臺灣博碩士論文加值系統

隨著台灣的密集式養殖，牛隻的腸道疾病成為現今養牛產業必須面對的一個棘手問題。牛隻的腸道疾病會影響牛隻對營養分吸收，嚴重甚至會導致牛隻死亡。而在各種腸道疾病中以細菌性感染最為常見，而傳統會使用抗生素進行治療，但近年來由於食安問題、藥物殘留及抗藥性的產生，各國政府已逐年公告禁止經濟動物使用大部分抗生素於飼料中添加，然而卻也尚未尋得一個可以完全替代抗生素的方案。因此若能找到一種具備預防性且是牛隻身體可以代謝無殘留問題的天然抑菌物質，將會是養牛產業的一大福祉。芽孢桿菌類被分類為公認安全（GRAS）的菌株，且被允許使用於食品工業及商業產品的添加中，同時地衣芽孢桿菌也被認為是具有改善腸道免疫功能，以及能與宿主建立良好的共生關係之潛力益生菌。因此本試驗之研究目的在於探討由地衣芽孢桿菌所分泌的代謝物脂肽是否能成為有效代替抗生素抑制仔牛下痢的效果。
本研究分為兩個部分，第一部分為體外發酵試驗，透過體外發酵將表面素脂肽與瘤胃菌株進行共培養，在不同的劑量處理之下，觀察瘤胃微生物組成以及微生物產物的變化。結果顯示不論是添加純化或是粗萃表面素脂肽皆會抑制格蘭氏陽性菌，進一步分析有觀察到些微抑制部分纖維分解菌，但不會抑制澱粉分解菌、蛋白質分解菌、或酸利用菌，預期有可能支持高精料飼糧的較佳利用效率。而第二部分則為動物餵食實驗，選用15隻剛出生的荷蘭牛仔牛分為三個組別，分別為1倍劑量組、10倍劑量組及對照組，於代乳粉中添加不同劑量的表面素脂肽並觀察仔牛的糞便菌相、健康狀況以及生長表現。結果顯示表面素脂肽僅有在臀寬及血溶比的項目有正面的影響，並且不會對於牛隻造成負面的影響。
總結來說，表面素脂肽相較於孟寧素需要較高的使用劑量，且效果也較孟寧素弱，但其優點為無殘留問題且添加後也對於牛隻無負面影響。未來還需要尋找提升粗萃表面素脂肽濃度的方法，才能以更小的添加劑量使用於飼糧，以提高應用於養牛產業的可行性。

With the intensive animal farming in Taiwan, intestinal diseases of cattle have become a major issue that the cattle industry must face today. Intestinal diseases will affect nutrient absorption, and if the disease became more serious cattle will even die. Bacterial infections are the most common cause among various intestinal diseases. Traditionally, antibiotics can be used for treatment. However, in recent years, due to food safety issue, drug residues and the emerging of drug resistance, many countries have banned most antibiotics use in cattle farming.
Therefore, if we can find a preventive natural bacteriostatic substance that can be metabolized by cattle without residue problems, it will be a great benefit for the dairy industry. Bacillus is classified as a recognized safe (general recognized as safe, GRAS) and allowed to be used in the food industry and commercial product additions. Bacillus licheniformis is also considered to have the function of improving intestinal immune function, and can establish a symbiosis relationship with the host. The purpose of this study is to investigate whether the lipopeptidesurfactin secreted by Bacillus licheniformis can be an effective alternative to antibiotics to effectively prevent calf diarrhea.
This study is divided into two parts. The first part is an in vitro fermentation trial, in which surfactinlipopeptide and rumen fluid inoculant are co-cultured to observe the changes in rumen microbial composition and fermentation products at different doses of lipopeptide. The results showed both purified and crude extract surfactinlipopeptide could inhibit Gram-positive bacteria. Further analysis showed that surfactinlipopeptide could suppress some cellulolytic bacteria, but not amylolytic bacteria, proteolytic bacteria, or acid-utilizing bacteria, it might improve utilization of high concentrate feed. The second part is an animal feeding experiment with 15 newborn calves randomly assigned to three groups: control group, 1x dose group, and 10x dose group. SurfactinLipopeptide was added into calves’ milk replacer to examine its effect on fecal microbiota, health status and growth performance. The results showed that surfactinlipopeptide only had a positive effect on the hip width, weight and hematocrit without any negative impact on calves.
In summary, surfactinlipopeptide required a higher effective dosage than monensin, and its effect was weaker than monensin, but its advantage was free of drug residue problem. In the future, we need to find a way to increase the concentration of surfactinlipopeptide in crude extract, so that it can be used in diets with a smaller dosage and increase the feasibility of applying in the dairy industry.

口試委員會審定書 i
謝辭 ii
中文摘要 iii
英文摘要 v
圖目錄 xi
表目錄 xiii
壹、 文獻探討 1
一、下痢對現今養牛產業的影響 1
二、反芻動物的消化與瘤胃微生物 1
（一）瘤胃的環境 1
（二）瘤胃內的微生物組成 2
二、腸道益菌與牛隻健康的關係 4
（一）產生有機酸 4
（二）分泌抗菌肽 4
（三）競爭 5
三、下痢的發生原因及判定方式 5
四、下痢的治療方法 8
五、抗生素治療的瓶頸 9
六、地衣芽孢桿菌與枯草芽孢桿菌（Bacillus subtilis） 11
七、地衣芽孢桿菌與表面素（Bacillus licheniformis and surfactin） 12
八、表面素脂肽(Surfactin） 13
九、次世代定序（NGS） 13
貳、 材料方法 15
體外試驗 15
一、體外發酵材料 15
（一）瘤胃液: 15
（二）體外發酵的樣品: 15
（三）表面素: 15
二、步驟 16
（一）瘤胃液的採集: 16
（二）體外發酵: 18
（三）採樣時間點及培養條件: 18
三、檢測項目及方法 19
（一）菌落培養 19
（二）氨態氮及揮發性脂肪酸濃度的測定 22
（三）原蟲和真菌動孢子數目 23
動物試驗 24
一、試驗場所 24
二、實驗牛選用及分組 25
三、採樣的項目時間點及方法 26
（一）糞便菌相分析及球蟲卵檢測 26
（二）下痢持續天數及糞便評分 29
（三）血液生化值 29
（四）生長表現: 32
（五）疾病紀錄 33
次世代基因定序 （NGS） 34
統計分析 35
參、 結果討論 36
體外試驗 36
一、菌落培養 36
二、氨態氮及揮發性脂肪酸濃度的測定 40
三、原蟲和真菌動孢子數目 43
四、NGS 45
（一）格蘭氏陰陽性菌數量 45
（二）Top10 Group 48
（三）三元相圖 56
（四）Venn圖 58
（五）PCA 分析及Welch's t-test 60
（六）LEfSe進化分支圖和LDA 分布柱狀圖 70
動物實驗 75
一、糞便菌相分析及球蟲卵檢測 75
（一）糞便菌相分析 75
（二）球蟲卵檢測: 79
二、下痢持續天數及糞便評分 80
三、血液生化值 80
（一）血容比 80
（二）仔牛白血球數 81
（三）血漿總蛋白 82
（四）血中IgG1濃度 83
（五）血酮濃度 84
四、生長表現 85
五、疾病紀錄 86
六、NGS 86
（一）Top 10 group 88
（二）三元相圖 92
（三）Venn圖 93
（四）PCA 分析及Welch's t-test 94
（五） LEfSe進化分支圖和LDA 分布柱狀圖 97
（六）與先前文獻之比較 98
肆、 結論 99
伍、 參考文獻 100

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