MW ROUND TABLE #3 - Advances in vinification processes, novel compounds and novel reactions

The role and quantification of vitamins in wine: what do we know?

Marie Sarah EVERS - Université Bourgogne Franche-Comté, AgroSup Dijon, PAM UMR A 02.102, Laboratoire VAlMiS-Institut Universitaire de la Vigne et du Vin, 2 rue Claude Ladrey, 21000 Dijon, France
SAS Sofralab, 79, Avenue A.A. Thévenet, BP 1031, Magenta, France
Chloé ROULLIER-GALL, Université Bourgogne Franche-Comté, AgroSup Dijon, PAM UMR A 02.102, Laboratoire VAlMiS-Institut Universitaire de la Vigne et du Vin, 2 rue Claude Ladrey, 21000 Dijon, France
Christophe MORGE, SAS Sofralab, 79, Avenue A.A. Thévenet, BP 1031, Magenta, France
Celine SPARROW, SAS Sofralab, 79, Avenue A.A. Thévenet, BP 1031, Magenta, France
Antoine GOBERT, SAS Sofralab, 79, Avenue A.A. Thévenet, BP 1031, Magenta, France
Hervé ALEXANDRE, Université Bourgogne Franche-Comté, AgroSup Dijon, PAM UMR A 02.102, Laboratoire VAlMiS-Institut Universitaire de la Vigne et du Vin, 2 rue Claude Ladrey, 21000 Dijon, France
Email contact: mariesarah[@]evers.fr

AIM: Vitamins are essential compounds to numerous organisms, including yeasts, and appear highly significant during winemaking processes. Acting as cofactors in major yeast metabolic pathways, such as those of alcohols, amino acids and fatty acids, it appears very likely that their involvement in fermentation courses, as well as in the development of aromatic compounds in wine is consequential.

METHODS: Numerous assays have been developed to determine and quantify vitaminic contents in grape musts and wines. Microbial assays, relying on the specific growth requirements of selected microorganisms, were the earliest methods used pursuing this goal, however poorly precise and accurate. Methods relying on vitamin properties, such as acid titrations and spectrophotometry have also been used to quantify vitamins in grape musts and wines, although they require specific physicochemical properties, and do not allow for simultaneous determination of several vitamin groups.

RESULTS: As a consequence, contemporary techniques, such as chromatography-based methods, stand as efficient means to quantify vitamins in grape musts. However, no method has recently been developed to assay vitamin contents in this specific matrix. Similarly, assays relying on spectroscopy and electrophoresis, proved efficient in simultaneously quantifying vitamins in several fruit matrixes, appear promising for extension towards the grape must and wine matrixes. In addition, winemaking processes, such as the addition of sulfites or clarifying agents, or vatting lengths have been shown to significantly impact vitamin contents.

CONCLUSION: The development of more methods to quantify vitamins in grape musts, relying on more sensitive and precise recent analytical techniques could offer ground for a broad range of prospects in the wine science field. Such developments could support better comprehensions of yeast requirements during winemaking, and allow for finer modulations of the processes, as well as elucidate the role of vitamins in the development of aroma in wines.

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First identification of a glycosylated fraction involved in mushroom-off-flavor in grapes: influence of B. cinerea, powdery mildew and C. subabruptus

Léa DELCROS, MHCS, Comité Champagne, Epernay, France
Teddy GODDET, SPO, Univ Montpellier INRAE, Institut agro, Montpellier, France
Sylvie COLLAS, Comité Champagne, Epernay, France
Marion HERVE, MHCS, Epernay, France
Bruno BLONDIN, SPO, Univ Montpellier INRAE, Institut agro, Montpellier, France
Aurélie ROLAND, SPO, Univ Montpellier, INRAE, Institut agro, Montpellier, France
Email contact: ldelcros[@]moethennessy.com

An organoleptic defect, called fresh mushrooms off-flavor, appeared in wines and spirits since the 2000’s. Numerous researches demonstrated that octen-3-one, octan-3-ol and octen-3-ol (C8 compounds) were involved in the mushroom off-flavor in wines (Pallotta et al., 1998), (Darriet et al., 2002). Botrytis cinerea and other molds played a role in the metabolism of such molecules directly on the grapes (La Guerche et al., 2006). Moreover, producers couldn’t detect this off-flavor in must but it appeared only in finished wines and the intensity can vary considerably during ageing of wines. Several biogenesis pathways have been proposed to explain the mushroom off-flavor in foods. In the fungus kingdom, the formation of C8 molecules came from the transformation of  linoleic acid under the action of several enzymes (Wurzenberger & Grosch, 1984). Glycosidic precursors of octen-3-ol have been identified in several plants such as recently in soybean (Matsui et al., 2018). However, under oenological conditions, no clear mechanism has been established yet. 

AIM: So, the aim of this work was to (i) identify glycosidic precursors of C8 compounds and (ii) to evaluate the influence of different rots on the glycosylated fractions of different grape varieties. For this purpose, we studied different grades of rot defined by visual intensity (healthy, 1-5%, 10-15% and 20-25% of rots) of grapes and musts of Meunier, Pinot noir and Chardonnay affected by Botrytis cinerea, Powdery mildew and Crustomyces subabruptus.

METHODS: From analytical point of view, glycosylated precursors were extracted on C18 cartridges (Lichlorut RP-18, 500 mg), then cleaved by a beta-glycosidase enzyme (Rapidase Revelation Aroma, 40°C, Overnight) and aglycones were analyzed by GC-MS (FS and SIM mode) as reported by Schneider in 2001.

RESULTS: For the first time, we identified a glycosylated fraction able to release fresh mushroom aroma (octen-3-one, octen-3-ol, octan-3-ol) in Meunier and Pinot noir musts. Indeed, contaminated musts of Pinot noir treated by beta glucosidase enzyme released three times more octen-3-one than control. In the samples infected with Powdery mildew, we didn’t observe any production of these glycosides. Botrytis cinerea seemed to decrease the level of octen-3-one glycoside(s) as soon as level contamination reached at least 1%. Finally, a Pinot noir must contaminated by Crustomyces subabruptus involved an increase of the glycosylated fraction responsible for the octen-3-one by 31% in comparison with uncontaminated sample.

CONCLUSION: In conclusion, the identification of a glycosylated fraction able to release fresh mushroom aroma compounds opens avenue to better understand the source of this specific taint and constitutes the first step to help winemakers to avoid off-flavor.

References:
Darriet, P., Pons, M., Henry, R., Dumont, O., Findeling, V., Cartolaro, P., Calonnec, A., & Dubourdieu, D. (2002). Journal of Agricultural and Food Chemistry, 50(11), 3277–3282. https://doi.org/10.1021/jf011527d
La Guerche, S., Dauphin, B., Pons, M., Blancard, D., & Darriet, P. (2006). Journal of Agricultural and Food Chemistry, 54(24), 9193–9200. https://doi.org/10.1021/jf0615294
Matsui, K., Takemoto, H., Koeduka, T., & Ohnishi, T. (2018). Journal of Agricultural and Food Chemistry, 66, 7409–7416.
Pallotta, U., Castellari, M., Piva, A., Baumes, R., & Bayonove, C. (1998). Die Wein Wissenschaft, 53, 32–36.
Schneider, R., Razungles, A., Augier, C., & Baumes, R. (2001). Journal of Chromatography A, 936(1–2), 145–157. https://doi.org/10.1016/S0021-9673(01)01150-5
Wurzenberger, M., & Grosch, W. (1984). Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism, 794(1), 25–30. https://doi.org/10.1016/0005-2760(84)90293-5

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Macromolecular characterization of disease resistant red wine varieties (PIWI)

Edward BREARLEY-SMITH, Matteo MARANGON - Department of Agronomy, Food, Natural Resources, Animals and Environment - University of Padova, Italy
Dan JACKSON, Gregory DUNN - Plumpton College, England
Tony MILANOWSKI - Rathfinny Wine Estate, England
Email contact: edward.brearley@unipd.it

AIM: Pilzwiderstandsfähige (PIWI) are disease resistant Vitis vinifera interspecific hybrid varieties that are receiving increasing attention for ability to ripen in  cool climates and their resistance to grapevine fungal diseases. Wines produced from these varieties have not been characterized, especially regarding their macromolecular composition. This study characterised and quantified colloid-forming molecules (proteins, polysaccharides and phenolics) of red PIWI wines produced in the UK.

METHODS: In 2019 6 wines were made from the PIWI varieties Rondo, Cabernet Jura, Cabernet Cortis, Cabernet Noir, Regent and Cabertin grown at the Plumpton Rock Lodge Vineyard in Sussex (UK) and harvested at similar level of maturity (TSS, pH and TA).  All juice was chaptalized to the same potential alcohol of 12%. Small scale winemaking (1L) was performed in quadruplicate using Bodum® coffee plungers to manage maceration [1]. Residual sugar content, pH, and titratable acidity were monitored during fermentation. For finished wines, the protein and polysaccharide content was measured by HPLC-SEC [2], while the total phenolic content was assessed using the Folin-Ciocalteau method [3]. The protein profile of the wines was further investigated by SDS-PAGE [4].

RESULTS: Fermentations (n=24) were all carried out to completion within 8 days. The resulting wines showed important differences in terms of their macromolecular composition. The total polysaccharide content ranged between 903-1217 mg/L and was higher than the typical content of red wines [5]. Also, the total phenolic content was greater than typical red wines from Vitis vinifera (range 2478-4678 mg/L), while the total protein concentration ranged between 114 -152 mg/L. Typical values for red wine range from 10-200 mg/L [4,6]. The electrophoresis analysis showed the presence of pathogenesis-related (defence) proteins, namely chitinases and thaumatin-like proteins in all wines, while a lipid transfer protein (LTP) was found in all wines except for Cabernet Cortis. This is noteworthy as LTPs can cause severe allergenic reactions [7].

CONCLUSIONS: Hybrid red grape varieties have the potential to produce wines with chemical and macromolecular composition in line with those from Vitis vinifera. This is a promising result for their future adoption in winegrowing regions subjected to difficult climatic conditions and high disease pressure. However, given that PIWI varieties are likely to over-produce pathogenesis-related proteins as a defence mechanism, future investigations should explore the role of these proteins with regard to colloidal and colour stability and allergenic potential.

References:
1. Sparrow, A.M.; Smart, R.E. Fermentation volume studies for red wine experimentation. South African J. Enol. Vitic. 2015, 36, 343–346.
2. González-Royo, E.; Esteruelas, M.; Kontoudakis, N.; Fort, F.; Canals, J.M.; Zamora, F. The effect of supplementation with three commercial inactive dry yeasts on the colour, phenolic compounds, polysaccharides and astringency of a model wine solution and red wine. J. Sci. Food Agric. 2017, 97, 172–181, doi:10.1002/jsfa.7706.
3. Singleton, V.L.; Orthofer, R.; Lamuela-Raventós, R.M. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent - ScienceDirect. In Methods in enzymology; Press, A., Ed.; 1999; pp. 152–178.
4. Smith, M.R.; Penner, M.H.; Bennett, S.E.; Bakalinsky, A.T. Quantitative colorimetric assay for total protein applied to the red wine Pinot Noir. J. Agric. Food Chem. 2011, 59, 6871–6876, doi:10.1021/jf200547u.
5. Ducasse, M.-A.; Canal-Llauberes, R.-M.; de Lumley, M.; Williams, P.; Souquet, J.-M.; Fulcrand, H.; Doco, T.; Cheynier, V. Effect of macerating enzyme treatment on the polyphenol and polysaccharide composition of red wines. Food Chem. 2010, 118, 369–376, doi:10.1016/J.FOODCHEM.2009.04.130.
6. Wigand, P.; Tenzer, S.; Schild, H.; Decker, H. Analysis of protein composition of red wine in comparison with rosé and white wines by electrophoresis and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). J. Agric. Food Chem. 2009, 57, 4328–4333, doi:10.1021/jf8034836.
7. Jaeckels, N.; Tenzer, S.; Rosfa, S.; Schild, H.; Decker, H.; Wigand, P. Purification and structural characterisation of lipid transfer protein from red wine and grapes. Food Chem. 2013, 138, 263–269, doi:10.1016/j.foodchem.2012.09.113.

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Oligosaccharides from Vitis Vinifera grape seeds: a focus on gentianose as a novel bioactive compound

Matteo BORDIGA, Department of Pharmaceutical Sciences, Università degli Studi del Piemonte Orientale “A. Avogadro”. Largo Donegani 2, 28100 Novara, Italy
Daniela IMPERIO, Department of Pharmaceutical Sciences, Università degli Studi del Piemonte Orientale “A. Avogadro”. Largo Donegani 2, 28100 Novara, Italy
Fabiano TRAVAGLIA, Department of Pharmaceutical Sciences, Università degli Studi del Piemonte Orientale “A. Avogadro”. Largo Donegani 2, 28100 Novara, Italy
Jean Daniel COÏSSON, Department of Pharmaceutical Sciences, Università degli Studi del Piemonte Orientale “A. Avogadro”. Largo Donegani 2, 28100 Novara, Italy
Luigi PANZA, Department of Pharmaceutical Sciences, Università degli Studi del Piemonte Orientale “A. Avogadro”. Largo Donegani 2, 28100 Novara, Italy
Marco ARLORIO, Department of Pharmaceutical Sciences, Università degli Studi del Piemonte Orientale “A. Avogadro”. Largo Donegani 2, 28100 Novara, Italy
Email contact: matteo.bordiga[@]uniupo.it

AIM. Grape seeds (Vitis vinifera) are among the main constituents of grape pomace, also exploited in ingredients for nutraceutics and cosmeceutics, particularly regarding the phenolic fraction. The macromolecules of grape/wine include polyphenols, proteins and polysaccharides. Polysaccharides have been comprehensively studied because of their importance (technological and sensory properties in wines). Unlike polysaccharides, oligosaccharides have only recently been characterised. Following a concise focus about the polysaccharide composition of grape seeds, in this work we describes the purification and the identification of low molecular weight saccharides contained in the aqueous extract of grape seeds.

METHODS. A sequential two-step purification by size exclusion chromatography was carried out to fractionate compounds according to molecular weights. Chemical characterization of the combined fractions was performed by Magnetic Resonance Spectroscopy analysis and by high-resolution accurate-mass (Orbitrap mass analyzer).

RESULTS. Apart from sucrose and glucose, a fraction containing primarily a trisaccharide has been detected. Acetylation allowed the purification of the trisaccharide by flash chromatography. Structural determination on the acetylated derivative revealed the trisaccharide gentianose, a predominant carbohydrate reserve in storage roots of perennial Gentiana lutea, poorly discovered in other genera.

CONCLUSIONS. The identification of gentianose, in grape seeds, could open new studies related to its biological functions, as well as to confirm its potential as prebiotic compound, as suggested by preliminary works.