Rapid Identification of Bifidobacteria in Dairy Products by Gene-targeted Species-specific PCR Technique and DGGE

Wei-Shung Hong,Ming-Ju Chen

doi:10.5713/ajas.2007.1887

Abstract

In this paper, a rapid and reliable gene-targeted species-specific polymerase chain reaction (PCR) technique based on a two-step process was established to identify bifidobacteria in dairy products. The first step was the PCR assay for genus Bifidobacterium with genus specific primers followed by the second step, which identified the species level with species-specific primer mixtures. Ten specific primer pairs, designed from nucleotide sequences of the 16-23S rRNA region, were developed for the Bifidobacterium species including B. angulatum, B. animalis, B. bifidum, B. breve, B. catenulatum, B. infantis, B. longum, B. minimum, B. subtile, and B. thermophilum. This technique was applied to the identification of Bifidobacterium species isolated from 6 probiotic products, and four different Bifidobacterium spp. (B. bifidum, B. longum, B. infantis, and B. breve) were identified. The findings indicated that the 16S-23S rDNA gene-targeted species-specific PCR technique is a simple and reliable method for identification of bifidobacteria in probiotic products. PCR combined with Denaturing Gradient Gel Electrophoresis (DGGE) for identification of the bifidobacteria was also evaluated and compared with the gene-targeted species-specific technique. Results indicated that for fermented milk products consistency was found for both species-specific PCR and PCR-DGGE in detecting species. However, in some lyophilized products, the bands corresponding to these species were not visualized in the DGGE profile but the specific PCR gave a positive result.

Highlights

Members of the genus Bifidobacterium are Grampositive, pleomorphic and strictly anaerobic bacteria, and major constituents of the human intestinal microflora as well as of other warm-blooded animals
Many attempts have been made to increase the number of Bifidobacterium cells in the intestinal tract by supplying certain bifidobacteria strains or food ingredients that stimulate the growth of bifidobacteria (Vaughn and Mollet, 1999; Chen et al, 2004; Chen et al, 2005; Chen et al, 2006)
Results demonstrated that a polymerase chain reaction (PCR) product was obtained The presence of Bifidobacterium, declared on the labels from all 6 probiotic products with 1,427 bp in size

Summary

INTRODUCTION

Members of the genus Bifidobacterium are Grampositive, pleomorphic and strictly anaerobic bacteria, and major constituents of the human intestinal microflora as well as of other warm-blooded animals. Detecting and identifying various species of bifidobacteria with rapid method is often important for quality control of probiotic products. Since directly identification of Bifidobacterium spp. from food products become an important quality issue, we compared the performance of two culture-independent approaches, including species-specific PCR method and PCR-DGGE, to identify the composition of dairy products labeled as containing Bifidobacterum directly from samples. In this present study, a novel series of species specific. Primers were designed that extend the number of probiotic Bilfidobacterium spp. to ten Among these 10 primers, B. regions of the 16S rDNA led to simultaneous detection of minimum, B. subtile, and B. thermophilum were never been several isolates of Bifidobacterium spp.

MATERIALS AND METHODS

RESULTS AND DISCUSSION

CONCLUSIONS