A unique case in which Kimoto-style fermentation was completed with Leuconostoc as the dominant genus without transitioning to Lactobacillus

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 in 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.

Sake is a traditional alcoholic beverage made from rice in Japan. In a preliminary stage 41 of a main brewing process, a fermentation starter with purely cultured yeast is produced to 42 prevent microbial contamination and poor fermentation and to promote smooth alcoholic 43 fermentation. The production process is as follows; First, Aspergillus oryzae, which secretes 44 amylases, is propagated on steamed rice to make koji. Then, koji, steamed rice, and water are 45 mixed in an open-top tank. Fermentation of this mixture produces a fermentation starter. The 46 fermentation starter is further mixed with koji, steamed rice, and water, and after a 3-5 week 47 fermentation process, the fermentation mash or starter is produced. The fermentation starter is 48 separated into sake and spent rice by a filter press to complete the sake. 49 The fermentation starter is divided into three styles, Sokujo, Kimoto and Yamahai. 50 Sokujo-style fermentation starter is a modern method to make the starter culture with the 51 addition of food-grade lactic acid. Kimoto-style fermentation starter is the traditional 52 preparation method of the starter culture and is manufactured under highly acidic conditions by 53 inducing the growth of nitrate-reducing bacteria and lactic acid bacteria properly. Yamahai- 54 style is similar to Kimoto-style but made without grinding rice. Lactic acid inhibits 55 contaminations of unintended yeasts and bacteria from external environments into the 56 fermentation starter. 57 The microbiome compositions during Kimoto-style fermentation starter production 58 show standard transitions as follows. Nitrate-reducing bacteria, which were reported to come 59 from water (1), initially grows and produces nitrite, thereby inhibiting the growth of 60 microorganisms that are less tolerant to nitrite. At the same time, lactic acid bacteria, especially 61 Leuconostoc spp., which grow at low temperatures and have fewer nutrient requirements, 62 increase, and then lactic acid bacteria such as Latilactobacillus sakei, which require strict 63 nutrients, occupy the microbiome compositions as a predominant species. These steps are 64 Microbial transition in the Kimoto-style starter 4 known as a common microbial transition in sake brewing (2-4). 65 However, it was reported that some fermentation starters brewed by Kimoto-style in 66 several sake breweries show distinctive microbial transitions and chemical changes and that 67 this is one of the reasons for producing unique sake flavors among breweries, even if they use 68 the same production process (5-8). A possible reason to explain this fact is differences in 69 Kuratsuki microorganisms (microorganisms living in sake breweries) and the introduction of 70 diverse microorganisms from outside of tanks during the sake brewing process (9,10).

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The Tsuchida Sake Brewery (Gunma, Japan) is one of a few breweries that produce 72 the Kimoto-style fermentation starter without adding yeasts and fully relies on Kuratsuki 73 microorganisms to produce the fermentation starter. In addition, compared to the Kimoto-style 74 fermentation in other sake breweries, this sake brewery is characterized by not using any food 75 additives such as brewers' alcohol, enzyme reagents, or activated charcoal. Furthermore, this 76 brewery does not use sake-brewing rice but table rice for sake brewing. For the above reasons, 77 the microbial community, and its transition in the fermentation starter of the brewery were 78 considered to be divergent from previous studies.

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In this study, we focused on the fermentation mechanism of the Kimoto-style 80 fermentation starter from the Tsuchida Sake Brewery and analyzed its microbial community 81 during the fermentation process in detail using its 16S ribosomal RNA amplicon sequencing.     deployed for this processing. The method described above was repeated to perform taxonomic 144 analysis. The depth of sequence reads (Features) differed among samples, and to normalize 145 them, we subsampled from each sample to 5,000 reads each. Samples with fewer than 5,000 146 reads (S22, 23, 24, 25, 26, 28) were excluded from the diversity analysis (Table S1). Sub-  (Table S2). Therefore, this investigation suggests that no significant changes in 153 microbial diversity are due to the subsampling of 5,000 reads from each sample. The genera with average relative abundances higher than 5% in the early stage (day 1 181 to day 7) of the fermentation starters were Methylobacterium-Methylorubrum (23.0%),

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The relative abundances of the genus Lactobacillus and Leuconostoc in each brewery 204 during the preparation are shown in the line graphs (Fig. 4). The genus Lactobacillus became

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A previous study revealed that L. sakei has a more stringent amino acid requirement 237 than L. mesenteroides, Lactobacillus sp., and di-tripeptides including asparagine, which is 238 produced by koji mold degradation, are growth factors, and that pH and temperature affect the 239 growth of L. sakei (8,28,29). In this study, the peak of nitrite concentration was relatively late 240 at day 11, and the increase in acidity (TA; Titratable acidity) was delayed accordingly, 241 suggesting a longer survival period of adventitious bacteria that were initially introduced (Fig.   242 2). Therefore, there is a possibility that the growth of L. sakei was inhibited by specific changes 243 in nutrients and temperature.  where Pseudomonas spp. were not detected and no nitrite production was observed (5,31). It is 257 considered that the genus Pseudomonas is easily lysed by exposure to alcohol due to its cell 258 surface structure (32). Therefore, we believe that Pseudomonas spp. was detected until the 259 middle stage, where the alcohol level rose relatively late, after day 20.

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The structure of the microbiome changed significantly before and after the production 261 of nitrite, suggesting that nitrate-reducing bacteria may also affect microbial transitions and 262 other factors in the early stage ( Fig. 1 and 2). This suggests that the presence or absence of  fermentation starter. 288 We found significant changes in relative abundances of these bacteria such as Rahnella1, 289 Serratia, Hafnia-Obesumbacterium during the early to the late stages (Fig. 2). But These 290 bacteria were not found during the early stage. Therefore, these bacteria may have been 291 contaminated outside tanks during the middle stage (days 9-13). And these bacteria maintained 292 high relative abundance and may have been grown in the fermentation starter for some time. Kuratuski microorganisms, and we believe that this scientific elucidation of the traditional 305 Japanese liquor will provide significant insights into food microbiology.