INFLUENCE OF WATER ACTIVITY ON FOODBORNE BACTERIA A REVIEW WILLIAM H.

. SPERBER ABSTRACT The influence of water activity on some characteristics of bacterial growth is presented. Bacteria are able to overcome the plasmolytic effect of reduced water activity by intracellularly accumulating compatible solutes such as glutamic acid or proline. In food systems, water activity is one of several preservative factors which interact to form a preservative system. Other preservative factors considered in this review are temperature, pH, O/R potential and chemical preservatives. Control of water activity in foods is receiving more attention as new food products and new technologies are developed.

This review has been organized to summarize current knowledge about the influence of water activity (aw) on bacterial growth and its importance in food bacteriology. The review does not include the influence of a w on bacterial toxin production or on the heat resistance of bacterial cells and spores. INFLUENCE OF WATER ACTIVITY ON BACTERIAL GROWTH Influence on growth rate A reduction in water activity has a dramatic effect on bacterial growth. One can observe, for instance, an increase in the lag phase and decreases in the growth rate and cell yield as water activity is reduced (50). Figure 1 shows the general response of bacterial growth rate to decreasing water activity. As the aw is decreased from 1.0, the growth rate increases (39). Many bacteria have their maximum growth rates between a w 0,990 and 0.995. From the maximum point, the growth rate diminishes until the minimum a w for growth in reached. The growth rate of Staphylococcus aureus at aw 0.90 is only 10% of its maximum growth rate (50). At a w values lower than the minimum for growth, cells either remain dormant or die.

Figure 1. Influence of water activity on bacterial growth rate. Generalized response.

Influence of type of solute The type of solute used to control aw has some influence on growth patterns. Generally NaCl, KCl, glucose and sucrose show similar patterns while glycerol permits growth at lower a w values. Several examples presented in Table 1 show similar minimum aw values for growth when NaCl or glucose are the solutes, but markedly lower values when glycerol is the solute.
Tabla 1. Influence of solute on the minimum water activity for bacterial growth.

Glycerol freely enters the bacterial cell and does not cause thee osmotic stress that most other solutes cause. Therefore, bacteria can grow at lower water activity values in the presence of glycerol than in the presence of most other solutes. An explanation of this phenomenon is presented later in this review. The examples for Clostridium sporogenes and Vibrio parahaemolyticus in table 1 show markedly higher minima for glucose than for NaCl. This is appararently related to the fact that the glucose was not sterilized separately from the media, resulting in production of inhibitory nonenzymatic-browning products in the glucose-containing media. Minimum values for growth The minimum aw values for growth of many foodborne bacteria are presented in Table 2. The values range from 0.99 for Moraxellal Acinetobacter type organism isolated from fresh meat, down to 0.86 for S. aureus. The minimum aw value for growth is a stationary property of bacteria, that is, they cannot mutate or adapt themselves to grow at lower aw value in the presence of a particular solute (50). The values in Table 2 are based on NaCl as the a w depressing solute since salt is commonly used in foods and yields results similar to other common solutes. Most of the bacteria listed would grow at lower aw values if glycerol were the solute. Glycerol, however, is used in few foods, but this phenomenon is worth bearing in mind if glycerol-containing foods are developed. Haas and Herman (25) have found several types of spoilage bacteria capable of growth at minimum aw values of 0.84 to 0.87 in intermediate moisture foods. Rodel et al. (48) have proposed that minimum a w tolerance may be a useful means of speciating the genus Vibrio.

Table 2. Minimum water activity (adjusted with NaCl) for growth of various foodborne bacteria

Denny et al. (19) proved that water activity, not moisture content, was the important factor in determining the safety of canned bread. Various kinds of breads were inoculated with 20,000 Clostridium botulinum types A and B spores/g and incubated at 85F for 1 year. None of 73 cans at a w values 0.950 became toxic while 28 of 56 cans at aw 0.955 did become toxic. A few reports, mostly unpublished, conflict with some of the minimum values presented in Table 2. The latter values, however, have been reproduced by many investigators. Conflicting values may indicate defects in the methods used to determinate water activity and illustrate the need to standardize a w methodology. Influence on sporulation and germination of spores Generally, the aw limits for sporulation appear to be the same as for growth (Table 3). Many have reported higher limits for sporulation than for growth, but Jakobsen and Murrell (30) suggest that this discrepancy is related to the method of cell preparation. In the experiments summarized in Table 3, cells were grown in a basal medium until they had advanced to the late stage III or early stage IV forespore i.e., the cells were irreversibly committed to sporulation. At this point the cells were subjected to the reduced aw media. It was also found that the reduction in aw affected only the quantity of spores produced. It did not affect spore properties such as heat resistance.
Table 3. Influence of water activity and solute on sporulation of Bacillus cereus.

In contrast to sporulation, spores can usually germinate at an a w substantially below that which will permit growth (Tables 4,5). Chyr et al. (18) found that although PA3679 could not grow at aw 0.95, germination of its spores was only slightly retarded at 0.95 in comparison to aw 0.99.
Table 4. Influenced of water activity on the germination and out growth of Clostridium botulinum spores.

Table 5. Influence of water activity and solute on germination and growth of Bacillus cereus var. mycoides.

MECHANISM OF RESOSTANCE TO REDUCED WATER ACTIVITY It has been known for many decades that reduction of water activity has a variable effect on different genera of bacteria. As water activity is reduced, some bacteria stop growing at high values, while others are able to grow at much lower values. An explanation of such differences is emerging from the research of the 1970s, and well undoubtedly learn a great deal more in the coming decade. The intracellular aw of cells is slightly lower than that of the external medium so that cells are able to maintain turgor pressure. When the a w of the external

medium is reduced, cells are subjected to osmotic shock and rapidly lose water, a process called plasmolysis. Koujima et al. (35) have shown that S. aureus loses about 50% of its intracellular water when switched from a medium of aw 0.995 to one of aw 0.950. Under similar conditions, Gibson (23) has shown that the cell volume of Salmonella typhimurium decreases 44%.
Table 6. Inhibition of various microbial functions by sodium chloride.

During plasmolysis a cell will not grow. It will either die or remain dormant. To grow, the cell must reduce its intracellular aw to regain its turgor. There are several hypothetical means of accomplishing this either the cell can admit the solute which has reduced the external aw, in which instance, the cells enzymes must be functional in the presence of that solute or the cell can accumulate or produce a different solute which will simultaneously reduced the intracellular aw and be compatible with enzyme functionality. The accumulating evidence supports that latter hypothesis. Measures (41) has shown that certain isolated enzymes are more susceptible to inhibition by NaCl than is growth of the host species (Table 6). Therefore, it would appear that NaCl does not enter the intact bacterial cell. Prior and Kenyon (46) have shown that Pseudomonas fluorescens could have a functional EntnerDoudoroff pathway at aw values below 0.80, even though the organism cannot grow below aw 0.957 (Table 7). Therefore, enzyme susceptibility is not necessarily the determinant in growth restriction by reduced water activity.
Table 7. Influence of water activity and solute on enzyme activity in the Entner-Doudaroff pathway

Examination of intracellular events suggets the mechanism depicted in Fig. 2. The cell on the far left is growing maximally at high a w, say 0.995. It is turgid and has a normal intracellular pool of such things as potassium ions and

glutamate ions. When the cell is switched to a medium of lower a w, say 0.950, it loses water and stops growing. The decreased amount of intracellular water effectively increases the internal potassium ion concentration since the original ions are confined more closely.

The increased potassium ion concentration activates glutamate dehydrogenase which reductively aminates - keto glutarate to glutamate, which them begins to accumulate in the cell. As glutamate builds up, water reenters the cell and growth resumes, though at a reduced rate. All of the common bacteria examined so far have been shown to accumulate an amino acid intracellularly when stressed osmotically. Those which accumulate glutamate only generally are unable to grow below a w 0,95. Glutamate, begin negatively charged at the cells pH, requires the presence of a counterbalancing cation, usually the potassium ion. When the glutamate concentration is sufficient to reduce the intracellular aw to 0.950, the potassium ion concentration becomes so high that it is toxic to some of the cells functions and growth ceases. Some bacteria, however, can decarboxylate glutamate to -aminobutyric acid (GABA) or they can reduce it to proline. These are relatively uncharged amino acids, which do not require the presence of a counterbalancing cation; therefore, cells which accumulate these amino acids will be able to grow at lower aw values than those which accumulate only glutamate.

This information has been assembled largely from the report of Measures (41) though many researchers have laid the groundwork for this hypothesis and continue to generate supporting evidence (8, 13- 16, 21, 24, 45, 56). Table 8 shows that those bacteria which accumulate principally proline are able to grow at much lower aw values than those which accumulate only glutamate. The relative increase in proline concentration varies only from one to four- fold when these particular bacteria are osmoticaly stressed; however, the absolute increase in proline concentration is quite great. Nonstressed cells have a 2 to 15 mM intracellular proline concentration while stressed cells have up to 1600 mM proline, a 100 to 1000- fold increase. At 1600 mM proline, the intracellular aw would be about 0.86 (11), and the cell would be so full of proline that one molecule in every 20 would be a proline molecule. This spatial limitation may explain why salt-stressed bacteria dont grow below aw 0.86.
Table 8. Intracellular accumulation of amino acids in NACl stressed bacteria.

The obligate halophilic bacteria such as Halobacterium sp. have not been included in this review. These bacteria can grow at aw 0.75, but they employ a different mechanism (7) are not common in food bacteriology, although they have been found in spoiled salt minced fie (62). Solutes such as proline are termed compatible solutes because they reduce the intracellular aw without themselves being toxic to enzyme activity and without accumulating an accompanying toxic moiety. Glycerol also functions a compatible solute. Though not produced by bacteria if present in the medium it will enter the cell and function just as proline does. Therefore, must bacteria can grow lower aw values when glycerol is the solute than when solute such as NaCl is used. This method of osmotic regulation is quite common other forms of life. It has been demonstrated, for example that algae, diatoms, clams, barley, grasses

and soybean also accumulate proline intracellularly when osmotical stressed (2, 4, 9, 17, 49, 52, 63). INFLUENCE ON SURVIVAL IN FOODS Reduced water activity has a preservative effect on man bacteria. For this reason, bacteria such as salmonellae can survive for long periods in dried foods. Table 9 shows the combined influence of temperature and water activity on survival of salmonellae in fish meat. The survival times are doubled or quadrupled at the temperatures studies as the a w is deceased from 0.71 to 0.34. A similar effect on salmonellae in milk powder at even lower a w values noted in table 10. The population is quite stable at a w 0.1, but dies relatively quickly at aw 0.4.
Table 9. Influence of water activity and temperature on survival of salmonellae in fishmeal.

Table 10. Influence of water activity on the survival of salmonellae in milk powder.

An extensive study by Uzelac and Stille (60) shows a similar effect on Escherichia coli and Streptococcus faecalis (Table 11). Both organisms die more rapidly at aw 0.53 than at aw 0.33 or 0.11 in several foods. Both die more rapidly in coffee than in dried potatoes and ice cream powder. The fact that S. faecalis survives dehydrated storage better than E. coli is advanced by the authors as an argument for using enterococci, rather than coloforms, as an index of sanitation, similar to the argument that has been advanced for frozen foods.

Table 11. Influence of water activity in several foods on the survival of Escherichia coli and Streptococcus faecalis.

INTERACTION WITH OTHER PRESERVATIVE FACTORS It is tempting to consider the influence of water activity as an isolated factor. In reality the aw of a food interacts with other preservative factors to create a preservative system. In studying foods, other factors such as oxidationreduction (O/R) potential, temperature, pH and chemical preservatives must be considered. Few illustrative examples are available because many researchers still repot solute concentrations and fail to measure water activity. Interaction with foodstuff It is often possible to note a degree of non-specific inhibition (or perhaps inhibition of unknown origin) when aw limits of an organism are tested in a food rather than in culture media. The minimum aw for growth of S. aureus in culture media is 0.86 (table 2). However, when S. aureus was inoculated into a sterilized shrimp product, Troller and Stinson (59) found that it could grow at aw 0.91 but not at 0.89. A similar trend has been noted in smoked snock (58). O/R potential Mead (40) has shown that as the amount of NaCl in culture media was increased, the maximum O/R potential which would permit growth of C. perfringens was decreased from 194 to 66 mv (Table 12).

Table 12. Influence of water activity and redox potential on growth of Clostridium perfringes

Temperature The interaction between aw and temperature is illustrated in Table 13. C. botulinum type E can grow at aw 0.970, provided that the temperature is optimum; and at 3.3 C, provided that the aw and other factors are optimum. In this example, ir grows well at aw 0.978 and 30C. The time required for outgrowth from spores to occur increases as temperature is lowered until at 7.2 C one of the two strains has not grown out in 6 months. Lotter and Leistner (37) have shown that the minimum a w for growth of S. aureus is 0.865 at 30 C, but increases at 0.878 with a reduction in temperature to 25 C. pH The data presented in table 14 to demostrate the combined influence of a w and pH on growth of C. botulinum type B is practically a response surface. In this report, the organism could grow at aw 0.960 and pH 7.0 or at pH 5.0 and aw 0.997. As the inhibitory between them is very clearly demonstrated. Because of the extreme importance in preventing the growth of C. botulinum in foods, the interaction between aw and pH has been given a great deal of attention in recent years (27, 42, 43).
Table 14. Influence of water activity and pH on growth of Clostridium-botulinum type B

Water activity and pH also interact, of course, to prevent growth of spoilage organisms. Jakobson and Jensen (29) found that butyric spoilage of canned pears, caused by Clostridium butyricum and C. pasteurianum, ossurred at pH 3,8 if the aw was greater than 0.98. If the aw was reduced below 0.97, spoilage did not occur at pH 4.5 during 30 days of storage at 30 C.

Chemical preservatives The interaction between chemical preservatives and a w has not been extensively documented to date. However, many of the earlier investigations which reported solute concentrations rather than aw are very instructive (47, Table 12). Varga et al. (62) found that spoilage of salt minced cod which occurs above aw 0.71 can be halted at 35 C if 0.3% sorbic acid is added to the fish. In a recent investigation of pasteurized process cheese spreads Tanaka et al. (55) found thatC. Botulinum types A and B did not grow at pH 5.8 and 30 C if the aw was 0.95 or less. It appeared that sodium phosphate used asa the emulsifying agent was more inhibitory than sodium citrate. In contrast, Kautteer et al. (34) have demonstrated C. botulinum toxin production in pasteurized process cheese spreads at pH 5,70 and a w 0.936. Clearly, this situation deserves more study, and it can best be done with standardized methods for the determination of water activity so that experiments can be reproduced in several laboratories. CONSIDERATION WITH INTERMEDIATE MOISTURE SHELF-STABLE FOODS Intermediate-moisture foods Intermediate-moisture foods (IMF) epitomize the interaction between water activity and other preservative factors (6, 26, 33). These are generally foods with an aw below 0.85, and are shelf-stable without receiving a heat treatment. The most significant commercial application of IMF technology has been with pet foods. IMF foods developed for human consumption have met with limited acceptance, provably because of the flavor and astringency characteristics of the high level of solutes required to achieve aw 0.85. One phenomenon wich has been noted with IMF is that of hysteresis (Fig. 3). Desorption-prepared foods are moist foods which have their aw lowered to the target value, e,g., fluid milk being dried. Adsorption-prepared foods are dried foods to which water is added to reach the target a w, e. g., dried milk being rehydrated. An important consideration which has been noted (44, 64), is that, at a given moisture level (line a) a desorption-prepared food can have a significantly lower aw than an adsorption-prepared food. Conversely, at a given aw (line b), a desorption prepared food can have a considerably higher moisture content than an adsorption-prepared food. MOISTURE AND HIGH-

This phenomenon was shown to be of significance in experimental work by Acott and Labuza (1). IMF pork prepared to aw 0.92 had a moisture content of 67.5% when prepared by desorption, but only 55.9% when prepared by adsorption. S. aureus grew readily in the former, but died in the latter. This and other examples have led to rather axiomatic IMF statement: At a given aw the foods with the highest moisture content will be the least stable (other factors being equal). High-moisture foods Partly because of flavor problems with IMF, there is emerging a new wave of food product development activity which is focused on foods of much higher aw and moisture content than typical IMF foods.

GMP regulations (57) require that all low-acid canned foods receive a sterilizing heat treatment to assure safety (Table 16). According to the code of federal regulations, canned foods above pH 4.6 would have to be lower than aw 0.85 to avoid the sterilizing heat treatment, i.e., they would be intermediate moisture foods. However, there are canned foods in widespread commercial usage which do not conform to these regulations and are nonetheless both safe and stable. Among these are the pasteurized process cheese spreads

which are higher than pH 5 and are in the a w range 0.90 to 0.95. The combination of reduce pH and reduce aw produces a food which does not permit growth of bacterial spores which survive the pasteurizing heat treatment.

Some researchers, e.g., Leistner et al. (36), are proposing potential guidelines which would permit acceptance of a much larger range of canned foods at high pH, high aw, and yet not require a sterilizing heat treatment. The sterilizing heat treatment, of course, severely alters product characteristics and in some instances, such as the cheese spread, simply cannot be used. Current proposals are that foods receive a pasteurizing heat treatment (up to 100 C) to kill all vegetative cells and that the surviving spores be prevented from growth by the control of both aw and pH. Strictly from a safety standpoint, we have seen that C. botulinum cannot grow below aw 0.95. However, to stabilize a food against that growth of all bacterial spores, Leistner at al, propose that foods above pH 6.3 be restricted to an aw value of less than 0.94. In practice this may turn out to be lower for some foods. Table 2 shows some spore formers capable of growth at aw 0.90, and pH 6.3 is rather favorable. Each food will need to be tested on an individual basis. The control of aw in foods will become more important as new foods are developed and as more energy-efficient processing methods are utilized. It is important that we as microbiologist learn to measure and control water activity and to understand its influence on the microbiology of the foods we produce.