臺灣博碩士論文加值系統

水產養殖業為最具潛力的產業之一，能夠滿足動物蛋白的需求，為人類提供營養和糧食。但是，集約化養殖對魚類的壓力越來越大，導致傳染病並最終造成經濟損失。目前，抗生素是減少水產養殖疾病的流行工具，但它可能造成環境污染並產生耐藥細菌。最近，被稱為益生菌的有益微生物已被用作替代品之一。它們最初被用作牛和豬的動物飼料添加劑，作用包括抵抗病原微生物，與病原體競爭粘附位點和養分，以及促進宿主的免疫反應。這項研究著重在了解透過乳酸菌發酵對飼料內菌相的改變，及其對白蝦成長與免疫之影響。這項實驗設計了四種飼料，其中包括商業飼料(C)，添加了乳酸菌的飼料(A)組，不添加乳酸菌發酵飼料(N)和乳酸菌介導發酵的飼料(F)。餵食六週後，結果顯示，A組平均最終體重為6.3±0.47 g，C組為3.9±0.38 g，部分發酵組的存活率為90％，空白組為60％。而飼料及乳酸菌粉內菌相的分析表明乳酸菌粉主要以三種乳酸桿菌屬所組成(Bifidobacterium 12%、Lactobacillus 8%及Enterococcus 78%)，而在A組飼料中則以Enterococcus(56%)數為主，而N組飼料內則以Bacillus(66%)為主，確定了Enterococcus能在發酵過程中存活並成為優勢菌種，也證實了添加乳酸菌與否對飼料內菌相有著重要的關聯。

Aquaculture is developing into one of the most viable and promising indus-tries that offers the needs of animal protein, provide nutrition for humans and food security. However, intensive farming is increasing stresses to the fish, leading to infectious diseases and ultimately causes financial losses. Currently, the antibiotics are popular tools to reduce diseases in aquaculture, but it may cause environmental pollution and produce drug-resistant bacteria. Recently, beneficial microorganisms called probiotics have been applied as one of the alternatives. Due to they were originally used as animal feed additives in cat-tle and pigs, including antagonizing pathogenic microorganisms, competing with pathogens for adhesion sites and nutrients, and altering the host's im-mune response. Here, this study focused on microbial populations of fer-mented feed mediated by lactobacteria and as potent nutritional candidates for shrimp. Three feeds were designed with commercial feed(C), lactobacte-ria-added feeds (A) blank fermentation feeds (N), and lactobacteria-mediated fermentation feeds (F). After six weeks of feeding, evidence shows the group F resulted in 6.3 g of the final body weight averaged, 2.5 g in the group N , and the survival rate was 90% in the partially fermented group and 60% in the blank group. Future work will screen the microbial populations of lactobacte-ria-mediated fermentation feeds to evaluate the potential effects on microbial community. However, the analysis of the microbial populations in feed and Lactobacillus powder showed that there were mainly three kinds of Lactoba-cillus (Bifidobacterium 12%, Lactobacillus 8% and Enterococcus 78%), In the group A, the dominant strain is Enterococcus (56%), and the group N is Ba-cillus (66%), It is confirmed that Enterococcus can be stored in the fermenta-tion process and become the dominant strain. It is also confirmed that the ad-dition of lactobacillus has an important relationship with the microbial popu-lations in the feed.

摘要 i
致謝 iv
縮寫代號指引 v
目錄 vi
表目錄 viii
圖目錄 viii
第１章、 前言 1
１.１、 全球白蝦養殖現況 1
１.２、 白蝦養殖常見疾病 3
１.３、 蝦類免疫系統 4
１.４、 蝦類養殖疾病發生與治療 6
１.５、 乳酸菌在水產養殖的應用 7
１.６、 乳酸菌介導發酵 8
１.７、 研究目的 9
第２章、 材料方法 10
２.１、 實驗設計 10
２.２、 實驗動物及材料 10
２.２.１、 實驗動物 10
２.２.２、 乳酸菌粉 11
２.２.３、 商業飼料 11
２.２.４、 培養液配置 11
２.２.５、 乳酸菌液態生長曲線 12
２.２.６、 乳酸菌固態生長曲線 12
２.２.７、 發酵飼料製備 13
２.２.８、 飼料溶失率測定 13
２.２.９、 水質檢測 14
２.２.１０、 成長指標 14
２.２.１１、 肝胰腺及血細胞採樣 14
２.２.１２、 總RNA提取及cDNA合成 15
２.２.１３、 聚合酶連鎖反應 16
２.２.１４、 飼料菌相分析 17
２.３、 統計分析 18
第３章、 結果 18
３.１、 水質數據 18
３.２、 成長指標及存活率 18
３.３、 免疫指標 19
３.３.１、 溶菌酶 19
３.３.２、 抗菌肽 20
３.３.３、 原酚氧化酵素 20
３.４、 生理指標 20
３.４.１、 血球總數 20
３.４.２、 肝體比 21
３.５、 飼料內菌相 21
３.５.１、 各組飼料16S RNA抽取 21
３.５.２、 物種豐富、多樣性比較 21
３.５.３、 樣品內細菌組成和群落結構 22
３.５.４、 細菌群落之間的關係 24
第４章、 討論 25
４.１、 發酵飼料對南美白對蝦成長及存活的影響 25
４.２、 發酵飼料對南美白對蝦免疫的影響 26
４.２.１、 溶菌酶 26
４.２.２、 抗菌肽 27
４.２.３、 原酚氧化酵素 28
４.３、 生理指標 28
４.４、 飼料內菌相 29
第５章、 結論與未來展望 31
第６章、 參考文獻 32



表目錄
表 一、商業乳酸菌粉標定成分 39
表 二、商業草蝦配合飼料標定成分 40
表 三、實驗使用引子 41
表 四、投餵發酵飼料後各組的成長指標 42
表 五、投餵發酵飼料後各組的生理指標 43
表 六、Alpha Diversity 指數 44
表 七、各組飼料的溶失率 45
圖目錄
圖 一、綜合乳酸菌生長曲線 46
圖 二、水質參數 47
圖 三、投餵發酵飼料後各組體重(FBW) 48
圖 四、投餵發酵飼料後各組體重增加率(WG) 49
圖 五、投餵發酵飼料後各組飼料轉換率(FCR) 50
圖 六、投餵發酵飼料後各組免疫基因電泳圖 51
圖 七、投餵發酵飼料後各組免疫基因相對表現量 52
圖 八、商業乳酸菌粉及發酵飼料經過16S RNA電泳圖 53
圖 九、OTU 的Venn 圖 54
圖 十、物種多樣性曲線 55
圖 十一、乳酸菌粉(LAB)物種注釋結果 56
圖 十二、無添加乳酸菌發酵飼料(BF)物種注釋結果 57
圖 十三、添加乳酸菌發酵飼料物種注釋結果 58
圖 十四、物種相對豐度 59
圖 十五、主座標分析 60
圖 十六、物種豐度聚類熱圖 61

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