Alexandre, H.

Chassagne, D.

Feuillat, M.

Massoutier, C.

Salles, C.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 39 (4), 177-178 (2000)  Research Note  The aroma glycosides composition of Burgundy Pinot noir must  D. CHASSAGNE, H. ALEXANDRE, C. MASSOUTIER, C. SALLES and M. FEUILLAT   K e y w o r d s : red wine, aroma precursors, chromatogra- phy, mass spectrometry.  Introduction: The sensory significance of hydrolysis products of glycosidically bound compounds to varietal aroma has been well established (WILLIAMS and FRANCIS 1996). Varieties such as Muscat (VOIRIN et al. 1990 and 1992), Syrah (RAZUNGLES et al. 1993) and Riesling (SKOUROUMOUNIS and WINTERHALTER 1994) have been  the most studied cultivars to establish databases of aroma glycosides. For Pinot noir, only volatile compounds re- leased after enzymatic hydrolysis of glycosidic extracts from clones were reported (PIGELLA et al. 1998). Tt has previously been found that enzymatic hydrolysis takes place sequentially (GUNATA et al. 1988). According to this find- ing and to the glycosidase specificity, the structure of aroma glycosides needs to be known to control the enzymatic hy- drolysis process for the release of volatile compounds. This study aimed at the identification of aroma glycosides from a must with the analysis of glycosidic extract by GC-MS after   trifluoroacetylation.  Material and Methods: Must was obtained from grapes (cv. Pinot noir) collected in 1997 at the vineyard of the ex- perimental station of the University of Burgundy. Tn order to remove polyphenolic compounds, grape juice was stirred for 30 min at 4 oC in the presence of PVPP (1:10, w/v). The glycosidic fraction was obtained according to the proce- dure described by WILLIAMS et al. (1982). Clear juice (2.0 l) obtained after centrifugation at 10,000 g for 15 min at 10 oC was passed through a column (200 x 30 mm i.d.) filled with C18 reversed-phase adsorbent (30 g). Water-soluble com- pounds were removed with 1.0 l of distilled water and the retained material was eluted with 300 ml of methanol at rates up to 2 ml·min-1. Enzymatic hydrolysis was performed on a glycosidic extract corresponding to 200 ml of clear juice. The sample concentrated to dryness was dissolved by 0.5 ml of 0.2 M citrate-phosphate buffer at pH 5 and extracted 5 times using pentane-dichloromethane (2:1) and 0.5 ml of an enzymatic extract (20 mg·ml-1 of enzymatic preparation AR2000 (Gist Brocades) in buffer 0.2 M citrate-phosphate, pH 5). After incubation at 40 oC for 16 h, 10 1-g of 5-nonanol were added as internal standard and the mixture was ex- tracted twice with 250 1-l of pentane/dichloromethane (2:1). This aglycon extract was directly analysed by GC-MS. Sug- ars of glycosides were converted into alditol acetates after  Correspondence to: Dr. D. CHASSAGNE, Institut Universitaire de la Vigne et du Vin, Universite de Bourgogne, Campus Montmuzard, rue Claude Ladrey, BP 27877, F-21078 Dijon cedex, France. Fax: +33-3-8039 6265. E-mail: dchassa@u-bourgogne.fr acid hydrolysis (2 M trifluoroacetic acid solution at 120 oC for 1.5 h) and analysed by GC (HARRIS et al. 1984). Synthetic glycosides and natural glycosides obtained from 40 ml must were trifluoroacetylated according to the method described by VOIRIN et al. (1992). GC was performed with Chrompack CP 9001 gas chro- matograph equipped with a HP-5 capillary column (30 m x 0.25 mm i.d.; 0.25 1-m bonded phase) for trifluoroacetylated glycosides and a Rtx®-2330 capillary column (15 m x 0.32 mm i.d.; 0.2 1-m bonded phase) for alditol acetates. The operat- ing conditions used for GC of trifluoroacetylated glycosides were those reported by VOIRIN et al. (1992). The operating conditions for alditol acetates: injector and detector tem- perature 265 oC; nitrogen carrier gas flow rate 1.4 ml·min-1. Column temperature was raised from 175 to 240 oC at 8 oC·min-1, hold for 1 min at 240 oC and then increased to 265 oC at 8 oC·min-1. ET mass spectra were recorded by coupling an HP 5890 gas chromatograph equipped with DB™Wax capillary col- umn (30 m x 0.32 mm i.d.; 0.5 1-m) for aglycons and a HP-5 capillary column (30 m x 0.32 mm i.d.; 0.25 1-m) for trifluoroacetylated derivatives and a splitless injector to a HP 5970 mass spectrometer. For trifluoroacetylated de- rivatives, column temperature programs were performed according to VOIRIN et al. (1992) and for GC of aglycons, column temperature was increased from 40 to 250 oC at 3 oC·min-1; the carrier gas was N2 at 2 ml·min-1. The injector temperature was maintained at 250 oC, the detector tempera- ture at 250 oC. Helium was used as carrier gas at 35 cm·s-1 at 200 oC. The mass range was scanned between 70 and 600 amu for trifluoroacetylated derivatives and between 28 and 300 amu for aglycons, ionisation voltage 70 eV.  Results and Discussion: GC analysis of alditol acetate from glycosidic moiety released by acidic hydrolysis of the glycosidic extract Pinot noir must showed the presence of glucose (77 %), apiose (12 %), arabinose (9 %) and rham- nose (2 %). These sugars are commonly reported as compo- nents of the sugar moiety of flavour glycosides in Vitis vinifera (VOIRIN et al. 1990, 1992). The high molar ratio of glucose can be explained by the presence of anthocyanins in the glycosidic extract obtained after elution of must on C18 column. From these results and with regard to glycosides already identified in other V. vinifera cvs (VOIRIN et al. 1992), the presence of glucoside, arabinosyl glucoside, rhamnosyl glucoside and apiosyl glucoside can be postulated. Glycosides have been characterized by means of GC and GC/ET-MS of their trifluoroacetylated derivatives (VOIRIN et al. 1990, 1992). Several constituents of the C18 fraction presented a mass spectrum containing ions typi- cally related to the electron impact mass fragmentation of trifluoroacetylated derivatives of glycosides (ions at m/z 193, 205, 207, 319). A comparison of the GC data reported by VOIRIN et al. (1992) for synthetic glycosides with the detected compounds of our trials revealed a dominance of disaccharides in the fraction analysed. The chromatographic and MS data for synthetic TFA glycosides and certain com- pounds detected in natural glycosidic extract were coinci- dent (Table). Thus, benzyl and 2-phenylethyl glucopyrano- sides and 6-0-(a-L-rhamnopyranosyl)-ß-D-glucopyrano-  Peak no.  Compound in Figure   unknown ref  1  benzyl Glcp  1.115  1.119  2 2-phenylethyl Glcp 1.259 1.266  3 hexoside 1.380   4 hexoside 1.452   5 benzyl Rhap-Glcp 1.727 1.714  6 benzyl Araf-Glcp 1.801 1.807  7 benzyl Apiof-Glcp 1.810   8 2-phenylethyl  Rhap-Glcp 1.825 1.836  9 benzyl pentosyl hexosides 1.988  Figure: Gas chromatograms of trifluoroacetylated glycosi10 hexoside 2.017  Pinot noir must (a) before enzymatic treatment and (b) a11 benzyl pentosyl hexoside 2.095  actio of AR2000. IS: internal standard (phenyl ß-D-gluc12 pentosyl hexoside 2.222  pyranoside); 1: benzyl ß-D-glucopyranoside; 2: 2-phenylethy 178 D. CHASSAGNE, H. ALEXANDRE, C. MASSOUTIER, C. SALLES and M. FEUILLAT  T a b l e  Trifluoroacetylated derivatives of glycosides identified or tenta- tively identified in Pinot noir must from Burgundy   RRT                    D-glucopyranoside; 5: benzyl a-L-rhamnopyranosyl ß-D-gluco- pyranoside; 6: benzyl a-L-arabinofuranosyl ß-D-glucopyranoside; RRT: Retention time relative to phenyl ß-D-glucopyranoside  sides and benzyl 6-0-(a-L-arabinofuranoside)-ß-D-gluco- pyranoside were identified unequivocal in Pinot noir must. The presence of benzyl ß-D-apiofuranosyl ß-D-gluco- pyranoside was highly probable with regard to the MS and GC data obtained in our study and those reported by VOIRIN et al. 1990. This confirms our results concerning the sugar moiety in the composition of glycosides. All identified com- pounds were also found in must of the other cultivars stud- ied for their bound aroma compounds composition (VOIRIN et al. 1990, 1992, LOPEZ-TAMAMES et al. 1997). Many unknown peaks in the chromatogram of TFA must glycosides (cv. Pinot noir) showed fragment ions characteristics of glyco- sides with osidic parts of hexoside and pentosyl hexoside (Table). Except for two peaks at RRT 1.988 and 2.095 for which ET-MS data correspond to glycosides with benzyl alcohol aglycone, characterization of their aglycone struc- tures was not possible due to the lack of published refer- ence mass spectra. The glycosidic fraction obtained from Pinot noir must was hydrolysed by the enzymatic preparation AR2000. The trifluoroacteylated glycosides present in the initial extract and after enzymatic action were analysed by GC and the results are given in the Figure. The chromatographic pro- file of the glycosidic extract was modified by action of enzymatic preparation AR2000. Tn the range of high reten- tion times, most of the detected peaks corresponding to mono- and diglycosides disappeared after action of enzym- atic preparation. Total hydrolysis of detected glycosides was then performed. Aliphatic and aromatic alcohols, phe- nols and C13 norisoprenoid released after enzymatic hy- drolysis of the glycosidic extract were identified by GC-MS and all have already been reported as constituents of glyco- sides in several Pinot noir clones by PIGELLA et al. (1998). Benzyl and 2-phenylethanol were the major components, which is in accordance with previous data given above for the composition of glycosides. 7: benzyl ß-D-apiofuranosyl ß-D-glucopyranoside; 8: 2-phenyl- ethyl a-L-rhamnopyranosyl ß-D-glucopyranoside; 3,4,9,10,11,12: Unknown glycosides.  We greatly acknowledge support by C. GINIES (Laboratoire de Recherches sur les Arömes, INRA, Dijon, France) for the GC-MS experiments and J. CROUZET, Laboratoire de Genie Biologique et Sciences des Aliments, Universite de Montpellier II, for providing reference compounds.  GÜNATA, Y. Z.; BITTEUR, S.; BRILLOUET J. M.; BAYANOVE, C. L.; CORDONNIER, R.; 1988: Sequential enzymatic hydrolysis of potentially aromatic glycosides from grapes. Carbohydr. Res. 184, 139-149. HARRIS, P. J.; HENRY, R. J.; BLAKENEY, A. B.; STONE, B. A.; 1984:An improved procedure for the methylation analysis of oligosaccharides and polysaccharides. Carbohydr. Res. 127, 59-73. LOPEZ-TAMAMES, E.; CARRO-MARINO, N.; GÜNATA, Z. Y.; SAPIS, C.; BAUMES, R.; BAYONOVE, C.; 1997: Potential Aroma in several varieties of spanish grapes. J. Agric. Food Chem. 45, 1729-1735. PIGELLA, R.; BOSSO A.; DI STEFANO R.; CORINO L.; MALERBA, G.; 1998: Pinot noir characterization by polyphenols and aroma precursors study. Riv. Vitic. Enol. 1, 45-62. RAZUNGLES, A.; GÜNATA, Z.; PINATEL, S.; BAUMES, R.; BAYONOVE C.; 1993: Etude quantitative de composes terpeniques, norisoprenoides et de leurs precurseurs dans diverses varietes de raisins. Sci. Aliments 13, 59-72. SKOUROUMOUNIS, G. K.; WINTERHALTER, P.; 1994: Glycosidically bound norisoprenoids from Vitis vinifera cv. Riesling. J. Agric. Food Chem. 42, 1068-1072. VOIRIN, S. G.; BAUMES, R. L.; BITTEUR, S. M.; GÜNATA, Y. Z.; BAYONOVE, C. L.; 1990: Novel monoterpene disaccharide glycosides of Vitis vinifera grapes. J. Agric. Food Chem. 38, 1373-1378. - -; - -; SAPIS, J. C.; BAYONOVE, C. L.; 1992: Analytical methods for monoterpene glycosides in grapes and wine. II. Qualitative and quantitative determination of monoterpene glycosides in grape. J.  Chromatogr.  595, 269-281. WILLIAMS, P. J.; FRANCIS, I. L.; 1996: Sensory analysis and quantitative determination of grape glycosides: the contribution of these data to winemaking and viticulture. In: G. R. TAKEOKA, R. TERANISHI, P. J. WILLIAMS, A. KOBAYASHI (Eds.): Foods Biotechnology for Im- proved Flavors, 124-133. ACS Symposium Series 637. American Chemical Society, Washington, DC. - -; STRAUSS, C. R.; WILSON, B.; 1982: Use of C18 reversed-phase liquid chromatography for the isolation of monoterpene glycosides and nor-isoprenoid precursors from grapes juice and wines. J. Chromatogr.  235,  471-480. 

The aroma glycosides composition of Burgundy Pinot noir must

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The aroma glycosides composition of Burgundy Pinot noir must

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