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Arquivo Brasileiro de Medicina Veterinária e Zootecnia

Print version ISSN 0102-0935On-line version ISSN 1678-4162

Arq. Bras. Med. Vet. Zootec. vol.54 no.6 Belo Horizonte Dec. 2002 





Identification of meat isolated bacteriocin-producing lactic acid bacteria using biotyping and ribotyping


[Identificação de bactérias láticas isoladas de produtos cárneos por meio de testes bioquímicos e ribotipagem]



E.C.P. De Martinis

Faculdade de Ciências Farmacêuticas de Ribeirão Preto da USP

Address for correspondence



Lactic acid bacteria (LAB) predominate the natural microflora of many foods where they serve as preservative or spoilage role. With the development of more sophisticated starter and biopreservative systems for an increasing number of foods, it is important to precisely identify lactic acid bacteria strains intended to be used in food systems (Stiles & Holzapfel, 1997). Schillinger & Lücke (1987) proposed identification schemes primarily based on differences in sugar fermentation patterns and other easily determinable physiological characteristics, allowing a rapid and simple identification of lactobacilli from meat and meat products.

Biochemical tests are very useful for a first assignment of lactic acid bacteria isolates, but for a more precise identification, methods based on molecular biology are recommended (Morishita & Shiromizu, 1986; Schillinger & Lücke, 1987). Ribotyping is a method that can identify and classify bacteria based upon differences in ribosomal RNA (rRNA). This technique generates a highly reproducible and precise fingerprint that can be used to classify bacteria beyond species level. Briefly, DNA is extracted from a colony of bacteria and then restricted into discrete-sized fragments. The DNA is then transferred to a membrane and probed with a region of the rRNA operon to reveal the pattern of rRNA genes. The patterns are compared in a database for identification of unknown strains (What…, [2000]).

This work was carried out with three strains of bacteriocinogenic lactic acid bacteria previoulsy isolated by De Martinis et al. (2001): isolate 1 (from pork sausage), isolate 5 (from hot dog) and isolate 16 (from bacon). They were maintained in MRS broth (Oxoid) at - 70oC with 20% glycerol. Cell morphology, carbohydrate fermentation pattern and gas production from glucose were previously reported (De Martinis et al., 2001). Additional tests proposed by Schillinger & Lücke (1987) were used for these strains: dextran production from sucrose, arginine hydrolysis, determination of enantiomeric form of lactic acid produced, acetoin and hydrogen sulfide production, growth in different pHs, temperature and sodium chloride levels.

Comparative analysis of 16S rRNA of strains 1, 5 and 16 was performed by Laboratory for Molecular Typing, Cornell University, USA, using RiboPrinterâ Microbial Characterization System (Qualicon - Du Pont).

De Martinis et al. (2001) could not identify strain 1 using only the API 50CH databank from bioMeriéux. However, in this work, it could be identified by complementing API 50 CH biochemical reactions with the tests proposed by Schillinger & Lücke (1987). The results were used to manually classify isolate 1. Strain 1 was a homofermentative rod, able to grow at 15oC, and at 8oC, produced isomers D and L of lactic acid and fermented ribose and melibiose but not mannitol. According to these characteristics, strain 1 was classified as Lactobacillus sake. This species is not in API 50 CH database which may explain why De Martinis et al. (2001) were unable to identify this strain. Morishita & Shiromizu (1986) also reported difficulties for identification of homofermentative lactic acid bacteria only on the basis of carbohydrate fermentation pattern.

Isolate 5 had been identified as Lactobacillus curvatus using the API 50 CH database (De Martinis et al., 2001). This identification was confirmed using the scheme proposed by Schillinger & Lücke (1987). Strain 5 was a Gram positive, homofermenter, grew at 8oC and 15oC, produced D and L lactate, and fermented ribose and maltose but not mannitol or melibiose.

Isolate 16 was previously identified as Lactobacillus curvatus using API 50 CH, and it was confirmed using the identification key of Schillinger & Lücke (1987). They were homofermentative rods, that grew at 8oC and 15oC, did not forme D and L lactate, fermented sucrose and ribose but did not ferment mannitol, melibiose, maltose or trehalose.

Ribotype patterns of strain 1 and 16 were similar to ribotype reference pattern DUP-4033, allowing their identification as Lactobacillus sake. The ribotype pattern of strain 5 was similar to ribotype pattern DUP-5019, and it was identified as Lactobacillus curvatus.

In conclusion, it is unsatisfactory to consider carbohydrate fermentation patterns as the only criteria for bacteria identification, because variable fermentations often occur. Biochemical tests are very useful for a preliminary identification of lactic acid bacteria but the readings may be subjective and techniques based on molecular biology are much less subject to error, and yield more rapid and accurate results. Therefore, the later are recommended for a conclusive identification of lactic acid bacteria from meats (Montel et al., 1991, Stiles & Holzapfel, 1997).

Keywords: Lactic acid bacteria, identification, meat


Três cepas de bactérias láticas previamente isoladas a partir de produtos cárneos foram caracterizadas comparando-se resultados de testes bioquímicos e ribotipagem. Os resultados obtidos foram concordantes para Lactobacillus sake 1 e Lactobacillus curvatus 5. A cepa 16 foi identificada como Lactobacillus curvatus por testes bioquímicos e como Lactobacillus sake por ribotipagem, demonstrando a diferença entre as técnicas utilizadas. Recomenda-se, no momento, a técnica de ribotipagem para a identificação de bactérias láticas isoladas de carnes.

Palavras-chave: Bactérias láticas, identificação, carnes



The author is grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo for financial support (Proc. 98/10759-2; 00/01458-0). The author also thank Prof. Thomas J. Montville, PhD (Rutgers, The State University of New Jersey, USA), for facilitating the ribotyping analysis and to Cláudia M. Rosa, PhD, for her help with maintenance of LAB strains.



What is ribotyping? Cornell: Cornell University, [2000]. Disponível em: [2000]        [ Links ]

DE MARTINIS, E.C.P.; PÚBLIO, M.R.P.; SANTAROSA, P.R. et al. Antilisterial activity of lactic acid bacteria isolated from vacuum-packaged Brazilian meat and meat products. Braz. J. Microbiol., v.32, p.32-37, 2001.        [ Links ]

MONTEL, M.C.; TALON, R.; FORNAUD, J. et al. A simplified key for identifying homofermentative Lactobacillus and Carnobacterium spp. from meat. J. Appl. Bacteriol., v.70, p.469-472, 1991.        [ Links ]

MORISHITA, Y.; SHIROMIZU, K. Characterization of lactobacilli from meats and meat products. Int. J. Food Microbiol., v.53, p.2683-2685, 1986.        [ Links ]

SCHILLINGER, U.; LÜCKE, F.-K. Identification of lactobacilli from meat and meat products. Food Microbiol., v.4, p.199-208, 1987.        [ Links ]

STILES, M.E.; HOLZAPFEL, W.H. Lactic acid bacteria of foods and their current taxonomy. Int. J. Food Microbiol., v.36, p.1-29, 1997.        [ Links ]



Address for correspondence
E.C.P. De

Avenida do Café, s/n
14040-903 - Ribeirão Preto, SP

Recebido para publicação em 30 de janeiro de 2002
Recebido para publicação, após modificações, em 9 de setembro de 2002

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