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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳明汝(Ming-Ju Chen) | |
dc.contributor.author | Hsin-Yu Tang | en |
dc.contributor.author | 湯芯瑜 | zh_TW |
dc.date.accessioned | 2021-07-10T21:36:26Z | - |
dc.date.available | 2021-07-10T21:36:26Z | - |
dc.date.copyright | 2016-10-14 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-07-18 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76761 | - |
dc.description.abstract | 本研究之目的為探討Lactobacillus kefiranofaciens M1對不同環境壓力包括熱、冷、酸、膽鹽、氯化鈉、乙醇和過氧化氫之適應性和交叉保護反應。測定壓力適應對L. kefiranofaciens M1生理特性 (生長、酸化活性、β-半乳糖苷酶活性、胞外多醣體產量、脂肪酸組成、蛋白質表現和菌體形態) 之影響。此外,測試壓力適應和未適應之菌體於噴霧乾燥、冷凍乾燥及後續貯存與模擬消化期間之存活。試驗中亦對噴霧和冷凍乾燥L. kefiranofaciens M1粉末之水分含量和形態及其於不同復水培養基之復甦情形進行探討。
結果顯示,除氯化鈉 (0.1 M) 適應外,熱 (37°C)、冷 (20°C)、酸 (pH 5.0)、膽鹽 (0.05%)、乙醇 (4%) 和過氧化氫 (100 ppm) 適應均可誘導L. kefiranofaciens M1之同源壓力忍受性。膽鹽、鹽、乙醇和過氧化氫適應可增加菌體之耐熱性 (52°C);酸、膽鹽、鹽、乙醇或過氧化氫適應可賦予菌體耐冷性 (-20°C);膽鹽和乙醇適應可增加耐酸性 (pH 3.0);所有壓力適應均可誘導對膽鹽 (0.2%) 之交叉保護作用;冷、乙醇或過氧化氫適應可提升對氯化鈉 (3.0 M) 和乙醇 (20%) 之抵抗力;熱、酸或膽鹽適應可導致菌體過氧化氫 (1000 ppm) 忍受性之提升。 壓力適應組菌體相較於未適應組菌體,具有相同之生長、酸化活性和胞外多醣體產量。熱和酸適應組菌體較未適應組菌體呈現較高之β-半乳糖苷酶活性。除冷適應外,所有壓力適應可增加L. kefiranofaciens M1菌體之飽和脂肪酸總量,減少不飽和脂肪酸總量。二維電泳分析顯示熱、冷、酸、膽鹽、氯化鈉、乙醇和過氧化氫適應處理分別改變菌體15、21、21、17、22、20和17種蛋白質之表現量,這些壓力蛋白質可被一個以上之壓力誘導,主要與壓力反應、蛋白質生合成、能量和醣類代謝功能相關。此外,與未適應組菌體相較,壓力適應組菌體在後續同源壓力挑戰下呈現較輕微之細胞表面傷害。 另外,試驗發現酸和膽鹽適應可增加L. kefiranofaciens M1於噴霧和冷凍乾燥後之存活。大致上,酸和膽鹽適應組菌體相較於未適應組菌體於後續貯存於-20、4和25℃及暴露模擬胃液 (pH 2.0) 和腸液 (pH 8.0) 期間可保持較高之存活菌數。此外,噴霧乾燥L. kefiranofaciens M1粉末呈現球狀,而冷凍乾燥粉末則呈現不規則狀。噴霧和冷凍乾燥粉末之水分含量分別為6.84和1.97%。試驗中所試8種復水培養基對噴霧和冷凍乾燥菌體呈現不同之復甦效果,其中菌體在0.85% NaCl、0.1% peptone、磷酸鹽緩衝液 (phosphate-buffered saline, PBS)、analog man (AM) buffer和10% 重組脫脂牛乳 (reconstituted skim milk, RSM) 中相較在dd H2O、maximum recovery diluent (MRD) 和20% sucrose中,可具有較高之復甦菌數。 | zh_TW |
dc.description.abstract | The objective of this study was to investigate the adaptive and cross-protective responses of Lactobacillus kefiranofaciens M1 to various environmental stresses, including heat, cold, acid, bile salts, sodium chloride, ethanol and hydrogen peroxide. The effects of stress adaptation on the physiological characteristics (growth, acidification activity, β-galactosidase activity, exopolysaccharide production, fatty acid composition, protein expression and cell morphology) of L. kefiranofaciens M1 were evaluated. Furthermore, survival of stress-adapted and non-adapted cells during spray drying, freeze drying and subsequent storage as well as simulated gastrointestinal digestion was examined. The moisture content and morphology of spray-dried and freeze-dried L. kefiranofaciens M1 powders and their recoveries in different rehydration media were also investigated.
The results showed that adaptation of L. kefiranofaciens M1 to heat (37°C), cold (20°C), acid (pH 5.0), bile salts (0.05%), ethanol (4%) and hydrogen peroxide (100 ppm) stresses, except for sodium chloride (0.1 M), could induce homologous tolerance. Adaptation to bile salts, sodium chloride, ethanol and hydrogen peroxide increased heat (52°C) tolerance. Acid, bile salts, sodium chloride, ethanol or hydrogen peroxide adaptation provided protection against cold (-20°C) stress. Adaptation to bile salts and ethanol led to increased resistance to acid (pH 3.0) challenge. All stress adaptation induced cross-protection against bile salts (0.2%). Cold, ethanol or hydrogen peroxide adaptation enhanced the resistance against sodium chloride (3.0 M) and ethanol (20%) challenges. Pretreatment with heat, acid or bile salts caused an increase in hydrogen peroxide (1000 ppm) tolerance. All stress-adapted cells showed similar growth, acidification activity and exopolysaccharide production when compared with non-adapted cells. Heat- and acid-adapted cells exhibited higher β-galactosidase activity than non-adapted cells. All treatments of stress adaptation, except for cold adaptation, increased the content of saturated fatty acids and decreased the content of unsaturated fatty acids in cells of L. kefiranofaciens M1. Two-dimensional SDS-PAGE analysis revealed that the expression levels of 15, 21, 21, 17, 22, 20 and 17 proteins were altered by heat, cold, acid, bile salts, sodium chloride, ethanol and hydrogen peroxide treatments, respectively. Several stress proteins involved in stress response, protein biosynthesis, energy and carbohydrate metabolism were induced by more than one treatment. In addition, stress-adapted cells of L. kefiranofaciens M1 showed a relatively minor cell surface damage under subsequent homologous stress challenge compared with their non-adapted cells. On the other hand, it was found that adaptation to acid and bile salts increased the survival of L. kefiranofaciens M1 after spray drying and freeze drying. In general, acid- and bile salt-adapted cells maintained higher bacterial counts during subsequent storage at -20, 4 and 25℃ and exposure to simulated gastric (pH 2.0) and intestinal juices (pH 8.0) than non-adapted cells. Moreover, spray-dried L. kefiranofaciens M1 powders appeared globular in shape while freeze-dried powders had angular shapes. The moisture content of spray- and freeze-dried powders was 6.84 and 1.97%, respectively. Eight rehydration media tested showed different capacities for recovery of spray- and freeze-dried L. kefiranofaciens M1. Among them, 0.85% NaCl solution, 0.1% peptone solution, phosphate-buffered saline (PBS), analog man (AM) buffer and 10% reconstituted skim milk (RSM) yielded greater recovery counts of L. kefiranofaciens M1 than double distilled water (dd H2O), maximum recovery diluent (MRD) and 20% sucrose solution. | en |
dc.description.provenance | Made available in DSpace on 2021-07-10T21:36:26Z (GMT). No. of bitstreams: 1 ntu-105-R03626006-1.pdf: 7115799 bytes, checksum: 96c98bffb64cc8b4ef29f5bd27e6fe23 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 誌謝 i
中文摘要 ii 英文摘要 iv 目錄 vi 圖目錄 xii 表目錄 xv 緒言 xvi 壹、 文獻回顧 1 一、 Lactobacillus kefiranofaciens M1 1 (一) 菌株之分離 1 (二) 生化特性 1 (三) 菌株之機能性 2 (四) 影響菌株生長之因子 3 1. pH值 3 2. 溫度 4 3. 膽鹽 4 4. 氯化鈉/滲透壓力 4 5. 乙醇 5 6. 過氧化氫/氧化壓力 5 7. 營養源 5 8. 其他 6 二、適應性壓力反應 (Adaptive stress response) 7 (一) 壓力反應 (Stress response) 7 (二) 壓力反應發生機制 13 三、適應性反應於益生菌生產保存之應用 19 (一) 噴霧乾燥和冷凍乾燥 19 (二) 噴霧乾燥和冷凍乾燥對菌體細胞之影響 19 (三) 乳酸桿菌於乾燥及後續貯存過程中之存活 20 貳、 材料與方法 25 一、 實驗架構 25 二、 材料與方法 26 (一) 菌株來源及保存 26 (二) 實驗菌液之製備 26 (三) L. kefiranofaciens M1 對各式壓力之敏感性 26 (四) L. kefiranofaciens M1之壓力適應處理 27 (五) L. kefiranofaciens M1於不同後續致死環境壓力中之存活 27 (六) L. kefiranofaciens M1於MRS培養液和重組脫脂牛乳之生長 27 (七) L. kefiranofaciens M1 酸化活性測定 28 (八) L. kefiranofaciens M1 β-半乳糖苷酶 (β-galactosidase) 活性測定 28 (九) L. kefiranofaciens M1 胞外多醣體產量測定 29 (十) L. kefiranofaciens M1菌體脂肪酸組成分析 29 1. 脂肪酸之萃取 29 2. 脂肪酸成分分析 29 (十一) L. kefiranofaciens M1 蛋白質表現分析 30 1. 菌體胞內粗蛋白質萃取 30 2. 蛋白質定量與濃度調整 31 3. 迷你膠體電泳分析 (Mini SDS-PAGE) 31 4. 二維膠體電泳分析 (Two-dimensional SDS-PAGE) 31 5. 蛋白質表現分析 34 6. L. kefiranofaciens M1壓力蛋白質鑑定 34 (1) 膠體內水解 (In-gel digestion) 34 (2) 蛋白質序列分析 (Protein sequence analysis) 35 (3) 資料庫搜尋 35 (十二) 噴霧乾燥和冷凍乾燥處理 36 (十三) L. kefiranofaciens M1於噴霧和冷凍乾燥後之存活實驗 36 (十四) 噴霧和冷凍乾燥L. kefiranofaciens M1粉末於不同溫度貯存期間之存活 36 (十五) 噴霧和冷凍乾燥L. kefiranofaciens M1粉末於模擬消化液中之存活 37 (十六) 掃描式電子顯微鏡觀察L. kefiranofaciens M1及其噴霧和冷凍乾燥粉末形態 37 (十七) 噴霧和冷凍乾燥L. kefiranofaciens M1粉末水分含量測定 38 (十八) 噴霧和冷凍乾燥L. kefiranofaciens M1粉末於不同復水培養基之復甦 38 (十九) 菌數測定 38 (二十) 統計分析 39 參、 結果與討論 40 第一節:L. kefiranofaciens M1對不同壓力之生長與忍受性 40 一、 L. kefiranofaciens M1對不同壓力之敏感性 40 (一) L. kefiranofaciens M1之熱敏感性 40 (二) L. kefiranofaciens M1之冷敏感性 41 (三) L. kefiranofaciens M1之酸敏感性 42 (四) L. kefiranofaciens M1之膽鹽敏感性 43 (五) L. kefiranofaciens M1之氯化鈉敏感性 44 (六) L. kefiranofaciens M1之乙醇敏感性 45 (七) L. kefiranofaciens M1之過氧化氫敏感性 46 二、 壓力適應處理對L. kefiranofaciens M1於後續致死壓力挑戰下存活之影響 47 (一) 壓力適應處理對L. kefiranofaciens M1於高溫下存活之影響 47 (二) 壓力適應處理對L. kefiranofaciens M1於低溫下存活之影響 49 (三) 壓力適應處理對L. kefiranofaciens M1於酸性環境下存活之影響 51 (四) 壓力適應處理對L. kefiranofaciens M1於膽鹽中存活之影響 53 (五) 壓力適應處理對L. kefiranofaciens M1於高鹽中存活之影響 55 (六) 壓力適應處理對L. kefiranofaciens M1於乙醇中存活之影響 57 (七) 壓力適應處理對L. kefiranofaciens M1於過氧化氫中存活之影響 59 (八) L. kefiranofaciens M1對不同環境壓力之適應性及交叉保護性反應 61 第二節:壓力適應對L. kefiranofaciens M1生理特性之影響 64 一、 壓力適應對L. kefiranofaciens M1生長之影響 64 二、 壓力適應對L. kefiranofaciens M1酸化活性之影響 66 三、 壓力適應對L. kefiranofaciens M1 β-半乳糖苷酶活性之影響 68 四、 壓力適應對L. kefiranofaciens M1胞外多醣體產生之影響 70 五、 壓力適應對L. kefiranofaciens M1脂肪酸組成之影響 72 六、 壓力適應對L. kefiranofaciens M1菌體蛋白質表現之影響 75 (一) 迷你膠體電泳分析 75 (二) 二維膠體電泳分析 77 (三) 壓力蛋白質鑑定 77 1. 胺基酸代謝 (Amino acid metabolism) 77 2. 醣類代謝 (Carbohydrate metabolism) 78 3. 能量代謝 (Energy metabolism) 82 4. 核苷酸代謝 (Nucleotide metabolism) 83 5. 肽聚醣生合成 (Peptidoglycan biosynthesis) 84 6. 壓力反應 (Stress response) 84 7. 轉錄和轉譯 (Transcription and translation) 85 七、 壓力適應及挑戰對L. kefiranofaciens M1菌體形態之影響 96 第三節:壓力適應對L. kefiranofaciens M1於噴霧乾燥和冷凍乾燥及後續貯存及消化期間存活之影響 106 一、 壓力適應對L. kefiranofaciens M1於噴霧和冷凍乾燥後存活之影響 106 二、 壓力適應對噴霧和冷凍乾燥L. kefiranofaciens M1粉末於不同溫度貯存期間存活之影響 109 三、 壓力適應對噴霧和冷凍乾燥L. kefiranofaciens M1粉末於模擬消化液中存活之影響 112 1. 噴霧和冷凍乾燥L. kefiranofaciens M1粉末於模擬胃液中之存活 112 2. 噴霧和冷凍乾燥L. kefiranofaciens M1粉末於模擬腸液中之存活 114 3. 噴霧和冷凍乾燥L. kefiranofaciens M1粉末於模擬胃腸液中之存活 116 第四節:噴霧乾燥和冷凍乾燥L. kefiranofaciens M1粉末特性分析 118 一、 噴霧和冷凍乾燥L. kefiranofaciens M1粉末之型態 118 二、 噴霧乾燥和冷凍乾燥L. kefiranofaciens M1粉末之水分含量 120 三、 噴霧和冷凍乾燥L. kefiranofaciens M1粉末於不同復水培養基中之復甦 122 肆、 結論 124 伍、 參考文獻 125 陸、 作者小傳 154 | |
dc.language.iso | zh-TW | |
dc.title | Lactobacillus kefiranofaciens M1對不同壓力之反應及其於噴霧乾燥和冷凍乾燥後存活之探討 | zh_TW |
dc.title | Investigation on the responses of Lactobacillus kefiranofaciens M1 to various stresses and its survival after spray drying and freeze drying | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 江明倫(Ming-Lun Chiang) | |
dc.contributor.oralexamcommittee | 周正俊,廖啟成,劉?睿 | |
dc.subject.keyword | Lactobacillus kefiranofaciens M1,壓力適應,交叉保護,噴霧乾燥,冷凍乾燥, | zh_TW |
dc.subject.keyword | Lactobacillus kefiranofaciens M1,stress adaptation,cross-protection,spray drying,freeze drying, | en |
dc.relation.page | 154 | |
dc.identifier.doi | 10.6342/NTU201600980 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2016-07-18 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 動物科學技術學研究所 | zh_TW |
顯示於系所單位: | 動物科學技術學系 |
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