Scientia Agricultura Sinica ›› 2015, Vol. 48 ›› Issue (9): 1845-1853.doi: 10.3864/j.issn.0578-1752.2015.09.18

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

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

LI Guan-nan1, XIA Xue-juan2, SENDEGEYA Parfait1, HE Shi-bao1, GUO Dong-dong1, ZHU Yong1   

  1. 1College of Biotechnology, Southwest University, Chongqing 400716
    2College of Food Science, Southwest University, Chongqing 400716
  • Received:2014-11-12 Online:2015-05-01 Published:2015-05-01

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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

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