Characterisation of bacteriocin genes and proteins from Lactobacillus plantarum B21 as potential new antimicrobial agents and natural food preservatives
Creators: name: Golneshin, Aida
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310509
subject: Genomics
classificationCode: 310704
subject: Microbial genetics
classificationCode: 340407
subject: Proteins and peptides

description: Lactic acid bacteria (LAB) have a long history of application in fermented foods because of their beneficial influence on nutritional, organoleptic, and shelf-life characteristics. The use of functional starter cultures of LAB to confer functionalities beyond acidification is attracting increasing interest. LAB produce a variety of compounds with antimicrobial activities, these include bacteriocins which are small proteins having antimicrobial activity against bacterial species closely related to the producer strain and / or other bacteria. As the demand for natural additive free and microbiologically safe food products grows, bacteriocin producing-LAB have considerable potential in the food industry as natural food preservatives.<br><br>Lactobacillus plantarum B21 was previously isolated from Vietnamese sausages (nem chua). The bacteriocin produced by this strain demonstrated inhibition of Gram-positive bacterial growth with good pH stability (pH 3.0 – 10.0) and moderate thermo-stability (up to 20 min at 90 °C). The B21 bacteriocin was selected for further characterization due to these properties and its rather unique source. The aim of this project was to study the genes responsible for bacteriocin production and to purify and characterise the bacteriocin produced by L. plantarum B21. The study was extended subsequently to other isolates from the same source.<br><br>The LAB strains were identified and classified as L. plantarum and L. brevis strains using 16S rRNA techniques. The pln locus responsible for production of class II bacteriocin(s) in L. plantarum strains was studied and the classical pln loci mapped in detail. This enabled the development of a classification for the new L. plantarum strains based on the pln locus organisation and structure. The pln structural genes from strain B21 and number of associated but non-bacteriocin related genes from other LAB strains were sequenced and studied at the bioinformatic level. In order to identify the similarities and differences between the pln locus from L. plantarum strains and the available reference genomes, whole genome sequencing using Next Generation Sequencing (NGS) platforms (Illumina and Ion Proton) was carried out and the Illumina-based genome sequencing of LAB B21 provided a new reference genome for the best bacteriocin producer. The highly plastic nature of the LAB genome was confirmed with evidence of recent impairment of classical pln locus.<br><br>The inhibitory activity of LAB strain B21 was evaluated against a range of indicator strains and the proteinaceous nature of the antimicrobial activity was confirmed. The functional bacteriocin protein from the B21 culture supernatant was purified and shown to consist of a single α-helical polypeptide of 5668 Da. A de novo sequencing approach using LC-MS/MS techniques was taken to identify the peptide sequence, allowing it to be mapped back to the genomic data. It was shown to be a novel cyclic bacteriocin (plantacyclin B21AG) produced from a new regulon in B21.