ProBacLab, Graduate School of Advanced Convergence, Handong Global University, Pohang, Gyeongbuk, 37554, Republic of Korea.
Probiotics Antimicrob Proteins. 2021 Aug;13(4):1195-1212. doi: 10.1007/s12602-021-09772-w. Epub 2021 Mar 15.
Bacteriocin production is considered a favorable property for various beneficial cultures. In addition to their potential as biopreservatives, bacteriocins are also promising alternatives for the control of multidrug-resistant pathogens and the inhibition of some viruses and cancer cells. The objective of this study was to screen and characterize a bacteriocin-producing strain with the aim of its future application for control of Listeria monocytogenes, an important food-borne pathogen. A total of 22 potentially bacteriocinogenic strains active against L. monocytogenes ATCC15313 were isolated from locally produced kimchi through a three-level approach. Pure cultures were obtained according to good microbiological practices and differentiated through RAPD-PCR using the primers OPL01, OPL09, and OPL11. Altogether, 5 strains were selected for further study. Specific focus was given to strain ST05DL based on its specific inhibitory activity against L. monocytogenes ATCC15313, while not affecting different strains belonging to the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella, most of which are beneficial microorganisms. The strain ST05DL was identified as Bacillus amyloliquefaciens based on its sugar fermentation profile obtained through API50CHB analysis and 16S rRNA partial sequencing. The antimicrobial compound produced by B. amyloliquefaciens ST05DL was found to be sensitive to pepsin and α-chymotrypsin, evidence of its proteinaceous nature. The presence of skim milk, NaCl, Tween 80, glycerol, and SDS did not affect the antimicrobial activity. The addition of 20% cell-free supernatant (CFS) obtained from a 24-h culture of B. amyloliquefaciens ST05DL to an exponentially growing culture of L. monocytogenes ATCC15313 successfully inhibited the test microorganisms during the monitored 10-h incubation. Optimal bacteriocin production by B. amyloliquefaciens ST05DL was observed during the stationary phase at 12 h (800 AU/mL) and remained stable for the next 15 h. The ratio between live and dead cells during this period was 74.37% and 25.66%, respectively, as determined by flow cytometry. The presence of the virulence genes hblA, hblB, hblC, nheA, nheB, and nheC was not detected in the total DNA of B. amyloliquefaciens ST05DL, and the strain was resistant only to ampicillin out of 10 tested antibiotics. Future evaluation of expressed bacteriocin/s by B. amyloliquefaciens ST05DL (amino acid sequence, molecular mass, cytotoxicity, detailed mode of action, etc.), will be the next step in the characterization and its potential application as biopreservative and/or pharmaceutical product.
细菌素的产生被认为是各种有益培养物的有利特性。除了作为生物防腐剂的潜力外，细菌素也有望替代控制多药耐药病原体和抑制某些病毒和癌细胞。本研究的目的是筛选和表征产细菌素的菌株，以期将来用于控制单核细胞增生李斯特菌，这是一种重要的食源性病原体。通过三级方法，从当地生产的泡菜中分离出 22 株对单核细胞增生李斯特菌 ATCC15313 具有活性的潜在细菌素产生菌。根据良好的微生物学实践获得纯培养物，并使用引物 OPL01、OPL09 和 OPL11 通过 RAPD-PCR 进行区分。总共选择了 5 株进行进一步研究。基于其对单核细胞增生李斯特菌 ATCC15313 的特异性抑制活性，特别关注 ST05DL 菌株，而不影响属于乳杆菌属、肠球菌属、明串珠菌属和魏斯氏菌属的不同菌株，其中大多数是有益微生物。基于 API50CHB 分析和 16S rRNA 部分测序获得的糖发酵谱，ST05DL 菌株被鉴定为解淀粉芽孢杆菌。发现解淀粉芽孢杆菌 ST05DL 产生的抗菌化合物对胃蛋白酶和α-糜蛋白酶敏感，证明其具有蛋白质性质。脱脂乳、NaCl、吐温 80、甘油和 SDS 的存在并不影响抗菌活性。将解淀粉芽孢杆菌 ST05DL 24 小时培养物获得的无细胞上清液（CFS）的 20%添加到单核细胞增生李斯特菌 ATCC15313 的指数生长期培养物中，在监测的 10 小时孵育期间成功抑制了测试微生物。在 12 小时（800 AU/mL）时观察到解淀粉芽孢杆菌 ST05DL 的最佳细菌素产生，并且在接下来的 15 小时内保持稳定。通过流式细胞术确定，在此期间活细胞和死细胞的比例分别为 74.37%和 25.66%。在解淀粉芽孢杆菌 ST05DL 的总 DNA 中未检测到毒力基因 hblA、hblB、hblC、nheA、nheB 和 nheC，并且该菌株仅对 10 种测试抗生素中的氨苄青霉素有抗性。下一步将对解淀粉芽孢杆菌 ST05DL 表达的细菌素/进行特征描述（氨基酸序列、分子量、细胞毒性、详细作用模式等），并将其作为生物防腐剂和/或药物产品的潜在应用进行评估。
