Chapter 2
Yeast-Mediated Formation of Pigmented Polymers in Red Wine 1
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Jeff Eglinton , Markus Griesser , Paul Henschke , Mariola Kwiatkowski , Mango Parker , and Markus Herderich 1,2
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The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, South Australia 5064, Australia Cooperative Research Centre for Viticulture, P.O. Box 154, Glen Osmond, South Australia 5064, Australia Current address: Biomolekulare Lebensmitteltechnologie, Technische Universität Müchen, D-85350 Freising-Weihenstephan, Germany *Corresponding author: telephone: +618-8303-6601; fax: +618-83036601; email:
[email protected] 2
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To gain full advantage of viticutural techniques that optimise anthocyanin levels in grapes, it is essential to identify factors that contribute to the stabilization of anthocyanins in wine and to characterise the reactions which are involved in the transformation of grape-derived anthocyanins into stable red wine pigments. HPLC analysis was used to determine the relative concentrations of anthocyanins, tannins and pigmented polymers in commercial-scale replicated fermentation trials, in the corresponding red wines during ageing, in small scale model ferments and in chemical model reactions. By closely monitoring the time course of anthocyanin degradation and pigmented polymer formation we were able to identify essential parameters for anthocyanin stability. In addition, studies of analyte profiles and reaction kinetics demonstrated that both condensed tannins and anthocyanins were required for the formation of pigmented polymers. Maximal formation of pigmented polymers was achieved in the presence of fermenting yeast, and soluble yeast metabolites were actively involved in the condensation reaction of anthocyanins with tannins. Finally, the results from the commercial-scale replicated fermentation trials could be confirmed by yeast-mediated biotransformation reactions employing purified substrates, and by chemical model reactions. © 2004 American Chemical Society In Red Wine Color; Waterhouse, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
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8 The wine industry has recognized the correlation between wine colour density and red wine quality scores. In general, everything else being equal deeper coloured wines are more likely to have greater flavour and body than lighter coloured wines (/, 2). In combination with other established compositional measures of grape quality such as total soluble solids, pH, titratable acidity, and glycosylated aroma compounds, methods to determine grape colour are essential for continued improvement of wine quality. As an example, robust and rapid measurements of grape colour based on near infrared spectroscopy are currently being implemented by the wine industry (2). The essential pigments responsible for red grape colour are the anthocyanins. These are, however, not only susceptible to bisulfite bleaching and pH-induced colour changes, but are also quite reactive molecules and degrade quickly in solution (5). Consequently, only small amounts of grape anthocyanins that have been extracted from skins during fermentation can be detected in aged red wine (4). Anthocyanins are largely responsible for the colour of young red wine and the concentration of individual anthocyanins can reflect grape composition. After less than twelve months of ageing, however, the concentration of anthocyanins has declined substantially and pigments that have been formed after crushing, during fermentation and during ageing are essential to maintain the red colour of wine (3-5). Two types of pigments contribute to the colour of red wines together with the anthocyanins: Pigmented polymers, a heterogenous group of pigments formed from anthocyanins and tannins (J), and pyranoanthocyanins, which are formed by the addition of vinylphenols or carbonyls to anthocyanins followed by successive cyclisation and oxidation reactions (6, 7). Pigmented polymers are of higher molecular weight than pyranoanthocyanins and they are resistant to S0 -bleaching (8). Pigmented polymers can be clearly separated from anthocyanins as well as from other S0 resistant pigments like the vitisins by HPLC analysis, but coelute with tannins (8). To differentiate by HPLC analysis between pigmented polymers and colourless tannins, tannins are detected at 280 nm, while pigmented polymers are detected at 520 nm. Based on spectrophotometric measurements, pigmented polymers are regarded to contribute as much as 90% to the color of red wine after 2 years of storage (0, 10 27) and according to the commonly accepted paradigm a gradual transition from monomelic anthocyanins through oligomers to polymeric pigments occurs during ageing (0). The focus of many viticultural research activities is to maximize grape colour and grape anthocyanin concentrations. To gain full advantage of enhanced grape anthocyanin levels it is, however, equally important to identify factors that contribute to the stabilization of anthocyanins in wine and to characterise the reactions which are involved in the transformation of grapederived anthocyanins into stable red wine pigments during winemaking. To address these objectives, we have studied parameters involved in anthocyanin 2
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In Red Wine Color; Waterhouse, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
9 degradation and pigmented polymer formation during replicated commercialscale winemaking trials and compared the outcomes to complementary model reactions involving purified anthocyanins and condensed tannins.
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Materials and Methods Grapes and Wines. Machine harvested Shiraz grapes from the Coonawarra region of South Australia (20 bins of approx. 400 kg) were used for commercialscale winemaking in the 2001 vintage. Two randomly selected bins of grapes per fermentation tank (either a 900 L rotary fermenter (RF) or a 1100 L Potter fermenter (JPF)) were destemmed and crushed. The must in each fermenter was individually adjusted to pH 3.3 with tartaric acid prior to inoculation with the active dry wine yeast Saccharomyces cerevisiae (Lalvin® E C U 18, 0.25 g/L ADWY), that had been previously rehydrated. Triplicated fermentations were conducted at 18°C (RF-18°C, PF-18°Q and duplicated fermentations were conducted at 25°C (RF-25°C, PF-25°Q. Fermenting must was pressed off skins after seven days (
