【作者】 胡晓清；

【导师】 陈福生；

【作者基本信息】 华中农业大学 ， 微生物学， 2003， 硕士

【摘要】 从红曲样品中分离筛选得到一株Monacolin K产率较高（高效液相色谱法，HPLC）而桔霉素产率为0.11ng／g（酶联免疫吸附法，ELISA）的红曲霉菌株，编号为M12。以之为出发菌株，通过诱变筛选，获得一株Monacolin K产率有明显提高而桔霉素产率仅为0.13ng／g（ELISA）的突变株，编号为M12-69。对该突变株的液态和固态发酵条件进行初步优化研究。具体包括以下五个方面的内容： 第一，从红曲样品分离纯化获得红曲霉菌株25株，以这些菌株和从中科院购得4株红曲霉菌株，共29株作为菌种，制备得29个（固态）红曲样品。 第二，以上述29个红曲样品为实验材料，对红曲中桔霉素的（单向）薄层层析（STLC）条件进行了研究。结果显示，以甲苯：乙酸乙酯：甲酸（6：3：1）为展开剂，桔霉素的分离效果较好。按照该方法，对上述29个红曲样品中的桔霉素进行了分析测定，筛选得到6个桔霉素含量低于0.2μg／g的红曲样品及其相应菌株。 以上述6个低桔霉素含量的红曲样品为材料，对Monacolin K的双向薄层层析（DTLC）条件进行了研究。结果显示，以正己烷：乙酸乙酯：无水乙醚：甲酸（20：1：5：0.5）为第一展开剂，正己烷：氯仿：无水乙醚（30：1：5）为第二展开剂，Monacolin K的分离效果较好。以该方法对6个样品中的Monacolin K进行了分析测定，得到1个Monacolin K含量最高为225μg／g（DTLC）的红曲样品及其相应菌株。 为了更准确地判断该红曲样品桔霉素和Monacolin K含量，采用ELISA对桔霉素进行了分析测定，结果显示，该样品中桔霉素含量仅为0.11ng／g。同时，采用HPLC对Monacolin K进行了分析测定，Monacolin K含量为213μg／g。将该样品对应的菌株编号为M12。 通过上述研究，获得1株固态发酵产物Monacolin K产率为213μg／g（HPLC），桔霉素含量为0.11ng／g（ELISA）的红曲霉菌株M12。 第三，以M12为诱变出发菌株，采用紫外线、⁶⁰Co—γ射线、硫酸二乙酯为诱变剂对其进行处理。突变株的桔霉素产率采用STLC分析，MonacolinK采用DTLC分析，最后获得一株桔霉素产率低且MonacofinK产率有明显提高的突变株M12一69。以M12一69为菌种制备（固态）红曲，其MonacolinK产率达2143协9/9（HPLC），较出发菌株M12提高906.10%，而桔霉素产率仅为0.13 ng/g （ELISA），和M12相比，变化不大。 第四，根据Hawkswo曲和Pitt关于红曲霉的分类方法，对M12及M 12一69的培养物进行了形态观察及分类鉴定。观察发现，M12一69与M12相比有较大的形态差别，但二者均为丛毛红曲菌（物nascusPl’lo拟5 Sato）。 第五，对红曲霉突变菌株M12一69产MonacofinK的液态和固态发酵条件进行了初步优化研究。 首先，研究了碳源、氮源、起始pH值、培养温度及时间等因素对M12一69液态发酵的影响。结果显示，当培养基配方为:4%葡萄糖，0.4%谷氨酸钠，0.3%蛋白膝，0.3%硝酸按，0.5%磷酸二氢钾，豆芽汁配制，起始pH值为5;培养条件为:25℃，15or/min，摇床培养12天时，MonacolinK产率达275“g/而（D几C），桔霉素产率为0.18n留ml（ELIsA）。在上述培养基和培养条件下，以14L模拟发酵罐进行扩大培养，接种量为10%（v/v），通气量为6L/min，转速为200r/min，25℃，培养7天时，MonacolinK产率达280协9/1111（DTLC），此时桔霉素产率为o.20ng‘ml（ELISA）。 然后，研究了大米吸水量、培养温度及时间等因素对M12一69固态发酵的影响。当固态发酵条件为:大米1009，鼓皮19，灭菌，趁热打散冷却后，添加20ml液体培养基，摇匀，接种，25oC，培养18天时，MonaeolinK产率达2400;9/9（DTLC），桔霉素产率仅为。.13n留g（ELISA），与国内外报道的MonacolinK高产菌株相比，以M12一69制备的固态红曲中MonacolinK含量与报道的最高水平持平:更多还原

【Abstract】 A strain of Monascus spp(M12) which produces high Monacolin K(HPLC) and low citrinin was isolated and screened from Hongqu products. Through a series of mutagen tests, A mutant strain of Monascus spp(M 12-69) from Ml2 was acquired, which can produce much higher Monacolin K than M12, and almost same citrinin level as M12. The liquid and solid fermentation conditions of M12-69 were also studied.The research was accomplished by a five-step procedure.Ⅰ . 29 strains of Monascus spp were acquired, of which 25 strains were isolated from different Hongqu products and 4 strain were bought from Institute of Microbiology. Chinese Academic of Science. Then 29 Hongqu Samples were prepared with these strains.Ⅱ. A single direction thin layer chromatography(STLC) was researched in order to detect citrinin from above samples,.The results revealed that ethyll acetate : toluene : formic acid (6:3:1) was a good developer, then the citrinin from the 29 Hongqu samples above were detected, and 6 samples with citrinin less than 0.20 μg/g(STLC) and their strains were screened.After that, the 6 samples were analysed by a double directions thin layer chromatography (DTLC) with hexane: ethyll acetate: aether: formic acid (20:1 :5:0.5) as the first developer and hexane: chloroform: aether(30:l:5) as the second developer. And the sample with the highest Monacolin K content and its relative strains were acquired .The strain was numbered M12.Finally, Monacolin K and citrinin in the sample above were precisely detected again by ELISA and HPLC respectively. The results showed that the content of citrinin and Monacolin K from the Hongqu samples produced by M12 were 0.11 ng/g, 213 ,μg/g respectively.Ⅲ. Physical mutagen treatments including ultraviolet radiation and 60CO- γ- ray and chemical mutagen diethyl suifate were applied to the original strain (M12). A final mutant strain (M12-69) producing low citrinin and 1 higher Monacolin k was obtained. The solid state Hongqu sample was manufactured by the strain M12-69. The Monacolin K content in the sample was 2143 μg/g (HPLC) which represented an increase of 9-time above that of M12 and the citrinin content was only 0.13ng/g (ELISA), almost same level as Ml2.Ⅳ. Studies on morphological observation and categorization showed that there were several differences between the strain M12 and the mutant strain M12-69, but they bothbelong to Monascus Pilosus Sato, according to Hawksworth and Pitt.Ⅴ. The conditions of the liquid state and solid state fermentation that M12-69 produced Monaclin K were studied.First, the factors which had an influence on the liquid-state tunning of M12-69 producing Monaclin K were studied, including carbon source, nitrogen source, initial pH value, culture temperature and time. The results indicated that Monaclin K content was 275 μg/ml(DTLC) and citrinin content was 0.18ng/ml when the culture medium was as follows: 4% glucose, 0.4% soda glutamate, 0.3% peptone. 0.3% NH4NO3, 0.5% NaH2PO4, dissolved with bean sprouts, initial pH was 5 and the culture condition was as follows: 25 ℃, 150r/min, incubated on shaker, 12 days. Then the same culture medium and fermentation condition were also applied to amplified fermentation in the modular research fermentater with the volume of 14L. Other conditions were as follows: inoculation concentration 10%(v/v), aeration quantity 6L/min, rotational speed 200r/min. 25℃. After 7 days incubation, it’s detected that the content of Monaclin K was 280 μ g/ml(DTLC) and citrinin content was 0.20ng/ml(ELISA).Second, the factors which had an influence on the solid-state tunning of M12-69 were studied too. including the water amount of rice, culture temperature and culture time. The results indicated that the content of Monaclin K was 2400 μ g/g(DTLC) and the content of citrinin was only 0.13ng/g(ELISA) when the solid-state fermentation conditions were as follows, 100g rice that absorbed 50g water, wheat bran 1g, dispersed immediately after steriliztation. then 20ml bacteria-free liquid-state medium were a更多还原