抑致病菌益生菌菌株及其胞外多糖抗TGEV作用的比较分析

发布时间：2018-05-17 13:58

本文选题：益生菌 + 筛选　；参考：《东北农业大学》2015年硕士论文

【摘要】：本研究选择猪粪便中初步分离的50株益生菌菌株,分别通过检测抑菌活性、抗猪传染性胃肠炎病毒(TGEV)活性、耐酸和耐胆盐活性,筛选出具有综合优势的益生菌菌株4株,并经16S r RNA深入鉴定,它们分别为食淀粉乳酸杆菌(Lactobacillus amylovorus)、屎肠球菌(Enterococcus Faecium)和两株枯草芽孢杆菌(Bacillus subtilis)。在上述基础上,本研究进一步应用这4株益生菌菌株,提取各株胞外多糖(Exopoly Saccharides,EPS)并检测其抗TGEV活性,分析比较各株益生菌培养上清的抗病毒活性与其胞外多糖的相关性,为探究益生菌抗TGEV的成分提供理论依据。在益生菌抑菌特性的检测中,选用临床上最常见的两种病原菌:以大肠杆菌O78(Escherichia coli O78)为代表的革兰氏阴性菌和以致病性金黄色葡萄球菌(Staphylococcus aureus)为代表的革兰氏阳性菌。采用牛津杯法(琼脂扩散法)来筛选具有抑制致病菌的益生菌菌株,确保益生菌具有对抗两株病原菌的特性。结果表明:50株益生菌均有不同程度的抑菌作用,对两株不同致病菌也存在抑制作用的差异,同一株益生菌抑制大肠杆菌O78作用强,但对致病性金黄色葡萄球菌(Staphylococcus aureus)表现为很强和/或很弱的抑制作用,缺乏规律性;不同益生菌菌株分别对大肠杆菌和金黄色葡萄球菌的抑制作用差异性显著(P0.05),存在着菌株差异性。在益生菌抗病毒特性的检测中,选用仔猪病毒性腹泻常见病毒——猪传染性胃肠炎病毒(TGEV)作为研究对象,以ST细胞建立体外TGEV感染细胞模型,采用MTT比色法检测益生菌的抗TGEV活性。检测结果表明:50株益生菌均对TGEV具有不同程度抑制作用,抑制程度不一;其中,具有显著差异抗病毒活性的菌株为S1、S4、S32、S45(p0.05)。为进一步评价这50株益生菌培养上清和其中4株益生菌胞外多糖的抗病毒特性,运用新城疫病毒的HA试验,检测了对NDV血凝特性的影响,结果表明:50株菌株中有13株菌株具有抗NDV血凝作用;S1和S4菌株的培养上清具有抗NDV血凝的作用,S32、S45、S15、S35跟生理盐水和MRS对照组相比无差别,不具有抗NDV血凝的作用,上述菌株所提取的胞外多糖均不具有抗NDV血凝的作用。为保证益生菌在胃肠道内能够存活、模拟胃肠道环境以及潜在的应用价值,对具有综合优势的菌株,选用p H 2.0的MRS培养以及含0.3%猪胆盐的MRS液体培养基分别处理益生菌1h和4h,同时设置正常MRS培养基作为对照组,检测益生菌的耐酸、耐胆盐特性。最终筛选出4株具有耐酸耐胆盐特性、抑制病原菌和抗病毒活性的益生菌:S1、S4、S32、S45。经过16S rRNA鉴定,S1为食淀粉乳杆菌(Lactobacillus amylovorus),S4为屎肠球菌(Enterococcus Faecium),S32和S45为枯草芽孢杆菌(Bacillus subtilis)。对上述4株益生菌菌株分别提取其培养上清中的胞外多糖,同样利用MTT比色法检测益生菌胞外多糖的抗TGEV活性。结果表明:上述4株益生菌菌株所提取胞外多糖均具有抗TGEV活性,不同益生菌株的胞外多糖抗TGEV效果不同,同一种属益生菌所分离的胞外多糖的抗病毒活性也不同。
[Abstract]:In this study, 50 prebiotic strains isolated from pig feces were selected and 4 strains of probiotic strains with comprehensive advantages were screened by detecting the antibacterial activity, anti infectious gastroenteritis virus (TGEV) activity, acid resistance and bile salt activity, and identified by 16S R RNA, respectively. They were Lactobacillus amylovor (Lactobacillus amylovor), respectively. Us), Enterococcus faecium (Enterococcus Faecium) and two strains of Bacillus subtilis (Bacillus subtilis). On the basis of the above, this study further applied the 4 strains of probiotics, extracted the extracellular polysaccharide (Exopoly Saccharides, EPS) and detected its anti TGEV activity, and compared the antiviral activity of the supernatant of the probiotics culture and the extracellular more. The correlation of sugar provides a theoretical basis for exploring the anti TGEV components of probiotics. In the detection of probiotic bacteriostasis, the two most common pathogenic bacteria were selected: Gram-negative bacteria represented by O78 (Escherichia coli O78) and gram-negative Staphylococcus aureus (Staphylococcus aureus) as the representative of gram. The positive bacteria were screened by the Oxford cup method (agar diffusion method) to screen the probiotic strains with inhibitory bacteria to ensure the characteristics of probiotics against two strains of pathogenic bacteria. The results showed that 50 probiotics had different degrees of bacteriostasis, and there were differences in inhibition for two strains of pathogenic bacteria. The same probiotics inhibited the Escherichia coli O. The 78 effect was strong, but it showed a strong and / or weak inhibitory effect on the pathogenic Staphylococcus aureus (Staphylococcus aureus). The inhibitory effects of different probiotics on Escherichia coli and Staphylococcus aureus were significantly different (P0.05). We selected the common virus infectious gastroenteritis virus (TGEV) of piglet viral diarrhea (TGEV) as the research object. The ST cells were used to build an external TGEV infection cell model, and the anti TGEV activity of probiotics was detected by MTT colorimetry. The results showed that all 50 probiotics had different degrees of inhibition on TGEV and the degree of inhibition was different. The strains with significant differences in antiviral activity were S1, S4, S32, S45 (P0.05). In order to further evaluate the antiviral properties of the 50 probiotics culture supernatants and 4 probiotic extracellular polysaccharides, the HA test of Newcastle disease virus was used to detect the effect of the NDV hemagglutination. The results showed that 13 strains of the 50 strains had anti NDV hemagglutination. The culture supernatant of S1 and S4 has the effect of anti NDV hemagglutination, S32, S45, S15, S35 is not different from the normal saline and MRS control group, and does not have the effect of anti NDV hemagglutination. The extracellular polysaccharide extracted by the above strain does not have the effect of anti NDV hemagglutination. To ensure the survival of probiotics in the stomach and intestines, the gastrointestinal environment and potential are simulated and potential. For the application value, the strains of P H 2 and the MRS liquid medium containing 0.3% pig bile salt were used to treat probiotics 1H and 4H respectively. At the same time, the normal MRS medium was set as the control group, and the acid resistance and bile salt resistance of probiotics were detected. Finally, 4 strains had the characteristics of acid resistance and bile salt, and the pathogenic bacteria and resistance were suppressed. The probiotics of viral activity: S1, S4, S32, S45. were identified by 16S rRNA, S1 was Lactobacillus amyloid (Lactobacillus amylovorus), S4 was Enterococcus faecium (Enterococcus Faecium), and it was used as Bacillus subtilis. The 4 strains of probiotics were extracted from the culture supernatant, respectively. The anti TGEV activity of probiotic extracellular polysaccharide was detected. The results showed that the extracellular polysaccharide extracted from the 4 probiotic strains had anti TGEV activity, and the effect of extracellular polysaccharide against TGEV was different, and the antiviral activity of the exo polysaccharide isolated from the same probiotics was different.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.6

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