Meat Spoilage and Evaluation of the Potential Storage Life of Fresh Meat

Transcription

1 444 Journal of Food Protection, Vol. 46, No.5, Pages (May 1983) Copyright~, International Association ot Milk, Food, and Environmental Sanitarians Meat Spoilage and Evaluation of the Potential Storage Life of Fresh Meat C.O.GILL Meat Industry Research Institute of New Zealand (Inc), Hamilton, New Zealand (Received for publication September 18, 1982) ABSTRACT Microbiological proccsses by which meat develops qualities unacceptable to consumers vary with the composition of the meat and spoilage microflora. Composition of the spoilage microflora is affected by meat composition and storage conditions. Aerobic spoilage microfloras are usually dominated by pseudomonads. With this type of microflora, spoilage occurs when glucose in meat is no longer sufficient for the requirements of the spoilage microflora and the bacteria start to degrade amino acids. When meat is deficient in glucose, spoilage becomes evident while bacterial numbers are relatively small. Anaerobic microfloras are usually dominated by lactobacilli which produce spoilage by the slow accumulation of volatile organic acids. Meat of high utimate ph packaged anaerobically spoils rapidly because the high ph allows anaerobic growth of bacterial species of higher spoilage potential than the lactobacilli. Before overt spoilage develops, the spoilage status of meat can be accurately assessed from the bacterial numbers on meat only when there is assumption or knowledge of meat composition, storage conditions and the types of bacteria present. Methods for estimating spoilage which depend upon detection of products of amino acid degradation have little predictive value as such products will only be present after attack on amino acids has commenced and are irrelevant to spoilage under anaerobic conditions. Estimation of the concentrations of other spoilage products may be the only method applicable to assessment of incipient spoilage of meat stored anaerobically. It is, therefore, unlikely that any single test can give unequivocal information on meat quality under all circumstances. but rapid tests for meat quality could be of value for specific commercial purposes. provided such tests are appropriate to the circumstances and the inherent limitations of any test are reeognized. Meat is spoiled by bacteria when the products of their metabolic activities make the food offensive to the senses of the consumer. The perception of a state of spoilage is. therefore, essentially a subjective evaluation which will vary with consumer expectations, but there would be few who do not acknowledge that the appearance of slime, gross discoloration and strong odors constitute spoilage of meat. It would be desirablc to identify the status of meat with respect to spoilage before its condition becomes evident to the senses. This would allow more accurate assessment of the meat's shelf-life. thus enabling appropriate action to be taken to prevent the loss of meat when spoilage was incipient but not overt, and could provide a basis for regulations controlling meat quality. For such a test to be of any practical value, it would have to be both simple and comparatively rapid. Numerous objective tests for spoilage have been suggested and claims made for their practical value. Most of this work has been concerned with establishing empirical relationships between spoilage and some readily measurable change in the composition of muscle foods. Such tests have been the subject of previous reviews, which have generally concluded that the reliability of most of the proposed methods has not been established (8, 37, 67). This conclusion is not surprising as the spoilage of meat is a variable phenomenon whose course is determined by both the composition of the meat and the types and relative numbers of bacterial species present in the spoilage microflora, The composition of the spoilage microflora is affected by the meat composition and also by storage conditions. No one method is likely to be applicable under all circumstances and attempts to assess the merits of any proposed test will be confused by differing circumstances unless the relationships between the factors being analysed and the spoilage processes are known. Since processes that are critical for spoilage development have now been more clearly defined, it is possible to understand at least some of these relationships. It is the objective of this review to use the current understanding of spoilage processes to describe the circumstances under which methods of estimating meat spoilage are likely to give results of practical value. THE SPOILAGE PROCESS Although there are recent reviews which give substantial accounts of current understanding of mcat spoilage processes (24,50,59), some description of these processes is necessary for any discussion of tests for spoilage. Accounts of earlier work and other aspects of meat spoilage have been the subjects of previous reviews (2,33,37). JOURNAL OF FOOD PROTECTION. VOL. 46, MAY 1983

2 MEAT SPOILAGE 445 The substrate The components of meat (44) tend to fall into one of three groups when considered as potential nutrients for microbial growth. The major components, proteins and fats, must be degraded before they can be utilized by bacteria. Most of these substances are insoluble and are, therefore, not readily available for microbial attack, but soluble protein is also present in abundance. The second group is composed of low molecular weight, soluble nitrogenous components. These include: (a) creatine and nucleotides, derived during the development of rigor from creatine phosphate and adenosine triphosphate, respectively, (b) amino acids, and (c) peptides, such as carnosine and anserine. These substances are always present in muscle tissue although their concentrations vary. The concentrations of amino acids and peptides tend to increase with time because of proteolysis. The third group of materials is derived from muscle glycogen which is degraded via glycolysis during the onset of rigor (4).The most abundant material formed by this process is lactic acid which can reach a final concentration of 9 mg/g. There can also be some residual glycogen and small quantities of glucose and glycolytic intermediates such as glucose-6-phosphate (14). The concentrations of these substances can vary widely. If muscle glycogen is depleted before slaughter because of exercise or stress, the amount of lactic acid formed can be less than half that which occurs in muscles well supplied with glycogen. There is, then, no residual glycogen and glucose, and glycolytic intermediates are also absent (14). The final muscle ph is determined by the amount of lactic acid that accumulates. In normal muscle, the ph will approach 5.5, but in glycogen-depleted muscle, the ph can remain above 6.0. Meat of high ultimate ph is often referred to as "dark, firm, dry" (DFD). The concentrations of some of the glycogen-derived substances can also vary with species. For example, the reported glucose concentrations in beef, mutton and pork are about 100, 300 and 900 IJ.-g/g, respectively (14, 21). Meat is comprised of fat as well as muscle tissue. Because carcass surfaces tend to lose moisture during chilling, fat surfaces of carcasses are often too dry to support bacterial growth. The bulk of the tissue is triglyceride, but low molecular weight soluble substances are also present at the surface. These appear to be derived from serum in the blood vessels within the fat tissue. Glucose is present in lower concentrations than in muscle, as are amino acids and lactic acid, but glucose-6-phosphate is absent and the ph is always close to neutrality (26). Growth of bacteria on muscle tissue Bacteria on uncomminuted muscle tissue are confined to the first few millimeters of the surface. It has been suggested that most meat harbors an "intrinsic" microflora within deep muscle tissues that is destinct from surface contaminants, but it is now clear that deep muscle tissue from animals killed under reasonably hygienic conditions is usually sterile (22). Bacteria from the surface cannot penetrate into muscle tissues until they produce proteolytic enzymes, which does not occur until late exponential growth (30). By that time, the muscle is usually covered by a slime layer and is obviously spoiled (11, 29). The microflora, therefore, develops to spoilage levels at the meat surface before invasion of deep tissues occurs. Bacteria on meat grow at the expense of low molecular weight soluble components. All bactera exposed to a rich source of nutrients will utilize some substrates preferentially. Usually, simple sugars, such as glucose, are used before other substrates. For example, a nutritionally versatile Enterobacter sp. growing on meat utilized substrates in the order of glucose, glucose-6-phosphate, amino acids (23). Some preferred substrates, such as glucose, are present only in comparatively low concentrations. As the bacterial cell density increases, the concentration of such substances at the meat surface declines. A concentration gradient will, therefore, develop from the surface and substrate will diffuse to the surface from within the meat. However, the rate of diffusion can become too slow to meet the increasing bacterial demand. The surface concentration of the preferred substrate will then be reduced to undetectable levels and the bacteria will attack secondary substrates even though a considerable amount of the preferred substrate is stili present in the deep tissue (21). The cell density at which the attack on secondary substrates begins depends on the initial concentration of the preferred substrate and whether degradation is via oxidative or fermentative metabolism. For aerobic growth, the cell yield with respect to the substrate should be about 50%. Hence any substrate should be able to support a cell mass at the meat surface approximately equivalent to its concentration in the tissue, i.e., a substrate concentration of 100 IJ.-g/g should support about 100 IJ.-g dry weight of cells/cm 2. Assuming that 1 g dry weight of bacteria contains about cells, then, with a preferred substrate at 100 IJ.-g/g, utilization of secondary substrates would start when the cell density reached about 10 8 bacterialcm 2 (21). The energy yield from fermentation is only about 10% of that from oxidation, so fermentation of a substrate present at 100 IJ.-g/g would support only about 10 7 bacterialcm 2. Bacteria oxidizing substrates still have excess secondary substrates available to them when growth ceases some time after spoilage has become evident. The final bacterial cell density, which is in excess of 10 9 /cm 2, seems to be determined by the limited rate of oxygen transfer into the slime layer that develops on the meat (23). The substrates available for fermentation by bacteria are very restricted, with most species being able to use only glucose and at most one or two other substrates. As these substrates are present only in low concentrations, their availability determines the final cell density of about 10 8 bacterialcm 2 (57). Growth of bacteria on fat tissue Bacteria are confined to the surface of fat tissue and attack on triglycerides plays no part in the onset of spoilage (26). Bacterial growth on moist fat surfaces occurs at the expense of the same low molecular weight soluble materials that are found in muscle tissue. Growth rates are the same on both moist fat and muscle. However, the nutrients JOURNAL OF FOOD PROTECTION. VOL. 46, MAY 1983

3 446 GILL in fat tissue are present in lower concentrations than in muscle and the rate of diffusion of fresh substrates to the surface is considerably slower. Substrates are, therefore, exhausted more rapidly on fat surfaces and final baeterial numbers are lower, with growth ceasing because of limited availability of substrates even with aerobie growth. Growth of bacteria in minced tissue Spoilage in minees develops in the same manner as in whole muscle tissue, but the total mass of any substrate is readily available to the bacteria which are distributed throughout the mince. Bacteria reach a higher number per gram of mince than per cm 2 of meat surface before spoilage is evident. Enhanced substrate availability and the dilution effect of the mince mass would account for this. Packing of the mince particles could affect bacterial growth as tight packing would prevent access of oxygen to the center of the mass, hence preventing growth of aerobic organisms there. Spoilage by aerobic organisms would still occur in the outer layers, however, so the time taken for spoilage to become evident in loosely and tightly packed minces would be about the same. THE SPOILAGE BACTERIA Aerobic micro flora Spoilage microfloras of meat stored in air are usually dominated by strictly aerobic gram-negative species belonging to the genera Pseudomonas, Mora."tella and Acinetobacter (33). The pseudomonads usually predominate on meat at chill temperatures. These organisms are unaffected by ph in the range that occurs in meat (28), and, at chill temperatures, grow faster than competing species (20). They preferentially utilize glucose and strongly suppress degradation of amino acids until glucose is exhausted, but are capable of using most of the naturally occurring amino acids for growth (35). Growth on the preferentially utilized amino acids is as rapid as on glucose (58). Many strains of the MomtelialAcinetobacter group are inhibited by the low ph of normal meat. Therefore, these organisms are less important than the pseudomonads on red meat of normal ultimate ph held at chill temperatures, but form a larger proportion of the microflora on meat of high ph or when meat is held at ambient temperatures because the effects of ph are then less pronounced (28). Most members of this group of organisms cannot utilize hexoses (42, 84) and so attack amino acids without delay when growing on mcat. In addition to the strict aerobes, facultative anaerobes belonging to the Enterobacteriaceae occur in aerobic spoilage microfloras. These organisms preferentially utilize glucose, and some will also utilize glucose-6-phosphate before degrading amino acids. At chill temperatures, these organisms grow far slower than the strict aerobes (18, 23) bul like the they produce malodorous byproduct~ 2.mino acids for growth (66). Another facultative anaerobe which can occur in spoilage microtloras is the gram-positive organism Brochothrix thermosphacta. This bacterium has a restricted range of substrates, and glucose is the only major one present in meat (10. 57). This organism is usually only a minor component of aerobic microfloras on meat, but it has been reported as a major component in some cases of lamb spoilage, either because of heavy initial loading with the organism or because the storage atmosphere limited growth of competitors (3,61). Microflora development in vacuum packages If conditions within a pack are anaerobic, the microtlora is usually dominated by Lactobacillus species. These organisms are unaffected by the ph of meat, can outgrow competitors at chill temperatures and producc antimicrobial agent(s) that inhibit potential competitors (57. 72). The final microflora is often composed almost entirely of these organisms. If oxygen is present in the package, pseudomonads can grow, although their growth may be slowed by limited availability of oxygen or, at higher oxygen levels, by CO 2 within the package (60). At higher temperatures, species of Enterobacteriaceae can dominate the microtlora (27). A number of organisms are prevented from growing by the ph of normal meat but can contribute to the microflora of high ultimate ph (DFD) meat which is packaged. There are two organisms of particular importance; B. thermosphacta which will not grow anaerobically on meat below ph 5.8 (31) and Altermonas putrefaciens which does not grow below ph 6.0. This latter organism degrades cysteine even in the presence of glucose and, under anaerobic conditions, H 2 S is released with deletrious effects upon meat color (25). THE ONSET OF SPOILAGE Most studies of spoilage have been concerned with meat held at chill temperature. There may be some differences in the process at higher temperatures because of changes in the composition of spoilage microfloras (27). Additional data are needed to assess the magnitude of any such changes, but they are likely to be minor. Spoilage at chill temperatures is of the greatest concern and all subsequent discussion will deal with meat held in this temperature range. Aerobic spoilage Bacteria utilizing a carbohydrate as the main carbon source do not usually produce metabolic products having very offensive odors or flavors (54). However, utilization of amino acids and other compounds containing nitrogen andlor sulfur often results in the formation of unpleasant metabolites (12, 50), The extent to which such offensive metabolites are produced depends not only on the substrate but also on the particular metabolic activities of the microorganism. In aerobic microfloras, the only microorganisms likely to be present in numbers sufficiently high to playa part in meat spoilage are the pseudomonads and the Moraxella! Acinetobacter group. The latter group utilizes amino acids JOURNAL OF FOOD PROTECTION, VOL. 46, MAY 1983

4 MEAT SPOILAGE 447 as preferred substrates when growing on meat and might be expected to be the major determinants of meat spoilage. However, these organisms do not appear to produce significant amounts of highly offensive metabolic by-products (47), hence, in practice, they contribute little to the spoilage process in a microflora dominated by pseudomonads. The onset of aerobic spoilage is a function of the metabolism of pseudomonads. Initially these bacteria grow at the expense of glucose and do not produce offensive byproducts. When the cell density is in excess of 1Q8/cm 2, the supply of glucose becomes insufficient to meet the bacterial demand and the microbes begin to attack amino acids. The ph of the meat then rises because ammonia is released as a consequence of amino acid degradation (46). Other undesirable products, such as organic amines (cadaverine, putrescine, isobutylamine) and sulfides (H 2 S, methyl sulfide), are formed in much smaller quantities, but the intensities of their odors and flavors more than compensate for their low concentrations (12, 52, 53). Because the bacterial cell density is already high, these undesirable compounds soon accumulate to levels where they can be detected organoleptically. Onset of spoilage is, therefore, rapid in normal-ph meat stored in air once glucose has been effectively exhausted at the meat surface. On DFD meat, pseudomonads are the determinant microorganisms for meat spoilage. Glucose can be absent from meat in this condition, which allows bacteria to use amino acids without delay. Under these conditions, spoilage will become evident when bacterial numbers are sufficiently high for undesirable by-products to be formed in quantities which can be detected. This occurs when bacterial numbers are somewhat in excess of 1Q6/cm 2, so aerobic spoilage of DFD meat occurs earlier than in normal meat (58). Similar considerations also apply to moist fat surfaces not bathed in muscle drip because the limited glucose supply allows amino acids to be degraded while cell densities are comparatively low (26). Spoilage in vacuum packages If defective packaging results in failure to exclude oxygen, growth of pseudomonads in vacuum packages will be possible, and these organisms will ultimately spoil the meat in the same manner as for aerobic storage. Such spoilage occurs at the minimum bacterial cell density of 10 6 bacterialcm 2 (60). This could be due to the available glucose having been fermented by lactobacilli or because amino acid degradation is not completely suppressed by glucose when pseudomonads are growing at submaximal rates in vacuum packages with oxygen as the rate-limiting substrate. When a microflora dominated by lactobacilli develops, spoilage is due to the accumulation of short chain fatty acids (82). Such spoilage develops slowly and is detectable only long after the maximum cell density has been attained (60. 81). If B. thermosphacta achieves significant numbers, spoilage occurs more rapidly than with lactobacilli, although spoilage by the former organism is also due to production of organic acids (9. 10). On meat of ph 6.0 and above, A. putrefaciens can become of critical importance. Under anaerobic conditions, this organism produces copious quantities of H 2 S and, if the numbers of this organism approach 10 6 cells/cm 2, the meat is rapidly spoiled because of the development of green sulfmyoglobin as a result of a combination of H 2 S with the meat pigments (25.63). DETECTION OF INCIPIENT SPOILAGE Bacterial cell density As spoilage must be related to some extent to the bacterial cell density, measurement of this parameter is perhaps the most obvious way of determining the spoilage status of meat. The usual method of determining bacterial density, by plate counting of colony-forming units, is of limited predictive value because 1 to 2 days are required for colony development on plates. This is unsatisfactory because the spoilage status of the meat could alter considerably in the time required to complete the test. This problem could be remedied by using a more rapid, indirect method of estimating bacterial numbers. Rapid methods for estimating bacterial numbers can be divided into those which employ sensitive measurement of components of bacteria present in the food, and those which determine numbers from the time taken for an inoculum to attain a cell density detectable in the system. In general, the first type of estimation should be capable of giving a more rapid result than the second when relatively small numbers of bacteria are present. This is because there is no necessity for bacterial growth to occur during the assay. Most methods of either type require specialized and costly equipment. Such methods include: (a) the determination of bacterial adenosine triphosphate (A TP) by the firefly bioluminescence reaction (7, 40, 76, 83); (b) measurement of decreases in electrical impedance resulting from microbial growth (6, 49); (c) microcalorimetric measurement of heat evolved during growth (15, 73);(d) detection of 14C0 2 released by metabolism of radioactive substrates (73); and (e) automated photometry (65). Most of these methods were originally developed for examination of clinical specimens, but some have subsequently been applied to foodstuffs. Any of these methods may potentially give acceptable results, but the equipment costs could only be justified by the routine examination of large numbers of samples. Less capital intensive methods would be desirable in many circumstances. Several early proposals for the rapid estimation of bacterial numbers in meat utilized dye reduction tests (70, ). These tests were derived from tests used for rapid estimation of milk quality and are based on the assumption that the time taken for a bacterial inoculum to alter the redox state of a standard medium to the point where an incorporated redox dye is reduced will be directly proportional to the number of bacteria in the inoculum. In fact, the time for dye reduction varies widely with the type of bacteria involved, and synergistic effects between bacteria can lead to gross overestimation of bacterial numbers (20). JOURNAL OF FOOD PROTECTION. VOL. 46, MAY 1983

5 448 GILL It is now recognized that dye reduction tests can only give crude estimates of microbiological quality and certainly cannot be used to determine bacterial cell density (1). Moreover, such tests are of little value unless large numbers of bacteria are present in the inoculum (16). Although dye reduction tests seem unlikely to be of real value for estimation of bacterial densities on meat, a method based on the same principle but using electrodes to follow redox changes has been claimed to give highly reproducible results with minces (5). However, further work on this mcthod is needed before its general utility can be assured. More recently, a method for estimating the concentration of gram-negative bacteria has been developed based upon a lysate of amoebocytes from the horseshoe crab. Limulus polyphemus, which will gel in the presence of small amounts of endotoxin from gram-negative bacteria whether the toxin is free or bound to living cells (41, 48). The Limulus amoebocyte lysate (LAL) assay is very sensitive and, with appropriate test conditions, it can rcliably estimate the cell density of gram-negative bacteria (39, 69, 75). No specialized equipment is required for this test moreover, a number of microtechniques have been proposed to reduce the amount of relatively expensive lysate required for testing serial dilutions (17). This assay may offer a convenient method for estimating the cell densities of aerobic spoilage microfloras as these are almost invariably dominated by gram-negative organisms. Although cell density is one of the fundamental factors for the evaluation of incipient spoilage, any test based on cell density alone will not give useful results in all cases. For normal meat stored aerobically, a cell density in excess of 10 8 bacterialcm 2 would indicate incipient spoilage, but for DFD meat the critical density is 10 6 bacterialcm 2, whereas with vacuum-packaged meat the cell density may give no indication of the remaining storage life unless bacterial numbers are below the maximum. Assessment of the spoilage status of meat from cell density measurements can only be made with some knowledge or assumptions regarding the meat composition and previous storage conditions. Estimation of spoilage products An obvious approach to the early detection of spoilage is measurement of the concentrations of the microbial products which are directly responsible for spoilage odors and flavors. Ammonia is probably the most prominent of theses substances as it is an inevitable product of amino acid degradation by bacteria. Ammonia in meat can be measured directly by colorimetric methods (34), by a membrane electrode (71), or as total volatile nitrogen by distillation of diffusion-over of the gas into standard acid and subsequent estimation by titration (68). Indirect measurements can also be used. As ammonia accumulates, the ph rises and this can readily be measured. It has been suggested that titration of meat homogenates with acid to an arbitary end-point gives a better correlation with spoilage (79), but it is difficult to see what real advantage this procedure has over direct ph measurement. The increase in meat ph also affects the ability of meat to retain water. All proteins in solution bind some quantity of water but much of the water in meat appears to be meehanically immobilized. The degree of meat hydration varies with the ph, a minimum value being attained at ph 5.0. Any increase in ph will result in enhanced water holding capacity (WHC). The WHC of meat is conveniently measured by the extract release volume (ERV), Le., the amount of water mechanically expressed from the meat. The actual amount of water expressed will depend to some extent on the method used, but with any method consistent results can be obtained (32). Jay (36) proposed that WHC could be used as an objective test for incipient spoilage. Further studies indicated that microbial products, such as exopolysaccharides which contain amino sugars, could be implicated in the decreased ER V associated with spoilage (78), but the contribution of such material was shown to be small (77). There are common problems for evaluation of meat spoilage by any of the above methods related to release of ammonia. The natural variation in ammonia content, meat ph and WHC means that comparatively high values for any of these factors are required before spoilage can be unequivocally identified. Moreover, with normal meat, aerobic spoilage will advance rapidly once amino acids start to be degraded because of the high bacterial concentration at which this commences. Any estimation of meat quality based on ammonia release will do little more than confirm subjective indications that the meat is spoiled; such tests have no predictive value for unspoiled meat. These tests would be inapplicable to DFD meat, where spoilage occurs at much lower bacterial numbers, or to meat carrying a gram-positive microflora, as significant amounts of ammonia would not be produced. Beeause spoilage of aerobically stored meat is usually due to the accumulation of volatile products of amino acid degradation, these volatile substances can be extracted and assayed for evaluation of spoilage. Detection of such substances has been used mainly to follow spoilage in fresh and-processed fish, but the methods appear to be equally applicable to other muscle foods. The volatiles can be separated from the bulk meat by diffusion, by flushing them from a homogenate with air or another gas, or by distillation at low temperatures under high vacuum (13, 51). The volatiles can be estimated as volatile reducing substances (VRS) by absorbing them in standard alkaline potassium permanganate solution and titrating the unreacted permanganate (13). Alternatively, they can be collected using a suitable absorbent, such as Porapak Q, or by condensation, and analysed and quantified by gas-liquid chromatography (GLC) (43, 53). Such studies on VRS are of considerable value in understanding the spoilage process but the time and equipment required for this type of estimation would limit their use for the practical evaluation of spoilage. Moreover, as with ammonia, the value of estimating these volatiles would be largely limited to confirming subjective judgements of aerobic spoilage since these substances can be detected organoleptically at very low concentrations and are only formed when amino acids are attacked. JOURNAl. OF FOOD PROTECTION, VOL. 46. MAY 1983

6 MEAT SPOILAGE 449 Although determination of aerobic spoilage products would seem to have little predictive value, this may not be true for anaerobic spoilage. In the latter case, bacterial numbers give little information about the spoilage status of the meat because spoilage by a microflora of lactobacilli only becomes evident well after maximum numbers have been attained. Spoilage by B. thermosphacta has recently been examined in some detail. This organism has a much greater spoilage potential than lactobacilli and can be important in both aerobic and anaerobic spoilage. Anaerobically, it will not grow on meat below ph 5.8 but in the presence of oxygen can grow down to ph 5.5 (31). Acetoin is a product of carbohydrate metabolism and volatile fatty acids, such as isobutyric and isovaleric, are produced from amino acids. These substances all contribute to spoilage odors and flavors. Unfortunately, for spoilage estimation, the relative proportions of these materials vary with the growth conditions (10). Spoilage by lactobacilli has not been investigated in such detail, but they also produce volatile acids from amino acids (55, 56) and impart acid or 'dairy' odors and flavors to meat (66, 82). Volatile fatty acids can be estimated by extraction with ether followed by steam distillation and titration. Estimation of total acids might not be of great value because of the presence of relatively large quantities of acetic acid which contribute little to spoilage odors and may not entirely be of microbial origin (9). Certain volatile acids would have to be estimated by GLC. Whether this type of analysis would be of any great value for predicting spoilage of vacuum-packaged meat can only be determined by further investigation. Estimation of other changes in meat composition Because microbial growth on meat was believed to involve the degradation of meat proteins, it was reasonable to suppose that the course of spoilage could be followed by monitoring the accumulation of products of protein degradation. Changes in total non-protein nitrogen (64) and individual amino acids (19, 38) have both been used for this purpose. The results invariably indicated that no significant changes occurred until spoilage had become evident. This is hardly surprising as bacteria usually only produce proteolytic enzymes in the late logarithmic phase of growth so no increase in the concentrations of these compounds due to microbial activity would occur until spoilage was well advanced (11). Alternatively, it has been suggested that the depletion of substrates utilized by bacteria could be used to indicate the onset of spoilage. There are two difficulties with such an approach. First, the variation in meat composition means that no single measurement can give any clear indication of prior bacterial activity, only evidence of a decrease over a period of time could be seen as significant. Secondly, until bacterial numbers approach spoilage levels, the bacterial biomass will be too small to significantly affect the concentration of any nutrient. Claims have been made that detectable decreases in nucleotide and individual amino acid concentrations occur before spoilage is evident (19, 38). In view of the primary utilization of glucose by most bacteria, it is unlikely that these observations are of general application. Glucose itself has had little consideration in this respect. It should be possible to detect glucose concentration gradients in uncomminuted meat before spoilage develops (21) but any such procedure would be somewhat impracticable because of the effort involved. In general, it can be concluded that before any changes in meat composition ascribable to bacterial activity rather that autolysis can be demonstrated, spoilage will be organoleptically evident (8, 45,46,80). APPLICATION OF MEmODS FOR ESTIMATING SPOILAGE To be of any practical value, methods for evaluating the spoilage status of meat must be rapid, economical and capable of giving some reliable estimation of the probable shelf-life under any particular storage conditions. The data required will vary depending on whether the meat is minced or in pieces up to carcass size, and on what storage conditions are applied. Aerobic spoilage of minces With a bulked mince, it can be assumed that the composition will approximate that of normal meat. Hence there is no necessity to determine the meat composition for the estimation of spoilage. The only factor that needs to be evaluated is the bacterial cell density since it can be assumed that spoilage is imminent once numbers exceed 10 8 cells/g. The shelf-life can be estimated from the cell density and any reasonable value for the growth rate of the spoilage flora at the anticipated holding temperature. The cell density need only be estimated to an order of magnitude, hence, in principle, any of the methods developed for rapid estimation of bacterial numbers could be used. The most likely source of error is the underestimation of shelf-life at chill temperature if the microflora is largely composed (>90%) of mesophilic bacteria that dominate before the growth of psychrotrophs commences and do not contribute to the spoilage flora. This problem would probably be reduced if the LAL test is used because only gram-negative bacteria are estimated, whereas most mesophilic contaminants on meat are gram-positive bacteria (62). With methods involving bacterial growth, conditions could be adjusted to minimize the effects of organisms that do not contribute to the spoilage micro flora. Aerobic spoilage of whole meat Examination of every piece of whole meat is clearly impractical, hence any sampling procedure for estimating shelf-life would involve taking enough samples to give some statistical assurance of the keeping qualities of meat. As with mince, some rapid method of estimating the cell density would be required, but an average value for this factor might not be sufficient because early spoilage of heavily contaminated pieces could result in tainting of meat which was not otherwise spoiled. With wide variations in bacterial loading, the estimated shelf-life would probably JOURNAL OF FOOD PROTECTION. VOL 46, MAY 1983

7 450 GILL have to be calculated on the basis of the highest levels of contamination. The same considerations would apply if meat composition varied widely because of early spoilage of any DFD meat present. Although fat can also spoil with lower cell densities than meat, this does not seem to be a common problem in commercial handling. However, this type of spoilage occurs in meat held in domestic refrigerators. There are two possible approaches to the problem of variation in meat composition. It could be assumed that some meat is likely to be DFD and will spoil when bacterial densities exceed 10 6 cells/cm 2. This would then define the maximum cell density for unspoiled meat. Unfortunately, this is close to acceptable levels of initial contamination of some carcasses and could lead to gross underestimation of shelf-life. Alternatively, some examination could be made of meat composition. The simplest method is determination of meat ph by means of a surface or probe electrode. However, this could still leave a high degree of uncertainty. The crucial factor for spoilage is the presence of glucose and, while glucose can be assumed to be absent if the meat ph is 6.4 or above, between ph 6.0 and 6.4 it mayor may not be present. This ambiguity could be overcome by assaying for the presence of glucose using a clinical assay system based on the reaction catalyzed by glucose oxidase. An accurate assay is unnecessary, with samples simply being scored positive or negative for glucose. Therefore, sampling need involve no more than pressing a dry swab onto the meat surface, shaking the swab with a minimal amount of dilute perchloric acid and testing a small quantity of the resultant extract. Vacuum-packaged meat As with aerobic spoilage, mince composition can be assumed to approximate that of normal meat. With vacuumpackaged cuts of meat, the problem of meat variation is simplified because ph is the determining factor and early spoilage of any pieces with a ph of 6.0 or above can be anticipated. The problem is to define incipient spoilage in normal meat. Enumeration of the microflora has limited value because the maximum cell density can be achieved long before spoilage becomes evident. However, the presence of high numbers of gram-negative bacteria (> 10 6 cells/cm2) would indicate faulty packaging and early spoilage. The presence of such microorganisms could be detected as discribed for meat stored aerobically. The only apparent general means of estimating the storage life of vacuum-packaged meat would require detection and quantification of the volatile fatty acids produced by microbes that usually predominate on such meat. Methods involving estimation of total volatile acids are inadequate while estimation of the concentrations of particular acids (9) is too demanding for routine application. Development of a simplified technique is necessary. CONCLUSIONS A rapid estimation of bacterial numbers is the most valu- able of possible tests for meat quality. Any additional information regarding the general composition of the microflora, i.e., predominantly gram-positive or -negative, mesophilic or psychrotrophic, would greatly increase the predictive value of such data. With information on the ph and glucose content of meat, a reasonable prediction of shelf-life under any particular storage condition could probably be made. However, the inherent variability of the meat-microbe system would suggest that it is not likely that a single, simple, unequivocal test could be devised to determine in all cases the spoilage status of meat before spoilage becomes evident to the senses. 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