| Tangerine peel has the functions of antitussive,antioxidant and anti-tumor,etc.Among them,Pericarpium citri reticulatae chachiensis(PCR-C)has the long history and is the unique Chinese medicine,which is dried from Pericarpium Citri Reticulatae‘Chachi’ peel.PCR-C,from Guangdong Province,is known as "the longer,the better".The main bioactive substances of PCR-C are flavonoids,and the aging process of PCRC is essential for the formation of its quality.Studies have shown that the composition and content of flavonoids change during the aging process of PCR-C,while a large number of fungi are present on the surface for metabolic activities.However,few studies have been reported on the correlation between active substances and the metabolic of surface fungi.Therefore,the aim of this study was to investigate systematically the changes in the composition of surface fungi,the trends of flavonoids and the mechanism of fungal metabolism on flavonoids during aging of PCR-C,using fresh peels,aged 1,3,5 and 10 years peels as experimental materials.The main results of the study are as follows.(1)Based on high-throughput sequencing technology and fungal pure culture isolation and identification experiments,the composition and changes of fungal communities on the surface of PCR-C in different aging years were investigated.The results showed that at the genus level,the dominant genus of fresh orange peel and aged1 year PCR-C were Aspergillus,and the dominant genus of aged 10 year old PCR-C were Xeromyces and Mortierell.The samples were divided into three groups: fresh peels and aged 1 year peels,aged 3 and 5 years peels,aged 10 years peels by Beta Diversity;18 strains of fungi were isolated from different PCR-C and Penicillium commune,Aspergillus niger and Rhodotorula mucilaginosa were present in all five groups.In conclusion,a variety of fungi existed during the storage of PCR-C,and the composition of the fungi changed significantly with the increase of storage time.(2)Based on analysing the extracts of PCR-C by HPLC-Q-TOF-MS/MS and UPLC experiments to investigate the trends of flavonoids in PCR-C with different aging years.The results showed that the total flavonoid content in PCR-C gradually increased from 4.58±0.02 mg RE/g in fresh peels to 15.30±0.20 mg RE/g in aged 10 years peels,and the highest growth rate was 3.12±0.08 mg RE/g-y in aged 0-1 years.The antioxidant activity increased gradually by ABTS,FRAP and DPPH assays,which was consistent with the trend of total flavonoid content.Meanwhile,a total of 17 flavonoids were identified among which 12 polymethoxyflavonoids(PMFs);in addition,the content of hesperidin in 10-year-old PCR-C was significantly lower than that in fresh peels,while the content of other 8 PMFs increased significantly during aging(p<0.05).In conclusion,the total flavonoid content and antioxidant activity increased and PMFs content accumulated during storage of PCR-C.(3)Based on different fungi on the fermentation of fresh peel during the process of determination of flavonoids,enzyme activity and other indicators,to study the different strains of different fermentation time on the effect of fresh peel flavonoids.The results showed that six species of fungi,namely Penicillium commune,Mucor circinelloides,Penicillium citrinum,Aspergillus niger,Rhizopus microspores and Rhodotorula mucilaginosa,could grow normally on fresh peel powder medium;the total flavonoid content and antioxidant activity of fresh peel fermented by liquid fermentation technology changed significantly,among which Penicillium commune,Penicillium citrinum and Rhodotorula mucilaginosa made the changes of both increased first and then decrease,while Mucor circinelloides,Aspergillus niger and Rhizopus microspores made the changes of both increased continuously.At the same time,most of the PMFs content have increased to some extent.Except Penicillium commune fermentation process,the other strains of fermentation of hesperidin content first increased and then decreased;In addition,the CMCase enzyme activity in Mucor circinelloides,Aspergillus niger and Rhizopus microspores was relatively high,the CMCase enzyme activity produced by the six species were higher than that of β-glucosidase.The Pearson correlation analysis between total flavonoids and hesperidin and the two enzyme activities showed that the changes in flavonoid content of the strains were significantly correlated with the enzyme activities to some extent;finally,the fermentation with hesperidin showed that Penicillium commune and Penicillium citrinum could gradually decompose hesperidin,with the former detecting a gradual increase in hesperetin and the latter detecting the production of hesperetin and HMG.The Mucor circinelloides,Aspergillus niger and Rhizopus microspores all made hesperidin increase in the early stage of fermentation,and then decomposed hesperidin to HMG and hesperetin.In conclusion,the fermentation of the strains had a significant effect on the flavonoid content and transformation of fresh peel,which was influenced by the activity of related enzymes during the fermentation process.In conclusion,a large number of fungi existed on the surface of PCR-C during aging and the fungal community composition changed from Aspergillus to Xeromyces and Mortierella;and the total flavonoid and antioxidant activities increased significantly during aging and the content of PMFs accumulated.Meanwhile,six different single strains isolated had different effects on the releasing and transformation of flavonoids from fresh peels.This study systematically analyzed the changes of fungi,flavonoids and the effects of surface fungi on flavonoids during aging process of PCRC,which provided a new idea for aging pretreatment of PCR-C and enriched the scientific concept of "the longer,the better" of PCR-C. |
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