Metagenomics analyses of microbial dynamics associated with putative flavor development in mash fermentation of Sake

Sake is one of the national representative alcoholic beverages in Japan. To examine the impacts of cofermentation on microbial compositions, liquor making with vs. without adding pineapple was conducted using rice of Oryza sativa ssp. Japonica. Dynamics of microbial communities across the fermentation course was investigated using targeted metagenomics of 16S rRNA and internal transcribed spacers (ITS). In regular (RS) and co-fermented (CS) sakes, the fungal microbiota was dominated by Saccharomyces. In contrast to the stable fungal composition, analyses of hierarchical clustering and non-metric multidimensional scaling identified two phases of bacterial succession, namely premature and maturing stages. Compared with the dividing point between phases on the 15th day in RS, the transition in CS was delayed till the 20th day. Nevetheless, in both experiments, Leuconostoc dominated the premature stage, while Erwinia dominated the maturing stage. In RS, bacterial succession was characterized by increasing Staphylococcus on the 7th day, decreasing Leuconostoc on the 10th day, and a dramatic decrease of Pseudomonas in the maturing stage; while in CS, a sharp decrease of Pseudomonas occurred earlier in the premature stage, followed by increasing Gluconobacter and decreasing Leuconostoc. Shannon index revealed a higher microbial diversity in CS than in RS, suggesting that adding fruits may have nurtured diverse bacteria that further enriched the sake flavor. Pathway analysis by PICRUSt2 revealed that sugar alcohol degradation was fundamental to the fermentation, while flavor rich in CS was likely attributable to the activation of biosynthesis of L-arginine and amino acids, and degradation of amine, carboxylate, polyamine, and secondary metabolites. Furthermore, Pearson correlation analysis revealed that Aerococcus was positively correlated with enzymes participating the phenylethyl metabolism.