臺灣博碩士論文加值系統

本研究以山藥為基質進行固態及液態培養，培養過程中藉控制 pH 值、加水量與添加低濃度的乙醇來降低 Monascus 生產紅麴時 citrinin 的生成量，並期望提高 monacolin K 生成量。同時比較玻璃缸與麴盤對紅麴次級代謝物 citrinin、monacolin K 生成之影響，以及不同品系山藥、不同培養條件、固態或搖瓶液態培養下紅麴次級代謝物生成量的不同。結果顯示，使用淮山藥為基質，利用 Monascus purpureus NTU 568 菌株以麴盤進行固態培養時有較好的效果，其 monacolin K 可達到 25,566 mg/kg，citrinin 之生成量為 3,166 μg/kg。而培養溫度、外添加水量與乙醇濃度對於固態發酵之紅麴山藥中 monacolin K 與 citrinin 的生成均有顯著影響。應用反應曲面法 (Response surface methodology, RSM) 進行最適培養條件的探討，結果當固態發酵之加水量 200 mL、溫度 34℃、乙醇濃度 0.53% 時可生成 39,800 mg/kg 之 monacolin K，且 citrinin 之生成量為 3,350 μg/kg。
本研究進一步以最適發酵條件生產高 monacolin K 產量與抗氧化效果佳的紅麴山藥，探討降血脂與抑制動脈粥狀硬化的效果。以高膽固醇倉鼠作為試驗動物，評估對血液總膽固醇、高密度脂蛋白膽固醇與低密度脂蛋白膽固醇濃度，以及對血漿中總抗氧化力與抗氧化酵素活性之影響。結果顯示，於不同劑量之山藥發酵紅麴所餵食之動物組，其血液中總膽固醇及低密度脂蛋白膽固醇濃度均顯著下降，高密度脂蛋白膽固醇濃度則無顯著差異。而血漿中總抗氧化狀態之測定，過氧化氫酶與超氧歧化酶活性分析，其結果均優於正常飲食控制組，並且有顯著差異。顯示山藥發酵紅麴對於降血脂以及抗粥狀動脈硬化具有良好的效果，應用在人體抗粥狀動脈硬化頗具潛力。

In this study, we used dioscorea as substrate to culture Monascus purpureus NTU 568 under solid and submerged condition. In addition, various dioscorea species, culture conditions (cultural temperature, pH value and extra ethanol) and culture apparatus (koji disk and glass jar) were selected to investigate the effect on monacolin K and citinin production. In the result, koji disk and Dioscorea batatas were chosen as the optimal culture apparatus and dioscorea species for high monacolin K production and low citrinin formation, respectively. In addition, cultural temperature, pH value and extra ethanol would significantly influence monacolin K and citrinin production of M. purpureus NTU 568 under solid dioscorea fermentation, so they were chosen as the factors of the response surface methodology (RSM) experiment in order to investigate the optimal culture conditions. From the result of statistical figure, we can gain the high monacolin K production at 39,800 mg/kg and low citrinin production at 3,350 μg/kg from red mold dioscorea when the culture conditions are as following: water added (200 mL), 37oC, and 0.53% ethanol.
This study also used jar fermentor to investigate the reason why dioscorea would stimulate monacolin K formation of Monascus species. In the result, the pH value of dioscorea powder media would retain at 3.0 during Monascus fermentation. When we used the acidic rice powder media (pH 3.0) as the substrate to fermentation, the monacolin K production would significantly increased. Therefore we suggested that the pH value of culture condition at 3.0 is one of the most important reasons why dioscorea stimulated monacolin K formation. Importantly, the dry cell weight would be increased when the pH value was adjusted at 3.0 as compared with other pH value. In the mechanism of dioscorea stimulating monacolin K formation, we can presume dioscorea controlled the pH value of culture condition near 3.0, which would further stimulate the growth of Monascus and monacolin K accumulation.
This study further uses the optimal Monascus fermented dioscorea with high monacolin K production to investigate the effect of hypolipidemic, anti-atherosclerosis and anti-oxidation in a hamster model of hyperlipidemia. In addition, plasma cholesterol, high density lipoprotein (HDL) and low density lipoprotein (LDL) would be the evaluative marker for hypolipidemic effect. As regards the evaluation of anti-atherosclerosis and anti-oxidation in vivo, this study will assay the improvement effect in total antioxidant status and the activity of anti-oxidative enzyme.
The results showed that the plasma level of total cholesterol and LDL were decreased significantly in the Monascus fermented dioscorea feeding group compared to the normal group. It also indicates that Monascus fermented dioscorea would perform a potent effect on reducing the risk of hypolipidemic via the increase of total antioxidant status (TAS), catalase and superoxide dismutase activity, and which are superior to the control significantly. Therefore, these results suggest that the antioxidant and antiatherogenic effects of Monascus fermented dioscorea can be useful in the prevention of the atherosclerotic formation.

目錄

目錄 I
縮寫表 IV
圖次 V
表次 VII
中文摘要 VIII
英文摘要 IX

第一章 前言 1
第一節 紅麴之菌種特性 1
第二節 紅麴之製作技術 3
第三節 紅麴生產之高價值二級代謝產物及其功效 5
第四節 山藥之背景與簡介 13
第五節 高血脂症及粥狀動脈硬化與自由基之關係 23
第六節 研究動機與目的 24
第二章 材料與方法 26
第一節 菌株之培養 26
第二節 儀器 27
第三節 藥品 29
第四節 固態培養方法與條件 30
第五節 搖瓶與發酵槽之液態培養 32
第六節 Monacolin K 與 citrinin 之分析方法 34
第七節 以反應曲面法探討最適培養條件之實驗設計 35
第八節 紅麴山藥改善高膽固醇與動脈粥狀硬化倉鼠模式之評估 40
第九節 預防動脈粥狀硬化之評估方法 45
第三章 結果與討論 47
第一節 山藥發酵之條件探討 47
1.1 Citrinin與monacolin K之分析 47
1.2 玻璃缸與麴盤對紅麴次級代謝物 citrinin、monacolin K生成之影響 47
1.3 以不同品系山藥作為基質對紅麴次級代謝物 citrinin、monacolin K生成之影響 47
1.4 培養條件對於紅麴次級代謝物生成之影響 51
1.4.1 培養溫度對於紅麴次級代謝物生成之影響 ….…51
1.4.2 pH值對於紅麴次級代謝物生成之影響 51
1.4.3 加水量對於紅麴次級代謝物之影響 51
1.5 反應曲面法 (Response surface methodology, RSM) 之試驗 56
1.5.1 固態發酵各因子在反應曲面法對於次級代謝物的影響 57
1.5.2 液態發酵各因子在反應曲面法對於次級代謝物的影響 57
1.6 山藥促進紅麴生產 monacolin K 之因素探討 63
1.6.1 紅麴菌株於山藥與在來米粉水溶液發酵對次級代謝物之影響 63
第二節 山藥發酵紅麴降血脂功效之評估 69
2.1 試驗動物體重與攝食量之變化 69
2.2 試驗動物之血液中總膽固醇濃度變化 69
2.3 試驗動物血液中三酸甘油酯濃度之變化………………………................73
2.4試驗動物血液中 HDL-C、LDL-C 濃度之變化…………………………..73
第三節 預防動脈粥狀硬化之評估 76
3.1 抗氧化酵素之活性分析 76
3.1.1 血液中 catalase 之活性分析 76
3.1.2 肝臟中 catalase 之活性分析 76
3.13 血液中 SOD 之活性分析 ..79
3.1.4 血漿中總抗氧化力分析 ..79
3.1.5餵食不同量紅麴對於倉鼠肝臟中脂質過氧化程度之影響…….……….....82
3.1.6餵食不同量紅麴對於倉鼠血液中脂質過氧化程度之影響…….……….....82
3.1.7餵食不同量紅麴對於倉鼠胸主動脈中脂質堆積程度之影響……………..83
第四章 結論 87
第五章 參考文獻 91

圖次

圖 1-1 紅麴菌之生活史 (蘇等，1970) 2
圖 1-2 八種紅麴色素之化學結構 6
圖 1-3 紅麴米中 monacolin 之結構 9
圖 1-4 研究流程 25
圖3-1 Citrinin 之 HPLC 檢量線 48
圖3-2 Monacolin K 之 HPLC 檢量線 49
圖3-3 加水量、培養溫度與乙醇濃度對紅麴山藥固態發酵時 monacolin K 生成量之反應曲面圖 58
圖3-4 加水量、培養溫度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 59
圖3-5 紅麴山藥固態培養之 citrinin 與 monacolin K生成量之反應曲面交疊圖 (water added = 200 mL) 60
圖3-6 乙醇濃度、pH 值與山藥基質濃度對紅麴山藥液態發酵時次級代謝物 citrinin 生成量之反應曲面圖 61
圖3-7 乙醇濃度、pH 值與山藥基質濃度對紅麴山藥液態發酵時 monacolin K 生成量之反應曲面圖 62
圖3-8 乙醇濃度、pH 值與山藥基質濃度對紅麴山藥液態發酵時紅麴菌體乾重之反應曲面圖 65
圖3-9 紅麴之米與山藥固態發酵物示意圖 66
圖3-10 紅麴以山藥與在來米粉為基質之發酵槽液態培養結果 67
圖 3-11 餵食不同量紅麴對於倉鼠血液中 catalase 活性之影響 77
圖 3-12 餵食不同量紅麴對於倉鼠肝臟中 catalase 活性之影響 78
圖 3-13 餵食不同量紅麴對於倉鼠血液中 SOD 活性之影響 80
圖 3-14 餵食不同量紅麴對於倉鼠血漿中總抗氧化力之影響 81
圖 3-15 餵食不同量紅麴對於倉鼠肝臟中脂質過氧化程度之影響 84
圖 3-16 餵食不同量紅麴對於倉鼠血液中脂質過氧化程度之影響 85
圖 3-17 餵食不同量紅麴對於倉鼠胸主動脈中脂質堆積程度之影響 86

表次

表 1-1 紅麴米、米與山藥之化學成分比較 20
表 2-1 固態發酵反應曲面法之三因子-三階層條件 36
表 2-2 固態發酵之三因子-三階層之中心旋轉組合設計 37
表 2-3 液態發酵反應曲面法之三因子-三階層條件 38
表 2-4 液態發酵之三因子-三階層之中心旋轉組合設計 39
表 3-1 玻璃缸或麴盤培養紅麴次級代謝物 citrinin、monacolin K生成之影響 50
表 3-2 不同品系山藥作為培養基質對紅麴次級代謝物 citrinin、monacolin K生成影響之比較 52
表 3-3 不同溫度條件對於 M. purpureus NTU568 生成 monacolin K 與 citrinin 之影響 53
表 3-4 不同 pH 值對於 M. purpureus NTU568 生成 monacolin K 與 citrinin 之影響 54
表 3-5 不同之加水量對於 M. purpureus NTU568 生成 monacolin K 與 citrinin 之影響 55
表 3-6 紅麴菌株於於酸性米粉水溶液中培養後對次級代謝物之影響 68
表 3-7各組實驗動物之平均體重 70
表 3-8 各組實驗動物之每日平均飼料攝食量 71
表 3-9 餵食不同量紅麴對於倉鼠血液中總膽固醇濃度之影響 72
表 3-10 餵食不同量紅麴對於倉鼠血液中三酸甘油酯濃度之影響 74
表 3-11 餵食不同量紅麴對於倉鼠血液中 HDL-C 與 LDL-C 濃度之影響 75

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