Heritage sustainable lactic acid fermentation in a byproduct of sake making

Heritage sustainable lactic acid fermentation in a byproduct of sake making | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Heritage sustainable lactic acid fermentation in a byproduct of sake making Daisuke Watanabe, Motomu Yoshioka, Yukihiko Masuda, Mariko Mori This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5456531/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Reusing byproducts has been an issue ever since sake production was established in ancient Japan. Sake kasu, an ethanol-containing solid byproduct remaining after pressing sake, is widely used as a marinade base to enhance flavor and preservation. Here, we isolated ethanolphiliclactic acid bacteria that dominate the microbial community in sake kasu-based traditional foods. This study highlights the potential existence of heritage, sustainable fermentation to transform byproducts into high-value foods. Biological sciences/Microbiology/Applied microbiology Biological sciences/Microbiology/Communities/Microbial ecology Figures Figure 1 Figure 2 Introduction Narazuke, a traditional Japanese preserved food, is prepared by repeatedly embedding salted gourds or other vegetables in aged sake kasu (Supplementary Fig. 1 and Fig. 1a) 1,2 . Its prototype was established by the early 8th century, when the capital of ancient Japan, Heijo-kyo, was located in present-day Nara (Supplementary Fig. 2). In the northeastern part of the Heijo Imperial Palace, there was an office responsible for fermentation, where sake and related fermented foods, such as rice vinegar, were systematically produced for presentations to the emperor and to entertain guests from overseas 3,4 . Thus, it is natural to assume that the byproducts of sake were reused in this region. Moreover, records of vegetables marinated in sake kasu have been found on wooden tablets excavated from the ruins of Prince Nagaya’s residence (684–729 CE) in Heijo-kyo (Fig. 1b) 5 . In orthodox narazuke making, vegetables are first exposed to a saturated salt solution and are then embedded in aged sake kasu, which contains an ethanol concentration of at least 8%, for a year or more. It remains unclear whether microbial fermentation occurs under such harsh conditions; however, several species of halotolerant yeast and lactic acid bacteria (LAB) have been isolated in a few previous reports 6 . It has not been determined whether these microorganisms actually grow and participate in the fermentation process during narazuke making. To investigate the microbial ecosystem in sake kasu, we conducted a microbiome analysis of samples obtained from the ingredients, in-process products, and finished products of narazuke (Supplementary Tables 1 and 2 and Fig. 1c). The samples were independently collected from two manufacturers located within 5 km of the Heijo Palace site, Factory N (Naraya Honten) and Factory M (Morinaraduketen), both of which continue to use traditional methods to prepare narazuke. Notably, the metagenomic profiles of the two manufacturers were similar, suggesting a common principle in the reproducible construction of microbial ecosystems in sake kasu environments. Fungal internal transcribed spacer (ITS) sequencing detected DNA from the genera Aspergillus (corresponding to koji) and Saccharomyces (corresponding to sake yeast), originating from the process of sake fermentation. Although these fungi were not isolated in a viable state, the halotolerant yeast Zygosaccharomyce rouxii was isolated from salted vegetables (Supplementary Table 3). Based on 16S rRNA gene sequencing, bacteria of the family Staphylococcaceae and various halotolerant bacteria and archaea were detected in aged sake kasu and salted vegetables, respectively. In contrast, a single LAB species from the Lactobacillaceae family, Fructilactobacillus fructivorans , dominated the microbial communities in the in-process and finished products. F. fructivorans was frequently isolated from narazuke samples using MRS and SI media (Supplementary Table 4 and Fig. 1d). Since this LAB was not present in the original ingredients, it may have been derived from the atmosphere in the factory or from the sake kasu used in the previous batch and propagated via the vegetables during the embedding process. The abundance of F. fructivorans temporarily decreased at the late stage in the in-process products due to the addition of new sake kasu. However, its abundance recovered in the finished products, suggesting the growth of LAB in sake kasu. Consistently, narazuke made from leafy celery showed a smaller dominance of F. fructivorans , likely due to the short embedding period. Thus, F. fructivorans was likely domesticated in each factory through repeated cycles of backslopping fermentation 7 . Under laboratory conditions, the F. fructivorans strains isolated from narazuke samples were not as tolerant to high NaCl concentrations as the type strain (Supplementary Fig. 3 and Fig. 2a). In contrast, narazuke strains were able to grow in environments containing ethanol concentrations of 15% or higher (Supplementary Fig. 4 and Fig. 2b). Furthermore, these isolates also exhibited ethanolphilicity 8 , showing faster growth in media containing 5–10% ethanol than in ethanol-free medium. The narazuke strain exhibited an abnormally elongated cell morphology, suggesting a possible link to growth delay in the absence of ethanol 8 (Fig. 1d). This high adaptation to ethanol likely allowed narazuke strains to exhibit dominant growth in sake kasu. The ethanol tolerance and ethanolphilicity of these narazuke strains were comparable to those of the F. fructivorans strains previously isolated from spoiled sake. However, they did not carry putative loss-of-function mutations in the accC2 gene, which are involved in the ethanolphilicity of sake strains 9 (Supplementary Fig. 5). A two-month laboratory-scale test using the in-process product of gourd narazuke and aged sake kasu reproduced embedding process #3 (Supplementary Fig. 6). We observed lactic acid production and the growth of ethanol-tolerant LAB, and F. fructivorans dominated the bacterial community at the end of the test (Supplementary Tables 5 and 6 and Fig. 2c-2f). When salted gourds before the embedding process were used instead, neither the growth of F. fructivorans nor the production of lactic acid was detected. These results suggest that lactic acid fermentation by F. fructivorans , which emerges as a result of backslopping, occurs in sake kasu. Moreover, beneficial metabolites not originally present in sake kasu but potentially produced by LAB, such as inosine, glutathione, and S -adenosylmethionine 10-12 , were detected in the samples after fermentation (Supplementary Table 7). Food byproducts are often used as animal feed, fuel, and fertilizer, but they can also be utilized in food, cosmetic, and pharmaceutical applications, enhancing their value 13,14 . This study focused on traditional foods prepared by reusing sake kasu, a major byproduct of sake fermentation. Although it is unclear when modern narazuke production was established, a technique for fermenting vegetables using sake kasu existed in ancient Japan. Ethanolphilic LAB, such as F. fructivorans , may have adapted to the high-ethanol niche environment of sake kasu, where most microorganisms cannot survive. LAB growth produces lactic acid and other metabolites, contributing to the flavor, nutritional profile, and preservation of fermented foods 15,16 . Hence, this research highlights the reuse of food byproducts that have been practiced since ancient times to reduce waste and create valuable, nutrient-rich products. By elucidating the microbial behaviors and dynamics in traditional foods, we can provide new sustainable value to these foods, which could pave the way for innovative approaches to food fermentation. Methods Amplicon sequencing analysis of narazuke samples The narazuke samples used in this study, ingredients (salted vegetables and sake kasu), in-process products, and finished products were provided by Naraya Honten (Factory N, Nara, Japan) and Morinaraduketen Co., Ltd. (Factory M, Nara, Japan). The salt water or sake kasu used to soak the vegetables was used for analysis. The in-process products at the early and late stages correspond to the samples during embedding processes #2 and #3, respectively. DNA extraction and ITS amplicon sequence analyses were performed by the Bioengineering Lab Co., Ltd. (Sagamihara, Japan), based on a previously reported method 17 , 18 . The ITS1 region was amplified using the ITS1F_KYO1/ITS2-KYO2 primer set. The V4 region of 16S rRNA was amplified using the 515f/806r primer set. Sequencing was conducted using a paired-end, 2×300 bp cycle run on a MiSeq sequencing system and a MiSeq reagent kit version 3 (Illumina). After quality filtering and chimera checking of the sequencing reads, operational taxonomic units (OTUs) were predicted using QIIME2. OTUs accounting for more than 1% of the total sequence reads were classified, while taxa with an abundance of less than 1% and those that could only be classified at the order level or higher were grouped together as “others.” Isolation and identification of microorganisms from narazuke samples To isolate eukaryotic microorganisms, 2 g of each sample was added to 15 mL of YPD medium (1% yeast extract, 2% casein peptone, and 2% glucose) supplemented with 0.01% chloramphenicol to inhibit bacterial growth, and incubated statically at 30°C for 3 days. Then, 100 µL of the diluted culture was spread on YPD plates supplemented with 0.01% chloramphenicol and incubated at 30°C for 3 days. Single colonies were isolated by repeated streaking onto YPD agar plates. The isolated microbes were identified by sequencing the ITS region of the rRNA gene using primers the ITS_1F (5′-GTAACAAGGTYTCCGT-3′) and ITS_1R (5′-CGTTCTTCATCGATG-3′) using genomic DNA as the template. To isolate LAB, 0.005% sodium azide and 0.005% cycloheximide were added to MRS broth (Biokar Diagnostics, France) to inhibit the growth of aerobic and eukaryotic microorganisms. Then, 2 g of the sample was added to 15 mL of this medium and cultured anaerobically at 30°C for 3 days using an AnaeroPack system (Mitsubishi Gas Chemical, Japan). Next, 100 µL of the diluted culture was spread on MRS plates supplemented with 0.005% sodium azide, 0.005% cycloheximide, and 0.5% CaCO 3 , followed by anaerobic incubation at 30°C for 3 days. Single colonies were isolated by repeated streaking onto MRS agar plates. To isolate ethanol-tolerant LAB, SI medium (1% yeast extract, 0.5% polypeptone, 2.5% glucose, 0.01% MgSO 4 ·7H 2 O, 0.00025% MnSO 4 ·4 ~ 6H 2 O, 0.00025% FeSO 4 ·7H 2 O, 0.005% sodium azide, 1% sodium acetate, 0.0005% mevalonic acid, 0.06% agar, pH 5.0) (The Brewing Society of Japan, Japan) containing 10% ethanol was also used. Two grams of the sample were added to 15 mL of SI medium and incubated anaerobically at 30°C for 7 days. Next, 100 µL of the diluted culture was spread on SI plates and incubated anaerobically at 30°C for 7 days. Single colonies were isolated by repeated streaking on SI agar plates. The isolated LAB were identified by sequencing the 16S rRNA V4 region using primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and PC3mod (5′-GGACTAHAGGGTATCTAAT-3′). Lab-scale narazuke fermentation test Salted vegetables (20 g, Factory N) or in-process products (early stage, Factory N) and 20 g of aged sake kasu (Factory N) were mixed in a 50 mL tube. The mixture was centrifuged at 3,000 rpm for 1 min to remove aged sake kasu from the inner walls of the test tubes. The mixture was cultured anaerobically at 25°C for 63 days. The mixture was stirred every seven days, and the sake kasu portion was sampled for subsequent measurements. To measure the lactic acid concentration, 0.1 g of each sample was suspended in 1 mL of RO water. The suspension was centrifuged at 12,000 rpm for 1 min and the supernatant was collected. The collected supernatant was adjusted to pH 8.0 using NaOH, and the lactic acid concentration was measured using Enzytec liquid d -/ l -lactic acid (R-Biopharm AG, Germany). The number of ethanol-tolerant LAB in the samples was measured using the drop-plate method. Briefly, a 0.05 g sample was suspended in 900 µL of 5% NaCl. A series of 10-fold dilutions was prepared by repeatedly suspending 100 µL of the suspension in 900 µL of 5% NaCl. Each dilution (10 µL) was spotted five times onto SI plates supplemented with 5% NaCl and 5% ethanol. After anaerobic incubation at 30°C for 14 days, colony counts were recorded to measure the number of viable bacteria. The general bacterial count of the samples was measured using the spread-plate method. Briefly, 0.05 g of each sample was suspended in 900 µL of 0.85% NaCl. A series of 10-fold dilutions was prepared by repeatedly suspending 100 µL of the suspension in 900 µL of 0.85% NaCl. Each dilution (100 µL) was spread on PCA plates (0.25% yeast extract, 0.5% casein peptone, and 0.1% glucose). After incubation at 30°C for 3 days, the colony count was recorded to measure the number of viable bacteria. Amplicon sequence analysis of the bacterial community was performed using the same method used for the narazuke samples (see above). LAB growth test The F. fructivorans strains used in the growth tests are listed in Supplementary Table 8. Briefly, LAB, other than the narazuke strains, were obtained from the NITE Biological Resource Center (NBRC, Japan) or the Japan Collection of Microorganisms (JCM) of the RIKEN BioResource Research Center (Japan). Each strain was anaerobically cultured at 30°C in SI medium supplemented with 0.1% Tween 80 and used for the experiments. LAB were added to 4 mL of SI medium containing 0.1% Tween-80 and ethanol at concentrations of 0%, 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, or 20%, or NaCl at concentrations of 0%, 2.5%, 5%, 7.5%, 10%, 12.5%, or 15% to adjust the optical density at 600 nm (OD 600 ) to 0.1. The samples were then cultured anaerobically at 30°C for 10 days. Scanning electron microscopy analysis Cultures were prepared by adding LAB to 2 mL of SI medium supplemented with 0.1% Tween-80 to adjust the OD 600 to 0.1, followed by anaerobic cultivation at 30°C. The bacterial culture (1 mL) in the late logarithmic phase was centrifuged at 10,000 rpm for 2 min and the supernatant was discarded. The pellet was mixed with 1 mL of 0.1 M phosphate buffer (pH 6.0) containing 1% glutaraldehyde and left to stand at room temperature for 2 h. The mixture was then centrifuged at 10,000 rpm for 2 min and the supernatant was discarded. The pellet was washed three times by adding 1 mL of 0.1 M phosphate buffer (pH 6.0) and left to stand for 15 min. After the final centrifugation, the supernatant was discarded, and the samples were observed under a Miniscope TM4000 Plus II tabletop scanning electron microscope (Hitachi, Japan). Declarations Data availability Data supporting the findings of this study are included in the article and its Supplementary Information files. All sequences determined in the amplicon sequence analysis were deposited in the DNA Data Bank of Japan (DDBJ) under accession numbers PRJDB19207-PRJDB19209. The 16S rRNA gene sequences of the narazuke strains (CS-2 and MS-5) were deposited in DDBJ under accession numbers LC848309 and LC848310, respectively. The accC2 gene sequences of the narazuke strains (CS-2 and MS-5) were deposited in DDBJ under accession numbers LC848311 and LC848312, respectively. Acknowledgements We would like to thank all the members of the Laboratory of Microbial Interaction at the Nara Institute of Science and Technology for their critical discussions and technical support. We would like to thank Editage for English language editing. This work was funded by the Foundation for the Nara Institute of Science and Technology (to Y.M.), Nara City (to M.M.), JST Program for co-creating startup ecosystem, Grant Number JPMJSF2315, Japan, and Nanto Bank, Ltd. (to D.W.). Additionally, this research received funding through generous contributions from supporters of our crowdfunding campaign. We deeply appreciate the support of Dr. Yoichiro Kanno (Sakenote Inc.) and all those who made this study possible. Author contributions Y.M., M.M., and D.W. designed and conceived the study. Y.M. and M.M. provided the experimental narazuke materials. M.Y. and D.W. performed the measurements, processed and analyzed the experimental data, and drafted the manuscript. All the authors have read and approved the final version of the manuscript. Competing interests The authors declare no competing interests. References Miyao, S. Japanese pickles “tsukemono” and their microorganisms. Jpn. J. Food Microbiol. 22 , 127–137 (2005) (Japanese). Nara Prefecture. Food culture of Nara. https://www3.pref.nara.jp/foodculture/index.htm (2024). Bamforth, C. W. Sake. In Food, Fermentation and Microorganisms , 143–153 (Blackwell Publishing Ltd., 2005). Kato, H. The world of sake, coming out “Fudoki” (4). J. Brew. Soc. Jpn. 102 , 352–363 (2007) (Japanese). Nara National Research Institute for Cultural Properties. Wooden Tablet Database. https://mokkanko.nabunken.go.jp/en/6AFITB11000362 (2018). Kitamura, Y. et al . Ethnic fermented foods and alcoholic beverages of Japan. In Ethnic Fermented Foods and Alcoholic Beverages of Asia , 193–236 (Springer India, 2016). Holzapfel, W. H. Appropriate starter culture technologies for small-scale fermentation in developing countries. Int. J. Food Microbiol. 75 , 197–212 (2002). Suzuki, K., Asano, S., Iijima, K. & Kitamoto, K. Sake and beer spoilage lactic acid bacteria — A review. J. Inst. Brew. 114 , 209–223 (2008). Wada, Y. & Mizoguchi, H. New insertion sequence in Lactobacillus fructivorans strains isolated from spoiled sake. J. Biosci. Bioeng. 103 , 399–405 (2007). Kilstrup, M., Hammer, K., Jensen, P. R. & Martinussen, J. Nucleotide metabolism and its control in lactic acid bacteria. FEMS Microbiol. Rev. 29 , 555–590 (2005). Pophaly, S. D., Singh, R., Pophaly, S. D., Kaushik, J. K. & Tomar, S. K. Current status and emerging role of glutathione in food grade lactic acid bacteria. Microb. Cell. Fact. 11 , 114 (2012). Chu, J., Qian, J., Zhuang, Y., Zhang, S. & Li, Y. Progress in the research of S -adenosyl-l-methionine production. Appl. Microbiol. Biotechnol. 97 , 41–49 (2013). Barba, F. J., Zhu, Z., Koubaa, M., Sant’Ana, A. S. & Orlien, V. Green alternative methods for the extraction of antioxidant bioactive compounds from winery wastes and byproducts: A review. Trends Food Sci. Technol. 49 , 96–109 (2016). Comunian, T. A., Silva, M. P. & Souza, C. J. F. The use of food byproducts as a novel for functional foods: Their use as ingredients and for the encapsulation process. Trends Food Sci. Technol. 108 , 269–280 (2021). Gänzle, M. G. Lactic metabolism revisited: metabolism of lactic acid bacteria in food fermentations and food spoilage. Curr. Opin. Food Sci. 2 , 106–117 (2015). Wang, Y., Zhang, C., Liu, F., Jin, Z. & Xia, X. Ecological succession and functional characteristics of lactic acid bacteria in traditional fermented foods. Crit. Rev. Food Sci. Nutr. 63 , 5841–5855 (2023). Caporaso, J. G. et al. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc. Natl. Acad. Sci. USA 108(supplement_1) , 4516–4522 (2011). Toju, H., Tanabe, A. S., Yamamoto, S. & Sato, H. High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples. PLoS One 7 , e40683 (2012). Additional Declarations No competing interests reported. Supplementary Files SupplementaryTables.xlsx SupplenemtaryFigs.pptx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5456531","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":412514389,"identity":"baa1625b-d090-4413-9a9e-9fa262ceb3b7","order_by":0,"name":"Daisuke Watanabe","email":"data:image/png;base64,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","orcid":"","institution":"Nara Institute of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Daisuke","middleName":"","lastName":"Watanabe","suffix":""},{"id":412514390,"identity":"b2bfb288-134d-495c-a260-89d85fcfd8de","order_by":1,"name":"Motomu Yoshioka","email":"","orcid":"","institution":"Nara Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Motomu","middleName":"","lastName":"Yoshioka","suffix":""},{"id":412514391,"identity":"bfa399e5-b3a7-464d-8282-5f65dceb526a","order_by":2,"name":"Yukihiko Masuda","email":"","orcid":"","institution":"Naraya Honten","correspondingAuthor":false,"prefix":"","firstName":"Yukihiko","middleName":"","lastName":"Masuda","suffix":""},{"id":412514392,"identity":"8a1ff905-367c-45cd-bbea-28c1b79ea88a","order_by":3,"name":"Mariko Mori","email":"","orcid":"","institution":"Morinaraduketen Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Mariko","middleName":"","lastName":"Mori","suffix":""}],"badges":[],"createdAt":"2024-11-14 22:38:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5456531/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5456531/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75890203,"identity":"31c3066d-c924-4a64-8159-353aa91182af","added_by":"auto","created_at":"2025-02-10 09:43:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":319298,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNarazuke making since ancient times, involving LAB. a,\u003c/strong\u003e Overview of how narazuke is made. The sake kasu, obtained as a byproduct of sake fermentation, is aged and used in making narazuke. Salted vegetables are repeatedly transferred into the aged sake kasu, during which the salt content is drawn out of the vegetables, and the flavor of the sake kasu is absorbed into them. The sake kasu used in embedding processes #2 and #3 is reused in the embedding processes #1 and #2 for the next production, respectively. Newly prepared sake kasu is only used in the embedding processes #3 and #4. \u003cstrong\u003eb,\u003c/strong\u003e Archaeological records of vegetables marinated in sake kasu. The wooden tablet excavated from the ruins of Prince Nagaya’s residence (684–729 CE)\u003csup\u003e5\u003c/sup\u003e records a list of gifts, such as vegetables marinated in sake kasu or ancient soy sauce. The letters indicated by the upper and lower dotted lines refer to gourds and eggplants marinated in sake kasu, respectively. \u003cstrong\u003ec,\u003c/strong\u003e Microbiota during narazuke making. The bars show relative abundance within each sample. The upper graph shows fungal ITS data at the genus level and the lower graph shows bacterial and archaeal 16S rDNA data at the family level. The narazuke samples, ingredients (salted vegetables and sake kasu), in-process products, and finished products were provided by Naraya Honten (Factory N; Nara, Japan) and Morinaraduketen Co., Ltd. (Factory M; Nara, Japan), both of which are located within 5 km of the Heijo Palace site. \u003cstrong\u003ed,\u003c/strong\u003e Scanning electron microscopy images of the type strain (NBRC 13954\u003csup\u003eT\u003c/sup\u003e, upper) and the narazuke strain (MS-5, lower) of \u003cem\u003eF. fructivorans\u003c/em\u003e logarithmically growing in SI medium supplemented with 0.1% Tween-80. The red arrow indicates the abnormally elongated cells observed only in the narazuke strain. Scale bars, 10 μm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5456531/v1/522d69271aa59d48fe43f35e.png"},{"id":75888619,"identity":"f557d0ae-1b2b-4411-b514-afe58edcfb52","added_by":"auto","created_at":"2025-02-10 09:35:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":138011,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacteristics and roles of the narazuke lactic acid bacteria (LAB) strain. a, \u003c/strong\u003eLAB growth under different concentrations of NaCl. The \u003cem\u003eF. fructivorans\u003c/em\u003e type strain (NBRC 13954\u003csup\u003eT\u003c/sup\u003e, left) and narazuke strain (MS-5, right) were cultured in SI medium supplemented with 0.1% Tween-80 containing NaCl at concentrations of 0% (green), 5% (blue), or 10% (pink). Data represent the average values ± standard deviations calculated from three independent experiments. \u003cstrong\u003eb, \u003c/strong\u003eLAB growth under different ethanol concentrations. The \u003cem\u003eF. fructivorans\u003c/em\u003e type strain (NBRC 13954\u003csup\u003eT\u003c/sup\u003e, left) and narazuke strain (MS-5, right) were cultured in SI medium with 0.1% Tween-80 containing ethanol at concentrations of 0% (green), 5% (blue), 10% (pink), or 15% (orange). Data represent the average values ± standard deviations calculated from three independent experiments. \u003cstrong\u003ec-e,\u003c/strong\u003e Lab-scale narazuke-making test using the in-process product of narazuke gourds (navy blue) and salted gourds before the embedding process (light blue). Data on the lactic acid concentrations (c), growth of ethanol-tolerant LAB (d), and growth of aerobic bacteria (e) are shown. Data represent the average values ± standard deviations calculated from three independent experiments. It should be noted that when salted gourds before the embedding process were used, ethanol-tolerant LAB were not detected for 63 days. \u003cstrong\u003ef, \u003c/strong\u003eMicrobiota in the lab-scale narazuke-making test at 63 days after inoculation. The graph shows bacterial and archaeal 16S rDNA data at the family level. Data represent the average values calculated from three independent experiments.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5456531/v1/56e54bcf66b5619a024cb882.png"},{"id":75890204,"identity":"e41955c4-d554-466e-839a-c2316749d014","added_by":"auto","created_at":"2025-02-10 09:43:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":903955,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5456531/v1/06a52bfd-f077-44e6-a075-bcd626d05d68.pdf"},{"id":75888617,"identity":"eed3b356-b2a5-4722-bdc6-48cc50b8a869","added_by":"auto","created_at":"2025-02-10 09:35:05","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":24329,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5456531/v1/b45b7b8bb7399786eab3465e.xlsx"},{"id":75888620,"identity":"e0fd4704-e123-470e-9aac-8e48e3599d1c","added_by":"auto","created_at":"2025-02-10 09:35:05","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16125489,"visible":true,"origin":"","legend":"","description":"","filename":"SupplenemtaryFigs.pptx","url":"https://assets-eu.researchsquare.com/files/rs-5456531/v1/6d48dd72ede3a4bc851b7f25.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Heritage sustainable lactic acid fermentation in a byproduct of sake making","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNarazuke, a traditional Japanese preserved food, is prepared by repeatedly embedding salted gourds or other vegetables in aged sake kasu (Supplementary Fig. 1 and Fig. 1a)\u003csup\u003e1,2\u003c/sup\u003e. Its prototype was established by the early 8th century, when the capital of ancient Japan, Heijo-kyo, was located in present-day Nara (Supplementary Fig. 2). In the northeastern part of the Heijo Imperial Palace, there was an office responsible for fermentation, where sake and related fermented foods, such as rice vinegar, were systematically produced for presentations to the emperor and to entertain guests from overseas\u003csup\u003e3,4\u003c/sup\u003e. Thus, it is natural to assume that the byproducts of sake were reused in this region. Moreover, records of vegetables marinated in sake kasu have been found on wooden tablets excavated from the ruins of Prince Nagaya’s residence (684–729 CE) in Heijo-kyo (Fig. 1b)\u003csup\u003e5\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn orthodox narazuke making, vegetables are first exposed to a saturated salt solution and are then embedded in aged sake kasu, which contains an ethanol concentration of at least 8%, for a year or more. It remains unclear whether microbial fermentation occurs under such harsh conditions; however, several species of halotolerant yeast and lactic acid bacteria (LAB) have been isolated in a few previous reports\u003csup\u003e6\u003c/sup\u003e. It has not been determined whether these microorganisms actually grow and participate in the fermentation process during narazuke making. To investigate the microbial ecosystem in sake kasu, we conducted a microbiome analysis of samples obtained from the ingredients, in-process products, and finished products of narazuke (Supplementary Tables 1 and 2 and Fig. 1c). The samples were independently collected from two manufacturers located within 5 km of the Heijo Palace site, Factory N (Naraya Honten) and Factory M (Morinaraduketen), both of which continue to use traditional methods to prepare narazuke. Notably, the metagenomic profiles of the two manufacturers were similar, suggesting a common principle in the reproducible construction of microbial ecosystems in sake kasu environments.\u003c/p\u003e\n\u003cp\u003eFungal internal transcribed spacer (ITS) sequencing detected DNA from the genera \u003cem\u003eAspergillus\u003c/em\u003e (corresponding to koji) and \u003cem\u003eSaccharomyces\u003c/em\u003e (corresponding to sake yeast), originating from the process of sake fermentation. Although these fungi were not isolated in a viable state, the halotolerant yeast\u0026nbsp;\u003cem\u003eZygosaccharomyce rouxii\u0026nbsp;\u003c/em\u003ewas isolated from salted vegetables (Supplementary Table 3). Based on 16S rRNA gene sequencing, bacteria of the family Staphylococcaceae and various halotolerant bacteria and archaea were detected in aged sake kasu and salted vegetables, respectively. In contrast, a single LAB species from the Lactobacillaceae family, \u003cem\u003eFructilactobacillus fructivorans\u003c/em\u003e, dominated the microbial communities in the in-process and finished products. \u003cem\u003eF. fructivorans\u0026nbsp;\u003c/em\u003ewas frequently isolated from narazuke samples using\u0026nbsp;MRS and SI media\u0026nbsp;(Supplementary Table 4 and Fig. 1d). Since this LAB was not present in the original ingredients, it may have been derived from the atmosphere in the factory or from the sake kasu used in the previous batch and propagated via the vegetables during the embedding process. The abundance of \u003cem\u003eF. fructivorans\u003c/em\u003e temporarily decreased at the late stage in the in-process products due to the addition of new sake kasu.\u0026nbsp;However, its abundance recovered in the finished products, suggesting the growth\u0026nbsp;of LAB in sake\u0026nbsp;kasu. Consistently, narazuke made from leafy celery showed a smaller dominance of \u003cem\u003eF. fructivorans\u003c/em\u003e, likely due to the short embedding period. Thus, \u003cem\u003eF. fructivorans\u003c/em\u003e was likely domesticated in each factory through repeated cycles of backslopping fermentation\u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eUnder laboratory conditions, the \u003cem\u003eF. fructivorans\u003c/em\u003e strains isolated from narazuke samples were not as tolerant to high NaCl concentrations as the type strain (Supplementary Fig. 3 and Fig. 2a). In contrast, narazuke strains were able to grow in environments containing ethanol concentrations of 15% or higher (Supplementary Fig. 4 and Fig. 2b). Furthermore, these isolates also exhibited ethanolphilicity\u003csup\u003e8\u003c/sup\u003e, showing faster growth in media containing 5–10% ethanol than in ethanol-free medium. The narazuke strain exhibited an abnormally elongated cell morphology, suggesting a possible link to growth delay in the absence of ethanol\u003csup\u003e8\u003c/sup\u003e (Fig. 1d). This high adaptation to ethanol likely allowed narazuke strains to exhibit dominant growth in sake kasu. The ethanol tolerance and ethanolphilicity of these narazuke strains were comparable to those of the \u003cem\u003eF. fructivorans\u003c/em\u003e strains previously isolated from spoiled sake. However, they did not carry putative loss-of-function mutations in the \u003cem\u003eaccC2\u003c/em\u003e gene, which are involved in the ethanolphilicity of sake strains\u003csup\u003e9\u003c/sup\u003e (Supplementary Fig. 5). A two-month laboratory-scale test using the in-process product of gourd narazuke and aged sake kasu reproduced embedding process #3 (Supplementary Fig. 6). We observed lactic acid production and the growth of ethanol-tolerant LAB, and \u003cem\u003eF. fructivorans\u003c/em\u003e dominated the bacterial community at the end of the test (Supplementary Tables 5 and 6 and Fig. 2c-2f). When salted gourds before the embedding process were used instead, neither the growth of \u003cem\u003eF. fructivorans\u003c/em\u003e nor the production of lactic acid was detected. These results suggest that lactic acid fermentation by \u003cem\u003eF. fructivorans\u003c/em\u003e, which emerges as a result of backslopping, occurs in sake kasu. Moreover, beneficial metabolites not originally present in sake kasu but potentially produced by LAB, such as inosine, glutathione, and \u003cem\u003eS\u003c/em\u003e-adenosylmethionine\u003csup\u003e10-12\u003c/sup\u003e, were detected in the samples after fermentation (Supplementary Table 7).\u003c/p\u003e\n\u003cp\u003eFood byproducts are often used as animal feed, fuel, and fertilizer, but they can also be utilized in food, cosmetic, and pharmaceutical applications, enhancing their value\u003csup\u003e13,14\u003c/sup\u003e. This study focused on traditional foods prepared by reusing sake kasu, a major byproduct of sake fermentation. Although it is unclear when modern narazuke production was established, a technique for fermenting vegetables using sake kasu existed in ancient Japan. Ethanolphilic LAB, such as \u003cem\u003eF. fructivorans\u003c/em\u003e, may have adapted to the high-ethanol niche environment of sake kasu, where most microorganisms cannot survive. LAB growth produces lactic acid and other metabolites, contributing to the flavor, nutritional profile, and preservation of fermented foods\u003csup\u003e15,16\u003c/sup\u003e. Hence, this research highlights the reuse of food byproducts that have been practiced since ancient times to reduce waste and create valuable, nutrient-rich products. By elucidating the microbial behaviors and dynamics in traditional foods, we can provide new sustainable value to these foods, which could pave the way for innovative approaches to food fermentation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eAmplicon sequencing analysis of narazuke samples\u003c/h2\u003e \u003cp\u003eThe narazuke samples used in this study, ingredients (salted vegetables and sake kasu), in-process products, and finished products were provided by Naraya Honten (Factory N, Nara, Japan) and Morinaraduketen Co., Ltd. (Factory M, Nara, Japan). The salt water or sake kasu used to soak the vegetables was used for analysis. The in-process products at the early and late stages correspond to the samples during embedding processes #2 and #3, respectively.\u003c/p\u003e \u003cp\u003eDNA extraction and ITS amplicon sequence analyses were performed by the Bioengineering Lab Co., Ltd. (Sagamihara, Japan), based on a previously reported method\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The ITS1 region was amplified using the ITS1F_KYO1/ITS2-KYO2 primer set. The V4 region of 16S rRNA was amplified using the 515f/806r primer set. Sequencing was conducted using a paired-end, 2\u0026times;300 bp cycle run on a MiSeq sequencing system and a MiSeq reagent kit version 3 (Illumina). After quality filtering and chimera checking of the sequencing reads, operational taxonomic units (OTUs) were predicted using QIIME2. OTUs accounting for more than 1% of the total sequence reads were classified, while taxa with an abundance of less than 1% and those that could only be classified at the order level or higher were grouped together as \u0026ldquo;others.\u0026rdquo;\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and identification of microorganisms from narazuke samples\u003c/h2\u003e \u003cp\u003eTo isolate eukaryotic microorganisms, 2 g of each sample was added to 15 mL of YPD medium (1% yeast extract, 2% casein peptone, and 2% glucose) supplemented with 0.01% chloramphenicol to inhibit bacterial growth, and incubated statically at 30\u0026deg;C for 3 days. Then, 100 \u0026micro;L of the diluted culture was spread on YPD plates supplemented with 0.01% chloramphenicol and incubated at 30\u0026deg;C for 3 days. Single colonies were isolated by repeated streaking onto YPD agar plates. The isolated microbes were identified by sequencing the ITS region of the rRNA gene using primers the ITS_1F (5\u0026prime;-GTAACAAGGTYTCCGT-3\u0026prime;) and ITS_1R (5\u0026prime;-CGTTCTTCATCGATG-3\u0026prime;) using genomic DNA as the template.\u003c/p\u003e \u003cp\u003eTo isolate LAB, 0.005% sodium azide and 0.005% cycloheximide were added to MRS broth (Biokar Diagnostics, France) to inhibit the growth of aerobic and eukaryotic microorganisms. Then, 2 g of the sample was added to 15 mL of this medium and cultured anaerobically at 30\u0026deg;C for 3 days using an AnaeroPack system (Mitsubishi Gas Chemical, Japan). Next, 100 \u0026micro;L of the diluted culture was spread on MRS plates supplemented with 0.005% sodium azide, 0.005% cycloheximide, and 0.5% CaCO\u003csub\u003e3\u003c/sub\u003e, followed by anaerobic incubation at 30\u0026deg;C for 3 days. Single colonies were isolated by repeated streaking onto MRS agar plates. To isolate ethanol-tolerant LAB, SI medium (1% yeast extract, 0.5% polypeptone, 2.5% glucose, 0.01% MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 0.00025% MnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;4\u0026thinsp;~\u0026thinsp;6H\u003csub\u003e2\u003c/sub\u003eO, 0.00025% FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 0.005% sodium azide, 1% sodium acetate, 0.0005% mevalonic acid, 0.06% agar, pH 5.0) (The Brewing Society of Japan, Japan) containing 10% ethanol was also used. Two grams of the sample were added to 15 mL of SI medium and incubated anaerobically at 30\u0026deg;C for 7 days. Next, 100 \u0026micro;L of the diluted culture was spread on SI plates and incubated anaerobically at 30\u0026deg;C for 7 days. Single colonies were isolated by repeated streaking on SI agar plates. The isolated LAB were identified by sequencing the 16S rRNA V4 region using primers 515F (5\u0026prime;-GTGCCAGCMGCCGCGGTAA-3\u0026prime;) and PC3mod (5\u0026prime;-GGACTAHAGGGTATCTAAT-3\u0026prime;).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLab-scale narazuke fermentation test\u003c/h3\u003e\n\u003cp\u003eSalted vegetables (20 g, Factory N) or in-process products (early stage, Factory N) and 20 g of aged sake kasu (Factory N) were mixed in a 50 mL tube. The mixture was centrifuged at 3,000 rpm for 1 min to remove aged sake kasu from the inner walls of the test tubes. The mixture was cultured anaerobically at 25\u0026deg;C for 63 days. The mixture was stirred every seven days, and the sake kasu portion was sampled for subsequent measurements.\u003c/p\u003e \u003cp\u003eTo measure the lactic acid concentration, 0.1 g of each sample was suspended in 1 mL of RO water. The suspension was centrifuged at 12,000 rpm for 1 min and the supernatant was collected. The collected supernatant was adjusted to pH 8.0 using NaOH, and the lactic acid concentration was measured using Enzytec liquid \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ed\u003c/span\u003e-/\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-lactic acid (R-Biopharm AG, Germany).\u003c/p\u003e \u003cp\u003eThe number of ethanol-tolerant LAB in the samples was measured using the drop-plate method. Briefly, a 0.05 g sample was suspended in 900 \u0026micro;L of 5% NaCl. A series of 10-fold dilutions was prepared by repeatedly suspending 100 \u0026micro;L of the suspension in 900 \u0026micro;L of 5% NaCl. Each dilution (10 \u0026micro;L) was spotted five times onto SI plates supplemented with 5% NaCl and 5% ethanol. After anaerobic incubation at 30\u0026deg;C for 14 days, colony counts were recorded to measure the number of viable bacteria.\u003c/p\u003e \u003cp\u003eThe general bacterial count of the samples was measured using the spread-plate method. Briefly, 0.05 g of each sample was suspended in 900 \u0026micro;L of 0.85% NaCl. A series of 10-fold dilutions was prepared by repeatedly suspending 100 \u0026micro;L of the suspension in 900 \u0026micro;L of 0.85% NaCl. Each dilution (100 \u0026micro;L) was spread on PCA plates (0.25% yeast extract, 0.5% casein peptone, and 0.1% glucose). After incubation at 30\u0026deg;C for 3 days, the colony count was recorded to measure the number of viable bacteria.\u003c/p\u003e \u003cp\u003eAmplicon sequence analysis of the bacterial community was performed using the same method used for the narazuke samples (see above).\u003c/p\u003e\n\u003ch3\u003eLAB growth test\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eF. fructivorans\u003c/em\u003e strains used in the growth tests are listed in Supplementary Table\u0026nbsp;8. Briefly, LAB, other than the narazuke strains, were obtained from the NITE Biological Resource Center (NBRC, Japan) or the Japan Collection of Microorganisms (JCM) of the RIKEN BioResource Research Center (Japan). Each strain was anaerobically cultured at 30\u0026deg;C in SI medium supplemented with 0.1% Tween 80 and used for the experiments. LAB were added to 4 mL of SI medium containing 0.1% Tween-80 and ethanol at concentrations of 0%, 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, or 20%, or NaCl at concentrations of 0%, 2.5%, 5%, 7.5%, 10%, 12.5%, or 15% to adjust the optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) to 0.1. The samples were then cultured anaerobically at 30\u0026deg;C for 10 days.\u003c/p\u003e\n\u003ch3\u003eScanning electron microscopy analysis\u003c/h3\u003e\n\u003cp\u003eCultures were prepared by adding LAB to 2 mL of SI medium supplemented with 0.1% Tween-80 to adjust the OD\u003csub\u003e600\u003c/sub\u003e to 0.1, followed by anaerobic cultivation at 30\u0026deg;C. The bacterial culture (1 mL) in the late logarithmic phase was centrifuged at 10,000 rpm for 2 min and the supernatant was discarded. The pellet was mixed with 1 mL of 0.1 M phosphate buffer (pH 6.0) containing 1% glutaraldehyde and left to stand at room temperature for 2 h. The mixture was then centrifuged at 10,000 rpm for 2 min and the supernatant was discarded. The pellet was washed three times by adding 1 mL of 0.1 M phosphate buffer (pH 6.0) and left to stand for 15 min. After the final centrifugation, the supernatant was discarded, and the samples were observed under a Miniscope TM4000 Plus II tabletop scanning electron microscope (Hitachi, Japan).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the findings of this study are included in the article and its Supplementary Information files. All sequences determined in the amplicon sequence analysis were deposited in the DNA Data Bank of Japan (DDBJ) under accession numbers PRJDB19207-PRJDB19209. The 16S rRNA gene sequences of the narazuke strains (CS-2 and MS-5) were deposited in DDBJ under accession numbers LC848309 and LC848310, respectively. The \u003cem\u003eaccC2\u003c/em\u003e gene sequences of the narazuke strains (CS-2 and MS-5) were deposited in DDBJ under accession numbers LC848311 and LC848312, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank all the members of the Laboratory of Microbial Interaction at the Nara Institute of Science and Technology for their critical discussions and technical support. We would like to thank Editage for English language editing. This work was funded by the Foundation for the Nara Institute of Science and Technology (to Y.M.), Nara City (to M.M.), JST Program for co-creating startup ecosystem, Grant Number JPMJSF2315, Japan, and Nanto Bank, Ltd. (to D.W.). Additionally, this research received funding through generous contributions from supporters of our crowdfunding campaign. We deeply appreciate the support of Dr. Yoichiro Kanno (Sakenote Inc.) and all those who made this study possible.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.M., M.M., and D.W. designed and conceived the study. Y.M. and M.M. provided the experimental narazuke materials. M.Y. and D.W. performed the measurements, processed and analyzed the experimental data, and drafted the manuscript. All the authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMiyao, S. Japanese pickles \u0026ldquo;tsukemono\u0026rdquo; and their microorganisms. \u003cem\u003eJpn. J. 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High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples. \u003cem\u003ePLoS One\u003c/em\u003e\u003cstrong\u003e7\u003c/strong\u003e, e40683 (2012).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5456531/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5456531/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eReusing byproducts has been an issue ever since sake production was established in ancient Japan. Sake kasu, an ethanol-containing solid byproduct remaining after pressing sake, is widely used as a marinade base to enhance flavor and preservation. Here, we isolated ethanolphiliclactic acid bacteria that dominate the microbial community in sake kasu-based traditional foods. This study highlights the potential existence of heritage, sustainable fermentation to transform byproducts into high-value foods.\u003c/p\u003e","manuscriptTitle":"Heritage sustainable lactic acid fermentation in a byproduct of sake making","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-10 09:35:00","doi":"10.21203/rs.3.rs-5456531/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"271b7558-3977-43d2-a330-d4887a674a52","owner":[],"postedDate":"February 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":43990180,"name":"Biological sciences/Microbiology/Applied microbiology"},{"id":43990181,"name":"Biological sciences/Microbiology/Communities/Microbial ecology"}],"tags":[],"updatedAt":"2025-02-10T09:35:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-10 09:35:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5456531","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5456531","identity":"rs-5456531","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}