The Analysis Of Several Secondary Metabolites Fermented By The Monascus Aurantiacus AS3.4384 And Its Gene Disrupted Mutants

Monascus-fermented rice?MFR?, also named as Hong Qu, is a commonly used traditional fermented food in China. And it is also famous as a mild folk medicine, which has the positive effect to depress blood pressure and blood-fat as well as the activity of antibacterial, antioxidant and anticancer etc. But the existence of citrinin in MFR greatly restricted its application. Fortunately, it was reported that the capacity of producing citrinin could be decreased by knocking out the genes involved in the biosynthesis of citrinin or changing the medium components. So in this study, we not only investigated the optimal extraction conditions for Monacolin K in MFR by using the response surface methodology, but also studied the secondary metabolites produced by the wild-type M.aurantiacus AS3.4384 and its gene disrupted strains in liquid fermentation. The main results were as follows:?1?When Monacolin K was detected by HPLC, the extract of red yeast rice fermented by pks CT-disrupted mutant was monascin corresponding to the peak of Monacolin K standard. And they could be seperated only when the mobile phase consisted of 52 % acetonitrile and 48 % water. Besides, 75 % acetonitrile and 25 % water could be acted as mobile phase to obtain a good separation and resolution when the functional red yeast rice was detected. In addition, the extraction conditions for Monacolin K were investigated by Response surface methodology, and their optimal extraction as following: ethanol concentration 67 %, the extracting time 65 min, temperature 43 ?, solid-liquid ratio 1:16.?2? The optimal cultivation medium were designed by Response surface methodology, which were as follows: 4.1 % rice powder, 0.64 % Na NO3, 0.05 % KH₂PO₄, 0.05 % K2HPO₄, 0.1 % Mg SO₄·7 H₂ O. In addition, the optimization of fermentation conditions for Monascus PHDS26 was also carried out to improve the yield of pigments and to reduce the yield of citrinin. And the best culture conditions were that the fermentation time 7 d, fungus age 48 h, the temperature 30 ?, medium volume 40 m L, initial p H 6.2 and inoculation amount 5 %. Under the above conditions, the yield of pigment reached up to 255.25 U/g, which was three times higher than that of unoptimized conditions. Besides, the yield of citrinin was decreased by 65.3 %.?3?Under the optimal conditions, the yield of red pigments produced by the wild type M.aurantiacus AS3.4384 was only 36.5 U/g and the yield of citrinin reached up to 8.61 ?g/m L. By contrast, the amount of red pigments fermented by the pks CT-disrupted mutant nearly increased by 6 times and the yield of citrinin decreased by 98.5%. What's more, the mutant could produce more components, such as the substance with m/z 425.3?51.091min?, Monasfluore A?56.025 min? and Monasfluore B?64.237 min?, Monascin?59.761min? and Ankaflavin?68.424 min?, Rubropunctatin?60.205 min? and Monascorabrin?69.689 min?. Compared with the original strain, the ctn G3-disrupted mutant could produce more yellow pigments,which was 2.8 times as high as the wild type strain. And the yield of red pigments was just increased by 4.875 U/g and the yield of citrinin was decreased by 23.6 %.?4? The output of secondary metabolites produced by Monascus PHDS26 varied in the medium containing different carbon source or nitrigen source. The yield of metabolites were much fewer when glucose was used as carbon source, especially the output of Monasflour A?11.578 min? and Monasflour B?13.470 min?. On the contrary, the yield of metabolites was significantly increased when peptone was selected as nitrigen source although the yield of Monascin?12.401 min?and Ankaflavin?14.154 min? was decreased.?5? The inorganic ions had a distinct effect on the secondary metabolites, especially the content of citrinin fermented by M.aurantiacus AS3.4384, Monascus PHDS26 and Monascus ctn G3. The ability of stimulating the synthesis of citrinin was Mg²⁺>Fe²⁺>Mn²⁺>Ca²⁺ in the fermentation by M.aurantiacus AS3.4384, and there was no growth in the medium containing Zn²⁺. During the fermentation process of pks CT-disrupted mutant, the output of citrinin was Fe²⁺>Zn²⁺>Ca²⁺ >Mn²⁺>Mg²⁺. And when ctn G3-disrupted mutant was fermented, the influence of inorganic ions on the synthesis of citrinin was Fe²⁺>Ca²⁺>Mg²⁺> Mn²⁺, no growth occured in the medium containing Zn²⁺.