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Nukadoko is the fermented rice bran bed traditionally used for pickling vegetables in Japan. Here, we investigate the bacterial community structure of nukadoko using several culture-independent methods. Denaturing gradient gel electrophoresis (DGGE) and sequence analysis of V2-V3 16S rRNA gene (16S rDNA) fragments amplified from a long-aged nukadoko bacterial community indicated seven predominant operational taxonomic units (OTUs) closely related to known Lactobacillus species. Phylogenetic analysis of these OTUs indicated a major cluster consisting of six OTUs including a dominant OTU closely related to Lactobacillus acidifarinae and one distinct OTU corresponding to Lactobacillus acetotolerans. L. acetotolerans was commonly detected as a dominant species in samples from different seasons. The succession of microbial community structure in the fermentation and ripening processes was investigated using a laboratory model nukadoko. The L. acidifarinae-like bacteria grew rapidly with a pH decrease in the first few days after inoculation, whereas L. acetotolerans grew slowly and became dominant after one week. Real-time quantitative polymerase chain reaction (Q-PCR) showed that the doubling time of L. acetotolerans was 12 h, while that of total bacteria was 4 h. Real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) targeting 16S rRNA showed a low metabolic activity of L. acetotolerans throughout the fermentation and ripening processes. Fluorescence in situ hybridization (FISH) showed that L. acetotolerans was a dominant bacterium in the ripening period and had a low metabolic activity. These results indicate that the slow-growing L. acetotolerans stably dominated nukadoko microbiota after the L. acidifarinae-like bacteria mainly contributed to the lactic acid fermentation of the rice bran. [Copyright &y& Elsevier]

In a Japanese paternity test, an alleged father was excluded only by reverse homozygosity of esterase D (ESD) phenotypes (mother, ESD 1; child, ESD 1; alleged father, ESD 2) out of 43 classical and DNA markers investigated. To solve the aberrant inheritance of the ESD phenotypes observed between them, fragments for all eight coding exons amplified by polymerase chain reaction (PCR) were subjected to DNA analysis. The child and alleged father shared a null allele, originating from ESD*1. It was characterized by having TGA for the stop codon instead of TCA for serine at codon 63. Thus, the sharing of a rare null gene, ESD*Q0yonago, increased the probability of paternity. [Copyright &y& Elsevier]