Effect Of Flavonoids On Citrinin Production By Monascus Based On Metabonomics

Monascus is a filamentous fungus,which can produce variety of beneficial metabolites.However,the production of the mycotoxin citrinin limits its application in industry.Scholars at home and abroad have done a lot of work on how to reduce the citrinin production by Monascus.In our previous study,we also found exogenous substances(flavonoids)had different effects on citrinin,but the relationship between the differential metabolites and citrinin in the synthesis pathway is not yet known.Therefore,this article is based on the metabonomics to study the effect of flavonoids on citrinin production by Monascus and the utilization and transformation of genistein in Monascus.(1)First of all,this article is based on ~1H NMR metabolomic to study the effects of four flavonoids on citrinin production by Monascus.It was found that adding different flavonoids(genistein?daidzein?apigenin and kaempferol),The content of citrinin were decreased by 78.13%,10.57%,45.62%,and 43.84%respectively.A total of 15 significantly differential metabolites were screened,the level of biotin,2-phosphoglycerate,glutamate,aspartate,chlorogenate,and glycocholate showed a decreasing trend compared with the control group,whereas the other nine compounds were higher than those in control group.The identified metabolites are mainly involved in lysine biosynthesis;arginine biosynthesis;alanine?glutamate and aspartate metabolism;biotin metabolism;arginine,glutathione metabolism and proline metabolism.In addition,after the addition of genistein,we identified two effective different metabolites(caprylate and ethanol),both of which showed an increasing trend in the experimental group.Finally,through the analysis of the KEGG pathway,we found that the down-regulation of glutamate,aspartate,biotin and 2-phosphoglycerate regulate the synthesis of acetyl-Co A,which is involved in the synthesis of citrinin.At the same time,it was also found that the four differential metabolites indirectly participate in the fatty acid synthesis pathway,thereby affecting the content of citrinin.(2)Secondly,we used the HPLC and UHPLC-Q-TOF MS/MS to analyze the effect of genistein on citrinin in different stages(6?9?12 d)of liquid fermentation of Monascus,the results showed the reduction effect of genistein on citrinin was 74.60%,74.26%and 78.02%at 6?9 and 12 days,respectively.In the positive ion mode,21differential metabolites changed in different stages of Monascus liquid fermentation,in the negative ion mode,only 4 differential metabolites were selected.Among these metabolites,L-asparagine?L-glutamine?L-lysine?citrulline?L-proline and nicotinamide may be related to the pathway of citrinin.These five metabolites were involved in the aspartate and glutamate pathways;tricarboxylic acid cycle;the pyruvate synthesis pathway and the lysine degradation pathway,and the content of these metabolites was lower than the control group.The down-regulation of these metabolites directly or indirectly regulate the synthesis of acetyl-Co A(related to the synthesis of citrinin),and ultimately lead to insufficient synthesis of acetyl-Co A,which affects the content of citrinin.(3)Finally,we added different levels of genistein to the liquid fermentation medium of Monascus,and tested its utilization and transformation after different days.Results shown that Monascus,like most fungi,can also produce genistein.When adding 0.1g/L genistein,its biomass has no obvious change compared with the control group.When 0.5 g/L,1.0 g/L and 2.0 g/L genistein were added respectively,the biomass showed an increasing trend under different days,which can be explained that genistein was not all used by Monascus,there was also a part of residue that caused the biomass were higher than that in the control group.The biomass reached the maximum on the6th day and then the biomass was decreased.Besides,there was a dose-effect relationship between the amount of genistein and its utilization rate,as the amount of addition increase,the more genistein was used.However,we found when adding 0.5g/L?1.0 g/L and 2.0 g/L genistein,the utilization of genistein gradually decreased at9th day,but when adding 0.1 g/L genistein,its utilization rate were gradually decreased at 12th day,moreover,the effect of genistein on citrinin reduction of Monascus was not obvious at the 6th and 9th days,which showed a decreasing trend at 12 day,and the decreasing effect was about 29%.