凡纳滨对虾肠道内产消化酶益生菌的分离与筛选

为获得具有消化酶活性且安全的益生菌,从凡纳滨对虾肠道中初步分离得到576株细菌,对菌株进行产蛋白酶、淀粉酶和脂肪酶能力的定性及定量测试,筛选出产酶种类多且产酶能力强的菌株11株。对筛选出的11株菌进行了幼虾浸浴实验、药敏性实验和溶血性实验,以评价其生物安全性。将11株菌的菌悬液添加到凡纳滨对虾幼虾的养殖水体中进行浸浴实验,浸浴结束后用鳗弧菌进行刺激,测定不同浸浴组幼虾相关免疫基因的相对表达量,以确定其对幼虾的保护效果。综合消化酶活性、菌株对幼虾的保护效果及生物安全性,筛选得到4株效果较好的菌株。菌株的16S r DNA分子鉴定结果表明,细菌1号、2号和4号分别与芽孢杆菌(Bacillus sp.PCSAS2-38,GQ284495.1)、蜡样芽孢杆菌(B.cereus strain N419,JN400121.1)及苏云金芽孢杆菌(B.thuringiensis strain EA26.1,KC758847.1)的相似性均为100%,9号菌株与荚膜红细菌(Rhodobacter capsulatus strain PSB-03,FJ866782.1)相似性达到99%,为后续益生菌制剂的开发奠定了前期基础。

Isolation and screeing of digestive enzyme producing probiotics from intestine of Litopenaeus vannamei

To get safe digestive enzyme producing probiotics from intestine of healthy Litopenaeus vannamei, 576 bacteria strains were isolated. Those bacteria strains, extracellular protease, amylase, and lipase activities were detected through qualitative assay and quantittive assay. 11bacteria strains with three kinds of digestive enzyme and high capacity of enzyme production were screened. Secondly, susceptibility and hemolysis of the11strains were tested to identify their security. In this study, vegetative cell suspensions of 11strains in equal proportions were added in the rearing water of juvenile shrimp(L. vannamei). Then shrimps were challenged with Vibrio anguillarum. After the challeng test, real-time RT-PCR was employed to determine the relative expression of several immune genes. Through the difference of the relative expression we could detemine the protective effect of these strains on juvenile shrimp. Considering digestive enzyme activity, biological safety and protective effect, 4 strains were selected. Similarity analysis of 16S rDNA sequences indicated that NO.1, 2 and 4 showed 100% similarity to Bacillus sp. PCSAS 2-38, B. cereus strain N419 and B. thuringiensis strain EA26.1, respectively, and NO.9 showed 99% similarity to Rhodobacter capsulatus strain PSB-03. This laid the foundation for the probiotic development.