一株海洋拮抗菌D-2的筛选及其发酵条件初探

Isolation and fermentation of a strain of antagonistic bacterium D-2

从西沙海域水样中分离了72株细菌，以副溶血弧菌Vibrioparahaemolyticus13150为病原指示菌，筛选到1株具有较好拮抗作用的海洋细菌，经形态观察、生理生化和16SrDNA测序鉴定为居幼虫普罗威登斯菌（Providenciavermicola），编号为D-2，此菌在NCBI的登录号为KJ660957。同时，通过卤虫浸泡实验和药敏试验检测该菌的安全性，并初步优化其发酵条件。结果表明，D-2对杀鲑气单胞菌、副溶血弧菌、哈氏弧菌等有拮抗作用；其对卤虫的72h半致死浓度（LC50）为108CFU/mL，而副溶血弧菌V.parahaemolyticus对卤虫的72hLC50为105CFU/mL；此外，药敏试验说明该菌只对20种常规抗生素中的卡那霉素、诺氟沙星2种产生抗性，其余的18种抗生素表现敏感性；优化后的发酵条件为：温度25℃-37℃，pH7.0，盐度4%。该菌作为一种新的益生菌，有望应用于海水养殖病害控制。

A probiotic strain D-2 has been isolated from seawater sample in this study. By observing its colony morphology, physiological and biochemical experiments, and the sequence analysis of its 16SrDNA, the strain was identified to be Providencia vermicola and was coded D-2, and the access number was KJ660957 in NCBI. The strain showed greater inhibition against the indicator pathogen V. parahaemolyticus13150, and it produced a wide zone of inhibition against several pathogens, such as V. parahaemolyticus, V. harveyi and Aeromonas salmonicida. The LC50 of D-2 was found to be 108 cfu/ml, and the LC50 of V. parahaemolyticus was 105 cfu/ml after 72 h treatment in Artemia nauplii. Moreover, drug sensitivity tests indicated that strain D-2 was just resistant to Kanamycin and Norfloxacin. At last, the optimized fermentation condition included temperature 25-37 ℃, pH 7.0 and 4% salinity. As a new native strain, it can be used in developing microbial ecological agents for the disease control of mariculture.