Studies On The Screening, Induction And Fermentation Conditions Of Monascus Spp With High-Monacolin K

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...