臺灣博碩士論文加值系統

疾病問題為吳郭魚養殖永續發展的主要阻礙之一，而利用抗生素治療或預防養殖疾病已衍生抗藥性致病菌與食用安全疑慮等問題，因此發展替代抗生素的疾病控制方式有其必要性。益生菌為水產養殖疾病預防控制之策略之一，本研究由吳郭魚腸道中分離出一株產蛋白酶、纖維素酶、木聚醣酶和澱粉酶之菌株，經由16S rDNA 序列鑑定與生化分析，確認此菌株為沙福芽孢桿菌 (Bacillus safensis)。抗生素敏感試驗結果顯示此菌株對於多種測試之抗生素無抗性。飼料中添加105 CFU/g (G1)、106 CFU/g (G2) 和107 CFU/g (G3) 餵食吳郭魚八週，評估該菌對吳郭魚的成長促進，腸道酵素活性、免疫調節功能。餵食八週後結果顯示，飼料中添加107 CFU/g沙福芽孢桿菌 (B. safensis) 餵食之吳郭魚，在體重增加與飼料效益上與控制組相較有顯著差異，腸道中之澱粉酶、纖維素酶與脂肪酶也顯著高於控制組。餵食吳郭魚有添加沙福芽孢桿菌 (B. safensis) 之飼料，則頭腎組織中之吞噬活性、呼吸爆與SOD活性及頭腎和脾臟之免疫相關基因 (IL-1、IL-8、TNF-和Lysozyme)分析均顯著高於未添加之控制組，而添加105 CFU/g之組別，血液中溶菌酶活性顯著高於控制組。感染實驗結果顯示沙福芽孢桿菌 (B. safensis) 有助於提升吳郭魚感染魚型鏈球菌 (Streptococcus iniae) 之存活率，上述結果證實吳郭魚每日餵食沙福芽孢桿菌 (B. safensis) 有助於成長促進、免疫調節與疾病抵抗，說明篩選之沙福芽孢桿菌 (B. safensis) 具有潛力開發作為水產養殖成長促進與疾病控制之益生菌。

Disease problems are a major contributor to block sustainable development of tilapia aquaculture. The use of antibiotics to treat or prevent diseases has caused issues such as the rapid spread of antibiotic-resistant pathogens and increased risk of food safety by residual antibiotic contamination. Thus, development of an alternative to antibiotics for disease prevention is necessary. Probiotics have been considered an efficient alternative to antibiotics for biocontrol in aquaculture. In this study, a bacterial strain producing protease, cellulase, xylanase and amylase was isolated from the intestine of Nile tilapia, which was identified as Bacillus safensis by 16S rDNA sequencing and biochemical analysis. The antibiotic susceptibility test showed that the B. safensis do not exhibit resistance to diverse tested antibiotics. The effects of B. safensis on growth performance, intestinal hydrolytic enzyme activities and innate immunity in Nile tilapia were evaluated after feeding basial diet (control) containing B. safensis at levels of 105 CFU/g (G1), 106 CFU/g (G2) and 107 CFU/g (G3) for 8 weeks. The results showed significantly increased weight gain (WG) and feed efficiency (FE) in Nile tilapia in G3 group compared to those in fish fed a control diet. Intestinal digestive enzymes such as amylase, cellulase, and lipase were also significantly increased in Nile tilapia in G3 group. Immune parameters such as the phagocytic activity, respiratory bursts, and superoxide dismutase of head kidney leukocytes; and expression of the cytokine genes of IL-1, IL-8, TNF- and lysozyme in head kidney and spleen were significantly elevated in fish fed B. safensis. Serum lysozyme activity was significantly increased in tilapia in G1 group than that in control group. Enhanced cumulative survival was exhibited in fish fed B. safensis after challenge with S. iniae. The results suggested that B. safensis can be used as a potential probiotic in tilapia aquaculture to enhance the growth and disease biocontrol.
Keywords: probiotics, B. safensis, Nile tilapia, growth enhancement, immune modulation

中文摘要 I
Abstract II
謝誌 III
目錄 IV
圖目錄 VII
表目錄 VIII
壹、前言 1
貳、文獻回顧 2
2.1 吳郭魚水產養殖現況 2
2.1.1 全球吳郭魚養殖 2
2.1.2 吳郭魚養殖面臨之困境 3
2.1.3 魚類鏈球菌 (Strptococcus spp.) 4
2.1.3 抗生素衍生之問題 5
2.2 益生菌 5
2.2.1 益生菌的定義 5
2.2.2 益生菌之條件特徵 6
2.2.3 益生菌的作用方式 7
2.2.4 益生菌在水產養殖上的應用 9
2.2.5 芽孢桿菌屬 (Bacillus) 9
2.3 魚類免疫系統 10
2.3.1 免疫器官介紹 11
2.3.2 先天性免疫系統 (Innate immune system) 11
2.4 研究目的 13
參、材料與方法 14
3.1. 實驗菌株 14
3.1.1 益生菌篩選 14
3.1.2 菌株鑑定 14
3.1.3 菌株活化與保存 14
3.1.4 菌株標準曲線 15
3.2. 菌株特性分析 15
3.2.1 菌株蛋白酶活性測定 16
3.2.2 菌株澱粉酶活性測定 16
3.2.3 菌株纖維素酶活性測定 16
3.2.4 菌株木聚醣酶活性測定 16
3.2.5 對於抗生素敏感性測定 16
3.3 實驗動物餵養實驗 17
3.3.1 自製飼料 17
3.3.2 實驗動物飼養及分組 19
3.3.3 吳郭魚體重變化 19
3.4 吳郭魚腸道消化酵素分析 19
3.4.1 腸道酵素活性樣本製備 19
3.4.2 蛋白濃度標準曲線 20
3.4.3 腸道蛋白酶活性測試 20
3.4.4 腸道還原糖活性測試 21
3.4.5 腸道脂肪酶活性測試 22
3.5 代謝及抗氧化基因分析 22
3.5.1 吳郭魚肝臟RNA抽取 22
3.5.2 反轉錄酶反應 23
3.7.3 即時定量聚合酶連鎖反應 (Real - time PCR) 23
3.6 吳郭魚免疫指標分析 24
3.6.1 巨噬細胞分離 24
3.6.2 超氧化物歧化酶 (Superoxide dismutase，SOD) 25
3.6.3 呼吸爆 (Respiratory burst，O2-) 25
3.6.4 吞噬活性 (Phagocytic activity，PA) 26
3.6.5 溶菌酶 (Lysozyme) 26
3.7 免疫基因分析 27
3.7.1 吳郭魚頭腎及脾臟RNA抽取 27
3.7.2 反轉錄酶反應 27
3.7.3 即時定量聚合酶連鎖反應 (Real - time PCR) 28
3.8 病原菌毒性測試 28
3.8.1 病原菌的製備及標準曲線 28
3.8.2 病原菌施打濃度曲線 29
3.8.3 吳郭魚腹腔注射 29
3.9統計方式 29
肆、結果 30
4.1 菌株篩選鑑定與特性分析 30
4.2 餵食吳郭魚B. safensis對成長之影響 37
4.3 吳郭魚餵食B. safensis對腸道消化水解酵素活性之影響 39
4.4 吳郭魚餵食B. safensis對肝臟營養代謝與生長基因表現之影響 41
4.5 吳郭魚餵食B. safensis對肝臟抗氧化基因表現之影響 43
4.6 吳郭魚餵食B. safensis對免疫調節之影響 45
4.7 吳郭魚餵食B. safensis對頭腎與脾臟免疫基因表現之影響 47
4.8 吳郭魚餵食B. safensis對疾病抵抗影響 50
伍、討論 52
陸、結論 57
柒、參考文獻 58

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