Effect Of MnSOD In Monascus On The Biosynthesis Of Monascus Pigments And Citrinin Of Monascus And Prokaryotic Expression Of MnSOD In Monascus

As an edible fungus,Monascus is an important microbial resource in China.As a natural edible pigment,Monascus has been widely used in food industry,medicine industry and other industries.However,Monascus can not only synthesize high-quality natural food pigment Monascus pigment,but also produce a kind of nephrotoxic mycotoxin citrinin.In this study,the effects of antioxidant substances such as ascorbic acid on the low citrinin production of Monascus were studied,and a prokaryotic expression method of MnSOD of Monascus rubber cctcc No.m2013082 was obtained.And then,The effect of Monascus MnSOD on Monascus was also discussed.1.Ascorbic acid was added to the fermentation medium of Monascus to detect the metabolites of Monascus.After detecting and analyzing,there are 5 kinds of fatty acids one more than intracellular fatty acid in the extracellular fermentation broth of Monascus.which is short-chain fatty acid succinic acid.The content of short-chain fatty acid succinic acid in the EAA(exogenous ascorbic acid,EAA)fermentation broth group was 26.731%,which was higher than 19.444% in the control group.It shows that the increase of short-chain fatty acids can promote the yield of Monascus pigment.At the same time,the antioxidant enzyme activities of the EAA group and the control group were detected and analyzed,and it was found that the antioxidant enzyme SOD activity in Monascus was mainly different,and combined with the results of citrinin,the SOD activity and citrinin in Monascus were found.The metabolism is closely related.2.In this study,a prokaryotic expression method of MnSOD in Monascus was obtained.By designing degenerate primers for Monascus H4000 MnSOD,the target gene fragment was obtained,and then full-length primers were designed to amplify the full-length sequence of Monascus MnSOD gene by PCR technology.After digested with Eco R Ⅰ and Hind Ⅲ,MnSOD Gene was ligated with the expression vector p ET28 a,and transformed into E.coli BL21 for induced expression.The cloned gene predicted to encode 152 amino acids,and the predicted relative molecular weight of the translated protein was 17 k D.Meanwhile,comparing the cloned MnSOD gene with NCBI database,it was found that the sequence had 99% similarity with the superoxide dismutase(SOD)gene of Monascus aurantiaaeus,and had a high similarity with the MnSOD gene of Colletotrichum gloeosporioides,Aspergillus oryzae and Aspergillus flavus.The molecular weight of MnSOD was estimated to be 19 k D by SDS-PAGE,which was consistent with the predicted molecular weight.The results also showed that the expressed protein had good acid resistance and still had enzyme activity after 1 h treatment with p H 2.0.3.In this study,the expression product of Monascus MnSOD in E.coli and sodium diethyldithiocarbamate(DDC,SOD inhibitor)were added to the fermentation medium of Monascus,and their related effects on Monascus metabolism were discussed.The results showed that the addition of MnSOD could reduce the citrinin produced by Monascus,while DDC,which inhibited SOD activity,increased citrinin production by52%.By detecting the free radical scavenging activity of Monascus,the results showed that the superoxide anion scavenging ability of Monascus fermentation broth with sod component was the highest,reaching 82%,while the superoxide anion scavenging ability of DDC component inhibiting SOD was as low as 56%.The results showed that the antioxidant capacity of Monascus could be improved by adding MnSOD,which affected the production of citrinin which is a secondary metabolite of Monascus.In addition,the content of extracellular pigment was the most in DDC group,which was35%higher than that in control group.In addition,different additive groups of Monascus intracellular pigment and intracellular pigment were analyzed.The results showed that the content of extracellular pigment in DDC group was the highest,which was 35%higher than that in control group.These results indicate that DDC can affect the ability of cell to transport pigment across the membrane,thus eliminating the inhibition of intracellular pigment metabolism and increasing the accumulation of intracellular and extracellular pigmentThese results indicate that MnSOD of Monascus can affect the formation of citrinin,a secondary metabolite of Monascus,and is a good regulatory factor,which provides a new idea for food safety Monascus pigment biosynthesis.