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

Abstract

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.

Keywords:

• Rice koji
• S. gallinarum
• bacterial flora
• microbial control

Acknowledgements

The authors thank Bioengineering Lab. Co., Ltd. (Sagamihara, Japan) for amplicon sequencing, Yukitaka Iizumi for the 16S rRNA gene sequencing data, FASMAC (Kanagawa, Japan) for direct sequence and all members of the Food & Liquor and Biotechnology Section of Yamanashi Industrial Technology Center for the opportunity to conduct this research.

Author’s contribution

M.H. and H.Y. supervised the team and the research. K.N., M.H. and H.Y. designed the methodology and the research. K.N., T.H., K.H., N.A., M.H. and H.Y. collected the data and performed data analysis. K.N., Y.N. and S.K. performed statistical analysis. K.N., T.H., K.H. and N.A. provided the study materials, reagents, materials, laboratory samples, instrumentation, computing resources. Y.N., and S.K. provided instrumentation, computing resources. K.N., T.H., K.H., N.A., M.H. and H.Y. verified that the results are reproducible. K.N. prepared and created figures, tables and wrote the manuscript. Y.N., S.K., M.H. and H.Y. critically reviewed the manuscript. M.H. and H.Y. performed the project administration. All authors read and approved the manuscript.

Data availability statements

All data that support the findings from this study are available from the corresponding author [K.N.] upon reasonable request.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.