Analysis of Bacterial Communities in Kusaya Gravy by Denaturing Gradient Gel Electrophoresis of PCR-Amplified Ribosomal DNA Fragments

Abstract

We analyzed the microbial communities in kusaya gravy obtained from three manufacturers by a combination of cultivating and molecular typing methods, PCR-RFLP and PCRDGGE. Viable counts were 1.2×10⁸-1.5×10⁹/g on TSA medium, BP₅G medium and ABCM medium. With PCR-RFLP, forty isolates obtained from BP₅G medium and ABCM medium were classified into six to nine groups. Representative strains of the isolates were identified as Pseudomonas, Marinobacter, Peptostreptococcus, Enterococcus etc. with 16S rDNA sequencing. DGGE analysis of 16S rDNA fragments amplified directly from each of three kinds of kusaya gravy showed a similar banding pattern each other. Several main fragments were recovered from DGGE-gel and were cloned and sequenced. The existence of Bacteroides, Flavobacterium, Fusobacterium, Eggerthela lenta and Clostridium was indicated from the analysis of fragments. Therefore, there are great differences between the dominant organisms obtained by culturing and by PCR-DGGE, suggesting that unculturable organisms prevail in kusaya gravy.