Robust Domination of Lactobacillus sakei in Microbiota During Traditional Japanese Sake Starter Yamahai-Moto Fermentation and the Accompanying Changes in Metabolites


The successful production of sake (Japanese rice wine) is brought about by drastic changes in microbial flora and chemical components during fermentation. In the traditional manufacturing process of sake starter (yamahai-moto), spontaneous growth of lactic acid bacteria suppresses inappropriate microorganisms and prepares the optimum environment for the alcohol fermentative yeast. In this study, we analyzed the changes in bacterial flora and chemical components of yamahai-moto. High-throughput next-generation sequencing (NGS) of the 16S ribosomal RNA gene V4 region revealed that various kinds of bacteria, including nitrate-reducing bacteria, existed in the early fermentation stage; however, Lactobacillus sakei then increased drastically to become dominant in the middle stage. Interestingly, this result was different from that obtained in the previous year at the same manufacturer; the early-stage major bacterium was Lactobacillus acidipiscis. Lactic acid, glucose, isomaltose, and total free amino acids increased throughout the fermentation process, which was attributable to the metabolism of L. sakei and the koji mold. It is noteworthy that significant ornithine accumulation and arginine consumption were observed from the middle to late stages. Thirty-eight percent of the L. sakei isolates from yamahai-moto exhibited significant ornithine production, indicating that the arginine deiminase pathway of L. sakei was working to survive the extremely low pH environment of the moto after the middle stage. This is the first report that includes concurrent analyses of the NGS-based bacterial flora and chemical components of yamahai-moto, providing further knowledge to help understand and improve the process of sake brewing.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3


  1. 1.

    Ashizawa T (1963) Yamahai shubo ni okeru biseibutsugaku-teki kenkyu (3) [Microbiological research on yamahai-shubo (3)]. J Brew Soc Jpn 58:543–548 (in Japanese)

    Article  Google Scholar 

  2. 2.

    Ashizawa T (1965) Yamahai shubo ni okeru biseibutsugaku-teki kenkyu (10) [Microbiological research on yamahai-shubo (10)]. J Brew Soc Jpn 60:900–903 (in Japanese)

    Article  Google Scholar 

  3. 3.

    Ashizawa T (1976) Nihonshu jyozo no shinpi [The mystery of Japanese sake brewing focused on kimoto]. J Brew Soc Jpn 71:424–427 (in Japanese)

    Article  Google Scholar 

  4. 4.

    Ashizawa T, Saito Y (1965) Yamahai shubo ni okeru biseibutsugaku-teki kenkyu (9) [Microbiological research on yamahai-shubo (9)]. J Brew Soc Jpn 60:803–807 (in Japanese)

    Article  Google Scholar 

  5. 5.

    Ashizawa T, Saito Y (1966) Yamahai shubo ni okeru biseibutsugaku-teki kenkyu (12) [Microbiological research on yamahai-shubo (12)]. J Brew Soc Jpn 61:638–642 (in Japanese)

    Article  Google Scholar 

  6. 6.

    Ashizawa T, Saito Y (1966) Yamahai shubo ni okeru biseibutsugaku-teki kenkyu (13) [Microbiological research on yamahai shubo (13)]. J Brew Soc Jpn 61:1033–1036 (in Japanese)

    Article  Google Scholar 

  7. 7.

    Bokulich NA, Ohta M, Lee M, Mills DA (2014) Indigenous bacteria and fungi drive traditional kimoto sake fermentations. Appl Environ Microbiol 80:5522–5529

    Article  Google Scholar 

  8. 8.

    Caporaso JG, Kuczynski J, Stombaugh J et al (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336

    CAS  Article  Google Scholar 

  9. 9.

    Champomier Vergès MC, Zuñiga M, Morel-Deville F, Pérez-Martínez G, Zagorec M, Ehrlich SD (1999) Relationships between arginine degradation, pH and survival in Lactobacillus sakei. FEMS Microbiol Lett 180:297–304

    Article  Google Scholar 

  10. 10.

    DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL (2006) Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 72:5069–5072

    CAS  Article  Google Scholar 

  11. 11.

    Fernández M, Zúñiga M (2006) Amino acid catabolic pathways of lactic acid bacteria. Crit Rev Microbiol 32:155–183

    Article  Google Scholar 

  12. 12.

    Iino T, Uchimura T, Komagata K (2003) Characterization of Lactobacillus sakei by the type of stereoisomers of lactic acid produced. J Gen Appl Microbiol 49:111–121

    CAS  Article  Google Scholar 

  13. 13.

    Kanauchi M (2013) SAKE alcoholic beverage production in Japanese food industry. In: Muzzalupo I (ed) Food industry, chap 3. InTech, London, pp 39–63

    Google Scholar 

  14. 14.

    Katagiri H, Kitahara K, Fukami K (1934) The characteristics of the lactic acid bacteria isolated from moto, yeast mashes for Saké manufacture Part IV. The classification of the lactic acid bacteria. Bull Agric Chem Soc Jpn 10:156–157

    CAS  Article  Google Scholar 

  15. 15.

    Kitagaki H, Kitamoto K (2013) Breeding research on sake yeasts in Japan: history, recent technological advances, and future perspectives. Annu Rev Food Sci Technol 4:215–235

    CAS  Article  Google Scholar 

  16. 16.

    Kitahara K (1966) Nyusankin no kenkyu [Research on lactic acid bacteria]. University of Tokyo Press, Tokyo

    Google Scholar 

  17. 17.

    Koyanagi T, Nakagawa A, Kiyohara M et al (2016) Tracing microbiota changes in yamahai-moto, the traditional Japanese sake starter. Biosci Biotechnol Biochem 80:399–406

    CAS  Article  Google Scholar 

  18. 18.

    Masuda Y, Noguchi T, Takahashi T, Iguchi A, Osawa R, Mizoguchi H (2012) DGGE and PFGE analysis of lactic acid bacterial succession during Kimoto making. Seibutsu-Kogaku Kaishi 90:684–690 (in Japanese)

    CAS  Google Scholar 

  19. 19.

    Masuda Y, Takahashi T, Yoshida K, Nishitani Y, Mizuno M, Mizoguchi H (2011) TLR ligands of Lactobacillus sakei LK-117 isolated from seed mash for brewing sake are potent inducers of IL-12. J Biosci Bioeng 112:363–368

    CAS  Article  Google Scholar 

  20. 20.

    Mizoguchi H (1998) Acquisition of ethanol tolerance by Saccharomyces cerevisiae in the sake brewing process and the tolerance determinants. Seibutsu-Kogaku Kaishi 76:122–130 (in Japanese)

    CAS  Google Scholar 

  21. 21.

    Mizoguchi H (2013) Quality of sake characterized by lactic acid bacterial flora in traditional yeast starter (kimoto). J Brew Soc Jpn 108:382–388 (in Japanese)

    CAS  Article  Google Scholar 

  22. 22.

    Mizoguchi H, Hara S (2010) A theoretical consideration of kimoto making. J Brew Soc Jpn 105:124–138 (in Japanese)

    CAS  Article  Google Scholar 

  23. 23.

    Momose H, Fujikura H (1996) Lactobacilli isolated from moto (Sake starter) prepared by traditional method. J Brew Soc Jpn 91:834–837 (in Japanese)

    Article  Google Scholar 

  24. 24.

    Montel MC, Champomier MC (1987) Arginine catabolism in Lactobacillus sake isolated from meat. Appl Environ Microbiol 53:2683–2685

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Narahara H, Mano F, Okawa H, Kawai Y, Matsuyama M (1969) Seishu koji ni kansuru kenkyu (1) [Research on koji used for Japanese sake]. J Brew Soc Jpn 64:915–918 (in Japanese)

    Article  Google Scholar 

  26. 26.

    Nishio A, Shigeru K (2008) Control of moto (Sake starter) prepared by traditional method by addition of lactic acid bacteria and nitrate reducing bacteria. Rep Tottori Inst Ind Technol 11:55–58 (in Japanese)

    Google Scholar 

  27. 27.

    Onda T (2003) Identification and characterization of lactic acid bacteria isolated from Sake-mush using ‘Kimoto’, prepared by a traditional brewing method. J Brew Soc Jpn 98:148–151

    CAS  Article  Google Scholar 

  28. 28.

    Rimaux T, Rivière A, Illeghems K, Weckx S, De Vuyst L, Leroy F (2012) Expression of the arginine deiminase pathway genes in Lactobacillus sakei is strain dependent and is affected by the environmental pH. Appl Environ Microbiol 78:4874–4883

    CAS  Article  Google Scholar 

  29. 29.

    Rimaux T, Vrancken G, Pothakos V, Maes D, De Vuyst L, Leroy F (2011) The kinetics of the arginine deiminase pathway in the meat starter culture Lactobacillus sakei CTC 494 are pH-dependent. Food Microbiol 28:597–604

    CAS  Article  Google Scholar 

  30. 30.

    Terasaki M, Fukuyama A, Takahashi Y, Yamada M, Nishida H (2017) Bacterial DNA detected in Japanese rice wines and the fermentation starters. Curr Microbiol 74:1432–1437

    CAS  Article  Google Scholar 

  31. 31.

    Terasaki M, Miyagawa S, Yamada M, Nishida H (2018) Detection of bacterial DNA during the process of sake production using sokujo-moto. Curr Microbiol.

    Article  PubMed  Google Scholar 

  32. 32.

    Uemura T, Furusaka C, Sudo T, Fujii Y, Takagi K, Miyagi T (1952) Studies on the sake yeast. Tohoku J Agric Res 3:185–218

    CAS  Google Scholar 

  33. 33.

    Umezu M, Shibata A, Maeda M (1977) Production of amines by nitrate reducing-bacteria and lactobacilli for sake-brewing. Hakkokogaku Kaishi 55:68–74 (in Japanese)

    CAS  Google Scholar 

  34. 34.

    Wakai Y, Shiraishi M, Maenaka M, Konishi Y (1990) Isolation and characteristics of nitrate-reducing bacteria in yamahai-moto. Hakkokogaku Kaishi 68:187–195 (in Japanese)

    CAS  Google Scholar 

  35. 35.

    Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267

    CAS  Article  Google Scholar 

  36. 36.

    Yamaji E, Furukawa K, Mizoguchi H, Hara S (2005) Growth factors required for the predominance of Lactobacillus sakei over Leuconostoc mesenteroides in kimoto. J Brew Soc Jpn 100:281–288 (in Japanese)

    CAS  Article  Google Scholar 

  37. 37.

    Yoshizawa K (1999) Sake: production and flavor. Food Rev Int 15:83–107

    CAS  Article  Google Scholar 

  38. 38.

    Zúñiga M, Champomier-Verges M, Zagorec M, Pérez-Martínez G (1998) Structural and functional analysis of the gene cluster encoding the enzymes of the arginine deiminase pathway of Lactobacillus sake. J Bacteriol 180:4154–4159

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Zúñiga M, Miralles Md Mdel C, Pérez-Martínez G (2002) The Product of arcR, the sixth gene of the arc operon of Lactobacillus sakei, is essential for expression of the arginine deiminase pathway. Appl Environ Microbiol 68:6051–6058

    Article  Google Scholar 

Download references


This work was partially supported by Faculty research funds from Ishikawa Prefectural University.

Author information



Corresponding author

Correspondence to Takashi Koyanagi.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 201 KB)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Tsuji, A., Kozawa, M., Tokuda, K. et al. Robust Domination of Lactobacillus sakei in Microbiota During Traditional Japanese Sake Starter Yamahai-Moto Fermentation and the Accompanying Changes in Metabolites. Curr Microbiol 75, 1498–1505 (2018).

Download citation