Study On Hyperchromicity And Metabolic Enhancement Strategies Of Monascus Yellow Pigments In Submerged Fermentation And Their Nano-compositing Modification

Monascus pigments are secondary metabolites with polyketide structures synthesized by Monascus,which are mainly intracellular hydrophobic pigments produced by submerged fermentation.The production efficiency of extracellular hydrophilic yellow pigments(HYPs)is low,and their maximum absorption wavelength is appeared in the ultraviolet region(fluorescence under ultraviolet light),which is fluorescent yellow pigments.In order to solve the key application problems of HYPs in Monascus ruber CGMCC 10910 fermentation,such as low production efficiency,unsatisfactory tonality and dye uptake as well as poor stability,this paper explored new efficient fermentation thechologies for HYPs production by microparticle-enhanced cultivation(MPEC)strategy,and investigated the regulation mechanism.A stable system of polyphenol-albumin-polysaccharide loaded with HYPs was constructed to improve its color property,physical and chemical stability and biological activity.Novel fermentation thechologies combined with chemical derivation were explored to produce natural bright HYPs and the regulation mechanism was also investigated.The synthesis of bright hydrophilic yellow pigment was enhanced by a cascade of non-enzymatic catalysis and microbial metabolism.The major conclusions are as follow:(1)The addition of sodium starch octenyl succinate(OSA-SNa)increased the permeability of cell membrane,resulted in enhanced metabolism and secretion of extracellular HYPs.Compared with conventional fermentation,OSA-SNa addition increased the production of extracellular HYPs by 69.68%,particularly in Y3 and Y4,and synergism of OSA-SNa and Triton X-100(10 g/L)further increased HYPs production by 134.06%.Improvement of hyphal morphology and membrane permeability,activities of enzymes involved in pentose phosphate pathway and oxidoreduction,as well as gene expression for yellow pigment biosynthesis(mpps A、D、E、R2 和 mp PKS5)are likely to be critical to increase yield of HYPs.(2)The presence of taurine in the medium can achieve the derivative fermentation to synthesize bright HYPs.Taurine was used as a fermentation substrate to synthesize three bright HYP derivatives YN1-YN3 by intracellular biotransformation.The maximum ultraviolet absorption wavelengths of YN1,YN2 and YN3 were 453 nm,458 nm and 463 nm,respectively.The analysis of pigment metabolism pattern showed that taurine competitively promoted the ammoniation of common precursor Y1 to synthesize bright HYPs,blocked the synthesis of fluorescent HYPs Y2-Y4,achieving the brightening of extracellular HYPs by derivative fermentation.In addition,taurine regulated the micromorphology of mycelium,enhanced the permeability of cell membrane to promote the production of HYPs.When the initial concentration of taurine was 15 g/L,the yield of fluorescent HYPs and bright HYPs reached 41.34AU350 /m L and 17.63AU450 /m L,respectively,and the total HYPs production increased by 18.37% compared with the control group.The bright HYPs showed good photostability and tolerance to high concentrations of Na Cl.However,it is easy to degrade at high temperature and isomerize into the azanigerone structure of red pigments under neutral or weak alkaline conditions and the color changed from yellow to purple red.(3)Multi-omics analysis showed that the mechanism of taurine promoting the biosynthesis and secretion of HYPs lies in up-regulating the expression levels of pigment synthetic genes,enhancing the precursor of pigment synthesis and energy metabolism,and improving the fluidity of cell membrane.The whole genome information and gene cluster composition of M.ruber CGMCC10910 were comprehensively analyzed by three-generation sequencing.The results of comparing transcriptome and metabolome analysis found that taurine promoted yellow pigments metabolism by up-regulating pigments biosynthetic genes,while coding genes and marker metabolites of pigments biosynthesis related pathways(carbon catabolism,amino acid metabolism,fatty acid metabolism and polyketone synthesis)were also up-regulated to provide precursors and energy for pigments metabolism.The up-regulation of secretion-related(membrane ergosterol biosynthesis and pigments transport)genes and the enhancement of cell membrane mobility also improve the transmembrane transport of extracellular HYPs.(4)The phosphates interfacing with microparticle enhancement can regulate the efficient fermentation of HYPs,catalyze the transformation of precursor substances and synthesize bright HYPs.Under the optimal initial p H of 4.0,phosphate(0.06M)competitively catalysed the transformation of precursor Y1 and synthesized the bright HYPs(YN1-YN3)by ammoniation derivative,and inhibit the synthesis of fluorescence Y3 and Y4 by feedback.High concentration of glucose(100 g/L)and taurine(30 g/L)increased the extracellular fluorescence HYPs and bright HYPs yields by 29.64% and 58.79%,respectively.OSA-SNa also showed potential micellar catalysis,which increased the yield of bright HYPs to60.09AU450/m L,about 2.5 times higher than that of conventional fermentation.(5)Loading by quercetin-Sn(Ⅱ)-albumin-chitosan can prepare bright HYPs and enhance their stability and bioactivity.A novel encapsulate system for HYPs were investigated through stable ternary nanocomplexes(252-382 nm)using quercetin-Sn(Ⅱ),bovine serum albumin(BSA),and chitosan(CS)in this work.A hyperchromic effect on HYPs was obtained since their maximum ultraviolet absorption wavelength redshifted from 352 nm to 408 nm,greatly improved the color tonality and dye-uptake.Layer-by-layer assembly preparation of HYPs-loaded quercetin-Sn(Ⅱ)-BSA-CS nanocomplexes(252-382 nm)were verified by structural characterization technique.Their conjugation had a great hyperchromicity and remitted HYPs degradation under various environmental stresses(thermal,light,and ascorbic acid treatment).Higher antioxidant and lipid-lowering activities were detected in the nanocomplexes than free HYPs.This paper explored novel preparation strategies for bright HYPs by high efficiency derivative fermentation and natural nanoencapsulation,which filled the blank of the preparation of natural bright HYPs,and provided a theoretical reference for the fermentation production of bright Monscus pigments and regulation strategy.