The Effect Of Flavonoids On Citrinin Production By Monascus Aurantiacus AS3.4384 And Its Related Omics Study

Monascus can produce a variety of beneficial secondary metabolites,such as Monascus pigment,Monacolin K,?-aminobutyric acid,etc.However,the existence of citrinin in Monascus restricted the wide application of Monascus products.Citrinin is a mycotoxin with strong nephrotoxicity,which has great damage to human health.Therefore,how to reduce the content of citrinin in Monascus is one of the focuses of Monascus research.Flavonoids are compounds with ketone carbonyl groups in nature,which have anti-oxidation and anti-aging effects.By adding flavonoids to the liquid fermentation of Monascus,the effect of flavonoids on citrinin and pigment production by Monascus was studied.The gene sequencing and transcriptomics were used to investigate the effect mechanism of flavonoids on the synthesis of citrinin from Monascus.The main research contents are as follows:(1)During the liquid fermentation process,the fermentation medium after adding genistein is optimized.Pearl rice was used as the carbon source for fermentation,the amount of rice flour added was 20 g/L,and the fermentation time was 12 d.The content of citrinin was reduced by about 73.2%compared with the blank group without drug addition.Among the optimization results of the types and contents of flavonoids,genistein has the best effect among nine flavonoids,which can reduce the content of citrinin by 80%.In studying the relationship between the structure and activity of nine flavonoid compounds,the different active sites that affected the production of citrinin from Monascus were preliminary explored.(2)Two flavonoid compounds were simultaneously added to the liquid fermentation medium of Monascus aurantiacus AS3.4384 to investigate its effect on the production of citrinin.After the addition of genistein and baicalein,the citrinin content decreased by 79.8%,while the addition of soybean isoflavone and baicalein reduced the citrinin content by 66.3%.After adding genistein and baicalein,the yellow,orange and red pigment contents of Monascus were 3.31,1.65 and 1.58 times of the original.In addition,genistein was added to the culture media of three strains of Monascus A,B,and C respectively.The results showed that the addition of genistein to Monascus A can reduce the content of citrinin by about 62.9%.In the liquid fermentation of Monascus B,genistein is added to reduce the content of citrinin to 90.1%.In Monascus C,the addition of genistein can reduce the content of citrinin by 94.8%and has no effect on the pigment content.When 2 g/L genistein and 0.1 g/L baicalein,2 g/L soybean isoflavone and 0.1 g/L baicalein were added at the same time,the citrinin content was reduced by 88.7%and 56%,respectively.(3)In order to explore the influencing factors of genistein on citrinin at the molecular biological level,the Monascus genome was sequenced first,and the data after the gene sequencing was quality-controlled to obtain better quality sequencing data.The total number of sequencing reads in the sequencing results was 55729,and the N50 length was 12558 bp.The quality score was 0.89,indicating good sequencing results.21 contigs were obtained from the gene assembly results after sequencing,and the GC content in the gene was 49.01%.After that,the coding genes,repeat sequences and non-coding RNA of the main components of the Monascus genome were predicted.The results showed that the number of coding genes was 6564,the number of interspersed repeat genes was 2443,the number of tandem repeat sequences was 5743,the number of non-coding RNA genes is 207.Finally,predict gene function,including functional analysis of GO,KEGG,KOG and NR.(4)Based on the completion of sequencing of the Monascus genome,transcriptome analysis was perforrued on the control group with genistein and the blank group without genistein.In the comparison of the differential genes between the two groups,it was found that there were 942 differential genes,including 564 up-regulated genes and 378 down-regulated genes.Among the down-regulated genes,we focused on genes related to citrinin biosynthesis and quantified them in real time PCR verification.It was found that orf5(A4928),pksCT(A4929),orf3(A4931),orfl(A4933),orf6(A4934),and ctnE(A4935)all showed a significant downward trend,indicating that the addition of genistein does affect citrin The synthesis of related genes prompts a significant drop in citrinin content.Then the difference genes of the two groups were analyzed by GO and KEGG functional enrichment.