Research On The Quality-related Chemical Constituents Of Raw Pu-erh Tea During Processing

Pu-erh tea is a kind of special tea made from the fresh leaves of Yunnan big leaves species(Camellia sinensis)through special processing technology,including raw Puerh tea(RPT)and ripen Pu-erh tea.RPT belongs to unfermented tea and possesses similar processing technology with sun-dried green tea,which mainly includes withering,de-enzyming,rolling,drying,and autoclaving–compressing.The complex chemical components contained in RPT significantly contribute to its the flavor quality and biological activity.Up to now,there have been many works performed on the chemical composition of different types of tea,but the chemical composition RPT is still not fully characterized,especially the changes of volatile and non-volatile components during manufacturing need to be further clarified.Therefore,it is of great importance for comprehensive analysis and characterization of volatile and non-volatile chemical components in RPT and in-depth study of their changes during processing,which is good for a better understanding of the material basis of flavor quality and biological activity of RPT and for clarification of the effect of processing technology on the quality and chemical composition of RPT.In this work,liquid/gas chromatography coupled with mass spectrometry(GC/LC-MS)techniques were used to characterize the volatile and non-volatile chemical components in RPT,and multivariate statistical analysis was used to study their changes during manufacturing.Detailed contents of current work are summarized as following:(1)Quantitative analysis and changes of main compounds in RPT.Based on ultrahigh performance liquid chromatography-triple quadrupole-mass spectrometry/mass spectrometry(UPLC-QQQ-MS/MS),this work established an effective analytical method that can simultaneously determine the accurate content of 35 chemical components including polyphenols,amino acids and alkaloids within only 10 min.The results of quantitative analysis indicated that EGCG,ECG,GCG,CG,caffeine,EGC,EC,C,theanine and GC were determined as the top 10 components(4085.44-76440.73 ?g/g DW)in RPT.Theanine(7634.76 ?g/g DW),caffeine(43441.11 ?g/g DW),EGCG(76440.73 ?g/g DW),rutin(416.80 ?g/g DW)and gallic acid(835.26?g/g DW)are the compounds with highest contents of amino acids,alkaloids,catechins,flavonoids and phenolic acids in RPT,respectively.The results of analysis of variance based on accurate content information showed that the manufacturing process,especially de-enzyming and autoclaving–compressing,had significant effects on the contents of 35 main chemical components(p < 0.05),but the changing trends of different types of compounds were inconsistent.(2)Non-targeted characterization and manufacturing-induced changes of nonvolatile components in RPT.By using ultra-high performance liquid chromatographyhigh resolution mass spectrometry(UPLC-HRMS),the RPT samples collected from withering,de-enzyming,rolling/drying and autoclaving–compressing was analyzed in a non-targeted manner.The results of principal component analysis showed that there were great differences in the non-volatile profile of RPT at different manufacturing stages.De-enzyming and autoclaving–compressing have more significant effects on the non-volatile components,while the effect of drying treatment seems to be negligible.Through partial least squares-discriminant analysis,91 differential compounds including catechins and their oligomers(34),phenolic acids(15),flavonoids and their glycosylated derivatives(10),free amino acids(15),alkaloids(7)and other substances(10)were further screened and identified.After processing,the content of 40 compounds increased significantly,while the content of 49 compounds decreased significantly.(3)Qualitative and quantitative analysis and manufacturing-induced changes of volatile components in RPT.Headspace solid phase microextraction(SPME)combined with gas chromatography-mass spectrometry(GC-MS)was used to establish a method for the analysis of volatile components in RPT samples at different processing stages.Aided by chemometrics,58 volatile compounds were qualitatively and quantitatively analyzed.Results show that de-enzyming and autoclaving–compressing have large effects on the volatile composition,with the total volatile contents sharply decreased by 76.68% and 35.77%,respectively.After de-enzyming,the relative content of aldehydes increased by 4.68-fold,whereas that of alkenes decreased by 2.65-fold;thus,the attractive fatty aroma must be enhanced while weakening the unpleasant grassy aroma.In addition,the relative contents of ?-terpineol,geraniol and 1-octene-3-ol with special aroma such as peppermint and rose increased by 1.42-1.98 times after autoclaving–compressing treatment.On the whole,de-enzyming and autoclaving–compressing were suggested as the key stages for the formation of characteristic aroma of RPT,because of their significant effects on the composition and content of volatile components.