本文選題：益生菌 + 篩選　； 參考：《東北農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：本研究選擇豬糞便中初步分離的50株益生菌菌株,分別通過檢測抑菌活性、抗豬傳染性胃腸炎病毒(TGEV)活性、耐酸和耐膽鹽活性,篩選出具有綜合優(yōu)勢的益生菌菌株4株,并經(jīng)16S r RNA深入鑒定,它們分別為食淀粉乳酸桿菌(Lactobacillus amylovorus)、屎腸球菌(Enterococcus Faecium)和兩株枯草芽孢桿菌(Bacillus subtilis)。在上述基礎(chǔ)上,本研究進(jìn)一步應(yīng)用這4株益生菌菌株,提取各株胞外多糖(Exopoly Saccharides,EPS)并檢測其抗TGEV活性,分析比較各株益生菌培養(yǎng)上清的抗病毒活性與其胞外多糖的相關(guān)性,為探究益生菌抗TGEV的成分提供理論依據(jù)。在益生菌抑菌特性的檢測中,選用臨床上最常見的兩種病原菌:以大腸桿菌O78(Escherichia coli O78)為代表的革蘭氏陰性菌和以致病性金黃色葡萄球菌(Staphylococcus aureus)為代表的革蘭氏陽性菌。采用牛津杯法(瓊脂擴(kuò)散法)來篩選具有抑制致病菌的益生菌菌株,確保益生菌具有對抗兩株病原菌的特性。結(jié)果表明:50株益生菌均有不同程度的抑菌作用,對兩株不同致病菌也存在抑制作用的差異,同一株益生菌抑制大腸桿菌O78作用強(qiáng),但對致病性金黃色葡萄球菌(Staphylococcus aureus)表現(xiàn)為很強(qiáng)和/或很弱的抑制作用,缺乏規(guī)律性;不同益生菌菌株分別對大腸桿菌和金黃色葡萄球菌的抑制作用差異性顯著(P0.05),存在著菌株差異性。在益生菌抗病毒特性的檢測中,選用仔豬病毒性腹瀉常見病毒——豬傳染性胃腸炎病毒(TGEV)作為研究對象,以ST細(xì)胞建立體外TGEV感染細(xì)胞模型,采用MTT比色法檢測益生菌的抗TGEV活性。檢測結(jié)果表明:50株益生菌均對TGEV具有不同程度抑制作用,抑制程度不一;其中,具有顯著差異抗病毒活性的菌株為S1、S4、S32、S45(p0.05)。為進(jìn)一步評價(jià)這50株益生菌培養(yǎng)上清和其中4株益生菌胞外多糖的抗病毒特性,運(yùn)用新城疫病毒的HA試驗(yàn),檢測了對NDV血凝特性的影響,結(jié)果表明:50株菌株中有13株菌株具有抗NDV血凝作用;S1和S4菌株的培養(yǎng)上清具有抗NDV血凝的作用,S32、S45、S15、S35跟生理鹽水和MRS對照組相比無差別,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。為保證益生菌在胃腸道內(nèi)能夠存活、模擬胃腸道環(huán)境以及潛在的應(yīng)用價(jià)值,對具有綜合優(yōu)勢的菌株,選用p H 2.0的MRS培養(yǎng)以及含0.3%豬膽鹽的MRS液體培養(yǎng)基分別處理益生菌1h和4h,同時(shí)設(shè)置正常MRS培養(yǎng)基作為對照組,檢測益生菌的耐酸、耐膽鹽特性。最終篩選出4株具有耐酸耐膽鹽特性、抑制病原菌和抗病毒活性的益生菌:S1、S4、S32、S45。經(jīng)過16S rRNA鑒定,S1為食淀粉乳桿菌(Lactobacillus amylovorus),S4為屎腸球菌(Enterococcus Faecium),S32和S45為枯草芽孢桿菌(Bacillus subtilis)。對上述4株益生菌菌株分別提取其培養(yǎng)上清中的胞外多糖,同樣利用MTT比色法檢測益生菌胞外多糖的抗TGEV活性。結(jié)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,同一種屬益生菌所分離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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.6

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本文編號：1901647