Antagonistic Bacteria of Bacillus subtilis: Screening, Identification and Antibacterial Substance Characterizing

This study aimed to screen an antimicrobial peptide (AMP)-produced lactic acid bacteria (LAB) against Bacillus subtilis from Kraut, and to analyze the properties and antibacterial effect of the antimicrobial peptide preliminarily. Bacillus subtilis B39 was used as the indicator, and a strain of LAB with strong inhibitory effect was obtained by primary screening with calcium-dissolving method and re-screening with double-layer agar diffusion method. Morphological observation, physiological and biochemical identification and phylogenetic analysis of 16S rRNA gene were subsequently carried out. The best purification method of the AMP was explored by ammonium sulfate precipitation and organic solvent extraction, then the purified AMP was characterized by UV full wavelength scanning, and a series of antibacterial properties were studied. Results showed that, a strain of AMP-produced LAB with strong inhibitory effect against B39 was screened from Kraut samples of Wuhan Ganghua market. The strain was identified as Lactobacillus plantarum by 16S rDNA sequence (GenBank accession number: MZ751041.1) and phylogenetic tree analysis, and was named L. plantarum WUH3. Through comparison, it was found that the AMP purified by organic solvent-ethyl acetate extraction method, could best retain its antibacterial activity. UV full-wavelength scanning showed that the peptide characteristic absorption peak of the purified substance was significant. According to the growth curve and antibacterial activity curve of WUH3, the strain could reach maximum yield of the AMP at stationary growth stage. The MIC of the AMP against B39 was 16 μg/mL measured by doubling dilution method, and the time-kill curve indicated the AMP had bactericidal effect on B39. This study can be helpful in exploring the biological antagonistic bacteria against Bacillus subtilis, and it lays a foundation for the development and application of L. plantarum WUH3 in the field of natural preservatives.