Differences in the bacterial microbiota of the traditional fermented rice beverage miki from the Okinawa region

Okinawa- miki is a traditional, porridge-like fermented beverage produced in Okinawa, Japan. Although it is recognised as being spontaneously fermented by lactic acid bacteria (LAB), its microbial properties remain largely uncharacterized. In this study, 15 samples were collected from five different manufacturing factories. These included nine products saccharified with dry germinated wheat (GW), two with barley koji (BK: malted with Aspergillus oryzae ), and four using rice soaking water (RW). Physicochemical properties, including water content, %Brix, viscosity, pH, and lactate concentration, were measured. Viable counts of LAB, catalase-positive bacteria, and yeast were also determined. Bacterial microbiota was analysed using 16S rDNA (V3–V4) amplicon sequencing, while typical LAB and yeast isolates were identified via 16S and 26S rRNA gene sequencing. Viable counts of LAB, catalase-positive bacteria, and yeast ranged from ND–9.1 log CFU/g, 4.9–8.9 log CFU/g, and ND–7.3 log CFU/g, respectively. Amplicon sequencing revealed that Leuconostoc predominated in GW- and RW- miki , whereas Weissella was dominant in BK- and RW- miki , with profiles varying over storage time. Enterococcus was also dominant in RW- miki samples. Regardless of saccharification method or natural starter used, Bacillus was one of the dominant genera in 10 of the samples. The genera Enterobacter and Kosaconia , both wheat seed endophytes, dominated GW- miki from specific factories, while Pantoea , another endophyte, was dominant in GW- miki during early fermentation. Among 12 LAB isolates, four were identified as Leuconostoc lactis , and among eight yeast isolates, three were Saccharomyces cerevisiae . Further studies are needed to clarify the functional roles of these indigenous microbes. • Okinawa- miki is made from germinated wheat (GW), berley koji (BK), or soaked rice (RW). • Leuconostoc dominated GW- and RW-miki, whereas Weissella dominated BK- miki . • ASVs of Bacillus spp. were varied by saccharification material. • The wheat seed endophytes Enterobacter, Kosaconia, and Pantoea were abundant in the GW- miki . • The isolates Ln. lactis and S. cerevisiae require further safety and function checks.