Characterisation & purification of class IIa bacteriocins from lactiplantibacillus plantarum

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dc.contributor.advisorBeukes, Mervynen_ZA
dc.contributor.advisorRautenbach, Marinaen_ZA
dc.contributor.authorBeukes, Cheyenne Tamikaen_ZA
dc.contributor.otherStellenbosch University. Faculty of Science. Dept. of Biochemistry.en_ZA
dc.date.accessioned2024-02-28T09:00:01Z
dc.date.accessioned2024-04-26T08:41:44Z
dc.date.available2024-02-28T09:00:01Z
dc.date.available2024-04-26T08:41:44Z
dc.date.issued2024-03
dc.descriptionThesis (MSc)--Stellenbosch University, 2024. en_ZA
dc.description.abstractENGLISH ABSTRACT: The rapid emergence of antibiotic resistance, coupled with the scarcity in the development of new drugs to combat resistance are threatening the food, medical and agricultural industries. This demands the development of novel alternative treatments, such as antimicrobial substances to target specific pathogenic micro-organisms. Lactic acid bacteria (LAB) have attracted attention in this research field, as LAB secrete antimicrobial peptides bacteriocins. Bacteriocins are ribosomally synthesized antimicrobial peptides that display promising potential as natural food preservatives and source of microbial inhibitors. The producer organism chosen for further research was Lactiplantibacillus plantarum (L. plantarum), a member of the LAB known to commonly produce multiple bacteriocins. The goal of this study was to perform the in-silico gene mining in conjunction with the in vitro molecular screening with the aim to isolate, and characterise bacteriocins from the producer organism, L. plantarum. The results obtained from the various assays and characterisation of bacteriocins will contribute to expanding the knowledge in the field. With potential application of such bacteriocins, specifically in the food industry as food preservative or health care sector, as further encouragement in this field. A combinatorial approach was adopted for the in silico gene mining of organisms of interest, alongside the in vitro studies. In silico screening methods included the use of bacteriocin genome mining tool version 4 (BAGEL4), National Centre for Biotechnology Information (NCBI) and Basic Local Alignment Search Tool (BLAST) for rapid identification of putative operons within the genomes of selected producer organisms. Initial in vitro screening of class IIa LAB from our local culture collection was conducted through bioassays. This included the colony-spot, spot-on-lawn, and microtiter plate growth inhibition assays, to monitor inhibitory activity. A producer organism was selected for subsequent isolation and purification studies. Inhibition zones surrounding colony spots were compared for a broad range of indicator organisms, including Listeria monocytogenes (L. monocytogenes), L. plantarum Ta10c, Micrococcus luteus (M. luteus), Staphylococcus aureus (S. aureus), Streptococcus milleri (S. milleri), and Enterococcus faecalis (E. faecalis). The highest sensitivity was observed towards L. monocytogenes. Further, another bioassay was used to confirm initial colony-spot and spot-on-lawn assays results. This included the microtiter plate growth inhibition assay providing inhibition results from continuous monitoring, instead of an end-point result. Production of bacteriocins was optimized with regards to type of media, pH and temperature. The optimal conditions were found to be in Man, Rogosa and Sharpe (MRS) medium (Neogen, USA), pH of 7, and at a temperature of 37°C. Genomic DNA (gDNA) of the producer organism was extracted, amplified through PCR with universal 16S rDNA primers, and the amplicons sequenced. A BLAST search was conducted for homology comparison to the NCBI DNA database, to confirm the producer organism as L. plantarum. Purification of bacteriocins was initiated with the use of Amberlite XAD-16N, for separation based on hydrophobic interactions; followed by cation exchange chromatography, for separation based on ionic charge; and, reversed-phase high-performance liquid chromatography (RP-HPLC), for separation based on hydrophobicity. Mass Spectrometry analysis at each purification step revealed three prominent peaks at 789, 1124.6282 and 2185 Daltons (Da). These display potential masses of bacteriocins as they fall within the range of molecular masses linked to inhibitory activity observed.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Die vinnige ontwikkeling van antibiotiese weerstand, tesame met die stadige en skaarste in die ontwikkeling van nuwe middels om weerstand te bekamp, bedreig die voedsel-, mediese-,en landboubedrywe. Dit vereis die ontwikkeling van nuwe alternatiewe behandelings, soos antimikrobiese stowwe om spesifieke patogene mikro-organismes te teiken. Melksuurbakterieë (MSB) het aandag getrek in hierdie navorsingsveld, aangesien MSB antimikrobiese peptiede, bakteriosiene afskei. Bakteriosiene is ribosomaal gesintetiseerde antimikrobiese peptiede wat belowende potensiaal toon as natuurlike voedselpreserveermiddels en bron van mikrobiese inhibeerders. Die produsente-organisme wat in hierdie studie vir verdere navorsing gekies is, was Lactiplantibacillus plantarum (L. plantarum), 'n lid van die MSB wat bekend is dat dit gewoonlik veelvuldige bakteriosiene produseer. Die doel van hierdie studie was om ‘n in siliko-geenmynbou uit te voer in samewerking met die in vitro molekulêre sifting met die doel om bakteriosiene van die produsentorganisme, te isoleer en te karakteriseer. Die resultate verkry uit die verskillende toetse en karakterisering van bakteriosiene sal bydra tot die uitbreiding van die kennis in die veld. Met potensiële toepassing van sulke bakteriosiene, spesifiek in die voedselbedryf as voedselpreserveermiddel of gesondheidsektor, as verdere aanmoediging op hierdie gebied. 'n Kombinatoriese benadering is aangeneem vir die in-siliko-geenmynbou van organismes van belang, saam met die in vitro-studies. In-siliko-siftingsmetodes het die gebruik van bakteriosien- genoom-ontginnings instrument weergawe 4 (BAGEL4), Nasionale Sentrum vir Biotegnologie- inligting (NCBI) en Basiese Plaaslike Belyningsoek instrument (BLAST) ingesluit vir die vinnige identifisering van vermeende operone binne die genome van geselekteerde produsente- organismes. Aanvanklike in vitro-sifting van klas IIa-melksuurbakterieë (MSB) uit ons plaaslike kultuurversameling is deur biotoetse uitgevoer. Dit het die kolonie-spot, spot-on-lawn en mikrotiter plaat inhibisie toetse ingesluit om inhibisie aktiwiteit te monitor. 'n Produsente organisme is gekies vir opvolgende isolasie- en suiweringstudies. Inhibisiesones, rondom kolonie kolle, is vergelyk vir 'n wye reeks indikator organismes, insluitend Listeria monocytogenes (L. monocytogenes), L. plantarum Ta10c, Mikrococcus luteus (M. luteus), Staphylococcus aureus (S. aureus), Streptococcus milleri (S. milleri), en Enterococcus faecalis (E. faecalis). Die hoogste sensitiwiteit is waargeneem teenoor Listeria monocytogenes. Verder is nog 'n bio-toets gebruik om die aanvanklike kolonie-spot- en spot-on-lawn-toetsresultate te bevestig. Dit het die mikrotiter plaat inhibisie toets ingesluit, wat intyds inhibisie resultate lewer, in plaas van 'n eindpunt resultaat. Produksie van bakteriosiene is geoptimaliseer met betrekking tot tipe media, pH en temperatuur. Daar is gevind dat die optimale toestande die beste is in MRS-media, neutrale pH van 7 en by 'n temperatuur van 37 °C. Genomiese DNA (gDNA) van die produsent organisme is onttrek, verveelvoudig deur PCR met universele 16S rDNA-primers, en die amplikone se DNA volgorde bepaal. 'N BLAST-soektog is uitgevoer vir homologievergelyking met die NCBI DNA-databasis, om die produsente-organisme as L. plantarum te bevestig. Suiwering van bakteriosien is begin met die gebruik van Amberlite XAD-16N, vir skeiding gebaseer op hidrofobiese interaksies; gevolg deur katioonuitruil chromatografie, vir skeiding gebaseer op ioniese lading; en, omgekeerde fase hoëprestasie vloeibare chromatografie (RP-HPLC), vir skeiding gebaseer op hidrofobiesheid. Massaspektrometrie-analise by elke suiweringstap het drie prominente pieke teen 789, 1124.6282 en 2185 Daltons (Da) aan die lig gebring. Hierdie potensiële peptiede massas vorm deel van die reeks bakteriosienmolekulêre massas wat aan inhibisiese aktiwiteit gekoppel is.af_ZA
dc.description.versionMastersen_ZA
dc.format.extentvii, 80 pages : illustrations (some color)en_ZA
dc.identifier.urihttps://scholar.sun.ac.za/handle/10019.1/130193
dc.language.isoen_ZAen_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshLactic acid bacteria -- Effect of stress onen_ZA
dc.subject.lcshBacteriocins -- Genetic aspectsen_ZA
dc.subject.lcshDrug resistance in microorganismsen_ZA
dc.subject.lcshPeptide antibioticsen_ZA
dc.subject.lcshGenotype-environment interactionen_ZA
dc.subject.lcshLactiplantibacillus plantarum -- Geneticsen_ZA
dc.subject.lcshLactic acid bacteria --Effect of temperature onen_ZA
dc.subject.nameUCTDen_ZA
dc.titleCharacterisation & purification of class IIa bacteriocins from lactiplantibacillus plantarumen_ZA
dc.typeThesisen_ZA
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