The Changes Of Pigments And Citrinin Of PksCT-disrupted Strain And Structural Identification Of Two New Orange Pigments

Red kojic rice, which is the traditional fermented food in our country, hasmedicinal and edible double effect. There are many metabolic products fromMonascus sp., which are biological and therapeutic, such as antihypertensive,hypolipidemic, antibacterial, antioxidative and anticarcinogenic activities. However,due to the prensence of citrinin, the application of Monascus was limited. The studyfound that knock-out genes related to produce citrinin, can reduce the ability ofproducing citrinin.In this study, the production of citrinin and pigments changes of Monascusaurantiacus and its pksCT gene disruption mutant in submerged fermentationan andsolid fermentation were analyzed. An accurate HPLC method for the simultaneousdetermination of rubropunctatin and monascorubrin has been established. And twonew Monascus orange pigments were studied. The major findings were expounded asfollows:1The submerged fermentation was performed with Monascus aurantiacus pksCTgene disruption mutant PHDS26in YES, powder and powder inorganic saltsmedia.The production,citrinin and red pigment of PHDS26were detected byHigh-performance liquid chromatography (HPLC) and Ultraviolet spectrophotometryrespectively. The results showed that the production of citrinin by PHDS26wasdecreased by97.24%、97.02%and39.18%, respectively, compared with theoriginal strain. At the same time, its capacities of producing pigments in powdermedium was decreased by62.4%but increased by60.2%in powder inorganic saltsmedium. In solid-fermentation, the production of citrinin by PHDS26was decreasedby97.9%, and its capacities of producing pigments increased by4times, comparedwith AS3.4384.2In this study, we established and validated an accurate HPLC method for thesimultaneous determination of rubropunctaine and monascorubrin in red yeast rice.The optimal chromatographic condition is selected. A250×4.6mm I.D.,5μmSymmetry C18column (Waters) was used as the chromatography column, andwater/acetonitrile (75:25, v/v) was used as the isocratic elution. However, the columntemperature was5℃. The average recoveries of the method for Rubropunctain andMonascorubrin were96.4%and97.6%, respectively. The linear equations ofRubropunctain and Monascorubrin were Y=69.41X+7.49and Y=75.48X-2.29 respectively. The correlation coefficient of Rubropunctain was0.9999, andMonascorubrin was0.9994. The linear ranges of Rubropunctaine and Monascorubrinewere from0.25-50μg/mL, respectively; and the RSD were4.1%and5.0%,respectively. Monascus aurantiacus AS3.4384and its pksCT gene disruption mutantPHDS26were determined in this way. We found that the maximum production ofRubropunctaine and Monascorubrine by PHDS26were were increased by5.43and14.6times, respectively.3Two new orange pigments with similar yellow fluorescence spectra，reactionproducts of Rubropunctatin and Monascorubrin with methanol or d4-methanol, wereisolated from Monascus sp. by chromatography on a silica gel column and purified bysemi-preparative HPLC. Based on ESI-MS, ESI-MS/MS, NMR, COSY and HMBCanalyses, their structures were elucidated. The molecular formulas were C22H26O6andC24H30O6. The structures of the two new orange pigments, namedmonasphilol-methoxy A (MMA) and monasphilol-methoxy B (MMB), were similarto rubropunctatin and monascorubrin, respectively. However, the hydroxyl andmethoxyl groups in compounds MMA and MMB, respectively, were substituted forthe conjugated ketone carbonyl in rubropunctatin and monascorubrin.