短小芽孢杆菌（Bacillus pumilus 4D-14）的分离与分子鉴定，抑菌性及微生态制剂应用研究
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摘要
本论文首先在微量肉汤稀释法基础上建立了实验室96孔板快速筛选方法；然后从疫病多发的养殖场土壤和市场动物微生态产品中分离到多株芽孢杆菌；选取部分从土壤中分离的芽孢杆菌菌株，利用鼠伤寒沙门氏菌（Salmonella typhimurium）ATCC14028作为指示菌进行芽孢杆菌抑菌能力初筛，得到来源于养殖场土壤的抑菌型芽孢杆菌7株；将抑菌效果较突出的7株芽孢杆菌和市场产品中分离的芽孢杆菌进一步用4株不同血清型动物源病原菌大肠杆菌（Escherichia coli）E1、E3、E4，及鸡沙门氏菌（Salmonella gallinarum）SC1，猪沙门氏菌（Salmonella choleraesuis）和SP1绿脓杆菌（pseudomonas aeruginosa）等进行了抑菌能力评价，发现来自土壤的芽孢杆菌菌株4D-14和J-14，及来自市场产品的芽孢杆菌菌株Y14和Y16等，体外抑菌效果与20ppm的硫酸粘杆菌素相当。
使用16SrRNA及gyrB基因分子鉴定法，对来自养殖场土壤和市场产品的抑菌型芽孢杆菌菌株进行了鉴定。发现养殖场土壤来源的抑菌型芽孢杆菌以蜡样芽孢杆菌、短小芽孢杆菌和蕈状芽孢杆菌为主，而市场微生态产品来源的抑菌型芽孢杆菌，以枯草芽孢杆菌为主。通过将菌株Bacillus subtilis Y16，Bacillus lincheniformis Y14和Bacilluscereus J14，Bacillus pumilus4D-14等4株芽孢杆菌上清液经蛋白酶、高温、不同pH处理后抑菌活性的变化观察，发现不同的菌株抑菌活性物的性质有所不同。Bacilluslincheniformis Y14的抑菌活性受蛋白酶和温度影响波动严重，上清液中的抑菌活性物质可能是蛋白质类。而B. subtilis Y16，B. cereus J14，B. pumilus4D-14的抑菌活性受蛋白酶和温度影响不大，特别是B. cereus J14，B. pumilus4D-14的抑菌物质具有热稳定性，表明其抑菌活性物可能是小肽类。
考虑到中国农业部对动物饲料和养殖用微生物菌种的限制性规定，和动物微生态国内外研究现状，选择短小芽孢杆菌B. pumilus4D-14做进一步的应用效果评价。同时以市场产品来源的抑菌型芽孢杆菌B. subtilis Y16为研究对照。
将B. pumilus4D-14及B. subtilis Y16使用20L小型发酵罐进行发酵，制备微生态制剂样品。制得B. subtilis Y16菌粉共2.7kg，菌粉菌量6.8×109CFU/g；B.pumilus4D-14菌粉共2.35kg，菌粉菌量1.47×1010CFU/g。设计肉鸡养殖试验进行B.pumilus4D-14等菌株的应用研究，以国内常用饲用促生长抗生素阿维拉霉素（Avilymacin）为正对照，以无任何添加剂的饲料组为负对照，以B.pumilus4D-14、B. subtilis Y16及市场代表性枯草芽孢杆菌微生态产品可速必宁为处理组进行养殖试验。通过对罗斯308肉公鸡生产性能、免疫指标及肠道形态的分析，发现短小芽孢杆菌（B. pumilus4D-14）能有效降低养殖后期料肉比并促使各段肠管隐窝变短，使上皮细胞成活率上升；并可替代阿维拉霉素，作为促生长剂应用于肉鸡饲料。
First, this paper established a rapid screening method of laboratory96pores plate based onminimum inhibitory concentration and screened Bacillus strains from farm soil and probioticproducts for animals. Then selected Bacillus strains using Salmonella typhimurium ATCC14028as the indicator bacteria to antibacterial ability of Bacillus, obtained7antibacterial Bacillusstrains from farm soil. The antibacterial effect of7Bacillus strains from farm soil and Bacillusstrains from probiotic products were evaluated with4strains of different serotypes were furtherused animal source pathogenic bacteria Escherichia coli E1, E3, E4, Salmonella gallinarum(Salmonella gallinarum) SC1, swine Salmonella (Salmonella choleraesuis) and SP1ofPseudomonas aeruginosa, found that the antibacterial effect of Bacillus strains4D-14, J-14from the soil and Bacillus strains Y14, Y16from the market products analogous to20ppmcolistin sulfate in vitro.
Bacillus cereus, Bacillus pumilus and Bacillus mycoides highlighted the Bacillus srtains in farmsoil and Bacillus subtilis highlighted the Bacillus srtains in market animal probiotic products,according to the bacteriostasis Bacillus identification with the use of molecular16SrRNA andgyrB gene method. The characters of Bacillus supernatant of the strain Bacillus subtilis Y16,Bacillus lincheniformis Y14and Bacillus cereus J14, Bacillus pumilus4D-14was observedthrough the changes of antibacterial activity of protease, temperature, different pH, and foundthat properties of antibacterial activity of different strains were different. Antibacterial activityof B. lincheniformis Y14fluctuations was affected by protease and temperature, antifungalactivity in the supernatant may be provided by proteins. B. subtilis Y16, B. cereus J14, B.pumilus4D-14antibacterial activity is not affected by protease and temperature, especially the B.cereus J14, B. pumilus4D-14antibacterial material has thermal stability, show its antibacterialactivity may be provided by small peptides. Considering the restrictive provision of microbial strains used for animal feed and breeding ofChina's agriculture ministry and the research status of animal probiotics, selected the strain B.pumilus4D-14to do further research in application effect in this paper. At the same time, thestrain B. subtilis Y16which screened from market animal probiotic product was selected as thecontrol study.
The probiotic samples of B. pumilus4D-14and B. subtilis Y16were prepared with20Lfermentation tank. Finally, prepared B.pumilus4D-14culture2.35kg, bacteria quantity was1.47×1010CFU/g; B. subtilis Y16culture2.7kg, bacteria quantity was6.8×109CFU/g.
In the application research, using Avilamycin as positive control, B. pumilus4D-14, B. subtilisY16and market representative Bacillus probiotics as treatment on broiler breeding test.Throughing the analysis of the Ross308male broilers production performance, immune indexand intestinal morphology, found that Bacillus pumilus (B. pumilus4D-14) can effectivelyreduce the late breeding ratio of feed and meat and to promote the intestines recess is shorter,the epithelial cell survival rate increased; and can replace Avilamycin, used as a growthpromoting agent used in broiler feed.
使用16SrRNA及gyrB基因分子鉴定法，对来自养殖场土壤和市场产品的抑菌型芽孢杆菌菌株进行了鉴定。发现养殖场土壤来源的抑菌型芽孢杆菌以蜡样芽孢杆菌、短小芽孢杆菌和蕈状芽孢杆菌为主，而市场微生态产品来源的抑菌型芽孢杆菌，以枯草芽孢杆菌为主。通过将菌株Bacillus subtilis Y16，Bacillus lincheniformis Y14和Bacilluscereus J14，Bacillus pumilus4D-14等4株芽孢杆菌上清液经蛋白酶、高温、不同pH处理后抑菌活性的变化观察，发现不同的菌株抑菌活性物的性质有所不同。Bacilluslincheniformis Y14的抑菌活性受蛋白酶和温度影响波动严重，上清液中的抑菌活性物质可能是蛋白质类。而B. subtilis Y16，B. cereus J14，B. pumilus4D-14的抑菌活性受蛋白酶和温度影响不大，特别是B. cereus J14，B. pumilus4D-14的抑菌物质具有热稳定性，表明其抑菌活性物可能是小肽类。
考虑到中国农业部对动物饲料和养殖用微生物菌种的限制性规定，和动物微生态国内外研究现状，选择短小芽孢杆菌B. pumilus4D-14做进一步的应用效果评价。同时以市场产品来源的抑菌型芽孢杆菌B. subtilis Y16为研究对照。
将B. pumilus4D-14及B. subtilis Y16使用20L小型发酵罐进行发酵，制备微生态制剂样品。制得B. subtilis Y16菌粉共2.7kg，菌粉菌量6.8×109CFU/g；B.pumilus4D-14菌粉共2.35kg，菌粉菌量1.47×1010CFU/g。设计肉鸡养殖试验进行B.pumilus4D-14等菌株的应用研究，以国内常用饲用促生长抗生素阿维拉霉素（Avilymacin）为正对照，以无任何添加剂的饲料组为负对照，以B.pumilus4D-14、B. subtilis Y16及市场代表性枯草芽孢杆菌微生态产品可速必宁为处理组进行养殖试验。通过对罗斯308肉公鸡生产性能、免疫指标及肠道形态的分析，发现短小芽孢杆菌（B. pumilus4D-14）能有效降低养殖后期料肉比并促使各段肠管隐窝变短，使上皮细胞成活率上升；并可替代阿维拉霉素，作为促生长剂应用于肉鸡饲料。
First, this paper established a rapid screening method of laboratory96pores plate based onminimum inhibitory concentration and screened Bacillus strains from farm soil and probioticproducts for animals. Then selected Bacillus strains using Salmonella typhimurium ATCC14028as the indicator bacteria to antibacterial ability of Bacillus, obtained7antibacterial Bacillusstrains from farm soil. The antibacterial effect of7Bacillus strains from farm soil and Bacillusstrains from probiotic products were evaluated with4strains of different serotypes were furtherused animal source pathogenic bacteria Escherichia coli E1, E3, E4, Salmonella gallinarum(Salmonella gallinarum) SC1, swine Salmonella (Salmonella choleraesuis) and SP1ofPseudomonas aeruginosa, found that the antibacterial effect of Bacillus strains4D-14, J-14from the soil and Bacillus strains Y14, Y16from the market products analogous to20ppmcolistin sulfate in vitro.
Bacillus cereus, Bacillus pumilus and Bacillus mycoides highlighted the Bacillus srtains in farmsoil and Bacillus subtilis highlighted the Bacillus srtains in market animal probiotic products,according to the bacteriostasis Bacillus identification with the use of molecular16SrRNA andgyrB gene method. The characters of Bacillus supernatant of the strain Bacillus subtilis Y16,Bacillus lincheniformis Y14and Bacillus cereus J14, Bacillus pumilus4D-14was observedthrough the changes of antibacterial activity of protease, temperature, different pH, and foundthat properties of antibacterial activity of different strains were different. Antibacterial activityof B. lincheniformis Y14fluctuations was affected by protease and temperature, antifungalactivity in the supernatant may be provided by proteins. B. subtilis Y16, B. cereus J14, B.pumilus4D-14antibacterial activity is not affected by protease and temperature, especially the B.cereus J14, B. pumilus4D-14antibacterial material has thermal stability, show its antibacterialactivity may be provided by small peptides. Considering the restrictive provision of microbial strains used for animal feed and breeding ofChina's agriculture ministry and the research status of animal probiotics, selected the strain B.pumilus4D-14to do further research in application effect in this paper. At the same time, thestrain B. subtilis Y16which screened from market animal probiotic product was selected as thecontrol study.
The probiotic samples of B. pumilus4D-14and B. subtilis Y16were prepared with20Lfermentation tank. Finally, prepared B.pumilus4D-14culture2.35kg, bacteria quantity was1.47×1010CFU/g; B. subtilis Y16culture2.7kg, bacteria quantity was6.8×109CFU/g.
In the application research, using Avilamycin as positive control, B. pumilus4D-14, B. subtilisY16and market representative Bacillus probiotics as treatment on broiler breeding test.Throughing the analysis of the Ross308male broilers production performance, immune indexand intestinal morphology, found that Bacillus pumilus (B. pumilus4D-14) can effectivelyreduce the late breeding ratio of feed and meat and to promote the intestines recess is shorter,the epithelial cell survival rate increased; and can replace Avilamycin, used as a growthpromoting agent used in broiler feed.
引文
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