具抗病毒能力之乳酸菌株篩選及其特性研究

Screening and characterization of lactic acid bacteria with antiviral activity

許琇涵(Hsiu-Han Hsu)

指導教授 ： 劉嚞睿

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201702580

摘要

直至今日，因病毒感染而導致農畜之損害仍為一不可忽視之議題。儘管疫苗目前為預防病毒侵害最為普及之方法，卻仍具有諸多潛在缺點，如安全疑慮、預防效率及成本高昂等，而藉由益生菌達到預防病毒侵入可作為一替代性方案。本試驗欲由腸道菌相中篩選出具有抗病毒特性之乳酸菌，並藉由Sindbis virus（SBV）感染幼年倉鼠腎臟細胞（baby hamster kidney cell, BHK cell）之模式來證實乳酸菌株之抗病毒能力。由10日齡嬰兒與未離乳仔豬之糞便中分離出106株乳酸菌，並依序測試菌體之胞內、胞外及胞壁等萃取物質對病毒活性之抑制能力。其中藉由胞內物質篩選出5株具有低細胞毒性及高抗病毒能力之乳酸菌，並藉由16S rDNA及DNA gyrase B基因定序，以及API生化套組測試，確認此5株菌為Enterococcus faecalis。為瞭解菌體對SBV病毒之抗性機制，進一步以此5株菌株中的E. faecalis HH53對倉鼠腎臟細胞進行不同時間點之處理。由流式細胞儀之分析結果，發現給予病毒的同時加入E. faecalis HH53胞內物之處理，其腎臟細胞在抵抗病毒感染之效率較佳（病毒感染率下降為53%）。但在即時定量聚合酶鏈鎖反應（quantitative real time PCR）中未能觀察到腎臟細胞在第一型干擾素相關基因IL 6、ISG 15、PKR及Mx 1的表現量上與未處理菌體胞內物之對照組有所不同。由此推測E. faecalis HH53之抗病毒機制可能不是藉由第一型干擾素之途徑，而可能是菌體直接抑制病毒對細胞的入侵，但相關之試驗仍待未來去證實。最後，在益生菌之特性分析上，證實E. faecalis HH53於pH 3環境下具有耐酸能力，較不具有耐膽鹽特性，但卻能抑制 Listeria monocytogenes BCRC 14930、L. monocytogenes BCRC 15387及L. monocytogenes BCRC 15338等病原菌之生長。此外，E. faecalis HH53對於ampicillin、nitrofurantonin、norfloxacin、penicillin、vancomycin及linezolid等抗生素物質不具抗性，在應用上較無抗藥性之疑慮。綜合上述，本試驗證實E. faecalis HH53具備極佳之抗病毒特性、低細胞毒性與益生菌特性，有潛力作為預防病毒感染之益生菌添加物。

關鍵字

益生菌 ； 乳酸菌 ； 抗病毒 ； Enterococcus faecalis ； Sindbis virus

並列摘要

Nowadays, viral diseases in farm animals are one of the major problems that cannot be ignored. Vaccination is the most common method to prevent viral diseases, but has some disadvantages such as safety issue, insufficient efficacy, expensive cost, and animal stress. Probiotic therapy may be an alternative method for antiviral infection. The aim of this study is to screen the potential lactic acid bacteria (LAB) strains with antiviral activity by using the Sindbis virus (SBV) infected baby hamster kidney (BHK) cell model. One hundred and six LAB strains were isolated from the pre-weaned piglet and 10-day-old infant feces and subsequently evaluated the antiviral activity of their extracellular media, intracellular extracts, and cell wall pellets. Amomg these LAB strains, 5 strains showed low cytotoxicity and high antiviral activity of their intracellular extracts. These 5 LAB strains were identified as Enterococcus faecalis by 16S rDNA and DNA gyrase B sequencing and API biochemical tests. To clarify the mechanism of virus inhibition, 1 of the LAB strains was further analyzed the time course of inhibition of SBV and the expression of type 1 interferon related genes of the BHK cells by using quantitative real time PCR. The results showed that intracellular extract of E. faecalis HH53 had the best antiviral activity when simultaneously added with SBV (virus infectivity diminished by 53%). However, there is no difference in the expression of the type 1 interferon related genes ISG15, PKR, and Mx1 of the BHK cells. The antiviral mechanism of the E. faecalis HH53 might not be through the type 1 interferon receptor system but directly inhibit the virus invasion. However, it needs further study to prove. The probiotic characteristics of E. faecalis HH53 were further investigated. The results showed that E. faecalis HH53 could survive in acidified medium under pH value of 3 but in medium contained 0.3% bile salt, and could inhibit the growth of Listeria monocytogenes BCRC 14930, L. monocytogenes BCRC 15387, and L. monocytogenes BCRC 15338. In addition, E. faecalis HH53 showed susceptibility to ampicillin, nitrofurantonin, norfloxacin, penicillin, vancomycin, and linezolid. With regard to a general concern about the safety of probiotics, such as potential transferability of resistance determinants, E. faecalis HH53 with low resistance may be thought of low safety risk. In conclusion, E. faecalis HH53 showed low cytotoxicity and high antiviral and probiotic activities, thus, has a potential to be used as a probiotic antiviral agent.

並列關鍵字

probiotic ； lactic acid bacteria ； anti-virus ； Enterococcus faecalis ； Sindbis virus

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