刺参(Apostichopus japonicus)大水面养殖池塘环境中优势益生菌筛选及其特性分析

Screening and Characteristic Analysis of Potential Probiotics from Large Water Ponds Used for Sea Cucumber (Apostichopus japonicus) Farming

中文关键词: 刺参  大水面池塘  腐皮综合征  益生菌  枯草芽孢杆菌  拮抗菌

英文关键词: Apostichopus japonicus  Large water pond  Skin ulcerative syndrome  Probiotic bacterium  Bacillus subtilis  Antagonistic bacteria

中文摘要:

      2014年12月–2015年12月，在大连地区刺参(Apostichopus japonicus)大水面养殖池塘进行了春、夏、秋、冬四季有益菌分离筛选，从其水体和底泥中共分离得到66株细菌。以刺参“腐皮综合征”主要病原菌——灿烂弧菌(Vibrio splendidus)和假交替单胞菌(Pseudoalteromonas nigrifaciens)为指示菌进行拮抗作用实验，利用选择培养基对菌株产淀粉酶和蛋白酶的能力进行测定，最后通过安全性实验得到潜在益生菌株YQ-2。结果显示，该菌株对灿烂弧菌和假交替单胞菌有较强的抑制作用，抑菌圈分别达到22 mm和24 mm；对淀粉和蛋白选择培养基水解圈的直径达到22 mm和36 mm。安全性实验显示，该菌株无论是在108 CFU/ml浸浴还是投喂108 CFU/g的粉末饲料感染，30 d内供试刺参没有发病和死亡现象，健康程度好，且相对于对照组的体重明显增长，108 CFU/g粉末饲料投喂组的相对增长率达到39.31%。此外，本研究对YQ-2菌株的生理生化指标、16S rDNA序列进行了分析，其同源性与枯草芽孢杆菌Bacillus subtilis strain KLP2015相似度达99%，故将该菌株鉴定为枯草芽孢杆菌。该株枯草芽孢杆菌在大水面刺参池塘四季水体中数量为140–280 CFU/ml，高于其他菌株；同时，该菌株在水体中还具有较高的优势度，优势度分别为4.2%、3.5%、2.6%、4.6%，冬、春季节的优势度明显高于夏、秋季节；它属于土著分离菌株，对引起刺参腐皮综合征的2株病原菌具有较强的抑制作用，这对刺参大水面生态养殖具有较大的应用潜力。

英文摘要:

      From December 2014 to December 2015, the flora population structural analysis and beneficial bacteria isolation and screening were carried out in large water ponds of a sea cucumber (Apostichopus japonicus) farm. Sixty-six bacterial strains were isolated from the water body and pond sediment. Two bacterial strains Pseudoalteromonas nigrifaciens and Vibrio splendidus associated with the “skin ulcerative syndrome” disease were used as indicators in the antagonistic effect trials. Based on the bacterial extracellular amylase, protease activities and challenge tests, one potential probiotic bacterium YQ-2 was obtained. The results showed that this strain had good ability to inhibit the proliferation of P. nigrifaciens and V. splendidus, evident by the diameter of inhibition zones, 22 mm and 24 mm, respectively; while the diameter of amylase and protease hydrolysis was 22 mm and 36 mm; In addition, the challenge tests revealed that the strain YQ-2 was safe to sea cucumber when it was challenged by bathing the bacterial strain at the concentration of 108 CFU/ml, or fed the food inoculated with the bacterial cells at the concentration of 108 CFU/g once daily for one month. The present study also found that among the sea cucumbers fed the food containing 108 CFU/g bacterial cells, the body weight increased significantly compared with the control group, with the relative growth rate as high as 39.31%. Homogenous analysis using 16S rDNA sequences indicated that the strain YQ-2 had 99% similarity with the Bacillus subtilis strain KLP2015. Thus, the strain YQ-2 was tentatively identified as Bacillus subtilis. The number of B. subtilis in the rearing water was counted in all four seasons and the year-round concentration was about 140–280 CFU/ml, which was higher than other strains. Meanwhile the dominance of YQ-2 was 4.2%, 3.5%, 2.6%, and 4.6% corresponding to Spring, Summer, Autumn and Winter. Furthermore, the dominance of YQ-2 was higher in Spring and Winter compared to Summer and Autumn. Nevertheless, the strain YQ-2 exhibited a strong antibacterial effect on the pathogens P. nigrifaciens and V. splendidus which were the main pathogenic agents causing the “skin ulcer syndrome” disease. Hence, the strain YQ-2 has great application for farming A. japonicus in large water ponds.