Study The Development Of The Microorganisms And Flavonoids Changes Of Citrus Reticulata ’chachi’ During Aging

Citri Reticulatae Pericarpium（CRP）,the dried ripe peel of Citrus reticulata Blanco and its cultivars,has been used in foods and drugs.The quality of the Citrus reticulata‘Chachi’is superior.The dried ripe pericarp of Citrus reticulata‘Chachi’,it is described as possessing the power to regulate qi,to dry dampness,and to resolve phlegm.At the same time,it can be made into the beverage and tea of Citri Reticulatae Pericarpium,additives,spices and so on.Flavonoids,as one of the major active constituents of CRP,have significant characteristics such as antioxidant,anti-inflammatory,anti-tumor,expectorant,antiasthmatic,spasmolysis,lowering blood lipid and neuroprotective effects.UPLC-Q-Orbitrap HRMS was used in this study to comprehensively identify the flavonoids in CRP under fixed sampling sites,collection seasons,processing methods,storage and curing conditions during 1-5 years.Theory of traditional Chinese medicine holds that“drugs processed long time ago have better effect”of Citri Reticulatae Pericarpium.The microbial transformation of Aspergillus Niger on the surface of CRP was correlated with the change of pharmacodynamic material basis of CRP.In addition,the microbial community structure is closely related to the aging environment changes.In this study,high-throughput sequencing technology was used to systematically study the structure and characteristics of bacteria and fungi on the surface of Citrus reticulata‘Chachi’with different aging times and regions.The correlation between flavonoids and dominant microbial genus on the surface of Citrus reticulata‘Chachi’at different aging time was studied,it provides a theoretical basis for the subsequent isolation and screening of dominant functional microorganisms,in-depth study of the aging mechanism of CRP during aging.Specific research contents are as follows:1 Analyze and identify the flavonoids of Pericarpium Citri Reticulatae（Citrus reticulata‘Chachi’）with different aging time by UPLC-Q-Orbitrap HRMSCompounds were separated on Agilent Extend-C18 column（3.0×100 mm,1.8μm）.The mobile phase was 0.1%acetic acid solution and 0.1%acetic acetonitrile solution（0～25 min,5%～95%B;25～30 min,95%B;30～30.1 min,95%～5%B;30.1～35 min,5%B）.The flow rate was 0.4 m L·min^-1,and the column temperature was set at 30℃.HRMS was performed using an electrospray ion source（ESI）,and scanned in positive and negative ion mode by means of full scan/data dependent secondary scan（Full MS/dd-MS2）.The multistage fragment ions was combined with mz Cloud network database,local high-resolution mass spectrometry database of traditional Chinese medicine components,literature information and relevant reference materials for accurate qualitative analysis.Totally 43 flavonoids in Citrus reticulata‘Chachi’were identified,including 24 flavone flavonoids,5 flavonols,13 dihydroflavones and1 chalcone.The flavonoids in Citrus reticulata‘Chachi’with different aging time were basically consistent in material types but the peak area was different.According to the comparison of relative content in peak area,it was found that the relative content of 30flavonoids showed an increasing trend with the increase of aging time.Among them,the relative content of 24 flavonoids,such as hesperidin,diosmin,isosinensetin,sinensetin,6-demethoxytangeretin,nobiletin and tangeretin increased significantly.There was no significant change in the relative content of the other 13 flavonoids,such as naringenin and neohesperidin.An efficient method was established in this work to identify flavonoids and the relative content changes of the flavonoids in Citrus reticulata‘Chachi’with different aging time rapidly and accurately.The findings provide a methodological reference for the study on pharmacodynamic material base and quality control of Citrus reticulata‘Chachi’,and it provides experimental basis that“drugs processed long time ago have better effect”of Citri Reticulatae Pericarpium.2 Variation of bacterial and fungal diversity on surface of Citrus reticulata‘Chachi’with different aging timeIn this study,high-throughput sequencing technology was used to systematically study the structure and characteristics of bacteria and fungi on the surface of Citrus reticulata‘Chachi’with different aging times,in which bacteria were sequenced by16S r DNA and fungi were sequenced by ITS1.The bacterial and fungal in Citrus reticulata‘Chachi’with different aging time were basically consistent in composition and structure but the relative abundance and the dominant microorganisms on the surface of Citrus reticulata‘Chachi’were different significantly.At the bacterial genus level were mainly unidentified＿Rickettsiales,Phyllobacterium,Methylobacterium,unidentified＿Cyanobacteri,Pseudomonas,Sphingomonas,Stenotrophomonas,Lactococcus and Bacillus.Among them,Methylobacterium and Sphingomonas were the dominant bacteria in the samples aged for 1 year.Bacillus was the dominant bacteria in the samples aged for 2 years.Pseudomonas,Stenotrophomonas were dominant bacteria in the samples aged for 4 years.Lactococcus was the dominant bacterium in the samples aged for 5 years.The level of fungal genus were mainly Xeromyces,Cladosporium,Zasmidium,Symmetrospora,Aureobasidium,Fusarium and Aspergillus.Among them,Zasmidium,Cladosporium,Symmetrospora and Fusarium were the dominant fungi in the samples aged for 1 year.Cladosporium,Aureobasidium and Aspergillus were the dominant fungi in the samples aged for 3 years.Xeromyces was the dominant fungus in the samples aged for 4 years.Based on the high-throughput sequencing technology,the structure and characteristics of microflora in Citrus reticulata‘Chachi’with different aging times were fully revealed,and the dominant microbial genera were detected detect in Citrus reticulata‘Chachi’with different aging times.It provides a theoretical basis for the subsequent isolation and screening of dominant functional microorganisms,in-depth study of the aging mechanism of Citri Reticulatae Pericarpium during aging.3 Variation of bacterial and fungal diversity on surface of Citrus reticulata‘Chachi’from different regions natural aged based on high-throughput sequencing technologySpecific climate and environmental conditions in different regions are the key factor that affects microbial community structure on the surface of CRP.To analyze the difference of bacterial and fungal community structure on the surface of Citrus reticulata‘Chachi’from different climatic conditions aged 1-year for high-throughout sequencing,namely,Monsoon Climate of Medium Latitudes（Beijing）,Subtropical Monsoon Humid Climate（Sichuan）,Subtropical Monsoon Climate（Guangdong）,Subtropical Plateau Monsoon Climate（Yunnan）.The structural composition of the bacterial and fungal communities was analyzed based on the analysis of Alpha&Beta diversity,in which bacteria were sequenced by 16S r DNA and fungi were sequenced by ITS1.The bacterial and fungal in Citrus reticulata‘Chachi’from different areas natural aged 1-year were basically consistent in composition and structure but the relative abundance and the dominant microorganisms on the surface of Citrus reticulata‘Chachi’were different significantly.At the bacterial genus level were mainly unidentified＿Chloroplast,unidentified＿Mitochondria,Methylobacterium-Methylorubrum,Sphingomonas,Mesorhizobium and Bacillus.Among them,Methylobacterium-Methylorubrum,Sphingomonas,Bacillus were the dominant bacteria in CD area.The level of fungal genus were mainly Zasmidium,Cladosporium,Aureobasidium,Symmetrospora,unidentified＿Cystobasidiomycetes＿sp and Fusarium.Among them,Aureobasidium was the dominant bacteria in BJ area.Symmetrospora,Aspergillus were the dominant bacteria in CD area.Zasmidium was the dominant bacteria in YN area.This research had determined the structural composition of the bacterial and fungal communities on the surface of Citrus reticulata‘Chachi’,with an aim to establish foundation for the screening of microorganisms suitable for aging of Citrus reticulata‘Chachi’,and provide theoretical basis for further study on the aging mechanism of Citri Reticulatae Pericarpium.