Component Analysis Of Monascus Pigment And Preparation,Characterization Of Oil-soluble Derivatives

Monascus pigment is the only natural pigments metabolized from microbial fermentation,with a wide source,simple production process,and low production costs.By modifying the structure of Monascus pigment and develop a kind of oil-soluble pigment will enrich the species of pigment and enlarge the application range.Monascus pigment contains a variety of low-polar components,including yellow components and fluorescent component.Understanding the structure of unknown fluorescence components and establishing the analytical testing methods have a certain theoretical and practical significance.This thesis used water-soluble Monascus pigment as raw material,through acidification,introducing low-polar groups to reduce the polarity of Monascus pigment and synthetic oil-soluble pigment.Structure characterization and performance characterization of fluorescence components and oil-soluble Monascus pigment are analyzed by TLC,HPLC,HPLC/MS and other modern analysis methods.The main contents and results are as follows:1.Preparation of acidizing Monascus pigment and analysis of liposoluble componentsThe liposoluble components can be effectively removed by pressure reducing column chromatography.The separation conditions of TLC and HPLC have been optimized,four fluorescence components were characterized by liquid chromatography-mass spectrometry.The results show that,with petroleum ether-acetone(5/1,v/v)as the developing solvent,five bands can be obtained,respectively,they are Rf-0.52 yellow bands,Rf-0.37 fluorescent band,Rf-0.33 fluorescent band,Rf-0.29 yellow band,Rf-0:15 fluorescence band.The optimal HPLC separation conditions were as follows:Agilent ZORBAX C18(5?m,4.6 × 150mm)as a separation column,UV detector,flow rate at 1.0mL/min,gradient elution:0-4min,methanol 70%-76%;4-15min,methanol 76%-81%;15-40min,methanol 81%-100%;40-60min,100%methanol.Four fluorescence components' molecular weight have been detected by liquid chromatography-mass spectrometry technique,respectively they are 330(MF1-A),358(MF1-B),356(MF2-A)and 384(MF2-B).By comparing the ion fragments between four fluorescence components and citrinin,it shows that they have no same ion fragments,that means the synthesis of four fluorescent components is not based on Citrinin.2.Synthesis and Characterization of oil-soluble Monascus pigmentThis chapter is attempting to introduce a non-polar group by esterification at the carboxyl terminus of Monascus pigment to change its solubility,and the performance characterization of the new products was analyzed as well.Using acidizing Monascus pigment and dicyclohexyl carbodiimide(DCC)as raw material,methylene chloride/N,N-dimethyl formamide(20:3,v/v)as a mixture solvent,the crude product were synthesized after 24h reaction under 35?.After the process of filtering,washing,drying,the oil-soluble Monascus pigment were extracted with petroleum ether-ethyl acetate(3/1,v/v).Solubility assay showed that the product can be dissolved in low polar solvents(e.g.carbon tetrachloride,petroleum)and oil,but can't dissolved in water.The result of stability test shows that the products was stable with the conditions which were as follows:pH was3?7,the temperature was50?100?,it also stable with carbohydrate and it has a good oxidation resistance and bad reduction resistance.The Zn2+ has considerable effect on increasing color and keeping pigments' stability while the Fe3+ will damage the pigments.Because of the poor tolerance to ultraviolet light,it should be stored in the dark to avoid UV.3.Separation,purification and structural characterization of oil-soluble Monascus pigmentThe separation conditions of TLC and HPLC have been optimized,the oil-soluble Monascus pigments were characterized by liquid chromatography-mass spectrometry.The results show that,with n-hexane-acetone(5/2,V/V)as the developing solvent,three red bands can be obtained,the Rf values are 0.400(R1),0.266(R2)and 0.137.Each component was achieved baseline separation by using acetonitrile-water(80/20,V/V)as a mobile phase.Ten components' molecular weight of oil-soluble Monascus pigments have been detected by liquid chromatography-mass spectrometry technique,respectively they are 659(R1-A)?707(R1-B)?673(R1-C)?687(R1-D)?735(R1-E)?701(R1-F)?617(R2-A)?631(R2-B)?645(R2-C)?659(R2-D).After analyzing the Tandem mass spectrometry,we find that each component share the same fragmentation pattern.They all have the ion fragments missing 206 molecular weight and 125 molecular weight,It means that the structure of oil-soluble Monascus pigments have connected dicyclohexyl carbodiimide structure.The products we achieved can be dissolved in oil.So we focus on the fragmentation pathway of R1-B and R1-E.