两株鹿源芽孢杆菌的分离鉴定及生物学特性研究
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摘要
为获得鹿源肠道菌群中的芽孢杆菌并研究其益生作用,对梅花鹿肠道分泌物85℃水浴处理15min以筛选芽孢杆菌。根据菌落形态、生理生化特征及分子生物学结果进行鉴定,通过抑菌试验、耐酸、耐胆盐试验及产纤维素酶试验等方法对其生物学特性进行研究。结果表明:试验共分离出4株芽孢杆菌,分别编号为XF-1、XF-2、XF-3、XF-4,对其中的两株进行鉴定,最终确定XF-1为地衣芽孢杆菌,XF-3为解淀粉芽孢杆菌。采用牛津杯法研究两株芽孢杆菌对4种致病菌的抑制作用,发现XF-1对大肠杆菌、链球菌、沙门氏菌都具有良好的抑制作用,XF-3对大肠杆菌、金黄色葡萄球菌、链球菌、沙门氏菌都具有良好的抑制作用。选择CMC-Na培养基检测这两株芽孢杆菌的产纤维素酶能力,试验表明,菌株XF-1的纤维素降解指标为D/d=2.18,菌株XF-3纤维素降解指标为D/d=3.18,XF-3的降解纤维素能力高于XF-1。试验中计算在不同酸性环境和胆盐环境中菌株的成活率,评价耐受能力的差异,结果表明:XF-1在p H值3.0环境中24h存活率是7.3%,在0.2%胆盐环境中24h存活率为8.57%,可产纤维素酶;XF-3在p H值3.0环境中24h存活率是12.5%,在0.2%胆盐环境中24h存活率为6.67%,可产纤维素酶。试验结果证明这两株芽孢杆菌有一定的抗逆性和益生能力,具有作为益生菌研究开发的潜力。
In order to obtain the Bacillus in the intestinal flora of deer and study its beneficial effects, intestinal secretion of Sika Deer was processed with 85℃ water-curing treatment for 15 min, and Bacillus were screened. Bacillus was identified according to the colony morphology, physiological and biochemical characteristics and molecular biological results. The biological characteristics were studied by the methods of antibacterial test, acid resistance, salt tolerance test and cellulase production test.Results showed that four strains of Bacillus were isolated from the experiment, named XF-1, XF-2, XF-3 and XF-4. Two of them were identified, XF-1 was Bacillus licheniformis and XF-3 was Bacillus amyloliquefaciens. The inhibitory effect of two strains of Bacillus on the four pathogenic bacteria of was studied by using the Oxford cup method. XF-1 had a good inhibitory effect on Escherichia coli, Streptococcus and Salmonella; XF-3 had a good inhibitory effect on Escherichia coli, Staphylococcus aureus, Streptococcus, Salmonella. CMC-Na medium was used to detect the cellulase producing ability of two strains of Bacillus.The cellulose degradation index of strain XF-1 was D/d=2.18, the cellulose degradation index of strain XF-3 was D/d=3.18, and the cellulose degrading capacity of XF-3 was higher than XF-1.In the experiment, the survival rate of bacteria in different acid environment and bile salt environment was calculated, and the difference of tolerance was evaluated. The results were indicated that after 24 h,the survival rate of XF-1 in the p H 3.0 environment was 7.3%, the survival rate was 8.57% in the 0.2% bile salt environment. Cellulase was produced after 24 h, the survival rate of XF-3 in the p H 3.0 environment was 12.5%, the survival rate was 6.67% in the 0.2% bile salt environment. The results showed that the two strains had a certain degree of resistance and probiotic ability. They had been used for research and development as probiotics.
In order to obtain the Bacillus in the intestinal flora of deer and study its beneficial effects, intestinal secretion of Sika Deer was processed with 85℃ water-curing treatment for 15 min, and Bacillus were screened. Bacillus was identified according to the colony morphology, physiological and biochemical characteristics and molecular biological results. The biological characteristics were studied by the methods of antibacterial test, acid resistance, salt tolerance test and cellulase production test.Results showed that four strains of Bacillus were isolated from the experiment, named XF-1, XF-2, XF-3 and XF-4. Two of them were identified, XF-1 was Bacillus licheniformis and XF-3 was Bacillus amyloliquefaciens. The inhibitory effect of two strains of Bacillus on the four pathogenic bacteria of was studied by using the Oxford cup method. XF-1 had a good inhibitory effect on Escherichia coli, Streptococcus and Salmonella; XF-3 had a good inhibitory effect on Escherichia coli, Staphylococcus aureus, Streptococcus, Salmonella. CMC-Na medium was used to detect the cellulase producing ability of two strains of Bacillus.The cellulose degradation index of strain XF-1 was D/d=2.18, the cellulose degradation index of strain XF-3 was D/d=3.18, and the cellulose degrading capacity of XF-3 was higher than XF-1.In the experiment, the survival rate of bacteria in different acid environment and bile salt environment was calculated, and the difference of tolerance was evaluated. The results were indicated that after 24 h,the survival rate of XF-1 in the p H 3.0 environment was 7.3%, the survival rate was 8.57% in the 0.2% bile salt environment. Cellulase was produced after 24 h, the survival rate of XF-3 in the p H 3.0 environment was 12.5%, the survival rate was 6.67% in the 0.2% bile salt environment. The results showed that the two strains had a certain degree of resistance and probiotic ability. They had been used for research and development as probiotics.
引文
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[2]谢琴,赵晓瑞,张学礼.猪肠道芽孢杆菌的分离鉴定、小鼠毒性以及体外颉颃试验[J].黑龙江畜牧兽医,2010(23):109-111.
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[4]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[J].中国畜牧兽医,2010,37(2):27-31.
[5]宋延龄,刘志涛.珍稀动物——梅花鹿及其研究[J].生物学通报,2005,40(7):1-3.
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[9]李玥莹.DNA体外扩增技术——聚合酶链式反应(PCR)[J].沈阳师范学院学报(自然科学版),2000(4):40-44.
[11]谭才邓,朱美娟,杜淑霞,等.抑菌试验中抑菌圈法的比较研究[J].食品工业,2016(11):122-125.
[12]刘冬梅,李理,杨晓泉,等.用牛津杯法测定益生菌的抑菌活力[J].食品研究与开发,2006(3):110-111.
[13]关心.黄曲霉毒素B1降解菌株的筛选鉴定、降解优化与特性研究[D].沈阳:沈阳农业大学,2016.
[14]布坎南R E,吉本斯N E.伯杰氏细菌鉴定手册[M].北京:科学出版社,1984:797-828.
[15]蒋磊,徐秀景.益生菌在畜禽生产中的应用[J].饲料广角,2017(1):45-46.
[16]DUC L H,HONG H A,BARBOSA T M,et al.Characterization of Bacillus probiotics available for human use[J].Appl Environ Microbiol,2004,70(4):2161-2171.
[17]张菊,李金敏,张志焱,等.地衣芽孢杆菌的研究进展[J].中国饲料,2012(17):9-11.
[18]张超,王祥红,徐涛,等.一株解淀粉芽孢杆菌的分离鉴定及其抑菌条件研究[J].生物技术通报,2015(9):170-176.
[19]杨柯,王傲,吕曼,等.基于NRPS功能基因筛选鉴定盐生海芦笋内生细菌的研究[J].食品工业科技,2017(1):175-182.
[20]SUTYAK K E,WIRAWAN R E,AROUTCHEVA AA,et al.Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the daily product-derived Bacillus amyloliquefaciens[J].Journal of Applied Microbiology,2008,104(4):1067-1074.
[21]沈勇涛.芽孢杆菌对母猪仔猪微生态的影响及其生长条件优化[D].武汉:华中农业大学,2011.
[22]罗佳捷,肖淑华,张彬,等.解淀粉芽孢杆菌的培养工艺及应用研究进展[J].饲料博览,2014(5):40-42.
[23]杜威,黄琴,付爱坤,等.解淀粉芽抱杆菌SC06对免疫抑制小鼠肠道细菌酶活性和肠粘膜屏障功能的影响[J].动物营养学报,2014,26(3):819-826.
[2]谢琴,赵晓瑞,张学礼.猪肠道芽孢杆菌的分离鉴定、小鼠毒性以及体外颉颃试验[J].黑龙江畜牧兽医,2010(23):109-111.
[3]张新雄,彭锋,毛光平,等.饲用芽孢杆菌研究与应用进展[J].应用与环境生物学报,2013,19(5):891-897.
[4]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[J].中国畜牧兽医,2010,37(2):27-31.
[5]宋延龄,刘志涛.珍稀动物——梅花鹿及其研究[J].生物学通报,2005,40(7):1-3.
[6]孙磊.十种常见栽培食用菌菌种保藏及菌种扩大工艺优化[D].烟台:鲁东大学,2016.
[7]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科技出版社,2001.
[8]陈新亮,邵启兵,王超,等.唾液乳杆菌的分离鉴定及生物特性研究[J].食品科学,2016(13):157-161.
[9]李玥莹.DNA体外扩增技术——聚合酶链式反应(PCR)[J].沈阳师范学院学报(自然科学版),2000(4):40-44.
[11]谭才邓,朱美娟,杜淑霞,等.抑菌试验中抑菌圈法的比较研究[J].食品工业,2016(11):122-125.
[12]刘冬梅,李理,杨晓泉,等.用牛津杯法测定益生菌的抑菌活力[J].食品研究与开发,2006(3):110-111.
[13]关心.黄曲霉毒素B1降解菌株的筛选鉴定、降解优化与特性研究[D].沈阳:沈阳农业大学,2016.
[14]布坎南R E,吉本斯N E.伯杰氏细菌鉴定手册[M].北京:科学出版社,1984:797-828.
[15]蒋磊,徐秀景.益生菌在畜禽生产中的应用[J].饲料广角,2017(1):45-46.
[16]DUC L H,HONG H A,BARBOSA T M,et al.Characterization of Bacillus probiotics available for human use[J].Appl Environ Microbiol,2004,70(4):2161-2171.
[17]张菊,李金敏,张志焱,等.地衣芽孢杆菌的研究进展[J].中国饲料,2012(17):9-11.
[18]张超,王祥红,徐涛,等.一株解淀粉芽孢杆菌的分离鉴定及其抑菌条件研究[J].生物技术通报,2015(9):170-176.
[19]杨柯,王傲,吕曼,等.基于NRPS功能基因筛选鉴定盐生海芦笋内生细菌的研究[J].食品工业科技,2017(1):175-182.
[20]SUTYAK K E,WIRAWAN R E,AROUTCHEVA AA,et al.Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the daily product-derived Bacillus amyloliquefaciens[J].Journal of Applied Microbiology,2008,104(4):1067-1074.
[21]沈勇涛.芽孢杆菌对母猪仔猪微生态的影响及其生长条件优化[D].武汉:华中农业大学,2011.
[22]罗佳捷,肖淑华,张彬,等.解淀粉芽孢杆菌的培养工艺及应用研究进展[J].饲料博览,2014(5):40-42.
[23]杜威,黄琴,付爱坤,等.解淀粉芽抱杆菌SC06对免疫抑制小鼠肠道细菌酶活性和肠粘膜屏障功能的影响[J].动物营养学报,2014,26(3):819-826.