Characterization and 16S rRNA-Based Identification of Antimicrobial Peptide-Producing Lactic Acid Bacteria from Traditional Nigerian Non-Dairy Fermentations

Characterization and 16S rRNA-Based Identification of Antimicrobial Peptide-Producing Lactic Acid Bacteria from Traditional Nigerian Non-Dairy Fermentations | 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 Characterization and 16S rRNA-Based Identification of Antimicrobial Peptide-Producing Lactic Acid Bacteria from Traditional Nigerian Non-Dairy Fermentations Oluwabukola Atinuke Popoola, Olubunmi Olaitan Agarry, Abimbola Ayodeji Orukotan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7419622/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 Background Lactic acid bacteria (LAB) produce antimicrobial peptides (AMPs) with activity against foodborne and clinically relevant pathogens, offering clean-label alternatives to chemical preservatives and potential tools to combat antimicrobial resistance. These bioactives have applications in food safety, medicine, and agriculture, contributing to global sustainability goals. Traditional Nigerian non-dairy fermented foods remain an underexplored source of such bacteria. This study aimed to isolate, identify, and evaluate AMP-producing LAB from soy milk, tigernut milk, and sorghum gruel for their antimicrobial activity, and to assess their potential for novel strain discovery. Methods Spontaneously fermented soy milk, tiger nut milk, and sorghum gruel were prepared under laboratory conditions, yielding 174 LAB isolates. These were characterized by Gram reaction, catalase, spore formation, motility, and carbohydrate fermentation tests. Antimicrobial activity was initially assessed using agar spot tests against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922. Active isolates underwent agar well diffusion assays against S. aureus , E. coli , Pseudomonas aeruginosa ATCC 27853, and Salmonella Typhi ATCC 6539. Neutralization with catalase and pH adjustment distinguished peptide-mediated inhibition. Six representative AMP-producing isolates were identified by 16S rRNA gene sequencing and phylogenetic analysis, with sequences deposited in GenBank (PV983358–PV983363). Results Of the 96 presumptive LAB screened, 58 (60.4%) inhibited at least one target pathogen. Agar well diffusion assays confirmed AMP production in 13 isolates, with inhibition zones of 10.33–15.00 mm. Peptide-mediated inhibition was retained in the 13 isolates following catalase and pH neutralization. Sequencing identified isolates as Lactiplantibacillus plantarum , Lactobacillus fermentum , and Limosilactobacillus fermentum . One strain, L. fermentum Atinukensis-TNA14, shared only 93.54% sequence identity with its closest type strain, indicating potential novelty and warranting whole genome sequencing. Conclusion Traditional Nigerian non-dairy fermented foods harbour diverse LAB with significant AMP-producing capacity, including a putative novel species, underscoring their potential as sources of natural food biopreservatives. Future studies will focus on whole genome sequencing and functional characterisation of the novel strain, as well as evaluating the stability and efficacy of its AMPs in model food systems. Lactic acid bacteria Antimicrobial peptides Non-dairy fermented foods Soy milk Sorghum gruel Biopreservation 16S rRNA sequencing Phylogenetic analysis Full Text Additional Declarations No competing interests reported. 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-7419622","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":511748371,"identity":"2925dd26-1bd4-485a-9a19-dc5b6de0eba1","order_by":0,"name":"Oluwabukola Atinuke Popoola","email":"data:image/png;base64,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","orcid":"","institution":"National Biotechnology Research and Development Agency (NBRDA)","correspondingAuthor":true,"prefix":"","firstName":"Oluwabukola","middleName":"Atinuke","lastName":"Popoola","suffix":""},{"id":511748372,"identity":"9ab4d707-d1e6-4f5d-b783-a7f90b2ebcf8","order_by":1,"name":"Olubunmi Olaitan Agarry","email":"","orcid":"","institution":"University of Abuja","correspondingAuthor":false,"prefix":"","firstName":"Olubunmi","middleName":"Olaitan","lastName":"Agarry","suffix":""},{"id":511748373,"identity":"1e7c7a74-3edb-47b2-9fbc-3fe4da89416f","order_by":2,"name":"Abimbola Ayodeji Orukotan","email":"","orcid":"","institution":"Lagos State University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Abimbola","middleName":"Ayodeji","lastName":"Orukotan","suffix":""}],"badges":[],"createdAt":"2025-08-20 17:08:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7419622/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7419622/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94172881,"identity":"9ecf50d6-511a-4a86-97ad-baf074a465f1","added_by":"auto","created_at":"2025-10-23 07:47:40","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":950089,"visible":true,"origin":"","legend":"","description":"","filename":"Characterisationand16SRNABasedIdentificationofAMPProducingLAB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7419622/v1_covered_d7937bb8-030c-4bb1-ab03-aee2427be629.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characterization and 16S rRNA-Based Identification of Antimicrobial Peptide-Producing Lactic Acid Bacteria from Traditional Nigerian Non-Dairy Fermentations","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"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":"Lactic acid bacteria, Antimicrobial peptides, Non-dairy fermented foods, Soy milk, Sorghum gruel, Biopreservation, 16S rRNA sequencing, Phylogenetic analysis","lastPublishedDoi":"10.21203/rs.3.rs-7419622/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7419622/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eLactic acid bacteria (LAB) produce antimicrobial peptides (AMPs) with activity against foodborne and clinically relevant pathogens, offering clean-label alternatives to chemical preservatives and potential tools to combat antimicrobial resistance. These bioactives have applications in food safety, medicine, and agriculture, contributing to global sustainability goals. Traditional Nigerian non-dairy fermented foods remain an underexplored source of such bacteria. This study aimed to isolate, identify, and evaluate AMP-producing LAB from soy milk, tigernut milk, and sorghum gruel for their antimicrobial activity, and to assess their potential for novel strain discovery.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eSpontaneously fermented soy milk, tiger nut milk, and sorghum gruel were prepared under laboratory conditions, yielding 174 LAB isolates. These were characterized by Gram reaction, catalase, spore formation, motility, and carbohydrate fermentation tests. Antimicrobial activity was initially assessed using agar spot tests against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923 and \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922. Active isolates underwent agar well diffusion assays against \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC 27853, and \u003cem\u003eSalmonella Typhi\u003c/em\u003e ATCC 6539. Neutralization with catalase and pH adjustment distinguished peptide-mediated inhibition. Six representative AMP-producing isolates were identified by 16S rRNA gene sequencing and phylogenetic analysis, with sequences deposited in GenBank (PV983358\u0026ndash;PV983363).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eOf the 96 presumptive LAB screened, 58 (60.4%) inhibited at least one target pathogen. Agar well diffusion assays confirmed AMP production in 13 isolates, with inhibition zones of 10.33\u0026ndash;15.00 mm. Peptide-mediated inhibition was retained in the 13 isolates following catalase and pH neutralization. Sequencing identified isolates as \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e, \u003cem\u003eLactobacillus fermentum\u003c/em\u003e, and \u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e. One strain, \u003cem\u003eL. fermentum\u003c/em\u003e Atinukensis-TNA14, shared only 93.54% sequence identity with its closest type strain, indicating potential novelty and warranting whole genome sequencing.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eTraditional Nigerian non-dairy fermented foods harbour diverse LAB with significant AMP-producing capacity, including a putative novel species, underscoring their potential as sources of natural food biopreservatives. Future studies will focus on whole genome sequencing and functional characterisation of the novel strain, as well as evaluating the stability and efficacy of its AMPs in model food systems.\u003c/p\u003e","manuscriptTitle":"Characterization and 16S rRNA-Based Identification of Antimicrobial Peptide-Producing Lactic Acid Bacteria from Traditional Nigerian Non-Dairy Fermentations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 20:10:11","doi":"10.21203/rs.3.rs-7419622/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":"3c52a683-713e-4951-a9d7-cee3dd7f1af7","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-23T07:39:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-12 20:10:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7419622","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7419622","identity":"rs-7419622","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}