Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, Serdang, Malaysia.
J Antibiot (Tokyo). 2012 Jun;65(6):289-94. doi: 10.1038/ja.2012.17. Epub 2012 Apr 11.
In this study, potential probiotic strains were isolated from fermented pickles based on antagonistic activity against two shrimp pathogens (Vibrio harveyi and Vibrio parahaemolyticus). Two strains L10 and G1 were identified by biochemical tests, followed by16S ribosomal RNA gene sequence analysis as Bacillus subtilis, and characterized by PCR amplification of repetitive bacterial DNA elements (Rep-PCR). Subsequently, B. subtilis L10 and G1 strains were tested for antibacterial activity under different physical conditions, including culture medium, salinity, pH and temperature using the agar well diffusion assay. Among the different culture media, LB broth was the most suitable medium for antibacterial production. Both strains showed the highest level of antibacterial activity against two pathogens at 30 °C and 1.0% NaCl. Under the pH conditions, strain G1 showed the greatest activity against V. harveyi at pH 7.3-8.0 and against V. parahaemolyticus at pH 6.0-8.0, whereas strain L10 showed the greatest activity against two pathogens at pH 7.3. The cell-free supernatants of both strains were treated with four different enzymes in order to characterize the antibacterial substances against V. harveyi. The result showed considerable reduction of antibacterial activity for both strains, indicating the proteinaceous nature of the antibacterial substances. A wide range of tolerance to NaCl, pH and temperature was also recorded for both strains. In addition, both strains showed no virulence effect in juvenile shrimp Litopenaeus vannamei. On the basis of these results and safety of strains to L. vannamei, they may be considered for future challenge experiments in shrimp as a very promising alternative to the use of antibiotics.
在这项研究中，根据对两种虾病原体（哈维氏弧菌和副溶血弧菌）的拮抗活性，从发酵泡菜中分离出潜在的益生菌株。通过生化试验鉴定了两个菌株 L10 和 G1，然后通过 16S 核糖体 RNA 基因序列分析鉴定为枯草芽孢杆菌，并通过重复细菌 DNA 元件（Rep-PCR）的 PCR 扩增进行了特征分析。随后，通过琼脂孔扩散法在不同的物理条件下（包括培养基、盐度、pH 和温度）测试枯草芽孢杆菌 L10 和 G1 菌株的抗菌活性。在不同的培养基中，LB 肉汤是最适合抗菌生产的培养基。两种菌株在 30°C 和 1.0%NaCl 下对两种病原体表现出最高水平的抗菌活性。在 pH 条件下，菌株 G1 对 V. harveyi 的活性最高在 pH 7.3-8.0，对 V. parahaemolyticus 的活性最高在 pH 6.0-8.0，而菌株 L10 对两种病原体的活性最高在 pH 7.3。用四种不同的酶处理两种菌株的无细胞上清液，以鉴定对 V. harveyi 的抗菌物质。结果表明两种菌株的抗菌活性均显著降低，表明抗菌物质的蛋白质性质。两种菌株对 NaCl、pH 和温度的耐受范围也很广。此外，两种菌株在幼虾凡纳滨对虾（Litopenaeus vannamei）中均无致病性。基于这些结果和对 L. vannamei 的菌株安全性，它们可能被考虑用于未来对虾的挑战实验，作为抗生素替代物具有很大的应用前景。
