As our foods are of plant and/or animal origin, it is worthwhile to consider those characteristics of plant and animal tissues that affect the growth of microorganisms. The plants and animals that serve as food sources have all evolved mechanisms of defense against the invasion and proliferation of microorganisms, and some of these remain in effect in fresh foods. By taking these natural phenomena into account, one can make effective use of each or all in preventing or retarding the growth of pathogenic and spoilage organisms in the products that are derived from them.
INTRINSIC PARAMETERS
The parameters of plant and animal tissues that are an inherent part of the tissues are referred to as intrinsic parameter.33These parameters are as follows:
1. pH
2. Moisture content
3. Oxidation–reduction potential (Eh) 4. Nutrient content
5. Antimicrobial constituents 6. Biological structures
Each of these substrate-limiting factors is discussed below, with emphasis placed on their effects on microorganisms in foods.
pH
It has been well established that most microorganisms grow best at pH values around 7.0 (6.6–7.5), whereas few grow below 4.0 (Figure 3–1). Bacteria tend to be more fastidious in their relationships
39
40 Modern Food Microbiology
Figure 3–1 Approximate pH growth ranges for some foodborne organisms. The pH ranges for L. monocytogenes and S. aureus are similar.
to pH than molds and yeasts, with the pathogenic bacteria being the most fastidious. With respect to pH minima and maxima of microorganisms, those represented in Figure 3–1 should not be taken to be precise boundaries, as the actual values are known to be dependent on other growth parameters.
For example, the pH minima of certain lactobacilli have been shown to be dependent on the type of acid used, with citric, hydrochloric, phosphoric, and tartaric acids permitting growth at a lower pH value than acetic or lactic acids. In the presence of 0.2 M NaCl, Alcaligenes faecalis has been shown to grow over a wider pH range than in the absence of NaCl or in the presence of 0.2 M sodium citrate (Figure 3–2). Of the foods presented in Table 3–1, it can be seen that fruits, soft drinks, vinegar, and wines all fall below the point at which bacteria normally grow. The excellent keeping quality of these products is due in great part to pH. It is a common observation that fruits generally undergo mold and yeast spoilage, and this is due to the capacity of these organisms to grow at pH values<3.5, which is considerably below the minima for most food-spoilage and all food-poisoning bacteria (see Table 3–2).
Intrinsic and Extrinsic Parameters of Foods That Affect Microbial Growth 41
Figure 3–2 Relationship of pH, NaCl, and Na citrate on the rate of growth of Alcaligenes faecalis in 1% peptone:
A= 1% peptone; B = 0.2 M NaCl; C = 1% peptone + 0.2 M Na citrate. Source: Redrawn from Sherman and Holm48; used with permission of the publisher.
It may be noted from Table 3–3 that most of the meats and seafoods have a final ultimate pH of about 5.6 and above. This makes these products susceptible to bacteria as well as to mold and yeast spoilage.
Most vegetables have lower pH values than fruits, and, consequently, vegetables should be subject more to bacterial than fungal spoilage.
With respect to the keeping quality of meats, it is well established that meat from fatigued animals spoils faster than that from rested animals and that this is a direct consequence of final pH attained upon completion of rigor mortis. Upon the death of a well-rested meat animal, the usual 1% glycogen is converted to lactic acid, which directly causes a depression in pH values from about 7.4 to about 5.6, depending on the type of animal. Callow11found the lowest pH values for beef to be 5.1 and the highest 6.2 after rigor mortis. The usual pH value attained upon completion of rigor mortis of beef is around 5.6.5 The lowest and highest values for lamb and pork were found by Callow to be 5.4 and 6.7, and 5.3 and 6.9, respectively. Briskey8reported that the ultimate pH of pork may be as low as
42 Modern Food Microbiology
Table 3–1 Approximate pH Values of Some Fresh Fruits and Vegetables
Product pH Product pH
Vegetables Fruits
Asparagus (buds and stalks) 5.7–6.1 Apples 2.9–3.3 Beans (string and Lima) 4.6–6.5 Apple cider 3.6–3.8
Beets (sugar) 4.2–4.4 Apple juice 3.3–4.1
Broccoli 6.5 Bananas 4.5–4.7
Brussels sprouts 6.3 Figs 4.6
Cabbage (green) 5.4–6.0 Grapefruit (juice) 3.0
Carrots 4.9–5.2; 6.0 Grapes 3.4–4.5
Cauliflower 5.6 Limes 1.8–2.0
Celery 5.7–6.0 Melons (honeydew) 6.3–6.7
Corn (sweet) 7.3 Oranges (juice) 3.6–4.3
Cucumbers 3.8 Plums 2.8–4.6
Eggplant 4.5 Watermelons 5.2–5.6
Lettuce 6.0
Olives 3.6–3.8
Onions (red) 5.3–5.8
Parsley 5.7–6.0
Parsnip 5.3
Potatoes (tubers and sweet) 5.3–5.6
Pumpkin 4.8–5.2
Rhubarb 3.1–3.4
Rutabaga 6.3
Spinach 5.5–6.0
Squash 5.0–5.4
Tomatoes (whole) 4.2–4.3
Turnips 5.2–5.5
approximately 5.0 under certain conditions. The effect of pH of this magnitude on microorganisms, especially bacteria, is obvious. With respect to fish, it is known that halibut, which usually attains an ultimate pH of about 5.6, has better keeping qualities than most other fish, whose ultimate pH values range between 6.2 and 6.6.42
Some foods are characterized by inherent acidity; others owe their acidity or pH to the actions of certain microorganisms. The latter type is referred to as biological acidity and is displayed by products such as fermented milks, sauerkraut, and pickles. Regardless of the source of acidity, the effect on keeping quality appears to be the same.
Some foods are better able to resist changes in pH than others. Those that tend to resist changes in pH are said to be buffered. In general, meats are more highly buffered than vegetables. Contributing to the buffering capacity of meats are their various proteins. Vegetables are generally low in proteins and, consequently, lack the buffering capacity to resist changes in their pH during the growth of microorganisms (see Tables 6–4 and 6–5 for the general chemical composition of vegetables).
The capacity of E. coli to grow in three retail mustards was assessed, and with an inoculum of 106 cfu/g of this pathogen, its growth was inhibited in all three products.31The organism was not detected in dijon-style mustard (pH 3.55–3.60) beyond 3 h at room temperature, and after 2 days at 5◦C. In yellow-and deli-style mustards (pH 3.30 yellow-and 3.38, respectively), the organism was not detectable beyond 1 h.31
Intrinsic and Extrinsic Parameters of Foods That Affect Microbial Growth 43
Table 3–2 Reported Minimum pH Values for the Growth of Some Foodborne Bacteria
Aeromonas hydrophila ca. 6.0
Asaia siamensis 3.0
Alicyclobacillus acidocaldarius 2.0
Bacillus cereus 4.9
Botrytis cinerea 2.0
Clostridium botulinum, Group I 4.6
C. botulinum, Group II 5.0
C. perfringens 5.0
Escherichia coli 0157:H7 4.5
Gluconobacter spp. 3.6
Lactobacillus brevis 3.16
L. plantarum 3.34
L. sakei 3.0
Lactococcus lactis 4.3
Listeria monocytogenes 4.1
Penicillium roqueforti 3.0
Propioniibacterium cyclohexanicum 3.2
Plesiomonas shigelloides 4.5
Pseudomonas fragi ca. 5.0
Salmonella spp. 4.05
Shewanella putrefaciens ca. 5.4
Shigella flexneri 5.5–4.75
S. sonnei 5.0–4.5
Staphylococcus aureus 4.0
Vibrio parahaemolyticus 4.8
Yersinia enterocolitica 4.18
Zygosaccharomyces bailii 1.8
The natural or inherent acidity of foods, especially fruits, may have evolved as a way of protecting tis-sues from destruction by microorganisms. It is of interest that fruits should have pH values below those required by many spoilage organisms. The biological function of the fruit is the protection of the plant’s reproductive body, the seed. This one fact alone has no doubt been quite important in the evolution of present-day fruits. Although the pH of a living animal favors the growth of most spoilage organisms, other intrinsic parameters come into play to permit the survival and growth of the animal organism.
Although acidic pH values are of greater use in inhibiting microorganisms, alkaline values in the range of pH 12–13 are known to be destructive, at least to some bacteria. For example, the use of CaOH2to produce pH values in this range has been shown to be destructive to Listeria monocytogenes and other foodborne pathogens on some fresh foods.
pH Effects
Adverse pH affects at least two aspects of a respiring microbial cell: the functioning of its enzymes and the transport of nutrients into the cell. The cytoplasmic membrane of microorganisms is relatively impermeable to H+and OH−ions. Their concentration in the cytoplasm therefore probably remains
44 Modern Food Microbiology
Table 3–3 Approximate pH Values of Dairy, Meat, Poultry, and Fish Products
Product pH Product pH
Dairy products Fish and shellfish
Butter 6.1–6.4 Fish (most species)∗ 6.6–6.8
Buttermilk 4.5 Clams 6.5
Milk 6.3–6.5 Crabs 7.0
Cream 6.5 Oysters 4.8–6.3
Cheese (American mild 4.9; 5.9 Tuna fish 5.2–6.1
and cheddar) Shrimp 6.8–7.0
Meat and poultry Salmon 6.1–6.3
Beef (ground) 5.1–6.2 White fish 5.5
Ham 5.9–6.1
Veal 6.0
Chicken 6.2–6.4
Liver 6.0–6.4
∗Just after death.
reasonably constant despite wide variations that may occur in the pH of the surrounding medium.45 The intracellular pH of resting baker’s yeast cells was found by Conway and Downey16 to be 5.8.
Although the outer region of the cells during glucose fermentation was found to be more acidic, the inner cell remained more alkaline. On the other hand, Pe˜na et al.37did not support the notion that the pH of yeast cells remains constant with variations in pH of the medium. It appears that the internal pH of almost all cells is near neutrality. Bacteria such as Sulfolobus and Methanococcus may be exceptions, however. When microorganisms are placed in environments below or above neutrality, their ability to proliferate depends on their ability to bring the environmental pH to a more optimum value or range. When placed in acid environments, the cells must either keep H+ from entering or expel H+ ions as rapidly as they enter. Such key cellular compounds as DNA and ATP require neutrality. When most microorganisms grow in acid media, their metabolic activity results in the medium or substrate becoming less acidic, whereas those that grow in high pH environments tend to effect a lowering of pH. The amino acid decarboxylases that have optimum activity at around pH 4.0 and almost no activity at pH 5.5 cause a spontaneous adjustment of pH toward neutrality when cells are grown in the acid range. Bacteria such as Clostridium acetobutylicum raise the substrate pH by reducing butyric acid to butanol, whereas Enterobacter aerogenes produces acetoin from pyruvic acid to raise the pH of its growth environment. When amino acids are decarboxylated, the increase in pH occurs from the resulting amines. When grown in the alkaline range, a group of amino acid deaminases that have optimum activity at about pH 8.0 and cause the spontaneous adjustment of pH toward neutrality as a result of the organic acids that accumulate.
With respect to the transport of nutrients, the bacterial cell tends to have a residual negative charge.
Therefore, nonionized compounds can enter cells, whereas ionized compounds cannot. At neutral or alkaline pH, organic acids do not enter, whereas at acid pH values, these compounds are nonionized and can enter the negatively charged cells. Also, the ionic character of side chain ionizable groups is affected on either side of neutrality, resulting in increasing denaturation of membrane and transport enzymes.
Among the other effects that are exerted on microorganisms by adverse pH is that of the interaction between H+and the enzymes in the cytoplasmic membrane. The morphology of some microorganisms
Intrinsic and Extrinsic Parameters of Foods That Affect Microbial Growth 45
can be affected by pH. The length of the hyphae of Penicillium chrysogenum has been reported to decrease when grown in continuous culture where pH values increased above 6.0. Pellets of mycelium rather than free hyphae were formed at about pH 6.7.45Extracellular H+and K+may be in competition where the latter stimulates fermentation, for example, while the former represses it. The metabolism of glucose by yeast cells in an acid medium was markedly stimulated by K+.46Glucose was consumed 83% more rapidly in the presence of K+ under anaerobic conditions and 69% more under aerobic conditions.
Other environmental factors interact with pH. With respect to temperature, the pH of the substrate becomes more acid as the temperature increases. Concentration of salt has a definite effect on pH growth rate curves, as illustrated in Figure 3–2, where it can be seen that the addition of 0.2 M NaCl broadened the pH growth range of Alcaligenes faecalis. A similar result was noted for Escherichia coli by these investigators. When the salt content exceeds this optimal level, the pH growth range is narrowed. An adverse pH makes cells much more sensitive to toxic agents of a wide variety, and young cells are more susceptible to pH changes than older or resting cells.
When microorganisms are grown on either side of their optimum pH range, an increased lag phase results. The increased lag would be expected to be of longer duration if the substrate is a highly buffered one in contrast to one that has poor buffering capacity. In other words, the length of the lag phase may be expected to reflect the time necessary for the organisms to bring the external environment within their optimum pH growth range. Analysis of the substances that are responsible for the adverse pH is of value in determining not only the speed of subsequent growth, but also the minimum pH at which salmonellae would initiate growth. Chung and Goepfert14 found the minimum pH to be 4.05 when hydrochloric and citric acids were used, but 5.4 and 5.5 when acetic and propionic acids were used, respectively. This is undoubtedly a reflection of the ability of the organisms to alter their external environment to a more favorable range in the case of hydrochloric and citric acids as opposed to the other acids tested. It is also possible that factors other than pH come into play in the varying effects of organic acids as growth inhibitors. For more information on pH and acidity, see Corlett and Brown.17
Moisture Content
One of the oldest methods of preserving foods is drying or desiccation; precisely how this method came to be used is not known. The preservation of foods by drying is a direct consequence of removal or binding of moisture, without which microorganisms do not grow. It is now generally accepted that the water requirements of microorganisms should be described in terms of the water activity (aw) in the environment. This parameter is defined by the ratio of the water vapor pressure of food substrate to the vapor pressure of pure water at the same temperature: aw= p/po, where p is the vapor pressure of the solution and pois the vapor pressure of the solvent (usually water). This concept is related to relative humidity (RH) in the following way: RH= 100 × aw.13Pure water has an awof 1.00, a 22%
NaCl solution (w/v) has an awof 0.86, and a saturated solution of NaCl has an awof 0.75 (Table 3–4).
The water activity (aw) of most fresh foods is above 0.99. The minimum values reported for the growth of some microorganisms in foods are presented in Table 3–5 (see also Chapter 18). In general, bacteria require higher values of awfor growth than fungi, with Gram-negative bacteria having higher requirements than Gram positives. Most spoilage bacteria do not grow below aw= 0.91, whereas spoilage molds can grow as low as 0.80. With respect to food-poisoning bacteria, Staphylococcus aureus can grow as low as 0.86, whereas Clostridium botulinum does not grow below 0.94. Just as yeasts and molds grow over a wider pH range than bacteria, the same is true for aw. The lowest reported value for foodborne bacteria is 0.75 for halophiles (literally, “salt-loving”), whereas xerophilic
46 Modern Food Microbiology
Table 3–4 Relationship between Water Activity and Concentration of Salt Solutions
Sodium Chloride Concentration
Water Activity Molal Percent, w/v
0.995 0.15 0.9
0.99 0.30 1.7
0.98 0.61 3.5
0.96 1.20 7
0.94 1.77 10
0.92 2.31 13
0.90 2.83 16
0.88 3.33 19
0.86 3.81 22
Source: From The Science of Meat and Meat Products, by the American Meat Institute Foundation. W.H. Freeman and Company, San Francisco; copyright c 1960.
Table 3–5 Approximate Minimum awValues for Growth of Microorganisms Important in Foods
Organisms aw Organisms aw
Groups Groups
Most spoilage bacteria 0.9 Halophilic bacteria 0.75
Most spoilage yeasts 0.88 Xerophilic molds 0.61
Most spoilage molds 0.80 Osmophilic yeasts 0.61
Specific Organisms Specific Organisms
Clostridium botulinum, type E 0.97 Candida scottii 0.92
Pseudomonas spp. 0.97 Trichosporon pullulans 0.91
Acinetobacter spp. 0.96 Candida zeylanoides 0.90
Escherichia coli 0.96 Geotrichum candidum ca. 0.9
Enterobacter aerogenes 0.95 Trichothecium spp. ca. 0.90
Bacillus subtilis 0.95 Byssochlamys nivea ca. 0.87
Clostridium botulinum, types A and B 0.94 Staphylococcus aureus 0.86
Candida utilis 0.94 Alternaria citri 0.84
Vibrio parahaemolyticus 0.94 Penicillium patulum 0.81
Botrytis cinerea 0.93 Eurotium repens 0.72
Rhizopus stolonifer 0.93 Aspergillus glaucus∗ 0.70
Mucor spinosus 0.93 Aspergillus conicus 0.70
Aspergillus echinulatus 0.64 Zygosaccharomyces rouxii 0.62 Xeromyces bisporus 0.61
∗Perfect stages of the A. glaucus group are found in the genus Eurotium.
Intrinsic and Extrinsic Parameters of Foods That Affect Microbial Growth 47
(“dry-loving”) molds and osmophilic (preferring high osmotic pressures) yeasts have been reported to grow at awvalues of 0.65 and 0.61, respectively (Table 3–5). When salt is employed to control aw, an extremely high level is necessary to achieve awvalues below 0.80 (see Table 3–4).
Certain relationships have been shown to exist among aw, temperature, and nutrition. First, at any temperature, the ability of microorganisms to grow is reduced as the awis lowered. Second, the range of awover which growth occurs is greatest at the optimum temperature for growth; and third, the presence of nutrients increases the range of awover which the organisms can survive.32The specific values given in Table 3–5, then, should be taken only as reference points, as a change in temperature or nutrient content might permit growth at lower values of aw.
Effects of Low aw
The general effect of lowering awbelow optimum is to increase the length of the lag phase of growth and to decrease the growth rate and size of final population. This effect may be expected to result from adverse influences of lowered water on all metabolic activities because all chemical reactions of cells require an aqueous environment. It must be kept in mind, however, that awis influenced by other environmental parameters such as pH, temperature of growth, and Eh. In their study of the effect of aw on the growth of Enterobacter aerogenes in culture media, Wodzinski and Frazier54 found that the lag phase and generation time were progressively lengthened until no growth occurred with a lowering of aw. The minimum awwas raised, however, when the incubation temperature was decreased. When both the pH and temperature of incubation were made unfavorable, the minimum awfor growth was higher. The interaction of aw, pH, and temperature on the growth of molds on jam was shown by Horner and Anagnostopoulos.24 The interaction between awand temperature was the most significant.
In general, the strategy employed by microorganisms as protection against osmotic stress is the intracellular accumulation of compatible solutes. Halophiles (e.g., Halobacterium spp.) maintain os-motic equilibrium by maintaining the concentration of KCl in their cytoplasm equal to that of the suspending menstruum, and this is referred to as the “salt in cytoplasm” response. Nonhalophiles accumulate compatible solutes (osmolytes) in a biphasic manner. The first response is to increase K+ (and endogenously synthesized glutamate), and the second is to increase, either by de novo synthesis or by uptake, compatible solutes. The latter are very soluble molecules that have no net charge at physiological pH, and they do not adhere to or react with intracellular macromolecules (see reference 49). The three most common compatible solutes in most bacteria are carnitine, glycine betaine, and proline. Carnitine may be synthesized de novo, but the other two are generally not. Proline is syn-thesized by some Gram-positive bacteria while it is transported by Gram negatives. The solubility of glycine betaine in 100 ml of water at 25◦C is 160 g; it is 162 g for proline. Glycine betaine is employed by more living organisms that the other two osmolytes noted.
The uptake of osmolytes is mediated by a transport system. In L. monocytogenes, glycine betaine is transporated by BetL (it couples betaine accumulation to a Na+-motive) and Gbu (transports betaine)
48 Modern Food Microbiology
whereas the transporter for carnitine is OpuC.1,49Although some Gram-positive bacteria accumulate proline, it is concentrated to higher levels by Gram-negative bacteria. The three transporter systems in E. coli and S. Typhimurium are PutP, ProP, and ProU, with ProP being the most effective. It has been shown that the overproduction of proline by mutants of L. monocytogenes did not lead to changes in mouse virulence.49Under salt stress, L. monocytogenes produces 12 proteins one of which is highly similar to the Ctc protein of B. subtilis, and it is involved in osmotic stress tolerance in the absence of osmoprotectants in the medium.21The sigma factor-B (δB; see Chapter 22) plays a major role in the regulation of carnitine utilization in L. monocytogenes, but it is not essential for betaine utilization.20 Because it can grow at 4◦C, evidence has been presented that low-temperature growth of L. monocy-togenes is aided by the accumulation of glycine betaine.29The same is true for Yersinia enterocolitica, where osmotically stressed as well as cold-stressed cells accumulated osmolytes including glycine betaine.36 Temperature downshock and osmotic upshock caused a 30-fold uptake of radiolabeled glycine betaine.36In at least one strain of L. monocytogenes, glycine betaine transport is mediated by Gbu and BetL; and to a lesser extent OpuC.1
With regard to specific compounds used to lower water activity, results akin to those seen with adsorption and desorption systems (see Chapter 18) have been reported. In a study on the minimum aw
for the growth and germination of Clostridium perfringens, Kang et al.28found the value to be between 0.97 and 0.95 in complex media when sucrose or NaCl was used to adjust awbut 0.93 or below when glycerol was used. In another study, glycerol was found to be more inhibitory than NaCl to relatively salt-tolerant bacteria, but less inhibitory than NaCl to salt-sensitive species when compared at similar levels of awin complex media.30In their studies on the germination of Bacillus and Clostridium spores, Jakobsen and Murrell25observed strong inhibition of spore germination when awwas controlled by NaCl or CaCl2, but less inhibition when glucose or sorbitol was used, and very little inhibition when glycerol, ethylene, glycol, acetamide, or urea, were used. The germination of clostridial spores was completely inhibited at aw= 0.95 with NaCl, but no inhibition occurred at the same awwhen urea, glycerol, or glucose was employed. In another study, the limiting awfor the formation of mature spores by B. cereus strain T was shown to be about 0.95 for glucose, sorbitol, and NaCl, but about 0.91 for glycerol.26Both yeasts and molds have been found to be more tolerant to glycerol than to sucrose.24 Using a glucose minimal medium and Pseudomonas fluorescens, Prior39found that glycerol permitted growth at lower awvalues than either sucrose or NaCl. It was further shown by this researcher that the catabolism of glucose, sodium lactate, and dl-arginine was completely inhibited by awvalues greater than the minimum for growth when awwas controlled with NaCl. The control of awwith glycerol allowed catabolism to continue at awvalues below that for growth on glucose. In all cases where NaCl was used by this investigator to adjust the aw, substrate catabolism ceased at an awgreater than the minimum for growth, whereas glycerol permitted catabolism at lower awvalues than the minimum for growth. In spite of some reports to the contrary, it appears that glycerol is less inhibitory to respiring organisms than agents such as sucrose and NaCl.
Osmophilic yeasts accumulate polyhydric alcohols to a concentration commensurate with their extracellular aw. According to Pitt,38the xerophilic fungi accumulate compatible solutes or osmoreg-ulators as a consequence of the need for high internal solutes if growth at a low awis to be possible. In a comparative study of xerotolerant and nonxerotolerant yeasts to water stress, Edgley and Brown19 found that Zygosaccharomyces rouxii responded to a low awcontrolled by polyethylene glycol by retaining within the cells increasing levels of glycerol. However, the amount did not change greatly, nor did the level of arabitol change appreciably by aw. On the other hand, a nontolerant S. cere-visiae responded to a lowering of awby synthesizing more glycerol but retaining less. The Z. rouxii response to a low awwas at the level of glycerol permeation/transport, whereas that for S. cerevisiae