Molecular identification of lactic acid bacteria involved in Ghanaian cocoa bean fermentation

Molecular identification of lactic acid bacteria involved in Ghanaian cocoa bean fermentation | 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 Molecular identification of lactic acid bacteria involved in Ghanaian cocoa bean fermentation Lawrence Asamoah, Hilary Domakyaara Zakpaa, Clement Osei This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7864986/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 Cocoa is a vital cash crop in Ghana, contributing approximately 15% of the country’s foreign exchange earnings and employing over 800,000 farmers. Fermentation significantly influences cocoa quality and flavour, yet controlling this process remains a challenge. This study investigated lactic acid bacteria (LAB) involved in Ghanaian cocoa fermentation by analyzing samples from the Eastern and Western North Regions. A combination of culture-dependent methods, 16S rRNA gene sequencing (Sanger platform), and phylogenetic analysis was used to identify and characterize LAB diversity. Bioinformatics tools, including BLASTN (NCBI) and R software (v4.4.0), were employed for taxonomy assignment and visualization, while phylogenetic analysis was performed using PhyML (v3.0) and iTOL. Three LAB species were identified: Limosilactobacillus fermentum , Lactiplantibacillus plantarum , and Lentilactobacillus hilgardii . L. fermentum dominated the fermentation process, forming two distinct phylogenetic clusters, suggesting genetic diversity. Temperature and pH analysis revealed similar trends across regions, with peak fermentation temperatures (47.1°C and 45.4°C) coinciding with the highest pH values (5.19 and 5.5) at 72 hours. Notably, L. plantarum was isolated at 72 hours, a stage characterized by a declining pulp pH (4.9) and a heap temperature of 45.4°C, suggesting its adaptation to intermediate fermentation conditions when acidification intensifies, and microbial succession progresses. Statistical analysis showed no significant regional differences in temperature (p > 0.05), but fermentation time significantly influenced both temperature and pH dynamics (p < 0.05). These findings enhance the understanding of LAB and physicochemical dynamics in cocoa fermentation, offering insights for optimizing fermentation processes to improve cocoa quality and chocolate flavour. Biotechnology and Bioengineering Cocoa fermentation Lactic acid bacteria Starter culture cocoa beans gene sequencing phylogenetic analysis Full Text Additional Declarations The authors declare no competing interests. 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. 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fermentation\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Kwame Nkrumah University of Science and Technology","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":"Cocoa fermentation, Lactic acid bacteria, Starter culture, cocoa beans, gene sequencing, phylogenetic analysis","lastPublishedDoi":"10.21203/rs.3.rs-7864986/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7864986/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCocoa is a vital cash crop in Ghana, contributing approximately 15% of the country\u0026rsquo;s foreign exchange earnings and employing over 800,000 farmers. Fermentation significantly influences cocoa quality and flavour, yet controlling this process remains a challenge. This study investigated lactic acid bacteria (LAB) involved in Ghanaian cocoa fermentation by analyzing samples from the Eastern and Western North Regions. A combination of culture-dependent methods, 16S rRNA gene sequencing (Sanger platform), and phylogenetic analysis was used to identify and characterize LAB diversity. Bioinformatics tools, including BLASTN (NCBI) and R software (v4.4.0), were employed for taxonomy assignment and visualization, while phylogenetic analysis was performed using PhyML (v3.0) and iTOL. Three LAB species were identified: \u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e, \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e, and \u003cem\u003eLentilactobacillus hilgardii\u003c/em\u003e. \u003cem\u003eL. fermentum\u003c/em\u003e dominated the fermentation process, forming two distinct phylogenetic clusters, suggesting genetic diversity. Temperature and pH analysis revealed similar trends across regions, with peak fermentation temperatures (47.1\u0026deg;C and 45.4\u0026deg;C) coinciding with the highest pH values (5.19 and 5.5) at 72 hours. Notably, \u003cem\u003eL. plantarum\u003c/em\u003e was isolated at 72 hours, a stage characterized by a declining pulp pH (4.9) and a heap temperature of 45.4\u0026deg;C, suggesting its adaptation to intermediate fermentation conditions when acidification intensifies, and microbial succession progresses. Statistical analysis showed no significant regional differences in temperature (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), but fermentation time significantly influenced both temperature and pH dynamics (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings enhance the understanding of LAB and physicochemical dynamics in cocoa fermentation, offering insights for optimizing fermentation processes to improve cocoa quality and chocolate flavour.\u003c/p\u003e","manuscriptTitle":"Molecular identification of lactic acid bacteria involved in Ghanaian cocoa bean fermentation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-16 11:58:48","doi":"10.21203/rs.3.rs-7864986/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":"e4cf62a2-f1b6-4182-a91c-ca25b2044b6f","owner":[],"postedDate":"October 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56325081,"name":"Biotechnology and Bioengineering"}],"tags":[],"updatedAt":"2025-10-16T11:58:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-16 11:58:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7864986","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7864986","identity":"rs-7864986","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}