bltenlational Journal of Food Microbioloffy. 16 (1992) 167-172 © 1992 Elsevier Science Publishers B.V. All rights reserved (1168-1605/92/$[15.lX1
167
FOOD ID515
Short communication
Classification of ropy slime-producing lactic acid bacteria based on D N A - D N A homology, and identification of Lactobacillus sake and Leuconostoc amelibiosum as dominant spoilage organisms in meat products P. M~ikel~i a, U. Schillinger b, H. Korkeala a and W.H. Holzapfel b '~ Deparmwnt of Food and Eneiromnental Hygiene. Cidlege of Veterinary Medicine, Helsinki. Finland and b blstilute of Hygiene and Toxieology. Federal Research Centre for Nutritim¢. garlsruhe. German).
(Received 8 May 1991;accepted 19 April Iq~2)
The classification of lactic acid bacteria able to cause ropy slime on vacuum-packed cooked meat products was carried out based on DNA-DNA homology. The ropy slime-producing lactoba¢ill[ wcrc identified as strains of Lactobacillus sake and the ropy slime-prc,ducing leuconostocs, such as Leucono~. toc amelibiosum and Leuconosto¢ mesenteroides. Key words: Identification; Taxonomy; DNA base composition; DNA homology; Lactobacillas sake; Leueoaostoc amelihiosnnl; Leut~mostoc me~,enteroides: Ropiness: Meat products, vacuumpacked
Introduction T h e f o r m a t i o n o f ropy slime o n v a c u u m - p a c k e d m e a t p r o d u c t s has b e c o m e a c o m m o n spoilage p r o b l e m in the l~'innish m e a t industry d u r i n g r e c e n t y e a r s ( K o r k e a l a et al., 1988). This ropiness has b e e n s h o w n by K o r k e a l a et al. (1988) to be c a u s e d by lactic acid b a c t e r i a c a p a b l e of p r o d u c i n g ropy slime. T h r e e different s l i m e - p r o d u c i n g lactic acid b a c t e r i a groups, two o f w h i c h w e r e lactobacilli a n d o n e o f w h i c h b e l o n g e d to the g e n u s L e u c o n o s t o c , w e r e isolated f r o m ropy m e a t p r o d u c t s . Subsequently, several o t h e r lactic acid b a c t e r i a strains c a p a b l e o f f o r m ing r o p y slime have b e e n isolated f r o m m e a t a n d various m e a t p r o d u c t s , o n e o f w h i c h is d e s c r i b e d by M~ikel~i a n d K o r k e a l a (1989).
Correspondence address: Dr Hannu Korkeala, Department of Food and Environmental Hygiene, College of Veterinary Medicine, P.O. Box 6, SF-00581 Helsinki, Finland. Phone, 358-0-3931702; Fax, 358-0-3931718. It should b¢ mentioned that publication of this manuscript have been delayed by reasons beyond the control of the authors.
168
Lactic acid bacteria are typically the major microbial group developing on vacuum-packed meat and meat products (Allen and Forster, 1960; Reuter, 1970b; Mol et al., 1971; Savell et al., 1981; Hitchener et al., 1982; Blickstad and Molin, 1983; Korkeala et al., 1987), and several recent studies have dealt with the identity of these lactic acid bacteria (Reuter, 1970a; Niemand and Holzapfel, 1984; Shaw and Harding, 1984; Holzapfel and Gerber, 1986; Morishita and Shiromizu, 1986; Hastings and Holzapfel, 1987a, b; Schillinger and Liicke, 1987; Borch and Molin, 1988; Korkeala and M~ikel~i, 1989). The ropy slime-producing bacterial strains of Korkeala et al. (1988) phenotypically resembled the lactic acid bacteria isolated from meat and meat products by other workers. The homofetmentative lactobacilli group 1 of Korkeala et al. (1988) was similar to group 2 of Hitchener et al. (1982) and to group S 4 / a of Morishita and Shiromizu (1986), while group 2 of Korkeala et al. (1988) appeared similar to group $ 6 / c of Morishita and Shiromizu (1986). The Leuconostoc group was identified as Leuconostoc mesenteroides using the scheme of Garvie (1979) and Sharpe (1979). The production of ropy slime caused by lactic acid bacteria in vacuum-packaged meat products is an interesting microbiological spoilage phenomenon, of considerable economic importance. More accurate information about the identity of these bacteria is therefore needed. In addition to the phenotypic characterization of the ropy slime-producing lactic acid bacteria strains concerned, their D N A homology was determined and used as basis for their taxonomy. Materials and Methods
Bacterial strains Strains A210, CI, D1, D2 and D3 have been isolated from ropy meat products by Korkeala et al. (1988). Strain A210 belonged to group 1, strain C1 t O group 2 and strains D1, D2 and I)3 formed group 3 of Korkeala et al. (1988). Strains A210 and CI were homofermentative lactobacilli and strains D1, D2 and D3 belonged to the genus Leuconostoc. The homofermentative Lactobacillus strain R51 was isolated from pork meat by Miikel/i and Korkeala (1989). Strains R152, R300 and R301 were isolated from cooked sausages. Strain R152 was a homofermentative Lactobacillus and strains R300 and R301 were leuconostocs. All strains studied were able to cause ropiness on vacuum-packed cooked meat products. Type strains of the following species were used as reference strains: Lactobacillus sake (DSM 20017), Leuconostoc mesenteroides subsp, mesenteroides (DSM 20343), Leuconostoc mesenteroides subsp, dextranicum (DSM 20484), Leuconostoc pseudomesenteroides (DSM 20193), Leuconostoc paramesenteroides (DSM 20288), Leuconostoc gelidum (NCFB 2775) and Leuconostoc carnosum (NCFB 2776). Additionally, Leuconostoc amelibiosum ATCC 10882, which is highly homologous to the type strain of this species, was used (Sehillinger et al., 1989). Culture conditions The bacterial strains were grown in MRS broth (Merck) at 30°C and were maintained on MRS agar slants at 4°C.
169 TABLE I Percentage of DNA-DNAhomologybetween roiayslime-producing LaclobaciUi strainsand reference strains Reference
strains
Laclobacitlus sake DSM 2iDI7 Lactobacillus sp. A210 Leuconostoc mesenwroides sabsp, mest'~teroides DSM 20343
Ropyslime-producingLacttdTacilli slrains A210 CI RSI 84 69 86 89 107
R152 90 ND
33
ND
ND
ND
ND, not done. DNA base composition and DNA-DNA hybridization DNA was prepared as described by Stackebrandt and Kandler (1979). The DNA base composition (mol% G + C) was estimated by the thermal denaturation method (Marmur and Dory, 1962). DNA homology values were determined from renaturation rates by using a Gilford spcctrophotometer (De Ley et al., 1970; Huss et al., 1983)
Results and Discussion DNA-DNA homology values between the slime-producing lactobacilli strains and the reference strains are presented in Table !. All the ropy slime producing lactobacilli strains sBzdied showed high DNA homology to the type strain of Lactobacillus sake. The mol% G + C content in the DNA of strains A210, Cl, R51 and R152 was determined as 43, 44, 43 and 45 mol%, respectively. The results for DNA-DNA homology between slime-producing leuconostoc strains and the reference strains are presented in Table II. Strains DI, D2 and D3 exhibited a very high DNA relatedness to Leuconostoc amelibiosum ATCC 10882 and strains R300 and R301 showed a high DNA homology to the type strains of Leuconostoc mesenteroides subsp, mesenteroides (DSM 20343) and Leueonostoc mesenteroides subsp, dextranicum (DSM 20484). The G + C content in the DNA of strains Dl, D2 and D3 was between 41 and 42 mol% and that of R300 and R301 was 41 and 40 mol%, respectively which corresponds to the values of 37-41% published by Garvie (1986) for Lawoaostoc mesenteroides. The tool% G + C of the DNA of the lactobacilli varied between 43 and 45, which is in correspondance with the values of 42-44 presented by Kandler and Weiss (1986) for Lactobacillus sake. The fatty acid composition of the cell walls of the strains A 210 and R 152 was also found to be very close to that of known L. sake strains (unpublished data). Strains of L. sake have been commonly found on vacuum-packod meat products, and they often form the predominant part of the lactic acid bacterial
70 TABLE II Percentage of DNA-DNAhomologybetween ropy slime-producingLeuconostoc strains and reference strains Reference~,Irains
DI
D2
D3
R300
R301
31
33
42
ND
39
95
ND
ND
105
96
97
~}
89
ND
ND
38
ND
ND
ND
ND
211 311 20
ND NO ND
ND ND ND
ND ND ND
ND ND ND
Leuconostoc mesenteroides subsp, meselrlcroides
DSM 2P343 Letlconoslo¢" rnest,nl~,roidt,s subsp, dextrunicum DSM 20484 Leuconostoc amelibk~sum ATCC 10882 Leuco~lostoc psetidotllesenlt, rokh, s DSM 20193 Lenconostoc paramesenteroides DSM 20288 Leucono.stoc geh~hun NCFB 2775 Leuconostoc carnosum NCFB 2776 ND. not done.
population (Holzapfel and Gerber, 1986; Champomier et al., 1987; Schillinger and Liicke, 19871. However, to our knowledge, the production of ropy slime in sausages without added sugars by Lactobacillus sake strains has not previously been reported. As one of the main causative agents of a highly offensive and commercially important spoilage phenomenon, Lactobacillus sake can thus be considered as a major spoilage bacterium in vacuum-packaged cooked meat products. The strains of group 3 (D1, D2 and D3) which had been tentatively identified as Leucolio~loc mesenterotdes on the basis of the API CH 50 Lactobacillus system (Korkeala eta[., 19881 were highly homologous, but exhibited only a low level of DNA-relatedness to the type strain of Leuconostoc mesenteroides. Similarly, there was no specific relationship to Leuconostoc carnosum and Leuconostoc gelidum, two newly described Leuconostoc species that seem to be frequently associated with chill-stored meats (Shaw and Harding, 1989). In contrast, strains D1, D2 and D3 exhibited high DNA-DNA homologies to Leuconostoc amelibiosum. To the authors' knowledge, strains of Leuconostoc amelibiosum have not previously been found associated with meat products. This, however, may be explained by the fact that this new species has only recently been described (Schillinger et al., 19891 This species may thus have significance as a spoilage bacterium on vacuum-packaged cooked sausages. The results demonstrate that phenotypic characterization, based on sugar fermentation patterns and conventional phenotypie properties may not always provide a sufficient basis for the reliable identification of Leuconostoc species. Strains of Leuconostoc mesenteroides and Leuconostoc amelibiosum, which may share the same habitat (meat products), show very similar sugar-fermentation abilities and both may produce ropy slime. Similarly, the numerical taxonomic study of Milliere et al. (19891, using 197 phenotypic characters, showed the difficulties in finding
171 p h e n o t y p i c c h a r a c t e r i s t i c s h e l p f u l f o r s t r a i n i d e n t i f i c a t i o n d o w n to s p e c i e s level. They therefore concluded that DNA-DNA hybridizations are needed for reliable classification of Leuconostoc strains. An absolutely reliable procedure for the rapid identification of Leuconostoc and Lactobacillus s t r a i n s f r o m m e a t a n d m e a t p r o d u c t s is n o t yet a v a i l a b l e . T h e u s e o f recombinant DNA technology for the development of genetic probes, however, m a y p r o v i d e a s a t i s f a c t o r y s o l u t i o n ( P c t r i c k e t al., 1988).
References Allen, J.F. and Forster, E.M. (19~O1. SImilage of vacuum-packed sliced prtv:essed meats during refrigerated storage. Food Res. 25, 19-25. Blickstad, E. and Molin, (3. (It183) The microbial flora of smoked la)rk loin and frankfurter sausage stored in different gas atmospheres at 4°C. J, Appl. Bacterial. 54, 45-5h. Borch, E. and Molin, G. (19881 Numerical taxonomy of ps3'chrotrophic lactic acid bacteria from prepacked meat and meat products. Anionic van Leeuwenhoc'k 54, 301-323. Champomier. M.-C.. Mantel M.-C.. Grimont, F. and Grimom, P.A.D. (1987) Genomle identification of meat lactobacilli as Lactobacillus sake. Ann. Inst. Pasteur Microbial. 138, 751-758. De Lcy, J., Cattoir, H. and Rcynaerts, A. (IqT0) The quantitative measurement of DNA hybridization from renaturation rates. Ear. J. Biochem. 12. 133-142. Garvi¢, E.I. (1979) Proposal on neotype strains for Leuconostoc mesenreroides (Tsenko~kii) van Tiegham, Leuconostoc dextranicum (Beijerinck) Hucker and Pederson. and Leueonosloc cremoris (Kundsen and Sorensen) Garvie. Int J. Syst. Baeteriol. 29, 149-151. Garvie. E.I. (19861 Genus Lenconosttx-. In: P.H.A. Sneath, r.!s. Mair, M.E. Sharpe and J.G, Holt (Eds.). lkrgcy's Manual of Systematic Bacteriology, Williams & Wilkins, Baltimore, MD. USA, pp. 1071- 1075. Hastings, J.W. and Holzapfel. W.H, oqgTa) Conventional taxonomy of lactobacilli surviving radurization of meat. J. Appl. Bacteriol. 62, 209-216. Hastings, J.W. and Holzapfel, W.H. (1987b) Numerical taxonomy of lactobacilli surviving radurization of meat. Int. J. Food Microbiol. 4. 33-49, Hitchener, B.J., Egan. A.F, and Rogers. P.J. (19821 Characteristics of lactic acid bacteria isolated from vacuum-packaged beef. J. Appl. Bacteriol. 52, 31-37. HolzapfeL W.H. and Gerber, E.S. (1986) Predominance of Lactol~lcilhLs curvams and Lactobacitlus sake in the spoilage ass~'miation of vacuum-packaged meat products. Abstracts of 32rid Europoan Meeting of Meat Research Workers, Ghent, Belgium, 26. Huss, V., Festl. H. and Schleifer, K.H. (19831 Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. System. Appl. Microbiol, 4, 184-192. Kandler, O. and Weiss, N. (19861 Genus Lact(dmcillus. In: P.H.A, Sheath, N.S, Malt, M.E. Shar0e and J.G. Holt (Eds.L Bergey's Manual of Systematic Bacteriology. Williams & Wilkins, Baltimore, MD, USA, pp. 1209-1234, Korkeala, H. and Miikelfi, P. (I 989) Characterization of lactic acid bacteria isolated from vacuum-packod cooked ring sausages. Int. J. Food Microbiol. q, 33-43. Korkeala, H., Lindroth, S. Ahvenainen. R. a~,d Alanko, T. (19871 Interrelationship between microbial numbers and other parameters in the spoilage of vacuum-packed cooked ring ~usages. Int, J. Food Microbiol. 5, 311-321. Korkeala. H., Suortti, T. and M~kel~i, P, (1988) Ropy slime formation in vacuum-packed cooked meat products caused by homofermentative laetobacilli and a Leneonostoc species. Int. J. Food Microbiol. 7, 339-347. M/ikelii, P. and Korkeala, H, (19891 A new Lacrobacillus strain capable of forming ropy slime on meat products. World Association of Veterinary Food Hygienists, Xth Symposium, Stockholm, Sweden, 2-7.7.1989, Abstracts of Posters, Appendix 2 to the Proceedings, p. 5.
172 Marmur. J. and Dory, P. (1962) Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J. Mol. Biol. 5, 109-118. Milliere, J.B., Mathot, A.-G., Schmitt, P. and Divies, C. (1989) Phcnotypic characterization of Leuconostoc species. J. Appl. Baeteriol. 67, 529-542. Mol, J.H.H., Hietbring, J.E.A.. Mollen, H.W.M. and van Tinteren, J. (1971) Observations on the microflora of vacuum packed sliced cooked meat products. J. Appl. Bacteriol. 34, 377-397. Morishita, Y. and Shiromizu, K. (1986) Characterization of lactobacilli isolated from meats and meat products. Int. J. Food Microbiol. 3, 19-29. Niemand, J.G. and Holzapf¢l, W.H. (1984) Characteristics of lactobacilli isolated from radurised meat. Int. J. Folxi Microbiol. I, 99-110. Petrick, tI.A.R., Ambrosio, R.E. and Holzapfel, W.H., (1988) Isolation of a DNA probe for Lactobacillus earl'alas. Appl. Environ. Microbiol., 54, No. 2, 405-408. R.eutcr, (3. (1970a) Laktobazillen and eng verwandte Mikroorgaaismcn in Fleisch uud Fleischerzcugni~ sun. 2. Mitteilung: Die Charakterisierung dcr isolierten Laktobazillenst[imme. (Lactobacilli and closely related micro-organisms in meat and meat products. 2. Characterisation of isolated lactobacilli strains). Fleischwirtschaft 50, 954-962. Reuter, G. (1970b) Untcrsuchungen zur Mikroflora yon vorverpackten, aufgeschnittenen Briih. und Kochwiirstcu. (I,lvcstigations on microflora in prcpacked sliced cooked sausages and sausages of bologna type). Arch. Lebe.;smittelhyg. 21. 257-264. Savell, J., Hanna, M.O., Vanderzant, C. and Smith. G.C. (1981) An incident of predominance of Leuconostoc sp. in vacuum-packaged beef strip loins: sensory and microbial profile of steaks stored in O ~ - C O 2 - N 2 athmospheres. J. Food Prof. 44, 742-745. Schillinger. U. and Liicke, F.-K. (1987) Lactic acid bacteria on vacuum-packaged meat and their influence on shelf life. Fleischwirlschaft 07, 1244-1249. Schillinger, U., Holzapfel, W. and Kandler, O. (1989) Nucleic acid studies on Leuconostoc and heteroferrnenlative lactobacilli and description of Leuconostoc amelibiosum sp. nov. System. Appl. Microhiol. 12, 48-55. Sharpe, M.E. (1979) Identification of the lactic acid bacteria. In: F.A. Skinner and D.W. Lo;'elock (Eds.), Identification Methods for Microbiologists. Academic Press, London, pp. 233-259. 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. Bacleriol. 56, 24-40. Shaw, B.G. and tia~ding, C.D. (1989) Leuconost¢u" gelidum sp. nov. and Leuconostoc carnosum sp, nov. from chill-stored meats. Int. J. System. Bactcriol. 39, 217-223. Stackebrandt, E. and Kandler, O. (1979) Taxonomy of the gel~us Ccllulomonas, based on phenotypic characters and deoxyribonucleic acid-deoxyribonucleic acid homology and proposal of seven ncotypc strains. Int. J. System. Bactcriol. 29, 273-282.
(Contents continued from back cover) C.-H. Ryu, S. Igimi, S, Inoue and S. Kumagai (Japan) The incidence of Listeria species in retail foods in Japan
157
P.M, M~kel~i and H.J, Korkeala (Finland) The ability of the ropy slime-producing lactic acid bacteria to form ropy colonies on different culture media and at different incubation temperatures and atmospheres 161 P. M&kel~, U. Schillinger, H. Korkeala and W.H. Holzapfel (Finland, Germany) Classification of ropy slime-producing lactic acid bacteria based on DNA-DNA homology, and identification of Lactobacillus sake and Leuconostoc amelibiosum as dominant spoilage organisms in meat products 167
167
FOOD ID515
Short communication
Classification of ropy slime-producing lactic acid bacteria based on D N A - D N A homology, and identification of Lactobacillus sake and Leuconostoc amelibiosum as dominant spoilage organisms in meat products P. M~ikel~i a, U. Schillinger b, H. Korkeala a and W.H. Holzapfel b '~ Deparmwnt of Food and Eneiromnental Hygiene. Cidlege of Veterinary Medicine, Helsinki. Finland and b blstilute of Hygiene and Toxieology. Federal Research Centre for Nutritim¢. garlsruhe. German).
(Received 8 May 1991;accepted 19 April Iq~2)
The classification of lactic acid bacteria able to cause ropy slime on vacuum-packed cooked meat products was carried out based on DNA-DNA homology. The ropy slime-producing lactoba¢ill[ wcrc identified as strains of Lactobacillus sake and the ropy slime-prc,ducing leuconostocs, such as Leucono~. toc amelibiosum and Leuconosto¢ mesenteroides. Key words: Identification; Taxonomy; DNA base composition; DNA homology; Lactobacillas sake; Leueoaostoc amelihiosnnl; Leut~mostoc me~,enteroides: Ropiness: Meat products, vacuumpacked
Introduction T h e f o r m a t i o n o f ropy slime o n v a c u u m - p a c k e d m e a t p r o d u c t s has b e c o m e a c o m m o n spoilage p r o b l e m in the l~'innish m e a t industry d u r i n g r e c e n t y e a r s ( K o r k e a l a et al., 1988). This ropiness has b e e n s h o w n by K o r k e a l a et al. (1988) to be c a u s e d by lactic acid b a c t e r i a c a p a b l e of p r o d u c i n g ropy slime. T h r e e different s l i m e - p r o d u c i n g lactic acid b a c t e r i a groups, two o f w h i c h w e r e lactobacilli a n d o n e o f w h i c h b e l o n g e d to the g e n u s L e u c o n o s t o c , w e r e isolated f r o m ropy m e a t p r o d u c t s . Subsequently, several o t h e r lactic acid b a c t e r i a strains c a p a b l e o f f o r m ing r o p y slime have b e e n isolated f r o m m e a t a n d various m e a t p r o d u c t s , o n e o f w h i c h is d e s c r i b e d by M~ikel~i a n d K o r k e a l a (1989).
Correspondence address: Dr Hannu Korkeala, Department of Food and Environmental Hygiene, College of Veterinary Medicine, P.O. Box 6, SF-00581 Helsinki, Finland. Phone, 358-0-3931702; Fax, 358-0-3931718. It should b¢ mentioned that publication of this manuscript have been delayed by reasons beyond the control of the authors.
168
Lactic acid bacteria are typically the major microbial group developing on vacuum-packed meat and meat products (Allen and Forster, 1960; Reuter, 1970b; Mol et al., 1971; Savell et al., 1981; Hitchener et al., 1982; Blickstad and Molin, 1983; Korkeala et al., 1987), and several recent studies have dealt with the identity of these lactic acid bacteria (Reuter, 1970a; Niemand and Holzapfel, 1984; Shaw and Harding, 1984; Holzapfel and Gerber, 1986; Morishita and Shiromizu, 1986; Hastings and Holzapfel, 1987a, b; Schillinger and Liicke, 1987; Borch and Molin, 1988; Korkeala and M~ikel~i, 1989). The ropy slime-producing bacterial strains of Korkeala et al. (1988) phenotypically resembled the lactic acid bacteria isolated from meat and meat products by other workers. The homofetmentative lactobacilli group 1 of Korkeala et al. (1988) was similar to group 2 of Hitchener et al. (1982) and to group S 4 / a of Morishita and Shiromizu (1986), while group 2 of Korkeala et al. (1988) appeared similar to group $ 6 / c of Morishita and Shiromizu (1986). The Leuconostoc group was identified as Leuconostoc mesenteroides using the scheme of Garvie (1979) and Sharpe (1979). The production of ropy slime caused by lactic acid bacteria in vacuum-packaged meat products is an interesting microbiological spoilage phenomenon, of considerable economic importance. More accurate information about the identity of these bacteria is therefore needed. In addition to the phenotypic characterization of the ropy slime-producing lactic acid bacteria strains concerned, their D N A homology was determined and used as basis for their taxonomy. Materials and Methods
Bacterial strains Strains A210, CI, D1, D2 and D3 have been isolated from ropy meat products by Korkeala et al. (1988). Strain A210 belonged to group 1, strain C1 t O group 2 and strains D1, D2 and I)3 formed group 3 of Korkeala et al. (1988). Strains A210 and CI were homofermentative lactobacilli and strains D1, D2 and D3 belonged to the genus Leuconostoc. The homofermentative Lactobacillus strain R51 was isolated from pork meat by Miikel/i and Korkeala (1989). Strains R152, R300 and R301 were isolated from cooked sausages. Strain R152 was a homofermentative Lactobacillus and strains R300 and R301 were leuconostocs. All strains studied were able to cause ropiness on vacuum-packed cooked meat products. Type strains of the following species were used as reference strains: Lactobacillus sake (DSM 20017), Leuconostoc mesenteroides subsp, mesenteroides (DSM 20343), Leuconostoc mesenteroides subsp, dextranicum (DSM 20484), Leuconostoc pseudomesenteroides (DSM 20193), Leuconostoc paramesenteroides (DSM 20288), Leuconostoc gelidum (NCFB 2775) and Leuconostoc carnosum (NCFB 2776). Additionally, Leuconostoc amelibiosum ATCC 10882, which is highly homologous to the type strain of this species, was used (Sehillinger et al., 1989). Culture conditions The bacterial strains were grown in MRS broth (Merck) at 30°C and were maintained on MRS agar slants at 4°C.
169 TABLE I Percentage of DNA-DNAhomologybetween roiayslime-producing LaclobaciUi strainsand reference strains Reference
strains
Laclobacitlus sake DSM 2iDI7 Lactobacillus sp. A210 Leuconostoc mesenwroides sabsp, mest'~teroides DSM 20343
Ropyslime-producingLacttdTacilli slrains A210 CI RSI 84 69 86 89 107
R152 90 ND
33
ND
ND
ND
ND, not done. DNA base composition and DNA-DNA hybridization DNA was prepared as described by Stackebrandt and Kandler (1979). The DNA base composition (mol% G + C) was estimated by the thermal denaturation method (Marmur and Dory, 1962). DNA homology values were determined from renaturation rates by using a Gilford spcctrophotometer (De Ley et al., 1970; Huss et al., 1983)
Results and Discussion DNA-DNA homology values between the slime-producing lactobacilli strains and the reference strains are presented in Table !. All the ropy slime producing lactobacilli strains sBzdied showed high DNA homology to the type strain of Lactobacillus sake. The mol% G + C content in the DNA of strains A210, Cl, R51 and R152 was determined as 43, 44, 43 and 45 mol%, respectively. The results for DNA-DNA homology between slime-producing leuconostoc strains and the reference strains are presented in Table II. Strains DI, D2 and D3 exhibited a very high DNA relatedness to Leuconostoc amelibiosum ATCC 10882 and strains R300 and R301 showed a high DNA homology to the type strains of Leuconostoc mesenteroides subsp, mesenteroides (DSM 20343) and Leueonostoc mesenteroides subsp, dextranicum (DSM 20484). The G + C content in the DNA of strains Dl, D2 and D3 was between 41 and 42 mol% and that of R300 and R301 was 41 and 40 mol%, respectively which corresponds to the values of 37-41% published by Garvie (1986) for Lawoaostoc mesenteroides. The tool% G + C of the DNA of the lactobacilli varied between 43 and 45, which is in correspondance with the values of 42-44 presented by Kandler and Weiss (1986) for Lactobacillus sake. The fatty acid composition of the cell walls of the strains A 210 and R 152 was also found to be very close to that of known L. sake strains (unpublished data). Strains of L. sake have been commonly found on vacuum-packod meat products, and they often form the predominant part of the lactic acid bacterial
70 TABLE II Percentage of DNA-DNAhomologybetween ropy slime-producingLeuconostoc strains and reference strains Reference~,Irains
DI
D2
D3
R300
R301
31
33
42
ND
39
95
ND
ND
105
96
97
~}
89
ND
ND
38
ND
ND
ND
ND
211 311 20
ND NO ND
ND ND ND
ND ND ND
ND ND ND
Leuconostoc mesenteroides subsp, meselrlcroides
DSM 2P343 Letlconoslo¢" rnest,nl~,roidt,s subsp, dextrunicum DSM 20484 Leuconostoc amelibk~sum ATCC 10882 Leuco~lostoc psetidotllesenlt, rokh, s DSM 20193 Lenconostoc paramesenteroides DSM 20288 Leucono.stoc geh~hun NCFB 2775 Leuconostoc carnosum NCFB 2776 ND. not done.
population (Holzapfel and Gerber, 1986; Champomier et al., 1987; Schillinger and Liicke, 19871. However, to our knowledge, the production of ropy slime in sausages without added sugars by Lactobacillus sake strains has not previously been reported. As one of the main causative agents of a highly offensive and commercially important spoilage phenomenon, Lactobacillus sake can thus be considered as a major spoilage bacterium in vacuum-packaged cooked meat products. The strains of group 3 (D1, D2 and D3) which had been tentatively identified as Leucolio~loc mesenterotdes on the basis of the API CH 50 Lactobacillus system (Korkeala eta[., 19881 were highly homologous, but exhibited only a low level of DNA-relatedness to the type strain of Leuconostoc mesenteroides. Similarly, there was no specific relationship to Leuconostoc carnosum and Leuconostoc gelidum, two newly described Leuconostoc species that seem to be frequently associated with chill-stored meats (Shaw and Harding, 1989). In contrast, strains D1, D2 and D3 exhibited high DNA-DNA homologies to Leuconostoc amelibiosum. To the authors' knowledge, strains of Leuconostoc amelibiosum have not previously been found associated with meat products. This, however, may be explained by the fact that this new species has only recently been described (Schillinger et al., 19891 This species may thus have significance as a spoilage bacterium on vacuum-packaged cooked sausages. The results demonstrate that phenotypic characterization, based on sugar fermentation patterns and conventional phenotypie properties may not always provide a sufficient basis for the reliable identification of Leuconostoc species. Strains of Leuconostoc mesenteroides and Leuconostoc amelibiosum, which may share the same habitat (meat products), show very similar sugar-fermentation abilities and both may produce ropy slime. Similarly, the numerical taxonomic study of Milliere et al. (19891, using 197 phenotypic characters, showed the difficulties in finding
171 p h e n o t y p i c c h a r a c t e r i s t i c s h e l p f u l f o r s t r a i n i d e n t i f i c a t i o n d o w n to s p e c i e s level. They therefore concluded that DNA-DNA hybridizations are needed for reliable classification of Leuconostoc strains. An absolutely reliable procedure for the rapid identification of Leuconostoc and Lactobacillus s t r a i n s f r o m m e a t a n d m e a t p r o d u c t s is n o t yet a v a i l a b l e . T h e u s e o f recombinant DNA technology for the development of genetic probes, however, m a y p r o v i d e a s a t i s f a c t o r y s o l u t i o n ( P c t r i c k e t al., 1988).
References Allen, J.F. and Forster, E.M. (19~O1. SImilage of vacuum-packed sliced prtv:essed meats during refrigerated storage. Food Res. 25, 19-25. Blickstad, E. and Molin, (3. (It183) The microbial flora of smoked la)rk loin and frankfurter sausage stored in different gas atmospheres at 4°C. J, Appl. Bacterial. 54, 45-5h. Borch, E. and Molin, G. (19881 Numerical taxonomy of ps3'chrotrophic lactic acid bacteria from prepacked meat and meat products. Anionic van Leeuwenhoc'k 54, 301-323. Champomier. M.-C.. Mantel M.-C.. Grimont, F. and Grimom, P.A.D. (1987) Genomle identification of meat lactobacilli as Lactobacillus sake. Ann. Inst. Pasteur Microbial. 138, 751-758. De Lcy, J., Cattoir, H. and Rcynaerts, A. (IqT0) The quantitative measurement of DNA hybridization from renaturation rates. Ear. J. Biochem. 12. 133-142. Garvi¢, E.I. (1979) Proposal on neotype strains for Leuconostoc mesenreroides (Tsenko~kii) van Tiegham, Leuconostoc dextranicum (Beijerinck) Hucker and Pederson. and Leueonosloc cremoris (Kundsen and Sorensen) Garvie. Int J. Syst. Baeteriol. 29, 149-151. Garvie. E.I. (19861 Genus Lenconosttx-. In: P.H.A. Sneath, r.!s. Mair, M.E. Sharpe and J.G, Holt (Eds.). lkrgcy's Manual of Systematic Bacteriology, Williams & Wilkins, Baltimore, MD. USA, pp. 1071- 1075. Hastings, J.W. and Holzapfel. W.H, oqgTa) Conventional taxonomy of lactobacilli surviving radurization of meat. J. Appl. Bacteriol. 62, 209-216. Hastings, J.W. and Holzapfel, W.H. (1987b) Numerical taxonomy of lactobacilli surviving radurization of meat. Int. J. Food Microbiol. 4. 33-49, Hitchener, B.J., Egan. A.F, and Rogers. P.J. (19821 Characteristics of lactic acid bacteria isolated from vacuum-packaged beef. J. Appl. Bacteriol. 52, 31-37. HolzapfeL W.H. and Gerber, E.S. (1986) Predominance of Lactol~lcilhLs curvams and Lactobacitlus sake in the spoilage ass~'miation of vacuum-packaged meat products. Abstracts of 32rid Europoan Meeting of Meat Research Workers, Ghent, Belgium, 26. Huss, V., Festl. H. and Schleifer, K.H. (19831 Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. System. Appl. Microbiol, 4, 184-192. Kandler, O. and Weiss, N. (19861 Genus Lact(dmcillus. In: P.H.A, Sheath, N.S, Malt, M.E. Shar0e and J.G. Holt (Eds.L Bergey's Manual of Systematic Bacteriology. Williams & Wilkins, Baltimore, MD, USA, pp. 1209-1234, Korkeala, H. and Miikelfi, P. (I 989) Characterization of lactic acid bacteria isolated from vacuum-packod cooked ring sausages. Int. J. Food Microbiol. q, 33-43. Korkeala, H., Lindroth, S. Ahvenainen. R. a~,d Alanko, T. (19871 Interrelationship between microbial numbers and other parameters in the spoilage of vacuum-packed cooked ring ~usages. Int, J. Food Microbiol. 5, 311-321. Korkeala. H., Suortti, T. and M~kel~i, P, (1988) Ropy slime formation in vacuum-packed cooked meat products caused by homofermentative laetobacilli and a Leneonostoc species. Int. J. Food Microbiol. 7, 339-347. M/ikelii, P. and Korkeala, H, (19891 A new Lacrobacillus strain capable of forming ropy slime on meat products. World Association of Veterinary Food Hygienists, Xth Symposium, Stockholm, Sweden, 2-7.7.1989, Abstracts of Posters, Appendix 2 to the Proceedings, p. 5.
172 Marmur. J. and Dory, P. (1962) Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J. Mol. Biol. 5, 109-118. Milliere, J.B., Mathot, A.-G., Schmitt, P. and Divies, C. (1989) Phcnotypic characterization of Leuconostoc species. J. Appl. Baeteriol. 67, 529-542. Mol, J.H.H., Hietbring, J.E.A.. Mollen, H.W.M. and van Tinteren, J. (1971) Observations on the microflora of vacuum packed sliced cooked meat products. J. Appl. Bacteriol. 34, 377-397. Morishita, Y. and Shiromizu, K. (1986) Characterization of lactobacilli isolated from meats and meat products. Int. J. Food Microbiol. 3, 19-29. Niemand, J.G. and Holzapf¢l, W.H. (1984) Characteristics of lactobacilli isolated from radurised meat. Int. J. Folxi Microbiol. I, 99-110. Petrick, tI.A.R., Ambrosio, R.E. and Holzapfel, W.H., (1988) Isolation of a DNA probe for Lactobacillus earl'alas. Appl. Environ. Microbiol., 54, No. 2, 405-408. R.eutcr, (3. (1970a) Laktobazillen and eng verwandte Mikroorgaaismcn in Fleisch uud Fleischerzcugni~ sun. 2. Mitteilung: Die Charakterisierung dcr isolierten Laktobazillenst[imme. (Lactobacilli and closely related micro-organisms in meat and meat products. 2. Characterisation of isolated lactobacilli strains). Fleischwirtschaft 50, 954-962. Reuter, G. (1970b) Untcrsuchungen zur Mikroflora yon vorverpackten, aufgeschnittenen Briih. und Kochwiirstcu. (I,lvcstigations on microflora in prcpacked sliced cooked sausages and sausages of bologna type). Arch. Lebe.;smittelhyg. 21. 257-264. Savell, J., Hanna, M.O., Vanderzant, C. and Smith. G.C. (1981) An incident of predominance of Leuconostoc sp. in vacuum-packaged beef strip loins: sensory and microbial profile of steaks stored in O ~ - C O 2 - N 2 athmospheres. J. Food Prof. 44, 742-745. Schillinger. U. and Liicke, F.-K. (1987) Lactic acid bacteria on vacuum-packaged meat and their influence on shelf life. Fleischwirlschaft 07, 1244-1249. Schillinger, U., Holzapfel, W. and Kandler, O. (1989) Nucleic acid studies on Leuconostoc and heteroferrnenlative lactobacilli and description of Leuconostoc amelibiosum sp. nov. System. Appl. Microhiol. 12, 48-55. Sharpe, M.E. (1979) Identification of the lactic acid bacteria. In: F.A. Skinner and D.W. Lo;'elock (Eds.), Identification Methods for Microbiologists. Academic Press, London, pp. 233-259. 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. Bacleriol. 56, 24-40. Shaw, B.G. and tia~ding, C.D. (1989) Leuconost¢u" gelidum sp. nov. and Leuconostoc carnosum sp, nov. from chill-stored meats. Int. J. System. Bactcriol. 39, 217-223. Stackebrandt, E. and Kandler, O. (1979) Taxonomy of the gel~us Ccllulomonas, based on phenotypic characters and deoxyribonucleic acid-deoxyribonucleic acid homology and proposal of seven ncotypc strains. Int. J. System. Bactcriol. 29, 273-282.
(Contents continued from back cover) C.-H. Ryu, S. Igimi, S, Inoue and S. Kumagai (Japan) The incidence of Listeria species in retail foods in Japan
157
P.M, M~kel~i and H.J, Korkeala (Finland) The ability of the ropy slime-producing lactic acid bacteria to form ropy colonies on different culture media and at different incubation temperatures and atmospheres 161 P. M&kel~, U. Schillinger, H. Korkeala and W.H. Holzapfel (Finland, Germany) Classification of ropy slime-producing lactic acid bacteria based on DNA-DNA homology, and identification of Lactobacillus sake and Leuconostoc amelibiosum as dominant spoilage organisms in meat products 167
