Pickeled cabbage. L. gelidum JCM 9697T. Vacuum-packaged beef ..... bacteria of vacuum packed bacon. J. Appl. Bacteriol., 26, 453â. 470. Collins, M. D. ...
J. Gen. Appl. Microbiol., 44, 153–159 (1998)
Characterization of Leuconostoc species isolated from vacuum-packaged ham Yimin Cai,* Yoshimi Benno, Akiko Takeda,1 Tsutomi Yoshida,1 Tamiko Itaya,2 and Takashi Nakase Japan Collection of Microorganisms, The Institute of Physical and Chemical Research, Wako 351–0198, Japan 1 Kagawa Women’s University, Sakado 350–0288, Japan 2 Saitama Chuo Meat Inspection Center, Yono 338–0001, Japan (Received January 21, 1998; Accepted April 27, 1998)
Thirty-six isolates of Leuconostoc spp. were isolated from yellow spots that occurred on the surface of vacuum-packaged ham. All isolates were Gram-positive, catalase-negative cocci that produced gas from glucose and formed more than 90% of their lactate as D() isomer. These isolates could grow at 4°C but not above 30°C and most strains produced yellow spots on the ham. The isolates were divided into three groups by sugar fermentation patterns. Representative strains from three groups showed intergroup DNA homology values of above 88.8%, showing that these groups were composed of a single species. This organism was positioned at a separate branch in the genus Leuconostoc on the phylogenetic tree based on 16S rRNA sequences, which was assigned to Leuconostoc gelidum on the basis of DNA-DNA relatedness. Key Words——characterization; Leuconostoc spp.; vacuum-packaged ham
Ham is now the most common preserved meat product in many countries, including Japan. The preservation of fresh ham depends upon vacuum packaging to inhibit the growth of undesirable microorganisms under low-temperature conditions. However, if Leuconostoc spp. contaminates the ham, they will contribute to spoilage because they are able to grow at low temperatures in vacuum-packaged meats. Therefore, the prevention of contamination with Leuconostoc spp. is important in the production of ham products. Leuconostocs are facultative anaerobes, and vacuum-packaged ham would be a selective niche for them. Several papers have reported that Leuconostoc spp. are a major component of the microbial flora which develops on various types of vacuum-packaged meats, and the prevalence of Leuconostoc spp. on meats stored in vacuum packs or under modified gas atmospheres containing carbon dioxide (Cavett, 1963; Gill and Newton, 1978; Hitchener et al., 1982; Shaw and Harding, 1984, 1989). Some isolates from meat have been identified as Leuconostoc mesenteroides subsp. mesenteroides or Weissella paramesen* Address reprint requests to: Dr. Yimin Cai, Japan Collection of Microorganisms, The Institute of Physical and Chemical Research, Wako 351–0198, Japan.
teroides (Cavett, 1963; Hitchener et al., 1982; Shaw and Harding, 1984). Shaw and Harding (1989) applied numerical taxonomic techniques to group leuconostocs obtained from chill-stored meats on the basis of cellular fatty acid analysis and DNA homology experiments, and added two new species of L. gelidum and L. carnosum. In this study we used a semi-automatic identification system (Benno, 1996) to group leuconostocs isolated from yellow spots occurring on the surface of sliced ham. Representative strains were also studied by performing a 16S RNA sequence analysis and DNA homology experiments. Materials and Methods
Strains studied. A total of 46 strains shown in Table 1 were isolated from yellow spots that occurred on the surface of fresh sliced ham, using Phenylethanol agar (PEA) (Difco, Laboratories, MI, USA) and Lactobacilli MRS broth (Difco) containing 1.5% agar incubated at 5°C for 14–21 days and 25°C for 2–4 days under aerobic and anaerobic conditions. The ham, without sodium nitrite addition, was made in a commercial meat company and stored under vacuum at 4°C at the Saitama Chuo Inspection Center, Yono, Japan. Each colony was purified twice by
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Strains used in this study.
Strain Group A (nb7) Group B (n30)
Source
44 (JCMa 10094), 47, 165 (JCM 10095), 170–172, 177 42 (JCM 10093), 43, 46, 50–53, 79, 94–102, 103 (JCM 10096), 107, 132, 133, 139, 160, 163, 167, 173–175, 178, 179 Group C (n9) 45 (JCM 10097), 48, 49, 161 (JCM 10098), 162, 164, 166, 168, 169 L. carnosum JCM 9695T L. citreum JCM 9698T L. fallax JCM 9694T L. gelidum JCM 9697T L. lactis JCM 6123T L. mesenteroides subsp. cremoris JCM 9889T L. mesenteroides subsp. dextranicum JCM 9700T L. mesenteroides subsp. mesenteroides JCM 6124T L. pseudomesenteroides JCM 9696T a b
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Vacuum-packaged ham Vacuum-packaged ham Vacuum-packaged ham Vacuum-packaged beef Food, blood Pickeled cabbage Vacuum-packaged beef Milk Dried starter powder Kefir Olives, silage, milk, blood Dairy food, blood
JCM, Japan Collection of Microorganisms. n, Number of strains tested.
streaking on MRS agar. The pure cultures were grown in MRS agar at 25°C for 24 h, collected with nutrient broth (Difco) and dimethyl sulfoxide at a ratio of 9 : 1, and stored as stock cultures at 80°C for further examination. As shown in Table 1, the type strains of Leuconostoc species were obtained from the Japan Collection of Microorganisms (JCM), The Physical and Chemical Research Institute, Japan. Reproduction test of yellow spots. Fresh sliced ham without preservative (sodium nitrite) was placed into sterile petri-dishes, and all isolates and type strains of Leuconostoc spp. were spotted onto the surface of the ham with a sterile toothpick. The dishes were put into plastic bags (Hiryuu KN type, 180260 cm, Asahikasei, Ltd., Tokyo, Japan) and the packages were sealed with a vacuum sealer (BH 950, Matsushita, Ltd., Tokyo, Japan). The packages were kept at 5°C for 14–21 days and at 25°C for 2–4 days, and three replicates were used for the reproduction test of yellow spots. Morphological, physiological and biochemical tests. Gram stain and cell morphology were examined after 24 h of incubation on MRS broth containing 1.5% agar. Gas production from glucose, ammonia production from arginine and dextran from sucrose were determined by a method described by Kozaki et al. (1992). Growth at different temperatures was observed in MRS broth after incubation at 5°C for 14 days, and 30 and 37°C for 7 days. Sugar fermentation was examined using a semi-automatic system for bacterial identification as described by Benno (1996). The isomers of lactate formed from glucose were determined enzymatically using reagents obtained from Boehringer GmbH, Mannheim, Germany. The enzyme activity tests were performed using the API ZYM system (API Products, Basingtoke, UK) according to the instructions of the manufacturer. Pigment production
was observed in wet packed cells harvested from MRS broth by centrifugation (Farrow et al., 1989). 16S rDNA sequencing. The 16S rDNA sequence coding region of Leuconostoc gelidum JCM 10093 was amplified by polymerase chain reaction (PCR) and performed in a TaKaRa PCR Thermal Cycler (Takara Shuzo Co., Ltd., Ohtsu, Japan) as described by Suzuki et al. (1996). The sequences of the PCR products were determined directly using an ALFexpressTM AutoCycleTM Sequence Kit (Pharmacia Biotech, CA, USA) with the primers as described by Suzuki et al. (1996). The 16S rDNA sequence of Leuconostoc gelidum JCM 10093 was deposited in the DNA Data Bank of Japan (DDBJ) under accession number AB004661. Neucleotide substitution rates (Knuc values) were calculated (Kimura and Ohta, 1972), and the phylogenetic tree was constructed by the neighbor-joining method (Saitou and Nei, 1987). The topology of trees was evaluated by bootstrap analysis of the sequence data with CLUSTAL W software (Thompson et al., 1994). This sequence was aligned with the following published sequence from DDBJ, GenBank, and EMBL: Leuconostoc mesenteroides subsp. mesenteroides DSM 20343T (M23035), L. mesenteroides subsp. cremoris DSM 20346T (M23034), L. carnosum NCFB 2776T (X95977), L. amelibiosum NCFB 2787 (X53963), L. pseudomesenteroides NCDO 768T (X95979), L. lactis DSM 20202T (M23031), Weissella kandleri NCDO 2753T (X52570), W. halotolerans DSM 20190T (M23037), W. minor NCDO 1973T (X52569), W. viridescens NCDO 1655T (X52568), W. confusa NCDO 1586T (X52567), W. hellenica NCFB 2973T (X95981), W. paramesenteroides NCDO 803T (X95982), Enterococcus sulfureus NCDO 2379T (X55133), E. columbae NCIMB 13013T (X56422) and Enterococcus seriolicida ATCC 49156T (L32813). Bacillus subtilis NCDO 1769T
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Leuconostoc gelidum from ham
(X60646) was used as the out-group organism. DNA base composition and DNA-DNA hybridization. DNA was extracted from the cells harvested from MRS broth incubated for 8 h at 30°C for type
strains of Leuconostoc and at 25°C for the isolates, and purified by the procedure of Saito and Miura (1963). DNA base composition was determined by the method of Tamaoka and Komagata (1984) using high-
Group B (n30)
Group C (n9)
L. carnosum JCM 9695T
L. citreum JCM 9698T
L. fallax JCM 9694T
L. gelidum JCM 9697T
L. lactis JCM 6123T
L. mesenteroides subsp. cremoris JCM 9889T
L. mesenteroides subsp. dextranicum JCM9700T
L. mesenteroides subsp. mesenteroides JCM 6124T
L. mesenteroides subsp. dextranicum JCM9700T
L. pseudomesenteroides JCM 9696T
Characteristics of Leuconostoc species.a
Group A (nb7)
Table 2.
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6d
28
1
w
w w w
w w w
w w w
w w
w w w w
w
w w
w
w
w w w
w
Characteristic
Growth at 5°C at 30°C at 37°C Yellow pigmentc Dextran formation Acid produced frome L-Arabinose Ribose D-Xylose Galactose D-Fructose D-Mannose L-Sorbitol Mannitol α-Methyl-D-glucoside N-Acetyl-glucosamine Amygdalin Esculine Salicine Cellobiose Maltose Lactose Melibiose Saccharose Trehalose D-Raffinose β-Gentiobiose D-Turanose Gluconate a
w w
All strains were Gram-positive, catalase-negative cocci that produced gas from glucose and formed more than 90% of their lactate as isomer. , 100% positive; , 100% negative; w, 100% weakly positive. b n, Number of strains tested. c Pigment detected in centrifuged cell pellet from MRS broth. d Reproducible strains, no. of yellow spots on ham. e Tests were performed using the Semi-Automatic Identification System, readings were determined at 7 days. All strains produced acid from D-glucose and failed to produce acid from glycerol, erythritol, D-arabinose, L-xylose, adonitol, β-methyl-xyloside, L-sorbitol, rhamnose, dulcitol, inositol, sorbitol, inulinmelezitose, amidon, glycogene, xylitol, D-lyxose, D-tagatose, D-fucose, and D-arabitol. D()
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α-Fucosidase
β-Glucuronidase
() () ()
N-Acetyl-β-glucosamidase α-Mannosidase
β-Glucosidase
β-Galactosidase
Phosphatase Naphthol-AS-BI-phosphohydrolase α-Galactosidase
Lipase Leucine arylamidase Valine arylamidase Cystine arylamidase Trypsin Chymotrypsin
() () ()
α-Glucosidase
Leuconostoc group A Leuconostoc group B Leuconostoc group C L. carnosum JCM 9695T L. citreum JCM 9698T L. fallax JCM 9694T L. gelidum JCM 9697T L. mesenteroides subsp. cremoris JCM 9889T L. mesenteroides subsp. dextranicum JCM 9700T L. mesenteroides subsp. mesenteroides JCM 6124T L. pseudomesenteroides JCM 9696T
Esterase lipase
Strain
Esterase
Enzymatic activities of Leuconostoc species.a
Alkaline phosphatase
Table 3.
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a
Enzymatic tests were performed with an API 20S system according to the instructions of the manufacturer. Quantity of hydrolyzed substrate: , 20 nmol; (), 11–20 nmol; , 6–10 nmol; and , 5 nmol.
performance liquid chromatography following enzymatic digestion of DNA to deoxyribonucleosides. The equimolar mixture of four deoxyribonucleotides in a Yamasa GC Kit (Yamasa Shoyu Co., Ltd., Choshi, Japan) was used as the quantitative standard. The DNA-DNA relatedness was determined by the method of Ezaki et al. (1989) using photobiotin and microplates. Results
Physiological and biochemical properties Six representative isolates (JCM 10093–10098) have been deposited in the JCM. The phenotypic characteristics and enzyme activity of the Leuconostoc species are shown in Tables 2 and 3, respectively. All isolates were Gram-positive, catalase-negative cocci that produced gas from glucose and formed more than 90% of the lactate as D() isomer. Dextran formation was common in all strains and no strain hydrolyzed arginine. All strains grew at 4°C, but not at temperatures above 30°C. These strains were divided into three groups on the basis of sugar fermentation patterns and enzyme activity: Group A produced acid from mannitol and amygdalin; Group B did not produce acid from amygdalin; and group C did not produce acid from mannitol. Group C was β-galactosidase positive, whereas groups A and B gave negative
reactions. The carbohydrate fermentation patterns of groups A, B, and C were similar to those of L. pseudomesenteroides JCM 9696T and L. gelidum JCM 9697T, but they are easily distinguished from the type strains of other Leuconostoc spp. Strains of groups A, B, and C were also α-galactosidase and αglucosidase negative, whereas L. pseudomesenteroides JCM 9696T and L. gelidum JCM 9697T gave positive reactions. Reproduction test of yellow spots Yellow spots were produced on the ham by 35 of the 46 strains tested. Group A included 6 strains that produced yellow spots, group B included 28 strains that produced yellow spots, and group C had only 1 strain that produced yellow spots. L. carnosum JCM 9695T, L. fallax JCM 9694T, L. gelidum JCM 9697T, L. mesenteroides subsp. dextranicum JCM 9700T, L. mesenteroides subsp. mesenteroides JCM 6124T and L. lactis JCM 6123T did not produce pigments in MRS broth or yellow spots on ham. However, L. citreum JCM 9698T, L. mesenteroides subsp. cremoris JCM 9889T, L. lactis JCM 6123T and L. pseudomesenteroides JCM 9696T produced pigments in MRS broth but did not produce yellow spots on ham. 16S rDNA sequence analysis The 16S rDNA of Leuconostoc sp. JCM 10093 was
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Fig. 1. Phylogenetic tree derived from 16S rDNA sequences. The tree was created using the neighbor-joining method and Knuc values. The numbers on the tree indicate bootstrap vlues for the branch points.
Table 4.
DNA base compositions and levels of DNA-DNA homology for Leuconostoc species.
determined for 1507 bases, as shown in the DDBJ database under accession number AB004661. The phylogenetic tree shown in Fig. 1 was reconstructed from evolutionary distances by the neighbor-joining method. This strain fell within the radiation of the cluster comprising the Leuconostoc genus. This cluster was recovered in 100% of bootstrap analysis, and the branch point of JCM 10093 and its closest relative, L. carnosum, was supported by 92% recovery in the bootstrap analysis. A comparison of the sequences of JCM 10093 and L. carnosum at 1333 unambiguous nucleotide positions showed that these organisms exhibited the highest similarity.
JCM 9695T
JCM 9696T
JCM 9697T
38.3 37.6 38.1 38.6 37.9 39.1 37.5 44.2 39.3 38.9 39.0
JCM 6124T
Group A (JCM 10094) Group B (JCM 10093) Group C (JCM 10097) L. carnosum JCM 9695T L. citreum JCM 9698T L. fallax JCM 9694T L. gelidum JCM 9697T L. lactis JCM 6123T L. mesenteroides subsp. dextranicum JCM 9700T L. mesenteroides subsp. mesenteroides JCM 6124T L. pseudomesenteroides JCM 9696T
Group C
GC content (mol%)
Group B
Strain
Group A
% DNA-DNA reassociation with
100 97.9 94.3 27.8 23.2 16.3 92.3 18.6 22.7 23.8 19.9
92.3 100 98.5 33.5 23.4 18.6 90 12.3 20.5 20.5 24.8
95.3 88.8 100 31.6 26.3 17.8 95.4 17.7 28.6 24.8 26.1
22.6 20.9 11.8 22.4 26.1 19.1 20.4 17.3 30.0 100 25.7
22.1 21.9 18.8 100 26.8 16.6 19.0 13.8 18.7 26.8 22.1
16.2 18.5 21.4 21.7 28.9 18.2 25.7 16.7 35.2 23.8 100
90.5 85.3 93.2 40.3 18.2 9.8 100 15.2 27.6 21.3 20
DNA base composition and DNA-DNA hybridization DNA base compositions and levels of DNA-DNA homology for Leuconostoc species are shown in Table 4. Representative strains from groups A, B and C had a GC content range of 37–38 mol%. The data are within the range of 37 to 44 mol% GC for the Leuconostoc genus. Strains in groups A, B, and C were 88.8 to 97.9% homologous with their atypical strains, and they showed a high level of DNA relatedness (89.6) to the reference strain L. gelidum JCM 9697T, whereas homology values were low (9.8 to 40.3%) against other reference strains from the type strains of the previously described species.
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Discussion
In this study, all isolates were predominantly isolated from spots occurring on the surface of sliced ham, which was processed without sodium nitrite and stored at 4°C for 2 to 3 weeks. Yellow spots were reproduced on the ham by most isolates, suggesting that the isolates were closely associated with the yellow spots on the ham. All isolates in this study were heterofermentative lactic acid bacteria and can grow at low temperatures under anaerobic conditions. The results in this study show that the inhibitory effect of anaerobic conditions on leuconostocs is not as great as that against aerobic bacteria, and that the isolates will grow during cold storage. The reason for this was that leuconostocs are facultative anaerobes, and anaerobic conditions are not expected to inhibit their growth. Therefore, when these strains contaminate ham, they may cause spoilage by souring. The strains of groups A, B and C were found to be similar to L. pseudomesenteroides JCM 9695T and L. gelidum JCM 9697T phenotypically. However, L. gelidum JCM 9697T was different from these strains for some of the enzyme production patterns and physiological characteristics, especially growth at 30°C, and it does not produce yellow spots on ham. L. pseudomesenteroides JCM 9695T was also different in terms of the enzyme activities of α-galactosidase and α-glucosidase. In this study, we found that the API ZYM system gave results which agree well with those obtained using other biochemical methods in the study of genus Leuconostoc, and proved very satisfactory, being simple to use and having good reproducibility. The API ZYM system proved useful in studying these organisms. The 16S rDNA sequence analysis results showed that these organisms were closely related to L. carnosum on the basis of phylogenetic tree. This is in agreement with Collins et al. (1993). The level of similarity of the 16S rRNA sequences (98.6%) of L. gelidum and L. carnosum supported this conclusion, whereas the 16S rDNA sequence of the L. gelidum type strain has not been deposited in the DDBJ. As shown in Table 3, the DNA-DNA hybridization results demonstrated that all three groups of leuconostocs from ham had intergroup DNA homology values of above 88.8%, showing that these groups were composed of a single species, which could be assigned to L. gelidum. The subdivision of three groups indicated by the sugar fermentation study was not supported by the DNA hybridization results, which showed that these strains formed a single homology group. This is reconcilable because the differences were only in the relative patterns of sugar fermentation, and would not be expected to affect genomic relatedness (Shaw and
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Harding, 1989). The results obtained in this study demonstrate the occurrence of atypical LAB on vacuum-packaged fresh ham. As L. gelidum was the dominant species present on this product at spoilage, more detailed studies of this species’ properties and how spoilage can be prevented are required. The authors wish to thank Dr. J. A. Hudson (Environmental Science and Research Ltd., Christchurch Science Centre, New Zealand) for reading the manuscript. References Benno, Y. (1996) A semi-automatic system for bacterial identification. Riken Rev., 12, 57–58. Cavett, J. J. (1963) A diagnostic key for identifying the lactic acid bacteria of vacuum packed bacon. J. Appl. Bacteriol., 26, 453– 470. Collins, M. D., Samelis, J., Metaxopoulos, J., and Wallbanks, S. (1993) Taxonomic studies on some leuconostoc-like organisms from fermented sausages: Description of a new genus Weissella for the Leuconostoc paramesenteroides group of species. J. Appl. Bacteriol., 75, 595–603. Ezaki, T., Hashimoto, Y., and Yabuuchi, E. (1989) Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int. J. Syst. Bacteriol., 39, 224–229. Farrow, J. A. E., Facklam, R. R., and Collins, M. D. (1989) Nucleic acid homologies of some vancomycin-resistant leuconostocs and description of Leuconostoc citreum sp. nov. and Leuconostoc pseudomesenteroides sp. nov. Int. J. Syst. Bacteriol., 39, 279–283. Gill, C. O. and Newton, G. K. (1978) The ecology of bacterial spoilage of fresh meat at chill temperatures. Meat Sci., 2, 207– 212. Hitchener, B. J., Egan, A. F., and Rogers, P. J. (1982) Characteristics of lactic acid bacteria isolated from vacuum-packaged beef. J. Appl. Bacteriol., 52, 31–37. Kimura, M. and Ohta, T. (1972) On the stochastic model for estimation of mutation distance between homologous proteins. J. Mol. Evol., 2, 87–90. Kozaki, M., Uchimura, T., and Okada, S. (1992) Experimental Manual for Lactic Acid Bacteria, Asakurashoten, Tokyo, pp. 29–72. Martinez-Murcia, A. J. and Collins, M. D. (1990) A phylogenetic analysis of the genus Leuconostoc based on reverse transcriptase sequencing of 16S rRNA. FEMS Microbiol. Lett., 70, 73– 84. Saito, H. and Miura, K. (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta, 72, 619–629. Saitou, N. and Nei, M. (1987) The neighbor-joining methods: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol., 4, 406–425. Shaw, B. G. and Harding, C. D. (1984) A numerical taxonomic study of lactic acid bacteria from vacuum-packed beef, pork, lamb and bacon. J. Appl. Bacteriol., 56, 25–40. Shaw, B. G. and Harding, C. D. (1989) Leuconostoc gelidum sp. nov. and Leuconostoc carnosum sp. nov. from chill-stored meats. Int. J. Syst. Bacteriol., 39, 217–223. Suzuki, K., Sasaki, J., Uramoto, M., Nakase, T., and Komagata, K. (1996) Agromyces mediolanus sp. nov., nom. rev., comb. nov.,
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a species for “Corynebacterium mediolanum” Mamoli 1939 and for some aniline-assimilating bacteria which contain 2,4-diaminobutyric acid in the cell wall peptidoglycan. Int. J. Syst. Bacteriol., 46, 88–93. Tamaoka, J. and Komagata, K. (1984) Determination of DNA base composition by reversed-phase high-performance liquid chro-
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