枯草芽孢杆菌拮抗菌的筛选鉴定及其抑菌特性研究

本研究旨在从泡菜中筛选一株对枯草芽孢杆菌具有拮抗作用的产抗菌肽乳酸菌，并对抗菌肽的性质及抑菌效果进行初步研究。以Bacillus subtilis B39为指示菌，通过溶钙圈法初筛、双层琼脂扩散法复筛得到一株具有较强抑制效果的乳酸菌，对其进行形态学观察、生理生化鉴定和16S rRNA基因的系统发育分析。利用硫酸铵沉淀法和有机溶剂萃取法探究抗菌肽的最佳提纯方法，并对该抗菌肽进行紫外全波长扫描定性和一系列抑菌特性的研究。结果表明，从武汉钢花菜市场泡菜样品中筛选到一株对B. subtilis B39具有较强抑制作用的产抗菌肽乳酸菌，通过16S rDNA序列（GenBank 登陆号:MZ751041.1）结合系统发育树分析，鉴定该菌株为植物乳杆菌（Lactobacillus plantarum）并将其命名为L. plantarum WUH3。通过对比发现，采用有机溶剂-乙酸乙酯萃取法可获得抗菌肽的最佳提纯效果，紫外全波长扫描结果显示，该纯化物质具有显著的肽类特征吸收峰。根据L. plantarum WUH3的生长曲线及抑菌活性曲线可知，该菌在生长稳定期时可达最大抗菌肽产量。二倍稀释法测得抗菌肽对B39的MIC为16 μg/mL。通过时间杀菌曲线可知，该抗菌肽对Bacillus subtilis B39具有杀菌作用。该研究在探索可抑制枯草芽胞杆菌的生物拮抗菌方面具有一定的借鉴意义，为L. plantarum WUH3在天然防腐剂领域的开发和应用奠定了基础。

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.