Study On The Regulation Mechanism Of Nitrates On Monascus Yellow Pigment Metabolic In Submerged Fermentation And Enhanced Pigment Dyeing

Monascus pigments is a kind of natural pigment produced by the filamentous fungi Monascus,which are mainly composed of three pigments:Monascus yellow,orange and red pigment.It is widely used in food and pharmaceutical industry due to its various biological activities and safety.Natural Monascus yellow pigment is bright and has anti-obesity,anti-oxidation,anti-tumor,anti-hyperglycemia and other biological activities,and have become a research hotspot in recent years.In this study,the effects of nitrates on the cell growth and the production of Monascus yellow pigments in Monascus ruber CGMCC 10910 were studied,the regulatory mechanism of nitrates on the high yield of water-soluble Monascus yellow pigment by Monascus was explored,and the application of Monascus yellow pigment in textile dyeing was discussed.Mycelial adhesion affects cell growth and the production of water-soluble extracellular yellow pigment(EYP)in submerged fermentation with Monascus ruber CGMCC 10910.Two nitrates,NaNO₃ and KNO₃,were used as nitrogen sources for mitigating mycelial adhesion and improving the production of EYP in this study.The results showed that the adhesion of mycelia in the fermentation broth significantly decreased by adding 5 g/L NaNO₃,which prevented mycelia from attaching to the inner wall of the Erlenmeyer flask.It was suggested that NaNO₃ reduced the total amount of extracellular polysaccharides,increased extracellular proteins and decreased the viscosity of the fermentation broth.Scanning electron microscopy(SEM)analysis revealed that the mycelial morphology was shorter and more dispersed and vigorous under NaNO₃ conditions than under the control conditions.The biomass increased by 49.2%and 45.4%with 5 g/L NaNO₃ and 6 g/L KNO₃ treatment,respectively,compared to that of the control,and the maximum production of EYP was 267.1 and 241.8 AU₃₅₀,which increased by 70.0%and 53.9%compared to that of the control,respectively.Simultaneously,the ratios of intracellular yellow pigment to orange pigment increased significantly with 5 g/L of NaNO₃ addition(p<0.05).Genetic analysis found that the expression levels of the key genes for Monascus pigment biosynthesis were significantly upregulated by NaNO₃ addition(p<0.05 or p<0.01).This study provides an effective strategy for the production of water-soluble Monascus yellow pigments.In order to further explore the regulatory mechanism of nitrates on the production of water-soluble Monascus yellow pigment by Monascus,two nitrates,NaNO₃ and NH₄NO₃,were used as nitrogen sources to produce Monascus yellow pigments.Transcriptomics and proteomics were combined to analyze the regulatory mechanism of nitrates on the biosynthesis of Monascus yellow pigment by Monascus ruber CGMCC 10910.The results showed that nitrates significantly affected the cell growth of and increased the production of water-soluble Monascus yellow pigment in Monascus ruber.Comparative transcriptome analysis found that the number of differentially expressed genes(DEGs)between different experimental groups-NA group(NaNO₃ group)vs control group(CK group)and NH group(NH₄NO₃ group)vs control group(CK group)were 1692 and 814,respectively.KEGG pathway enrichment analysis showed that nitrates(NaNO₃ and NH₄NO₃)regulated carbon and nitrogen metabolism in Monascus,including the biosynthesis and biodegradation of amino acids and fatty acids,which provided more energy and precursors substances for the biosynthesis of Monascus yellow pigments.NR database analysis found that the addition of nitrates could up-regulated the gene transcription level of ctnG and ctnF,which are the transcription factors associated with Monascus pigments biosynthesis.The nitrates as nitrogen source also regulated the gene transcription level of pyruvate decarboxylase,which is the precursor of Monascus pigments,and up-regulated the genes transcription level of polyketide synthase-nonribosomal peptide synthase(PKS-NRPS synthase),it could be key regulatory genes involved in the biosynthesis of Monascus yellow pigments.In addition,the addition of nitrates also affected the genes expression of?-1,3-glucan and glycosylphosphatidylinositol(GPI),which are involved in cell morphology,and down-regulated the expression levels of genes related to meiosis and cell cycle,which may affect the cell morphology of Monascus.Proteomic analysis showed that the number of differentially expressed proteins(DEPs)in NA vs CK and NH vs CK groups was 102 and 72,respectively.The GO functional database analysis showed that the nitrates(NaNO₃and NH₄NO₃)significantly affected the redox reaction in Monascus.DEPs were mainly involved in carbon and nitrogen metabolism,energy transport and production.It was found that acyltransferase was a key protein in the biosynthesis pathway of Monascus yellow pigments by Monascus.The addition of nitrates up-regulated the expression levels of proteins that involved in the metabolic pathway of glyoxylate and dicarboxylate,so we suggested that glyoxylate and dicarboxylate metabolic pathway provided the key precursors such as hydrophilic groups for the abundant biosynthesis of water-soluble Monascus yellow pigment.Finally,a natural water-soluble Monascus yellow pigment was developed for textile dyeing.The water-soluble Monascus yellow pigment was used to dye silk,and the dyeing process,such as dyeing bath ratio,dye concentration,dyeing temperature and time,was optimized to improve the dyeing rate.Metal salt and biological mordant were used to optimize the dyeing process to improve the dyeing efficiency and dyeing property of yellow pigment dyed silk.Monascus yellow pigment has the biological activity of antimicrobial,and the silk has the biological activity of antimicrobial after dyeing.The dyed silk with tannic acid as mordant has the stronger antimicrobial property.In this study,an environmental protection,energy saving and biological activity dyeing process with water-soluble Monascus yellow pigment for silk was developed.