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Title: | 具抗病毒特性與益生菌潛力之乳酸菌株的分離與篩選 Isolation and screening of lactic acid bacteria strains with antiviral activity and probiotic potential |
Authors: | Yang-Ming Chen 陳揚明 |
Advisor: | 劉嚞睿(Je-Ruei Liu) |
Keyword: | 益生菌,乳酸菌,Lactobacillus agilis,Lactobacillus salivarius,豬流行性下痢病毒,抗病毒, Probiotics,Lactic acid bacteria,Lactobacillus agilis,Lactobacillus salivarius,Porcine epidemic diarrhea virus,Antiviral agent, |
Publication Year : | 2020 |
Degree: | 碩士 |
Abstract: | 近年來,經濟動物的產業規模化面臨一系列管理與健康上的挑戰,在密集式的畜牧產業中,疾病的傳播變得十分容易,一旦如此,必然會造成嚴重的經濟損失。人類使用乳酸菌已有長遠的歷史,其主要應用於食品的發酵,而在種類眾多的乳酸菌中,有一部分具有在腸胃消化道定殖的能力,這種能力被視為益生菌所應具備的特性,在過去也有研究指出,以益生菌作為飼料添加物除了可以增進宿主健康之外,也可以作為預防及對抗病毒性疾病的潛力,因此,本研究以綠猴腎細胞平台(Cercopithecus aethiops kidney cells, Vero cells)篩選具有抗豬流行性下痢病毒(porcine epidemic diarrhea virus, PEDV)的乳酸菌,並評估其作為益生菌的潛力。首先,本研究分離出60株乳酸菌菌株,並以這60株乳酸菌之胞內萃取物篩選出具有最佳抗病毒潛力的菌株,試驗後篩選出6株乳酸菌具有低細胞毒性及高抗病毒能力,再經由16S rRNA及rpoA的基因序列鑑定後,這些乳酸菌屬於Lactobacillus agilis 以及 Lactobacillus salivarius這兩種菌種,為了分析乳酸菌樣品對PEDV的抗病毒機制,本研究以即時定量聚合酶連鎖反應試驗分析病毒感染時Vero細胞之相關抗病毒基因及細胞激素之表現量,並協同樣品與細胞在感染之時間效應分析其抗病毒原因。在即時定量聚合酶連鎖反應試驗中,Vero細胞之抗病毒基因並未經由額外添加乳酸菌樣品而有所調節,但是乳酸菌樣品卻可以感染後48小時顯著降低病毒複製組裝所需的核蛋白之編碼RNA表達量;在時間效應試驗結果顯示,添加乳酸菌樣品無法顯著影響病毒感染進程,因此抗病毒機制還需進一步探討;綜合上述試驗,本研究篩選出Lb. agilis YM22 及 Lb. salivarius YM33為抗豬流行性下痢病毒之潛力菌株。在乳酸菌特性分析上,Lb. agilis YM22 及 Lb. salivarius YM33具有抑制病原菌生長的特性,Lb. salivarius YM33具有能在pH 2環境下維持菌量的耐酸性,而Lb. agilis YM22在腸道貼附性試驗有明顯的貼附能力,總結來說,Lb. agilis YM22 及 Lb. salivarius YM33具有抗豬流行性下痢病毒的特性,且各具備益生菌所需之特性,因此兩株乳酸菌株皆有作為抗病毒之飼料添加物的潛力。 Nowadays, large scale of animal farming in livestock industries is facing a series of management problems and health concerns. In highly-populated animals, diseases spread rapidly and may lead to significant economic loss. Lactic acid bacteria (LAB) are human-related with a long history in food fermentation. A group of them, presenting the ability to colonize at intestinal tract of animals, is generally recognized as probiotic. Several reports have provide a new strategy of using probiotics as feed additive to control and prevent viral infections. Therefore, the aim of this study is to screen and isolate LAB with antiviral activity by using Vero cell (Cercopithecus aethiops kidney cells) model, and to evaluate the probiotic potential of the LAB. First, 60 LAB strains were isolated and the intracellular fractions of LAB with presenting higher anti-porcine epidemic diarrhea virus (PEDV) activity were selected. Among these LAB strains, 6 strains showed low cytotoxicity and high antiviral activity These bacterial strains were found to belong to Lactobacillus agilis and Lactobacillus salivarius by the identification results based on 16S rRNA and rpoA sequence analysis. To elucidate the mechanisms of the anti-PEDV activity of the bacterial isolated strains, time-of-addition assay were conducted and the expression levels of the antiviral-related genes and cytokines in Vero cells were determined by qPCR. Although the bacterial sample did not significantly up-regulate the expression of antiviral genes, the expression level of PEDV nuclear protein coding RNA showed significant down-regulation at 48 hours post infection. In addition, the results of time-of-addition assay showed low influence on both LAB sample, thus the antiviral effects require further investigation Overall the results showed that Lb. agilis YM22 and Lb. salivarius YM33 present higher anti-PEDV activity. Moreover, these two isolates showed anti-pathogenic activities and were tolerant to low pH value. The results of intestinal adhesion assay showed that Lb. agilis YM22 present better adhesion ability. In conclusion, Lb. agilis YM22 and Lb. salivarius YM33 were found to possess anti-PEDV activity and probiotic potential. Thus, these two isolated strains are promising candidates to be used as antiviral additives in animal feed. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68805 |
DOI: | 10.6342/NTU202003673 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生物科技研究所 |
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