[in Japanese]

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The microflora of human intestinal tract is highly diverse and plays an important role in nutrition, immunomodulation, and colonization resistance against pathogens, etc. To understand the relationship between the function of intestinal microflora and human health, it is important to develop an accurate method to analyze the diverse structure of the microbial community. Since it has been confirmed that the 16S rRNA gene sequences are quite useful not only for analyzing the microbes phylogenetically but also for identifying and classifying them rapidly and accurately, molecular biological techniques have had a rapid development. With the rise of technology, culture-independent methods for analyzing intestinal microflora, such as FISH, DGGE/TGGE, 16S rDNA-clone library, quantitative PCR, or T-RFLP, have been improved significantly. This paper describes the brief information about molecular biology-based methods, which are widely used in the analysis of human intestinal microflora, their application and remaining issues.

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Recently, it has become possible to sequence the bacterial genome in a short time and the genome data of bacteria has rapidly increased as a result of the improved performance of sequencers and computers. At the time of writing (May 2007), 19 complete genome sequences of non-pathogenic lactic acid bacteria (LAB) representing 14 species from the order Lactobacillales are available. The comparative genomic analysis of LAB might elucidate new functions in species/strains. Furthermore, transcriptome analysis using microarrays has been applied to the study of LAB. The LAB genome data is an important resource for obtaining new knowledge.

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Human health benefits are expected of the anti-estrogenic or estrogenic effects of soy isoflavone. Recently, the biological effects of one of the metabolites of soy isoflavone, equol, has been focused. Equol is an active metabolite produced by intestinal bacteria. However, in humans, non-equol producers exist owing to differences in intestinal microbiota among individuals. Therefore, it is considered that non-equol producers will not benefit from soy isoflavone, even when they ingest it. The purpose of this study was to look for the lactic acid bacteria which metabolize daidzein to equol which could be used in foods. We tested 213 strains (Bifidobacterium spp and Lactobacillus spp), screening for the lactic acid bacteria which metabolize daidzein to equol, but none were found. We also tried to isolate human intestinal bacteria capable of metabolizing daidzein to equol using the feces of healthy humans, and succeeded in newly isolating 3 strains of equol-producing bacteria. Only one strain, Lactococcus 20-92, was the equol-producing lactic acid bacterium. It was identified as Lactococcus garvieae by the 16S rDNA homology. It was confirmed that equol production by Lactococcus 20-92 occurrs after the steady state of growth has been achieved. This is the first report of lactic acid bacteria capable of metabolizing daidzein to equol directly, and the itilization of Lactococcus 20-92 is expected in functional foods.

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Equol producing lactic acid bacterium, Lactococcus 20-92, isolated from healthy human feces was identified as homologous to Lactococcus garvieae. To evaluate the safety of Lactococcus 20-92, we investigated the dietary history of Lactococcus garvieae and its existence in the human intestine. Detection of Lactococcus garvieae in traditional Italian cheeses and feces collected from Japanese healthy women were analyzed by the RT-PCR method using species-specific primers. Lactococcus garvieae was detected in 7 types (21 samples) of traditional Italian cheese, Toma Piemontase, Raschera, Bra duro, Bra tenero, Murazzano, Castelmagno and Robiola di Roccaverano collected in Italy and Japan. It was also detected in 49 samples out of 135 samples of human feces (detection rate 36.3%). In this study, the dietary history of Lactococcus garvieae became clear through the confirmation of the existence of Lactococcus garvieae in some traditionally cheeses eaten. Moreover, it was clear that Lactococcus garvieae commonly exists in the healthy human intestine. These results suggest that Lactococcus garvieae is highly safe. Therefore, we consider that the human feces derived Lactococcus 20-92 is also highly safe bacteria, and its utilization in foods of the future is anticipated.

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