Traditional microbial control methods used in sake brewing effectively suppress predominant bacteria emerging during production of rice koji

Rice koji is a raw material used in the production of sake; however, details regarding the microbial flora and their dynamics in rice koji during the production process are poorly understood. Clarifying these issues can contribute to proposing a method and evaluation that will improve the quality of rice koji and sake. The aim of this study was to determine the microflora in rice koji and the effectiveness of the traditional microbial control techniques used in the sake production process. We analyzed the diversity and changes in bacterial flora during rice koji production by amplicon sequencing of the 16S rRNA gene. The predominant taxon in all rice koji samples was family Staphylococcaceae. The microbial population and the changes in its distribution for five consecutive stages in rice koji production were examined by direct colony counting. Bacteria counts in all samples were below the limit of detection initially, then increased rapidly toward the final stage. The predominant bacterial colonies from all samples were yellow and were identified as Staphylococcus gallinarum through 16S rRNA gene sequence similarity. The S. gallinarum isolates exhibited faster growth in pregelatinized rice medium. Interestingly, the growth of S. gallinarum isolates was suppressed by low temperature (12 °C), ethanol concentration (≥6%) and the addition of lactic acid, which are traditional microbial control methods used during sake fermentation. Therefore, proper control of the traditional sake production process can effectively inhibit the growth of undesirable bacteria such as S. gallinarum that emerge during the production of rice koji.