Anaerobic Germination and Bacteriocin-like Inhibitory Substance (BLIS) Production by Bacillus subtilis BSC35 Enable Inhibition of Clostridioides difficile and Clostridium perfringens

Anaerobic Germination and Bacteriocin-like Inhibitory Substance (BLIS) Production by Bacillus subtilis BSC35 Enable Inhibition of Clostridioides difficile and Clostridium perfringens | 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 Research Article Anaerobic Germination and Bacteriocin-like Inhibitory Substance (BLIS) Production by Bacillus subtilis BSC35 Enable Inhibition of Clostridioides difficile and Clostridium perfringens Yong Hwi Kwon, Beomseok Park, Seung Hyeon Ji, Myeong Seong Cha, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8485171/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 Bacteriocin-like inhibitory substances (BLIS) from spore-forming probiotics are promising natural antimicrobials for food safety and gut microbial management. While BLIS produced by Bacillus subtilis BSC35 has previously been shown to inhibit Clostridium perfringens, the production and activity of BLIS from spore-initiated cultures remain unexplored. In this study, we first evaluated the antibacterial activity of a bacteriocin-like inhibitory substance (BLIS BSC35) produced by B. subtilis BSC35 against Clostridiodes difficile of multiple ribotypes. Cell-free supernatants (CFS) of BSC35 exhibited consistent and quantifiable inhibition zones (≥18 mm) in well diffusion assays and inhibited bacterial growth. Transmission electron microscopy revealed bactericidal effects, including disruption of cell walls, membrane separation, and leakage of cytoplasmic contents. Secondly, we show that B. subtilis BSC35 efficiently produced heat-resistant spores under both aerobic and anaerobic conditions. And finally, CFS from spore-derived cultures retained antibacterial activity against C. difficile and C. perfringens. These findings demonstrate that spore-initiated B. subtilis BSC35 produces stable BLIS with broad inhibitory activity against the two clostridial species, supporting its potential application as a probiotic or bioprotective culture in food matrices and gastrointestinal contexts. C. difficile B. subtilis BSC35 spore BLIS C. perfringens Full Text Additional Declarations Competing interest reported. Kwang-Pyo Kim reports financial support was provided by National Research Foundation of Korea. Kwang-Pyo Kim has patent pending to 1020230178292. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Supplementary Files SupplementaryFigureS1.docx 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8485171","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":611461595,"identity":"45e1e346-9573-400d-a8ab-b15627129876","order_by":0,"name":"Yong Hwi Kwon","email":"","orcid":"","institution":"Jeonbuk National University","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"Hwi","lastName":"Kwon","suffix":""},{"id":611461596,"identity":"643493a6-415c-4ba7-9f85-d4d3ce9af4ac","order_by":1,"name":"Beomseok Park","email":"","orcid":"","institution":"Jeonbuk National University","correspondingAuthor":false,"prefix":"","firstName":"Beomseok","middleName":"","lastName":"Park","suffix":""},{"id":611461597,"identity":"62f86ddc-b350-4ffd-87b7-c17e7956e4a8","order_by":2,"name":"Seung Hyeon Ji","email":"","orcid":"","institution":"Jeonbuk National University","correspondingAuthor":false,"prefix":"","firstName":"Seung","middleName":"Hyeon","lastName":"Ji","suffix":""},{"id":611461598,"identity":"b366e989-33e7-4446-a224-0207eaf37448","order_by":3,"name":"Myeong Seong Cha","email":"","orcid":"","institution":"Jeonbuk National University","correspondingAuthor":false,"prefix":"","firstName":"Myeong","middleName":"Seong","lastName":"Cha","suffix":""},{"id":611461599,"identity":"a84d7b78-4e46-4d3c-9f39-064655126d0d","order_by":4,"name":"Manasa Gaddapara","email":"","orcid":"","institution":"Jeonbuk National University","correspondingAuthor":false,"prefix":"","firstName":"Manasa","middleName":"","lastName":"Gaddapara","suffix":""},{"id":611461600,"identity":"366fb325-e049-45b6-b3a6-957cb086c7ce","order_by":5,"name":"Arun K. 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While BLIS produced by Bacillus subtilis BSC35 has previously been shown to inhibit Clostridium perfringens, the production and activity of BLIS from spore-initiated cultures remain unexplored. In this study, we first evaluated the antibacterial activity of a bacteriocin-like inhibitory substance (BLIS BSC35) produced by B. subtilis BSC35 against Clostridiodes difficile of multiple ribotypes. Cell-free supernatants (CFS) of BSC35 exhibited consistent and quantifiable inhibition zones (≥18 mm) in well diffusion assays and inhibited bacterial growth. Transmission electron microscopy revealed bactericidal effects, including disruption of cell walls, membrane separation, and leakage of cytoplasmic contents. Secondly, we show that B. subtilis BSC35 efficiently produced heat-resistant spores under both aerobic and anaerobic conditions. And finally, CFS from spore-derived cultures retained antibacterial activity against C. difficile and C. perfringens. 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