Study On The Separation And Purification,Structural Characterization And Stability Of Pigments From Red Yeast Glutinous Rice

Red yeast pigment in this study is a kind of natural pigment extracted from red yeast glutinous rice.Due to its security,low cost and stability,monascus pigments get a lot of attention.However,there are few studies on monascus pigment's components in red yeast glutinous rice.With homemade red yeast glutinous rice as raw material,this study was based on the separation and purification of water-extracted monascus pigments and alcohol-extracted monascus pigments.In this study,water-extracted monascus pigments and alcohol-extracted monascus pigments were separated by silica gel column chromatography and then further separated by thin layer chromatography.A large number of experiments were conducted to determine the eluent and the ratio of running solvent,and then mass spectrometry and nuclear magnetic resonance technology were used to identify the structure of the obtained monascus pigment's monomers.And finally the effect of temperature and p H value on the stability of water-extracted monascus pigments,alcohol-extracted monascus pigments and two monascus pigment's monomers.The main research results are as follows:1.The separation and purification method of water-extracted monascus pigments was established.Firstly,silica gel column chromatography was used for preliminary separation with chromatographic methanol as eluent,and seven kinds of monascus pigments of different colors were obtained.Then thin layer chromatography was used for further separation.The optimized mixture of 13:7?v/v?ethyl acetate and methanol was used as the running solvent.Finally,a water-extracted monascus pigment's monomer was obtained.2.The separation and purification method of alcohol-extracted monascus pigments was established.Firstly,silica gel column chromatography was used for preliminary separation with chromatographic ethyl alcohol as eluent,and five kinds of monascus pigments of different colors were obtained.Then thin layer chromatography was used for further separation.The optimized mixture of 4:6?v/v?ethyl acetate and ethyl alcohol was used as the running solvent.Finally,a alcohol-extracted monascus pigment's monomer was obtained.3.UPLC-Q-TOF-MS and NMR were used to identify the structure of water-extracted and alcohol-extracted monascus pigment's monomer.The molecular mass of this pigment was determined to be 353.2 m/z[M-H]^-,therefore the molecular weight was 354 u.Its structure comprise an azaphilone body and two following side chains:a)the acyl five-membered chain;b)two connected unsaturated CH.This pigment was confirmed as rubropunctatin.The molecular mass of alcohol-extracted monascus pigment's monomer was determined to be 415.1773 m/z[M-H]^-,therefore its molecular weight was 416 u.According to the literatures,it was speculated that the pigment was a novel monascus pigment,which was named as Pigment R and the molecular formula was C₂₃H₂₈O₆.4.The study about the effect of temperature on the stability of water-extracted monascus pigments,alcohol-extracted monascus pigments,rubropunctatin and Pigment R found that:Macro analysis,the pigment retention rate of alcohol-extracted monascus pigments treated with heat was higher than that of water-extracted monascus pigments,thus the stability of alcohol-extracted monascus pigments was better.And according to the analysis,alcohol-extracted monascus pigments was more suitable for food production requiring pasteurization.When the temperature is between 70 and 100?,macroscopically,the pigment retention rate of rubropunctatin was generally higher than that of Pigment R,thus rubropunctatin was relatively stable and more suitable for pasteurization between 70and 100?.According to the liquid phase analysis,the amount of other substances which were alcohol-extracted monascus pigments transformed into was less than that of water-extracted monascus pigments under the environment of high temperature,indicating that alcohol-extracted monascus pigments were more resistant to high temperature.And it has a better stability.This corresponded to the macro analysis results.Similarly,with the increase of temperature,Pigment R will be transformed into more and more new substances,while rubropunctatin was more stable than Pigment R.This also corresponded to the macro analysis results.5.The study about the effect of p H value on the stability of water-extracted monascus pigments,alcohol-extracted monascus pigments,rubropunctatin and Pigment R found that:Macro analysis,the pigment retention rate of alcohol-extracted monascus pigments treated with acid and alkali was higher than that of water-extracted monascus pigments,thus the stability of alcohol-extracted monascus pigments was better than that of water-extracted monascus pigments.And according to the analysis,alcohol-extracted monascus pigments was more suitable for food processing with p H value of about 5.Under the p H value of 1-11,the pigment retention rate of rubropunctatin was higher than that of Pigment R,and the stability is better.From the microscopic point of view,the amount of other substances which were alcohol-extracted monascus pigments transformed into was less than that of water-extracted monascus pigments under the environment of very acid and very alkali,indicating that alcohol-extracted monascus pigments were more resistant to acid or alkali.This corresponded to the macro analysis results.According to the liquid phase analysis,we also knew that rubropunctatin and Pigment R can exist in a neutral environment,with little influence from acid and alkali.