Mechanism Study Of Environmental Factor On Water-soluble Yellow Pigment Metabolic In Submerged Fermentation By Monascus Ruber

Monascus pigments,a kind of polyketide secondary metabolites produced by filamentous fungi Monascus,which used as natural edible pigments has tremendous application prospect.Monascus yellow pigment not only used as colorant,a large number of studies reported it also has a variety of biological activity.The current commercially available Monascus yellow pigment was produced by chemical conversion of Monascus red pigment,which is a potential risk in food applications.But researches on production of Monascus yellow pigment is still at the laboratory level,therefore the research and development of the Monascus yellow pigment,especially the water-soluble Monascus yellow pigment has a huge market prospects.In this study,the external stress control used to improve Monascus yellow pigment by Monascus ruber CGMCC 10910 in submerged fermentation were investigated,the fermentation method and related mechanism also investigated,which provide theoretical guidance for the industrial production of Monascus yellow pigment.In this study,submerged fermentation of Monascus yellow pigments by Monascus ruber CGMCC 10910 under high glucose stress was investigated.The increase of lipid content was the major contributor to the increase of dry cell weight（DCW）,and the lipid-free DCW was only slightly changed under high glucose stress,which benefited the accumulation of intracellular hydrophobic pigments.The fatty acid composition analysis in Monascus cell membranes showed that high glucose stress significantly increased the ratio of unsaturated/saturated fatty acid and the index of unsaturated fatty acid（IUFA）value,which would improve the fluidity and permeability of the cell membrane.As a consequence,high glucose stress increased extracellular yellow pigments production by enhancing secretion and trans-membrane conversion of intracellular pigments to the broth.Under high glucose stress fermentaion,intracellular yellow pigment yield reached 195 AU₄₁₀,increased by 101%,and extracellular water-soluble yellow pigment yield reached 147 AU₃₅₀,increased by 194%.The total yield of extracellular and intracellular yellow pigments per unit of lipid-free DCW increased by 94.86 and 26.31%under high glucose stress compared to conventional fermentation,respectively.A real-time quantitative PCR analysis revealed that the expression of the pigment biosynthetic gene cluster was up-regulated under high glucose stress.The gene mppE,which is associated with yellow pigment biosynthesis,was significantly up-regulated.These results indicated that high glucose stress can shift the Monascus pigment biosynthesis pathway to accumulate yellow pigments and lead to a high yield of both extracellular and intracellular yellow pigments.These findings have potential application in commercial Monascus yellow pigment production.In order to further improve the glucose utilization and yellow pigments yield under high glucose stress fermentation,H₂O₂ and dithiothreitol（DTT）were used to change the oxidoreduction potential for investigating the effects of oxidative or reductive substances on Monascus yellow pigment production by Monascus ruber CGMCC 10910.The extracellular ORP could be controlled by H₂O₂ and DTT.Both cell growth and extracellular water-soluble yellow pigment production were enhanced under H₂O₂-induced oxidative（HIO）conditions and were inhibited under dithiothreitol-induced reductive conditions.By optimizing the amount of H₂O₂ added and the timing of the addition,the yield of extracellular water-soluble yellow pigments significantly increased and reached a maximum of 209 AU₃₅₀,increased by 42%,and intracellular yellow pigments production reached 236AU₄₁₀,increased by 35%,one of intracellular main yellow pigment Monascin yield reached467.75μg/mL,when 10 mM H₂O₂ was added on the 3rd day of fermentation with Monascus ruber CGMCC 10910.Under HIO conditions,the ratio of NADH/NAD+was much lower than that in the control group,and the expression levels of relative pigment biosynthesis genes were up-regulated;moreover,the activity of glucose-6-phosphate dehydrogenase（G6PDH）was increased while 6-phosphofructokinase（PFK）activity was inhibited.Oxidative conditions induced by H₂O₂ increased water-soluble yellow pigment accumulation via up-regulation of the expression levels of relative genes and by increasing the precursors of pigment biosynthesis through redirection of metabolic flux.In contrast,reductive conditions induced by dithiothreitol inhibited yellow pigment accumulation.This experiment provides a potential strategy for improving the production of Monascus yellow pigments.Finally,the effect of fermentation temperature on the extracellular water-soluble Monascus yellow pigment metabolism was investigated.Four types of extracellular water-soluble yellow pigments（Y1-Y4）were generated by submerged fermentation with Monascus ruber CGMCC 10910,of which Y1 was identified as the reported pigment synthesis intermediates Azanigerone E,Y3 and Y4 had strong yellow fluorescence.The composition of the pigment mixtures was closely related to the fermentation temperature.The dominating pigments changed from Y1 to Y3 and Y4 when fermentation temperature increased from 30 to 35℃.Increasing the temperature to 35℃changed the metabolic pathways of the pigments,which inhibited the biosynthesis of Y1 and enhanced the biosynthesis of Y3 and Y4.Moreover,the yield of Y1 reduced insignificantly,while the yields of Y3 and Y4 increased by 98.21 and 79.31%respectively under two-stage temperature fermentation condition.The two-stage temperature strategy is a potential method for producing water-soluble Monascus yellow pigments with strong yellow fluorescence.