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研究生:王志傑
研究生(外文):Jyh-Jye Wang
論文名稱:紅麴菌二級代謝產物之生產與應用研究
論文名稱(外文):Studies on the fermentative production and application of secondary metabolites of Monascus
指導教授:潘子明潘子明引用關係
指導教授(外文):Tzu-Ming Pan
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:微生物與生化學研究所生物工業組
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2003
畢業學年度:92
語文別:中文
論文頁數:197
中文關鍵詞:紅麴菌紅麴米橘黴素monacolin Kγ-胺基丁酸反應曲面法山藥抗氧化能力
外文關鍵詞:MonascusRed mold riceCitrininMonacolin KGABAResponse surface methodologyAntioxidant activityFree radical
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紅麴菌是中國傳統使用之醱酵菌株,其培養物或醱酵產品可被當作一種天然的機能性膳食補充品 (natural dietary supplement)。紅麴菌之二級代謝產物有monacolin K 及γ-胺基丁酸 (γ-aminobutyric acid, GABA) 等,其分別被使用為降低膽固醇藥物或降血壓之物質。橘黴素 (citrinin) 是紅麴菌的另一種代謝產物,由於其為黴菌毒素,也因此減低人類對紅麴醱酵產物之接受度。本研究利用Monascus purpureus NTU 601 紅麴菌株進行固態醱酵培養,並探討其最適培養條件。在外加碳源中,當培養基中添加 0.5% ethanol,則紅麴米中 citrinin 之生成量可由 813 ppb 降至 561 ppb,而 monacolin K 之含量可由 136 mg/kg 提高至 383 mg/kg,GABA 的含量由 1060 mg/kg 提高至 7450 mg/kg 。另外再以反應曲面法 (response surface methodology, RSM) 探討最適培養條件。其結果顯示當溫度在 30℃添加 0.3% ethanol 及 145 mL 加水量可使 monacolin K 之生成量由136 mg/kg提高至 530 mg/kg,GABA含量則由1060 mg/kg提高至 5000 mg/kg,另外citriinin的生成量則由813 ppb降至 460 ppb。
由於 citrinin 是一種黴菌毒素,並具有肝毒與腎毒效應 (hepatoxic and nephrotoxic effect) 因此減低人類對紅麴之接受度。在本研究中我們利用citrinin對Bacillus subtilis具有抗菌活性 (antibacterial activity),並於菌落外圍形成抑制環 (inhibition zone) 之原理,設計一種簡單且快速的方法以篩選出低 citrinin 生成的變異株。其中M. purpureus N301變異菌株生成的 citrinin 為 230 ppb,較母株 (parent strain) 減少 50 % ,而 monacolin K 為 481 mg/kg,仍保有 90% 的產量。M. purpureus N310變異株生成的 citrinin 為 275 ppb,較母株減少 40% ,而 monacolin K 為 526 mg/kg,則無太大改變。此外兩變異菌株之 γ-aminobutyric acid 含量皆在 5000 mg/kg 左右,與母株之生成量相同。因此顯示此方法可作為篩選 citrinin 低生成之紅麴菌株的方法。
自由基與疾病有著相互密切之關係,而抗氧化劑可消除過多的自由基以確保人體正常之生理代謝。本研究以米和山藥 (dioscorea) 為基質,以紅麴菌進行固態醱酵後探討其產物之抗氧化特性。研究中以清除α,α-diphenyl-β-picrylhydrazyl (DPPH) 自由基能力之測定法篩選具抗氧化能力之紅麴菌株。結果以在來米為醱酵基質進行固態培養後之產物,以Monascos sp. NTU801及M. anka M-13清除自由基的能力最佳,其清除自由基之百分率分別為82.5% (0.10 g rice/mL) 及 85.2% (0.15 g rice/mL)。以山藥為醱酵基質進行固態培養所得產物,以M. purpureus NTU601及M. anka M-13之清除能力最佳,其清除自由基之百分率分別為73.9% (0.20 g dioscorea/mL) 及 67.1% (0.25 g dioscorea/mL)。由於評估抗氧化的機制有多種,因此本研究再以共軛雙烯法 (formation of conjugated diene)、清除超氧陰離子能力之測定法 (SOD-like activity)、螯合鐵能力之測定法 (chelating ability on iron(II)) 及還原能力 (reducing power) 之測定法分別評估紅麴菌醱酵產物之抗氧化形式與能力,證實紅麴確實有不錯之抗氧化能力,因此可提供為傳統食物中抗氧化機能成份。另外;本研究發現紅麴菌以山藥為基質經醱酵後可產生大量的monacolin K。
至於紅麴產品之新應用。本研究探討於雞隻飼糧中添加不同濃度之紅麴米 (red mold rice) 對蛋黃與血清中膽固醇及血清中三酸甘油脂、高密度蛋白 (high density lipoprotein, HDL) 及低密度脂蛋白 (low density lipoprotein, LDL) 之影響。研究中取48週之海蘭 (Hy-line) 蛋雞48隻,並區分四組每一處理組12隻,試驗除對照組外另分三組分別添加2.0%, 5.0%及8.0%紅麴米 (monacolin K分析濃度分別為0.0145%, 0.035%及0.056%) 進行六週試驗,實驗期間每日紀錄蛋重、產蛋率並於每週採蛋測定其蛋黃中膽固醇含量及採血測定其血清中膽固醇、三酸甘油脂、HDL及LDL含量。實驗結果顯示,雞蛋中膽固醇含量各處理明顯低於控制組 (0%: 194±8, 2%: 167±4, 5%: 168±9, 8%: 183±7 mg/egg; p<0.05),另外血清中三酸甘油脂 (0%: 1494±178, 2%:1280±174, 5%: 1189±248, 8%: 1381±218 mg/dL; p<0.05) 及LDL (0%: 36.8±5.5, 2%: 32.2±7.9, 5%: 30.0±4.3, 8%: 28.8±4.1 mg/dL; p<0.05) 亦有下降之現象但是對HDL則無明顯之改變 (0%: 36.0±3.9, 2%: 36.2±5.3, 5%: 33.1±3.6, 8%: 31.4±4.2 mg/dL; p<0.05)。此外於雞隻飼糧中添加紅麴可抑制血清脂質過氧化所產生之malondialdehyde (MDA) (0%: 27.4±0.5, 2%: 25.6±0.7, 5%: 24.3±0.5, 8%: 23.6±0.4 μM; p<0.05)。另外;利用HPLC分析雞蛋中並無monacolin K之殘留。
Monascus, one of the Chinese traditional fermentation fungus, was used to be the natural dietary supplement. The metabolic product of Monascus species i.e. monacolin K and γ-aminobutyric acid (GABA) were individually approved to be a cholesterol-lowering drug and a hypotensive agent. Citrinin, another secondary metabolites of Monascus species, is toxic to human. So the receivability of monascus product is restricted. In this study, Monascus purpureus NTU 601 was fermented in the solid culture medium that contained extra chemical compounds. While added 0.5% ethanol, the production of citrinin would be decreased from 813 ppb to 561 ppb, monacolin K increased from 136 mg/kg to 383 mg/kg, and GABA increased from 1060 mg/kg to 7450 mg/kg. Optimum culture condition was determined by response surface methodology (RSM) using a design of three factors (temperature, the amount of water and ethanol added) and three levels. The optimum culture conditions were determined as follows: temperature, 30℃; water added, 145 mL; ethanol, 0.3%. The production of monacolin K would be increased from 136.03 mg/kg to 530 mg/kg; GABA from 1060 mg/kg to 5000 mg/kg. Citrinin would be decreased from 813 ppb to 460 ppb.
Since citrinin is a mycotoxin and has nephrotoxic and hepatoxic effect, thus it has diminished the acceptance of red mold rice by people. In this research, citrinin was used the antibacterial activity of Bacillus subtilis and inhibition zone around the outside of Monascus strain to design a simple and quick selection method for mutant strains with low citrinin production. M. purpureus N301 only produced 230 ppb citrinin, and was 50% less than the parent strain; monacolin K was 481 mg/kg while maintained 90% productivity. M. purpureus N310 produced 275 ppb citrinin, and was 40% less than the parent strain; monacolin K was 526 mg/kg, and showed no significant changes. The GABA content of two strains was 5000mg/kg, and was similar to the parent strain. The results showed the method could be used to select red mold rice with low citrinin production.
Free radicals are closely related to illness, and antioxidant activity can get rid of excess radicals to ensure the regular metabolism of human bodies. This study discusses the antioxidant activity produced using both rice and dioscorea through fermentation of Monascus species. The study uses α,α-diphenyl-β-picrylhydrazyl (DPPH) free radical-scavenging method to screen the fermented rice and dioscorea that possesses antioxidant activity. The result shows that among the materials produced by rice as fermentation substrate, M. sp. NTU801 and M. anka M-13 have the best ability to scavenge the radicals at percentage of 82.5% (0.10 g rice/mL) and 85.2% (0.15 g rice/mL), respectively. Among the materials produced by dioscorea as fermentation substrate, M. purpureus NTU601 and M. anka M-13 have the best scavenging activity at the percentage of 73.9% (0.20 g dioscorea/mL) and 67.1% (0.25 g dioscorea/mL), respectively. In addition, the methods of which formation of conjugated diene form, superoxide dismutase-like (SOD-like) activity, chelating activity on iron (II), and reducing power were used to evaluate the formation and ability of antioxidant activity. It was found that fermentated rice and dioscorea using Monascus species possessed considerable antioxidant ability to provide antioxidant ingredients in traditional foods. In addition, this work concluded that discorea was the optimum source for cultivating Monascus species because it resulted in the highest productivity of monacolin K.
Monacolin K is a secondary metabolite of Monascus species, and has effect on reducing cholesterol level. The objective of this study was to investigate the effect of red mold rice supplementation with different concentration on egg quality, laying performance, serum cholesterol, serum triglyceride, high density lipoprotein (HDL), low density lipoprotein (LDL), serum lipid peroxidation and egg yolk cholesterol in Hy-line laying hens. The research studied 48 Hy-line laying hens of 48 weeks old by dividing them into 4 groups. Except for the control group, the feed for three other groups contained 2.0%, 5.0% and 8.0% red mold rice (monacolin K concentration is 0.0145%, 0.035% and 0.056%). The experiment lasted for 6 weeks. During this period, egg weight and egg production were recorded everyday, and cholesterol concentration, triglyceride, HDL, and LDL in serum were measured weekly as well. The result showed that cholesterol level in eggs produced by experimental groups was lower than that of control group (0%: 194±8, 2%: 167±4, 5%: 168±9, 8%: 183±7 mg/egg; p<0.05), the triglyceride (0%: 1494±178, 2%: 1280±174, 5%: 1189±248, 8%: 1381±218 mg/dL; p0.05) and LDL level (0%: 36.8±5.5, 2%: 32.2±7.9, 5%: 30.0±4.3, 8%: 28.8±4.1 mg/dL; p<0.05) were also significantly lowered in the experimental groups. However, HDL level did not show significant change for both control and experimental groups (0%: 36.0±3.9, 2%: 36.2±5.3, 5%: 33.1±3.6, 8%: 31.4±4.2 mg/dL; p<0.05). Besides, adding red mold rice also helps on inhibiting production of malondialdehyde (MDA) in serum lipid oxidation (0%: 27.4±0.5, 2%: 25.6±0.7, 5%: 24.3±0.5, 8%: 23.6±0.4 μM; p<0.05). HPLC analysis showed that no monacolin K residues were detected in egg yolk and albumen extracts from hens fed red mold rice.
目 錄
目錄……………………………………………………………………………….. Ⅰ
縮寫表……………………………………………………………………..……… Ⅲ
圖次…………………………………………………………………………..…… Ⅴ
表次………………………………………………………………………………. Ⅶ
中文摘要………………………………………………………………………….. Ⅸ
英文摘要………………………………………………………………………….. ⅩⅡ
第一章 緒言………………………………………………………………..…… 1
第二章 文獻回顧……………………………………………………………….. 4
第一節 古今有關紅麴功效之研究及記載………………………………… 5
第二節 紅麴之菌種特性………………………………………………….. 6
第三節 紅麴之製作技術……………………………………………………. 7
第四節 紅麴在傳統食品上的應用…………………………………………. 10
第五節 紅麴生產之高價值二級代謝產物…………………………………. 17
第六節 Monacolin K 與 GABA 之研究…………………………………. 22
第七節 紅麴中的黴菌毒素─Citrinin…………………………………..…. 37
第八節 紅麴之抗氧化性質之探討…………………………………………. 46
第九節 Monacolin K對蛋雞體內與雞蛋中膽固醇之調控……………….. 51
第十節 紅麴專利現況………………………………………………………. 57
第三章 材料與方法……………………………………………………….…….. 64
第一節 菌株之培養………………………………………………………… 65
第二節 儀器…………………………………………………………………. 67
第三節 藥品…………………………………………………………………. 68
第四節 固態培養方法與條件……………………………………………… 69
第五節 Citrinin、monacolin K及GABA分析方法……………………… 71
第六節 培養環境調控對二級代謝產物生成之影響………………………. 75
第七節 利用變異法篩選低citrinin生成紅麴菌株並比較二級代謝產物
之生成量………………………………………………………….. 79
第八節 體外法評估紅麴米及山藥抗氧化特性之研究………………….. 80
第九節 蛋雞飼糧中添加紅麴米對雞蛋及血清中膽固醇之影響………... 82
第四章 結果與討論……………………………………………………………. 94
第一節 Citrinin、monacolin K與GABA分析方法之建立…………….. 95
第二節 培養條件對二級代謝產物生成之影響………………………….. 107
第三節 以反應曲面法 (response surface methodology, RSM) 探討
最適培養條件組合……………………………………………….. 114
第四節 利用變異法篩選低citrinin生成紅麴菌株………………………. 125
第五節 體外法評估紅麴米及紅麴山藥抗氧化特性…………………….. 134
第六節 蛋雞飼糧中添加紅麴米對雞蛋及血清中膽固醇之影響……….. 146
第五章 結論……………………………………………………………………. 155
第一節 citrinin、monacolin K與GABA分析方法之建立……………… 156
第二節 培養條件對二級代謝產物生成之影響………………………….. 156
第三節 利用變異法篩選低citrinin生成紅麴菌株………………………. 158
第四節 體外法評估紅麴米及山藥抗氧化特性………………………….. 160
第五節 蛋雞飼糧中添加紅麴米對雞蛋及血清中膽固醇之影響……….. 161
第六章 參考文獻……………………………………………………………… 164
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