Investigation of Microbial Community Shifts under the Mizumoto Japanese Traditional Sake Brewing Process Using Chemical Analyses and High-throughput Sequencing

Over the past 10 centuries, sake brewing methods have been developed in stages, including doburoku, mizumoto, kimoto, yamahaimoto, and sokujyomoto. Mizumoto-sake is considered the oldest prototype. The brewing process involves lactic acid fermentation and multiple parallel saccharification and alcoholic fermentation by indigenous microbes, which has been operated based on a sense of craftsmanship. The processes involved lead to the creation of extreme conditions characterized by low pH levels and high alcohol concentrations. The characteristic feature of mizumoto-sake is that it begins with fermentation by indigenous lactic acid bacteria to produce acidic water for yeasts to ferment alcohol by inhibiting the growth of undesirable microbes. In the present study, we investigated changes in the microbial community and the transition of metabolites that affect taste and flavor during processes from the initiation of mizumoto-sake brewing to the final product. In the lactic acid fermentation phase, bacteria, including those in the genera Lactococcus, Leuconostoc, and Lactobacillus, produced lactic acid and contributed to the production of acidic water (pH of approximately 4) called soyashimizu. A heating process, known as "Anka", which increased the brewing temperature, then switched the relative abundance of 18S rRNA from 75.0% Pichia to 72.3% Saccharomycetaceae. Alcohol fermentation was accelerated by the Saccharomyces family (relative abundance: 89.8%), reaching alcohol concentrations >15%.