Optimization Of Culture Conditions For Monascus Liquid Fermentation To Produce Pigment Based On Metabonomics

As a natural edible pigment produced by Monascus secondary metabolism,Monascus pigment has a variety of biological activities such as antibacterial,antioxidant and lipid-lowering.Monascus pigment has great commercial prospects in meat products,flour products,condiments and other industries.Due to the complex metabolic system of Monascus,the production of Monascus pigment is easily disturbed by external conditions,so it is difficult to effectively regulate the fermentation to improve the yield of Monascus pigment.Based on this,this subject aims to analyze the metabolic regulation mechanism of Monascus pigment production by Monascus using a systematic biological method combining metabome and weighted gene co-expression network analysis,find the potential targets and ways to improve Monascus pigment production,and then carry out directional transformation on Monascus pigment metabolism.(1)Fine tune the liquid fermentation conditions of Monascus to disturb the fermentation process.Firstly,Monascus was fermented with basic fermentation medium.The results showed that 60 h fermentation was the key observation point to distinguish the biosynthesis of different Monascus pigments.Therefore,60 h fermentation was used as the sampling point for metabolic disturbance.An additional 17 amino acids were added to the basic fermentation medium respectively to interfere with Monascus metabolism.The results showed that the variation coefficients of the six Monascus pigments(R1,R2,Y1,Y2,O1and O2)were between 0.3459 and 0.6397,indicating that the phenotypic variation of Monascus induced by metabolic disturbance was moderate and did not lead to metabolic collapse.Then,the metabolomics technology based on UHPLC-MS was used for sampling.A total of 68 metabolites were isolated and identified.Taking the abundance of all metabolites as independent variables and six pigments as dependent variables,WGCNA analysis was carried out to find the key metabolites fumaric acid and malic acid in the module significantly related to pigment synthesis,and both of them belong to important metabolites in the tricarboxylic acid cycle.(2)Subsequently,exogenous key metabolites were added to verify the metabolic perturbation of the tricarboxylic acid cycle.The results showed that the addition of 0.05 g/L fumaric acid,malic acid and oxaloacetic acid was beneficial to improve the yield of red pigment and bacterial volume.The yield of red pigment was 1.18 times,1.19 times and1.15 times that of the control group.The biomass in fumaric acid group was significantly higher than that in control group(P<0.05),increased by 37.1%.The addition of fumaric acid and malic acid promoted the conversion of more orange pigment to yellow pigment,indicating the effectiveness of adding key metabolites in regulating the production of Monascus pigment.(3)The production of NAD(P)~+,NAD(P)H and ATP in cells after the addition of fumaric acid,malic acid and oxaloacetic acid were investigated.The results showed that NADH/NAD~+in malic acid group and oxaloacetic acid group were 1.3 times and 2.2 times higher than those in control group,respectively.The ratio of NADPH/NADP~+in the experimental group was higher than that in the control group,especially in the malic acid group and oxaloacetic acid group.Compared with the control group,after adding 0.05 g/L fumaric acid and malic acid,the concentration of ATP increased by 112%and 158%respectively,indicating the feasibility of regulating intracellular redox state through tricarboxylic acid cycle.(4)The expression of Monascus pigment synthesis gene cluster after exogenous addition of fumaric acid,malic acid and oxaloacetic acid was detected by RT-q PCR.The results showed that the expression of the gene encoding oxidoreductase changed greatly.The expression of Mpig C in fumaric acid and malic acid group was down-regulated,while the expression of oxaloacetic acid group was up-regulated;The expressions of Mpig F and Mpig N were up-regulated in the experimental group;Mpig H was up-regulated in malic acid and oxaloacetic acid group,but the expression multiple of fumaric acid group did not change significantly.These results indicate that the expression of gene cluster is not a limiting factor in the synthesis of Monascus pigment under the present study conditions.In conclusion,this study identified the key pathway affecting Monascus pigment synthesis tricarboxylic acid cycle and central metabolites fumaric acid and malic acid through metabolic perturbation of Monascus fermentation process and WGCNA analysis.The directional transformation of Monascus pigment produced by Monascus fermentation was realized through rational addition strategy,The research results will provide theoretical guidance for Monascus liquid fermentation to produce pigment.