凡纳滨对虾养殖体系中群体感应淬灭菌的筛选、安全性评估及发酵条件优化

Screening, Safety Evaluation and Fermentation Conditions Optimization of Quorum Quenching Bacteria from Litopenaeus vannamai Culture System

中文关键词: 凡纳滨对虾  细菌性病害防治  群体感应淬灭菌株  安全性  条件优化

英文关键词: Litopenaeus vannamai  Bacterial disease control  Quorum quenching bacteria  Safety  Condition optimization

中文摘要:

      从凡纳滨对虾(Litopenaeus vannamai)养殖体系中筛选得到具有群体感应淬灭(Quorum quenching, QQ)活性的潜在功能菌，并对菌株进行16S rDNA鉴定、安全性评估及发酵条件优化。采用根癌农杆菌(Agrobacterium tumefaciens) A136液体X-gal法进行活性菌株筛选，发现2株细菌(BDZ5和W1B)的发酵液对信号分子己酰基高丝氨酸内酯(C6-HSL)具有显著的降解作用，但经沸水浴处理后均丧失降解活性。经16S rDNA基因序列分析，菌株BDZ5和W1B分别被鉴定为芽孢杆菌属(Bacillus)和科贝特氏菌属(Cobetia)。安全性评估结果显示，菌株BDZ5和W1B均无溶血性，且对多数抗生素具有敏感性，注射2株活性菌未提高凡纳滨对虾死亡率，且对凡纳滨对虾血清免疫酶活性无显著影响。单因素条件优化结果显示，在起始pH为7.0、盐度为20、0.0005%的MnCl2条件下培养48 h后，菌株BDZ5达到最大生长量；在起始pH为6.0、盐度为40、0.05%的CaCl2条件下培养48 h，菌株W1B达到最大生长量。2株菌的最适信号分子C6-HSL降解条件与其生长条件有所不同，在起始pH为7.5、盐度为30、0.0005%的MnCl2条件下培养48 h，2株活性菌对信号分子C6-HSL的降解率达到最大值。研究表明，菌株BDZ5和W1B可作为益生菌应用于水产养殖，为基于QQ角度防治对虾细菌性病害提供优良菌种。

英文摘要:

      With the development of shrimp culture industry, bacterial diseases are becoming very serious. To explore the new methods of controlling bacterial diseases in shrimps through quorum quenching (QQ), potential probiotics with QQ activity from the culture system of Litopenaeus vannamei were screened and identified in the present study. The safety of QQ strains was evaluated and fermentation conditions were optimized. The Agrobacterium tumefaciens A136 liquid X-gal method was used to screen QQ strains. Two bacterial fermentation broths showed significant degradation of hexanoyl homoserine lactone (C6-HSL), while the degradation activities were both lost after boiling water bath treatment. The strains were subjected to 16S rDNA gene sequencing analysis, and BDZ5 and W1B were identified as Bacillus sp. and Cobetia sp., respectively. The safety evaluation results showed that strains BDZ5 and W1B were not hemolyzed and were not sensitive to most antibiotics. After injection of the two strains, the cumulative mortality of shrimp did not increase, and the serum immune and antioxidase enzyme activities in shrimp did not change significantly, indicating that the two strains are safe for shrimp. Single factor condition optimization results showed the optimal growth conditions of BDZ5 as follows: initial pH 7.0, salinity 20, and 0.0005% MnCl2 for 48 h culture time. Moreover, the optimal growth conditions of W1B were as follows: initial pH 6.0, salinity 40, and 0.05% CaCl2 for 48 h. Except for the culture time, the optimal C6-HSL degradation conditions of two strains were different from their growth conditions. Under the culture condition of initial pH 7.5, salinity 30, and 0.0005% MnCl2 for 48 h, the degradation rate of the signal molecule C6-HSL by the action of the two active bacteria reached the maximum. The results of the study can provide excellent strains for controlling bacterial diseases in shrimp based on QQ and provide reference materials for large-scale fermentation and subsequent application of the strains.