Phylogenomic analysis in Latilactobacillus sakei by using polymorphisms detected by next-generation sequencing.

Latilactobacillus sakei is a lactic acid bacterium used to produce a wide range of fermented food products. To understand their characteristics and adaptability to various nutrient sources, we applied strain-specific, nucleotide-concatenated (SSC) sequences to the phylogenetic analysis of 32 L. sakei strains isolated from various locations and products. SSC sequences were developed by concatenating the polymorphisms detected by whole-genome sequencing. This enabled us to use sufficient polymorphisms and avoid the bias caused by selecting partial sequences, such as that in core genome and multi-locus sequence typing. SSC sequence-based analysis revealed that the phylogenetic relations for L. sakei are based on the different nutrition sources rather than geographical distance.

Co-cultivation of sake yeast and Kocuria isolates from the sake brewing process.

Kocuria isolates collected from the sake brewing process have inhabited the Narimasa Sake Brewery in Toyama, Japan. To investigate the effect of these actinobacterial isolates on the growth and metabolism of sake yeast, co-cultivation of sake yeast and Kocuria isolates was performed in a medium containing tryptone, glucose and yeast extract (TGY), and a solution containing koji (steamed rice covered with Aspergillus oryzae) and glucose. In the TGY medium, the ethanol concentration and the number of living cells of each microorganism were measured. In the koji solution, the concentrations of ethanol and organic acids (citric acid, lactic acid and succinic acid) were measured. The results showed that in TGY media, the growth of each Kocuria isolate in the co-culture of the two Kocuria isolates was similar to that in each monoculture. However, the growth of both Kocuria isolates was inhibited in the co-cultures of sake yeast and Kocuria isolates. On the other hand, the growth and ethanol productivity of sake yeast did not differ between its monoculture and co-cultures with Kocuria isolates. In the koji solution, Kocuria isolates TGY1120_3 and TGY1127_2 affected the concentrations of ethanol and lactic acid, respectively. Thus, Kocuria isolates affected the microbial metabolism, but the effects were not identical between the two isolates. This strongly suggests that bacteria inhabiting a sake brewery may influence the flavor and taste of sake products of the brewery.

Genomic information of Kocuria isolates from sake brewing process.

Bacteria belonging to the genus Kocuria were identified as bacteria peculiar to a sake brewery in Toyama, Japan. Comparison of the 16S rRNA gene sequences revealed two groups of Kocuria isolates. Among known species, one group was similar to K. koreensis (Kk type), and the other, K. uropygioeca (Ku type). We determined complete genomic DNA sequences from two isolates, TGY1120_3 and TGY1127_2, which belong to types Kk and Ku, respectively. Comparison of these genomic information showed that these isolates differ at the species level with different genomic characters. Isolate TGY1120_3 comprised one chromosome and three plasmids, and the same transposon coding region was located on two loci on the chromosome and one locus on one plasmid, suggesting that the genetic element may be transferred between the chromosome and plasmid. Isolate TGY1127_2 comprised one chromosome and one plasmid. This plasmid encoded an identical transposase coding region, strongly suggesting that the genetic element may be transferred between these different isolates through plasmids. These four plasmids carried a highly similar region, indicating that they share a common ancestor. Thus, these two isolates may form a community and exchange their genetic information during sake brewing.

Chemical and Bacterial Components in Sake and Sake Production Process.

Together with the worldwide Washoku (traditional Japanese foods and drinks) boom, interest in sake, a traditional Japanese alcoholic drink, is increasing around the world. There are few scientific analyses and studies on the production of sake or the final product itself. We show the diversity of bacterial contaminants during sake production and investigated the effects of different ingredients on sake (for example, amino acids). The koji mold Aspergillus oryzae converts rice starch into sugars, and then, the sake yeast Saccharomyces cerevisiae converts the sugars to ethanol. Comparative studies of the bacterial flora of different sakes have shown that various bacterial species are detected, but that there are few frequently detected bacteria. In addition, the bacterial flora does not vary much during the process of sake brewing, after the koji (steamed rice covered with koji mold) and moto (fermentation starter) are mixed, suggesting that most bacteria contaminate the sake during the process of koji and moto production. Thus, there is the possibility that the contaminating bacteria may grow due to a relationship with the koji mold and/or the sake yeast. The flavor, taste, and quality of sakes differ, even between the same brands of sakes, which may be attributed to variations in the contaminating bacteria during sake production.

Detection of Bacterial DNA During the Process of Sake Production Using Sokujo-Moto.

There are two types of starter cultures used in Japanese rice wine (sake) fermentation, namely, sokujo-moto and yamahai-moto. Analyses of microbiota changes during sake production using yamahai-moto have already been reported. In this study, we analyzed microbiota changes during sake production using sokujo-moto. In addition, we sequenced bacterial DNA from the water used in sake production. The Lactobacillus DNA sequences, which are frequently detected during sake production using yamahai-moto, were not detected during sake production using sokujo-moto, indicating that the Lactobacillus DNA detected in sakes made from yamahai-moto is from the fermentation starter. Most bacterial DNA sequences detected in water were not found in the production samples of sake suggesting that these bacteria do not proliferate during sake production. Thus, most of the bacterial DNA sequences detected during the production may be from the bacterial contamination during the production process.

Bacterial DNA Detected in Japanese Rice Wines and the Fermentation Starters.

As Japanese rice wine (sake) brewing is not done aseptically, bacterial contamination is conceivable during the process of sake production. There are two types of the fermentation starter, sokujo-moto and yamahai-moto (kimoto). We identified bacterial DNA found in various sakes, the sokujo-moto and the yamahai-moto making just after sake yeast addition. Each sake has a unique variety of bacterial DNA not observed in other sakes. Although most bacterial DNA sequences detected in the sokujo-moto were found in sakes of different sake breweries, most bacterial DNA sequences detected in the yamahai-moto at the early stage of the starter fermentation were not detected in any sakes. Our findings demonstrate that various bacteria grow and then die during the process of sake brewing, as indicated by the presence of trace levels of bacterial DNA.