Microbial Evaluations

When microbial degradation is the major source of spoilage in the food, it is common practice to analyze for total numbers of bacteria in the food. The food may be rinsed to determine surface count or it may be ground/mixed with a specially formulated liquid medium of which diluted portions are taken and spread onto an agar plate. One of the drawbacks with regard to this type of analysis is that it takes a minimum of one to two days for the bacteria to grow on the agar and generate visible colonies for counting. The second even more important drawback is the realization that total count of bacteria does not always correlate to degree of spoilage. When spoilage is evident as visible growth (mold, pigmented or nonpigmented, slimy bacterial colonies), there is a direct relationship between total numbers of microorganisms and degree of spoilage. More often, though, spoilage is a result of the production of off-odors and off-flavors caused by bacterial metabolism. In these cases, there is no correlation between total numbers of bacteria and spoilage since only a fraction of the total flora participates in the spoilage. Thus, it becomes advantageous to identify which organisms are responsible for the spoilage and concentrate on enumerating their populations by employing selective media. No specific spoilage bacteria, however, have been identified in the case of vacuum-packaged beef or with vacuum-packaged cold-smoked salami (18). Even when the specific spoilage organisms are well established, there are instances where their enumeration is of limited value. For example, spoilage occurs at lower cell densities on high pH (>6.0) meat than on normal pH (<5.8) meat (19). Therefore, in those cases, it has been more appropriate to concentrate on identifying and quantifying the metabolic products responsible for the spoilage.

Other more advanced methods exist to identify spoilage organisms, and these rely on the immunochemical and genetic characteristics of the microorganisms. For detecting molds in grains, for example, antibodies to specific fungi may be linked directly or indirectly to a fluorescent dye. This technique is suitable for work on individual grains, but difficulties arise in assessment of molds in large samples of grain and in quantifying levels of molds present. Genetic detection techniques, on the other hand, are generally based on the hybridization of genomic DNA with a specific DNA probe. Hybridization may be accomplished by a variety of methods, but the most popular is the polymerase chain reaction (PCR). Such tools have proven useful in the identification of wild yeast belonging to the genus Sac-charomyces that are biochemically and physiologically very similar to the culture yeast used in beer. By and far, the largest number of PCR applications have been directed at specific food pathogens, where it is essential to confirm the causative agent from a public health standpoint. Spoilage organisms, however, are of concern primarily from an economic significance, hence it is not necessary to detect them individually in foods. Consequently, primers may be designed for a conserved region of the DNA that allows detection of the majority of spoilage organisms as a group for a given commodity. Using such an approach for the PCR-ELISA assay, a detection threshold of 102 cfu/cm2 was found (20). The advantage to using PCR rather than traditional plating is that the detection of the microorganisms is not dependent on the organisms' state of growth or on environmental influences. Limitations of PCR, however, are the inability to differentiate between viable and nonviable microorganisms, and substances in the food variably affect the activity of the DNA polymerase.

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