The Kimoto-style fermentation starter is a traditional preparation method of sake brewing. In this process, specific microbial transition patterns have been observed within nitrate-reducing bacteria and lactic acid bacteria during the production process of the fermentation starter. We have characterized phylogenetic compositions and diversity of the bacterial community in a sake brewery performing the Kimoto-style fermentation. Comparing the time-series changes with other sake breweries previously reported, we found a novel type of Kimoto-style fermentation which the microbial transition differed significantly from other breweries during the fermentation step. Specifically, the lactic acid bacteria, Leuconostoc spp. was a predominant species in the late stage in the preparation process of fermentation starter, on the other hand, Lactobacillus spp., which plays a pivotal role in other breweries, was not detected in this analysis. The discovery of this new variation of microbiome transition in Kimoto-style fermentation has further deepened our understanding of the diversity of sake brewing.

IntroductionIn Kimoto-style fermentation, a fermentation starter is produced before the primary brewing process to stabilize fermentation. Nitrate-reducing bacteria, mainly derived from brewing water, produce nitrite, and lactic acid bacteria such as Leuconostoc can proliferate because of their tolerance toward low temperature and their low nutritional requirements. Later, Lactobacillus becomes the dominant genus, leading to weakly acidic conditions that contribute to control yeasts and undesired bacterial contaminants. However, the sources of these microorganisms that play a pivotal role in Sake brewing have not yet been revealed. Thus, comprehensive elucidation of the microbiome is necessary.MethodsIn this study, we performed 16S rRNA amplicon sequencing analysis after sampling from floor, equipment surfaces, and raw materials for making fermentation starters, including koji, and water in Tsuchida Sake brewery, Gunma, Japan.ResultsAmplicon sequence variants (ASVs) between the external environments and the fermentation starter were compared, and it was verified that the microorganisms in the external environments, such as built environments, equipment surfaces, and raw materials in the sake brewery, were introduced into the fermentation starter. Furthermore, various adventitious microbes present in the fermentation starter of early days and from the external environments were detected in a nonnegligible proportion in the starter, which may impact the taste and flavor.DiscussionThese findings illuminate the uncharacterized microbial dark matter of sake brewing, the sources of microbes in Kimoto-style fermentation.

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In Kimoto-style fermentation, a fermentation starter is produced before the primary brewing process to stabilize fermentation. Nitrate-reducing bacteria, mainly derived from brewing water, produce nitrite, and the molecule proliferates lactic acid bacteria such as Leuconostoc spp. due to its characteristics of tolerance against low temperatures and fewer nutritional requirements. Later, Lactobacillus spp. is switched to the dominant genus, creating a weakly acidic condition to control wild yeasts and unexpected bacterial contamination. However, the sources of these microorganisms that play a pivotal role in Sake brewing have not yet been revealed. Thus, comprehensive elucidation of the microbiome is necessary. In this study, we performed 16S rRNA amplicon sequencing analysis after sampling from architectural surfaces, tools, and raw materials for making fermentation starters, including koji, and water in Tsuchida Sake brewery, Gunma, Japan. Amplicon sequence variants (ASVs) between the external environments and the fermentation starter were compared, and it was verified that the microorganisms in the external environments, such as built environments, tools, and raw materials in the sake brewery, were introduced into the fermentation starter. Furthermore, various adventitious microbes confirmed in the fermentation starter of early days and from the external environments were detected in a nonnegligible proportion in the starter, which may impact the sake' taste and flavor. These findings illuminate the uncharacterized microbial dark matter of sake brewing, the sources of microbes in Kimoto-style fermentation.