蚕用益生芽孢杆菌SWL-19的筛选鉴定及其对肠道菌群多样性的影响  

Screening and Identification of Silkworm Probiotic Bacillus SWL-19 and Its Effect on Intestinal Microflora Diversity

作　　者：李冠楠[1] 夏雪娟[2] SENDEGEYA Parfait 何石宝 郭东东[1] 朱勇[1] 

机构地区：[1]西南大学生物技术学院,重庆400716 [2]西南大学食品科学学院,重庆400716

出　　处：《中国农业科学》2015年第9期1845-1853,共9页Scientia Agricultura Sinica

基　　金：国家农业科技成果转化资金(2012GB2F100376);西南大学基本科研业务费专项资金(XDJK2014D039)

摘　　要：【目的】筛选对家蚕(Bombyx mori)具有益生作用的芽孢杆菌,改善家蚕肠道微环境,促进蚕业生产的发展。【方法】采用高温选育法对家蚕肠道内的芽孢杆菌进行初筛;以产胞外消化酶(蛋白酶、脂肪酶、纤维素酶和淀粉酶)及能在肠道定殖为基础,结合溶血试验、抗生素药敏试验和产消化酶能力大小等对芽孢杆菌进行复筛。通过形态、生理生化特征和16S r DNA序列分析鉴定菌种,再利用变性梯度凝胶电泳(DGGE)技术检测其在家蚕肠道内定殖情况及其对肠道内微生物菌群多样性的影响。【结果】共从57株细菌中筛选出两株能同时产蛋白酶、脂肪酶、纤维素酶和淀粉酶且不产生溶血现象的芽孢杆菌SWL-17和SWL-19。14种抗生素药敏纸片检测结果表明,二者对绝大多数抗生素敏感,且敏感程度基本相同。除SWL-19不能利用蔗糖发酵外,两个株菌的革兰氏染色、芽孢染色、产酸产气、柠檬酸利用、葡萄糖发酵、硝酸盐还原和V-P等生理生化特性均相同。二者生长速度相同,但菌落形态特征差异较大。SWL-17的菌落形态为淡黄色,圆形隆起,表面光滑湿润,边缘整齐,而SWL-19的菌落形态为乳白色不透明,表面扁平干燥,边缘不整齐。SWL-17产蛋白酶、脂肪酶、纤维素酶和淀粉酶的能力分别为(1.62±0.04)、(2.12±0.11)、(1.87±0.03)和(1.43±0.03),SWL-19的产酶能力分别为(2.91±0.05)、(2.43±0.04)、(3.24±0.12)和(3.48±0.10)。因此SWL-19具有较高的产消化酶能力,且除脂肪酶外,其产蛋白酶、纤维素酶和淀粉酶的能力均显著性高于SWL-17(P<0.05)。细菌16S r DNA序列比对和系统发育分析表明SWL-17和SWL-19分别为芽孢杆菌(Bacillus sp.)和枯草芽孢杆菌(B.subtilis),且SWL-19具有更清楚的遗传背景,适于进行后续试验。进一步DGGE试验结果表明,自然条件下家蚕肠道内的优势菌群为肠球菌属(Enterococcus)细菌,而外源性添加菌株SWL-19后能使家�Objective] The objective of this study is to screen theBacillius probiotic for silkworm, which could be used to improve silkworm intestinal micro-environment and promote the development of sericulture production.[Method] Heat treatment was used for primary screening ofBacillusin the gut of silkworm. Further screening were conducted based on extracellular production of protease, lipase, cellulose and amylase, combined with hemolysis test, antibiotic susceptibility assay and abilities of producing digestive enzymes, moreover, colonized in the silkworm gut. Strains’ identification was carried out based on morphological, physiological and biochemical and 16S rDNA sequence analyse. In addition, denaturing gradient gel electrophoresis （DGGE） technology was used to detect the colonization of the strain and its effect on microflora diversity in the gut of silkworm.[Result] Two strains termed as SWL-17 and SWL-19 were screened from 57 bacterial strains. Both of them did not cause hemolysis and were able to produce protease, lipase, cellulose and amylase, simultaneously. Kirby-Bauer disc diffusion method was employed to perform antimicrobial susceptibility testing by using 14 kinds of paper disc, which showed that both SWL-17 and SWL-19 were sensitive to most of the drugs. The physiological and biochemical characteristics, like gram’s staining, spore staining, production of gas and acid, citrate utilization, glucose fermentation, nitrate reduction and V-P test etc. of the two strains were the same, except that SWL-19 couldn’t ferment sucrose. The growth rates of SWL-17 and SWL-19 were similar, while the colony morphologies were different. The colony morphology of SWL-17 was light yellow, round, ridgy, humid and tidy edge, while that of SWL-19 was white opaque, flat, dry and jagged edge. The protease, lipase, cellulose and amylase producing abilities of SWL-17 were （1.62±0.04）, （2.12±0.11）, （1.87±0.03） and （1.43±0.03）, respectively; those of SWL-19 were （2.91±0.05）, （2.43±0.04）, （3.

关 键 词：益生菌 筛选 家蚕 肠道菌群 芽孢杆菌 巢式PCR—DGGE 

分 类 号：S88[农业科学—特种经济动物饲养]