Abstract
Lactiplantibacillus plantarum B21 isolated from Vietnamese sausage (nem chua) has previously displayed broad antimicrobial activity against Gram-positive bacteria including foodborne pathogens Listeria monocytogenes and Clostridium perfringens. This study successfully identified the antimicrobial agent as plantacyclin B21AG, a 5668 Da circular bacteriocin demonstrating high thermostability, resistance to a wide range of pH, proteolytic resistance and temporal stability. We report a reverse genetics approach to identify and characterise plantacyclin B21AG from first principles. The bacteriocin was purified from culture supernatant by a three-step process consisting of concentration, n-butanol extraction and cation exchange chromatography. A de novo peptide sequencing using LC-MS/MS techniques identified two putative peptide fragments which were mapped to the genome sequence of L. plantarum B21. This revealed an ORF corresponding to a putative circular bacteriocin with a 33-amino acid leader peptide and a 58-amino acid mature peptide encoded on a native plasmid pB21AG01. The bacteriocin is shown to be a small cationic predominantly α-helical protein (69%). The corresponding gene cluster, consisted of seven genes associated with post-translational circularisation, immunity and secretion. Whilst plantacyclin B21AG is 86% identical to the newly published plantaricyclin A it is more highly cationic having a net charge of +3 due to an additional basic residue in the putative membrane interaction region. This and other substitutions may well go some way to explaining functional differences. The robust nature of plantacyclin B21AG, its antimicrobial activity and associated machinery for cyclisation make it an interesting biotechnological target for development, both as a food-safe antimicrobial or potentially a platform technology for recombinant protein circularisation.
Highlights
Food borne disease due to pathogen contamination is a major concern in the food industry [1]
In later work we demonstrated that only a single bacteriocin is present and by way of calibration, a strong positive result for a sensitive strain such as L. plantarum A6 corresponds to a MIC value of 0.156 μM measured with pure bacteriocin
cell free supernatant (CFS) showed strong antimicrobial activity against one other Lactic acid bacteria (LAB)'s tested in this study, Lactococcus lactis 345-18, but it is only weakly active against L. brevis 19012 (Table 1)
Summary
Food borne disease due to pathogen contamination is a major concern in the food industry [1]. Lactic acid bacteria (LAB) have long been associated with food and feed fermentation, as a starter culture in the production of fermented meat, vegetables, fruit, alcoholic beverages, dairy products and silage [6, 7]. The preservative effect of LAB is due to the production of antimicrobial substances, including organic acids, hydrogen peroxide, diacetyl, bacteriocins and bacteriocin-like antimicrobial substances [10, 11] Among these antimicrobial components, bacteriocins have received particular attention in recent years because of their application in the food industry as natural preservatives, and as potential antimicrobial peptides or antibiotic-like molecules targeting multi-drug resistance pathogens [5, 12]
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