Development of an immunochemical method to detect Lactobacillus kefir

Abstract Kefir is a fermented milk elaborated with kefir grains. It constitutes an ecological system composed by a protein-polysaccharide matrix on which a complex microbiota is naturally immobilized. The objective of this work was to develop an antibody-based method in order to achieve a simple and rapid way to quantify Lactobacillus kefir in a complex environment. Rabbit antisera against isolated S-layer proteins from L. kefir and L. parakefir were obtained. Both antisera recognized the 21 strains of L. kefir and the three strains of L. parakefir analysed by dot-blot. By indirect ELISA, specific antiserum against L. kefir S-layer showed reactivity against pure cultures of both L. kefir and L. parakefir, but did not react with L. brevis cells. Competitive ELISA allowed quantification of L. kefir at concentrations ranging from 5×105 to 108 bacteria ml−1, but neither L. parakefir nor L. brevis pure cultures produced inhibition. Through this approach it was possible to detect 108 L. kefir ml−1 in samples of milk fermented for 48 h at 20°C with different kefir grains. Competitive ELISA developed in our laboratory is a suitable method to detect and quantify L. kefir in milk and in consequence it could be applied to analyse the relationship between the composition of microflora and probiotic and technological characteristics of the products.

[1]  R. Bibiloni,et al.  Lactobacilli isolated from kefir grains: evidence of the presence of S-layer proteins , 2004, Journal of Dairy Research.

[2]  G. Antoni,et al.  Chemical and microbiological characterisation of kefir grains , 2001, Journal of Dairy Research.

[3]  G. L. Garrote,et al.  Inhibitory power of kefir: the role of organic acids. , 2000, Journal of food protection.

[4]  U. Sleytr,et al.  S-Layer Proteins , 2000, Journal of bacteriology.

[5]  K. Watanabe,et al.  Survival of a probiotic, Lactobacillus casei strain Shirota, in the gastrointestinal tract: selective isolation from faeces and identification using monoclonal antibodies. , 1999, International journal of food microbiology.

[6]  G. Antoni,et al.  Characteristics of kefir prepared with different grain[ratio ]milk ratios , 1998, Journal of Dairy Research.

[7]  S. Ricke,et al.  Detection and quantification of poultry probiotic bacteria in mixed culture using monoclonal antibodies in an enzyme-linked immunosorbent assay. , 1997, International journal of food microbiology.

[8]  R. P. Ross,et al.  An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147 , 1996, Applied and environmental microbiology.

[9]  T. Yasui,et al.  Analysis of S-layer proteins of Lactobacillus brevis. , 1995, FEMS microbiology letters.

[10]  N. Kalkkinen,et al.  A Collagen-Binding S-Layer Protein in Lactobacillus crispatus , 1995, Applied and environmental microbiology.

[11]  P. G. Sarra,et al.  Competitive exclusion of Salmonella kedougou in kefir fed chicks , 1994 .

[12]  V. Marshall.  Starter cultures for milk fermentation and their characteristics , 1993 .

[13]  L. Angulo,et al.  Microflora present in kefir grains of the Galician region (North-West of Spain) , 1993, Journal of Dairy Research.

[14]  L. Nuotio,et al.  Adhesion of Lactobacillus acidophilus to avian intestinal epithelial cells mediated by the crystalline bacterial cell surface layer (S-layer). , 1993, The Journal of applied bacteriology.

[15]  S. Ricke,et al.  Characterization of Egg Yolk Antibodies for Detection and Quantification of Selenomonas ruminantium by Using an Enzyme-Linked Immunosorbent Assay , 1990, Applied and environmental microbiology.

[16]  D. Pum,et al.  Crystalline Bacterial Cell Surface Layers , 1988, Springer Berlin Heidelberg.

[17]  K. H. Kang,et al.  Differentiation of ruminal bacterial species by enzyme-linked immunosorbent assay using egg yolk antibodies from immunized chicken hens , 1988, Applied and environmental microbiology.

[18]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[19]  M. Rogosa,et al.  A MEDIUM FOR THE CULTIVATION OF LACTOBACILLI , 1960 .

[20]  U. Sleytr,et al.  Characterization of the S-Layer Glycoproteins of Two Lactobacilli , 1993 .

[21]  Juan A. Torres,et al.  Producción y aspectos fisicoquímicos de un hidrocoloide obtenido por fermentación en sistema modelo y suero ácido de leche con Lactobacillus plantarum , 1991 .

[22]  H. Otani,et al.  Binding properties of lactic acid bacteria isolated from kefir milk with mutagenic amino acid pyrolyzates , 1990 .

[23]  A. Zourari,et al.  Le kéfir. Caractères physico-chimiques, microbiologiques et nutritionnels. Technologie de production. Une revue , 1988 .

[24]  P. Tijssen,et al.  Practice and theory of enzyme immunoassays , 1985 .

[25]  V. Tsang,et al.  Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. , 1983, Methods in enzymology.