Macrogenome Analysis Of Red Vinasse Acid And Study On Screening And Liquid Fermentation Of High Pigment Yielding Strain Monascus

Red vinasse acid is a traditional fermented food widely spread in Guangxi,which is made of rice fermented and steamed by Monascus spp.supplemented with pickled vegetables such as melons and beans.The complex microbial metabolic activity in red vinasse acid gives it a unique flavor and bright red color,which is why it is often used in Chinese kitchens for cooking meat and vegetables.However,at present,the production mode of red vinasse acid is mainly household manual production or small workshop production,the pigment production capacity of Monascus used for fermentation is uneven,and the production process is not standardized,which leads to the unknown composition of microorganisms in the fermented finished products and no guarantee for product safety.Therefore,analyzing the microbial composition and structure in red vinasse acid,screening high-yield Monascus pigment and optimizing the fermentation process are not only conducive to stabilizing the quality of red vinasse acid,but also of great significance to promote the industrial production of Monascus pigment.In this study,the microbial diversity of red vinasse acid was analyzed based on metagenomic technology,the functional genes and metabolic pathways were explored,and the functional strain Monascus purpureus D21 with high pigment yield in red vinasse acid was screened out.Further,by optimizing the liquid fermentation medium and fermentation process,the yield of Monascus pigment was increased by 20.95 times.On this basis,by transcriptome sequencing analysis,the regulatory mechanism of different nitrogen sources on Monascus red and Monascus yellow pigment in the medium was clarified and the key genes were summarized.The main research contents and results are as follows:(1)Macrogenome-based microbial diversity analysis of red vinasse acid and screening of functional strain Monascus: Macrogenome sequencing analysis of red vinasse acid samples from 3 different regions showed that 4,019 species of microorganisms were detected,belonging to 69 phyla,88 classes,191 orders,407 families and 1,057 genera.At the phylum level,Proteobacteria,Ascomycota and Firmicutes were the main dominant flora,accounting for more than 70%.At the genus level,Burkholderia spp.,Pediococcus spp.,Lactobacillus spp.,Aspergillus spp.,Acetobacter spp.and Monascus spp.were the dominant genera.At the species level,B.cepacia,B.multivorans,A.wentii,M.purpureus were abundant.9 Monascus strains were isolated from red vinasse acid,and a strain named M.purpureus D21 with high Monascus pigment production was selected as the starting strain for further study.(2)Macrogenome-based analysis of functional genes and metabolic pathways of red vinasse acid: The non-redundant gene set of red vinasse acid was functionally annotated,and 16,093,49,652,3,328 genes were annotated in egg NOG,KEGG and CAZy databases,among which the most abundant metabolic activities in egg NOG database were carbohydrate metabolism,inorganic ion transport and metabolism,amino acid transport and metabolism.The two most important metabolic processes annotated in KEGG database were carbohydrate metabolism and amino acid metabolism,and glycosyl transferase and glycoside hydrolase were the most annotated in CAZy database.Based on the above annotation results,the carbohydrate metabolism and amino acid metabolism pathways were analyzed emphatically.In the carbohydrate metabolism pathway,sugar transport system genes such as mannitol,fructose,mannose and sucrose were annotated,as well as key enzyme genes that catalyze intracellular D-1-phosphate mannitol,D-1-phosphate fructose,D-6-phosphate mannose.The amino acid metabolic pathway was annotated to 32 genes controlling branched-chain aminotransferases,27 aromatic transaminases,60 ketoacid converting enzymes,123 alcohol/aldehyde dehydrogenases,and 27 acetyl esterases,which are closely related to amino acid flavor formation.(3)Optimization of liquid fermentation and pigment extraction process of functional strain M.purpureus D21: The liquid fermentation and extraction process of M.purpureus D21 were optimized.The results of single factor experiment showed that the total color value was the highest when maltose was used as carbon source,calcium chloride was used as inorganic salt,peptone was used as organic nitrogen source and sodium nitrate was used as inorganic nitrogen source.The orthogonal experiment method was used to further optimize the medium composition.The results showed that the optimal medium composition was 60 g/L maltose,0.05 g/L calcium chloride,15 g/L peptone,1.5 g/L sodium nitrate and 1 g/L potassium dihydrogen phosphate,and the total color value was1,780.48 U/g,which was 12.99 times higher than that before optimization.On this basis,the fermentation conditions and extraction process were further optimized.When the fermentation conditions were temperature 30℃,inoculation amount 8% and liquid volume 125 m L,the total color value was 2,410.98 U/g.When the extraction process is ethanol concentration of 75%,extraction temperature of 60℃ and extraction time of 2 h,the total color value is 2,794.13 U/g,which is 10.80% higher than that before optimization,and 20.95 times higher than that of the original fermentation and extraction process.(4)Transcriptome sequencing-based analysis of key genes for Monascus red and Monascus yellow pigments: The transcriptome sequencing of Monascus mycelia producing different Monascus pigments under the regulation of different nitrogen sources was performed.The COG annotation results showed that the functional categories with the most annotated genes in the three subgroups were carbohydrate transport and metabolism;The results of GO analysis showed that the synthesis of Monascus pigment was related to biofilm,while the synthesis of Monascus red and yellow pigment was related to iron ion and zinc ion,respectively.The results of KEGG analysis showed that the formation of Monascus red pigment was mainly related to glycerol metabolism,valine,leucine and isoleucine degradation pathways,Mpig H,and UMF1 genes.The formation of Monascus yellow pigment was mainly related to propionate metabolism,glycine,serine and threonine metabolism pathways,Mpig I,Mpig F,Mpig H,CTR,and HNM1 genes.