A dark matter in sake brewing: Origin of microbes producing a Kimoto-style fermentation starter

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.