Effect of Lactic Acid Concentration on Growth on Meat of Gram-Negative Psychrotrophs from a Meatworks

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1982, p. 284-288 0099-2240/82/020284-05$02.00/0 Vol. 43, No. 2 Effect of Lactic Acid Concentration on Growth on Meat of Gram-Negative Psychrotrophs from a Meatworks C. 0. GILL* AND K. G. NEWTONt Meat Industry Research Institute of New Zealand (Inc.), Hamilton, New Zealand Received 27 July 1981/Accepted 25 September 1981 The inhibitory effect of the lactic acid in meat on gram-negative psychrotrophs appears to be due mainly to the decrease in ph, not to action of the undissociated acid. Species of Pseudomonas were essentially unaffected by the ph of normal meat. Other gram-negative psychrotrophs isolated from a meatworks included a large number of strains which would not grow on meat of normal ph at chill temperatures. Raising either the ph or the incubation temperature allowed many of the ph-sensitive strains to initiate growth. However, the growth rates of phinsensitive strains were not affected by increasing the ph, and there were no significant differences in the composition of the spoilage floras which developed on chilled meat of normal and high ph. The ultimate ph of muscle tissue can vary between 5.5 and 7, the value being largely dependent upon the amount of glycogen present in the tissue at slaughter. After death, glycogen is converted to lactic acid via glycolysis, and if glycogen reserves are high, a lactic acid concentration of about 0.9% (wet weight) is attained with a concomitant low ph. If glycogen reserves are depleted before slaughter, the final lactic acid concentration can be less than half the normal value, and the meat is described as dark, firm, and dry with a ph of >6.0 (24). It has been suggested that growth of many bacteria of importance in spoilage may be partially or totally inhibited when ph values of meat approach 5.5 (13). Both the undissociated acid and the low ph can affect some spoilage bacteria (6, 7, 10). The importance of meat ph in the development of anaerobic floras is well established, since on meat of high ph, species of high spoilage potential, such as Brochothrix thermosphacta and Alteromonas putrefaciens, can grow and cause early spoilage in vacuum packages (7, 10). The effect of meat ph on aerobic spoilage floras is not as clear. It has long been known that dark, firm, dry meat spoils more rapidly than meat of normal ph, and this has been ascribed to faster growth of the flora on dark, firm, dry meat (14, 16). Although there is some evidence for this suggestion (24), at least some strains of aerobic spoilage bacteria are unaffected by ph in the range found in meat, and early spoilage of dark, firm, dry meat is due mainly to spoilage becoming evident at lower cell densities than with normal meat (21). Variations in the compot Present address: Australian Government Analytical Laboratory (N.S.W.), Royal Exchange 2000, Svdney, Australia. sitions of aerobic spoilage floras with meat ph have also been described for both red meats and muscle tissues such as fish and chicken leg muscle that are normally of high ph (1, 18, 25). However, the significance of these differences has not been established. Aerobic spoilage floras are almost invariably dominated by gram-negative psychrotrophs, species of Pseudomonas, Acinetobacter, and Moraxella being the major organisms present on normal meat (6). However, other psychrotrophic species might contribute to the flora when the ph is high, as any species present in the meatworks environment is likely to occur in the initial flora (22). Only a few strains of some of the relevant species have been examined for their responses to ph and lactic acid concentration. We have therefore investigated the effects of these variables on the growth of gram-negative psychrotrophs isolated from a meatworks with the objective of obtaining better understanding of the selective effect on the aerobic spoilage flora of the lactic acid content of meat. MATERIALS AND METHODS Isolation and identification of bacteria. Gram-negative bacteria which grew on nutrient agar within 2 weeks when incubated at 2 C were isolated at a meatworks, as described by Newton et al. (23). Isolates were obtained from work surfaces, carcasses, and meat at all stages of processing. When possible, strains were identified to the species level: Pseudomonas by the criteria of Bergeys Manual (4), Davidson et al. (5), and Lahellec et al. (15); Alteromonas by the criteria of Lee et al. (17); and Enterobacteriaceae as previously described by Newton et al. (22). Moraxella and Acinetobacter were identified by the criteria of Bergeys Manual (4), Baumann et al. (2, 3), and Henriksen (11). Strains with the characteristics of Morax- 284

VOL. 43, 1982 ella which hydrolyzed gelatin or starch and produced yellow or orange pigments were considered to be Flavobacterium. Response to ph. Plates of Difco nutrient agar, the ph of which had been adjusted after sterilization to 5.5 or 6.0 with lactic, hydrochloric, or acetic acid, were inoculated from cultures growing logarithmically in nutrient broth at 25 C. Visible growth was recorded from plates which were incubated in air for 3 days at 25 C or 6 weeks at 2 C. Sensitivity to lactate. Bacterial strains which grew at 2 C on media adjusted to ph 5.5 with lactic acid were further inoculated onto the same agar containing either O.9o lactate or glycerol to adjust the water activity (0.997) to that of the high lactate medium. Growth on meat. Five strains which grew in the presence of lactate at ph 5.5 and 2 C were selected from each group of bacteria, except Aeromonas and Alteromonas. Cultures of each strain were grown in nutrient broth and suitably diluted in 0.9%o saline. Sterile slices of beef (ca. 5 by 2 by 1 cm; 10 g) were cut from two striploins of ph 5.6 and 6.4, using sterile instruments. The growth rates of the bacteria growing in pure culture on high- and low-ph meats were determined by inoculating each culture at an initial density of 103 to 104 cells per cm2 onto one surface of each slice of meat in separate series of seven pieces of high- and low-ph meat. The slices were incubated at 2 C in a humid atmosphere, slices from each series being removed at zero time and at subsequent intervals of 2 days. The slices were macerated with 20 ml of peptone water, using a stomaching machine, and suitably diluted samples were spread on nutrient agar plates which were incubated at 25 C for 48 h. Composition of spoilage floras. Steaks were cut from the ends of three beef rib eye muscles obtained from a meatworks. The steaks, one of low (5.5) and two of high (6.4) ph, were wrapped in stretch polyethylene and stored in air at 2 C. At intervals of 0, 7, 12, and 17 days, pieces (5 cm2) were removed from steak surfaces TABLE 1. LACTIC ACID AND MEAT MICROFLORA 285 to Stomacher bags and homogenized with 20 ml of 0.1% (wt/vol) peptone water. The homogenates were plated on nutrient agar and incubated at 25 C for 3 days. At each sampling time, 100 colonies from each flora were randomly selected, isolated, and identified. RESULTS Reactions to ph. Numbers and identities of strains in seven groups are shown in Table 1. About half the strains of four of the bacterial groups and all strains of Alteromonas did not grow at 2 C on the medium adjusted to ph 5.5 with lactic acid. About 20% of the strains of Enterobacteriaceae were inhibited, but these included only a few (3 of 59) strains of Serratia liquefaciens. Only 2% of the Pseudomonas strains did not initiate growth. When the medium ph was 6.0, 40%o of Alteromonas strains were still inhibited, but few strains of any other group failed to grow at 20C. Raising the incubation temperature to 250C allowed most strains to grow on the medium adjusted to ph 5.5 with lactic acid, although significant numbers of Moraxella, Aeromonas, and Alteromonas were still inhibited (Fig. 1). Lactic acid and HCI inhibited similar proportions of all groups at ph 5.5 and 2 C, but all groups were more severely inhibited by acetic acid. Pseudomonas and Acinetobacter were the only groups with significant numbers of strains able to initiate growth in the presence of acetic acid (Fig. 2). Both of the other acids had inhibitory effects similar to those of lactic acid at ph 6.0 and 20C. At ph 5.5 and 25 C inhibition by HCI was similar to that of lactic acid, but acetic acid still produced considerable inhibition in all groups except the Enterobacteriaceae (Fig. 2). Identities of gram-negative species isolated at a meatworks Family or genus of strains Species isolates Pseudomonas 189 P. fluorescens 87 P. putida 16 P. fragi 26 Unidentified 60 Enterobacteriaceae 178 Enterobacter aerogenes 55 Serratia liquefaciens 59 Erwinia caratovora 16 Yersinia enterocolitica 12 Klebsiella pneumoniae 9 Hafnia alvei 5 Citrobacter freundii 2 Citrobacter amalonica 3 Proteus rettgeri 3 Serratia marcescens 14 Moraxella 96 Unidentified 96 Aeromonas 44 Unidentified 44 Acinetobacter 25 A. calocoaceticus 25 Alteromonas 23 A. putrefaciens 23 Flavobacterium 19 Unidentified 19

286 GILL AND NEWTON l00r 501 a Hrir APPL. ENVIRON. MICROBIOL. 100O 50F 0 100 501- b C _-AENN v a.. - * - 1 2 3 4 5 6 7 FIG. 1. Percentage of bacterial strains unable to initiate growth on nutrient agar with the ph adjusted by addition of lactic acid: (a) ph 5.5, 2 C; (b) ph 6.0, 2 C; (c) ph 5.5, 25 C. (1) Pseudomonas; (2) Enterobacteriaceae; (3) Moraxella; (4) Aeromonas; (5) Acinetobacter; (6) Flavobacterium; (7) Alteromonas. None of the acids prevented growth of a significant proportion of the strains from any group on media of ph 6.0 with incubation at 25 C. Sensitivity to lactate. Several strains of Enterobacteriaceae and Flavobacterium failed to grow on either lactate- or glycerol-containing medium at 2 C, so they were apparently sensitive to the slightly reduced water activity under the growth conditions used. Only strains of Aeromonas (9 of 15) showed lactate sensitivity in that they grew on the glycerol- but not on the lactatecontaining medium. Growth on meat. Only Aeromonas grew appreciably faster on high-ph meat. The growth rates of the other genera were similar on normaland high-ph meat (Table 2). A lag phase was not apparent before growth of the bacteria began on high-ph meat, but Flavobacterium and Moraxella strains showed a lag phase on normal-ph meat. Pseudomonas strains grew faster than the other bacteria on both types of meat. Composition of spoilage floras. There were no significant differences between the compositions of any of the spoilage floras. The initial floras contained large numbers of micrococci and staphylococci but these were displaced by gramnegative organisms, with Pseudomonas strains predominating. When bacterial densities reached 107/cm2, >90% of the isolates from all flora were pseudomonads. These organisms formed 97 to 99% of the final floras (Table 3). loor b 50 IOOrc 50t H F'l 1 2 3 4 5 6 7 FIG. 2. Percentage of bacterial strains unable to grow at ph 5.5 and 2 C on nutrient agar with the ph adjusted by addition of (a) HCI and (b) acetic acid. (c) Percentage of bacterial strains unable to grow at 2 C on nutrient agar with the ph adjusted to 6.0 with acetic acid. (1) Pseudomonas; (2) Enterobacteriaceae; (3) Moraxella; (4) Aeromonas; (5) Acinetobacter; (6) Flavobacterium; (7) Alteromonas. DISCUSSION The strong inhibitory action of acetic acid on bacteria is largely due to the undissociated acid (9), whereas HCl inhibits only by reducing the ph. Since the pattern of inhibition produced by lactic acid was very similar to that produced by HCl and markedly different from that due to acetic acid, it appears that the lactic acid in meat exerts any selective effect on the gram-negative flora mainly by reducing the ph. This seems to be confirmed by the observation that all groups except Aeromonas were unaffected by increasing concentrations of lactic acid. TABLE 2. Mean growth rates of five lactic acidtolerant strains of each genus on meat slices held at 2 C under aerobic conditions Generation time (h) Family or genus at meat ph: 5.6 6.4 Pseudomonas 11.8 11.4 Enterobacteriaceae 21.2 20 Moraxella 13 13.2 Aeromonas 25 17.6 Acinetobacter 15.2 15.5 Flavobacterium 20.3 21 F

VOL. 43, 1982 LACTIC ACID AND MEAT MICROFLORA 287 TABLE 3. Development of the spoilage flora on a ph 6.4 steak stored in air at 2 C Composition (%) Time Cell Entero- (days) density Pseudo- Acineto- Moraxella B. ther- bacteri- Micro- Staphy- Others monas bacter mosphacta aceae coccus lococcus 0 1 x 103 1 5 6 13 1 53 10 11 7 2 x 105 56 36 4 2 1 1 12 6 x 107 99 1 17 4 x 109 97 2 1 The aerobic flora of meat is usually dominated by species of Pseudomonas (6), the group least affected by the ph of normal meat. This apparent advantage would not apply when the ph was greater than 6.0, so it might be expected that pseudomonads would form a smaller proportion of the spoilage flora on meat of high ph. In fact, meat ph had no effect on the composition of the spoilage floras. This contrasts with studies of chicken meat which showed that breast muscle (low ph) supported a flora of pseudomonads whereas on leg muscle (high ph) organisms of the Acinetobacter/Moraxella group formed a large fraction of the flora at all stages of development and flavobacteria were of importance in the early stages of bacterial growth (19, 20). The difference between the floras on high-ph beef and poultry probably arises from differences in the initial floras. Although the initial densities of the two floras may be similar, the poultry flora, derived from the water used to chill the carcasses, is composed mainly of psychrotrophs, whereas the beef flora is predominantly mesophilic. This means that selective effects are exerted on several more generations of the beef than of the chicken psychrotrophic microflora so that the composition of the initial flora is unlikely to be reflected in the final beef flora as it can be in the final chicken flora. The data from chicken leg muscle tend to confirm this interpretation since the proportion of Pseudomonas in the flora increased as the flora developed (20), indicating that these organisms were still growing faster than their competitors on the high-ph meat. The possibility that growth rates of many spoilage organisms are slowed rather than totally inhibited on normal-ph meat at chill temperatures has not been completely resolved. However, our results indicate that a significant proportion of the organisms able to grow at all at ph 5.5 and 2 C will be doing so at their maximum rate. Apart from the observations of Rey et al. (25), all other evidence indicates that the Pseudomonas species occurring in meat spoilage floras are unaffected by ph over the range found in meat (1, 2, 6, 9, 20). After Pseudomonas species, the other group least affected by ph changes were the members of the Enterobacteriaceae. Because this group is composed of a large number of distinct species, any generalization must be treated with caution in the absence of more precise data on individual species. However, it seems that these organisms tend to grow very much slower than Pseudomonas at chill temperatures (6) and so do not usually contribute to the aerobic spoilage flora despite their apparent insensitivity to the ph of normal meat. Suboptimal temperatures tend to enhance the inhibitory effect of low ph (12). Raising the storage temperature should therefore allow a greater proportion of nonpseudomonads to appear in the flora. This seems to occur, because at elevated temperatures the pseudomonads are progressively displaced by species of Acinetobacter and Enterobacteriaceae, which include psychrotrophic as well as mesophilic strains (8). However, the lack of effect on the spoilage flora of raising the ph at chill temperatures indicates that the change in flora composition associated with elevated temperature is not due only to more strains of nonpseudomonads initiating growth, but probably also results from an increase in their growth rates relative to that of the pseudomonads. Since a low meat ph does not necessarily reduce the growth rates of ph-insensitive strains, the failure of many of these organisms to emerge in the spoilage flora is likely to be due to their growth rates at chill temperatures being slow relative to those of the Pseudomonas species. The initiation of growth on meat of high ph by a larger proportion of the nonpseudomonads clearly has no significant effect upon the spoilage flora. The pseudomonads are essentially unaffected by the ph of meat, but their advantage in growth rate at chill temperatures is probably the decisive factor in their dominance on aerobically stored chilled meats. ACKNOWLEDGMENTS We thank K. M. DeLacey for technical assistance. LITERATURE CITED 1. Barnes, E. M., and C. S. Impey. 1968. Psychrophilic spoilage bacteria of poultry. Appl. Bacteriol. 31:97-107.

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