SIMULTANEOUS DETERMINATION OF GLYCEROL, FRUCTOSE AND GLUCOSE FOR QUALITY CONTROL OF WINES

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1 SIMULTANEOUS DETERMINATION OF GLYCEROL, FRUCTOSE AND GLUCOSE FOR QUALITY CONTROL OF WINES Patrizia Restani, Andrea Persico, Cinzia Ballabio, Dalia Fuggetta and Enzo Moro Dept. Pharmacological Sciences, State University of Milano via Balzaretti 9, 2133 Milano, Italy Key words: Glycerol,,, quality control, HPLC Abstract Glycerol is the major by-product of ethanol fermentation and could be associated with wine quality. A simple method of assaying, and in wine by highperformance liquid chromatography using a modified polar bonded NH 2 -silica phase column and a refractive index detector was used to determine their concentrations in high- and standard-quality wines. Results show., and levels ranged from 1.65 to 9.54, non-detect to 2.57 and non-detect to 1.63 g/l, respectively, for high-quality wines. For standard-quality wines, these levels ranged from 2.67 to 8.43, 1.29 to 9.12, and.62 to 3.6 g/l, respectively. Fructose and levels (76.2±3.8 and 43.9±18. g/l, respectively) were much higher in sweet and sparkling wines. Introduction Glycerol is predominant among several polyols usually found in wine, which also contains erythritol, arabitol, mannitol, sorbitol, mesoinositol and 2,3-butanediol. Glycerol is the major by-product of ethanol fermentation and results from the reduction of dihydroxyacetone phosphate to 3-phosphate concomitant with NADH oxidation by NAD + -dependent 3-phosphate dehydrogenase (Gancedo et al. 1968). Several factors are involved in the final content of in wine: ripeness of the grapes, the micro-organisms present, the type of cellar equipment, the ph and temperature of fermentation, the source of nitrogen and the strain of yeast used in fermentation (Cronwright et al., 22). The concentration in wine is rarely higher than 12 g/l unless the grapes are massively infected by the fungus Botrytis cinerea, when the concentration can be as high as 25 g/l (Ribereau-Gayon et al.,1998). Several authors have determined the concentration in investigating its correlation with wine quality: the results were inconclusive (Nieuwoudt et al., 22; Sehovic et al., 24). However, the presence of in wines can acquire new interest in wine control also considering that this ingredient is used as an adulterant to improve the sweetness, body, and fullness of wines. In this research we simultaneously determine, and, comparing their concentrations in high and standard-quality wines. The analytical technique used is a simple liquid-chromatography which uses a polar bonded silica (-NH 2 modified) stationary phase connected to a refractive index detector (Moro et al. 27). Material and Methods Chemicals and Reagents Glycerol, and of analytical grade were purchased from Carlo Erba (Milan, Italy).

2 Wines Analysis was performed on certified (EC Regulation 22) and uncertified wines listed below. High-Quality Red Wines Standard-Quality Red Wines Barbera d Alba DOC 24 (BA-4) Bricchello Rosso (BRR) Barbera del Monferrato DOC 23 (BM-3) Cabernet del Veneto (CV) Barbera del Monferrato DOC 25 (BM-5) Merlot del Veneto (MV) Castel del Monte Rosso DOC 23 (CMR-3) Tavernello Rosso 1 (TR1) Chianti Colli Senesi DOCG 22 (CS-2) Tavernello Rosso 2 (TR2) Montepulciano d Abruzzo DOC 23 (MA-3) Vigna dei Colli Rossi (VCR) Rosso di Puglia ITG 23 (RP-3) Rosso di Sardegna DOC 1998 (RS-98) Sangiovese Umbria ITG 24 (SN-4) Sassoalloro ITG 24 (SS-4) Sudtiroler Lagrein Rosso DOC 2 (SUL-) Teroldego Rotaliano DOC 24 (TR-4) Velletri Rosso DOC 2 (VR-) High-Quality White Wines Standard-Quality White Wines Bianco di Custoza DOC 23 (BC-3) Bianco Frizzante (BF) Frascati Superiore DOC 22 (FS-2) Bricchello Bianco (BRB) Frascati Superiore DOC 23 (FS-3) Chardonnay Frizzante Secco (CFS) Lugana Superiore DOC 23 (LS-3) Garzellino (GZ) Sudtiroler Sauvignon DOC 23 (SS-3) Tavernello Bianco 1 (TB1) Tavernello Bianco 2 (TB2) Others Asti Spumante DOCG 25 Bracchetto d Acqui Fragolino Moscato Moscato d Asti Vin Santo (AS) (BA) (FR) (MS) (MSA) (VS) High Performance Liquid Chromatography An 88-PU Liquid Chromatographic Pump (Jasco Corporation, Tokyo, Japan) connected to a refractive index (RI) detector RI-231 (Jasco Corp.) was used for the analysis of the wines. The analytical column was a 25x4 mm LiChrosorb (Merck, Darmstadt) packed with a 5-µm diameter NH 2 -silica phase and was protected by a RP 18 guard column (Merck). The mobile phase was acetonitrile water (85:15, v/v) pumped under constant flow at 1. ml/min at room temperature. Samples were injected via a Model 7125 injector valve equipped with a 2 µl loop (Rheodyne, Cotati, CA, USA). On-line data acquisition and calculations were performed by a Data Jet Integrator (Thermo Separation Products, Riviera Beach, FL, USA) and Winner/386 Autolab software (Thermo Separation Products). Wine samples were injected as such. Statistical analysis The significance of the difference between the mean values was calculated by analysis of variance (ANOVA) and then by Bonferroni multiple comparison test.

3 Results Figure 1A shows a typical chromatogram of a standard solution containing 5.3 g/l (peak 4), 2.5 g/l (peak 5) and 3. g/l (peak 6). The calibration graphs calculated for were linear over the concentration range.1 12 g/l, with correlation coefficients r 2 >.999. Similar good linearity was obtained for and in the concentration range.1 15 g/l. In all cases, the limit of detection and quantification was.1 g/l. A B C Figure 1 Chromatograms of, and A = standard solution ( 5.3 g/l, 2.5 g/l, 3. g/l) B = High-quality wine (BM-5) C = Standard-quality wine (TR-2) Figure 1 B and C show the chromatograms of two different samples of wine, where the differences between standard- and high-quality wines is easily observable: the high-quality wine (Figure 1B) contains very small amounts of and while the two sugars are easily measurable in standard wines (Figure 1C). On the other hand, concentrations of are similar in both samples. Figures 2 and 3 show the concentrations of, and in high and standard quality wines, respectively. The contents of the three analytes between high- and standard-quality wines were compared and statistically evaluated.

4 g/l BA-4 BM-3 BM-5 CMR-3 CS-2 MA-3 RP-3 RS-98 SN-4 SS-4 SUL- TR-4 VR- BC-3 FS-2 FS-3 LS-3 SS-3 Red Wines White Wines Figure 2 Concentration (g/l) of, and in high-quality wines g/l BRR CV MV TR1 TR2 VCR BF BRB CFS GZ TB1 TB2 Red wines White wines Figure 3 Concentration (g/l) of, and in standard-quality wines g/l AS BA FR MS MSA VS Wines Figure 4 Concentration (g/l) of, and in sparkling and sweet wines

5 Results show., and levels ranged from 1.65 to 9.54, non-detect to 2.57 and non-detect to 1.63 g/l, respectively, for high-quality wines. For standard-quality wines, these levels ranged from 2.67 to 8.43, 1.29 to 9.12, and.62 to 3.6 g/l, respectively. There was a highly significant statistical difference between high- and standard-quality wines in concentration and concentration but no significant difference in content (Table 1). Table 1 Glycerol, and concentration in high- and standard-quality wines Analyte Glycerol Fructose Glucose High-quality wines (m±sd) 6.23± ±.73.32±.44 Standard-quality wines (m±sd) 4.98± ± ±1.16 Statistical difference p>.5 p<.1 p<.1 Important differences can be found also when the ratio between / (p<.5) and / (p<.1) concentrations are considered. The characteristic concentration of, and in some sweet and sparkling wines is shown in Figure 4; in this case, and levels (76.2±3.8 and 43.9±18. g/l, respectively) were much higher. In conclusion, this research shows that, and contents can represent a new tool to differentiate high- and standard-quality wines. To improve the significance of this determination, a larger sampling is required; moreover, ranges of the suitable amount of and sugars in each high-quality wine should be established. Further analyses are necessary to better define if the ratio between and sugar substrates could be used as a marker of wine quality and if these parameterss could be used to identify adultered wines due to the addition of or sugars. References Cronwright G.R., J.M. Rohwer and B.A. Prior. 22. Metabolic control analysis of synthesis in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 68: EC Regulation 753/22, Commission Regulation (EC) No 753/22 of 29 April 22 laying down certain rules for applying Council Regulation (EC) No 1493/1999 as regards the description, designation, presentation and protection of certain wine sector products. Official Journal L118, dated Gancedo, C., J.M. Gancedo, and A. Sols Glycerol metabolism in yeast. Pathways of utilization and production. Eur. J. Biochem. 5: Moro E., R. Majocchi, C. Ballabio, S. Molfino, and P. Restani. 27. A rapid and simple analytical method for the simultaneous determination of, and in wine. Am J Enology and Viticulture 58 (2) in press. Nieuwoudt H., B. Prior, S. Pretorius, and F. Bauer. 22. Glycerol and wine quality: fact and fiction. In: Wynboer: a technical Guide for Wine Producers, nov. 22 ( Ribereau-Gayon J., Y. Glories, A. Maujean, and D. Dubourdieu The microbiology of wine and vinification. In: Handbook of Enology, vol. II, 1st edition, John Wiley and Sons Ltd., New York. Sehovic D., V. Petravic, and V. Maric. 24. Glycerol and the wine industry - Glycerol determination in grape must and wine. Kemija u industriji 53: