Study on the optimal culture condition of Monascus-fermented dioscorea rich in GABA and ACEI, and the evaluation of reducing the risk of hypertension

高血壓是造成代謝症候群的危險因子之一，須長期靠降壓物質控制。紅麴發酵產物含有降血壓功效物質 γ-胺基丁酸 (γ-aminobutyric acid, GABA)，為抑制性神經傳導物質，可引起血管的擴張。本研究建立了紅麴發酵產物酒萃物中 acid form 及 lactone form monacolin K、citrinin、黃色素 monascin 及 ankaflavin 的同步分析方法，以及水萃物 GABA 的衍生化分析方法，再以 Monascus purpureus NTU 568 固態發酵方式篩選出最適培養方式及基質，以厭氧處理時間、厭氧處理起始時間及乙醇添加量，藉由三因子三階層的反應曲面法提高 GABA 含量。結果顯示，以山藥為基質生產之 GABA 較米高，發酵過程 GABA 含量在第六天最高，在第八天開始厭氧處理 60 小時及 3% 乙醇添加下可使 GABA 含量從 134 mg/kg 提高至 513 mg/kg，citrinin 從 30.81 ppm 降低至 6.35 ppm，趨勢顯示厭氧可造成 GABA 含量提升及 citrinin 含量下降。蛋白質經蛋白酶水解或微生物發酵生成之短鏈胜肽，具有血管張力素轉換酶抑制劑 (angiotensin I converting enzyme inhibitor, ACEI) 的活性，ACEI 為一降血壓物質。本研究又利用固態發酵得到的紅麴米與紅麴山藥進行ACEI活性分析，再以自發性高血壓大鼠 (spontaneous hypertensive rat, SHR) 經過連續八週管餵紅麴米或紅麴山藥，以評估短效性與長效性降血壓效果。結果顯示，單一次餵食後紅麴米可降低收縮壓 8 mmHg 與舒張壓 9 mmHg，紅麴山藥顯著降低收縮壓 20 mmHg 與舒張壓 17 mmHg (p<0.05)，且持續至八小時後仍有效果；餵食八週後紅麴米顯著降低收縮壓 20 mmHg 與舒張壓 18 mmHg (p<0.05)，紅麴山藥顯著降低收縮壓 26 mmHg 與舒張壓 22 mmHg (p<0.05)，且不會造成肝、腎、肌肉、心律影響及體內電解質不平衡，紅麴山藥在動物試驗結果顯示較紅麴米有更顯著降血壓效果。Hypertension is one critical factor of metabolic symdromes, and it has to be controlled by anti-hypertensive substances. Red mold fermented products contain an antihypertensive substance, γ-aminobutyric acid (GABA). GABA is one of the inhibitory neurotransmitters, and it can cause blood vessel relaxation. In this study, we established a rapid synchronous analysis method for detection of acid form and lactone form of monaolin K, citrinin, monascin, and ankaflavin levels in red mold fermented products ethanol extracts. And we also established an analysis method for the detection of GABA level in red mold fermented products aqueous extracts. In addition, various cultivation containers (tray and packed-bottle), substrate, culture conditions (pH, cultural time, ethanol addition and anaerobic treatment time) were selected to investigate the effect on GABA and citrinin production. In the result, tray and Dioscorea batatas were chosen as optimal culture apparatus and substrate, respectively. urthermore, cultural time, ethanol addition and anaerobic treatment time significantly influenced both GABA and citrinin production of M. purpureus NTU 568 under solid state dioscorea fermentation, so they were chosen as the factors of the response surface methodology (RSM) experiment in order to investigate the optimal culture condition. From the result of statistical figures, we can gain the GABA production at 513 mg/kg and citrinin production at 6,350 μg/kg from red mold dioscorea when the culture conditions are as following: culture 8 days, anaerobic treatment 60 hours, and adding 3% ethanol.e further used the Monascus fermented dioscorea with high GABA production to investigate the effect on anti-hypertension in rat model of spontaneous hypertension. The result showed that the systolic and diastolic blood pressure levels were decreased significantly after eight-hour daily oral administration, but the heart rates were not affected. The systolic and diastolic blood pressure levels were decreased significantly after eighth-week long-term oral administration, but the heart rates were not affected. From the result of tissue section staining of the aorta elastin fiber, rats with orally administrated red mold dioscorea had elastin fiber more straight than the control group. Therefore, these results suggest that anti-hypertensive effect of Monascus fermented dioscorea can prevent hypertension effectively.目錄一章 文獻回顧............................................1一節 紅麴菌之文獻回顧...................................1.1 古今有關紅麴功效之研究及記載..........................1.2 紅麴菌之特性..........................................2.3 紅麴生產之高價值二級代謝產物..........................5.4 紅麴機能性食品之開發.................................13.5 橘黴素 Citrinin 之研究...............................16二節 山藥與紅麴之關係..................................23三節 厭氧培養對真菌 GABA 之影響........................25四節 胺基酸分析........................................25.1 衍生化方法：.........................................25.2 常用的胺基酸衍生化試劑與衍生化反應...................26五節 高血壓之回顧與研究................................28.1 高血壓與代謝症候群...................................28.2 血壓之調控...........................................29.3 應用於改善高血壓疾病之相關研究.......................31六節 哺乳類小動物的血壓量測............................32.1 侵入式血壓計.........................................32 .2 非侵入式血壓計.......................................33二章 研究動機與目的....................................36三章 材料與方法........................................38一節 儀器..............................................38.1 固態培養相關設備.....................................38.2 萃取及分析相關設備...................................38.3 其他.................................................39二節 藥品..............................................39.1 標準品...............................................39.2 ㄧ般試藥.............................................39.3 萃取分析溶劑.........................................39.4 培養基...............................................40三節 紅麴菌菌株來源、培養方法..........................40.1 試驗菌株.............................................40.2 紅麴菌之種菌培養方法.................................40四節 紅麴菌固態發酵方法與條件..........................41五節 建立紅麴產物中初級代謝產物胺基酸含量之分析方法....42.1 紅麴產物水萃物中胺基酸之萃取方法.....................42.2 胺基酸 PITC 衍生化之 HPLC 分析條件...................43.3 胺基酸 OPA 衍生化之 HPLC 分析條件....................43六節 建立紅麴次級代謝產物 monacolin K、citrinin、monascin 及 ankaflavin 之同步分析方法..............................44.1 紅麴產物酒萃物之萃取方法.............................44.2 標準液之配製.........................................44.3 紅麴中 monacolin K lactone form、acid form 及 citrinin 濃度之同步分析方法........................................44.4 黃色素 (monascin、ankaflavin) 濃度之測定.............45七節 以反應曲面法探討最適培養條件之實驗設計............45八節 降血壓功效評估....................................46.1 紅麴發酵產物體外 ACEI 活性之測定.....................46.2 紅麴發酵產物降血壓之體內功效性評估試驗...............49四章 結果與討論........................................53一節 紅麴發酵產物之代謝物分析...........................53.1 建立紅麴酒萃物次級代謝物 monacolin K、citrinin、monascin 與 ankaflavin 之同步分析方法..............................59.2 建立紅麴水萃物 GABA 之分析方法.......................67二節 紅麴發酵產物之培養................................72.1 麴盤與填充瓶式培養方式對紅麴代謝產物 GABA、citrinin 生成之影響....................................................72.2 不同基質對紅麴代謝產物 GABA 生成之影響...............72.3 培養條件對於紅麴代謝產物 GABA 生成之影響.............78.4 三因子三階層反應曲面法 (Response surface methodology) 之試驗......................................................84.5 厭氧處理促進紅麴生產 GABA 之影響.....................86三節 紅麴米及紅麴山藥降血壓功效評估...................100.1 紅麴米與紅麴山藥之基本成分分析......................100.2 紅麴米與紅麴山藥之體外 ACEI 活性比較................100.3 試驗動物體重變化....................................101.4 試驗動物之單一餵食後 24 小時內收縮、舒張血壓及心律短期變化.......................................................101.5 試驗動物餵食8週後之收縮、舒張血壓及心律長期變化.....102.6 基本安全性..........................................103.7 弓動脈組織切片染色..................................104五章 結論.............................................117考文獻.................................................120次 1-1 紅麴菌之生活史......................................4 1-2 紅麴色素之結構......................................6 1-3 Citrinin 之結構式..................................19 1-4 M. ruber 合成citrinin 及紅色色素之途徑.............21 1-5 紅麴菌中 citrinin 之生成途徑.......................22 1-6 (A) OPA 衍生物之螢光全波長圖 (B) PITC 衍生物之 UV-VIS 全波長圖..................................................27 1-7 ACE 調節血壓之機制圖...............................30 1-8 非侵入式血壓計.....................................35 2-1 研究大綱...........................................37 4-1 李等同步分析方法中 monacolin K lactone form 和 monascin..................................................56 4-2 紅麴產物中 monascin 及 ankaflavin 之同步分析方法圖譜 ..........................................................57 4-3 UV 全波長吸收圖....................................58 4-4 紅麴發酵產物中monacolin K lactone form、monacolin K acid form、citrinin、monascin 及 ankaflavin 之同步分析方法圖譜........................................................59 4-5 紅麴產物質譜圖.....................................60 4-6 Monacolin K acid form之 HPLC 標準檢量線............61 4-7 Monacolin K lactone form之 HPLC 標準檢量線.........62 4-8 Citrinin 之 HPLC 標準檢量線........................63 4-9 Monascin 之 HPLC 標準檢量線........................64 4-10 Ankaflavin 之 HPLC 標準檢量線.....................65 4-11 紅麴米水萃物以 PITC 衍生化後之 HPLC 圖譜..........69 4-12紅麴米水萃物以 OPA 衍生化後之 HPLC 圖譜............70 4-13 GABA 之 HPLC 標準檢量線...........................71 4-14 以不同容器培養紅麴次級代謝物 citrinin、GABA 生成之影響........................................................73 4-15 紅麴發酵產物......................................75 4-16 紅麴發酵產物......................................76 4-17 GABA 含量每日變化.................................83 4-18 厭氧處理起始時間、厭氧處理時間與乙醇濃度對紅麴山藥固態發酵時 citrinin 生成量之反應曲面圖......................89 4-19 厭氧處理起始時間、厭氧處理時間與乙醇濃度對紅麴山藥固態發酵時 GABA 生成量之反應曲面圖..........................90 4-20 厭氧處理起始時間、厭氧處理時間與乙醇濃度對紅麴山藥固態發酵時 GABA 生成量之反應曲面圖..........................91 4-21 紅麴山藥固態培養之 citrinin 與 GABA 生成量之反應曲面交疊圖 (Ethanol conc. = 3%)...............................92 4-22 厭氧處理起始時間、厭氧處理時間與乙醇濃度對紅麴山藥固態發酵時 GABA 生成量之反應曲面圖..........................93 4-23 厭氧處理起始時間、厭氧處理時間與乙醇濃度對紅麴山藥固態發酵時 GABA 生成量之反應曲面圖..........................94 4-24 紅麴山藥固態培養之 citrinin 與 monascin 生成量之反應曲面交疊圖 (Ethanol conc. = 1%)...........................95 4-25 厭氧處理起始時間、厭氧處理時間與乙醇濃度對紅麴山藥固態發酵時 GABA 生成量之反應曲面圖..........................96 4-26 厭氧處理起始時間、厭氧處理時間與乙醇濃度對紅麴山藥固態發酵時 GABA 生成量之反應曲面圖..........................97 4-27 紅麴山藥固態培養之 citrinin 與 monascin 生成量之反應曲面交疊圖 (Ethanol conc. = 1%)...........................98 4-28 紅麴發酵產物水萃物 ACEI 活性 HPLC 圖譜...........106 4-29 單一次餵食紅麴產物後收縮血壓變化.................1084-30單一次餵食紅麴產物後舒張血壓變化...................109 4-31單一次餵食紅麴產物後心律變化......................110 4-32 餵食紅麴產物八週間收縮血壓變化...................111 4-33餵食紅麴產物八週間舒張縮血壓變化..................112 4-34 餵食紅麴產物八週間心律變化.......................113 4-35 實驗動物弓動脈切片鏡檢結果.......................116次 1-1 紅麴米、米之化學成分比較...........................24 3-1 固態發酵反應曲面法之三因子-三階層條件..............47 3-2 固態發酵之三因子-三階層之中心旋轉組合設計..........48 4-1 市售產品 MKL、MKA、CT、M 及 AF 含量................66 4-2 以填充瓶或麴盤培養對紅麴代謝物 GABA 及 citrinin 生成之影響......................................................74 4-3 不同品系山藥作為培養基質對紅麴代謝物 GABA 生成影響之比較........................................................77 4-4 不同之乙醇添加量對於 M. purpureus NTU 568 生成 GABA 之影響......................................................79 4-5 不同 pH 值對於 M. purpureus NTU 568 生成 GABA 之影響 ..........................................................80 4-6 不同培養時間對於 M. purpureus NTU 568 生成 GABA 之影響 ..........................................................81 4-7 不同厭氧處理時間對於 M. purpureus NTU 568 生成 GABA 之影響......................................................82 4-8 反應曲面法之因子與階層.............................87 4-9 紅麴代謝產物之三因子三階層反應曲面法實驗過程.......88 4-10 不同培養條件對紅麴產物 GABA、citrinin、monascin 及 ankaflavin 之影響.........................................99 4-11 紅麴米及紅麴山藥基本成分組成.....................105 4-12 各組實驗動物之平均體重...........................107 4-13 紅麴產物對實驗動物血清中 GOT 與 GPT 之影響.......114 4-14 紅麴產物對實驗動物血清中 CPK 與 電解質之影響.....11