CAJ | 학술논문

【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。【目的】篩選對傢蠶（Bombyx mori）具有益生作用的芽孢桿菌，改善傢蠶腸道微環境，促進蠶業生產的髮展。【方法】採用高溫選育法對傢蠶腸道內的芽孢桿菌進行初篩；以產胞外消化酶（蛋白酶、脂肪酶、纖維素酶和澱粉酶）及能在腸道定殖為基礎，結閤溶血試驗、抗生素藥敏試驗和產消化酶能力大小等對芽孢桿菌進行複篩。通過形態、生理生化特徵和16S rDNA序列分析鑒定菌種，再利用變性梯度凝膠電泳（DGGE）技術檢測其在傢蠶腸道內定殖情況及其對腸道內微生物菌群多樣性的影響。【結果】共從57株細菌中篩選齣兩株能同時產蛋白酶、脂肪酶、纖維素酶和澱粉酶且不產生溶血現象的芽孢桿菌SWL-17和SWL-19。14種抗生素藥敏紙片檢測結果錶明，二者對絕大多數抗生素敏感，且敏感程度基本相同。除SWL-19不能利用蔗糖髮酵外，兩箇株菌的革蘭氏染色、芽孢染色、產痠產氣、檸檬痠利用、葡萄糖髮酵、硝痠鹽還原和V-P等生理生化特性均相同。二者生長速度相同，但菌落形態特徵差異較大。SWL-17的菌落形態為淡黃色，圓形隆起，錶麵光滑濕潤，邊緣整齊，而 SWL-19的菌落形態為乳白色不透明，錶麵扁平榦燥，邊緣不整齊。SWL-17產蛋白酶、脂肪酶、纖維素酶和澱粉酶的能力分彆為（1.62±0.04）、（2.12±0.11）、（1.87±0.03）和（1.43±0.03），SWL-19的產酶能力分彆為（2.91±0.05）、（2.43±0.04）、（3.24±0.12）和（3.48±0.10）。因此SWL-19具有較高的產消化酶能力，且除脂肪酶外，其產蛋白酶、纖維素酶和澱粉酶的能力均顯著性高于SWL-17（P＜0.05）。細菌16S rDNA序列比對和繫統髮育分析錶明SWL-17和SWL-19分彆為芽孢桿菌（Bacillus sp.）和枯草芽孢桿菌(B.subtilis)，且SWL-19具有更清楚的遺傳揹景，適于進行後續試驗。進一步 DGGE 試驗結果錶明，自然條件下傢蠶腸道內的優勢菌群為腸毬菌屬(Enterococcus)細菌，而外源性添加菌株SWL-19後能使傢蠶腸道內的芽孢桿菌屬和腸桿菌屬細菌成為優勢菌群，說明芽孢桿菌SWL-19能在傢蠶腸道內定殖，併且能改變傢蠶腸道內微生物菌群的結構和多樣性。【結論】傢蠶益生芽孢桿菌的篩選為研髮蠶用複閤微生態製劑提供菌株資源，在蠶業生產上具有重要的應用價值。
【목적】사선대가잠（Bombyx mori）구유익생작용적아포간균，개선가잠장도미배경，촉진잠업생산적발전。【방법】채용고온선육법대가잠장도내적아포간균진행초사；이산포외소화매（단백매、지방매、섬유소매화정분매）급능재장도정식위기출，결합용혈시험、항생소약민시험화산소화매능력대소등대아포간균진행복사。통과형태、생리생화특정화16S rDNA서렬분석감정균충，재이용변성제도응효전영（DGGE）기술검측기재가잠장도내정식정황급기대장도내미생물균군다양성적영향。【결과】공종57주세균중사선출량주능동시산단백매、지방매、섬유소매화정분매차불산생용혈현상적아포간균SWL-17화SWL-19。14충항생소약민지편검측결과표명，이자대절대다수항생소민감，차민감정도기본상동。제SWL-19불능이용자당발효외，량개주균적혁란씨염색、아포염색、산산산기、저몽산이용、포도당발효、초산염환원화V-P등생리생화특성균상동。이자생장속도상동，단균락형태특정차이교대。SWL-17적균락형태위담황색，원형륭기，표면광활습윤，변연정제，이 SWL-19적균락형태위유백색불투명，표면편평간조，변연불정제。SWL-17산단백매、지방매、섬유소매화정분매적능력분별위（1.62±0.04）、（2.12±0.11）、（1.87±0.03）화（1.43±0.03），SWL-19적산매능력분별위（2.91±0.05）、（2.43±0.04）、（3.24±0.12）화（3.48±0.10）。인차SWL-19구유교고적산소화매능력，차제지방매외，기산단백매、섬유소매화정분매적능력균현저성고우SWL-17（P＜0.05）。세균16S rDNA서렬비대화계통발육분석표명SWL-17화SWL-19분별위아포간균（Bacillus sp.）화고초아포간균(B.subtilis)，차SWL-19구유경청초적유전배경，괄우진행후속시험。진일보 DGGE 시험결과표명，자연조건하가잠장도내적우세균군위장구균속(Enterococcus)세균，이외원성첨가균주SWL-19후능사가잠장도내적아포간균속화장간균속세균성위우세균군，설명아포간균SWL-19능재가잠장도내정식，병차능개변가잠장도내미생물균군적결구화다양성。【결론】가잠익생아포간균적사선위연발잠용복합미생태제제제공균주자원，재잠업생산상구유중요적응용개치。
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), (3.24±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 toBacillus sp. andB. 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 wasEnterococcus spp., after exogenous adding strain SWL-19, theBacillus sp. andEnterobacter 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 ofBacillus probiotic is believed to have an important application value in the development of sericulture compound micro-ecological agents.