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.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
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)
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)
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)
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)
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)
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)
Bokulich NA, Ohta M, Lee M, Mills DA (2014) Indigenous bacteria and fungi drive traditional kimoto sake fermentations. Appl Environ Microbiol 80:5522–5529
Caporaso JG, Kuczynski J, Stombaugh J et al (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336
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
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
Fernández M, Zúñiga M (2006) Amino acid catabolic pathways of lactic acid bacteria. Crit Rev Microbiol 32:155–183
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
Kanauchi M (2013) SAKE alcoholic beverage production in Japanese food industry. In: Muzzalupo I (ed) Food industry, chap 3. InTech, London, pp 39–63
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
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
Kitahara K (1966) Nyusankin no kenkyu [Research on lactic acid bacteria]. University of Tokyo Press, Tokyo
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
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)
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
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)
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)
Mizoguchi H, Hara S (2010) A theoretical consideration of kimoto making. J Brew Soc Jpn 105:124–138 (in Japanese)
Momose H, Fujikura H (1996) Lactobacilli isolated from moto (Sake starter) prepared by traditional method. J Brew Soc Jpn 91:834–837 (in Japanese)
Montel MC, Champomier MC (1987) Arginine catabolism in Lactobacillus sake isolated from meat. Appl Environ Microbiol 53:2683–2685
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)
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)
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
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
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
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
Terasaki M, Miyagawa S, Yamada M, Nishida H (2018) Detection of bacterial DNA during the process of sake production using sokujo-moto. Curr Microbiol. https://doi.org/10.1007/s00284-018-1460-x
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
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)
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)
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
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)
Yoshizawa K (1999) Sake: production and flavor. Food Rev Int 15:83–107
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
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
This work was partially supported by Faculty research funds from Ishikawa Prefectural University.
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.
About this article
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). https://doi.org/10.1007/s00284-018-1551-8