Characterization Of The Uniqueness And Correlation Of Bacterial Community And Phytochemicals In Pericarpium Citri Reticulatae ‘Chachiensis'

Pericarpium Citri Reticulatae ‘Chachiensis'(PCR-C),a famous traditional Chinese medicinal material and dietary supplementary,is regarded as superior Pericarpium Citri Reticulatae(PCR).In order to explore uniqueness of PCR-C,fifty PCR-C samples and six other PCR samples which were originated from Si Chuan(C),Zhe Jiang(Z)and Guang Xi(G)were analyzed by 16 S rRNA sequencing to explore the microbiota in PCR.Phytochemicals and physiochemical characters such as pH and moisture were also measured.The correlation among the microbiota,the phytochemicals and physiochemical characters were analyzed to attain the following conclusion:Firstly,the polyphenol flavonoids extracted from PCR were extracted and characterized with internal standards rutin and vanillic acid.They were analyzed by high performance liquid chromatography(HPLC).For PCR-C,in the free phenolic acid,salicylic acid was still the dominant one,followed by ferulic acid and coumaric acid.Among the five bound phenolic acids,salicylic acid had the highest content,followed by ferulic acid,coumaric acid,chlorogenic acid and caffeic acid,while chlorogenic acid and caffeic acid were only detected in bound fraction.Among the seven free flavonoids,the content of tangerine,hesperidin and nobiletin was high,while the content of hesperidin,neohesperidin and naringin in the bound flavonoids was high.PCR-C and other PCR were different in the content of polyphenol flavonoids.Though there was no significant difference between PCR-C and other PCR in terms of the free polyphenol flavonoids,PCR-C and PCR were obviously separated in principal component analysis(PCA)analysis in terms of the bound polyphenol flavonoids,reflecting in lowere content of naringin,hesperidin,neohesperidin and naringenin in PCR-C.Thus the bound polyphenol flavonoids contributed significantly to the special quality of PCR-C.In addition,partial least squares discrimination analysis(PLS-DA)indicated that naringin,neohesperidin,hesperidin,coumaric acid,salicylic acid,nobiletin and tangerine had significant contribution to the difference between PCR-C and PCR in the first two aging years(VIP > 1)and could be research objects for further exploring the differences between PCR-C and other PCR.The volatile oil was extracted by steam distillation.Volatile oil content extracted from same amount of PCR-C is much higher than the other PCR(P?0.03).Gas Chromatography-Mass Spectrometer(GC-MS)analysis identified 111 volatile oil components,while 48 components were identified for the first time.The compositions of volatile oil were statistically analyzed using PCA and heatmap.Significant difference was found between PCR-C and the other PCR,as not only in the high relatively content of D-limonene,?-terpinene and benzoic acid,2-(methylamino)-,methyl ester,but also in some low relatively content of ?-farnesene,terpinol and caryophyllene.PLS-DA of volatile oil in PCR aged for one and two years,indicated that other than components discovered in PCA and heatmap,(-)-Carvone,2,6,9,11-Dodecatetraenal,2,6,10-trimethyl-,(E,E,E),1-Cyclohexene-1-carboxaldehyde,4-(1-methylethenyl)-,Bicyclo[3.1.0]hex-2-ene,4-methyl-1-(1-methylethyl)-and Bicyclo[3.1.1]heptane,6,6-dimethyl-2-methylene-,(1S)-also contributed significantly to the difference between the PCR-C and the other PCR.Moreover,PCA analysis was carried on the volatile oil of high-quality PCR-C from one to five aging years.The content of volatile oil from PCR-C aged for one year was significantly different from PCR-C that have been aged for two to five years,which suggested that the first two aging years were a key period in the aging of PCR-C,also a key point for the flavor formation of PCR-C.In addition,there were not only abundant pectin but also stable protease activity detected in PCR,prompting the existence of viable bacteria in PCR.The total DNA of 56 samples of PCR was analyzed by using 16 S rRNA amplicon sequencing.It was found that 16 dominant OTUs were identified as Proteobacteria,Firmicutes and Bacteroidetes at the phylum level.The PCR of aging in the first year and PCR which were aged two to five years were clearly separated on the level of OTUs,with major associated OTU in eight genus: Enhydrobacter,Bradyrhizobium,Sphingobium,Caulobacter,Rhizobium,Singulisphaera,Novosphingobium and Chryseobacterium,the relative abundance of these OTU in the second to fifth years was higher than that in the first aging year.The physiochemical properties of PCR,such as pH and moisture,were determined.The correlation among OTU,phytochemical composition and physical and chemical properties was analyzed by canonical correspondence analysis(CCA)and pearson correlation analysis.The decreased pH through aging years was related to OTU683 and OTU20,both of which were identified as Methylobacterium.The moisture was related to OTU1558,which is identified as Enterobacter.There was also a correlation between the phytochemical composition and OTUs,for examples,OTUs from Bacillus,Stenotrophomonas,Acidisoma,Sphingomonas,Enterobacter,Neochlamydia,and Methylobacterium were associated with a variety of volatile oil components.OTUs identified as the Novosphingobium,Microbacterium and bacillus was associated with many bound polyphenol flavonoids.Here in this thesis,through correlation analysis of microbiota and phytochemicals from PCR,it was found that there were significant differences in OTUs and volatile oil content between the PCR aged for one year and PCR aged for two to five years,indicating that OTU in the early aging year were particularly critical to the whole aging process of PCR and might contributed to the volatile oil.Thus it provided foundation for further exploring the aging mechanism of PCR.