Isolation, Identification And Pigment Production Research Of Monascus From Red Yeast Rice

Red yeast rice,known for containing natural monascus pigment,is a great invention of Chinese ancient people. Application of red yeast rice in food pigmentation,food fermentation such as making vintage and vinegar,food antisep,Chinese traditional medicine,etl.,have been more than one thousand year.In recent years,with intensive study of monascus,a variety of available metabolic product used in food,medicine,etc.field,such as natural monascus pigment,glucoamylase,Monacolin K,gamma-amino butyric acid(GABA),etc.,has been discoveried.That have drawn the attention of the international community.A number of domestic and international researcher have used modern biotechnology for production and application research of monascus,obtained a lot of new achievements.This subject get pure culture of monascus strains by isolation of monascus from Red yeast rice,then use traditional morphological method to classify and identify the pure culture.On that basis coordinate with using method of ribosome LSU D1/D2 region and ITS region sequence analysis to construct phylogenetic relationship tree in order to identify their genetic homology and phylogenetic relationship.For identification of the traditional morphological method has long cycle,subjective,great affected by the environmental factor , and development of molecular technology provide helpful means to research genetic relationships of species and phylogenetic classification.There is need to utilize molecule mark technology to set up a fast effective monascus identification method.And probe the probability of using relevant ribosomes gene sequences analysis to identification monascus strains and probe identification ability of different gene sequences at different levels:genara,species and mutation.The 15 strains of monascus were identified to 4 species of monascus:7 strains were M. anka,5 strains were M. rubber,2 strains were M. purpureus and 1 strain was M. barkeri by observation clone pattern characteristic on malt ager culture medium of monascus cultured at 25℃for 7 days and refer to taxa key on literature to identify monascus.This shows most monascus identificate from red yeast rice are M. anka and M. rubber.The 15 strains of monascus were identified base on rRNA LSU D1/D2 and ITS region gene sequences , by following these steps : DNA isolation , PCR amplification,sequencing,sequence analysis.Two sequences analysis consistently classify the 15 strains into two groups:M1,M4,M7, M8,M9, M10, M13, M14, M15 in first group and M2 , M3 , M5 , M6 , M11 , M12 in other group.Moreover,the 15 strain are identified to monascus genus,that consist with morphological identification,but not enough identified to monascus species.Research result shows,traditional morphological method can classify and identify monascus,while relevant ribosomes gene sequences analysis can identify monascus to genus,but not suitable to species.Therefore,rDNA can use in classify and identify monascus to some extent and provide support and supplement for morphological identification of monascus.Research on pigment production of separated Monascus found that there are 9 monascus strains producing pigment among 15 strains,and that the highest pigments value 1532 u/g is produced by M4 strain after fermentation on rice solid culture medium.The liquid fermentation conditions of M4 strain for pigment production were optimized as follows:culture media with the components of 8 % rice powder, 3 % peptone, 0.1 % MgSO4,0.15% KH2PO4,original pH 6.0(adjusted with lactic acid),inoculation volumes 8%, filling quantity of 80/500mL flask,fermentation temperature 32℃and rotate speed 180r/min.Under the optimized conditions for 10 days M4 will produce high level of monascus pigment color value in the liquid fermentation, which could reach 124 u/mL.