加州鲈肠道芽孢杆菌的筛选及其功能研究

Screening and functional study of Bacillus from intestines of largemouth bass

为筛选对加州鲈(Micropterus salmoides)有益生效果的芽孢杆菌,从加州鲈肠道分离芽孢杆菌,检测其溶血性、产酶能力、生物被膜形成能力、对多种常见抗生素的敏感性、耐受人工胃肠液及体外拮抗诺卡氏菌的能力等,并将菌株拌料饲喂加州鲈后测定加州鲈体质量增长和炎症因子的表达。结果显示,从鉴定到的94株芽孢杆菌中筛选出无溶血性且产酶能力广的4株枯草芽孢杆菌K-3、K-12、K-13、K-22,4株菌均是强生物被膜形成株,对多种常见抗生素敏感,其中K-3、K-13菌株能在体外拮抗诺卡氏菌。4株菌在人工胃液(pH 4)和人工肠液中2 h的存活率达到80%以上。K-13试验组的体质量增长率显著高于对照组,K-12、K-13试验组肠道蛋白酶、脂肪酶活性显著高于对照组。此外,与对照组相比,K-13试验组头肾、脾、后肠中的促炎因子表达水平下调,抑炎因子表达水平上调,表明K-13菌株具有减轻加州鲈肠道炎症发生的潜力。综合分析,本研究分离筛选的枯草芽孢杆菌K-13株具有安全性高、产酶能力强、拮抗诺卡氏菌、促进加州鲈生长的特性,可作为加州鲈饲料添加的益生菌制剂。

In order to screen Bacillus strains which are beneficial to largemouth bass,the Bacillus strains from the intestine of largemouth bass were isolated,their hemolysis,enzymatic activity,biofilm for⁃mation ability,sensitivity to various common antibiotics,tolerance to gastric and intestinal fluids,and in vitro antagonism against Nocardia were detected.These strains were added to the feed of largemouth bass and weigh gain and expression of inflammatory cytokine were detected.A total of 94 Bacillus strains were identified.Four Bacillus subtilis strains,K-3,K-12,K-13,and K-22,were selected based on their nonhemolytic and broad enzymatic activity.These four strains all formed strong biofilms and were sensitive to various common antibiotics.Strains K-3 and K-13 showed in vitro antagonism against Nocardia.The 2-hour survival rates of the four strains in artificial gastric and intestinal fluids were above 80%.After feeding for 42 days,the weight gain of largemouth bass of K-13 group was significantly higher than that of the con⁃trol group,while protease and lipase activities in the intestines in K-12 and K-13 groups were significantly higher than that of the control group.In addition,compared with the control group,the expression levels of pro-inflammatory factors in the head kidney,spleen,and hind gut of the K-13 experimental group were down-regulated,while the expression levels of anti-inflammatory factor were up-regulated,indicating that K-13 has the potential to reduce intestinal inflammation in largemouth bass.Overall,the B.subtilis strain K-13 selected in this study has high safety,broad enzymatic activity,antagonism against Nocardia,and promotes the growth of largemouth bass,making it a potential probiotic additive for largemouth bass feed.