Chemical Synthesis,Isolation and Analysis of Condensed Tannins Derived from Wine Grapes

Author:Ma Zuo

Supervisor:li hua pierrelouis teissedre


Degree Year:2016





Condensed tannin is a group of nature products widely spread in the plant kingdom.They are active and their structure is complex.Thanks to its great contribution to astringency and bitterness perception in wine,condensed tannins is considered as the ―sole‖ of the red wine.However,the investigations concerning to the polymeric condensed tannins in grapes still remain in the beginning.The purified polymeric condensed tannins were always a mixture with a range of degree of polymerization.The pure condensed tannins with the targeted polymerization degree were hardly obtained.In the present study,both chemical synthesis and nature products isolation approaches were applied to search for the targeted pure condensed tannins.The astringency response of condensed tannins with diversified polymerization degree was observed.And characterizations of condensed tannins in Vitis vinifera and Vitis vinifera Sauvignon grapes seeds and skins from six vine-growing areas of Bordeaux were studied.The principle results are as follows:(1)Chemical synthesis of dimeric condensed tannins and its chemical identificationsB-type procyanidin dimers are the most common and the simplest condensed tannins derived from grapes and wine.Based on the Lewis acid catalyzation,four B-type procyanidins B1,B2,B3 and B4 were synthesized through an approach of the stoichiometric coupling of two protected monomeric units.Their purities were 99.1%,99.2%,99.5% and 99.0%,respectively.This synthesis pathway well controlled the flavan-3-ol linkage,the degree of polymerization and the stereo structures.The products were also with a pleasant yield.Procyanidin dimers B1,B2,B3 and B4 were identified by the techniques of both NMR and UHPLC-Q-ToF.(2)Fractionation,purification and identification of condensed tannins from grape seedsThe strategies to fractionate oligomeric,polymeric and a targeted flavan-3-ol were developed.And the CPC(centrifugal partition chromatography)solvent systems were ethyl acetate: ethanol: water(6:1:5,v/v/v),ethyl acetate: acetone: water(2:2:1,v/v/v)and Hexane: ethyl acetate: methanol: water(1:4:3:1,v/v/v/v),respectively.19 oligomeric condensed tannin molecules(with the polymerization degree of between 1 and 5)and 12 polymeric condensed tannin molecules(with the polymerization degree of between 1 and 5)were identified by the approach of UHPLC-Q-To F.A new flavan-3-ol(-)-Epicatechin-3-(3’’-O-methyl)gallate was discovered in grape seed.It was examined by the techniques of both NMR and HRMSn.The molecule mass in negative mode was m/z = 439.1011 and the MSMS fragments are m/z = 289.0703,m/z = 271.0594,m/z = 167.0335.The MSMS fragments in negative mode were interpreted and its possible cleavage pathway was proposed.(3)The test on salivary protein binding ability of condensed tannins with diversified degree of polymerizationThe mechanism of wine astringency perception is the non-coupled interaction between condensed tannins and salivary protein.In the present investigation,astringency intensity of condensed tannins with diversified degree of polymerization(DP)was studied by the test of interaction between the purified condensed tannins(their DP ranged from 1 to 5)and human salivary proteins in model wine,which implicates their effect on astringency perception.The results suggested a positive nonlinear relationship between the DP of condensed tannin and salivary protein affinity.(4)Characterizations of condensed tannins in grapes seeds and skins from vine-growing areas of Bordeaux,FranceUsing the condensed tannin molecule standards synthesized and purified above,we investigated the characteristics of condensed tannin composition from two grape varieries in six vine-growing areas(seven Chateaux)of Bordeaux,France in vintage 2014.Their global phenolic compounds,small molecule tannins,oligomeric condensed tannins and polymeric tannins were studied.The results indicated that: 1)A significant difference of condensed tannin composition between seeds and skins was found.The content of both total phenols and total tannins in seeds were higher than those in skins.Anthocyanidins and prodelphinidins only presented in skins;The average DP of condensed tannins in skins(21.69~61.51)was significantly higher than those in seeds(8.9~15.4).2)There was a significant difference of condensed tannin composition between Vitis vinifera and Vitis vinifera Sauvignon.In skins,the content of small tannins molecules from Merlot was higher than that from Cabernet Sauvignon,and both DP and prodelphinidin from Merlot was lower than those from Cabernet Sauvignon.3)There was a significant difference of condensed tannin composition among different appellations of Bordeaux.A significant difference was observed in DP,galloylated degree and prodelphinidin content of condensed tannins among different appellations in Bordeaux.The DP of seeds(15.4),the DP of skins(61.51)and the content of prodelphinidin(46.64%)in Chateau V1 were significately higher than those in the other Chateaux.