【作者】 付海平；

【导师】 何煜波； 林亲录；

【作者基本信息】 湖南农业大学 ， 食品科学， 2004， 硕士

【摘要】 从1979年日本学者远腾章发现红色红曲霉（M. ruber）产生强降胆固醇物质Monacolin K之后，国内外学者掀起了降脂红曲霉的研究热潮，对红曲霉功能因子的分子结构和安全性等方面进行了广泛而深入的研究，它的药用价值开始逐渐得到人们的重视。1987年美国默沙东（MERCK）开发出新一代降胆固醇的药物美降之，随后美国和日本又开发出一系列的他汀类药物。但是我国对降脂红曲霉的研究相对落后，1996年我国才首次生产洛伐他汀原料药及其片剂。目前我国降胆固醇功能红曲霉的生产还是普遍存在产量偏低、生产成本偏高等问题。究其原因，一方面是培养工艺的不足，另一方面是缺乏优良的红曲霉菌株。因此进行红曲霉菌株的筛选研究具有非常重要的现实意义。本实验对高产Monacolin K的菌株进行了筛选，并对Monacolin K的检测条件，红曲霉M32菌株的发酵条件进行了研究。 试验结果如下： 1．建立了高效液相色谱法对Monacolin K进行定量分析。选用色谱柱：Kromasil C₁₈5um，250*46；柱温为35℃；检测波长238nm；流动相：V（甲醇）：V（水）=87：13，流速为1ml/min，样品经0.45um有机膜过滤后用HPLC测定。该方法对Monacolin K的测定具有良好的线性关系，重现性好，相对标准偏差和平均回收率分别为0.3896、99.23％。 2．从60个样品中分离到45株红曲霉，其中产Monacolin K的红曲霉菌株有9株，其中M32菌株产生Monacolin K最多，其余36株菌产生少量或不产生Monacolin K。 3．本实验对筛选的红曲霉菌株M32进行了菌落形态观察，并进行了红曲霉M32菌株的生理特性试验，结果表明，红曲霉M32菌株的生长温度范围为：25—35℃；孢子萌发和菌丝生长最适的起始pH范围为：4.0-6.5；而且其孢子耐酒精浓度能力较强，最高耐酒精浓度达14％。 4．通过单因素和正交实验确定了红曲霉M32液态发酵的培养基组成，还发现在培养基中添加适量硫酸镁、硫酸锌和玉米粉可以提高发酵液中Monacolin K的含量。对红曲霉M32菌株产Monacolin K的培养基进行了进一步优化，确定了其最佳的培养基配方：甘油90g/l，大米粉水解液20g/l，蛋白胨10g/l，硝酸钠5g/l，MgSO₄.7H₂O1g/l，ZnSO₄.7H₂O₂g/l，KH₂PO₄2.5g/1。最佳发酵条件为：培养温度：25℃，起始pH：5.0，500ml三角瓶装液量为200ml，摇床转速：150r/min，发酵培养12天。用最佳发酵培养基和最佳发酵条件进行深层液态发酵，最终发酵液中MonacolinK的产量为700一soomg/l。更多还原

【Abstract】 In 1979, A. endo in Japan found that M.rubber could produce Monacolin K , which is cholesterol synthesizing inhibitor. Researchers both in china and abroad have been making study of it since then. The molecular structure and the safety of physiological active materials were studied in details. Its value of medicine has been given especial attention to among researchers in the world. After MERCK developed Mevacor that can decrease the content of the cholesterol, a series of statins were developed. However, the study of Monascus of decreasing cholesterol falls behind relatively in our country. The source of Lovstatin was produced firstly in 1996. Up to now, reasons that the production of Monacolin K is low and operating costs is high in our country are the lack of Monacolin K Monascus strains and the lack of qualified industrial fermentation. Therefore, the isolation of Monascus that is over-production Monacolin K is very important. And the detection of Monacolin K and fermentation condition were studied. The results as follows:1. The detection methods of Monacolin K concent in Monascus products were investigated. The content of Monacolin K was determined by HPLC. Monacolin K was separated on a kromasil C18 column (5um,250 X4. 6mm) using methanol-water (87:13 V/V) as the mobile phase and flow rate 0. 6ml/min with detection at 238nm. Under such chromatographic condition, Monacolin K can be determined. The method demonstrated a good linear relationship , separation degree and repeatability. The average rate of recovery of Monacolin K was 99.23%, the relative standard deviation(RSD) was 0. 38%.2.45 strains of Monascus were isolated from 60 samples mainly from the red rice and so on. 9 strains were proved to produce Monacolin K. Among them, M32 can produced higher Monacolin K.3.In the paper, colony characteristics, optic microscopic observation thestrains and the biological features of the M32 Monascus strains were studied. Its growth temperature was 25-35C, the optimum original pH of spore budding and mycelium growth was 4. 0-6. 5, the resistance ability to ethanol was as high as 14%. An optimum medium was obtained through single factor tests and optimization tests. During the research, we found that the addition of 1g/1 MgSO4 and 2g/l ZnSO4 could improve the Monacolin K value. And we got the optimum medium: glycerine90g/l, rice meal20g/l, amylaselg/1,peptone10g/1, NaNO35g/l, MgSO41g/1, ZnS042g/l, KH2PO42.5g/l. The most moderate breeding condition for fermentation is: Temperature 25C,the original pH for medium is 5. 0, The volume of fermentation solution is 200ml in 500ml Erlenmeyer flash , the rocking bed velocity is 150r/min, fermentation time is 12 days. In submerged fermentation with optimum medium and optimum fermentation condition, The concentration of Monacolin K can be reached to 700-800mg/l.更多还原