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研究生： 楊佳叡
Chia-Jui Yang
論文名稱： 探討光照對Rhizopus oligosporus轉換豆漿中異黃酮醣苷之影響
Effect of light condition on bioconversion of isoflavone glycosides in soymilk by Rhizopus oligosporus
指導教授： 徐敬衡
口試委員:
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程與材料工程學系
Department of Chemical & Materials Engineering
論文出版年： 2020
畢業學年度： 108
語文別： 中文
論文頁數： 93
中文關鍵詞： 異黃酮 、少孢根黴菌 、兩階段 、大豆苷元 、染料木黃酮 、光照
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    • 苷元型異黃酮(isoflavone aglycones)因具有降低心血管疾病、預防骨質疏鬆、抗腫瘤、改善肥胖症等特性，因此被廣泛地討論。本實驗利用微生物發酵法，以豆漿取代傳統大豆作為基質，透過Rhizopus oligosporus將醣苷型異黃酮(isoflavone glucosides)轉化成苷元型異黃酮(isoflavone aglycones)。而在微生物發酵中，不同光源也會影響微生物的生理狀態和發酵行為，因此本研究主要的目的是在於探討「光照」環境因子對於Rhizopus oligosporus生產苷元型異黃酮之影響，探討不同光源以及不同光照度對於菌體的生長及產物的變化。

    本研究結果顯示在光照的條件下，紅光200 lux最有利於Rhizopus oligosporus的菌體生長，能將菌體重量提升至避光組的1.38倍，而在藍光100 lux的情況下，則有最佳的單位菌重轉化產物能力，約為控制組的1.35倍。因此，本研究設計了以紅光接續藍光的兩階段光照的策略，以提升最佳的菌體重量與產物產量。在兩階段光照的條件下，大豆苷元的產量可以高達24.5 µg/ml，與單純紅光照相比及單純藍光照相比，皆提升了約26%。此外，經由兩階段光照條件下的染料木黃酮產量，可提升至20.26 µg/ml，與單純紅光照相比，提升了約15%。
     

    Isoflavone aglycones have been reported to help in the prevention of cancer, cardiovascular diseases, osteoporosis, obesity . It plays an important role in human health and has been discussed for several years. Isoflavone aglycones can be produced from isoflavone glucosides. In this work, a fermentation process with soymilk which replaces traditional soybean as substrate has been performed. Besides, different light conditions will also has a strong connection with microbial growth and fermentation behavior.

    In this work, we have explored the effects of light condition on bioconversion of isoflavone glucosides into isoflavone aglycones using Rhizopus oligosporus as well as develop strategies to enhance isoflavone aglycones production. Experimental results showed that at red light 200 lux condition performed the optimum cells growth, which was 38% higher than dark group. But at blue light condition, which light intensity was 100 lux, was more favorable for product yield based on biomass. It could perform 35% higher than the dark group. Therefore, two-stage shake flask fermentation by light control was conducted. Daidzein concentration using two-stage fermentation was 24.5µg/ml which corresponds to 26 % higher than single-light fermentation at red and blue light. On the other hand, Genisrein concentration using two-stage fermentation was 20.26µg/ml which corresponds to 15 % higher than single-light fermentation at red light.

    目錄 摘要 ii Abstract iii 誌謝 iv 目錄 v 表目錄 viii 圖目錄 ix 第一章 緒論 1 1-1研究動機 1 1-2研究目的 2 第二章 文獻回顧 3 2-1大豆 3 2-2天貝 4 2-3豆漿 5 2-4大豆異黃酮 7 2-4-1異黃酮基本性質介紹 7 2-4-2異黃酮特性及應用 8 2-4-3轉換醣苷型異黃酮的β-葡萄糖苷酶(beta-glucosidase) 9 2-4-4大豆苷元(daidzein)與金雀異黃酮(genistein) 10 2-5少孢根黴(Rhizopus oligosporus)菌種介紹 13 2-6 影響發酵之因素 14 2-6-1 培養基組成 14 2-6-2 pH值 16 2-6-3 通氣速率 16 2-6-4 溫度 16 2-6-5 光 17 第三章 材料與方法 23 3-1 實驗規劃 23 3-2 實驗材料與設備 24 3-2-1 實驗菌株 24 3-2-2 實驗藥品 25 3-2-3 實驗儀器與設備 26 3-2-4 數據呈現之參數符號設定 28 3-3 實驗方法 29 3-3-1 分離菌種 29 3-3-2 培養基組成 31 3-4 分析方法 32 3-4-1 液態培養菌重測定 32 3-4-2 固態培養菌重測定 33 3-4-3異黃酮分析 34 第四章 實驗結果與討論 39 4-1 搖瓶實驗之避光組與紅光強度對少孢根黴菌生產大豆異黃酮發酵動力曲線影響 39 4-1-1 紅光強度對少孢根黴菌生產菌體影響 44 4-1-2 紅光強度對少孢根黴生產大豆苷元之影響 44 4-1-3 紅光強度對少孢根黴生產染料木黃酮之影響 46 4-2 搖瓶實驗之避光組與藍光強度對少孢根黴菌生產大豆異黃酮發酵動力曲線之影響 48 4-2-1藍光強度對少孢根黴生產菌體之影響 53 4-2-2藍光強度對少孢根黴生產大豆苷元之影響 53 4-2-3藍光強度對少孢根黴生產染料木黃酮之影響 55 4-3搖瓶實驗之避光組與兩階段光照對少孢根黴菌生產大豆異黃酮發酵動力曲線之影響 57 4-3-1兩階段光照對少孢根黴生產菌體之影響 58 4-3-2兩階段光照對少孢根黴生產大豆異黃酮之影響 60 4-3-3兩階段光照對少孢根黴生產染料木黃酮之影響 61 4-4固態培養與搖瓶實驗對少孢根黴菌生產大豆異黃酮發酵動力曲線之影響 63 4-4-1固態培養對少孢根黴生產菌體之影響 65 4-4-2固態培養對少孢根黴每單位黃豆所含反應物之影響 66 4-4-3固態培養對少孢根黴每單位黃豆可生成產物之影響 67 4-4-4固態培養對少孢根黴每單位菌重可生成產物之影響 68 第五章 結論及建議 69 5-1 結論 69 5-2 建議 70 參考文獻 71   表目錄 表2- 1每100g大豆中所含營養成分 4 表2-2金雀異黃酮對相關疾病影響 12 表2-3工業發酵常用碳源與氮源 15 表2-4光波長影響植物發育關係 17 表2-5光接收器種類 19 表3-1 藥品清單 25 表3-2 實驗設備清單 26 表3-3 數據符號定義 28 表4-1避光與紅光照射下搖瓶發酵參數值 43 表4-2避光與藍光照射下搖瓶發酵參數值 52 表4-3避光下固態發酵與搖瓶發酵參數值 64   圖目錄 圖2-1天貝外觀 5 圖2-2各式豆漿製程 6 圖2-3異黃酮化學結構式 7 圖2-4β-葡萄糖苷酶對異黃酮醣苷的反應機制 10 圖2-5大豆苷元藥理作用 11 圖3-1實驗規劃示意圖 23 圖3-2市售天貝菌粉 24 圖3-3 Rhizopus oligosporus生長於PDA外觀 30 圖3-4搖瓶光照配置 32 圖3-5各產物之HPLC圖譜 35 圖3-6大豆醣苷標準品濃度之檢量線 36 圖3-7染料木苷標準品濃度之檢量線 36 圖3-8大豆苷元標準品濃度之檢量線 37 圖3-9染料木黃酮標準品濃度之檢量線 37 圖3-10葡萄糖胺標準品濃度檢量線 38 圖3-11菌重濃度對葡萄糖胺檢量線 38 圖4-1 Rhizopus oligosporus在避光0 lux對大豆苷元發酵曲線 39 圖4-2 Rhizopus oligosporus在避光0 lux對染料木黃酮發酵曲線 40 圖4-3 Rhizopus oligosporus在紅光100 lux對大豆苷元發酵曲線 40 圖4-4 Rhizopus oligosporus在紅光100 lux對染料木黃酮發酵曲線 41 圖4-5 Rhizopus oligosporus在紅光200 lux對大豆苷元發酵曲線 41 圖4-6 Rhizopus oligosporus在紅光200 lux對染料木黃酮發酵曲線 42 圖4-7 Rhizopus oligosporus在紅光300 lux對大豆苷元發酵曲線 42 圖4-8 Rhizopus oligosporus在紅光300 lux對染料木黃酮發酵曲線 43 圖4-9不同紅光強度對Rhizopus oligosporus最大菌重圖 44 圖4-10不同紅光強度對Rhizopus oligosporus生產最大大豆苷元圖 45 圖4-11不同紅光強度對Rhizopus oligosporus單位菌重對大豆苷元轉化率 45 圖4-12不同紅光強度對Rhizopus oligosporus生產最大染料木黃酮圖 46 圖4-13不同紅光強度對Rhizopus oligosporus單位菌重對染料木黃酮轉化率 47 圖4-14 Rhizopus oligosporus在避光0 lux對大豆苷元發酵曲線 48 圖4-15 Rhizopus oligosporus在避光0 lux對染料木黃酮發酵曲線 49 圖4-16 Rhizopus oligosporus在藍光100 lux對大豆苷元發酵曲線 49 圖4-17 Rhizopus oligosporus在藍光100 lux對染料木黃酮發酵曲線 50 圖4-18 Rhizopus oligosporus在藍光200 lux對大豆苷元發酵曲線 50 圖4-19 Rhizopus oligosporus在藍光200 lux對染料木黃酮發酵曲線 51 圖4-20 Rhizopus oligosporus在藍光300 lux對大豆苷元發酵曲線 51 圖4-21 Rhizopus oligosporus在藍光300 lux對染料木黃酮發酵曲線 52 圖4-22不同藍光強度對Rhizopus oligosporus最大菌重圖 53 圖4-23不同藍光強度對Rhizopus oligosporus生產最大大豆苷元圖 54 圖4-24不同藍光強度對Rhizopus oligosporus單位菌重對大豆苷元轉化率 54 圖4-25不同藍光強度對Rhizopus oligosporus生產最大染料木黃酮圖 55 圖4-26不同藍光強度對Rhizopus oligosporus單位菌重對染料木黃酮轉化率 56 圖4-27 Rhizopus oligosporus在兩階段光照對大豆苷元發酵曲線 57 圖4-28 Rhizopus oligosporus在兩階段光照對染料木黃酮發酵曲線 58 圖4-29兩階段光照與不同紅光強度對Rhizopus oligosporus最大菌重圖 59 圖4-30兩階段光照與不同蘭光強度對Rhizopus oligosporus最大菌重圖 59 圖4-31兩階段光照與不同紅光強度對Rhizopus oligosporus生產最大大豆苷元圖 60 圖4-32兩階段光照與不同藍光強度對Rhizopus oligosporus生產最大大豆苷元圖 61 圖4-33兩階段光照不同紅光強度對Rhizopus oligosporus生產最大染料木黃酮圖 62 圖4-34兩階段光照不同藍光強度對Rhizopus oligosporus生產最大染料木黃酮圖 62 圖4-35固態培養避光對Rhizopus oligosporus對大豆苷元發酵曲線 63 圖4-36固態培養避光對Rhizopus oligosporus對染料木黃酮發酵曲線 64 圖4-37固態培養與搖瓶培養對Rhizopus oligosporus最大菌重產率圖 65 圖4-38固態培養與搖瓶培養對Rhizopus oligosporus單位黃豆所含大豆醣苷及單位黃豆所含染料木苷圖 66 圖4-39固態培養與搖瓶培養對Rhizopus oligosporus單位黃豆可生成大豆苷元及單位黃豆可生成染料木黃酮圖 67 圖4-40固態培養與搖瓶培養對Rhizopus oligosporus單位菌重可生成大豆苷元及單位菌重可生成染料木黃酮圖 68

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