Antimicrobial Properties of A Postbiotic Strain and Gene Cluster Mining of Its Bacteriocin

The aim of this study was to screen a postbiotic strain against Aeromonas hydrophila, and analyze its antibacterial characteristics and bacteriocin related gene cluster (RiPPs). The pathogen Aeromonas hydrophila (Ah2) from Andrias davidianus was used as an indicator for the screening of an excellent lactic acid bacterium (LAB), then the antibiotic susceptibility of the LAB to 10 common antibiotics was evaluated. The crude extract of bacteriocin (CEB) produced by the LAB was preliminarily purified by organic solvent extraction. Antibacterial properties of the CEB were then evaluated by 6 metrics below: pH, temperature and digestive enzyme tolerance, storage stability, antimicrobial spectrum, minimum inhibitory concentration (MIC) and maximum inhibitory concentration (MBC). CEB of two concentrations above was used for hemolysis and cytotoxicity experiment to further verify the bacteriocin’s effect on Ah2, and for scanning electron microscope (SEM) observation to preliminary explore the antimicrobial mechanism of the bacteriocin. Then through UV full-wavelength scanning and Tricine-SDS-PAGE, the qualitation and molecular weight of the bacteriocin was seprately identified and estimated. Finally, the whole genome sequence of the LAB was determined and then its potential bacteriocin gene cluster (RiPPs) mining was predicted in the core peptide database BAGEL4. Results showed that the excellent bacteriocin-producing strain M4L1 was isolated and screened from the Sufu sold in Qingdao, and was identified as Lactiplantibacillus plantarum (L. plantarum). The bacteriocin of L. plantarum M4L1 was half-purified and named as LP01. Bacteriocin LP01 exhibited good digestive enzyme tolerance and relatively stable antibacterial activity at pH2~12, −20~121 ℃ and 9 months storage, it also presented a wide antimicrobial spectrum on 14 Gram positive and negative strains including 7 aquatic pathogens, such as Listeria monocytogenes, Citrobacter freundii and etc. Moreover, the MIC and MBC of LP01 against Ah2 was 12.94 and 25.88 μg/mL, respectively. After treatment with LP01, the hemolytic activity and cytotoxicity caused by Ah2 were significantly relieved as confirmed, which presented bacteriocin LP01’s excellent antibacterial effect. Especially, the SEM results showed that LP01 could inhibit or kill Ah2 by damaging cell wall. LP01 showed significantly characteristic peak at 200~220 nm corresponding to peptides, and electrophoresis band indicated the molecular weight range of LP01 was 3.3~4.0 kDa. LP01 was identified as small-peptide bacteriocin. There are 2 bacteriocin gene clusters (RiPPs) corresponding to the class II bacteriocins, L. plantarum Plantaricin K and Plantaricin E, were found in the genome of strain M4L1. In all, the bacteriocin-producing L. plantarum M4L1 not only owned bacteriocin gene clusters, but was also equipped with excellent antimicrobial characteristics and could inhibit a variety of aquatic pathogens, which has the potential to be developed as a postbiotic candidate strain in aquaculture.