- 學位論文
克弗爾與其分離菌株抗病毒活性之研究
Antiviral Potential of Kefir and the Bacterial Strains Isolated from Kefir Grains
摘要
近年來，密集型的畜牧產業導致病毒性疾病快速傳播，造成嚴重的經濟損失。部分研究證實益生菌除了有利於宿主健康，亦可用於預防及控制病毒感染。由於在不同動物實驗當中已評估過傳統發酵乳製品克弗爾有作為飼料添加物的潛力，因此本論文的主旨為探討克弗爾是否具有抗病毒之潛力，分析克弗爾發酵乳上清液及克弗爾粒分離菌株在綠猴腎細胞（Cercopithecus aethiops kidney cells, Vero cells）上抗豬流行性下痢病毒（porcine epidemic diarrhea virus, PEDV）之活性，並進一步探討可能的機制。本研究先進行菌種鑑定，並根據預防模式中抗豬流行性下痢病毒活性的結果篩選出胞內萃取物具有最佳抗病毒潛力之菌株。為探討其抗豬流行性下痢病毒活性之機制，以即時定量聚合酶鏈鎖反應進行抗病毒相關基因表現量之測定，並利用時間效應分析在不同時間添加樣品對抗病毒活性之影響。根據16S rRNA及rpoA基因序列進行菌種鑑定之結果發現，由克弗爾粒中分離出的乳酸菌株包含四個乳酸菌菌種，其中Leuconostoc mesenteroides之胞內萃取物不但沒有細胞毒性，還具有較佳的抗豬流行性下痢病毒活性，因此選擇該菌種中生長速度最快的L. mesenteroides YPK30作為最有抗病毒潛力之菌株。另一方面，作為發酵乳控制組之還原乳可抑制細胞受到豬流行性下痢病毒之感染，且效果與發酵乳相當，因此後續分別進行還原乳與L. mesenteroides YPK30之抗病毒機制探討。由即時定量聚合酶鏈鎖反應之實驗結果發現還原乳及L. mesenteroides YPK30胞內萃取物的預處理皆無法顯著提升Vero細胞之抗病毒基因表現量。而時間效應分析結果發現還原乳及L. mesenteroides YPK30於治療模式中也具有抗病毒活性，且還原乳可能透過抑制病毒的附著及進入達到預防性抗病毒效果，而L. mesenteroides YPK30之抗病毒機制則尚待釐清。綜合以上結果，克弗爾與其分離菌株具有抗豬流行性下痢病毒之潛力，而菌株L. mesenteroides YPK30之活性成分及其體內試驗之功效，仍需未來進一步確認。
並列摘要
Nowadays, significant economic loss has been caused by the rapid spread of viral disease due to intensive animal industries. It was shown in several reports that probiotics can exert their beneficial effect on the host to provide control and prevention of viral infections. The potential of kefir, a traditional fermented dairy product, as feed additive has been studied in different animal models. Therefore, the aim of this study is to evaluate the potential of kefir and the bacterial strains isolated from kefir grains as antiviral agents against the infection of porcine epidemic diarrhea virus (PEDV) in Cercopithecus aethiops kidney cells (Vero cells), and further investigate the possible mechanisms. Firstly, several bacterial isolated strains were identified, and the strain with the highest antiviral potential was determined according to its preventive anti-PEDV activity. To elucidate the anti-PEDV mechanisms, the expression levels of antiviral-related genes of Vero cells were determined using quantitative PCR (qPCR). Time-of-addition assay was also performed to evaluate the effects of different addition time points. The identification results based on 16S rRNA and rpoA sequence analysis indicated that the isolated strains belonged to four species. Among them, Leuconostoc mesenteroides presented higher anti-PEDV activity without cytotoxicity. Therefore, Leuconostoc mesenteroides YPK30 was selected as the strain with the highest antiviral potential due to its higher growth rate. However, the supernatant of milk inhibited the infection of PEDV in Vero cells to the same extent as kefir. Therefore, the mechanisms of the anti-PEDV effects of milk and L. mesenteroides YPK30 were further investigated. The results indicated that treatment of Vero cells with milk or L. mesenteroides YPK30 did not significantly up-regulate the expression of antiviral genes of Vero cells. In addition, the results of time-of-addition assay showed that both milk and L. mesenteroides YPK30 exerted therapeutic anti-PEDV activity. The preventive anti-PEDV activity of milk was attributed to its effect on viral attachment and entry, while the antiviral mechanism of L. mesenteroides YPK30 remains unknown. In conclusion, kefir and the isolated strain showed anti-PEDV potential. The bioactive component of L. mesenteroides YPK30 and its in vivo antiviral effects need to be further investigated.
參考文獻
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