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Formulation and surface activity of natural antimicrobial peptides produced by Bacillus species
Thesis (MSc)--Stellenbosch University, 2025.
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Stellenbosch : Stellenbosch University
2025
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| _version_ | 1867614101459435520 |
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| access_status_str | Open Access |
| author | Bezuidenhout, Sherwin Heinrich |
| author2 | Rautenbach, Marina |
| author_browse | Bezuidenhout, Sherwin Heinrich Rautenbach, Marina |
| author_facet | Rautenbach, Marina Bezuidenhout, Sherwin Heinrich |
| author_sort | Bezuidenhout, Sherwin Heinrich |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2025. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/132070 |
| institution | Stellenbosch University (South Africa) |
| last_indexed | 2026-06-10T12:46:41.344Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2025 |
| publishDateRange | 2025 |
| publishDateSort | 2025 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/132070 Formulation and surface activity of natural antimicrobial peptides produced by Bacillus species Bezuidenhout, Sherwin Heinrich Rautenbach, Marina Stellenbosch University. Faculty of Science. Dept. of Biochemistry. Bacillus (Bacteria) -- Genetics Peptides -- Mechanism of action Peptide antibiotics -- Mechanism of action Biofilms UCTD Thesis (MSc)--Stellenbosch University, 2025. Bezuidenhout, S. H. 2025. Formulation and surface activity of natural antimicrobial peptides produced by Bacillus species. Unpublished masters thesis. Stellenbosch: Stellenbosch University [online]. Available: https://scholar.sun.ac.za/items/e0aed5c2-b144-449d-b8c0-8acbe4d40895 ENGLISH ABSTRACT: This study aimed to characterize antimicrobial peptide production in Brevibacillus laterosporus and assess the solid surface antimicrobial activity of the produced peptides. Electrospray ionisation mass spectrometry (ESI-MS) revealed that all in-culture productions were either categorised as a high loloatin production with minimal bogorols, a high bogorol production with minimal loloatins, or a varying ratio of both peptides in the culture extracts. The production of loloatins in a Luria-Betrani (LB) medium was significantly higher on day 7 than on day 1. However, the overall production on a solid medium (LB agar) was significantly lower when compared to production in liquid culture. The production of loloatins LolG and LolH also remained low, while LolA production increased over the seven-day period. Amino acid supplementation of the growth media with 10 mM and 20 mM Tyr significantly increased LolA production compared to 20 mM Phe supplementation, while significantly greater amounts of LolH were produced with the 10 mM and 20 mM Phe supplementations. Trp-rich peptide analogues were detected exclusively in the Trp-supplementation of culture media. The successful purification and/or enrichment of LolA, LolG, LolH, and LeoAf was done using semipreparative high performance liquid chromatography. The loloatin purity and composition, well as sequence and identity were confirmed by high resolution ESI-MS and tandem MS. The solid surface antimicrobial activity of loloatins against Staphylococcus aureus was found to vary with ethanol (EtOH) concentration during pre-incubation, before depositing and drying of the peptide preparations on a polystyrene surface. Optimal activity was observed at 50-80% EtOH, with a poor activity at 5-30% EtOH and 90% EtOH. Extending the peptide incubation time in 70% EtOH to 16 hours was found to significantly enhance the surface activity compared to the 1-hour incubation. Moreover, the study found that a short incubation at 4 °C and 37 °C significantly improved peptide activity compared to a short incubation at 18 °C in 70% EtOH, while extending the incubation time to 16 hours diminished the effect of incubation temperature. Our hypothesis is that the surface-derived antimicrobial activity of the loloatins requires the formation of seed structures, probably small oligomers (dimers, trimers and tetramers), that when deposited on the surface direct the layering or assembly of peptides in an active metastable conformation. Future studies should investigate the antimicrobial activity of different surfaces and materials treated with loloatins, as well as the co-produced bogorols. Robustness testing of the surfaces and materials treated with these peptides would be crucial for future applications and the development of self-sterilising surfaces and materials. AFRIKAANSE OPSOMMING: Hierdie studie het ten doel gehad om antimikrobiese peptiedproduksie in Brevibacillus laterosporus te karakteriseer en die vaste oppervlak antimikrobiese aktiwiteit van die geproduseerde peptiede te bepaal. Elektrosproei-ionisasie massaspektrometrie (ESI-MS) het aan die lig gebring dat alle in- kultuur produksies óf gekategoriseer is as 'n hoë loloatienproduksie met minimale bogorole, 'n hoë bogorolproduksie met minimale loloatiene, of 'n wisselende verhouding van beide peptiede in die kultuurekstrakte. Die produksie van loloatiene in 'n Luria-Betrani (LB) medium was betekenisvol hoër op dag 7 as op dag 1. Die algehele produksie op 'n vaste medium (LB agar) was egter aansienlik laer in vergelyking met produksie in vloeibare kultuur. Die produksie van loloatiene LolG en LolH het ook laag gebly, terwyl LolA-produksie oor die sewe dae tydperk toegeneem het. Aminosuuraanvulling van die groeimedia met 10 mM en 20 mM Tyr het LolA-produksie aansienlik verhoog in vergelyking met 20 mM Phe-aanvulling, terwyl aansienlik groter hoeveelhede LolH geproduseer is met die 10 mM en 20 mM Phe-aanvullings. Trp-ryke peptiedanaloë is uitsluitlik in die Trp-aanvulling van kultuurmedia opgespoor. Die suksesvolle suiwering en/of verryking van LolA, LolG, LolH en LeoAf is gedoen deur gebruik te maak van semipreparatiewe hoë werkverrigting vloeistofchromatografie. Die loloatien suiwerheid en samestelling, sowel as volgorde en identiteit is bevestig deur hoë resolusie ESI-MS en tandem MS. Daar is gevind dat die vaste oppervlak antimikrobiese aktiwiteit van loloatiene teen Staphylococcus aureus wissel met etanol (EtOH) konsentrasie tydens pre-inkubasie, voordat die peptiedpreparate op 'n polistireen oppervlak gedeponeer en gedroog word. Optimale aktiwiteit is waargeneem by 50-80% EtOH, met 'n swak aktiwiteit by 5-30% EtOH en 90% EtOH. Die uitbreiding van die peptied-inkubasietyd in 70% EtOH na 16 uur is gevind om die oppervlakaktiwiteit aansienlik te verbeter in vergelyking met die 1-uur inkubasie. Verder het die studie bevind dat 'n kort inkubasie by 4 °C en 37 °C peptiedaktiwiteit aansienlik verbeter het in vergelyking met 'n kort inkubasie by 18 °C in 70% EtOH, terwyl die verlenging van die inkubasietyd na 16 uur die effek van inkubasietemperatuur verminder het. Ons hipotese is dat die oppervlak-afgeleide antimikrobiese aktiwiteit van die loloatiene die vorming van saadstrukture vereis, waarskynlik klein oligomere (dimere, trimere en tetramere), wat wanneer dit op die oppervlak neergelê word, die lae of samestelling van peptiede in 'n aktiewe metastabiele konformasie rig. Toekomstige studies behoort die antimikrobiese aktiwiteit van verskillende oppervlaktes en materiale wat met loloatiene behandel is, sowel as die mede-geproduseerde bogorole te ondersoek. Robuustheidstoetsing van die oppervlaktes en materiale wat met hierdie peptiede behandel is, sal deurslaggewend wees vir toekomstige toepassings en die ontwikkeling van selfsteriliserende oppervlaktes en materiale. Masters 2025-05-22T08:06:32Z 2025-05-22T08:06:32Z 2025-03 Thesis https://scholar.sun.ac.za/handle/10019.1/132070 Stellenbosch University 135 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Bacillus (Bacteria) -- Genetics Peptides -- Mechanism of action Peptide antibiotics -- Mechanism of action Biofilms UCTD Bezuidenhout, Sherwin Heinrich Formulation and surface activity of natural antimicrobial peptides produced by Bacillus species |
| title | Formulation and surface activity of natural antimicrobial peptides produced by Bacillus species |
| title_full | Formulation and surface activity of natural antimicrobial peptides produced by Bacillus species |
| title_fullStr | Formulation and surface activity of natural antimicrobial peptides produced by Bacillus species |
| title_full_unstemmed | Formulation and surface activity of natural antimicrobial peptides produced by Bacillus species |
| title_short | Formulation and surface activity of natural antimicrobial peptides produced by Bacillus species |
| title_sort | formulation and surface activity of natural antimicrobial peptides produced by bacillus species |
| topic | Bacillus (Bacteria) -- Genetics Peptides -- Mechanism of action Peptide antibiotics -- Mechanism of action Biofilms UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/132070 |
| work_keys_str_mv | AT bezuidenhoutsherwinheinrich formulationandsurfaceactivityofnaturalantimicrobialpeptidesproducedbybacillusspecies |