Masters Degrees (Biochemistry)

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Browsing Masters Degrees (Biochemistry) by Author "Beukes, Cheyenne Tamika"

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    Characterisation & purification of class IIa bacteriocins from lactiplantibacillus plantarum
    (Stellenbosch : Stellenbosch University, 2024-03) Beukes, Cheyenne Tamika; Beukes, Mervyn; Rautenbach, Marina; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH 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.