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

doi:10.3864/j.issn.0578-1752.2015.09.18

摘要/Abstract

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 rDNA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 rDNA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后能使家蚕肠道内的芽孢杆菌属和肠杆菌属细菌成为优势菌群，说明芽孢杆菌SWL-19能在家蚕肠道内定殖，并且能改变家蚕肠道内微生物菌群的结构和多样性。【结论】家蚕益生芽孢杆菌的筛选为研发蚕用复合微生态制剂提供菌株资源，在蚕业生产上具有重要的应用价值。

关键词: 益生菌, 筛选, 家蚕, 肠道菌群, 芽孢杆菌, 巢式PCR-DGGE

Abstract: 【Objective】 The objective of this study is to screen the Bacillius 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 of Bacillus in 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), (3.24±0.12) and (3.48±0.10), respectively. The protease, cellulose and amylase producing abilities of SWL-19 were significantly higher (P＜0.05) than that of SWL-17. Through homology analysis of 16S rDNA sequence, strains SWL-17 and SWL-19 were assigned to Bacillus sp. and B. subtilis,respectively. However, genetic background of SWL-19 were more clearly, which indicated that it was more suitable for further study. The dominant microflora of control group was Enterococcus spp., after exogenous adding strain SWL-19, the Bacillus sp. and Enterobacter sp. became the dominant microfloras. These results suggested that strain SWL-19 were able to permanent colonizes in the gut of silkworm and change the diversity of bacteria. 【Conclusion】 Screening of Bacillus probiotic is believed to have an important application value in the development of sericulture compound micro-ecological agents.

Key words: probiotics, screening, Bombyx mori, intestinal microflora, Bacillus, nested PCR-DGGE

李冠楠，夏雪娟，SENDEGEYA Parfait，何石宝，郭东东，朱勇. 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[J]. 中国农业科学, 2015, 48(9): 1845-1853.

LI Guan-nan, XIA Xue-juan, SENDEGEYA Parfait, HE Shi-bao, GUO Dong-dong, ZHU Yong. Screening and Identification of Silkworm Probiotic Bacillus SWL-19 and Its Effect on Intestinal Microflora Diversity[J]. Scientia Agricultura Sinica, 2015, 48(9): 1845-1853.

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参考文献

[1] Li G H, Tang Q, Chen H Q, Yao Q, Ning D G, Chen K P. Display of Bombyx mori nucleopolyhedrovirus GP64 on the Bacillus subtilis spore coat. Current Microbiology, 2011, 62(5): 1368-1373.

[2] 李冠楠, 夏雪娟, 赵欢欢, 隆耀航, 李姣蓉, 朱勇. 氟化物对家蚕耐氟和氟敏感品种肠道中留存产酶菌群落组成和多样性的影响. 昆虫学报, 2014, 57(2): 187-193.

Li G N, Xia X J, Zhao H H, Long Y H, Li J R, Zhu Y. Effects of fluoride on the community and diversity of the retained enzyme- producing bacteria in the intestina of silkworm (Bombyx mori). Acta Entomologica Sinica, 2014, 57(2): 187-193. (in Chinese)

[3] Liang X, Fu Y M, Tong L, Hong L. Microbial shifts of the silkworm larval gut in response to lettuce leaf feeding. Applied Microbiology and Biotechnology, 2014, 98: 3769-3776.

[4] 张剑飞, 王林玲, 周泽扬. 家蚕K₁₀₀E₂₃肠道微生物初探. 四川蚕业, 2002(3): 8-12.

Zhang J F, Wang L L, Zhou Z Y. Microflora in gut of the silkworm K₁₀₀andE₂₃. Sichuan Sericulture, 2002(3): 8-12. (in Chinese)

[5] Kim K, Kim M, Kim D, Park Y, Kang J. Characterization of Bacillus polyfermenticus KJS-2 as a probiotic. Journal of Microbiology and Biotechnology, 2009, 19(9): 1013-1018.

[6] Lutful K S M. The role of probiotics in the poultry industry. International Journal of Molecular Sciences, 2009, 10(8): 3531-3546.

[7] Guo X H, Li D F, Lu W Q, Piao X S, Chen X L. Screening of Bacillus strains as potential probiotics and subsequent confirmation of the in vivo effectiveness of Bacillus subtilis MA139 in pigs. Antonie Van Leeuwenhoek, 2006, 90(2): 139-146.

[8] Kreuzer S, Machnowska P, Aβmus J, Sieber M, Pieper R, Schmidt M F, Brockmann G A, Scharek-Tedin L, Johne R. Feed of the probiotics bacterium Enterococcus faecium NCIMB 10415 differentially affects shedding of enteric viruses in pigs. Veterinary Research, 2012, 43(1): 58.

[9] Thankappan B, Ramesh D, Ramkumar S, Natarajaseenivasan K, Anbarasu K. Characterization of Bacillus spp. from the gastrointestinal tract of Labeo rohita-towards to identify novel probiotics against fish pathogens. Applied Biochemistry and Biotechnology, 2015, 175(1): 340-353.

[10] Ozkan A D, Han D, Umu O C O, Angun P, Senturk B, Yasa O, Tekinay T. Screening and selection of novel animal probiotics isolated from bovine chime. Annals of Microbiology, 2013, 63(4): 1291-1300.

[11] Tao H P, Shen Z Y, Zhu F, Xu X F, Tang X D, Xu L. Isolation and identification of a pathogen of silkworm Bombyx mori. Current Microbiology, 2011, 62(3): 876-883.

[12] 相辉, 李木旺, 赵勇, 赵立平, 张月华, 黄勇平. 家蚕幼虫中肠细菌群落多样性的PCR-DGGE和16S rDNA文库序列分析. 昆虫学报, 2007, 50(3): 222-233.

Xiang H, Li M W, Zhao Y, Zhao L P, Zhang Y H, Huang Y P. Bacterial community in midguts of the silkworm larvae estimated by PCR-DGGE and 16S rDNA gene library analysis. Acta Entomologica Sinica, 2007, 50(3): 222-233. (in Chinese)

[13] Ercolini D. PCR-DGGE fingerprinting: novel strategies for detection of microbes in food. Journal of Microbiological Methods, 2004, 56(3): 297-314.

[14] 刘灿, 生吉萍, 申琳. 变性梯度凝胶电泳技术及其在食品微生物研究中的应用. 食品科学, 2009, 30(11): 266-373.

Liu C, Sheng J P, Shen L. Review on denaturing gradient gel electrophoresis and its application in food microbiology. Food Science, 2009, 30(11): 266-373. (in Chinese)

[15] 高绘菊, 路国兵, 查传勇, 刘敬瑞, 牟志美. 家蚕肠道产酶菌的分离与筛选. 蚕业科学, 2007, 33(2): 228-233.

Gao H J, Lu G B, Cha C Y, Liu J R, Mu Z M. Isolation and screening of the enzyme-producing bacteria in the intestine of silkworm. Acta Sericologica Sinica, 2007, 33(2): 228-233. (in Chinese)

[16] 东秀珠, 蔡妙英. 常见细菌系统鉴定手册. 北京: 科学出版社, 2001.

Dong X Z, Cai M Y. Manual of Systematic Determinative Bacteriology. Beijing: Science Press, 2001. (in Chinese)

[17] 沈萍, 范秀容, 李广武. 微生物学实验. 3版. 北京: 高等教育出版社, 1999.

Shen P, Fan S R, Li G W. Microbiology Experiment. 3rd ed. Beijing: Higher Education Press, 1999. (in Chinese)

[18] Nakatsu C H, Torsvik V, Ovreas L. Soil community analysis using DGGE of 16S rDNA polymerase chain reaction products. Soil Science Societyof America Journal, 2000, 64(4): 1382-1388.

[19] Hou Y, Ma Z, Dong S Z, Chen Y H, Yu X. Analysis of yeast-like symbiote diversity in the brown planthopper (BPH), Nilaparvata lugens Stål, using a novel nested PCR-DGGE protocol. Current Microbiology, 2013, 67(3): 263-270.

[20] Lefebvre T, Miambi E, Pando A, Diouf M, Lefèvre C R. Gut-specific actinobacterial community structure and diversity associated with the wood-feeding termite species, Nasutitermescorniger (Motschulsky) described by nested PCR-DGGE analysis. Insectes Sociaux, 2009, 56(3): 269-276.

[21] MalagoJ J,Koninkx. Probiotic Bacteria and Enteric Infections: Cytoprotection by Probiotic Bacteria. Springer, 2011. J F,Marinsek-Logar R

[22] FAO/WHO. Report on joint FAO/WHO expert consultation on evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria 2001. Cordoba, Argentina, 2001.

[23] 郭小华, 赵志丹. 饲用益生芽孢杆菌的应用及其作用机理的研究进展. 中国畜牧兽医, 2010, 37(2): 27-31.

Guo X H, Zhao Z D. Research progress on the application and mechanisms of Bacilli-derived probiotics. Chinese Animal Husbandry and Veterinary Medicine, 2010, 37(2): 27-31. (in Chinese)

[24] Fuller R. Probiotics in man and animals. Journal of Applied Bacteriology, 1989, 66(5): 365-378.

[25] Tokuda G, Saito H, Watanabe H. A digestive β-glucosidase from the salivary glands of the termite, Neotermes koshunensis (Shiraki): distribution, characterization and isolation of its precursor cDNA by 5′ and 3′-RACE amplifications with degenerate primers. Insect Biochemistry and Molecular Biology, 2002, 32(12): 1681-1689.

[26] 王在贵, 杨文静, 刘朝良, 朱保健, 魏国清, 邹昌瑞. 家蚕肠道产蛋白酶菌株的分离与鉴定及其发酵条件. 农业生物技术学报, 2011, 19(1): 149-156.

Wang Z G, Yang W J, Liu C L, Zhu B J, Wei G Q, Zou C R. Isolation and identification of the protease-producing bacteria from gut of silkworm (Bombyx mori) and its suitable fermentation condition. Journal of Agricultural Biotechnology, 2011, 19(1): 149-156. (in Chinese)

[27] 杨文静, 王在贵, 刘朝良, 魏国清, 朱保健, 邹昌瑞. 家蚕肠道产淀粉酶细菌的分离鉴定及其酶基因的克隆与表达. 激光生物学报, 2011, 20(2): 219-229.

Yang W J, Wang Z G, Liu C L, Wei G Q, Zhu B J, Zou C R. Isolation and identification of the amylase-producing bacteria in silkworm intestine and cloning and expression of their amylase genes. Acta Laser Biology Sinica, 2011, 20(2): 219-229. (in Chinese)

[28] 王在贵, 韩薇, 刘朝良, 许晓丹. 家蚕肠道环境对外源纤维素酶活力稳定性影响的研究. 激光生物学报, 2012, 21(1): 31-35.

Wang Z G, Han W, Liu C L, Xu X D. The stability of exogenous cellulase activity influenced by the intestine environment of silkworm. Acta Laser Biology Sinica, 2012, 21(1): 31-35. (in Chinese)

[29] Feng W, Wang X Q, Zhou W, Liu G Y, Wan Y J. Isolation and characterization of lipase-producing bacteria in the intestine of the silkworm, Bombyx mori, reared on different forage. Journal of Insect Science, 2011, 11(135): 1-10.

[30] Subramanian S, Gadhave K R, Mohanraj P, Thangamalar A. Use of 16S rRNA probes for characterization of gut microflora of silkworm (Bombyx mori L.) breeds. Karnataka Journal of Agricultural Sciences, 2009, 22(3): 476-478.

[31] Ceuppens S, Boon N, Uyttendaele M. Diversity of Bacillus cereus group strains is reflected in their broad range of pathogenicity and diverse ecological lifestyles. FEMS Microbiology Ecology, 2013, 84(3): 433-450.

[32] Orhan E, Omay D, Güvenilir Y. Partial purification and characterization of protease enzyme from Bacillus subtilis and Bacillus cereus. Applied Biochemistry and Biotechnology, 2005, 121/124: 183-194.

[33] Schäfer A, Konrad R, Kuhnigk T, Kämpfer P, Hertel H, König H. Hemicellulose-degrading bacteria and yeasts from the termite gut. Journal of Applied Bacteriology, 1996, 80(5): 471-478.

[34] 向芸庆, 王晓强, 冯伟, 周围, 谢洪霞, 万永继. 不同饲料饲养家蚕其肠道微生态优势菌群类型的组成及差异性. 生态学报, 2010, 30(14): 3875-3882.

Xiang Y Q, Wang X Q, Feng W, Zhou W, Xie H X, Wan Y J. Comparative analysis of the composition of dominant intestinal microflora in silkworm reared with different forages. Acta Ecologica Sinica, 2010, 30(14): 3875-3882. (in Chinese)