红曲霉发酵及其生长趋向性的初步研究
摘要
Monacolin K是某些红曲霉菌株的次级代谢产物，对合成胆固醇的限速酶(HMG-CoA还原酶)有特异性的抑制作用，因此具有降血脂、降血糖、抗肿瘤等作用。本文以红曲霉为生产菌株，以提高Monacolin K产量以及红曲霉生长过程中的趋向性为主要研究目标。
1．实验确定了紫外分光光度计法检测Monacolin K的含量，确定最佳提取溶液为70％乙醇，最佳提取时间为8h，检测波长为236nm。该方法具有准确性高、重现性好、操作方便等优点。
2．通过单因素试验确定了红曲霉发酵生产Monacolin K的最佳碳源、氮源及其最适浓度，结果表明甘油和蛋白胨分别为最佳碳源和氮源，甘油最佳浓度范围为14％～16％，蛋白胨最佳浓度为3.0％。
3．红曲霉与酵母菌的混合培养与非混合培养的试验证明：两种培养条件下酵母菌对红曲霉的菌体量和Monacolin K的生成都有显著的促进作用。但是混合培养中酵母菌对红曲霉产物合成的促进作用优于非混合培养，可能原因是混合培养中酵母菌对红曲霉的作用更为直接。
4．研究分别添加三种诱导剂(酵母、酵母破壁液和酵母滤液)于红曲霉培养基中分别进行液态、固态和膜面培养对红曲霉代谢产物Monacolin K合成的影响。结果表明：液态培养中最佳诱导剂为酵母破壁液、固态培养中为酵母滤液、膜面中为酵母。
5．确定了红曲霉液态培养时破壁液的添加时间和添加量为分别为与红曲霉种子培养液同时接入和2.67％(v／v)，此时Monacolin K的产量达61.99mg／L。并且对添加破壁液后红曲霉的形态进行了观察，结果表明酵母破壁液对红曲霉孢子形态具有显著的影响。
6．探讨了不同营养源对红曲霉生长趋向性的影响，结果发现菌体对麦芽汁(提供碳源和氮源)培养基的趋向性好于葡萄糖(提供碳源)，最次为水(无碳源和氮源)。通过三组验证实验确定营养物也是红曲霉趋向性生长的必要条件。
Monacolin K is secondary metabolite of some Monascus, which is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis, thereby is effective in the therapy of hypercholesterolemia, besides, Monacolin K has recently been indicated as potential therapeutic agent for the treatment of various type of tumors.
In the essay M.ruber was considered as tool of production, in order to improving the productivity of Monacolin K and researching the trend growth of microorganism which close with nutrition.
1. Measure the content of Monacolin K by Uvspectrophotometer. From a series of experiments we draw a conclusion that use 70% ethanol as solution to extract Monacolin K 8hrs and 236nm is the maximal wavelength of Monacolin K in 70% ethanol solvent.
2. Factor test indicated that glycerol and peptone benefited obviously the synthesis of monacolin K. Furthermore we studied the optimal concentration of glycerol and peptone.
3. From co-culture and non-coculture of Mruber and Saccharomyces cerevisiae, we found that during the two culture condition Saccharomyces cerevisiae could increase cell mass and the yield of monacolin K, but the co-culture is better than the co-culture to improve the Monacolin K of M.ruber, the possible reason was during co-cultured the effect of Saccharomyces cerevisiae became direct.
4. During the liquid, solid and surface fermentation of Monascus, respectively adding Yeast lysate, Yeast and Yeast filtrate, indicated that the best inducer in liquid, solid, and surface culture was Yeast lysate, Yeast and Yeast. Furthermore, when Monascus cultivate in liquid method, adding Yeast filtrate in the beginning of Monascus fermentation and the quantity is 2.7% (v/v), the yield of Monacolin K reaches 61.93mg/L. Study on variance of Monascus biomass show Yeast filtrate could improve the biomass of Monascus and metabolize path, so increase the yield of Monacolin K.
5. In the test M.ruber was considered as tool of study, a series of experiment testified that atmosphere was essential condition of the trend growth of microorganism.
6. we research that different nutrition effect the trend growth of microoganism, the result was the best nutrition is malt extraction, secondly glucose, the worst
is water. Three experiments testified that nutrition was also essential condition of the trend growth of microorganism.
1．实验确定了紫外分光光度计法检测Monacolin K的含量，确定最佳提取溶液为70％乙醇，最佳提取时间为8h，检测波长为236nm。该方法具有准确性高、重现性好、操作方便等优点。
2．通过单因素试验确定了红曲霉发酵生产Monacolin K的最佳碳源、氮源及其最适浓度，结果表明甘油和蛋白胨分别为最佳碳源和氮源，甘油最佳浓度范围为14％～16％，蛋白胨最佳浓度为3.0％。
3．红曲霉与酵母菌的混合培养与非混合培养的试验证明：两种培养条件下酵母菌对红曲霉的菌体量和Monacolin K的生成都有显著的促进作用。但是混合培养中酵母菌对红曲霉产物合成的促进作用优于非混合培养，可能原因是混合培养中酵母菌对红曲霉的作用更为直接。
4．研究分别添加三种诱导剂(酵母、酵母破壁液和酵母滤液)于红曲霉培养基中分别进行液态、固态和膜面培养对红曲霉代谢产物Monacolin K合成的影响。结果表明：液态培养中最佳诱导剂为酵母破壁液、固态培养中为酵母滤液、膜面中为酵母。
5．确定了红曲霉液态培养时破壁液的添加时间和添加量为分别为与红曲霉种子培养液同时接入和2.67％(v／v)，此时Monacolin K的产量达61.99mg／L。并且对添加破壁液后红曲霉的形态进行了观察，结果表明酵母破壁液对红曲霉孢子形态具有显著的影响。
6．探讨了不同营养源对红曲霉生长趋向性的影响，结果发现菌体对麦芽汁(提供碳源和氮源)培养基的趋向性好于葡萄糖(提供碳源)，最次为水(无碳源和氮源)。通过三组验证实验确定营养物也是红曲霉趋向性生长的必要条件。
Monacolin K is secondary metabolite of some Monascus, which is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis, thereby is effective in the therapy of hypercholesterolemia, besides, Monacolin K has recently been indicated as potential therapeutic agent for the treatment of various type of tumors.
In the essay M.ruber was considered as tool of production, in order to improving the productivity of Monacolin K and researching the trend growth of microorganism which close with nutrition.
1. Measure the content of Monacolin K by Uvspectrophotometer. From a series of experiments we draw a conclusion that use 70% ethanol as solution to extract Monacolin K 8hrs and 236nm is the maximal wavelength of Monacolin K in 70% ethanol solvent.
2. Factor test indicated that glycerol and peptone benefited obviously the synthesis of monacolin K. Furthermore we studied the optimal concentration of glycerol and peptone.
3. From co-culture and non-coculture of Mruber and Saccharomyces cerevisiae, we found that during the two culture condition Saccharomyces cerevisiae could increase cell mass and the yield of monacolin K, but the co-culture is better than the co-culture to improve the Monacolin K of M.ruber, the possible reason was during co-cultured the effect of Saccharomyces cerevisiae became direct.
4. During the liquid, solid and surface fermentation of Monascus, respectively adding Yeast lysate, Yeast and Yeast filtrate, indicated that the best inducer in liquid, solid, and surface culture was Yeast lysate, Yeast and Yeast. Furthermore, when Monascus cultivate in liquid method, adding Yeast filtrate in the beginning of Monascus fermentation and the quantity is 2.7% (v/v), the yield of Monacolin K reaches 61.93mg/L. Study on variance of Monascus biomass show Yeast filtrate could improve the biomass of Monascus and metabolize path, so increase the yield of Monacolin K.
5. In the test M.ruber was considered as tool of study, a series of experiment testified that atmosphere was essential condition of the trend growth of microorganism.
6. we research that different nutrition effect the trend growth of microoganism, the result was the best nutrition is malt extraction, secondly glucose, the worst
is water. Three experiments testified that nutrition was also essential condition of the trend growth of microorganism.
引文
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[2] 王利彦，李红宝，车胜荣．HMG-CoA还原酶抑制剂新进展及其临床应用．华西药学杂志，1996，11(3)：168～169．
[3] 王伟平，夏福宝，吴思方．红曲霉发酵法生产Monacolin K研究进展．药物生物技术2002，9(5)：301～304．
[4] 朱文译，沈迪校．高胆固醇血症的现代治疗．美国医学会杂志中文版，1990，9(2)：105～112．
[5] 沈寅初．胆固醇合成抑制剂的研究开发．上海化工，23(11)：31～34．
[6] 张俊杰，赵树欣，赵华等．红曲霉及其生理活性物质．食品研究与开发，1998，19(2)：14～18．
[7] 刘奕琼，张灏，田丰伟．微生物降胆固醇作用研究进展．食品与机械，2003，1：6～9．
[8] 徐勇，李天德．HMG-CoA还原酶抑制剂的作用机制及临床应用前景．心血管病学进展，1995，15(5)：260～263．
[9] Endo A, M Kuroda, Y Tsujita. ML-236A, ML-236B and ML-236C, new inhibitors of cholesterogensis produced by Penicillium citrinum, J.Antibiotics 1976;29: 1346～1348.
[10] Brown AG, TC Smale, TJ King, et al. Crystal and molecular structure of compactin, a new antifungal metabolite from Penicillium brevicompactum. J C S Perkin I 1976; 1165～1170.
[11] Endo A, Monacolin K, a new hypocholesterolemic agent produced by a Monascus species[J]. J.Antibiotics. 1979;32: 852-854.
[12] Alberts AW, Chen J, KuronG, et al. Mevinolin, a highly potent competitive inhibitor of 3-hydroxy-3-methylgutaryl-coenzyme A reductase and a cholesterol lowering agent[J]. Proc Natl Acad.Sci.USA 1980;77: 3957～3961.
[13] Endo A. Dihydromonacolin L and monacolin.X, new metabolites those inhibit cholesterol biosynthesis[J]. Antibiotics 1985;3: 321～331.
[14] Endo A. Monacolins J L, New Inhibitors of cholesterol biosynthesis produced by Monascus ruber[J].Antibiotics 1985; 420～422.
[15] 郑土才．HMG-COA还原酶抑制剂类血脂调节药物的合成与应用．有机氟工业．1996，2：20～26．
[16] 李小燕．它汀类降脂的应用与开发．国外医药-合成药、生化药、制剂分册，2000，23(3)：135～138．
[17] 洪智勇，毛宁．红曲霉降胆固醇有效成分的研究．中国调味品，2002，6：6～8．
[18] 中国科学院微生物研究所编．常见与常用真菌．北京：科学出版社．1973．
[19] 张纪忠等．微生物分类学．上海：复旦大学出版社．1990．
[20] 中国科学院微生物研究所中国微生物菌种保藏管理委员会．中国菌种目录．北京：科学出版社．1983．
[21] 马美荣，王正祥，诸葛建．红曲有效生理活性物质的研究现状与进展．酿酒科技，1999,95(5)：26～29．
[22] Hadfield JR,Holker JSE, Stanway DN.the biosynthesis of fungal metabolites Part Ⅱ. The β-oxo-lactone equivalences in rubropunctatin and monascorubrin. J.Chem.Soc 1967; 19: 751～755.
[23] Kuromo M,Nakanishi K, Tada M.Biosynthesis monascorubrin and monascoflavin. Chem.Pharm.Bull.Tokyo 1963; 11: 358～362.
[24] 陈义光，彭德姣，田宏现．红曲及红曲霉的研究与应用．湖北农学院学报，2000，20(20)：188～191．
[25] 董明胜等．红曲霉抑菌作用的探讨．中国调味品，1991，1：12～15．
[26] 王雅芬，傅月华．红曲霉的有效生理活性物质及应用．杭州科技双月刊，2000，5：25-27．
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