臺灣博碩士論文加值系統

本研究由吳郭魚腸道中篩選出一株具澱粉酶、蛋白酶、纖維素酶及木聚醣酶活性之菌株，經過16S rDNA序列鑑定及生化分析，確認該菌株為沙福芽孢桿菌 (Bacillus safenesis)。抗生素敏感性試驗結果顯示此菌株對於數種抗生素均無抗藥性。為評估其是否具有作為水產養殖益生菌之潛力，在自製飼料中添加105 CFU/g、106 CFU/g與107 CFU/g的B. safenesis，並餵食白蝦8週，分析該菌株對白蝦的生長促進、免疫調節、疾病抵抗及肝胰腺菌相之改善。結果顯示，添加106 CFU/g與107 CFU/g的組別，其體重增加及飼料效益方面皆顯著高於未添加B. safenesis的控制組。在免疫指標分析中，攝食107 CFU/g的B. safenesis的白蝦，其血液中酚氧化酶、超氧化物歧化酶、呼吸爆、溶菌酶及吞噬活性皆顯著高於控制組；另外，105 CFU/g及106 CFU/g組別在酚氧化酶、呼吸爆及吞噬活性分析中，亦有提升活性的趨勢。在免疫基因分析中，以real-time PCR分析免疫相關基因prophenoloxidase、TNF-α、astakine以及lysozyme的表現量，其中106 CFU/g與107 CFU/g的組別在四種基因表現量皆顯著高於控制組，105 CFU/g的組別在TNF-α、astakine及lysozyme的表現量相較於控制組亦有顯著提升。以溶藻弧菌 (Vibrio alginolyticus) 注射白蝦感染7天後，結果顯示餵食106 CFU/g與107 CFU/g的組別，其累積死亡率顯著低於控制組。次世代定序結果說明白蝦攝食B. safenesis有助於改變其菌相。綜上所述，每日以每克飼料含106 CFU之B. safenesis餵食白蝦，有助於白蝦之生長促進、免疫調節、疾病抵抗以及肝胰腺菌相之變化，說明B. safenesis具有潛力開發作為應用於水產養殖之益生菌。

In the present study, a strain with amylase, protease, cellulase and xylanase activities, was isolated from the intestine of Nile tilapia and characterized as Bacillus safensis by 16S rDNA sequencing and biochemical analysis. In order to evaluate the efficacy of B. safensis as a probiotic on improvement of growth performance, immune modulation, disease resistance and gut microbiota of white shrimp (Litopenaeus vannamei), shrimp was fed with diet containing 105 CFU/g (G1), 106 CFU/g (G2) or 107 CFU/g (G3) for eight weeks. The results showed that weight gain and feed efficiency in the shrimps of G2 and G3 groups were significantly increased than those in shrimp of control group. Immune parameters including phenoloxidase, superoxide dismutase (SOD), respiratory burst, lysozyme and phagocytic activities in shrimp fed diet containing 107 CFU/g of B. safenesis were significantly higher than those in shrimp of control group. In addition, shrimps of G1 and G2 groups were also showed significant difference from the shrimp of control group in phenoloxidase, respiratory burst and phagocytic activity. The expression level of prophenoloxidase, TNF-α, astakine and lysozyme genes in hepatopancreas of shrimps in G1, G2 and G3 groups were significantly higher than those in shrimp of control group. A significant decreasing of cumulative mortality was exhibited in white shrimp fed diet containing 106 CFU/g and 107 CFU/g of B. safenesis after challenging with Vibrio alginolyticus. The shrimps dietary supplemented with B. safensis revealed an improved microbiota in hepatopancreas of white shrimp were performed by next generation sequencing (NGS). In summary, dietary feeding of diet containing 106 CFU/g of B. safenesis could improve growth performance, immune modulation, disease resistance and microbiota of white shrimp. The results suggested that B. safenesis has potential to be developed as a probiotic for aquaculture.

摘 要 I
Abstract II
謝誌 III
目錄 IV
圖目錄 VIII
表目錄 IX
第一章 前言 1
第二章 文獻回顧 3
2.1. 白蝦的簡介 3
2.1.1. 白蝦的生長特性 3
2.1.2. 台灣白蝦發展史 3
2.1.3. 台灣白蝦養殖現況及面臨問題 4
2.1.4. 弧菌 (Vibrio spp.) 4
2.2. 益生菌 5
2.2.1. 益生菌的定義 5
2.2.2. 益生菌於水產養殖之應用 6
2.2.3. 芽孢桿菌 (Bacillus) 7
2.2.4. 沙福芽孢桿菌 (B. safensis) 8
2.3. 白蝦免疫系統 9
2.3.1. 細胞性免疫 9
2.3.2. 體液性免疫 11
2.4. 研究目的 12
第三章 材料與方法 13
3.1. 實驗菌株 13
3.1.1. 芽孢桿菌 (Bacillus) 篩選 13
3.1.2. 菌株之活化與保存 13
3.1.3. 菌株特性分析 14
3.1.3.1. 蛋白酶試驗 (Protease) 14
3.1.3.2. 澱粉酶試驗 (Amylase) 14
3.1.3.3. 纖維水解酶試驗 (Cellulase) 15
3.1.3.4. 木聚糖酶試驗 (Xylanase) 15
3.1.3.5. 菌株對抗生素之敏感性 16
3.1.4. 菌株鑑定 16
3.1.5. 菌株標準曲線 16
3.2. B. safensis作為飼料補充物對白蝦之生長評估 17
3.2.1. 自製基礎飼料 17
3.2.2. 飼料組成分析 19
3.2.2.1. 水分測定 (Moisture) 19
3.2.2.2. 灰分測定 (Ash) 19
3.2.2.3. 粗蛋白測定 (Crude protein) 19
3.2.2.4. 粗脂質測定 (Ether extract) 20
3.2.3. 實驗動物分組與飼養 21
3.2.4. 白蝦體重成長之變化 21
3.3. 白蝦免疫指標分析 22
3.3.1. 白蝦血液樣本採集 22
3.3.2. 酚氧化酶活性分析 (Phenoloxidase activity, PO) 22
3.3.3. 呼吸爆 (Respiratory burst) 22
3.3.4. 超氧化物歧化酶活性分析 (Superoxide dismutase activity, SOD) 23
3.3.5. 溶菌酶標準曲線 23
3.3.6. 溶菌酶活性分析 (Lysozyme activity) 24
3.3.7. 吞噬活性 (Phagocytic activity) 24
3.4. 白蝦免疫基因分析 25
3.4.1. 白蝦肝胰腺RNA抽取 25
3.4.2. 反轉錄酶反應 (Reverse Transcriptase, RT) 25
3.4.3. 即時定量聚合酶連鎖反應 (Real-time PCR) 25
3.5. 病原菌之毒性測試 28
3.5.1. 病原菌之活化與標準曲線 28
3.5.2. 病原菌注射 28
3.6. 白蝦肝胰腺菌群分析 28
3.6.1. 肝胰腺細菌genomic DNA抽取 28
3.6.2. 次世代定序 (Next-Generation Sequencing, NGS) 29
3.7. 統計分析 29
第四章 結果 30
4.1. 菌株篩選鑑定與特性分析 30
4.2. 餵食B. safensis對白蝦成長之影響 37
4.3. 餵食B. safensis對白蝦免疫調節之影響 39
4.4. 餵食B. safensis對白蝦免疫基因表現之影響 41
4.5. 餵食B. safensis對白蝦疾病抵抗之影響 43
4.6. 餵食B. safensis對白蝦肝胰腺菌相之影響 45
第五章 討論 49
第六章 結論 53
第七章 參考文獻 54

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