Microbiological Degradation

Organisms and Factors Affecting Growth. The types of microorganisms that are responsible for spoiling foods include bacteria, molds, and yeasts. Like other living things, microorganisms require nutrients, water, and minerals to survive and grow, and to accomplish these activities, the microorganisms degrade the foods. Many factors affect the growth and biochemical activities of microorganisms in foods and these are the chemical composition of the food, the conditions of processing and storage, and the inherent properties of the microbial species present. With regard to the latter point, different types of microorganisms have growth habitats for which they are best suited. For example, bacteria are adapted to fast growth in nonacidic (pH 5.0-8.0) and moist conditions, whereas molds grow more slowly but are better adapted to growth in relatively acidic (pH 2.5) or dry conditions. Yeasts are similar to molds, but many also tolerate conditions containing high concentrations of sugar.

The microbial flora in a food will be initially dictated by the product's environment or by contaminants picked up during processing, handling, packaging, and storage of that product. For example, freshly caught fish from warm or tropical waters carry a microbial population primarily of mesophilic bacteria (bacteria that grow best from 15 to 40°C) such as Micrococcus coryneforms and Bacillus. In contrast, cold and temperate water fish species harbor predominantly psychrotrophic bacteria (bacteria that grow at refrigeration temperatures) that includes the genera Moraxella /Acinetobacter, Pseudomonas, Flavobacterium, and Vibrio. Upon refrigerated storage, however, the psy-

chrotrophic bacteria in both the warm and cold water fish become the predominant species as a result of natural competition. Since a greater concentration of psychrotrophic organisms are initially found in seafood harvested from temperate waters, their spoilage could be expected to be quicker than seafood harvested from tropical waters. Such conjecture has at least been substantiated with organoleptic evaluations of tropical and temperate shrimp stored in ice (1). Another important intrinsic factor in fish that has played an important role in determining the microbial flora during storage is the pH. Most fish contain only very little carbohydrate (<0.5%) in the muscle tissue, and thus only small amounts of lactic acid are produced postmortem. As a result, fish muscle contains a high postmortem pH (>6.0), and under these conditions the pH-sensitive spoilage bacteria Shewanella putrefaciens may grow. In the case of bivalves such as oysters and clams that store energy in their tissues as glycogen, lactic acid may be produced in much greater quantities. As a result, the reduction in pH favors multiplication by fermentative-type microorganisms such as Lactobacilli, Streptococci, and yeasts.

In other meat items stored at refrigeration temperatures, only approximately 10% of the initial bacteria present are able to grow, and of those microorganisms able to grow, the fraction causing spoilage is even lower. Hence, it is important to identify these "specific spoilage organisms" in each food so treatments can be optimized to minimize their growth. Influencing the composition of those specific spoilage organisms on meat, however, will be the available moisture. In a high humidity environment, bacterial spoilage in fresh meats is dominant, with coalescence of colonies producing a slime layer on the surface. When the surface of the meat is too dry, as it is with many dried and fermented meat products, mold tends to predominate as the spoilage organisms. On the other hand, the application of vacuum or modified atmosphere packaging to cooked meat products generates conditions conducive to dominance by lactic acid bacteria. Producing acids such as lactic acid, acetic acid, and formic acid, the bacteria generate spoiled products described as sour and acid. When these products are stored aerobically following their anaerobic storage, additional obnoxious odors may be produced that are described as slightly sweet and cheesy.

Molds are able to grow on many other kinds of food in addition to meat. These include cereals/grains, milk, fruit, vegetables, and nuts. As a result of the mold growth, several kinds of food spoilage may arise: off-flavors, toxins, discolorations, and rotting. Of these, the most important spoilage problem associated with molds is the formation of mycotoxins. Mycotoxins are secondary metabolites of molds that are toxic to vertebrate animals in small amounts when introduced via a natural route. The most important toxic effects are different kinds of cancer and immune suppression. While some mycotoxins are only present in the mold, most of them are excreted in the foods. The high resistance of mycotoxins to physical and chemical treatments makes them difficult to remove from foods once they are present. Hence, it is important to limit mold growth on food items.

The well-accepted conclusion that molds are mostly responsible for the spoilage of fruit is based on the visible presence of fungal mycelium and spores on the surface of these products at the time of rotting. Yeasts, however, commonly occur on the surface of freshly harvested fruits at populations of 103 to 105 cells per square centimeter (2). These yeasts in most circumstances are considered to remain relatively inactive as they do not produce the appropriate enzymes to degrade the skin of the fruit and establish infection. Physical damage of the skin, however, by overripening, mechanical injury, or fungal attack exposes the fruit tissue upon which yeast can rapidly grow to produce secondary spoilage.

In products, such as beer and wine, that are dependent on microbial action, spoilage may also ensue when growth of undesirable microorganisms occurs. Even in chocolate-covered confectionery products, spoilage may result from growth of foodborne Chrysosporium species. Growth of these xerophilic fungi has produced a white surface discoloration that for all purposes was similar in appearance to the effects observed from recrystallization of cocoa butter (3).

Enzymatic/Biochemical Activity of Microorganisms. During their growth, microorganisms utilize food constituents and generate metabolic end products. As a consequence, the physical, chemical, and sensory properties of the food are substantially changed. Generally, the nature of these changes is not well described, making it difficult to explain spoilage on a biochemical basis. These biochemical activities, however, often include enzyme activity of a pectino-lytic, proteolytic, and/or lipolytic nature.

In fruits and vegetables, pectolytic fluorescent pseudo-monads, mainly P. fluorescens and P. viridiflava, account for substantial proportions of postharvest rot in cold storage and at wholesale and retail markets. The ability of these pseudomonads to cause maceration of plant tissues is primarily due to their ability to produce an extracellular pectate lyase capable of degrading pectin components of plant cell walls.

Grain is also subject to breakdown by carbohydrases from spoilage fungi. These fungi have been shown through in vitro studies to produce cellulase, polygalacturonase, pectin methyl esterase, l-4-/?-glucanase, /i-glucosidase, and /?-xylosidase (4). Environmental conditions such as water activity (Aw) and temperature, however, influence the production of these enzymes.

A key metabolic reaction of most yeasts when cultured under favorable anaerobic conditions is the fermentation of sugars such as glucose, fructose, sucrose, and maltose to produce ethanol and carbon dioxide. In addition to the loss of sweetness, this activity produces gassy products with a distinctive alcoholic aroma and flavor. Many hundreds of other secondary end products are formed during fermentation, and these also have a significant impact on the sensory properties. Such products include higher alcohols, organic acids, esters, aldehydes, and ketogenic substances that, although produced in small concentrations, have very low flavor and aroma thresholds. Unfortunately, with the exception of Saccharomyces cerevisiae, the pro duction and sensory relevance of these secondary metabolites by yeasts are not well known (2).

Fragmentary knowledge exists on yeast metabolism of nitrogen compounds. As one example where this type of biochemical activity is of significance, metabolism of lysine during the growth of the yeast Brettanomyces intermedins in wine produced mousy taints due to the production of substituted tetrahydropyridines (2). It is also well documented in the brewing and wine literature that some, but not all, strains of S. cerevisiae can produce objectionable concentrations of hydrogen sulfide and sulfur dioxide and that these properties are linked to their metabolism of amino acids and inorganic sulfur compounds in the growth medium.

Yeast can both synthesize and metabolize organic acids. These activities change the acidities and flavor profiles of the product. In addition, when oxidative utilization of organic acids by yeast occurs via the tricarboxylic acid cycle, the pH of the product may rise to values that allow the growth of spoilage bacteria.

In foods, microbial proteolysis is another key spoilage reaction. Initially, it leads to the development of bitter flavors, followed by the production of strong ammonia odors and putrefaction. These proteases, secreted into the food to break down the proteins, include both endoproteases and exoproteases (aminopeptidases and carboxypepti-dases). Thus, volatile fatty acids originate from proteins by deamination of amino acids while volatile bases arise from decarboxylation of amino acids and deamination of adenylates in the food. The degradation products, in turn, serve as a nutrient source for the microorganisms. When ample concentrations of low molecular weight nitrogen constituents (free amino acids, creatine, taurine, etc) are already present in the food product, however, as they are in fresh fish, protease synthesis and excretion is inhibited. In later stages of spoilage when most of the free amino acids have been depleted, then proteolysis becomes important. Even so, it appears that some level of low molecular weight peptides must be present to activate the synthesis of the extracellular proteases in P. fragi, an active spoiler of muscle food (5). Other factors in the food medium have also been shown to affect synthesis and excretion. For example, in milk supplemented with iron, the size ofthePseudomonas population when extracellular enzymes were produced was 10 times larger in the supplemented milk than in the nonsupplemented milk (6). The types of proteases produced by a microorganism, however, will vary. Thus, P. aeruginosa secretes two proteases, V. alginolyticus secretes a collagenase and five alkaline serine proteases, and Aero-monas hydrophila secretes two proteinases and one ami-nopeptidase. In general, these proteases have molecular weights ranging from 20 to 50 kDa, consist of only a single polypeptide, and lack the cysteine amino acid. The latter two characteristics are especially important because they contribute to the high heat stability of the extracellular proteases. Hence, even though the vegetative cells may be killed by a heat treatment, extracellular microbial enzymes continue to exert their activity. To reduce the possibility of this type of activity in milk, thresholds have been set in raw milk at approximately 2 x 106 colony forming units/mL (7). Levels above this threshold would thus gen erate pasteurized milk that would spoil quickly despite bacterial counts being reduced greatly. Product deterioration by proteolytic enzymes generated from psychrotrophic sporeformers, such as Bacillus spp., however, is also receiving attention in the dairy industry. When in the spore state, these microorganisms easily survive the typical range of pasteurization conditions with subsequent germination and outgrowth of vegetative cells. Consequently, during their growth, activity by degradative enzymes, including proteases, leads to gelation of ultra-high temperature (UHT) milk, sweet curdling of milk, bitter and unclean off-flavors in cheese, decreases in cheese yield, and textural and body defects such as "wheying off" in cultured dairy products. Interestingly, it has been noted that the optimum temperature for production of degradative enzymes by these microorganisms is usually lower than the optimum temperature for cell division (8). Thus, it is possible for milk held at refrigeration temperatures for extended periods of time to develop off-flavors through mi-crobially produced enzymes even though the observed microbial population might remain below that normally associated with formation of microbial defects.

Lipases are another group of hydrolytic enzymes released by microorganisms to break down the food. In this case, the enzymes cleave fatty acids from triglycerides (a molecule containing three fatty acids esterified to a glycerol backbone). For those lipases found in milk, a preference occurs for cleavage at the 3-position in the triglyceride where butyric and caproic fatty acids reside. Upon hydrolysis, these volatile fatty acids at very small concentrations (1.5 mEq/100 g of fat) have distinctly unpleasant smells and tastes, which is termed hydrolytic rancidity. Also, in milk, some thermoduric psychrotrophs have been shown to produce phospholipases, particularly phospholipase C, that hydrolyse the ester linkage between the glycerol backbone and the phosphoryl group of a phospholipid. In milk, this attack takes place at the fat globule membrane where phospholipids are found. The lipolysis at the fat globule membrane results in an increased susceptibility of the exposed milkfat to the subsequent action of lipases. It has also been suggested that the degradation of the fat globule membrane by phospholipase C results in the aggregation of fat globules that leads to the bitty cream defect frequently observed in cream products.

Not all lipolytic action results directly in flavor defects. Only when the free fatty acids (FFAs) are short to medium range (C4 to C12) do they contribute directly. In many cases, FFAs react with protein constituents in the food producing textural defects in the product, but rarely are these defects of a severity to render the product spoiled. In cases where the FFAs are of an unsaturated nature (contain one or more double bonds in their structure), they may increase or decrease the susceptibility of the food to undergo lipid oxidation. When the FFA is cleaved from a membrane phospholipid early in storage, it will increase the oxidative stability of the product, but release of FFA from membranes later in storage will decrease oxidative stability. FFAs released from storage triglycerides almost always are more susceptible to lipid oxidation than they were when esterified. This process of lipid oxidation, in turn, leads to the presence of off-flavors and off-odors in the product. Lipid oxidation will be described in more detail in the next section.

The last group of microbial enzymes to be discussed in this section is one that leads to trimethylamine oxide (TMAO) degradation. In marine fish, TMAO is believed to be involved in the fish's osmotic regulation, and levels in the fish will be dictated by the levels required for counteracting the osmotic pressure of seawater as well as amounts present in the food. Degradation of this compound by psy-chrotrophic bacteria, such as Achromobacter, apparently involves both a triamineoxidase for activation of the TMAO substrate and a dehydrogenase for reduction of the compound to trimethylamine (TMA). In turn, when TMA reacts with the fat in the muscle, it creates a characteristic fishy odor. Odors appear at TMA levels as low as 2.9 mM of muscle extract, while definite odors have been observed at levels of 7.2 mM in the muscle extract (9). Significant amounts of TMA, however, will not be observed until after the bacterial lag phase that extends from the onset of rigor to its resolution, due to a postulated bacteriostatic effect of rigor mortis.

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