| Since ancient times,CRP has the saying of “The longer the better”,one of the six Chinese medicines,mainly used for the prevention and treatment of digestive and respiratory diseases.A large number of studies have shown that the better the CRP is,the better the accumulation of active substances in the process of aging.The previous research of this research group showed that the material basis of CRP "The longer the better" is the accumulation of polymethoxylated flavonoids(PMFs),but the cause or mechanism of its accumulation is still unclear;according to relevant reports,microbes are involved in the tangerine peel.The aging process and the ability to transform the compounds in the tangerine peel to increase the active substance content.Based on this,this study explores the accumulation mechanism of the active material of the tangerine peel from the microbial perspective by analyzing the relationship between the flavonoids and the microbial diversity of the tangerine peel and the aging time of the tangerine peel.In this experiment,different years(2017,2013,and 2003)of Xinhui Chenpi were used as raw materials.The HPLC method was used to analyze the major flavonoid components.The ABTS,FRAP,and DPPH methods were used to analyze the antioxidant properties of total flavones in CRP.Ability,HPLC-Q-TOF-MS analysis of components in flavonoid extract of CRP;results showed that a total of 13 compounds were detected in flavonoid extract of CRP in three years,with the storage time prolonged,there was no significant difference in flavonoid extract composition,the total The flavonoid content increased significantly.The total flavonoid content in 2003 increased by 69% compared to2017.Its ABTS,FRAP,and DPPH values increased significantly(p <0.05),indicating that its antioxidant capacity was also enhanced.Hesperidin and The contents of major flavonoids such as Chuanchen peel and hesperetin changed significantly,increasing by2.61 times,1.63 times,and 1.56 times,respectively.High-throughput sequencing was used to sequence the bacterial 16 S rDNA(V3 + V4)and the fungal ITS: ITS1 variable region to study the relationship between the storage time of tanned peel and its microbial diversity;the results showed that four different years(2017,2013,2003,and 1993)There were 2402 bacterial OTUs and 2981 fungal OTUs in the tanned skin samples.There was no significant difference in the bacterial Ace index and Chao1 index of the tanned skin in different years,but the fungal Ace index and Chao1 index were significantly different.Correlation analysis of sample communities showed that there was no correlation between the bacterial diversity of Chen Pei and storage time,and its fungal diversity had a significant correlation with storage time.With the increase of storage time,the abundance of Xeromyces and Xerochrysium increased,the abundance of Symmetrospora decreased,and the abundance of Mortierella increased first and then decreased.Isolated and purified Chenpi fungi,and identified the fungal species in Chenpi by 18 S rDNA.As a result,18 strains in 2017,13 strains in 2013 and 8 strains in 2003 fungi were isolated from Chenpi,which were obtained in different years The number and types of fungi decreased with the prolongation of the aging period of the peels.After removing the common fungi in each year,32 different fungi were obtained from the peels in 3 years.Hesperidin was used as the sole carbon source for 32 isolated Chenpi fungi,and the biotransformation of hesperidin was measured.The change of hesperidin content before and after the transformation of the strains was analyzed by HPLC,and the combination of product peaks was used to analyze Chenophyte and orange.The transformation of hesperidin,screened 8 fungi that have significant transformation effect on hesperidin,respectively: Aspergillus brasiliensis,Aspergillus niger,Aspergillus tubingensis,Aspergillus aculeatus,Penicillium simplicissimum,Alternaria alternata,Aureobasidium pullulans,and Clavispora lusitaniae.Each strain has different time points for the maximum conversion rate of hesperidin.The 8 strains of fungi were disrupted.Phosphate buffer was used to extract the invertase of the strain.The conversion of hesperidin was analyzed to determine the position of the invertase in the strain Intracellular,extracellular,or cellular debris,as a result,the invertases of Aspergillus brasiliensis,Alternaria alternata,and Clavispora lusitaniae were in the cell.Based on HPLC and HPLC-Q-TOF-MS(LC-MS),the products of 8 hesperidin fungi transformed into hesperidin were analyzed.The HPLC preliminary analysis confirmed the target transformation product.HPLC-Q-TOF-MS identified the transformation product,which could transform the orange Strains that have been converted to the target product by Aspergillus;as a result Aspergillus brasiliensis,Aspergillus niger and Aspergillus aculeatus can transform hesperidin to obtain the product hesperetin-7-O-glucoside.Aspergillus brasiliensis,Aspergillus tubingensis,Aspergillus aculeatus,Penicillium simplicissimum,Alternaria alternata,Aureobasidium pullulans,and Clavispora lusitaniaecan convert hesperidin to produce hesperetin.Can convert hesperidin to hesperidin-7-O-glucoside and hesperidin are Aspergillus brasiliensis and Aspergillus aculeatus.In summary,there were significant differences in the total flavonoids and main flavonoids content in the rinds of different years.There was a certain correlation between the fungi diversity of rinds and the aging time of rinds.Thirty-two fungi were isolated from the rinds.The glycoside conversion effect is significant,specifically Aspergillus brasiliensis,Aspergillus niger,Aspergillus tubingensis,Aspergillus aculeatus,Penicillium simplicissimum,Alternaria alternata,Aureobasidium pullulans,and Clavispora lusitaniae.The transformation products of these 8 fungi include hesperetin,gallic acid,hesperidin-7-O-glucoside and methyl hesperidin.Among them,the transforming enzymes of Aspergillus brazil,Alternaria alternans and Corynebacterium portugalis were in the cell. |
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