Diversity of Bacillus Isolates from the Sake Brewing Process at a Sake Brewery

Yamagishi

et al. 2022

Preprint

The Kimoto-style fermentation starter is a traditional preparation method of sake brewing. In this process, specific microbial transition patterns have been observed within nitrate-reducing bacteria and lactic acid bacteria during the production process of the fermentation starter. We have characterized phylogenetic compositions and diversity of the bacterial community in a sake brewery performing the Kimoto-style fermentation. Comparing the time-series changes with other sake breweries previously reported, we found a novel type of Kimoto-style fermentation which the microbial transition differed significantly from other breweries during the fermentation step. Specifically, the lactic acid bacteria, Leuconostoc spp. was a predominant species in the late stage in the preparation process of fermentation starter, on the other hand, Lactobacillus spp., which plays a pivotal role in other breweries, was not detected in this analysis. The discovery of this new variation of microbiome transition in Kimoto-style fermentation has further deepened our understanding of the diversity of sake brewing.

“…It is reported that Bacillus spp. may live in sake breweries as Kuratsuki bacteria (24). Bacillus spp.…”

Section: Resultsmentioning

confidence: 99%

Section: Processing For Comparative Microbiome Analysis Sequence Data...mentioning

confidence: 99%

A unique case in whichKimoto-style fermentation was completed withLeuconostocas the dominant genus without transitioning toLactobacillus

Ito

Yamagishi

et al. 2022

Preprint

“…In addition, several Kuratsuki microbes have also been isolated, including Staphylococcus sp. and Bacillus sp., as well as lactic acid bacteria considered necessary for Sake production ( Terasaki and Nishida, 2020 ; Kanamoto et al, 2021 ; Takahashi et al, 2021 ). As an alternative method to identify Kuratsuki microbes, a study using amplicon sequencing was reported in 2014 ( Bokulich et al, 2014 ).…”

Section: Introductionmentioning

confidence: 99%

A dark matter in sake brewing: Origin of microbes producing a Kimoto-style fermentation starter

Ito¹,

Kobayashi³

et al. 2023

Front. Microbiol.

IntroductionIn Kimoto-style fermentation, a fermentation starter is produced before the primary brewing process to stabilize fermentation. Nitrate-reducing bacteria, mainly derived from brewing water, produce nitrite, and lactic acid bacteria such as Leuconostoc can proliferate because of their tolerance toward low temperature and their low nutritional requirements. Later, Lactobacillus becomes the dominant genus, leading to weakly acidic conditions that contribute to control yeasts and undesired bacterial contaminants. However, the sources of these microorganisms that play a pivotal role in Sake brewing have not yet been revealed. Thus, comprehensive elucidation of the microbiome is necessary.MethodsIn this study, we performed 16S rRNA amplicon sequencing analysis after sampling from floor, equipment surfaces, and raw materials for making fermentation starters, including koji, and water in Tsuchida Sake brewery, Gunma, Japan.ResultsAmplicon sequence variants (ASVs) between the external environments and the fermentation starter were compared, and it was verified that the microorganisms in the external environments, such as built environments, equipment surfaces, and raw materials in the sake brewery, were introduced into the fermentation starter. Furthermore, various adventitious microbes present in the fermentation starter of early days and from the external environments were detected in a nonnegligible proportion in the starter, which may impact the taste and flavor.DiscussionThese findings illuminate the uncharacterized microbial dark matter of sake brewing, the sources of microbes in Kimoto-style fermentation.

“…In the past, studies have yet comprehensively examined changes in microbes sharing between different sample types (floor surfaces and equipment surfaces) and Moto in a brewery during the fermentation process with a lens of amplicon sequencing. Previous studies have isolated Kocuria and Bacillus as viable microbes (Kanamoto et al 2021; Terasaki et al 2021), indicating that certain microbes can live in Moto , such as lactic acid bacteria and yeast, and may be involved in Sake brewing. The possibility of horizontal gene transfer among microorganisms in Sake breweries (Terasaki et al 2021) has also been demonstrated; instead of targeting only specific bacteria, such as lactic acid bacteria, the community-level structure should be targeted as Kuratsuki microbiota.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, several Kuratsuki microbes have also been isolated, including Staphylococcus sp. and Bacillus sp., as well as lactic acid bacteria considered necessary for Sake production (Terasaki & Nishida 2020; Kanamoto et al 2021; Takahashi et al 2021). As an alternative method to identify Kuratsuki microbes, a study using amplicon sequencing was reported in 2014 (Bokulich et al 2014).…”

Section: Introductionmentioning

confidence: 99%

A dark matter insakebrewing: Origin of microbes producing aKimoto-style fermentation starter

Ito¹,

Kobayashi³

et al. 2022

Preprint

In Kimoto-style fermentation, a fermentation starter is produced before the primary brewing process to stabilize fermentation. Nitrate-reducing bacteria, mainly derived from brewing water, produce nitrite, and the molecule proliferates lactic acid bacteria such as Leuconostoc spp. due to its characteristics of tolerance against low temperatures and fewer nutritional requirements. Later, Lactobacillus spp. is switched to the dominant genus, creating a weakly acidic condition to control wild yeasts and unexpected bacterial contamination. However, the sources of these microorganisms that play a pivotal role in Sake brewing have not yet been revealed. Thus, comprehensive elucidation of the microbiome is necessary. In this study, we performed 16S rRNA amplicon sequencing analysis after sampling from architectural surfaces, tools, and raw materials for making fermentation starters, including koji, and water in Tsuchida Sake brewery, Gunma, Japan. Amplicon sequence variants (ASVs) between the external environments and the fermentation starter were compared, and it was verified that the microorganisms in the external environments, such as built environments, tools, and raw materials in the sake brewery, were introduced into the fermentation starter. Furthermore, various adventitious microbes confirmed in the fermentation starter of early days and from the external environments were detected in a nonnegligible proportion in the starter, which may impact the sake' taste and flavor. These findings illuminate the uncharacterized microbial dark matter of sake brewing, the sources of microbes in Kimoto-style fermentation.