請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90755完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳明汝 | zh_TW |
| dc.contributor.advisor | Ming-Ju Chen | en |
| dc.contributor.author | 劉世穩 | zh_TW |
| dc.contributor.author | Esmond Sie Wen Lau | en |
| dc.date.accessioned | 2023-10-03T17:28:36Z | - |
| dc.date.available | 2023-11-10 | - |
| dc.date.copyright | 2023-10-03 | - |
| dc.date.issued | 2023 | - |
| dc.date.submitted | 2023-08-11 | - |
| dc.identifier.citation | A.O.A.C. (1980) Official Methods of Analysis, 13th ed. Association of Official Analytical Chemists. Washington D.C. 376-384.
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Detection of infectious agents causing neonatal calf diarrhea on two large dairy farms in Yangxin County, Shandong Province, China. Frontiers in Veterinary Science, 7, 589126. https://doi.org/10.3389/fvets.2020.589126 Yan, S., Yang, B., Ross, R. P., Stanton, C., Zhang, H., Zhao, J., & Chen, W. (2020). Bifidobacterium longum subsp. longum YS108R fermented milk alleviates DSS induced colitis via anti-inflammation, mucosal barrier maintenance and gut microbiota modulation. Journal of Functional Foods, 73, 104153. https://doi.org/10.1016/j.jff.2020.104153 Yoon, I. K., & Stern, M. D. (1995). Influence of direct-fed microbials on ruminal microbial fermentation and performance of ruminants–A Review. Asian-Australasian Journal of Animal Sciences, 8(6), 533–555. https://doi.org/10.5713/ajas.1995.553 Zhang, C., Yu, Z., Zhao, J., Zhang, H., & Zhai, Q. (2019). Colonization and probiotic function of Bifidobacterium longum. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90755 | - |
| dc.description.abstract | 益生菌的精準餵食方法可以解決益生菌菌株、宿主及其微生物組中固有的異質性。實驗室先前研究發現，Bifidobacterium longum 和 Ruminococcus flavefaciens 在腸道中的相對豐富度對牛隻的健康有顯著的影響。然而，目前 R. flavefaciens 並非行政院農委會所認可的飼料添加劑。先前有研究顯示餵食牛隻 Saccharomyces cerevisiae 可以顯著提高其瘤胃中 R. flavefaciens 的相對豐富度。因此，S. cerevisiae 和 B. longum 可能是促進牛隻健康之潛力益生菌。
首先，我們從健康仔牛糞便中分離出乳酸菌及從水果中分離出 S. cerevisiae。從仔牛糞便中分離出共 57 株乳酸菌，並透過 16S rRNA 基因序列進行定序。 初步篩選乳酸菌菌株的條件為具有抑制牧場常見之病原菌。結果顯示，共 10 個菌株分别具有抑制 Salmonella enterica 及 Staphylococcus aureus 的能力。但其中只有 6 株乳酸菌目前是被行政院農委會認可的飼料添加劑。接續使用 RAW264.7 细胞在脂多糖刺激之下與乳酸菌菌株共培養作為篩選潛力益生菌株。結果發現，Ligilactobacillus salivarius K108 具有同時抑制兩個病原菌及免疫調節的能力。此外，從水果中分離出57株酵母菌，經由 18S-26S ITS來進行鑑定。結果顯示其中 14 株被鑑定為 S. cerevisiae。進一步進行瘤胃體外發酵試驗來進行篩選 S. cerevisiae。在 24 小時的體外發酵過程中，S. cerevisiae T15 表現出較高的 pH 值和產生較多的微生物菌體蛋白。另外，本實驗室先前分離出 5 株 B. longum，其中 B. longum APL 30具有較好的免疫調節和抑制病原菌之能力。在進行益生菌單株篩選後，將 B. longum APL 30、L. salivarius K108 和 S. cerevisiae T15 組合並檢測其對免疫調節及瘤胃體外發酵之影響。結果發現，細胞在LPS 刺激之下，組合益生菌顯示出較高的 IL-10 和較低的 TNF-α 分泌量。在 24 小時瘤胃體外發酵後，有較高的丁酸產生；而在 48 小時瘤胃體外發酵後，有較高的丙酸和较低的乙酸/丙酸比例。 接著，使用 DSS 誘導小鼠結腸炎模式來釐清組合益生菌對抗發炎的能力。結果顯示，組合益生菌有能力緩解 DSS 誘導造成的下痢、糞便潛血及腸道通透性等症狀。給予高劑量組合益生菌可以增加抗發炎細胞素 IL-10 的分泌量、提高腸道中 Bifidobacterium 的菌數及腸道中丙酸的含量。此外，給予低劑量組合益生菌可以降低腸道中 Clostridium perfringens 的菌數。未來還需在乳牛場進行試驗，以進一步驗證此混合菌株對乳牛健康益處的影響。 | zh_TW |
| dc.description.abstract | A targeted method towards probiotics could effectively tackle the natural diversity present in probiotic strains, the hosts, and their microbiomes. Our previous research found that the abundance of Bifidobacterium longum and Ruminococcus flavefaciens in intestinal tracts positively affects dairy cattle health. However, R. flavefaciens is not yet allowed to be applied as a feed supplement by the Council of Agriculture, Taiwan. Several studies indicated that supplementation of Saccharomyces cerevisiae could significantly increase R. flavefaciens relative abundance. Thus, S. cerevisiae and B. longum might be potential probiotics for promoting dairy cows’ health.
First, we aimed to isolate lactic acid bacteria (LAB) from healthy dairy calf feces and S. cerevisiae from fruits. Fifty-seven LAB were isolated and identified through the sequencing of 16S rRNA gene from calves’ feces. Isolates with the ability to inhibit common pathogens in dairy farms as the preliminary screening criteria. The results showed that 10 strains have the ability to inhibit Salmonella enterica and Staphylococcus aureus, respectively. Only 6 strains of LAB were allowed to be applied as feed supplements by the Council of Agriculture, Taiwan. In the in vitro screening, RAW264.7 cells stimulated by lipopolysaccharide (LPS) were used as a model. We found that Ligilactobacillus salivarius K108 has the ability to inhibit pathogens and immunomodulation. Besides, 57 isolates were obtained from fruits, using the 18S-26S ITS region to identify them. The results showed that 14 strains were confirmed as S. cerevisiae. We further evaluated the functionality of S. cerevisiae by in-vitro rumen fermentation. S. cerevisiae T15 showed higher pH value and microbial crude protein (MCP) production in 24-hour period fermentation. Additionally, 5 strains of B. longum were previously isolated in our lab, and B. longum APL 30 showed better immunomodulatory and anti-pathogen properties. After single strain screening, the combination of B. longum APL30, L. salivarius K108, and S. cerevisiae T15 was tested for their effects on immunomodulatory and in-vitro rumen fermentation. The probiotic mixture showed a higher IL-10 production and lower TNF-α when stimulated by LPS, higher butyrate after 24-hour in-vitro rumen fermentation, and a higher propionate and lower acetate/propionate ratio after 48-hour in-vitro rumen fermentation. Moreover, to clarify the ability of anti-inflammatory of the probiotic mixture, the induction of colitis using DSS was carried out in the mouse model. The findings indicated that the probiotic mixture has the ability to alleviate symptoms such as diarrhea, fecal bleeding, and gut leakage, which were induced by DSS. Administration of a high-dosage probiotic mixture has been shown to enhance the secretion of the anti-inflammatory cytokine IL-10, increase the bacterial count of Bifidobacterium, and elevate the level of propionic acid in the colon content of DSS-induced mice. However, administration of a low-dosage probiotic mixture has been shown to decrease the bacterial count of Clostridium perfringens. The field trial in dairy farms is also needed to verify the health benefits in dairy cattle. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-10-03T17:28:36Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2023-10-03T17:28:36Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | Acknowledgments i
中文摘要 iii Abstract v Table of Contents vii List of Figures xi List of Tables xiii Chapter 1 Literature review 1 1.1 Common dairy cattle disease related to inflammation 1 1.1.1 Mastitis 1 1.1.2 Diarrhea 2 1.1.3 Lameness 3 1.1.4 Pneumonia 3 1.2 Factors affecting dairy cattle’s health 5 1.2.1 Nutrition 5 1.2.2 Genetics 6 1.2.3 Housing 6 1.2.4 Environmental conditions 7 1.2.5 Gut microbiota 7 1.3 Lipopolysaccharide 10 1.3.1 Interaction between LPS and gut microbiota dysbiosis 13 1.3.2 Interaction between LPS and intestinal barrier function 13 1.4 The role of selected probiotics strain 16 1.4.1 Bifidobacterium longum 17 1.4.1.1 Intestinal barrier function 17 1.4.1.2 Immunomodulatory 18 1.4.1.3 Antimicrobial activity 19 1.4.1.4 Interactions among microorganisms influencing gut microbial metabolism 19 1.4.2 Saccharomyces cerevisiae 22 1.4.2.1 Effect on rumen microbial composition 24 1.4.2.2 Rumen volatile fatty acid composition 24 1.4.2.3 Milk quality and production performance 25 1.4.2.4 Nitrogen metabolism 26 1.4.3 Ligilactobacillus salivarius 29 Chapter 2 Research Motivation and Objectives 30 2.1 Objectives 30 2.2 Experimental design 31 Chapter 3 Materials and Methods 32 3.1 Materials 32 3.1.1 Chemicals and reagents 32 3.1.2 Biological medium 35 3.1.3 Antibody 36 3.1.4 Kits 36 3.1.5 Equipment 37 3.1.6 Cell line 39 3.1.7 Animal 39 3.1.8 Software 39 3.2 Isolation, purification, and identification of target microorganism 42 3.2.1 Isolation of Saccharomyces cerevisiae 42 3.2.2 Isolation of lactic acid bacteria 42 3.2.3 Microorganism purification 42 3.2.4 Microorganism preservation 43 3.2.5 DNA extraction 43 3.2.6 Microorganism identification 44 3.2.7 Phylogenetic tree construction 44 3.3 In-vitro screening experiment 50 3.3.1 Preparation of microorganism strain 50 3.3.2 Antimicrobial activity 51 3.3.3 Coculture of isolated strain with RAW 264.7 cells 54 3.3.4 In-vitro rumen fermentation 56 3.4 Dextran sodium sulfate (DSS) induced intestine inflammation mice model 59 3.4.1 Bacterial strain and culture condition 59 3.4.2 Animal experimental design 59 3.4.3 Disease activity index (DAI) 60 3.4.4 Fecal bleeding assessment 60 3.4.5 Colon length measurement 60 3.4.7 Measurement of intestinal permeability 62 3.4.8 Preparation of colonic homogenates 62 3.4.9 Measurement of cytokine and immunoglobulin 63 3.4.10 Measurement of tight junction proteins and myeloperoxidase 63 3.4.11 Short-chain fatty acid analysis 64 3.4.12 DNA extraction of cecum content 65 3.4.13 Analysis of cecum microbiota 66 3.5 Statistical analysis 66 Chapter 4 Results 70 4.1 Isolation and identification of lactic acid bacteria from the feces of healthy dairy calves 70 4.2 Identification of Saccharomyces cerevisiae 74 4.3 Screening of lactic acid bacteria 77 4.3.1 Antimicrobial activity 77 4.3.2 Immunomodulatory effect 81 4.4 Screening of Saccharomyces cerevisiae 85 4.4.1 In-vitro rumen fermentation 85 4.4.2 Antimicrobial activity 90 4.4.3 Immunomodulatory effect 92 4.5 Screening of Bifidobacterium longum 95 4.5.1 Antimicrobial activity 95 4.5.2 Immunomodulatory effect 97 4.6 The effect of probiotic mixture in-vitro 100 4.6.1 Immunomodulatory effect 100 4.6.2 In-vitro rumen fermentation of the probiotic mixture 102 4.6.3 In-vitro rumen fermentation of the single strain 107 4.7 The effect of the probiotic mixture on DSS-induced mice colitis 112 4.7.1 Average daily weight gain (ADG) 112 4.7.2 Disease activity index (DAI) 112 4.7.3 Incidence of hemoccult 112 4.7.4 Gut permeability 117 4.7.5 Colon length 117 4.7.6 Immunomodulatory 117 4.7.7 H&E staining 121 4.7.8 Myeloperoxidase (MPO) 121 4.7.9 Tight junction protein 121 4.7.10 Cecal microbiota 122 4.7.11 Short-chain fatty acid 122 Chapter 5 Discussion 128 5.1 Isolation of microorganisms 128 5.2 In-vitro experiment for screening potential probiotic strains 129 5.3 In-vitro experiment for selecting the optimal combination of a probiotic mixture 131 5.4 The effect of probiotic mixture in-vivo 133 Chapter 6 Conclusion 136 References 138 | - |
| dc.language.iso | en | - |
| dc.title | 利用體外及小鼠試驗篩選促進乳牛健康之潛力益生菌組合 | zh_TW |
| dc.title | Screening potential probiotic mixture in vitro and in vivo for promoting dairy cattle health | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 111-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 王翰聰;莊士德;陳詠宗;謝睿純 | zh_TW |
| dc.contributor.oralexamcommittee | Han-Tsung Wang;Shih-Te Chuang;Yung-Tsung Chen;Jui-Chun Hsieh | en |
| dc.subject.keyword | 乳牛,益生菌組合,腸道屏障保護,結腸炎, | zh_TW |
| dc.subject.keyword | Dairy cattle,Probiotic mixture,Intestinal barrier protection,Colitis, | en |
| dc.relation.page | 158 | - |
| dc.identifier.doi | 10.6342/NTU202304094 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2023-08-13 | - |
| dc.contributor.author-college | 生物資源暨農學院 | - |
| dc.contributor.author-dept | 動物科學技術學系 | - |
| 顯示於系所單位： | 動物科學技術學系 | |
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