also, changes in titratable acidity are not always evident from pH measurements. Not only are the rates of growth of microorganisms affected by pH, so are the rates of survival during storage, heating, drying, and other forms of processing. Also, the initial pH may be suitable, but because of competitive flora or growth of the organism itself, the pH may become unfavorable. Conversely, the initial pH may be restrictive, but the growth of a limited number of microorganisms may alter the pH to a range that is more favorable for the growth of many other microorganisms. ## Moisture Requirement: The Concept of Water Activity Microorganisms have an absolute demand for water, for without water no growth can occur. As might be expected, the exact amount of water needed for growth of microorganisms varies. This water requirement is best expressed in terms of available water or water activity, $a_w$, the vapor pressure of the solution (of solutes in water in most foods) divided by the vapor presure of solvent (usually water). The $a_w$ for pure water would be 1.00, and for a 1.0 m solution of the ideal solute the $a_w$ would be 0.9823. The $a_w$ (× 100) would be in equilibrium with the relative humidity (RH) of the atmosphere about the food. In other words, $a_w$ × 100 = equilibrium relative humidity (ERH) (%), or $ERH= 100 \times a_w$. A relative humidity about a food corresponding to an $a_w$ lower than that of the food would tend to dry the surface of the food; conversely, if the relative humidity were higher than that corresponding to the $a_w$, the latter would be increased at the surface of the food. The $a_w$ for many groups of foods is summarized in Table 1.1. Water is made unavailable in various ways: 1. Solutes and ions tie up water in solution. Therefore, an increase in the concentration of dissolved substances such as sugars and salts is in effect a drying of the material. Not only is water tied up by solutes, but water tends to leave the microbial cells by osmosis if there is a higher concentration of solute outside the cells than inside. 2. Hydrophilic colloids (gels) make water unavailable. As little as 3 to 4 percent agar in a medium may prevent bacterial growth by leaving too little available moisture. 3. Water of crystallization or hydration is usually unavailable to microorganisms. Water itself, when crystallized as ice, no longer can be used by microbial cells. The $a_w$ of water-ice mixtures (vapor pressure of ice divided by vapor pressure of water) decreases with a decrease in temperature below 0 C. The $a_w$ values of pure water are 1.00 at 0 C, 0.953 at -5 C, 0.907 at -10 C, 0.846 at 15 C, 0.823 at -20 C, and so on. In a food, as more ice is formed, the concentration of solutes in the unfrozen water is increased, lowering its $a_w$. The reduction of $a_w$ by a solute depends primarily on the total concentration of dissolved molecules and ions, each of which is surrounded by water molecules held more or less firmly. The solution then has a lower freezing point and a lower vapor pressure than does pure water. The organisms must compete with these particles for water molecules. The decrease in vapor pressure for an ideal solvent follows Raoult's law: The vapor pressure of the solution relative to that of the pure solvent is equal to the mole fraction of the solvent; that is, $p/p_o$ = n2 / n1 + n2, where p and $p_o$ are the vapor pressures of the solution and solvent and n1 and n2 are the number of moles of solute and solvent, respectively. Although a varies with temperature, the variations are only slight within the range of temperatures permitting microbial growth. However, as the concentration of solutes increases, a variation in temperature becomes more important in regard to $a_w$, because of an increasing effect on ionization. | **$a_w$ Values** | **Food** | |---|---| | 0.98 and above | Fresh meat and fish | | | Fresh fruits and vegetables | | | Milk and most beverages | | | Canned vegetables in brine | | | Canned fruits in light syrup | | 0.93-0.98 | Evaporated milk | | | Tomato paste | | | Processed cheese | | | Bread| | | Canned cured meats | | | Fermented sausage (not dried)| | | Canned fruits in heavy syrup | | | Gouda cheese | | 0.85-0.93 | Dry or fermented sausage | | | Dried beef | | | Raw ham | | | Aged cheddar cheese | | | Sweetened condensed milk | | 0.60-0.85 | Dried fruit | | | Flour | | | Cereals | | | Jams and jellies | | | Nuts | | | Some aged cheeses | | | Intermediate-moisture foods | |Below 0.60 | Chocolate | | | Confectionery | | | Honey | | | Biscuits| | | Crackers | | | Potato chips | | | Dried eggs, milk, and vegetables | Source: Adapted from Christian, 1980. Each microorganism has a maximal, optimal, and minimal $a_w$ for growth. This range depends upon factors discussed subsequently. As the $a_w$ is reduced below the optimal level, there is a lengthening of the lag phase of growth, a decrease in the rate of growth, and a decrease in the amount of cell substance synthesized- changes that vary with the organism and with the solute employed to reduce $a_w$. Factors that may affect $a_w$ requirements of microorganisms include the following: 1. The kind of solute employed to reduce the $a_w$. For many organisms, especially molds, the lowest $a_w$ for growth is practically independent of the kind of solute used. Other organisms, however, have lower limiting $a_w$ values with some solutes than with others. Potassium chloride, for example, usually is less toxic than sodium chloride, and it in turn is less inhibitory than sodium sulfate. 2. The nutritive value of the culture medium. In general, the better the medium for growth, the lower the limiting $a_w$. 3. Temperature. Most organisms have the greatest tolerance to low $a_w$ at about optimal temperatures. 4. Oxygen supply. Growth of aerobes takes place at a lower $a_w$ in the presence of air than in its absence, and the reverse is true of anaerobes. 5. pH. Most organisms are more tolerant of low $a_w$ at pH values near neutrality than in acid or alkaline media. 6. Inhibitors. The presence of inhibitors narrows the range of $a_w$ for growth of microorganisms. Methods for the control of $a_w$ are (1) equilibration with controlling solutions, (2) determination of the water-sorption isotherm for the food (Iglesias and Chirife, 1976), and (3) addition of solutes. Methods for measuring or establishing $a_w$ values of foods include freezing-point determinations, manometric techniques, and electrical devices. Freezing-point determinations are feasible only on liquid foods with high $a_w$ values. The measurement is based on the Clausius-Clapeyron equation for dilute solutions (Strong et al., 1970). A manometric technique for directly measuring the vapor pressure in the vapor space surrounding a food is considered very accurate. This technique and the device are described in detail by Labuza (1974). Various electrical devices for measuring $a_w$ indirectly have been employed. The most common use sensors which measure relative humidity in the vapor space surrounding a food based on electrical resistance. One such device is shown in [Figure 1.1] . The probes vary in their sensitivity to relative humidity ranges and must be selected on the basis of expected results. The food is placed in a jar, and equilibration of the water in the food and vapor space is usually obtained in one to several hours. As the current is passed through the salt-coated filament of the probe, the resistance is determined and a readout is shown on the dial. Calibration charts are used to convert readout values to $a_w$ or percent relative humidity. A collaborative study conducted on a comparison of $a_w$ methodology (Labuza et al., 1976) showed manometric vapor-pressure determinations to be the most accurate. Most bacteria grow well in a medium with a water activity $a_w$ approaching 1.00 (at 0.995 to 0.998, for example); i.e., they grow best in low concentrations of sugar or salt, although there are notable exceptions that will be mentioned later. Culture media for most bacteria contain not more than 1 percent sugar and 0.85 percent sodium chloride (“physiological salt solution"); as little as 3 to 4 percent sugar and 1 to 2 percent salt may inhibit some bacteria. The optimal $a_w$ and the lower limit for growth vary with the bacterium as well as with food, temperature, pH, and the presence of oxygen, carbon dioxide, and inhibitors; they are lower for bacteria able to grow in high concentrations of sugar or salt. Examples of reported lower limits of $a_w$ for growth of some food bacteria are 0.97 for Pseudomonas, 0.96 for Escherichia coli, 0.95 for Bacillus subtilis, 0.945 for Enterobacter aerogenes, 0.86 for Staphylococcus aureus, and 0.93 for Clostridium botulinum. Other bacteria will grow with the $a_w$ below 0.90. These figures would be different under other conditions of growth than those used in obtaining the values. Some optimal $a_w$ figures reported for food bacteria are 0.99 to 0.995 for Staph. aureus and Salmonella spp., 0.995 for E. coli, and 0.982 for Streptococcus faecalis. Molds differ considerably in optimal $a_w$ and range of $a_w$ for germination of the asexual spores. The range for spore germination is greater at temperatures near the optimum for germination and in a better culture medium. The minimal $a_w$ for spore germination has been found to be as low as 0.62 for some molds and as high as 0.93 for others (e.g., Mucor, Rhizopus, and Botrytis). Each mold also has an optimal $a_w$ and range of $a_w$ for growth. Examples of optimal $a_w$ are 0.98 for an Aspergillus sp., 0.99 to 0.98 for a Rhizopus sp., and 0.99 for a Penicillium sp. The $a_w$ would have to be below 0.62 to stop all chances for mold growth, although an $a_w$ below 0.70 inhibits most molds causing food spoilage and an $a_w$ below 0.94 inhibits molds such as Rhizopus and below 0.85 inhibits Aspergillus spp. The reduction of the $a_w$ below the optimum for a mold delays spore germination and reduces the rate of growth and therefore can be an important factor in food preservation. Many of the molds can grow in media with an $a_w$ approaching 1.00 (pure water). A consideration of the moisture requirements of microorganisms leads to some general considerations: 1. Each organism has its own characteristic optimal $a_w$ and its own range $a_w$ for growth for a given set of environmental conditions. Factors affecting the moisture requirements of organisms are (a) the nutritive properties of the substrate, (b) its pH, (c) its content of inhibitory substances, (d) availability of free oxygen, and (e) temperature. The range of $a_w$ permitting growth is narrowed if any of these environmental factors is not optimal and is narrowed still more if two or more conditions are not favorable. 2. An unfavorable $a_w$ will result not only in a reduction in the rate of growth but also in a lowered maximal yield of cells. 3. The more unfavorable the $a_w$ of the substrate, the greater the delay (lag) in initiation of growth or germination of spores. This often is as important in food preservation as is reduction in the rate of growth of the organism. 4. In general, bacteria require more moisture than yeasts, and yeasts more than molds, as shown in Table 1.2, which shows lower limits of $a_w$ for bacteria yeasts, and molds. There are notable exceptions to this generalization, however, as some molds have a higher minimal $a_w$ for growth (and spore germination) than do many yeasts and some bacteria. | **Group of microorganisms** | **Minimal $a_w$ value** | |---|---| | Many bacteria | 0.91 | | Many yeasts | 0.88 | | Many molds | 0.80 | | Halophilic bacteria | 0.75 | | Xerophilic fungi | 0.65 | | Osmophilic yeasts | 0.60 | Source: After Mossel and Ingrams, 1955. 5. Microorganisms that can grow in high concentrations of solutes, e.g., sugar and salt, obviously have a low minimal $a_w$. Halophilic bacteria require minimal concentrations of dissolved sodium chloride for growth. Osmophilic yeasts grow best in high concentrations of sugar. (See Chapter 2.) ## Oxidation-Reduction Potential The oxygen tension or partial pressure of oxygen about a food and the O-R potential, or reducing and oxidizing power of the food itself, influence the type of organisms which will grow and hence the changes produced in the food. The O-R potential of the food is determined by (1) the characteristic O-R potential of the original food, (2) the poising capacity, i.e., the resistance to change in potential, of the food, (3) the oxygen tension of the atmosphere about the food, and (4) the access which the atmosphere has to the food. Air has a high oxygen tension, but the head space in an "evacuated" can of food would have a low oxygen tension. From the standpoint of ability to use free oxygen, microorganisms have been classified as aerobic when they require free oxygen, anaerobic when they grow best in the absence of free oxygen, and facultative when they grow well either aerobically or anaerobically. Molds are aerobic, most yeasts grow best aerobically, and bacteria of different kinds may be aerobic, anaerobic, or facultative. From the standpoint of O-R potential, a high (oxidizing) potential favors aerobes but will permit the growth of facultative organisms, and a low (reducing) potential favors anaerobic or facultative organisms. However, some organisms that are considered aerobic can grow (but not well) at surprisingly low O-R potentials. Growth of an organism may alter the O-R potential of a food enough to restrain other organisms. Anaerobes, for example, may lower the O-R potential to a level inhibitory to aerobes. The O-R potential of a system is usually written Eh and measured and expressed in terms of millivolts (mV). A highly oxidized substrate would have a positive Eh, and a reduced substrate a negative Eh. Aerobic microorganisms including bacilli, micrococci, pseudomonads, and acinetobacters require positive Eh values or positive mV O-R potentials. Conversely, anaerobes including clostridia and bacteriodes require negative Eh values or negative mV O-R potentials. Most fresh plant and animal foods have a low and well-poised Q-R potential in their interior: the plants because of reducing substances such as ascorbic acid and reducing sugars and animal tissues because of -SH (sulfhydryl) and other reducing groups. As long as the plant or animal cells respire and remain active, they tend to poise the O-R system at a low level, resisting the effect of oxygen diffusing from the outside. Therefore, a piece of fresh meat or a fresh whole fruit would have aerobic conditions only at and near the surface. The meat could support aerobic growth of slime-forming or souring bacteria at the surface at the same time that anaerobic putrefaction was proceeding in the interior. This situation may be altered by processing procedures. Heating may reduce the poising power of the food by means of destruction or alteration of reducing and oxidizing substances and also allow more rapid diffusion of oxygen inward, either because of the destruction of poising substances or because of changes in the physical structure of the food. Processing also may remove oxidizing or reducing substances; thus clear fruit juices have lost reducing substances by their removal during extraction and filtration and therefore have become more favorable to the growth of yeasts than was the original juice containing the pulp. In the presence of limited amounts of oxygen the same aerobic or facultative organisms may produce incompletely oxidized products, such as organic acids, from carbohydrates, when with plenty of oxygen available complete oxidation to carbon dioxide and water might result. Protein decomposition under anaerobic conditions may result in putrefaction, whereas under aerobic conditions the products are likely to be less obnoxious. ## Nutrient Content The kinds and proportions of nutrients in the food are all-important in determining what organism is most likely to grow. Consideration must be given to (1) foods for energy (2) foods for growth, and (3) accessory food substances, or vitamins, which may be necessary for energy or growth. - **Foods for Energy** The carbohydrates, especially the sugars, are most commonly used as an energy source, but other carbon compounds may serve, e.g., esters, alcohols, peptides, amino acids, and organic acids and their salts. Complex carbohydrates, e.g., cellulose, can be utilized by comparatively few organisms, and starch can be hydrolyzed by only a limited number of organisms. Microorganisms differ even in their ability to use some of the simpler soluble sugars. Many organisms cannot use the disaccharide lactose (milk sugar) and therefore do not grow well in milk. Maltose is not attacked by some yeasts. Bacteria often are identified and classified on the basis of their ability or inability to utilize various sugars and alcohols. Most organisms, if they utilize sugars at all, can use glucose. The ability of microorganisms to hydrolyze pectin, which is characteristic of some kinds of bacteria and many molds, is important, of course, in the softening or rotting of fruits and vegetables or fermented products from them. A limited number of kinds of microorganisms can obtain their energy from fats but do so only if a more readily usable energy food, such as sugar, is absent. First, the fat must be hydrolyzed with the aid of lipase to glycerol and fatty acids, which then can serve as an energy source for the hydrolyzing organism or other organisms. In general, aerobic microorganisms are more commonly involved in the decomposition of fats than are anaerobic ones, and the lipolytic organisms usually are also proteolytic. Direct oxidation of fats containing unsaturated fatty acids usually is chemical. Hydrolysis products of proteins, peptides, and amino acids, for example, serve as an energy source for many proteolytic organisms when a better energy source is lacking and as foods for energy for other organisms that are not proteolytic. Meats, for example, may be low in carbohydrate and therefore decomposed by proteolytic species, e.g., Pseudomonas spp., with successive growth of weakly proteolytic or nonproteolytic species that can utilize the products of protein hydrolysis. Organisms differ in their ability to use individual amino acids for energy. This is because it is the number of molecules (or moles) of sugar which affects aw, and a percentage is usually expressed as weight per unit volume. Not only is the kind of energy food important but also its concentration in solution and hence its osmotic effect and the amount of available moisture. For a given percentage of sugar in solution, the osmotic pressure will vary with the weight of the sugar molecule. Therefore, a 10% solution of glucose has about twice the osmotic pressure of a 10% solution of sucrose or maltose; i.e., it ties up twice as much moisture. In general, molds can grow in the highest concentrations of sugars and yeasts in fairly high concentrations, but most bacteria grow best in fairly low concentrations. There are, of course, notable exceptions to this generalization: Osmophilic yeasts grow in as high concentrations of sugar as molds, and some bacteria can grow in fairly high concentrations of sugar. Of course, an adequate supply of foods for growth will favor utilization of the foods for energy. More carbohydrate will be used if a good nitrogen food is present in sufficient quantity than will be the case if the nitrogen is poor in kind or amount. Organisms requiring special accessory growth substances might be prevented from growing if one or more of these "vitamins" were lacking, and thus the whole course of decomposition might be altered. - **Foods for Growth** Microorganisms differ in their ability to use various nitrogenous compounds as a source of nitrogen for growth. Many organisms are unable to hydrolyze proteins and hence cannot get nitrogen from them without help from a proteolytic organism. One protein may be a better source of nitrogenous food than another because of different products formed during hydrolysis, especially peptides and amino acids. Peptides, amino acids, urea, ammonia, and others simpler nitrogenous compounds may be available to some organisms but not to others or may be usable under some environmental conditions but not under others. Some of the lactic acid bacteria grow best with polypeptides as nitrogen foods, cannot attack casein, and do not grow well with only a limited number of kinds of amino acids present. The presence of fermentable carbohydrate in a substrate usually results in an acid fermentation and suppression of proteolytic bacteria and hence in what is called a "sparing" action on the nitrogen compounds. Also, the production of obnoxious nitrogenous products is prevented or inhibited. Many kinds of molds are proteolytic, but comparatively few genera and species of bacteria and very few yeasts are actively proteolytic. In general, proteolytic bacteria grow best at pH values near neutrality and are inhibited by acidity, although there are exceptions, such as proteolysis by the acid-proteolytic bacteria that hydrolyze protein while producing acid. Carbon for growth may come partly from carbon dioxide, but more often it comes from organic compounds. The minerals required by microorganisms are nearly always present at the low levels required, but occasionally an essential mineral may be tied up so that it is unavailable, lacking, or present in insufficient amounts. An example is milk, which contains insufficient iron for pigmentation of the spores of *Penicillium roqueforti*. Bacteria causing septicemia usually have the ability to bind some of the iron in blood. Only strains which can compete for transferring iron are able to grow well in human blood. ## Accessory Food Substances, Orvitamins Some microorganisms are unable to manufacture some or all of the vitamins needed and must have them furnished. Most natural plant and animal foodstuffs contain an array of these vitamins, but some may be low in amount or lacking. Thus meats are high in B vitamins and fruits are low, but fruits are high in ascorbic acid. Egg white contains biotin but also contains avidin, which ties up biotin, making it unavailable to microorganisms and eliminating as possible spoilage organisms those which must have biotin supplied. The processing of foods often reduces the vitamin content. Thiamine, pantothenic acid, the folic acid group, and ascorbic acid (in air) are heat-labile, and drying causes a loss in vitamins such as thiamine and ascorbic acid. Even storage of foods for long periods, especially if the storage temperature is elevated, may result in a decrease in the level of some of the accessory growth factors. Each kind of bacterium (or other microorganism) has a definite range of food requirements. For some species the range is wide, and growth takes place in a variety of substrates, as is true for coliform bacteria; but for others, e.g., many of the pathogens, the range is narrow and the organisms can grow in only a limited number of kinds of substrates. Thus, bacteria differ in the foods that they can utilize for energy: Some can use a variety of carbohydrates, e.g., the coliform bacteria and *Clostridium* spp., and others only one or two, while some can use other carbon compounds such as organic acids and their salts, alcohols, and esters (*Pseudomonas* spp.). Some can hydrolyze complex carbohydrates, although others cannot. Likewise, the nitrogen requirements of bacteria such as *Pseudomonas* spp. may be satisfied by simple compounds such as ammonia or nitrates; or more complex compounds such as amino acids, peptides, or proteins may be utilized or even required, as is true for the lactics. Bacteria also vary in their need for vitamins or accessory growth factors; some (*Staph. aureus*) synthesize part and others (*Pseudomonas* or *E. coli*) all of the factors needed, and still others must have them all furnished (the lactics and many pathogens). It should be emphasized that in general, the better the medium for an organism, the wider the ranges of temperature, pH, and $a_w$ over which growth can take place. ## Inhibitory Substances and Biological Structure Inhibitory substances, originally present in the food, added purposely or accidentally, or developed there by growth of microorganisms or by processing methods, may prevent growth of all microorganisms or, more often, may deter certain specific kinds. Examples of inhibitors naturally present are the lactenins and anticoliform factor in freshly drawn milk, lysozyme in egg white, and benzoic acid in cranberries. A microorganism growing in a food may produce one or more substances inhibitory to other organisms, products such as acids, alcohols, peroxides, and even antibiotics. Propionic acid produced by the propionibacteria in Swiss cheese is inhibitory to molds; alcohol formed in quantity by wine yeasts inhibits competitors; and nisin produced by certain strains of *Streptococcus lactis* may be useful in inhibiting lactate-fermenting, gas-forming clostridia in curing cheese and undesirable in slowing down some of the essential lactic acid streptococci during the manufacturing process. There also is the possibility of the destruction of inhibitory compounds in foods by microorganisms. Certain molds and bacteria are able to destroy some of the phenol compounds that are added to meat or fish by smoking or benzoic acid added to foods; sulfur dioxide is destroyed by yeasts resistant to it; and lactobacilli can inactivate nisin. Heating foods may result in the formation of inhibitory substances: Heating lipids may hasten autoxidation and make them inhibitory, and browning concentrated sugar sirups may result in the production of furfural and hydroxymethyl furfural, which are inhibitory to fermenting organisms. Long storage at warm temperatures may produce similar results. The effect of the biological structure of food on the protection of foods against spoilage has been noted. The inner parts of whole, healthy tissues of living plants and animals are either sterile or low in microbial content. Therefore, unless opportunity has been given for their penetration, spoilage organisms within may be few or lacking. Often there is a protective covering about the food, e.g., the shell on eggs, the skin on poultry, the shell on nuts, and the rind or skin on fruits and vegetables, or we may have surrounded the food with an artificial coating, e.g., plastic or wax. This physical protection of the food not only may help its preservation but also may determine the kind, rate, and course of spoilage. Layers of fat over meat may protect that part of the flesh, or scales may protect the outer part of the fish. On the other hand, an increase in exposed surface brought about by peeling, skinning, chopping, or comminution may serve not only to distribute spoilage organisms but also to release juices containing food materials for the invaders. The disintegration of tissues by freezing may accomplish a similar result. In meat the growth of spoilage bacteria takes place mostly in the fluid between the small meat fibers, and it is only after rigor mortis that much of this food material is released from the fibers to become available to spoilage organisms. ## Combined Effects of Factors Affecting Growth Each of the compositional factors of foods- $a_w$, pH, O-R potential, and nutrient content- can significantly affect the resulting microbial flora. Many of these factors interact, and therefore one must be concerned with the total ecology of the food. For example, a microorganism growing near its optimal pH, will be more tolerant to changes in $a_w$ than will one growing close to its minimal or maximal pH. Therefore, a combined inhibitory effect of an unfavorable pH and $a_w$ can be noted. To prevent or retard growth, several of these factors can be manipulated rather than adjusting one to an inhibitory level. Factors affecting the germination of spores of *Clostridium botulinum* have indicated interactions or combined effects involving $a_w$, pH, temperature, O-R potential, and sodium chloride and sodium nitrate concentrations. The difficulty of collecting numerous data points over a wide range of variables may result in the need to construct mathematical models to predict a suitable preservation system. Techniques for describing the effect of two factors affecting growth (pH and $a_w$) plus temperature have been used to predict the level of a possible hazard resulting from the growth of *Staph. aureus* and *Salmonella typhimurium* (Broughall and Brown, 1984). ## Review Questions 1. What are the factors in a food that influence microbial activity? 2. Discuss the role of water in the growth of microorganisms. 3. What are the factors affecting $a_w$? 4. Discuss the role of biological structures in the protection of foods against spoilage. 5. How does the O-R potential of a food influence microbial growth? 6. Write notes on inhibitory substances present in foods. 7. Write notes on foods as energy sources for microorganisms. 8. Discuss how water is made unavailable to microbes. 9. Write notes on accessory food substances. 10. Write notes on the combined effects of factors affecting microbial growth in foods. ## Multiple-Choice Questions 1. Nisin is produced by strains of a. *Streptococcus lactis* b. *Pseudomonas* c. *E. coli* d. *Clostridium* 2. The inner parts of whole healthy tissues of living plants and animals are a. low in microbial content b. sterile c. high in microbial content d. both a & b 3. *Pseudomonas* grows well in food containing a. vitamins b. organic acids c. antibiotics d. nitrates 4. Many kinds of molds are a. proteolytic b. inhibit breakdown of proteins c. lipolytic d. none of these 5. Proteolytic organisms utilize hydrolysis products of a. proteins b. peptides c. amino acids d. all of the above 6. Osmophilic organisms like yeasts grow best in a. low concentrations of sugar b. high concentrations of sugar c. low concentrations of salt d. high concentrations of salt 7. The $a_w$ for pure water is a. 1.00 b. 9.99 c. 0.9823 d. 0.1 8. Organisms that grow over a wide range of pH are a. bacteria b. yeasts c. thermophillic anaerobes d. molds 9. Avidin is present in a. meat b. fish c. eggs d. fruits 10. Browning of sugar syrups results in the production of a. phenols b. furfural c. alcohol d. ketones ## Answers 1. (a) 2. (d) 3. (d) 4. (a) 5. (d) 6. (a) 7. (a) 8. (d) 9. (c) 10. (b)