Étude des composés impliqués dans la rétention des tanins ...

© Pamela Nicolle, 2019

Étude des composés impliqués dans la rétention des tanins des vins rouges de cépages hybrides

interspécifiques cultivés en climat froid

Thèse

Pamela Nicolle

Doctorat en sciences et technologie des aliments

Philosophiæ doctor (Ph. D.)

Québec, Canada

Étude des composés impliqués dans la rétention des tanins des vins rouges de cépages hybrides

interspécifiques cultivés en climat froid

Thèse

Paméla Nicolle

Sous la direction de :

Paul Angers, directeur de recherche

Karine Pedneault, codirectrice de recherche

iii

Résumé

La vigne Vitis vinifera, appelée aussi « vigne européenne », est l’espèce la plus cultivée

mondialement pour la production de vin. Dans les régions froides comme le Québec, les vins sont

majoritairement produits à partir de cépages hybrides interspécifiques (CHI) qui offrent une plus

grande tolérance au froid et aux maladies. Les vins rouges issus des CHI sont généralement décrits

comme étant moins astringents et plus colorés que les vins rouges européens issus de V. vinifera, ce

qui ne répond pas toujours aux goûts des consommateurs. Ces caractéristiques sont attribuables à

des différences de composition entre les baies de CHI et de V. vinifera. Une meilleure

compréhension des molécules affectant la rétention et la composition en tanins des vins de CHI

rouges et des procédés de vinification affectant leurs profils pourrait permettre de modifier leur

astringence et contribuer à augmenter la compétitivité des vins rouges issus de CHI sur le marché.

Trois études ont été conduites sur ce thème dans le cadre de cette thèse.

La première étude avait pour but d’étudier l’impact de l’utilisation de marc de raisin blanc (MRB) en

co-fermentation avec du marc de raisin rouge (MRR) sur la teneur en tanins des vins, en utilisant les

CHI Frontenac et Vidal. Les résultats ont montré que les vins produits avec du MRB présentaient

plus de flavan-3-ols monomériques et oligomériques et davantage de terpènes. La manipulation du

ratio MRR/MRB a permis de modifier le profil en anthocyanes des vins finis, résultant dans certains

cas en des vins plus clairs. L’utilisation d’un ratio MRR/MRB approprié (30% MRR/6% MRB) a permis

une meilleure stabilisation de la couleur des vins sans affecter significativement la couleur.

L’utilisation de MRB en co-fermentation avec du MRR s’est avérée un outil intéressant pour moduler

la couleur des vins ainsi que leur composition phénolique et volatile.

La seconde étude avait pour objectif d’étudier l’impact de différents traitements seuls ou en

combinaison (traitements pré-fermentaire du moût, fermentation en présence et absence de marc,

addition de tanins œnologiques) sur la teneur en tanins, en protéines et en pigments des vins de

Frontenac. L’élimination des protéines par chauffage du moût ou ajout de bentonite n’a pas permis

une meilleure rétention des tanins dans le vin. Néanmoins, fermenter le moût sans marc de raisin a

amélioré significativement leur rétention dans le vin, notamment celle des flavan-3-ols polymériques

(jusqu’à 27,8%). L’addition de 3 g/L de tanins œnologiques dans les vins, fermentés en présence et

iv

absence de marc, s’est montrée nécessaire pour augmenter significativement la concentration en

tanins des vins de Frontenac.

La troisième étude consistait en une étude exploratoire et comparative de la teneur et de la nature

des polysaccharides des vins de CHI Frontenac et Frontenac blanc avec celles du V. vinifera

Cabernet Sauvignon. Les vins de Frontenac ont montré une concentration plus élevée en

polysaccharides totaux et des polysaccharides plus ramifiés que les vins de Cabernet Sauvignon.

Ces différences pourraient contribuer à la faible astringence des vins rouges issus de CHI par rapport

à celle des vins rouges de variété V. vinifera.

Ce projet a permis d’apporter de nouvelles connaissances sur le profil d’extraction et la rétention des

tanins dans les vins de CHI cultivés au Québec. Une meilleure compréhension des facteurs

impliqués dans la rétention des tanins dans ces vins a permis de fournir des pistes à envisager pour

élaborer des procédés de vinification adaptés à la composition physico-chimique atypique des CHI

rouges afin de produire des vins plus riches en tanins, ayant un potentiel accru de répondre aux

goûts des consommateurs.

v

Abstract

Vitis vinifera is the most cultivated grapevine species for wine production, worldwide. But in cold

areas such as Quebec, Canada, most wine is produced from interspecific hybrid grape varieties

(CHI) that better respond to harsh growing conditions such as cold temperature and high disease

pressure. Red CHI wines are generally described as less astringent and more colourful than

European V. vinifera red wines, but those characteristics do not fully align with consumers taste and

preferences. The low astringency of red CHI wine is largely attributable to differences between the

respective chemical composition of CHI and V. vinifera berries. A better understanding of the

molecules affecting tannin retention and composition of red CHI wines, and of the winemaking

processes that affect their tannin profiles could provide solutions to impact their astringency and

improve their competitiveness on the market. Three studies conducted on this topic as part of this

thesis are presented in this manuscript.

The first study aimed at investigating the impact of the co-fermentation of white (WP) and red (RP)

grape pomace on the tannin content of red wine, using the CHI varieties Frontenac and Vidal. The

results showed that wines produced with WP contained higher levels of monomeric and oligomeric

flavan-3-ols and terpenes than wines issued from the RP treatment. Modifying the ratio of RP to WP

during alcoholic fermentation modified the anthocyanin profile of the wines, sometimes resulting in

lighter coloured wines. A ratio of 30% RP to 6% WP improved colour stabilisation without significantly

affecting wine colour. The use of WP in co-fermentation with RP proved to be an interesting tool to

modulate wine colour as well as its phenolic and volatile composition.

The second study aimed at exploring the impact of different winemaking treatments (pre-fermentative

treatment of the must, fermentation with and without pomace, addition of enological tannins), alone or

in combination, on the tannin, protein and pigment content of Frontenac wines. Wine protein removal

by heat or bentonite addition did not improve tannin retention in wine. In contrast, fermenting the

must without pomace significantly improved tannin retention, especially for polymeric flavan-3-ols (up

to 27.8%). With or without pomace, the addition of enological tannins at a minimal rate of 3 g/L, was

necessary to increase tannin concentration in Frontenac wine significantly.

The third study explored the content and, to some extent, the molecular weight and structure of the

polysaccharides from the CHI Frontenac and Frontenac Blanc, in comparison with wines from the V.

vinifera variety Cabernet Sauvignon. Frontenac wines showed a higher concentration of total

polysaccharides with more branched polysaccharides than wines from Cabernet Sauvignon. Results

showed that significant differences exist between the polysaccharides content and structure of the

vi

studied CHI varieties and Cabernet Sauvignon variety. Those differences could contribute to the low

astringency of CHI wines when compared with red wines from V. vinifera varieties.

This project has brought new knowledge on the tannin, protein and polysaccharide content of red CHI

varieties grown in Quebec and how they impact tannin retention in CHI wines. Understanding the

chemistry of phenolic compounds and macromolecules in CHI wines made possible the development

of new winemaking processes adapted to the atypical biochemical composition of red CHI wine. As

such, results from this study provide new venues to vary and improve the style and the quality of CHI

wines and better meet consumers taste and preferences.

vii

Table des matières

Résumé............................................................................................................................................. iii

Abstract ............................................................................................................................................. v

Table des matières.......................................................................................................................... vii

Liste des tableaux ............................................................................................................................. x

Liste des figures ............................................................................................................................. xii

Liste des annexes ........................................................................................................................... xv

Liste des abréviations, sigles et acronymes ................................................................................ xvi

Remerciements ............................................................................................................................. xvii

Avant-propos ................................................................................................................................. xix

Introduction ....................................................................................................................................... 1

Chapitre 1. Revue de littérature ....................................................................................................... 5

1.1. L’industrie vinicole du Québec ......................................................................................................... 5 1.1.1. La place de l’industrie vinicole du Québec dans le monde ............................................................ 5

1.1.2. Les cépages cultivés au Québec .................................................................................................. 7

1.1.2.1. La classification botanique de la vigne .................................................................................. 7

1.1.2.2. Les variétés de vigne au Québec .......................................................................................... 7

1.1.3. Les vins québécois et l’astringence ............................................................................................ 12

1.2. La composition phénolique des baies de raisin ............................................................................ 13 1.2.1. Les anthocyanes ........................................................................................................................ 17

1.2.2. Les flavan-3-ols et proanthocyanidines....................................................................................... 17

1.3. La composition macromoléculaire de la baie de raisin ................................................................. 19 1.4. Du raisin au vin ................................................................................................................................ 20

1.4.1. Le processus de vinification ....................................................................................................... 20

1.4.2. Extraction et évolution des composés phénoliques durant la vinification ..................................... 22

1.4.2.1. Extraction ........................................................................................................................... 22

1.4.2.2. Évolution ............................................................................................................................ 23

1.4.2.3. Interactions des tanins avec les protéines et polysaccharides ............................................. 24

1.4.3. Impact des procédés vinicoles sur les tanins .............................................................................. 25

1.5. Problématique, hypothèse et objectifs ........................................................................................... 26 1.5.1. Problématique ............................................................................................................................ 26

1.5.2. Hypothèse ................................................................................................................................. 28

1.5.3. Objectifs .................................................................................................................................... 28

Chapitre 2. Co-fermentation of red grapes and white pomace: a natural and economical process to modulate red hybrid wine composition ...................................................................... 29

2.1. Avant-propos ................................................................................................................................... 29 2.2. Résumé ............................................................................................................................................ 29 2.3. Abstract ........................................................................................................................................... 30 2.4. Introduction ..................................................................................................................................... 30

viii

2.5. Material and Methods ...................................................................................................................... 33 2.5.1. Chemicals .................................................................................................................................. 33

2.5.2. Grape materials ......................................................................................................................... 33

2.5.3. Winemaking trials ....................................................................................................................... 34

2.5.4. Phenolic compound analysis ...................................................................................................... 35

2.5.4.1. Phenol estimation ............................................................................................................... 35

2.5.4.2. Anthocyanin analysis .......................................................................................................... 35

2.5.4.3. Flavan-3-ol analysis ............................................................................................................ 36

2.5.5. Colour analysis .......................................................................................................................... 37

2.5.6. Volatile compound analysis ........................................................................................................ 37

2.5.7. Statistical analysis ...................................................................................................................... 38

2.6. Results and Discussion .................................................................................................................. 39 2.6.1. Effect on phenolic compounds ................................................................................................... 39

2.6.1.1. Effect on flavan-3-ols .......................................................................................................... 39

2.6.1.2. Effect on pigments .............................................................................................................. 42

2.6.2. Effect on wine colour and colour evolution .................................................................................. 48

2.6.3. Effect on the volatile composition of bottled wines ...................................................................... 49

2.7. Conclusion ....................................................................................................................................... 53

Chapitre 3. Pomace limits tannin retention in Frontenac wines .................................................. 55

3.1. Avant-propos ................................................................................................................................... 55 3.2. Résumé ............................................................................................................................................ 55 3.3. Abstract ........................................................................................................................................... 56 3.4. Introduction ..................................................................................................................................... 56 3.5. Material and Methods ...................................................................................................................... 59

3.5.1. Chemicals .................................................................................................................................. 59

3.5.2. Experimental design................................................................................................................... 59


3.5.3.1. Grape material.................................................................................................................... 60

3.5.3.2. Winemaking ....................................................................................................................... 60

3.5.4. Tannin analysis .......................................................................................................................... 61

3.5.4.1. HPLC-fluorescence ............................................................................................................ 61

3.5.4.2. Protein precipitation ............................................................................................................ 62

3.5.5. Pigment analysis ........................................................................................................................ 63

3.5.6. Protein analysis ......................................................................................................................... 64


3.6. Results and discussion ................................................................................................................... 65 3.6.1. Relevance of the method used for tannin quantification .............................................................. 65

3.6.2. Impact of winemaking processes on protein concentration ......................................................... 67

3.6.3. Impact of winemaking processes on wine polyphenol concentration ........................................... 73

3.6.3.1. Impact on pigment concentration ........................................................................................ 73

3.6.3.2. Impact on tannin concentration ........................................................................................... 76

3.6.3.3. Relevant observations ........................................................................................................ 80

3.7. Conclusion ....................................................................................................................................... 80

ix

Chapitre 4. Evaluation of flavan-3-ols and polysaccharides in musts and wines from Vitis vinifera Cabernet Sauvignon and cold-hardy Vitis sp. Frontenac ............................................... 82

4.1. Avant-propos ................................................................................................................................... 82 4.2. Résumé ............................................................................................................................................ 82 4.3. Abstract ........................................................................................................................................... 83 4.4. Introduction ..................................................................................................................................... 83 4.5. Material and Methods ...................................................................................................................... 85

4.5.1. Grape material ........................................................................................................................... 85


4.5.3. Sugars analysis ......................................................................................................................... 86

4.5.4. Flavan-3-ol analysis ................................................................................................................... 87

4.5.5. Polysaccharide analysis ............................................................................................................. 87

4.5.5.1. Total polysaccharide precipitation and quantification ........................................................... 87

4.5.5.2. Total polysaccharide characterisation ................................................................................. 87


4.6. Results and discussion ................................................................................................................... 88 4.6.1. Flavan-3-ols ............................................................................................................................... 88

4.6.2. Total polysaccharides ................................................................................................................ 93

4.6.3. Polysaccharide profile ................................................................................................................ 94

4.7. Conclusion ....................................................................................................................................... 97

Conclusion & perspectives ............................................................................................................ 99

Annexes......................................................................................................................................... 104

Références bibliographiques ....................................................................................................... 125

x

Liste des tableaux

Tableau 1.1. Génétique (en % de chaque espèce de vigne), susceptibilité aux maladies et

tolérance/résistance au froid des principaux cépages cultivés au Québec (d’après Dubé &

Turcotte (2011); Pedneault & Provost (2016)). ......................................................................... 11

Tableau 1.2. Composition chimique (en % du poids frais) de la pellicule, de la pulpe et des pépins de

raisin (d’après Flancy (1998); Gros & Yerle (2014)). ................................................................. 14

Tableau 1.3. Teneur moyenne en anthocyanes et tanins des pellicules et pépins de baies de

cépages V. vinifera et de cépages hybrides interspécifiques (en mg/kg baie). .......................... 19

Table 2.1. Concentration of anthocyanin compounds (mean ± standard deviation, in mg/L cyanidin-3-

glucoside, delphinidin-3-glucoside, malvidin-3-glucoside, pelargonidin-3-glucoside, and

peonidin-3-glucoside equivalent depending of the aglycone) in control (50% RP), RP/WP-

treated (30% RP/6% WP, 30% RP/12% WP, and 30% RP/18% WP), and WP-treated (23% WP)

wines after 395 days of bottling and ESI-MS m/z values (molecular ion; product ions) of

anthocyanins detected in red Frontenac wines. ........................................................................ 45

Table 2.2. CIELab parameters (mean ± standard deviation, in CIELab unit) in control (50% RP),

RP/WP-treated (30% RP/6% WP, 30% RP/12% WP, and 30% RP/18% WP), and WP-treated

(23% WP) wines at different winemaking stages. ..................................................................... 50

Table 3.1. Repeated measures analysis of variance (ANOVA) for protein, tannin, total pigment (Tpg),

and co-pigmented, monomeric, and polymeric anthocyanin (CA, MA, and PP, respectively).

Main effects are: must protein treatment (MT; untreated, bentonite-treated, and heat-treated

must); pomace (P; must fermented with and without pomace); tannin addition (TA; 0, 1, 3, and

9 g/L); and time of maceration (TM; 0, 4, and 11 days after the end of alcoholic fermentation). 70

Table 3.2. Protein concentration (mean ± standard deviation (SD), mg/L BSA equivalent) in

experimental Frontenac wines made with untreated (control), bentonite-treated, and heat-

treated must, fermented with (WP) or without (WOP) pomace, and with different doses of tannin

addition (0, 1, 3, and 9 g/L) at the end of alcoholic fermentation (e-AF) and on the 4th and the

11th day following the e-AF (corresponding to days 4, 8, and 15 of the winemaking process,

respectively). ............................................................................................................................ 71

Table 4.1. Composition of musts and wines made from the cold-hardy Vitis sp. Frontenac blanc,

Frontenac, and V. vinifera Cabernet Sauvignon (Primary fermentable sugars, g/L; alcohol

concentration, % v/v; titratable acidity, g tartaric acid eq./L; pH; primary amino nitrogen, mg/L;

and ammonia, mg/L). ............................................................................................................... 86

Table 4.2. Average molecular weight values (number average molecular weight, Mn; weight average

molecular weight, Mw; Z average molecular weight, Mz; and molecular weight polydispersity

ratio, Mw/Mn), and intrinsic viscosity (IV) for the ethanol-precipitated polysaccharide of must,

fermented must (middle of alcoholic fermentation, mid-AF), and wine made from the cold-hardy

Vitis sp. Frontenac blanc, Frontenac, and V. vinifera Cabernet Sauvignon. .............................. 95

xi

Table S2.1. Composition (mean ± standard deviation) of the must and control wines and RP/WP-

(30% RP/6% WP, 30% RP/12% WP, and 30% RP/18% WP) and WP-treated wines (23% WP)

after 395 days of bottling. ....................................................................................................... 106

Table S2.2. Monomeric, oligomeric (2 to 5 flavan-3-ol units), and polymeric (≥ 6 flavan-3-ol units)

flavan-3-ol compound concentration (mean ± standard deviation, in mg/L epicatechin

equivalent) in control (50% RP), RP/WP-treated (30% RP/6% WP, 30% RP/12% WP, and 30%

RP/18% WP), and WP-treated (23% WP) wines at different winemaking stages. ................... 107

Table S2.3. Phenol estimation (mean ± standard deviation, in absorbance unit) in control (50% RP),


(23% WP) wines at different winemaking stages. ................................................................... 109

Table S2.4. Volatile compound parameters for GC-MS-SPME analysis. ......................................... 111

Table S3.1. Total pigment (Tpg) and co-pigmented, monomeric, and polymeric anthocyanin (CA, MA,

and PP, respectively) estimation (mean ± standard deviation (SD)) in experimental Frontenac

wines made with untreated (control), bentonite-treated, and heat-treated must, fermented with

(WP) and without (WOP) pomace, and with different doses of tannin addition (0, 1, 3, and 9 g/L)

at 11 days after the end of alcoholic fermentation (corresponding to day 15 of the winemaking

process). ................................................................................................................................ 118

Table S3.2. Kinetic of tannin concentration (mean ± standard deviation (SD), mg/L epicatechin

equivalent) in experimental Frontenac wines made with untreated (control), bentonite-treated,

and heat-treated must, fermented with (WP) and without (WOP) pomace, and with different

doses of tannin addition (0, 1, 3, and 9 g/L) at the end of alcoholic fermentation (e-AF) and on

the 4th and the 11th day after the e-AF (corresponding to days 4, 8, and 15 of the winemaking

process, respectively). ............................................................................................................ 120

Table S4.1. Monomeric, oligomeric (2-5 flavan-3-ol units), and polymeric (≥ 6 flavan-3-ol units)

flavan-3-ol (mean ± standard deviation (SD), mg/L epicatechin equivalent), polysaccharide

(mean ± SD, mg/L galactose equivalent), and ethanol (%, v/v) concentration during the

alcoholic fermentation of V. vinifera Cabernet Sauvignon and cold-hardy Vitis sp. cultivars

Frontenac and Frontenac blanc. ............................................................................................. 122

xii

Liste des figures

Figure 1.1. Principaux bassins viticoles dans le monde, incluant les vignobles canadiens, et les types

de climats associés à ces régions (d’après Carbonneau & Escudier (2017)). ............................. 6

Figure 1.2. Famille des Vitacées. ....................................................................................................... 8

Figure 1.3. Encépagement des membres du Conseil des vins du Québec de 2012 à 2017 (d’après

AVQ, 2017). ............................................................................................................................. 10

Figure 1.4. Structure ((a), d'après Kennedy (2002)) et organisation tissulaire ((b), d’après Fougère-

Rifot & Cholet (1996)) d’une baie de raisin à maturité. Organisation structurale de la paroi

végétale primaire ((c), d’après Koolman, Röhm, Wirth, & Robertson (2005)) et structure des

principaux polysaccharides de la paroi : (d), cellulose ; (e), pectine (d’après Scheller, Jensen,

Sørensen, Harholt, & Geshi (2007)) et (f), hémicellulose. ......................................................... 15

Figure 1.5. Exemple de composés phénoliques non-flavonoïdes et flavonoïdes présents dans les

baies de raisin. ......................................................................................................................... 16

Figure 1.6. Schéma de vinification traditionnelle du vin en rouge (étapes détaillées en Appendix A).

................................................................................................................................................. 21

Figure 1.7. Cinétique d’extraction des anthocyanes (bleu, point carré), tanins (orange, tiret point) et

polysaccharides (vert, tiret cadratin-point) du raisin lors des étapes de la vinification en

rouge (Mpf, macération pré-fermentaire ; FA, fermentation alcoolique ; et MPF, macération

post-fermentaire) et évolution de l’intensité colorante (noir, uni) chez Vitis vinifera (d’après

Ribéreau-Gayon, Glories, et al. (2006)). ................................................................................... 23

Figure 1.8. Les différents mécanismes potentiels d'interactions entre polyphénols et protéines

(d’après (Le Bourvellec & Renard, 2012). ................................................................................. 25

Figure 2.1. Monomeric (a), oligomeric (2-5 flavan-3-ol units) (b), polymeric ( 6 flavan-3-ol units) (c),

and total (d) flavan-3-ol concentration (mean ± standard deviation, mg/L epicatechin equivalent)

in control (50% RP), RP/WP-treated (30% RP/6% WP, 30% RP/12% WP, and 30% RP/18%

WP), and WP-treated (23% WP) wines at different winemaking stages: PFM, after the pre-

fermentative cold maceration; 1-FAM, m-FAM, and e-FAM, during the fermentative alcoholic

maceration (at days 1, 4, and 8); MLF, after the malolactic fermentation (day 45); and BW, after

bottling (day 395). Means comparison using Tukey’s honest significant difference test at the

0.05 probability level is shown in Appendix B. .......................................................................... 41

Figure 2.2. Phenol parameters including pigments at acidic pH (PpH < 1) (a), wine pigments corrected

(WPcor) (b), pigments resisting to sulphite bleaching (PRSO2) (c), and colour intensity corrected

(CIcor) (d) (mean ± standard deviation, absorbance unit) for control (50% RP), RP/WP-treated

(30% RP/6% WP, 30% RP/12% WP, and 30% RP/18% WP), and WP-treated (23% WP) wines

at different winemaking stages: 1-FAM, m-FAM, and e-FAM, during the fermentative alcoholic

maceration (at days 1, 4, and 8); MLF, after the malolactic fermentation (day 45); and BW, after

bottling (day 395). Means comparison using Tukey’s honest significant difference test at the

0.05 probability level is shown in Appendix B. .......................................................................... 43

xiii

Figure 2.3. Principal component analysis of flavan-3-ol and phenol (by spectrophotometry-UV) profile

(a) in control (50% RP), RP/WP-treated (30% RP/6% WP, 30% RP/12% WP, and 30% RP/18%

WP), and WP-treated (23% WP) wines after 395 days of bottling (b). Variables: PpH<1, pigments

at acidic pH; WPcor, wine pigments corrected; PRSO2, pigments resisting to sulphite bleaching;

CIcor, colour intensity corrected; H, hue..................................................................................... 47

Figure 2.4. Principal component analysis of the volatile compound profile (a) in control (50% RP),


(23% WP) wines after 395 days of bottling (b). ......................................................................... 52

Figure 3.1. Regression of tannin concentration of experimental Frontenac wines as measured by

protein precipitation and HPLC-fluorescence. Wine samples from the end of alcoholic

fermentation (e-AF) and day 11 after the e-AF (corresponding to days 0 and 15 of the

winemaking process, respectively) were included in the analysis. ............................................ 67

Figure 3.2. Heat map of the protein and tannin concentration of experimental Frontenac wines made

with untreated (control, CT), bentonite-treated (BE), and heat-treated (HT) must, fermented with

(WP) and without (WOP) pomace, and with different doses of tannin addition (0, 1, 3, and 9 g/L)

at 0, 4, and 11 days after the end of alcoholic fermentation (e-AF). Red and blue colours

represent the highest and the smallest concentration of proteins or tannins in wines. Data are

ranked from the lowest to the highest protein concentration, 11 days after the e-AF (a), and from

the highest to lowest tannin concentration, 11 days after the e-AF (b). ..................................... 68

Figure 3.3. Total pigment (a) and co-pigmented (b), monomeric (c), and polymeric anthocyanin (d)

estimation (in absorbance unit), in experimental Frontenac wines made with untreated (control),

bentonite-treated, and heat-treated must, fermented with and without pomace, and with different

doses of tannin addition (0, 1, 3, and 9 g/L) at 11 days after the end of alcoholic fermentation. 75

Figure 3.4. Oligomeric (2–5 units of flavan-3-ols) and polymeric (>5 units of flavan-3-ols) flavan-3-ol

concentration (mean, mg/L epicatechin equivalent) in experimental Frontenac wines made with

untreated (control, CT), bentonite-treated (BE), and heat-treated (HT) must, fermented with

(WP) and without (WOP) pomace, and with different doses of tannin addition (0, 1, 3, and 9 g/L)

at the end of alcoholic fermentation (e-AF; a) and on the 4th (b) and the 11th (c) day following the

e-AF (corresponding to days 4, 8, and 15 of the winemaking process, respectively). For a given

combination of Dose*Treatment*Day, small letters compare the oligomeric flavan-3-ol

concentration and capital letters compare the polymeric flavan-3-ol concentration of pomace

treatment at the 0.05 probability level. The statistics for the other combinations are available in

Appendix B. .............................................................................................................................. 78

Figure 4.1. Changes in the concentration of monomeric, oligomeric (2-5 flavan-3-ol units), and

polymeric (≥ 6 flavan-3-ol units) flavan-3-ols (mean ± standard deviation, mg/L, epicatechin

equivalent) and polysaccharides (mean ± standard deviation, mg/L, galactose equivalent)

during the alcoholic fermentation of the cold-hardy Vitis sp. Frontenac blanc (a), Frontenac (b),

and V. vinifera Cabernet Sauvignon (c). Mean comparison using Tukey’s honest significant

difference test at the 0.05 probability level is shown in Appendix D. ......................................... 90

Figure 4.2. Kinetic of fermentable sugar consumption during the fermentation of the cold-hardy Vitis

sp. Frontenac blanc (a), Frontenac (b), and V. vinifera Cabernet Sauvignon (c). ...................... 91

xiv

Figure 4.3. Monomeric (a), oligomeric (b; 2-5 flavan-3-ol units), and polymeric (c; ≥ 6 flavan-3-ol

units) flavan-3-ols (mean ± standard deviation, mg/L epicatechin equivalent) extraction as a

function of increasing ethanol concentration (mean ± standard deviation; %, v/v) during the

alcoholic fermentation of the cold-hardy Vitis sp. Frontenac blanc, Frontenac, and V. vinifera

Cabernet Sauvignon. Mean comparison using Tukey’s honest significant difference test at the

0.05 probability level is shown in Appendix D. .......................................................................... 93

Figure 4.4. Mark–Houwink–Sakurada plot for must (a) and wine (b) polysaccharide samples of the

cold-hardy Vitis sp. Frontenac blanc (must, green; wine, maroon), Frontenac (must, grey; wine,

blue), and V. vinifera Cabernet Sauvignon (must, orange; wine, yellow). .................................. 97

Figure 4.5.1. Proposition d’itinéraires technologiques adaptés à la vinification en rouge des cépages

hybrides interspécifiques cultivés en climat froid. ................................................................... 103

Figure S2.1. Anthocyanin (a) (mg/L cyanidin-3-glucoside, delphinidin-3-glucoside, malvidin-3-

glucoside, pelargonidin-3-glucoside, and peonidin-3-glucoside equivalent depending of the

aglycone) and flavan-3-ol compound (b) (mg/L epicatechin equivalent) profiles in control wines

and RP/WP- (30% RP/6% WP, 30% RP/12% WP, and 30% RP/18% WP) and WP-treated

wines (23% WP) after 395 days of bottling. ............................................................................ 112

Figure S2.2. CIELab parameters including L, lightness (a); a, red-green (b); b, blue-yellow (c); C,

chroma (d); H, hue (e) (mean ± standard deviation) for control wines and RP/WP- (30% RP/6%

WP, 30% RP/12% WP, and 30% RP/18% WP) and WP-treated wines (23% WP) at different

winemaking stages: 1-FAM, m-FAM, and e-FAM, during the fermentative alcoholic maceration

(day 1, 4, and 8); MLF, after the malolactic fermentation (day 45); and BW, after bottling (day

395). ....................................................................................................................................... 114

Figure S2.3. Colour representation of control wines and RP/WP- (30% RP/6% WP, 30% RP/12%

WP, and 30% RP/18% WP) and WP-treated wines (23% WP) from the CIELab data at different

winemaking stages: 1-FAM, m-FAM, and e-FAM, during the fermentative alcoholic maceration

(day 1, 4, and 8); MLF, after the malolactic fermentation (day 45); and BW, after bottling (day

395). The colour representation was obtained from the website: http://colorizer.org/. ............. 115

Figure S3.1. Factorial experimental design used to produce experimental Frontenac wines, including

the following factors: must protein treatment (untreated, bentonite-treated, and heat-treated

must), pomace (must fermented with and without pomace), tannin addition (0, 1, 3, and 9 g/L),

and time of maceration (0, 4, and 11 days after the end of alcoholic fermentation, corresponding

to days 4, 8, and 15 of the winemaking process, respectively). .............................................. 116

xv

Liste des annexes

Appendix A. Étapes de la vinification en rouge traditionnelle ......................................................... 104

Appendix B. Supplementary material - Chapter 2 .......................................................................... 106

Appendix C. Supplementary material - Chapter 3 .......................................................................... 116

Appendix D. Supplementary material - Chapter 4 .......................................................................... 122

xvi

Liste des abréviations, sigles et acronymes

AF

ANOVA

AOC

BSA

BW

CHI

ET

FAM

FD

GC-MS-SPME

GPC/SEC

HPLC-FLD

IFV

IGP

IV

LOQ

LSD

LSMEAN

Mn

Mz

Mw

mDP

MHS

MLF

MRB/MRR

MRM

MS

OIV

PCA

PFM

PG

PP

Protéine PR

Rh

RP/WP

SAQ

SO2

UPLC-MS/MS

UV-Vis

VQA

WP/WOP

Alcoholic fermentation

Analysis of variance

Appellation d’origine contrôlée

Bovine serum albumin

Bottled wine

Cépage hybride interspécifique/Interspecific hybrid grape variety

Enological tannin

Fermentative alcoholic maceration

Dilution factor

Gas chromatography - mass spectrometry - solid phase microextraction

Gel permeation/size exclusion chromatography

High performance liquid chromatography – fluorescence detector

Institut français de la vigne et du vin

Indication géographique protégée

Intrinsic viscosity

Limit of quantification

Least significant difference

Least-squares means

Number average molecular weight

Z average molecular weight

Weight average molecular weight

Mean degree of polymerisation

Mark-Houwink-Sakurada

Malolactic fermentation

Marc de raisin blanc/rouge

Multiple reactions monitoring

Mass spectrometry

Organisation internationale de la vigne et du vin

Principal component analysis

Pre-fermentative cold maceration

Depolymerisation using phloroglucinolysis

Protein precipitation

Protéine liée à la pathogenèse

Hydrodynamic radius

Red/White pomace

Société des alcools du Québec

Sulphur dioxide

High performance liquid chromatography - tandem mass spectrometry

Ultraviolet - visible

Vintners quality alliance

With/Without pomace

xvii

Remerciements

Je tiens tout d’abord à remercier mon directeur et ma codirectrice de thèse, Paul Angers et Karine

Pedneault, pour m’avoir offert l’opportunité de réaliser ce doctorat. Merci de m’avoir permis de mener

à bien ce projet avec une grande liberté de décision. Un merci tout particulier à Karine Pedneault

pour le temps passé sur la correction des articles et son soutien durant cette dernière année de

thèse difficile sur le plan émotionnel.

Je souhaite ensuite remercier pour le financement de cette thèse par l’obtention d’une bourse en

milieu pratique : le Centre de développement bioalimentaire du Québec (CDBQ), le Fonds de

recherche du Québec – Nature et technologies, et le Conseil de recherches en sciences naturelles et

en génie du Canada. Merci également au Ministère de l'Agriculture, des Pêcheries et de

l'Alimentation du Québec et à Agriculture and Agri-Food Canada pour le financement de ce projet.

J’adresse aussi mes remerciements pour leur soutien technique à l’équipe du CDBQ (Sabrina

Després, Mélissa Beaulieu, Daphné McNicoll, Réjean Deschênes, Charles Lavigne et Katy Dumont),

l’équipe de la plateforme analytique de l’INAF (Pascal Dubé, Perrine Feutry et Véronique Richard),

l’équipe du laboratoire de technologie alimentaire de la FSAA (Pierre Côté, Pascal Lavoie et Mélanie

Martineau) et, bien évidemment, l’indispensable Diane Gagnon. Merci également à mes collègues

Annabelle Veillette, Geneviève Montminy et Charlène Marcotte pour leur aide durant cette thèse.

Je remercie bien évidement Jean-Louis Escudier de l’INRA de Pech Rouge pour sa disponibilité, ses

conseils et son expertise de qualité dans le domaine vitivinicole. Merci également pour le temps

passé à relire nos manuscrits, aux réviseurs de Food Chemistry. Nous apprécions sincèrement tous

les commentaires et suggestions qui nous ont permis d’améliorer la qualité des articles.

Mes sincères remerciements à mes amis du bureau (Abdel, Agathe, Attara, Élodie, Emna, Guida,

Mathilde, Ourdia, Raquel et tous les autres) d’avoir rendu mon séjour au Québec, et plus

spécifiquement au pavillon Comtois, plus agréable. Merci pour votre soutien !

Enfin, mes plus gros remerciements vont à ma moitié, Mayank. Sa patience, nos discussions

personnelles et scientifiques, ainsi que son amour et son soutien inconditionnel m’auront finalement

permis d’achever cette thèse !

xviii

« Le vin met à jour les secrets cachés de

l'âme » — Horace

xix

Avant-propos

La présente thèse a été soumise à la Faculté des études supérieures de l’Université Laval pour

satisfaire aux exigences de l’obtention du grade Philosophiae Doctor (Ph.D.) en Sciences des

Aliments (STA) de la Faculté des Sciences de l’Agriculture et de l’Alimentation (FSAA).

Cette thèse s’articule en quatre chapitres, précédés d’une introduction et suivis d’une conclusion.

L’introduction présente le contexte global du projet et l’introduit brièvement. Le premier chapitre fait

un état des lieux de la littérature pertinente en lien avec le sujet et aborde des notions qui ont été

utilisées dans l’analyse des résultats. La problématique ainsi que les hypothèses et objectifs de thèse

sont présentés en dernière section de ce chapitre. Les chapitres 2, 3 et 4 représentent chacun un

article qui répond à un des objectifs proposés. Finalement, la thèse se termine par une conclusion

générale du projet incluant des perspectives qu’il serait intéressant d’étudier.

Les chapitres 2, 3 et 4 sont présentés sous forme d’articles scientifiques et rédigés en anglais :

• Une version du chapitre 2, intitulée « Co-fermentation of red grapes and white pomace: A

natural and economical process to modulate hybrid wine composition », a été publiée dans le

journal Food Chemistry (https://doi.org/10.1016/j.foodchem.2017.09.053) ;

• Une version du chapitre 3, intitulée « Pomace limits tannin retention in Frontenac wines », a

été publiée dans le journal Food Chemistry (https://doi.org/10.1016/j.foodchem.2018.10.116) ;

• Et une version du chapitre 4, intitulée « Evaluation of flavan-3-ols and polysaccharides in

musts and wines made from Vitis vinifera Cabernet Sauvignon and cold-hardy Vitis sp.

Frontenac », sera soumise au journal Food Chemistry sous la forme d’une « short

communication ».

L'implication de chacun des auteurs dans les articles cités est la suivante :

• Paméla Nicolle, Candidate au doctorat : planification et réalisation des expériences, analyses

des résultats, rédaction des articles scientifiques et publication des articles scientifiques ;

• Charlène Marcotte, Étudiante en 1er cycle : accompagnement ponctuel dans la préparation

des échantillons ;

xx

• Dre Kyle Williams, Applications Scientist chez Malvern Instruments Ltd : analyses

chromatographiques des polysaccharides, aide dans l’interprétation des résultats, correction

et révision de l’article ;

• Dre Karine Pedneault, Co-directrice de thèse : élaboration du projet de recherche,

supervision de l'étudiante, correction et révision des articles scientifiques et publication des

articles scientifiques ;

• Et Dr Paul Angers, Directeur de thèse : collaboration au projet.

1

Introduction

L’intérêt des Québécois pour le vin est de plus en plus marqué. En 2017, les ventes de vin au

Québec atteignaient 2,3 milliards de dollars canadiens, soit 79% des ventes d'alcool en volume, les

vins rouges représentant à eux seuls 63,9% des ventes (SAQ, 2017). Malgré un regain de popularité

pour les produits locaux, les vins québécois occupaient cette même année moins de 3% des parts de

marché, bien moins que les vins français et italiens qui eux détenaient 54% des parts (SAQ, 2017).

Ces faibles résultats peuvent s’expliquer en partie par une internationalisation de la population

québécoise via l’immigration (habitude de consommation) et/ou l’accès à de nombreux vins de styles

différents provenant de diverses régions du monde (concurrence directe). Mais outre le prix, la

qualité et le goût sont parmi les premiers critères pris en considération par la majorité des Québécois

lors de l’achat d’un vin (MAPAQ, 2016).

La qualité et le goût d’un vin rouge reposent en grande partie sur son acidité, son astringence et son

degré d’alcool, qui contribuent à l’équilibre de sa structure (Blouin & Cruège, 2013). Une structure

équilibrée, une belle robe ainsi qu’une harmonie des arômes au nez et en bouche sont autant de

signes d’un bon vin. Parmi les nombreuses molécules qui contribuent au goût du vin, les tanins

occupent une place prépondérante puisque leur concentration a été positivement corrélée avec la

perception de la qualité des vins (Kassara & Kennedy, 2011; Mercurio, Dambergs, Cozzolino,

Herderich, & Smith, 2010). Les tanins sont naturellement présents dans le raisin et sont cédés au vin

au cours du processus de vinification, plus spécifiquement lors de l’étape de macération moût-marc,

mais l’élevage en fût de chêne contribue aussi à augmenter la teneur en tanins des vins (Casassa &

Harbertson, 2014; Herderich & Smith, 2005).

Par définition, les tanins sont des substances de poids moléculaire variable (300-3000 g.mol-1)

capables de former une combinaison stable avec les protéines via des interactions hydrophobes et

des ponts hydrogènes (Gawel, 1998). Ils jouent conséquemment un rôle important dans la perception

de l’astringence en modifiant les propriétés lubrifiantes des protéines salivaires lors de l’ingestion du

vin (Soares, Brandão, Mateus, & De Freitas, 2017). L'intensité astringente augmente avec la teneur

en tanins des vins et s’accroit de façon non linéaire avec le degré de polymérisation et/ou le poids

moléculaire des tanins (W. Ma et al., 2014).

2

Dans les provinces de l’Est du Canada, telle que la province de Québec, la production vinicole

repose essentiellement sur la culture des cépages hybrides interspécifiques (CHI) (Dubé &

Pedneault, 2014). Le développement et la commercialisation de ces cépages a permis d’accroitre

considérablement l’essor de l’industrie vitivinicole québécoise ces quarante dernières années (Dubé

& Pedneault, 2014). Issus de croisements entre différentes espèces de vignes dont la vigne

européenne Vitis vinifera et les vignes nord-américaines (ex. : V. riparia, V. labrusca), les CHI

montrent une forte résistance aux conditions climatiques extrêmes et aux maladies fongiques, les

rendant mieux adaptés que leurs homologues européens aux conditions environnementales

limitantes de l’Est canadien (Pedneault & Provost, 2016).

La génétique distincte des CHI confère également à leurs baies une composition chimique différente

de celle des cépages Vitis vinifera, ce qui a des conséquences significatives sur les qualités

organoleptiques des vins qu’ils produisent. Bien que les raisins de CHI rouges permettent d’obtenir

des vins présentant des arômes agréables, leur teneur élevée en acide malique accroit leur acidité

tandis que leur forte teneur en anthocyanes leur donne une couleur foncée (Manns, Coquard Lenerz,

& Mansfield, 2013; Pedneault, Dorais, & Angers, 2013). Par ailleurs, leur teneur réduite en tanins

résulte en des vins peu astringents, présentant un manque de structure et peu de longueur en

bouche (Manns et al., 2013; Pedneault et al., 2013).

Une conduite appropriée de la vigne (ex. : palissage en hauteur, éclaircissage) et de la vinification

(ex. : levures désacidifiantes, désacidification microbiologique ou par procédés) et/ou l’assemblage

avec d’autres cépages moins acides permettent de gérer la forte acidité des CHI et de trouver les

équilibres requis (Aubry, 2018). Néanmoins, l’astringence demeure une composante encore peu

maitrisée chez les vins issus de CHI rouges. Sa modulation se fait généralement de façon empirique

par les vinificateurs québécois, via des tanisages soutenus.

Des résultats relativement récents sur la vinification des CHI rouges montrent une inefficacité

partielle ou générale de nombreux traitements pré-fermentaires tels que l’ajout de tanins exogènes,

le traitement par le froid, la thermovinification et l’enzymage pectique pour l’extraction et la rétention

des tanins dans les vins de CHI (Manns et al., 2013). De fait, ceux-ci présentent en moyenne jusqu’à

six fois moins de tanins que les cépages européens en dépit du fait que leurs baies en contiennent

en moyenne seulement deux fois moins que les cépages européens. Les travaux de Springer &

3

Sacks (2014) ont en partie attribué ces résultats par une plus forte teneur en protéines et

polysaccharides des raisins de CHI rouges.

À l’instar des tanins, d’autres molécules d’importances telles que les protéines et les polysaccharides

sont relarguées à partir du raisin vers le moût/vin durant le procédé de vinification. Ces

macromolécules jouent un rôle majeur dans la rétention des tanins, avec un impact significatif sur

l’astringence des vins rouges. Les protéines précipitent en présence de tanins, limitant alors la

rétention des tanins dans les vins de CHI rouges (Springer, Sherwood, & Sacks, 2016). Leur

élimination par chauffage ou par ajout de bentonite a récemment montré des résultats positifs sur la

rétention des tanins dans les vins de CHI (Springer, Chen, Stahlecker, Cousins, & Sacks, 2016). Par

ailleurs, le rôle des polysaccharides sur la rétention des tanins est plus ambigu. Les polysaccharides

du raisin ont montré un effet sur le comportement colloïdal des tanins et sur les interactions tanins-

protéines, néanmoins, ces effets varient selon la nature et la concentration du polysaccharide

(structure, taille, charge, etc.) ainsi que la force ionique et le pourcentage d’éthanol de la matrice

(Hanlin, Hrmova, Harbertson, & Downey, 2010).

Les anthocyanes jouent également un rôle prépondérant sur l’astringence des vins rouges. Ces

composés sont extraits lors de la pré-macération alcoolique ou tôt lors de la fermentation alcoolique

(Casassa & Harbertson, 2014). Ils favorisent l’extractabilité des tanins de la pellicule en interagissant

de façon concurrentielle avec les polysaccharides des parois cellulaires du raisin (Bautista-Ortín,

Martínez-Hernández, Ruiz-García, Gil-Muñoz, & Gómez-Plaza, 2016). Au cours de la vinification, les

anthocyanes réagissent avec les tanins et donnent naissance à des adduits anthocyane-tanin, aussi

appelés « pigments polymériques » (Cheynier, Souquet, Fulcrand, Sarni, & Moutounet, 1998),

stabilisant la couleur des vins d’une part, et bloquant la polymérisation des tanins et possiblement

leur précipitation d’autre part (Casassa & Harbertson, 2014). Chez les CHI, la présence

d’anthocyanes diglucosides en forte proportion tend à ralentir ces réactions entre tanins et

anthocyanes (Burtch, Mansfield, & Manns, 2017). Pour des degrés moyens de polymérisation

similaires, les pigments polymériques montrent une astringence moindre comparativement aux tanins

(Vidal, Francis, et al., 2004).

Au vu des connaissances actuelles, il apparait que les constituants macromoléculaires de la paroi

cellulaire végétale (protéines et polysaccharides) du raisin et les anthocyanes jouent un rôle

prépondérant sur l’extraction/rétention des tanins dans les vins de CHI durant la vinification et, par

4

conséquent, influencent la concentration en tanins des vins et leur astringence. Dans cette optique,

l’objectif général de cette thèse vise (1) à développer un procédé de vinification adapté à la

composition physico-chimique atypique des CHI rouges afin de produire des vins plus riches en

tanins, ayant un potentiel accru de satisfaire les goûts des consommateurs québécois et,

conjointement, (2) à clarifier le rôle des constituants de la paroi cellulaire du raisin (polysaccharides

et protéines) et des anthocyanes sur la rétention des tanins dans les vins de CHI.

Pour répondre à ces objectifs, nous avons dans un premier temps étudié l’effet du ratio moût/marc

durant la fermentation alcoolique et du ratio tanins/anthocyanes dans le vin, en remplaçant

partiellement ou totalement le marc de raisin de CHI rouge (pauvre en tanins et riche en

anthocyanes) par du marc de raisin de CHI blanc plus riche en tanins mais pauvre en anthocyanes

(chapitre 2). Cette alternative aux tanins œnologiques commerciaux s’inscrit parfaitement dans un

contexte de vitiviniculture durable en réutilisant le marc, résidu majoritaire de la vinification en blanc.

Cette démarche environnementale de plus en plus sollicitée par les consommateurs pourraient

apporter une valeur ajoutée au produit. Dans un second temps, nous avons étudié conjointement

l’impact de deux traitements pré-fermentaires du moût visant les protéines (bentonite et chaleur), de

l’absence/présence de marc de raisin pendant la vinification et de l’ajout de tanins œnologiques

commerciaux sur l’extraction et la rétention des tanins dans le vin (chapitre 3). Cette approche vise à

garder intacte la typicité aromatique du vin. Enfin, une étude comparative des polysaccharides des

vins de CHI rouges avec ceux issus de V. vinifera a été effectuée afin d’explorer l’impact potentiel de

ces derniers sur la rétention des tanins et la perception d’astringence des vins rouges issus de CHI

(chapitre 4). Les CHI rouges Frontenac et Marquette, ainsi que les CHI blancs Vidal et Frontenac

blanc ont été utilisés au cours de ces études, étant parmi les cépages les plus cultivés au Québec.

5

Chapitre 1. Revue de littérature

1.1. L’industrie vinicole du Québec

1.1.1. La place de l’industrie vinicole du Québec dans le monde

L’Organisation International de la vigne et du vin (OIV) a estimé récemment la production viticole

mondiale à 7,6 millions d’hectares (ha) ; près de 45% de cette production était alors attribuable au

continent européen (ex. : Espagne, 967 milliers ha ; France, 787 milliers ha ; Italie, 695 milliers ha ;

Portugal, 194 ha ; OIV, 2018). Non loin de 50% des raisins cultivés sont dédiés à la production de

vin, alors que le reste est utilisé pour la production de raisins de table (36%), de raisins secs (8%) et

de produits dérivés (ex. : jus, vinaigre, huile de pépins) (OIV, 2017). En 2017, la production mondiale

de vin était de 250 millions d’hectolitres (Miohl), la majeure partie de la production provenant de

l’Italie (42,5 Miohl), de la France (36,7 Miohl) et de l’Espagne (32,1 Miohl) (OIV, 2018). Le climat

méditerranéen (été chaud et sec) est propice à la culture de la vigne, toutefois, elle pousse

également dans des régions plus chaudes, comme l’Australie et l’Afrique du Sud, et des régions plus

froides, comme le Nord-Est et Nord-Ouest des États-Unis, le Sud-Est et Sud-Ouest du Canada, le

Nord-Est de la Chine et le Japon (Fig. 1.1 ; Carbonneau & Escudier (2017)).

Depuis les années quatre-vingt, la culture de la vigne connait un essor considérable dans la province

de Québec (Canada) où les conditions climatiques sont pourtant peu favorables à sa culture (hiver

très froid, très long et humide) (Dubé & Pedneault, 2014). En 2017, d’après l’Association des

vignerons du Québec (AVQ1), la province de Québec comptait 467 hectares de vignes en culture,

dont 377 hectares en production, soit une hausse de 12% par rapport à 2016 (AVQ, 2017). La

superficie du vignoble québécois représentait alors près de 3,8% du vignoble canadien, ce qui le

plaçait en troisième position derrière l’Ontario et la Colombie-Britannique (AVQ, 2017).

En 2017, 2 500 tonnes de raisins ont été foulés résultant en 2,3 millions de bouteilles de vin

embouteillées. Les ventes de vin ont totalisé 24,8 M$, une croissance de 33,3% par rapport à l’année

précédente (AVQ, 2017). Cependant, les vins québécois ne représentent que 3% des parts de

marché, bien moins que les vins français et italiens qui eux occupent 54% des parts (SAQ, 2017).

1 L’AVQ est devenue le Conseil des vins du Québec en 2018.

6

Figure 1.1. Principaux bassins viticoles dans le monde, incluant les vignobles canadiens, et les types de climats associés à ces régions (d’après Carbonneau &

Escudier (2017)).

7

1.1.2. Les cépages cultivés au Québec

1.1.2.1. La classification botanique de la vigne

Le genre Vitis sp., est une plante grimpante de la famille des Vitacées (Fig. 1.2). Il se subdivise en

deux sous-genres, Muscadinia et Vitis, qui se distinguent en fonction de leurs caractéristiques

morphologiques, anatomiques et cytologiques (Mullins, Bouquet, & Williams, 1992). Le sous-genre

Vitis, dont la majeure partie des vignes cultivées fait partie, comprend trois groupes de vignes qui se

définissent par leur origine géographique : américaine, euro-asiatique et asiatique stricto sensus. La

vigne euro-asiatique ne comprend que l’espèce V. vinifera, divisée en deux sous-espèces, la vigne

sauvage V. vinifera ssp sylvestris et la vigne cultivée V. vinifera ssp sativa (This, Lacombe, &

Thomas, 2006). Parmi les espèces asiatiques, V. amurensis est probablement l’espèce la plus

connue, notamment pour sa très grande résistance au froid (–40°C) (L. Liu & Li, 2013; Wan et al.,

2008). Les espèces américaines V. aestivalis, V. riparia, V. rupestris et V. berlandieri ont également

une grande résistance aux conditions climatiques extrêmes (ex. : –36°C pour V. riparia), ainsi qu’une

grande résistance aux maladies fongiques (Keller, 2015; Pedneault & Provost, 2016).

1.1.2.2. Les variétés de vigne au Québec

L’évolution du genre Vitis résulte à la fois de la sélection naturelle et de la domestication

(principalement effectuée sur l’espèce V. vinifera) par l’être humain (This et al., 2006). Chez la vigne,

un croisement résulte en de nombreux descendants qui sont tous, par définition, des hybrides.

Lorsque les parents appartiennent à la même espèce (ex. : V. vinifera X V. vinifera), on parle

d’hybrides intraspécifiques alors que lorsque les parents appartiennent à deux espèces différentes

(ex. : V. vinifera X V. riparia), on parle d’hybrides interspécifiques. Dans les deux cas, chaque

descendant représente une variété génétiquement spécifique (génotype). Le terme « cépage » est

habituellement utilisé pour désigner les variétés sélectionnées pour leurs caractéristiques

spécifiques ; un nom vernaculaire leur est alors attribué (ex. : Cabernet Sauvignon). Un cépage

donné est généralement constitué d’un ensemble de clones.

8

Figure 1.2. Famille des Vitacées.

9

Le profilage génotypique des cépages européens V. vinifera suggère qu’il en existe près de 5 000

(This et al., 2006). Ce sont les cépages les plus communément utilisés en viticulture et les plus

importants sur le plan économique. Les cépages hybrides interspécifiques franco-américains sont le

second groupe le plus représenté. Ils sont le résultat de croisements entre les vignes européennes V.

vinifera et les vignes sauvages américaines telles que V. riparia, V. rupestris et V. labrusca. Ils sont

de plus en plus utilisés en raison de leur grande tolérance aux maladies et au gel, dans les zones

froides et humides comme l’Est du Canada, le Nord-Est et Centre-Ouest des États-Unis ainsi que le

Nord-Est de l’Europe, mais également dans le Sud-Est des États-Unis, en raison de leur résistance à

la maladie de Pierce (Ehrhardt, Arapitsas, Stefanini, Flick, & Mattivi, 2014; Kamas, Stein, & Nesbitt,

2010; J. Liu et al., 2015; Manns et al., 2013; Zhang, Petersen, Liu, & Toldam-Andersen, 2015). Les

cépages V. vinifera issus de climats chauds sont généralement très sensibles aux blessures

provoquées par la rudesse du climat et nécessitent pour leur culture en climat froid l’utilisation de

systèmes de protection hivernale (Stafne, 2007).

Au Québec, en 2017, on dénombrait une soixantaine de cépages cultivés, dont près de 90% de

cépages hybrides interspécifiques (CHI ; AVQ, 2017). Deux cépages V. vinifera et dix CHI semi-

rustiques à rustiques2 représentaient près de 70% de la superficie totale cultivée : (i) les cépages V.

vinifera Pinot noir et Chardonnay (respectivement rouge et blanc) ainsi que les CHI (ii) rouges :

Frontenac noir, Marquette, Maréchal Foch, St. Croix et Sabrevois ; (iii) blancs : Vidal, Seyval blanc,

Frontenac blanc et St. Pépin ; et (iv) gris : Frontenac gris (Fig. 1.3). La génétique, susceptibilité aux

maladies et tolérance/résistance au froid de ces CHI sont présentées Tableau 1.1.

Au Québec, les températures hivernales peuvent atteindre de –25 à –40°C. La période de

débourrement des différents cépages s’échelonne donc généralement plus tardivement entre le

début et la fin du mois de mai avec des températures pouvant occasionnellement passer sous le

seuil des 0ºC (gel printanier). La saison de croissance, c’est à dire le nombre de jours sans gel, est

donc relativement courte, variant de 140 à 185 jours. La majorité des cépages cultivés au Québec,

tels que le Maréchal Foch et le Seyval blanc, présentent une période de maturité dite hâtive (Wolf,

2008). En d’autres termes, ces cépages sont en mesure de compléter leur cycle végétatif et leur

cycle reproducteur dans un laps de temps limité, coincé d’une part par les gels printaniers et d’autre

2 Par définition, la rusticité est la capacité d’une vigne en dormance à survivre aux températures hivernales et automnales

froides (Zabadal, Dami, Goffinet, Martinson, & Chien, 2007).

10

part par les gels d’automne. D’autres cépages tels que le Frontenac ou le Vidal blanc présentent une

période de maturité intermédiaire voire tardive pouvant ainsi conduire à une maturité incomplète du

fruit (Wolf, 2008).

Figure 1.3. Encépagement des membres du Conseil des vins du Québec de 2012 à 2017 (d’après AVQ,

2017).

11

Tableau 1.1. Génétique (en % de chaque espèce de vigne), susceptibilité aux maladies et tolérance/résistance au froid des principaux cépages cultivés au

Québec (d’après Dubé & Turcotte (2011); Pedneault & Provost (2016)).

Génétique Susceptibilité aux maladies et résistance au froid a

Cépage V

. vin

ifer

a

V. r

up

estr

is

V. r

ipar

ia

V. l

abru

sca

V. c

iner

ea

V. b

erla

nd

ieri

V. a

esti

valis

Po

llin

isat

ion

lib

re

Mild

iou

de

vig

ne

Bla

nc

de

vig

ne

Po

urr

itu

re g

rise

Po

urr

itu

re n

oir

e

An

thra

cno

se

Bra

s m

ort

no

ir

Tu

meu

r d

u c

olle

t

Eu

typ

iose

To

léra

nce

au

fro

id b

Frontenac noir 25,4 10,2 50,4 2,3 0 7,8 2,3 0 -/+ ++ ++ ++ + ++ –29 à –34°C

Marquette 63,1 7,7 19,3 4,3 0,4 0 3,4 1,6 - + + + +++ –29 à –34°C

Maréchal Foch 50 25 25 0 0 0 0 0 + ++ + ++ + +++ +++ –26 à –31°C

Seyval noir 54,7 31,2 0 0 0 0 14,1 0 + ++ –23 à –29°C

Sabrevois 42,2 14,1 9,4 25,4 0 0 6,6 2,3 + + + + –29 à –34°C

Vidal 75 15,6 0 0 0 0 9,4 0 + ++ -/+ + + + +++ + –20 à –26°C

Seyval blanc 54,7 31,2 0 0 0 0 14,1 0 + +++ ++ ++ + + ++ + –23 à –29°C

St. Croix 42,2 14,1 9,4 25,4 0 0 6,6 2,3 ++ ++ ++ + + –29 à –34°C

St. Pepin 39,8 28,1 6,3 12,5 0 0 13,3 0 + + + + –29 à –34°C

Frontenac blanc 25,4 10,2 50,4 2,3 0 7,8 2,3 0 -/+ + ++ ++ –29 à –34°C

Frontenac gris 25,4 10,2 50,4 2,3 0 7,8 2,3 0 -/+ + ++ + –29 à –34°C

a Échelle de résistance et susceptibilité aux maladies : −/+, résistant et peu susceptible ; −, résistant ; +, assez susceptible ; ++, modérément susceptible ; +++, hautement

susceptible. b Classes de rusticités des cépages : « very tender », –15 à –20°C ; « tender », –17 à –22°C ; « moderately tender », –20 à –23°C ; « moderately hardy », –23 à –26°C ; « hardy »,

–26 à –29°C « very hardy » –29 à –34°C (d’après Wolf (2008); Zabadal et al. (2007)).

12

D’un point de vue historique, les croisements interspécifiques de vignes européennes et

américaines (ex. : V. riparia et V. rupestris), naturellement résistantes au phylloxéra racinaire,

ont été introduits en Europe à la fin XIXème siècle afin de remédier aux pathologies et ravageurs

(ex. : oïdium, phylloxéra et pourriture noire). Originellement arrivés via l’importation des

cépages américains en Europe, ces pathologies et ravageurs ont successivement affecté le

vignoble européen (This et al., 2006). Fortement productifs, les CHI ont été à l’origine entre-

deux-guerres d’une surproduction de vin à faible coût. Les pays environnants tels que l’Algérie

et l’Espagne ont contribué également à la crise des vignobles européens en produisant de forte

quantité de vins issus de cépages V. vinifera à faible coût. Une démarche de qualité (AOC,

appellation d’origine contrôlée) a de ce fait été mise en place afin d’écarter entre autre les CHI

qualifiés de médiocres pour la production de vin comparativement à leurs homologues

européens (Trotignon, 2015). Les hybrides franco-américains ont alors été abandonnés (et

même proscrits des années cinquante jusqu’à tout récemment où certains cépages ont été

réintroduits dans le catalogue des variétés autorisées) au détriment du greffage de V. vinifera

sur des porte-greffes de vignes nord-américaines. Cette dernière option présentait l’avantage

de conserver la typicité et qualité des cépages européens tout en intégrant, via le porte-greffe

nord-américain la tolérance au phylloxera. Au Québec, les hybrides furent dans les années

quatre-vingt, la pierre angulaire de l’industrie vinicole québécoise, lorsque les premiers

vignobles commerciaux ont été implantés.

1.1.3. Les vins québécois et l’astringence

Les vins rouges québécois issus des CHI sont généralement décrits comme des vins souples,

parfois plus frais et plus colorés que leurs homologues européens ; des caractéristiques

sensorielles qu’il pourrait être souhaitable de diversifier afin de contribuer à une meilleure

compétitivité sur le marché. La qualité et le goût d’un vin rouge reposent en grande partie sur

son acidité, son astringence et son degré d’alcool, qui contribuent à l’équilibre de sa structure

(Blouin & Cruège, 2013). Parmi les nombreuses molécules qui contribuent au goût du vin, les

tanins occupent une place prépondérante puisque leur concentration et leur structure ont un

impact sur l’astringence des vins (Kassara & Kennedy, 2011; Mercurio, Dambergs, Cozzolino,

Herderich, & Smith, 2010).

L’astringence se définit comme une sensation tactile qui se correspond à une sensation de

rétrécissement, d’étirement et de plissement de l’épithélium buccal (Testing & Materials, 1978).

13

L'augmentation de la friction, la viscosité salivaire, les interactions entre les protéines salivaires

et les tanins ou encore entre les récepteurs du goût acide et les petits tanins condensés

seraient impliqués dans le développement de l’astringence (Bajec & Pickering, 2008).

Le mécanisme d’astringence le plus étudié est celui impliquant les interactions protéines-

tanins. Il est reconnu que les interactions et/ou la précipitation des protéines salivaires

(principalement celles riches en proline) avec les polyphénols du vin, majoritairement les tanins

condensés, jouent un rôle important dans la perception de l’astringence des vins. Ces

interactions modifient les propriétés lubrifiantes des protéines salivaires, conduisant ainsi à une

sensation tactile d’astringence au niveau de la paroi buccale (sentiment de sècheresse, de

rudesse ou encore de rugosité en bouche) (McRae & Kennedy, 2011; Scollary, Pásti, Kállay,

Blackman, & Clark, 2012; Soares et al., 2017).

L'intensité astringente d’un vin dépend de la teneur et de la structure de ses tanins (W. Ma et

al., 2014). Elle est corrélée positivement à la teneur en tanins (Harbertson, Kilmister, Kelm, &

Downey, 2014; Kallithraka, Kim, Tsakiris, Paraskevopoulos, & Soleas, 2011) et croit

également, sans être linéaire, avec le degré de polymérisation et/ou le poids moléculaire des

tanins (Sarni-Manchado, Cheynier, & Moutounet, 1999; Sun et al., 2013). Poncet-Legrand,

Cartalade, Putaux, Cheynier, & Vernhet (2003) observent que les tanins présentant un degré

de polymérisation de 8 à 10, permettent une combinaison stable avec les protéines salivaires

alors que les tanins ayant un degré de polymérisation supérieure à 15 présentent une meilleure

solubilité (Scollary et al., 2012). Le degré de galloylation, c’est à dire la proportion d’unités

flavan-3-ols portant un acide gallique estérifié au noyau flavan-3-ol, augmente l'intensité

astringente (Poncet-Legrand et al., 2003). Les tanins retrouvés dans les vins proviennent en

grande partie des baies de raisin ; leur concentration, de même que leur structure, varient

selon les cépages.

1.2. La composition phénolique des baies de raisin

Le raisin est une baie charnue constituée de pépins et d’un péricarpe, lequel est composé d'un

épicarpe (pellicule), d'un mésocarpe (pulpe) et d'un endocarpe (Fig. 1.4a) (Kennedy, 2002). La

composition chimique globale des principaux constituants de la baie est présentée au Tableau

1.2. La pellicule est formée d’un cuticule riche en cires lipidiques, des cellules de l'épiderme et

des cellules de l'hypoderme (Pinelo, Arnous, & Meyer, 2006) (Fig. 1.4b). Elle est le lieu de

synthèse et d'accumulation de nombreux composés d'intérêt œnologique, notamment les

14

tanins et les anthocyanes (Gagné, Saucier, & Gény, 2006). La pulpe, constituée de 25 à 30

assises cellulaires à maturité, est majoritairement constituée d'eau et contient la majeure partie

des sucres et des acides du raisin (Diakou & Carde, 2001). L'endocarpe contient une fine

couche de cellules en contact avec les pépins. Les pépins, constitués d'un embryon, d'un

albumen et d'un tégument, sont riches en tanins et en lipides (Winkler, 1974).

Tableau 1.2. Composition chimique (en % du poids frais) de la pellicule, de la pulpe et des pépins de

raisin (d’après Flancy (1998); Gros & Yerle (2014)).

Pellicule Pulpe Pépins

Eau 78 - 80% 70 - 80% 20 - 50%

Sucres - 10 - 25% -

Sels (hydrogénotartrate) - 1% -

Matières lipidiques 1 - 2% - 10 - 20%

Matières tanniques 0,4 - 3% - 7 - 8%

Pigments 0 - 0,5% - -

Matières acides 0,8 - 1,6% 1% 1%

Matières azotées 1,5 - 2% - 5%

Matières minérales 1,5 - 2% - 1 - 5%

Matières hydrocarbonées - - 30-35%

Les composés phénoliques présents dans le raisin peuvent se répartir en deux classes : (i) les

composés non-flavonoïdes incluant les acides phénols, divisés en acides benzoïques et

hydroxycinnamiques (structure en C6-C1 et C6-C3), ainsi que d’autres dérivés phénoliques tels

que les stilbènes (structure en C6-C2-C6) et (ii) les composés flavonoïdes (structure en C6-C3-

C6) comprenant les anthocyanes, les flavonols et les flavan-3-ols (Fig. 1.5). L'espèce de vigne,

le type de cépage, le millésime (température, ensoleillement, stress hydrique ou encore le

rendement de la parcelle) et la maturité déterminent en grande partie le potentiel phénolique du

raisin (Del Rio & Kennedy, 2006; Kennedy, Matthews, & Waterhouse, 2000; Springer & Sacks,

2014). Outre le fait d’être impliqués dans les processus de défenses des plantes contre les UV

et les attaques pathogènes, les composés phénoliques, et plus particulièrement les

anthocyanes et flavan-3-ols, ont un impact majeur en œnologie puisqu’ils sont notamment

responsables des différences de couleur et de saveur entre les vins (Kennedy, Saucier, &

Glories, 2006; Moreno-Arribas & Polo, 2009).

15

Figure 1.4. Structure ((a), d'après Kennedy (2002)) et organisation tissulaire ((b), d’après Fougère-Rifot & Cholet (1996)) d’une baie de raisin à maturité. Organisation

structurale de la paroi végétale primaire ((c), d’après Koolman, Röhm, Wirth, & Robertson (2005)) et structure des principaux polysaccharides de la paroi : (d), cellulose ; (e),

pectine (d’après Scheller, Jensen, Sørensen, Harholt, & Geshi (2007)) et (f), hémicellulose.

16

Figure 1.5. Exemple de composés phénoliques non-flavonoïdes et flavonoïdes présents dans les baies de raisin.

17

1.2.1. Les anthocyanes

Les anthocyanes sont les pigments rouges des raisins. Ils sont localisées dans les vacuoles

des cellules épidermiques de la pellicule de raisin et, dans le cas des cépages dits

‘‘teinturiers’’, dans les cellules de la pulpe (Amrani Joutei & Glories, 1995).

Les anthocyanes sont formés d’une partie aglycone, l’anthocyanidine, attachée à un ou deux

sucres, généralement un glucose (Chira, Suh, Saucier, & Teissèdre, 2008); (Ribéreau-Gayon,

Glories, Maujean, & Dubordieu, 2006). Les anthocyanidines les plus couramment rencontrés

chez Vitis sont la pelargonidine, la cyanidine, la delphinidine, la péonidine et la malvidine. Elles

ne sont présentes en quantité significative que chez les cépages rouges ou gris.

Les CHI rouges présentent des teneurs en anthocyanes généralement élevées, de l’ordre de

990 à 1 260 mg/kg baie (Sun, Sacks, Lerch, & Heuvel, 2011). Chez les V. vinifera, les teneurs

peuvent être plus ou moins importantes dépendamment du cépage et varier de 500 à 3 000

mg/kg baie (Chira et al., 2008; Ribéreau-Gayon, Glories, et al., 2006). Le Tableau 1.3 reprend

de façon synthétique ces données. Aussi, les CHI rouges présentent une forte proportion

d’anthocyanes diglucosylées qui peuvent représenter jusqu’à 100% des anthocyanes totaux

(Balík, Kumšta, & Rop, 2013; Van Buren, Bertino, Einset, Remaily, & Robinson, 1970). Chez V.

vinifera, ils ne sont présents qu’à l’état de traces (Ribéreau-Gayon, Glories, et al., 2006).

1.2.2. Les flavan-3-ols et proanthocyanidines

Par définition, les tanins condensés du raisin, également appelés proanthocyanidines, sont des

substances capables de former des combinaisons stables avec les protéines (Bate Smith &

Swain, 1965) ou encore les polysaccharides (Riou, Vernhet, Doco, & Moutounet, 2002).

Les tanins sont principalement localisés dans les pépins et la pellicule du raisin. Dans la

pellicule, les tanins se trouvent sous forme libre dans les vacuoles et liés aux protéines et

polysaccharides de la paroi cellulaire (Amrani & Mercierz, 1994; Gagné et al., 2006). Dans les

pépins, ils sont présents dans les enveloppes internes et externes, sous la cuticule et les

cellules épidermiques.

Les flavan-3-ols sont présents dans le raisin sous formes de simples monomères de (+)-

catéchine et (–)-épicatéchine et aussi sous formes polymérisées (oligomères et polymères de

18

flavan-3-ols) formant alors les tanins condensés (Ribéreau-Gayon, Dubourdieu, Donèche, &

Lonvaud, 2006). Les oligomères de flavan-3-ols sont formés de 2 à 5 unités de (+)-catéchine

ou (–)-épicatéchine alors que les polymères de flavan-3-ols sont formés de 6 unités et plus. La

condensation des flavanols est réalisée en position 4 et positions 6 et/ou 8, ce qui conduit à la

formation de liaisons interflavanes C4/C8 et/ou C4/C6 (Fig. 1.5 ; Hemingway, Foo, & Porter

(1982)). Les flavan-3-ols peuvent également être estérifiés avec l’acide gallique ou bien

hydroxylés pour former des gallocatéchines comme l’épicatéchine gallate, l’épigallocatéchine

et l’épigallocatéchine gallate ainsi que des gallotanins.

Les pépins de raisin des cépages V. vinifera et CHI rouges présentent des teneurs en

proanthocyanidines beaucoup plus importantes que la pellicule de raisin (Gagné, 2016;

Lorrain, Ky, Pechamat, & Teissedre, 2013). Ils présentent également un plus faible degré de

polymérisation (nombre moyen d’unités monomériques) que les pellicules de raisin (Tableau

1.3). Ce degré peut varier chez les V. vinifera de 2 à 17 unités de flavan-3-ols et il est

généralement plus faible, et de l’ordre de 3, chez les CHI rouges (Gagné, 2016; Lorrain et al.,

2013). Les pépins de raisin des cépages V. vinifera contiennent également une forte

proportion, de l’ordre de 30%, d’épicatéchine-3-gallate et de catéchine-3-gallate (Cheynier et

al., 1998).

La pellicule de raisin des cépages V. vinifera rouges présente un degré moyen de

polymérisation compris entre 3 et 83 (Brossaud, Cheynier, & Noble, 2001; Cheynier et al.,

1998; Gagné, 2016; Monagas, Gomez-Cordoves, Bartolome, Laureano, & Ricardo da Silva,

2003; Souquet, Veran, Mané, & Cheynier, 2006). La pellicule de raisin des CHI rouges contient

des tanins de plus de 9 unités flavan-3-ols et montre généralement une teneur en tanins

inférieure ou égale à celles des cépages V. vinifera : 0,35 mg/g baie chez les CHI rouges et

0,25-0,75 mg/g baie chez les V. vinifera (Harbertson, Kennedy, & Adams, 2002; Springer &

Sacks, 2014; Sun, Sacks, et al., 2011; Sun, Sacks, Lerch, & Heuvel, 2012). Le Tableau 1.3

reprend de façon synthétique ces données (pellicule et pépins).

19

Tableau 1.3. Teneur moyenne en anthocyanes et tanins des pellicules et pépins de baies de cépages

V. vinifera et de cépages hybrides interspécifiques (en mg/kg baie).

Cépages V. vinifera Cépages hybrides interspécifiques

Anthocyanes Pellicule 500-3 000 mg/kg baie 1 990-1 260 mg/kg baie 2

Flavan-3-ols Pellicule 100 à 750 mg/kg baie 3 ≤ 350 mg/kg baie 4

3-83 unités flavan-3-ols 5 ≥ 9 unités flavan-3-ols 6

Pépins 1 000-6 000 mg/kg baie 1 2-17 unités flavan-3-ols 7

200-1 200 mg/kg baie 6 ~3 unités flavan-3-ols 6

1 (Chira et al., 2008; Ribéreau-Gayon, Glories, et al., 2006)

2 (Sun, Sacks, et al., 2011)

3 (Chira et al., 2008; Harbertson et al., 2002)

4 (Springer & Sacks, 2014; Sun, Sacks, et al., 2011; Sun et al., 2012)

5 (Brossaud et al., 2001; Cheynier et al., 1998; Monagas et al., 2003; Souquet et al., 2006)

6 (Gagné, 2016)

7 (Lorrain et al., 2013)

1.3. La composition macromoléculaire de la baie de raisin

La paroi cellulaire du raisin est un réseau complexe de macromolécules : elle est constituée de

90% de polysaccharides de haut poids moléculaires (celluloses, hémicelluloses et pectines) et

de 10% de protéines structurales et enzymes qui contribuent à la fermeté des baies,

notamment pendant le processus de mûrissement (Fig. 1.4.c-f) (Carpita & Gibeaut, 1993;

Pinelo et al., 2006).

Chez V. vinifera, les baies de raisin perdent en fermeté durant le processus de maturation

(Maury et al., 2009; Robin, Abbal, & Salmon, 1997). Chez plusieurs CHI, les baies de raisin

présentent une pellicule plus épaisse et une plus grande fermeté même à pleine maturité

(Pedneault, Dubé, & Turcotte, 2011). La paroi cellulaire de la pellicule des baies des CHI

rouges, comparativement à leurs homologues européens, présente une teneur particulièrement

élevée en pectine (Apolinar-Valiente, Gomez-Plaza, Terrier, Doco, & Ros-Garcia, 2017; Lee,

Robinson, Van Buren, Acree, & Stoewsand, 1975; Springer & Sacks, 2014) De plus, la teneur

en protéines de la paroi cellulaire de leur pulpe est généralement plus élevée que celle des

cépages V. vinifera (Springer & Sacks, 2014). Les protéines de pathogenèse (protéines PR)

ont été trouvées en plus grande quantité dans les baies de ces cépages (Springer, Sherwood,

et al., 2016). Les protéines PR contribuent à la résistance accrue des cépages hybrides aux

maladies fongiques (Agarwal & Agarwal, 2014; J.-J. Liu & Ekramoddoullah, 2006; Singh et al.,

2014).

20

1.4. Du raisin au vin

1.4.1. Le processus de vinification

La vinification est l’ensemble des techniques mises en œuvre pour transformer le raisin en vin.

Le procédé de vinification est habituellement raisonné en fonction du cépage (ex. : maturités

technologique et phénolique, état sanitaire), du style et de la qualité du vin recherché, mais il

inclut généralement une extraction mesurée des composés désirables de la pellicule

(composés phénoliques et aromatiques) vers le moût.

Au Québec, la vinification traditionnelle de vin en rouge est la méthode de vinification la plus

utilisée par les vinificateurs. Les étapes de la vinification traditionnelle en rouge sont

schématisées en Fig. 1.6 et détaillées dans l’Appendix A. Si la macération et la fermentation

alcoolique sont souvent effectuées en simultané (vinification traditionnelle), elles peuvent aussi

être dissociées comme lors de la macération carbonique et de la thermovinification.

La vinification en rouge se distingue de la vinification en blanc par une utilisation soutenue de

processus de macérations ayant pour objectif d’extraire différents composés contribuant à la

qualité des vins comme les anthocyanes et les tanins. Les premiers contribuent à la couleur et

les seconds à l’astringence et à l’amertume. Toutefois, la macération conduit également à

l’extraction de macromolécules, telles que les protéines et les polysaccharides, qui peuvent

interférer avec la stabilisation de la couleur, l’astringence et l’amertume.

21

Figure 1.6. Schéma de vinification traditionnelle du vin en rouge (étapes détaillées en Appendix A).

22

1.4.2. Extraction et évolution des composés phénoliques durant la

vinification

1.4.2.1. Extraction

Les anthocyanes sont extraites préférentiellement en phase aqueuse lors de la macération pré-

fermentaire et de la fermentation alcoolique. Leur extraction arrive généralement à terme en

quelques jours, une fois un certain degré d'alcool atteint lors de la fermentation alcoolique (Ribéreau-

Gayon, Glories, et al., 2006). Les anthocyanes s’extraient plus ou moins facilement dépendamment

de la composition en pectines, en cellulose et en glucanes des parois des cellules de la pellicule

(Ortega-Regules, Romero-Cascales, Ros-García, López-Roca, & Gómez-Plaza, 2006). Les vins de

CHI peuvent contenir jusqu’à 600 mg/L d’anthocyanes dans le cas de cépage très coloré comme le

Frontenac ou le St. Croix (Gagné, 2016).

Les tanins condensés sont extraits pendant la fermentation alcoolique et la macération post-

fermentaire. Chez V. vinifera, les tanins de la pellicule formés de 2 à 3 unités de flavan-3-ols sont

extraits dès le début de la fermentation alcoolique tandis que les tanins oligomériques s’extraient au

cours de la vinification avec l’augmentation du degré d’alcool (González-Manzano, Santos-Buelga,

Pérez-Alonso, Rivas-Gonzalo, & Escribano-Bailón, 2006). Les tanins de haut poids moléculaires

(degré moyen de polymérisation > 20) sont plus difficilement extractibles puisqu’ils sont impliqués

dans des interactions hydrophobes avec les composants de la paroi cellulaire (protéines et

polysaccharides) pendant la macération. Toutefois, ces interactions s’affaiblissent avec

l'augmentation de la concentration en éthanol pendant la fermentation alcoolique (Casassa &

Harbertson, 2014). La solubilisation des tanins des pépins s’effectue plus tardivement,

principalement en phase post-fermentaire, une fois la cuticule dissoute par l’éthanol (Ribéreau-

Gayon, Glories, et al., 2006; Springer & Sacks, 2014). L’étape de post-fermentation donne par contre

des résultats contradictoires chez V. vinifera (Smith, McRae, & Bindon, 2015) : la composition

chimique du raisin, dépendante du cépage, du millésime et de la maturité, impacte grandement la

teneur initiale en tanins des baies de raisin ainsi que leur extraction et rétention dans le vin (Del Rio

& Kennedy, 2006; Kennedy et al., 2000; Springer & Sacks, 2014). Les vins de CHI contiennent des

tanins de faible degré moyen de polymérisation (dmp 4) et une concentration en tanin inférieure à

150 mg/L (Manns et al., 2013).

23

Les cinétiques d'extraction des tanins et des anthocyanes (Fig. 1.7) durant le processus de

fermentation suggèrent qu'un ratio tanins/anthocyanes défaillant initialement ne peut être modifié par

d'autres pratiques traditionnelles que l'addition de tanins exogènes. En effet, les méthodes favorisant

l'extraction des tanins, favorisent aussi l’extraction des anthocyanes.

Figure 1.7. Cinétique d’extraction des anthocyanes (bleu, point carré), tanins (orange, tiret point) et

polysaccharides (vert, tiret cadratin-point) du raisin lors des étapes de la vinification en rouge (Mpf, macération

pré-fermentaire ; FA, fermentation alcoolique ; et MPF, macération post-fermentaire) et évolution de l’intensité

colorante (noir, uni) chez Vitis vinifera (d’après Ribéreau-Gayon, Glories, et al. (2006)).

1.4.2.2. Évolution

Les anthocyanes, une fois extraites, voient leur concentration diminuer (Ribéreau-Gayon, Glories, et

al., 2006). Ces molécules instables se combinent par interactions moléculaires (liaisons de faibles

énergies) avec des copigments ou cofacteurs tels que des acides phénols, des flavonols, des

flavones ou encore des tanins (principalement des monomères et dimères). Ce phénomène est

appelé co-pigmentation (Boulton, 2001) .

Les tanins sont très réactifs et peuvent former par réaction avec eux-mêmes ou avec des

anthocyanes de nombreux dérivés tanins-tanins et anthocyanes-tanins dans le vin. La liaison des

anthocyanes sur les unités terminales des tanins a pour conséquence un arrêt de la polymérisation

24

des petits tanins entre eux (Boulton, 2001; Zoecklein, Fugelsang, Gump, & Nury, 1990) et engendre

donc une baisse de l'astringence des vins (McRae, Schulkin, Kassara, Holt, & Smith, 2013). Chez les

CHI, la présence d’anthocyanes diglucosides en forte proportion tend à ralentir les réactions entre

tanins et anthocyanes (Burtch, Mansfield, & Manns, 2017). De ce fait, la co-pigmentation contribue

davantage à la couleur des vins chez les CHI comparativement aux vins de cépages V. vinifera. Les

tanins présentent également des propriétés physico-chimiques spécifiques et peuvent former des

agrégats et interagir avec les protéines et les polysaccharides.

1.4.2.3. Interactions des tanins avec les protéines et polysaccharides

Deux types de polysaccharides sont susceptibles d’être extraits pendant la vinification : les

polysaccharides pectiques, qui proviennent principalement de la paroi cellulaire des baies de raisin,

et les mannoprotéines, des glycoprotéines provenant principalement des parois cellulaires des

microorganismes impliqués dans la fermentation (Dols-Lafargue et al., 2007; Z. Guadalupe &

Ayestarán, 2007; Vidal, Williams, Doco, Moutounet, & Pellerin, 2003). Les mécanismes d’interactions

entre les tanins et les polysaccharides sont mal connus, néanmoins, des interactions hydrophobes et

des ponts hydrogène semblent entrer en jeu (Le Bourvellec & Renard, 2012). Ces phénomènes

d’association ont un effet sur le comportement colloïdal des tanins et sur les interactions tanins-

protéines (et possiblement l’astringence), néanmoins, ces effets varient selon la nature et la

concentration du polysaccharide (structure, taille, charge, etc.) ainsi que la force ionique et le

pourcentage d’éthanol de la matrice (Hanlin et al., 2010).

Tout comme les polysaccharides, les protéines du vin rouge proviennent en partie de la baie et en

partie des microorganismes (glycoprotéines) (Springer, Sherwood, et al., 2016). Les interactions

hydrophobes (forces attractives de Van der Waals) ainsi que les ponts hydrogène occupent

également une place importante dans les interactions tanins-protéines (Fig. 1.8 ; (Le Bourvellec &

Renard, 2012).

25

Figure 1.8. Les différents mécanismes potentiels d'interactions entre polyphénols et protéines (d’après (Le

Bourvellec & Renard, 2012).

1.4.3. Impact des procédés vinicoles sur les tanins

Les travaux de Manns et al. (2013) montrent que les pratiques œnologiques favorisant

habituellement l’extraction des tanins dans le moût chez les cépages V. vinifera ne présentent pas de

réel intérêt pour la vinification des CHI rouges. Brièvement, les vins rouges de CHI obtenus après

macération pré-fermentaire (à froid ou à chaud) ainsi qu’après enzymage (pectinase) du moût ne

présentent pas de différences significatives au niveau des teneurs en tanins et de l’astringence des

vins par rapport à des vins issus d’une fermentation en rouge classique. Ces auteurs constatent

également qu'un ajout de tanins œnologiques à une dose commerciale en début de fermentation n'a

aucun impact sur le profil sensoriel des vins rouges hybrides L’addition de tanins exogènes

(tannisage) est une méthode largement utilisée lors de la vinification en rouge pour compenser une

carence en tanins des baies ou du vin fini et par conséquent, pour augmenter l’astringence d’un vin

fini (Kyraleou et al., 2015; Kyraleou et al., 2016). Le tannisage permet également de corriger un

26

rapport tanin-anthocyanes déficient, qui pourrait compromettre la stabilisation de la couleur d’un vin

rouge : chez Vitis vinifera, une bonne stabilisation de la couleur correspond à un ratio compris entre 1

et 4, avec un ratio optimal de 2 à 3 pour une bonne structure en bouche (Zoecklein et al., 1990). Cet

objectif est souvent atteint par l’utilisation de tanins œnologiques du commerce, soit des tanins

hydrolysables, traditionnellement dérivés du bois ou de la châtaigne, ou des tanins condensés,

principalement issus de la pellicule et/ou des pépins de raisin (Versari, du Toit, & Parpinello, 2013).

L’extractibilité des tanins des CHI par rapport à ceux des cépages V. vinifera s’explique en partie par

des interactions entre les tanins et les protéines solubles de la pulpe (protéines PR telles que la β-

glucanase, le précurseur de la chitinase, la protéine de type thaumatine (VVTL1), le précurseur de

VVTL1 et la peroxidase-4) et, dans une moindre mesure, entre les tanins et les pectines de la

pellicule (Springer, Chen, et al., 2016; Springer & Sacks, 2014). Le retrait de ces protéines par ajout

de bentonite ou chauffage en amont de la fermentation a conduit à une rétention des tanins

améliorée dans les vins de CHI mais l’effet a tout de même été limité (Springer, Chen, et al., 2016).

Le collage à la bentonite est couramment utilisé dans l’industrie vinicole pour éliminer les protéines

instables du vin blanc afin d’éviter les troubles. La bentonite est une argile colloïdale chargée

négativement au pH du vin ; elle interagit électrostatiquement avec les protéines du vin chargées

positivement, les faisant alors floculer (Ferreira, Piçarra-Pereira, Monteiro, Loureiro, & Teixeira,

2001). Le chauffage, quant à lui, entraîne des modifications conformationnelles des protéines en

éliminant l'eau, ce qui entraîne leur dénaturation et leur agrégation (Dufrechou, Poncet-Legrand,

Sauvage, & Vernhet, 2012).

1.5. Problématique, hypothèse et objectifs

1.5.1. Problématique

Les goûts des consommateurs québécois en matière de vin sont extrêmement diversifiés, d’une part

à cause de l’internationalisation de la population québécoise via l’immigration, mais aussi parce que

les Québécois ont accès à de nombreux vins de styles différents, provenant de diverses régions du

monde.

27

Les vins rouges québécois sont majoritairement produits à partir de cépages hybrides

interspécifiques rustiques. Ils sont généralement décrits comme plus souples, parfois plus frais et

plus colorés que leurs homologues européens ; des caractéristiques sensorielles qu’il pourrait être

souhaitable de diversifier afin de contribuer à une meilleure compétitivité sur le marché.

Les différences organoleptiques observées sur les vins s’expliquent par des différences génétiques

entre les cépages qui se reflètent dans la composition chimique de leurs raisins : Les raisins de CHI

présentent par exemple des teneurs en polysaccharides, protéines et anthocyanes plus élevées mais

des teneurs en tanins plus faibles que dans les cépages Vitis vinifera.

Les procédés de vinification traditionnellement utilisés sur les cépages rouges européens pour

augmenter la teneur en tanins des vins (donc l’astringence) ne conviennent pas aux CHI dû à leur

composition chimique atypique. Une étape d’élimination des protéines post-fermentation alcoolique

semble nécessaire pour une meilleure rétention des tanins dans le vin. Le chauffage et le collage à la

bentonite ont montré récemment des résultats encourageants, néanmoins les modalités d’utilisation

restent encore peu concluantes. Le collage des protéines par ajout de tanins exogènes à des teneurs

supérieures à la dose commerciale n’a quant à lui pas été étudié. D’autre part, le rôle des

polysaccharides doit être défini ; les informations à ce sujet (ex. teneur, composition) restent

fragmentaires chez les CHI. Quant aux anthocyanes, le rôle du ratio tanins/anthocyanes est connu

pour avoir un impact sur la couleur et le goût des vins rouges mais leur rôle n’a jamais été étudié

directement chez les CHI.

Cette thèse vise (1) à développer un procédé de vinification adapté à la composition physico-

chimique atypique des CHI rouges afin de produire des vins plus riches en tanins, ayant un potentiel

accru de répondre aux goûts des consommateurs et, conjointement, (2) à clarifier le rôle des

constituants de la paroi cellulaire du raisin (polysaccharides et protéines) et des anthocyanes sur la

rétention des tanins dans les vins rouges de CHI.

28

1.5.2. Hypothèse

Ce projet repose sur l’hypothèse que le développement de procédés de vinification adaptés à la

composition chimique des baies des cépages hybrides, incluant notamment l’ajout de tanins

exogènes en quantité suffisante et la réduction des interactions avec les constituants de la paroi

cellulaire du raisin (protéines et polysaccharides) durant la vinification, permettra d’augmenter

significativement la teneur en tanins des vins rouges issus de CHI.

1.5.3. Objectifs

Afin de vérifier l’hypothèse proposée dans ce projet, trois objectifs ont été définis. Chacun des

objectifs a fait l’objet d’un chapitre à part entière, sous forme d’article (chapitres 2, 3 et 4).

Objectif I : Étudier l’effet du ratio moût/marc durant la fermentation alcoolique et du ratio

tanins/anthocyanes dans le vin sur les profils phénolique (tanins et pigments), colorimétrique et

aromatique des vins, en remplaçant partiellement ou totalement le marc de raisin rouge Vitis sp.

Frontenac (pauvre en tanins et riche en anthocyanes) par du marc de raisin3 blanc Vitis sp. Vidal

(source naturelle et écologique de tanins mais pauvre en anthocyanes).

Objectif II : Étudier conjointement l’impact de deux traitements pré-fermentaires du moût

visant les protéines (bentonite et chaleur), de l’absence/présence de marc de raisin pendant la

vinification et de l’ajout de tanins œnologiques commerciaux sur l’extraction et la rétention des

composés phénoliques (tanins et pigments) et protéines des vins de Vitis sp. Frontenac en fin et

post-fermentation.

Objectif III : Étudier et comparer la cinétique d’extraction ainsi que la teneur et le profil des

polysaccharides des cépage Vitis sp. Frontenac avec le cépage Vitis vinifera Cabernet Sauvignon.

3 Le marc de raisin est le sous-produit majoritaire de la vinification. Il est constitué de pellicule, de pépins et de rafles. Il

est riche en composés phénoliques et aromatiques. Le marc représente 20 % du poids des raisins avant vinification, ce

qui correspond à un peu plus de 500 tonnes/an au Québec. Actuellement, ces résidus sont compostés et retournés aux

champs comme fertilisants organiques. Le développement de pratiques durables en agroalimentaire passe par la

valorisation de tels sous-produits.

29

Chapitre 2. Co-fermentation of red grapes and

white pomace: a natural and economical process

to modulate red hybrid wine composition

2.1. Avant-propos

Ce chapitre répond à l’objectif 1 qui vise à étudier l’effet du ratio moût/marc et du ratio

tanins/anthocyanes, en remplaçant partiellement ou totalement le marc de raisin de CHI rouge

(pauvre en tanins) par du marc de raisin de CHI blanc (riche en tanins mais pauvre en anthocyanes)

pendant la macération alcoolique. Les vins produits avec différentes proportions de marc rouge et

marc blanc (MRR/MRB) furent caractérisés et comparés sur leurs teneurs en tanins (flavan-3-ols), en

anthocyanes et en composés volatils. Les vins ont été analysés durant la vinification et après une

période de vieillissement. Les travaux de ce chapitre ont été publiés dans le journal Food Chemistry :

Nicolle, P., Marcotte, C., Angers, P., & Pedneault, K. (2018). Co-fermentation of red grapes and

white pomace: A natural and economical process to modulate hybrid wine composition. Food

Chemistry, 242, 481-490.

2.2. Résumé

L’impact de l’utilisation de marc de raisin blanc (MRB) en co-fermentation avec du marc de raisin

rouge (MRR) sur la composition chimique des vins a été étudié en utilisant les cépages hybrides

interspécifiques Frontenac et Vidal. Les teneurs en flavan-3-ols et en anthocyanes des vins finis ont

respectivement été analysées par chromatographie liquide haute performance couplée à un

détecteur de fluorescence et par chromatographie liquide à ultra-haute performance couplée à un

spectromètre de masse en tandem. Les caractéristiques chromatiques selon l’espace CIELab et la

composition en composés volatils des vins finis ont respectivement été analysées par spectrométrie-

UV et par micro extraction sur phase solide et chromatographie gazeuse couplée à un spectromètre

de masse. Les résultats ont montré que les vins produits avec du MRB présentaient plus de flavan-3-

ols monomériques et oligomériques et davantage de terpènes. La manipulation du ratio MRB/MRR a

conduit à une modification du profil des anthocyanes des vins finis, résultant, lors d’ajout excessif de

MRB, en l’obtention de vins plus clairs. L’utilisation d’un ratio MRB/MRR approprié (30% MRR/6%

30

MRB) a permis une meilleure stabilisation de la couleur des vins sans impacter de façon importante

la couleur. En conclusion, l’utilisation de MRB en co-fermentation avec du MRR s’avère être un outil

intéressant pour moduler la couleur des vins ainsi que leur composition en phénoliques et volatils.

2.3. Abstract

The impact of co-fermenting white grape pomace (WP) and red grape pomace (RP) on the

composition of interspecific hybrid wine was studied using the Vitis sp. ‘Frontenac’ and ‘Vidal’. The

flavan-3-ol and anthocyanin content of the resulting wines were analysed by HPLC-fluorescence and

UPLC-MS/MS, respectively. The CIELab parameters and volatile compounds were analysed using

spectrophotometry-UV and GC-MS-SPME, respectively. The WP addition increased the

concentration of monomeric and oligomeric flavan-3-ols and terpenes in the wines. The manipulation

of the WP/RP ratio efficiently modulated the anthocyanin profile of the wines, resulting in a faded red

colour, a desirable achievement in hybrid red wine, which is usually perceived as too dark. An

appropriate ratio (30% RP/6% WP) improved the colour stability of the wines without a significant

impact on wine colour. Addition of WP proved to be a suitable tool to modulate the colour and the

phenolic and volatile composition of interspecific hybrid wine.

2.4. Introduction

The development and commercialisation of cold-hardy interspecific hybrid grape cultivars have

contributed to the vast expansion of northern wine production, notably in Quebec, Canada. The

province of Quebec is the third largest wine producer in Canada, and interspecific hybrid cultivars,

such as Frontenac and Marquette, account for up to 90% of local grape production. Most of the

interspecific hybrid grapes are crosses between Vitis vinifera and wild North American native species

such as Vitis riparia, Vitis labrusca, and Vitis rupestris (Pedneault & Provost, 2016).

The wine market is a fast-expanding sector in the province of Quebec. In 2016, wine sales reached

CA$ 2.3 billion and accounted for 79.3% of alcohol sales in volume (wine, beer, cider, spirit, and

others). Red wine alone accounted for 65.8% of alcohol sales, but the clear majority (> 75%) of red

wine currently sold in Quebec is imported from Europe, and wine made in Quebec struggles to find its

place with less than 3% of the market share (SAQ, 2016).

31

Red wines produced from hybrid grapes can exhibit atypical organoleptic characteristics when

compared to the Vitis vinifera wines the consumers are used to. For instance, certain hybrid red

wines have been shown to carry higher concentrations of eugenol, cis-3-hexenol, 1,8-cineole,

nonanal, and (E,Z)-2,6-nonadienal that may contribute to undesirable vegetative and earthy aromas

(Slegers, Angers, Ouellet, Truchon, & Pedneault, 2015; Sun, Gates, Lavin, Acree, & Sacks, 2011).

This and other factors, reviewed by Pedneault & Provost (2016), have led to the assumption that

interspecific hybrids produce low-quality wines. In terms of mouthfeel, hybrid wines often have high

titratable acidity (Pedneault et al., 2013) and low astringency due to a small concentration of

polymeric flavan-3-ols (Manns et al., 2013; Springer, Chen, et al., 2016).

With respect to colour, hybrid wines do not undergo the colour evolution from purple towards orange

hues typical of Vitis vinifera wines and are less likely to form stable colour during ageing (Alcalde-

Eon, Escribano-Bailón, Santos-Buelga, & Rivas-Gonzalo, 2006; Li et al., 2016; Manns et al., 2013).

Indeed, hybrid cultivars are known to have high anthocyanin content resulting in deeply coloured red

wines. In addition, the anthocyanin profile of hybrid red wines is dominated by anthocyanin-3,5-

diglucosides (Manns et al., 2013). When compared to the anthocyanin monoglucosides typically

found in Vitis vinifera wines, anthocyanin diglucosides have been found to be less likely to react with

flavan-3-ols to form stable polymeric pigments at wine pH (Burtch et al., 2017; He et al., 2012b;

Manns et al., 2013). In contrast, short-term reactions such as self-association and copigmentation

between anthocyanin diglucosides and other components such as flavonols and flavan-3-ols are

thought to play a significant role in the colour development of hybrid red wines (He et al., 2012a;

Manns et al., 2013). Anthocyanins also promote the retention of polymeric proanthocyanins (≥ 5

flavan-3-ol units) in wine and therefore modulate wine mouthfeel by decreasing the sensory

perception of astringency (Casassa & Harbertson, 2014).

In order to improve hybrid wines acceptability among consumers, winemakers must adapt their

products to the market trend and constantly innovate to offer high-quality wines. Wine quality is

largely related to its chemical composition, especially regarding phenolic and volatile compounds

(Ribéreau-Gayon, Dubourdieu, et al., 2006; Sáenz-Navajas et al., 2015). Some studies have shown a

positive correlation between high tier wines and wines showing higher content in total phenolic

compounds and tannins (Kassara & Kennedy, 2011; Mercurio et al., 2010). Appropriate winemaking

techniques, mainly based on the duration and temperature of maceration, contribute to optimising the

32

extraction of varietal aromas and phenolic compounds in wine (Sacchi, Bisson, & Adams, 2005). The

use of additives is also a common practice to improve wine quality. For example, the addition of

enological tannins and wood chips are typically used to compensate for tannin deficiency and

contributes to improving colour stabilisation, wine structure, and aroma (Chen et al., 2016; Kyraleou

et al., 2016).

Severe restrictions regarding organic waste management are pushing the industry toward

sustainable development to improve cost-effectiveness and meet customers demand for naturally

and plant-sourced additives. One of the major by-products of the wine industry is grape pomace,

which consists of a mixture of berry skins, seeds, and stalks. The valorisation of this industrial waste

has received extensive attention from both the scientific and industry communities over the past few

years (García‐Lomillo & González‐SanJosé, 2017). Recently, non-aromatic vine-shoot extracts used

as biostimulants in viticulture successfully improved wine pH and colour intensity and diversified the

volatile and phenolic composition of wines (Sánchez-Gómez, Zalacain, Pardo, Alonso, & Salinas,

2017). Addition of enzymatic hydrolysate of grape seeds has also been proposed as a suitable

alternative to prevent colour losses during red wine fermentation (Cejudo-Bastante et al., 2016). Use

of fresh and dehydrated white grape by-products as wine additives in red winemaking at appropriate

levels was also shown to improve the phenolic potential of young red wines, therefore, contributing to

preserving wine colour during ageing (Gordillo et al., 2014; Pedroza, Carmona, Alonso, Salinas, &

Zalacain, 2013).

Using fresh grape pomace as an additive in the wine industry requires fast turn-around because of its

perishability, making such management difficult for winemakers. In addition, enormous volumes of

grape pomace are produced on a weekly basis, during a very busy period of the year, making it even

more complicated to manage. On the other hand, processing grape pomace into ready-to-use

concentrates using extraction-concentration technology also results in significant costs. In small-scale

winemaking as that occurring in the emerging cold climate wine industry, using fresh pomace

remains the most cost-effective solution, when concomitant harvest dates make it feasible.

This work aimed to study the impact of white pomace c.v. ‘Vidal’ during the fermentation of red Vitis

sp. berries c.v. ‘Frontenac’. White grape pomace was chosen as a natural additive that could improve

the concentrations of tannins and other non-anthocyanin compounds, including volatiles, in hybrid red

wines while preventing the extraction of additional anthocyanins as occurs with typical skin

33

maceration. Different proportions of red and white grape pomace were assayed to produce different

compositional profiles, and wines were evaluated for phenolic, colour, and volatile compounds during

winemaking and wine ageing.

2.5. Material and Methods

2.5.1. Chemicals

Polyphenol analysis: Acetic acid (HPLC grade), hydrochloric acid (37% solution in water), and

acetaldehyde were purchased from Fisher Scientific (Ottawa, ON, Canada). Methanol and

acetonitrile (HPLC grade) were purchased from EMD Millipore (Toronto, ON, Canada). (−)-

Epicatechin standard, trifluoroacetic acid (HPLC grade), and sodium bisulphite were purchased from

Sigma-Aldrich (Oakville, ON, Canada). Cyanidin-3-glucoside, delphinidin-3-glucoside, malvidin-3-

glucoside, pelargonidin-3-glucoside, and peonidin-3-glucoside standards were purchased from

Extrasynthèse (Lyon, France) and Alkemist Labs (Costa Mesa, CA, USA). Purified water was

obtained from a MiliQ filtration system.

Volatile compound analysis: Absolute ethanol was purchased from Commercial Alcohols (Brampton,

ON, Canada) and sodium chloride (NaCl) from Fisher Scientific (Fair Lawn, NJ, USA). Deuterated

standards (d8-ethyl acetate, d13-hexanol, 3-methyl-1-butyl alcohol-d4, d5-2,3,4,5,6-benzyl alcohol, and

2-phenyl-d5-ethanol) were purchased from C/D/N Isotopes Inc. (Pointe-Claire, QC). β-Myrcene was

purchased from MP Biomedicals (Santa Ana, CA, USA). Ethyl hexanoate and ethyl propanoate were

purchased from Nu-Chek-Prep (Elysian, MN, USA). Other reagents and standards were purchased

from Sigma-Aldrich (St. Louis, MO, USA) (Slegers et al., 2015).

2.5.2. Grape materials

The white Vitis sp. variety c.v. ‘Vidal’ (Rayon d’Or (S. 4986) × Ugni blanc) and the red Vitis sp. variety

c.v. ‘Frontenac’ (Landot (L. 4511) × Vitis riparia 89) were used for this study. These cultivars were

selected because they are largely grown in Quebec, Canada, and they typically ripen around the

same time (mid-October), hence facilitating their use in co-fermentation. Frontenac grapes (1.5 T;

21.4 °Brix; 14.2 g/L as tartaric acid equivalent; pH 3.3) were obtained from a commercial grower

located in St-Rémi (QC, Canada; 45° 16′ 0″ N, 73° 37′ 0″ W). Vidal pomace (approx. 350 kg) was

34

obtained from a commercial grower located in Dunham (QC, Canada; 45° 7′ 60″ N, 72° 48′ 0″ W).

The Vidal grapes were processed for traditional white winemaking at maturity as follows: Grapes

were partly destemmed, preserving 10% intact clusters, and pressed. The residue was recovered,

and the remaining stems were removed manually, yielding the white grape pomace (WP), which

majorly contained skins and seeds. In order to prevent spoilage, the WP was stored at 4°C and

treated with 20 mg/kg of sulphur dioxide (SO2) until further use.

2.5.3. Winemaking trials

Frontenac grapes were destemmed, crushed, treated with 30 mg/kg of SO2, and cold-soaked in

stainless steel tanks under an inert atmosphere at 12°C overnight. The juice and red grape pomace

(RP) were recovered by pressing the grape mash (1.8 bar) and kept separately until winemaking. The

WP was pressed (1.8 bar) to remove the sulphated solution.

Five co-fermentation treatments were prepared using different proportions of WP and RP co-

fermented in red Frontenac juice: a) 50% w/w of RP in juice as control; b) 30% w/w of RP and 6%

w/w WP in red juice; c) 30% w/w of RP and 12% w/w WP in red juice; d) 30% w/w of RP and 18%

w/w of WP in red juice; and e) 23% w/w WP in red juice. Each treatment was carried out in 4

replicates (n = 4). Fermentations were conducted in 100 L stainless steel tanks equipped with floating

lids as follows: Alcoholic fermentation was induced by a commercial dry yeast Saccharomyces

cerevisiae (Anchor NT50; Scott Laboratories, Pickering, ON, Canada) at 25 g/hL and carried out at

24°C until dryness. The cap was punched down twice a day. The progression of alcoholic

fermentation was monitored daily by measuring specific gravity. The wine was pressed at the end of

alcoholic fermentation. Free-run and pressed wines were combined and stored in 22 L glass carboys

equipped with air locks in the dark. The wine was then inoculated with Oenococcus oeni lactic acid

bacteria (MBR31; Scott Laboratories, Pickering, ON, Canada) at 1 g/hL to induce malolactic

fermentation. Malolactic fermentation was controlled by paper chromatography using the protocol of

Institut Français de la Vigne et du Vin (IFV, 2017). At the end of malolactic fermentation (45 days),

the wine was racked, treated with SO2 (50 mg/L), and cold stabilised (0°C, 1 month). The level of free

SO2 was then readjusted at 50 mg/L. Wines were filtered over 0.45 μm, bottled, and stored at 4°C

until analyses. The composition of the final wines (alcohol concentration, % v/v; titratable acidity, g

tartaric acid equivalent/L; pH; free SO2, mg/L) is provided in the Table S2.1 of the Appendix B.

35

Wines sampling begun after the pre-fermentative cold maceration (PFM) and were conducted on

days 1, 4, and 8 of the fermentative alcoholic maceration (1-FAM, m-FAM, and e-FAM, respectively);

after the malolactic fermentation (MLF, day 45); and after bottling (BW, day 395). The samples were

stored at −30°C until analysis.

2.5.4. Phenolic compound analysis

2.5.4.1. Phenol estimation

Phenol estimation was carried out as described by Ducasse et al. (2010). Absorption readings were

done on a Shimadzu UV–Vis spectrophotometer UV-2700 with 1-cm path length glass cells and a

high absorbance kit including a partition plate and a dark filter (Shimadzu, Kyoto, Japan). Samples

were centrifuged at 10,000 rpm (4°C, 15 min) before spectrophotometric analyses. Briefly,

absorbance measurements at 420, 520, and 620 nm (A420, A520, and A620, respectively) were

done; hue (H), colour intensity (CI), and wine pigments (WP) at wine pH were calculated as

A420/A520, A420 + A520 + A620, and A520, respectively. Same measurements were done 30 min

after addition of aqueous acetaldehyde solution (10% w/v); hue corrected (Hcor), colour intensity

corrected (CIcor), and wine pigments corrected (WPcor) for bisulphite content at wine pH were

calculated. Colour due to phenolic derivatives resistant to sulphite bleaching (PRSO2) was determined

at 520 nm, 30 min after addition of a solution of sodium metabisulphite (20% w/v). Total wine

pigments at pH < 1 (PpH<1) and total polyphenol index (TPI) were measured at 520 and 280 nm,

respectively, after diluting samples 100-fold in HCl (1 M) and maintaining them at room temperature

for 4 h. Analyses were performed in duplicates.

2.5.4.2. Anthocyanin analysis

Samples were filtered through 0.22 μm nylon syringe filters (25 mm diam.; Silicycle, Quebec City,

QC, Canada) and diluted in a solution of methanol:water (1:4, v/v) containing 0.1% trifluoroacetic

acid. Two different dilution factors (DF = 2 and 25) were used to allow proper quantification of both

minor and major compounds. Anthocyanins were characterised as described by Martí et al. (2010)

using a Waters Acquity H-class UPLC–MS/MS (Waters, Mississauga, ON, Canada) equipped with a

quaternary pump system (Waters, Mississauga, ON, Canada). The separation was achieved using

an Acquity High-Strength silica (HSS) T3 column (100 mm × 2.1 mm i.d.; 1.8 μm particle size;

36

Waters, Mississauga, ON, Canada) maintained at 30°C. The mobile phases consisted of 10% v/v

acetic acid in water (solvent A) and 100% acetonitrile (solvent B) with the following gradient: 5–35%

B, from 0 to 10 min; 35–80% B, from 10 to 10.1 min; 80% B isocratic, from 10.1 to 11 min; 80–5% B,

from 11 to 11.1 min; and 5% B isocratic, from 11.1 to 13 min. The flow rate was set at 0.4 mL/min.

The mass spectrometry (MS) analyses were carried out in positive mode using a Xevo TQD mass

spectrometer (Waters, Mississauga, ON, Canada) equipped with a Z-spray electrospray interface.

The MS parameters were set as follows: Electrospray capillary voltage at 2.5 kV, source temperature

at 150°C, cone and desolvation gas flow rate at 50 L/h and 800 L/h, respectively, and desolvation

temperature at 400°C. Nitrogen (99% purity) and argon (99% purity) were used as nebulising and

collision gases, respectively. Data were acquired through multiple reactions monitoring (MRM) using

the Waters Masslynx V4.1 software (Waters, Mississauga, ON, Canada). Results were quantified as

cyanidin-3-glucoside, delphinidin-3-glucoside, malvidin-3-glucoside, pelargonidin-3-glucoside, and

peonidin-3-glucoside equivalent, depending on the aglycone.

2.5.4.3. Flavan-3-ol analysis

Samples were filtered through 0.45 μm PTFE syringe filters (25 mm diam., Silicycle, Quebec City,

QC, Canada) prior HPLC analyses. Flavan-3-ol content and composition analyses were carried out

as described by Wallace & Giusti (2010) using an Agilent 1260 Infinity system (Agilent, Santa Clara,

CA, USA) equipped with a fluorescence detector (G1321C, Agilent, Santa Clara, CA, USA). The

separation was performed on a Develosil Diol column (250 mm × 4.6 mm; 5 μm particle size)

protected with a Cyano SecurityGuard column (Phenomenex, Torrance, CA, USA) and maintained at

35°C. A gradient of acetonitrile:acetic acid (98:2, v/v; solvent A) and methanol:water:acetic acid

(95:3:2, v/v; solvent B) was used with the following gradient: 0–40% B, from 0 to 35 min; 40–100% B,

from 35 to 40 min; isocratic at 100% B, from 40 to 45 min; and 100–0% B, from 45 to 50 min. The

flow rate was maintained at 0.8 mL/min. Compounds were separated according to their degree of

polymerisation (1–9 units of flavan-3-ols and polymers (> 10 units)). Compounds were detected by

fluorescence at 230 and 321 nm for excitation and emission wavelengths, respectively, and

quantified based on an external calibration curve of (−)-epicatechin (0–100 mg/L). Correction factors

were used to adjust the respective responses of small to large proanthocyanidins in fluorescence

(Prior & Gu, 2005).

37

2.5.5. Colour analysis

Samples were centrifuged at 10,000 rpm (4°C, 15 min) prior analyses. Colour measurements were

carried out using a Shimadzu UV–Vis spectrophotometer UV-2700, with 1-cm path length glass cells

and a high-absorbance kit including a partition plate and a dark filter (Shimadzu, Kyoto, Japan). The

visible spectrum was recorded from 400 to 600 nm, with 2 nm increments, taking the illuminant D65

and 10-degree observer as references. Distilled water was used as a blank. Analyses were

performed in duplicates. The colour definition was made using the CIELab space. The CIELab

parameters (L, a, b, C, and H) were determined by using the colour measurement software

(Shimadzu, Kyoto, Japan). The L coordinate represents the lightness (L is related to the darkest

black at L = 0 and the brightest white at L = 100). The a axis represents the green-red opponent

colours (a < 0 values relate to greenness and a > 0 values relate to redness) and the b axis, the blue-

yellow opponent colours (b < 0 values relate to blueness and b > 0 values relate to yellowness). C

and H are the chromaticness and the hue angle, respectively. The colour difference (ΔE) between

samples was calculated to evaluate the impact of WP on the colour of bottled wines and colour

evolution. The colour difference was calculated as the Euclidean distance between two points in the

three-dimensional space defined by L, a, and b: . A colour

difference superior to 3 in wine is visually perceivable by the human eye (Habekost, 2013; Martínez,

Melgosa, Pérez, Hita, & Negueruela, 2001).

2.5.6. Volatile compound analysis

Wine samples were prepared and analysed as described by Slegers et al. (2015) using an Agilent

6890 Series gas chromatograph-mass spectrophotometer equipped with a time-of-flight (Pegasus HT

TOFMS; Leco, Saint Joseph, MI, USA) and a Gerstel Solid-Phase microextraction system (Linthicum,

MD, USA) connected to a computer with the Leco ChromaTOF software (Leco, Saint Joseph, MI,

USA). Briefly, wine samples (3 mL) were placed in a 20 mL vial containing 3 mL of distilled water,

sodium chloride (3 g), and a solution of deuterated standards (50 μL) including 2-phenyl-d5-ethanol,

hexanol-d13, benzyl alcohol-d5, ethyl acetate-d8, and 3-methyl-1-butyl alcohol-d4. Volatile compounds

were extracted using a 2-cm grey fibre coated with 50–30 μm

Divinylbenzene/Carboxen/Polydimethylsiloxane. Extraction was performed at 60°C (25 min, 500

rpm). Volatile compounds were desorbed for 5 min to an open tubular DB-Wax column (Polyethylene

38

glycol, 60 m × 0.25 mm i.d.× 0.25 μm film thickness; SGE, Austin, TX, USA) using a splitless injector

set at 270°C. The oven temperature was programmed as follows: isothermal at 30°C for 1 min;

increased to 40°C at a rate of 10°C/min; increased to 240°C at a rate of 3.5°C/min; isothermal for 2

min; increased to 250°C at a rate of 20°C/min, and isothermal for 5 min. Helium was used as carrier

gas under constant flow (1 mL/min). Volatile compounds were identified by comparing retention time,

retention indices, and the mass abundance of selected ions with those of authentic standards and by

matching spectral data with the NIST Spectral Library as described by Slegers et al. (2015). Analyte

relative concentrations were estimated using the ratio of the surface area for each analyte to the

surface area of the selected deuterated standard (Table S2.4 of Appendix B).

2.5.7. Statistical analysis

Flavan-3-ol compounds, phenols (spectral analysis), and CIELab parameters were analysed with the

SAS software (version 3.5 Basic Edition; SAS Institute Inc., Cary, NC, USA) using analysis of

variance (ANOVA) methods with PROC MIXED statement, analysing the main and interaction effects

of the 2 factors: treatment and day. Since each wine was sampled during FAM and ageing, a

repeated-measures model was used, along with the DIFF option in a least-squares means

(LSMEANS) statement. Means were compared using the PDMIX macro formatting tool developed by

Saxton (1998) and a user-specified Tukey’s honest significant difference comparison test (at the 0.05

probability level) to assign letter groupings.

ANOVA on anthocyanin and volatile compound data at the BW stage was done using the MIXED

procedure of the SAS software (version 3.5 Basic Edition; SAS Institute Inc., Cary, NC, USA). The

DIFF option in a LSMEANS statement was used, and means were compared as described earlier.

Principal component analyses (PCA) were carried out to compare the volatile and phenolic

compound profile of the different treatments. Variables for the PCA were selected based on the result

of the ANOVA, and only significant variables were used for the PCA. The PCA were carried out using

the SPSS software (version 20; SPSS Inc., Chicago, IL, USA).

39

2.6. Results and Discussion

2.6.1. Effect on phenolic compounds

The kinetics of flavan-3-ols, anthocyanins, and others phenolic parameters such as spectral analyses

of pigments are shown in Figs 2.1 and 2.2 and in Table 2.1 (Additional representations of data, as

well as values and statistics, are available in Appendix B).

2.6.1.1. Effect on flavan-3-ols

At the BW stage, the RP/WP- and WP-treated wines contained significantly higher concentrations of

monomeric and oligomeric flavan-3-ols (2–5 units of flavan-3-ols) compared to the control wines and

these concentrations increased as the proportion of WP increased, reaching values 3–5 times higher

than those of control wines (Fig. 2.1a and b). In contrast, the addition of WP had little to no impact on

the content in polymeric flavan-3-ols (≥ 6 units of flavan-3-ols, Fig. 2.1c). A significant difference was

only observed between the control wines and the wines treated with 18% and 23% of WP; the wines

with excessive WP addition (> 18%) and less RP addition (< 30%) showed smaller polymeric flavan-

3-ol content.

The kinetic curves of flavan-3-ol compound concentration during winemaking showed that the content

in polymeric flavan-3-ols increased later at the MFL stage, whereas monomeric and oligomeric

flavan-3-ol content increased earlier at the FAM stage. Until pressing, at the e-FAM stage, the

phenolic compounds were extracted from the pomace, while during MLF and until bottling change of

colour and phenolic compounds were likely attributable to chemical reactions. This observation is

consistent with the results of González-Manzano et al. (2006) which showed an extraction of

monomers to trimers at the end of the cold pre-fermentative maceration and continuous extraction of

oligomers in further winemaking stages. Indeed, high molecular weight proanthocyanidins (average

degree of polymerisation > 20) are involved in hydrophobic interactions with the cell wall components

during maceration; these interactions are weakened by the increasing ethanol concentration

occurring during the alcoholic fermentation (Casassa & Harbertson, 2014). In interspecific hybrids,

cell wall components from berries are known to bind tannins at higher rates than cell wall

components from V. vinifera berries (Springer & Sacks, 2014). The main molecules responsible for

this binding appear to be pathogenesis-related proteins which have been detected in high

40

concentration in hybrid cultivars (Springer, Sherwood, et al., 2016). In wine, oligomeric and polymeric

flavan-3-ols are known to be responsible for the development of astringency, whereas the monomeric

flavan-3-ols likely impact the perception of bitterness (Soares et al., 2017).

41

Figure 2.1. Monomeric (a), oligomeric (2-5 flavan-3-ol units) (b), polymeric ( 6 flavan-3-ol units) (c), and total

(d) flavan-3-ol concentration (mean ± standard deviation, mg/L epicatechin equivalent) in control (50% RP),

RP/WP-treated (30% RP/6% WP, 30% RP/12% WP, and 30% RP/18% WP), and WP-treated (23% WP) wines

at different winemaking stages: PFM, after the pre-fermentative cold maceration; 1-FAM, m-FAM, and e-FAM,

during the fermentative alcoholic maceration (at days 1, 4, and 8); MLF, after the malolactic fermentation (day

45); and BW, after bottling (day 395). Means comparison using Tukey’s honest significant difference test at the

0.05 probability level is shown in Appendix B.

c)

42

2.6.1.2. Effect on pigments

At the BW stage, the RP/WP- and WP-treated wines showed a significantly lower concentration of

total pigments at acidic pH (PpH<1) than the control wines (Fig. 2.2a). In addition, the concentration of

PpH<1 was found to decrease significantly as the amount of RP decreased (3.6 times less for WP-

treated wines and up to 1.4 times for RP/WP-treated wines). Such pigment loss had a significant

impact on wine colour given that monomeric anthocyanins largely contribute to the colour of young

red wines (He et al., 2012a).

43

Figure 2.2. Phenol parameters including pigments at acidic pH (PpH < 1) (a), wine pigments corrected (WPcor)

(b), pigments resisting to sulphite bleaching (PRSO2) (c), and colour intensity corrected (CIcor) (d) (mean ±

standard deviation, absorbance unit) for control (50% RP), RP/WP-treated (30% RP/6% WP, 30% RP/12%

WP, and 30% RP/18% WP), and WP-treated (23% WP) wines at different winemaking stages: 1-FAM, m-FAM,

and e-FAM, during the fermentative alcoholic maceration (at days 1, 4, and 8); MLF, after the malolactic

fermentation (day 45); and BW, after bottling (day 395). Means comparison using Tukey’s honest significant

difference test at the 0.05 probability level is shown in Appendix B.

44

HPLC–MS/MS analyses of 26 anthocyanins showed variable levels on anthocyanins, including

primarily free anthocyanins, in the control wines when compared to WP-treated wines (1153.01,

708.35, 706.93, 677.97, and 231.56 mg/L anthocyanin-3,5-diglucoside equivalent; from 0 to 23% of

WP, respectively; Table 2.1) at the BW stage. Also, the absence of RP during the FAM modified the

proportions of cyanidin, delphinidin, malvidin, peonidin, and petunidin derivatives. Indeed, in the WP-

treated wines, malvidin and peonidin derivatives were extracted primarily during the pre-maceration

stage, whereas other derivatives released slowly during the FAM. Differences in the anthocyanin

profile have a direct impact on wine colour as recently demonstrated by Burtch et al. (2017).

Experimental wines showed relatively small amounts of polymeric pigments. The treatments involving

WP showed significantly lower concentration in polymeric pigments based on the pigment resisting to

sulphite bleaching to wine pigment corrected ratio (PRSO2/WPcor = 16.7% for WP-treated wines

versus 28.3% for the control wines, based on the data presented in Fig. 2.2b and c).

HPLC–MS/MS analysis showed that the proportion of glucoside/diglucoside was almost 1:1 in the

bottled wines, with malvidin derivatives in their glucoside and diglucoside form as major compounds

(Table 2.1). Diglucosylated anthocyanins are known to be more stable than monoglucosides (He et

al., 2012b). Thus, their concentrations have been found to decrease more slowly than the ones of the

monoglucosides in model wine solutions containing both (Burtch et al., 2017). Moreover, in the

presence of diglucosides, monoglucosides have slower reaction rates than monoglucosides alone

(Burtch et al., 2017) This observation could probably explain the low transformation rate of

anthocyanins in polymeric pigments observed in the experimental wines. The incorporation of

anthocyanins into polymers modulates the perception of astringency in wines (Casassa &

Harbertson, 2014) but also contributes to increasing the solubility and the retention of oligomeric and

polymeric flavan-3-ols via the formation of polymeric pigments (Casassa & Harbertson, 2014).

In order to resume the effect of the WP addition on the phenolic composition of the bottled wines, a

PCA was carried out on the flavan-3-ol compounds and the phenol parameters determined by

spectrophotometry-UV (Fig. 2.3). The principal component 1 (PC 1) explained 66.2% of the variation

of the phenolic composition and the principal component 2 (PC 2) explained 13.8%.

45

Table 2.1. Concentration of anthocyanin compounds (mean ± standard deviation, in mg/L cyanidin-3-glucoside, delphinidin-3-glucoside, malvidin-3-glucoside,

pelargonidin-3-glucoside, and peonidin-3-glucoside equivalent depending of the aglycone) in control (50% RP), RP/WP-treated (30% RP/6% WP, 30% RP/12%

WP, and 30% RP/18% WP), and WP-treated (23% WP) wines after 395 days of bottling and ESI-MS m/z values (molecular ion; product ions) of anthocyanins

detected in red Frontenac wines.

Anthocyanins a MS; MS2 (m/z) RT (min)

50% RP (control)

30% RP/ 6% WP 30% RP / 12% WP 30% RP/ 18% WP 23% WP

Cy-3,5-diglc 611; 449; 287 1.4 19.51 ± 1.10 b a 13.42 ± 2.04 b 13.01 ± 0.59 b 12.38 ± 0.25 b 5.35 ± 0.94 c

Cy-3-cmglc 595; 287 6.7 0.31 ± 0.06 a 0.21 ± 0.10 a,b 0.23 ± 0.06 a 0.18 ± 0.02 a 0.03 ± 0.01 b

Cy-3-glc 449; 287 2.74 3.74 ± 0.39 a 1.67 ± 0.64 b 2.05 ± 0.44 b 1.65 ± 0.24 b 0.35 ± 0.04 c

Sum cyanidin derivatives (mg/L cy-3-glc equivalent)

23.57 ± 1.44 a 15.30 ± 2.64 b 15.28 ± 1.01 b 14.20 ± 0.49 b 5.73 ± 0.97 c

2.04% 2.16% 2.16% 2.09% 2.48%

Dp-3,5-diglc 627; 465; 303 0.93 100.85 ± 25.69 a 38.18 ± 20.55 b 43.51 ± 18.19 b 44.41 ± 22.01 b 2.38 ± 3.68 b

Dp-3-cmglc 611; 303 6.13 5.78 ± 1.17 a 3.88 ± 1.91 a,b 4.30 ± 1.05 a 3.60 ± 0.29 a 0.47 ± 0.13 b

Dp-3-cmglc -5-glc

773; 611; 465; 303

4.74 5.11 ± 0.37 a 3.10 ± 0.71 b 3.39 ± 0.29 b 3.07 ± 0.12 b 0.63 ± 0.05 c

Dp-3-glc 465; 303 2.1 91.64 ± 22.66 a 32.90 ± 18.31 b 40.02 ± 14.58 b 35.19 ± 14.58 b 0.71 ± 1.35 c

Sum delphinidin derivatives (mg/L dp-3-glc equivalent)

203.38 ± 49.44 a 78.06 ± 41.40 b 91.22 ± 33.33 b 86.27 ± 36.67 b 4.19 ± 5.04 c

17.64% 11.02% 12.90% 12.73% 1.81%

Mv-3,5-diglc 655; 493; 331 2.47 336.41 ± 7.91 a 232.71 ± 37.02 b 221.59 ± 15.59 b 218.60 ± 6.02 b 93.33 ± 14.07 c

Mv-3-acglc 535; 331 6.19 0.85 ± 0.03 a 0.50 ± 0.14 a,b,c,d 0.52 ± 0.02 b 0.46 ± 0.02 c 0.14 ± 0.01 d

Mv-3-acglc-5-glc 697; 655; 493; 331

8.96 1.02 ± 0.07 a 0.72 ± 0.10 b 0.73 ± 0.06 b 0.65 ± 0.12 b 0.31 ± 0.02 c

Mv-3-cfglc 655; 331 2.5 338.05 ± 6.03 a 232.20 ± 35.80 b 225.68 ± 9.15 b 217.10 ± 7.40 b 93.06 ± 14.54 c

Mv-3-cmglc 639; 331 7.66 2.30 ± 0.29 a 1.48 ± 0.63 a,b,c 1.51 ± 0.27 b 1.26 ± 0.07 b 0.25 ± 0.06 c

Mv-3-cmglc-5-glc

801; 639; 493; 331

6.06 0.18 ± 0.01 a 0.12 ± 0.03 a,b 0.13 ± 0.00 b 0.12 ± 0.00 b 0.04 ± 0.00 c

Mv-3-glc 493; 331 4.02 79.01 ± 5.64 a 49.68 ± 12.91 a,b 51.35 ± 0.96 b 47.31 ± 3.80 b 16.01 ± 2.75 c

Mv-3-glc-4-PA 561; 399 4.6 0.68 ± 0.03 a 0.64 ± 0.12 a 0.44 ± 0.05 b 0.39 ± 0.03 b 0.16 ± 0.05 c

46

Pinotin A 625; 463 2.34 2.74 ± 0.12 a 2.00 ± 0.34 b 1.82 ± 0.09 b 1.81 ± 0.10 b 1.00 ± 0.15 c

Sum malvidin derivatives (mg/L mv-3-glc equivalent)

761.25 ± 18.58 a 520.04 ± 86.66 b 503.77 ± 24.02 b 487.71 ± 16.95 b 204.30 ± 29.92 c

66.02% 73.42% 71.26% 71.94% 88.23%

Pn-3,5-diglc 625; 463; 301 2.34 20.18 ± 0.55 a 14.75 ± 1.91 b 13.89 ± 0.41 b 13.28 ± 0.18 b 7.28 ± 1.07 c

Pn-3-acglc 505; 301 6.04 0.02 ± 0.00 a 0.01 ± 0.00 b 0.01 ± 0.00 b 0.01 ± 0.00 b 0.00 ± 0.00 c

Pn-3-cmglc 609; 301 7.59 0.05 ± 0.01 a 0.04 ± 0.02 a,b,c 0.04 ± 0.01 a,b 0.03 ± 0.00 b 0.01 ± 0.00 c

Pn-3-glc 463; 301 2.96 1.82 ± 0.16 a 1.01 ± 0.27 b 1.16 ± 0.16 b 0.95 ± 0.09 b 0.32 ± 0.06 c

Sum peonidin derivatives (mg/L pn-3-glc equivalent)

22.07 ± 0.71 a 15.81 ± 2.15 b 15.10 ± 0.50 b 14.27 ± 0.24 b 7.61 ± 1.06 c

1.91% 2.23% 2.14% 2.10% 3.29%

Pt-3,5-diglc 641; 479; 317 1.42 65.02 ± 4.86 a 38.91 ± 7.86 b 38.49 ± 4.79 b 36.81 ± 4.76 b 6.11 ± 4.27 c

Pt-3-acglc 521; 317 5.34 1.12 ± 0.07 a 0.64 ± 0.16 b 0.65 ± 0.03 b 0.56 ± 0.02 b 0.12 ± 0.01 c

Pt-3-acglc-5-glc 683; 641; 479; 317

3.21 1.12 ± 0.07 a 0.64 ± 0.16 b 0.65 ± 0.03 b 0.56 ± 0.02 b 0.12 ± 0.01 d

Pt-3-cmglc 625; 317 6.94 3.34 ± 0.59 a 2.10 ± 0.96 a,b,c 2.20 ± 0.48 a.b 1.82 ± 0.13 b 0.28 ± 0.06 c

Pt-3-cmglc -5-glc

787; 625; 479; 317

5.48 3.61 ± 0.24 a 2.38 ± 0.54 a,b 2.47 ± 0.19 b 2.35 ± 0.04 b 0.54 ± 0.05 c

Pt-3-glc 479; 317 2.96 68.54 ± 8.61 a 34.46 ± 12.31 b 37.11 ± 5.48 b 33.42 ± 6.41 b 2.56 ± 2.17 c

Sum petunidin derivatives (mg/L mv-3-glc equivalent)

142.75 ± 14.16 a 79.13 ± 21.74 b 81.57 ± 10.57 b 75.52 ± 11.14 b 9.73 ± 6.45 c

14.16% 11.17% 11.54% 11.14% 4.20%

Sum anthocyanin derivatives (mg/L anthocyanin-3-glc equivalent)

1153.01 ± 84.33 a 708.35 ± 154.59 b 706.93 ± 69.43 b 677.97 ± 65.50 b 231.56 ± 43.44 c

100% 100% 100% 100% 100%

a Abbreviations for anthocyanins: dp, delphinidin; cy, cyanidin; pt, petunidin; pn, peonidin; mv, malvidin; diglc, diglucoside; glc, glucoside; cmglc, (6-coumaroyl)-glucoside; acglc, (6-

acetyl)-glucoside; cfglc, (6-caffeoyl)-glucoside; 4-PA, 4-pyruvic acid. b Values in the same row followed by different letters are significantly different according to Tuckey’s honest significance test at the 0.05 probability level.

47

Figure 2.3. Principal component analysis of flavan-3-ol and phenol (by spectrophotometry-UV) profile (a) in

control (50% RP), RP/WP-treated (30% RP/6% WP, 30% RP/12% WP, and 30% RP/18% WP), and WP-

treated (23% WP) wines after 395 days of bottling (b). Variables: PpH<1, pigments at acidic pH; WPcor, wine

pigments corrected; PRSO2, pigments resisting to sulphite bleaching; CIcor, colour intensity corrected; H, hue.

48

2.6.2. Effect on wine colour and colour evolution

The CIELab parameters of the treated wines were significantly different from the control wines at all

winemaking stages, indicating a significant impact of the RP/WP and WP treatments on pigment

colour and stability (Table 2.2).

Until the BW stage, the control wines had a deeper colour intensity, lower redness, and higher

yellowness (represented by lightness, chroma, and hue values, respectively) compared to the treated

wines (Table 2.2). At the BW stage, the difference in redness and yellowness decreased, and all

wines became lighter than those from previous stages. An increase of the yellow hue indicates the

contribution of other pigments than anthocyanins, which may be due to the formation of polymeric

pigments. The ΔE occurring from the e-FAM to BW stage showed lower colour difference value in

RP/WP-treated wines (ΔE between 26.4 and 39.6 u) compared to the control wines. Lower values of

colour difference indicate lower colour variation and, thus, higher colour stability. On the contrary, the

control wines and the WP-treated wines exhibited higher ΔE values (ΔE = 53.0 and 44.1 u,

respectively) meaning that they are more prone to change in colour than the RP/WP-treated wines.

According to the CIELab values for each treatment at e-FAM and BW stage, the values of a and b

mainly explained the higher ΔE observed previously. The colour of the WP-treated wines became

less red and less yellow between the e-FAM and BW stages (ΔL = −33.3 u, Δa = 28 u, Δb = 7.8 u,

ΔC = 26.8 u, ΔH = −12 u), whereas the one of the control wines became more red and more yellow

(ΔL = −32.0 u, Δa = −30.7 u, Δb = −29.0 u, ΔC = −40.6 u, ΔH = −18.1 u). The RP/WP-treated wines

presented little colour evolution between the e-FAM and BW stages (ΔL between −26.3 u and −38.5

u, Δa between −2.1 u and 5.0 u, Δb between −9.7 u and 0.0 u, ΔC = −7.5 u and 4.3 u, ΔH between

−9.6 and 1.4 u). Moreover, more than 60% of the colour loss occurred between MLF and BW stage

and was partly attributable to the addition of SO2 in the wines at bottling, which likely resulted in the

formation of colourless anthocyanin-SO2 adducts (Jackson, 2008).

At the BW stage, the control wines exhibited the lowest value of lightness and the highest value of

chroma (L = 35.55 ± 7.72 u and C = 62.50 ± 6.03 u, respectively). On the contrary, the wines treated

with 23% WP had the highest value of lightness and the lowest value of chroma (L = 65.50 ± 12.29 u

and C= 39.00 ± 7.75 u, respectively). Indeed, the control wines were darker and more saturated than

the ones treated with WP, which is consistent with the higher anthocyanin content found in the control

49

wines at the BW stage. No significant impact on the hue parameter was observed between wines.

The calculation of the colour difference (ΔE) between the control wines and the wines treated with

WP allowed to evaluate the effect of the WP addition on wine colour. The lowest ΔE was obtained

between the control wines and the wines treated with 6% of WP (ΔE = 13.5 u), whereas the highest

ΔE occurred between the control wines and the wines treated with 23% of WP alone (ΔE = 38.9 u).

As expected, the wines treated with 12 and 18% of WP showed a higher colour difference than the

wines treated with 6% of WP but a lower colour difference than the wines treated with 23% of WP

(ΔE = 18.3 and 23.9 u, respectively).

Colour representation showed notable differences between the control wines and the treated wines

(Fig. S2.3 of Appendix B). The control wines exhibited a ruby colour with violet nuance, whereas the

wines treated with both RP and WP had a deep salmon colour. On the contrary, the wines treated

only with WP showed a very light salmon-like colour, which is typical of rosé wines. Its colour

intensity (CIcor) closed to 3, a value often used as the upper limit to distinguish rosé from red wines,

confirmed this observation (Fig. 2.2d).

2.6.3. Effect on the volatile composition of bottled wines

The volatile compound analyses of the bottled wines resulted in the semi-quantification of 27

compounds including (i) C6 and other fatty acid degradation products, (ii) C13-norisoprenoids, (iii)

terpenes, and (iv) fermentation compounds such as higher alcohols, higher esters, and free fatty

acids (Table S2.4 of Appendix B). Among those, nine compounds resulted significantly to the

ANOVA (p ≤ 0.05): cis-hexen-3-ol, hexanol, phenylethyl acetate, linalool, geraniol, α-terpineol, ethyl

decanoate, ethyl isovalerate, and β-myrcene. In order to give an overall picture of the effect of the

WP addition on the volatile composition of the bottled wines, a PCA was carried out on these

compounds (Fig. 2.4). The principal component 1 (PC 1) explained 62.6% of the variation of the

volatile composition and the principal component 2 (PC 2) explained 18.0%.

50

Table 2.2. CIELab parameters (mean ± standard deviation, in CIELab unit) in control (50% RP), RP/WP-treated (30% RP/6% WP, 30% RP/12% WP, and 30%

RP/18% WP), and WP-treated (23% WP) wines at different winemaking stages.

Par

amet

ers

a

Stages b 50% RP (control) 30% RP / 6% WP 30% RP / 12% WP 30% RP / 18% WP 23% WP

L 1-FAM 1.43 ± 0.78 c A c 34.87 ± 3.37 B a 22.20 ± 4.07 B b 25.50 ± 5.74 B b 27.50 ± 2.52 D a,b

m-FAM 5.58 ± 3.41 A,B d 14.62 ± 1.24 A c 7.85 ± 5.64 A c,d 25.50 ± 3.11 B b 35.00 ± 1.41 B,C a

e-FAM 3.47 ± 1.33 A,B c 15.28 ± 1.01 A b 12.12 ± 0.85 A b 17.00 ± 1.15 C b 32.25 ± 1.50 C,D a

MLF 10.25 ± 1.71 B c 18.25 ± 3.77 A b 21.00 ± 3.46 B b 22.00 ± 0.82 BC b 41.75 ± 0.50 B a

BW 35.50 ± 7.72 C c 41.60 ± 13.93 B b 50.50 ± 13.53 C b 55.50 ± 2.38 A b 65.50 ± 12.29 A a

C 1-FAM 9.51 ± 5.09 D b 64.06 ± 1.76 A a 59.61 ± 4.12 A a 61.75 ± 6.50 A a 64.50 ± 1.73 A a

m-FAM 28.94 ± 9.28 C c 49.48 ± 2.40 B b 30.97 ± 13.54 C c 63.00 ± 2.00 A a 62.75 ± 2.22 A a

e-FAM 21.89 ± 7.23 C d 51.14 ± 1.84 B b,c 44.97 ± 1.39 B c 53.75 ± 1.50 B b 65.75 ± 0.96 A a

MLF 41.50 ± 3.11 B b 52.50 ± 3.87 B a 55.00 ± 0.82 A a 56.50 ± 0.58 A,B a 52.75 ± 1.71 B a

BW 62.50 ± 1.29 A a 50.50 ± 6.03 B b 52.50 ± 8.66 A b 49.50 ± 1.91 B b 39.00 ± 7.75 B c

H 1-FAM 14.99 ± 0.17 C c 23.64 ± 4.20 B b 33.43 ± 0.51 A,B a 32.50 ± 1.00 A a 33.00 ± 1.63 A,B a

m-FAM 18.26 ± 4.89 B,C b 30.04 ± 1.07 A,B a 21.12 ± 6.01 C a 32.75 ± 2.22 A b 21.75 ± 2.50 C,D b

e-FAM 15.64 ± 0.92 C b 30.55 ± 0.94 A a 27.43 ± 0.96 B,C a 31.75 ± 0.96 A a 27.75 ± 3.20 B,C a

MLF 16.25 ± 0.96 B b 30.50 ± 1.29 A,B a 29.75 ± 3.86 A,B a 30.50 ± 0.58 A a 24.75 ± 2.63 D a

BW 33.75 ± 1.50 A a,b 32.00 ± 5.29 A b 37.00 ± 9.27 A a,b 35.00 ± 4.97 A a,b 39.75 ± 17.63 A a

a 1-FAM 9.19 ± 4.91 51.82 ± 14.48 49.70 ± 3.54 52.00 ± 5.35 54.00 ± 2.16

m-FAM 27.20 ± 7.73 42.80 ± 1.60 28.51 ± 11.48 52.75 ± 2.63 58.00 ± 1.63

e-FAM 21.06 ± 6.86 44.03 ± 1.18 40.16 ± 0.87 45.50 ± 1.29 58.00 ± 0.82

MLF 37.25 ± 2.22 45.50 ± 3.32 47.50 ± 1.73 48.50 ± 0.58 50.50 ± 1.29

BW 51.75 ± 2.06 42.75 ± 3.40 42.25 ± 12.01 40.50 ± 4.04 30.00 ± 12.99

51

b 1-FAM 2.47 ± 1.34 23.19 ± 9.53 32.97 ± 2.47 33.00 ± 3.46 35.25 ± 0.96

m-FAM 9.59 ± 5.81 24.80 ± 2.01 11.91 ± 8.54 34.00 ± 1.63 23.25 ± 2.99

e-FAM 5.98 ± 2.31 26.02 ± 1.64 20.84 ± 1.29 28.25 ± 1.50 30.75 ± 3.86

MLF 17.50 ± 3.11 26.50 ± 3.11 27.25 ± 2.87 28.25 ± 0.50 15.00 ± 1.15

BW 35.00 ± 0.82 27.00 ± 6.63 30.50 ± 0.58 28.25 ± 1.89 23.00 ± 2.16

a Abbreviations for parameters: L, lightness; a, red-green; b, blue-yellow; C, chroma; H, hue. b Abbreviations for stages: PFM, after the pre-fermentative cold maceration; 1-FAM, m-FAM, and e-FAM, during the fermentative alcoholic maceration (at days 1, 4, and 8); MLF,

after the malolactic fermentation (day 45); and BW, after bottling (day 395). c Values in the same row (lower-case letters) and the same column (capital letters) followed by different letters are significantly different according to Tuckey’s honest significance

test at the 0.05 probability level.

52

Figure 2.4. Principal component analysis of the volatile compound profile (a) in control (50% RP), RP/WP-

treated (30% RP/6% WP, 30% RP/12% WP, and 30% RP/18% WP), and WP-treated (23% WP) wines after

395 days of bottling (b).

53

The concentration of phenylethyl acetate, C6-compounds such as hexanol and cis-3-hexenol, and

terpenes such as linalool, β-myrcene, and α-terpineol increased significantly as the proportion of WP

increased, whereas the content in geraniol and ethyl decanoate tended to decrease. Previous studies

have shown that fatty acid degradation products, mainly C6 compounds, are major contributors to the

volatile compounds of Vidal grape and wine (Slegers, Angers , & Pedneault, 2017). C6 compounds

occur from the chemical and enzymatic oxidation of unsaturated fatty acids, mostly in the pre-

fermentative stages of winemaking, but prolonged contact with the skin usually increases their

concentration in wine (Gomez, Martinez, & Laencina, 2015). C6 compounds may negatively impact

wine aroma if their concentration reaches over their olfactory threshold (Slegers et al., 2015). On the

other side, Vidal cultivar also exhibits a high concentration of terpenes (Slegers et al., 2015), hence

the high level of terpenes such as linalool, β-myrcene, and α-terpineol found in the RP/WP- and WP-

treated wines. The presence of linalool and α-terpineol has been related to desirable lavender and

floral notes in Vidal wine (Chisholm, Guiher, Vonah, & Beaumont, 1994). The differences in ethyl

decanoate concentration between the control wines and the RP/WP- and WP-treated wines might

relate to the modulation of the amino nitrogen content due to the treatments. Indeed, ethyl decanoate

is biosynthesised from amino acids during fermentation. Thus, the modification of the amino acid

profile of grape must have a direct impact on the ethyl ester profile of wine (Carrau et al., 2008).

2.7. Conclusion

The addition of WP increased the contents in monomeric and oligomeric flavan-3-ols but did not

increase the polymeric flavan-3-ol content of the wines. An excessive ratio of WP/RP (23% WP,

without red pomace) tended to decrease the content in polymeric flavan-3-ols. In contrast, the

modification of the polyphenol content via the manipulation of the WP/RP ratio resulted in

modifications of colour and colour stability of the wines. An appropriate ratio (30% RP/6% WP)

improved the stability of the wines without a significant impact on wine colour, whereas higher

WP/RP ratio tended to lighten wine colour and resulted in wines that were mostly related to rosés

wines than red wines. The addition of WP also modified the aromatic profile of the experimental

wines by adding certain C6-alcohols and terpenes from WP.

Blending white and red grapes, either musts or wines, is a common practice in wine production but is

subject to certain guidelines depending on local legislations. In the European Union, blending is

54

strictly regulated by the International Organisation of Vine and Wine (OIV) regulation and is thus

rarely allowed in wines produced under certain specifications such as Protected Designation of Origin

(AOP) and Protected Geographical Indication (IGP). In Canada, the Vintners Quality Alliance (VQA)

regulation does not limit blending red and white varieties, and therefore the potential of such blending

in Eastern Canada is immense. Indeed, red hybrid cultivars such as Frontenac carrying high

anthocyanin content are known to produce dark coloured reds as well as highly coloured rosés that

are unsuited for customer preferences. Addition of WP proves to be a useful tool to modulate the

phenolic, volatile, and colour profile of wine, which may result in a positive impact on wine mouthfeel,

aroma, and appearance. In this respect, this process could be used to produce an extensive range of

wine styles.

55

Chapitre 3. Pomace limits tannin retention in

Frontenac wines

3.1. Avant-propos

Ce chapitre répond à l’objectif 2 qui vise à étudier conjointement l’impact de deux traitements pré-

fermentaires du moût visant les protéines du vin (bentonite et chaleur), d’une vinification en absence

de marc de raisin et de l’ajout de tanins œnologiques commerciaux sur l’extraction et la rétention des

tanins, pigments et protéines du vin (chapitre 3). Les travaux de ce chapitre ont été publiés dans le

journal Food Chemistry :

Nicolle, P., Marcotte, C., Angers, P., & Pedneault, K. (2019). Pomace limits tannin retention

in Frontenac wines. Food chemistry, 277, 438-447.

3.2. Résumé

L’impact de différentes combinaisons de traitements (traitement pré-fermentaire du moût : non traité,

traité à la bentonite et à la chaleur ; addition de marc de raisin : moût fermenté en présence et

absence de marc ; addition pré-fermentaire de tanins œnologiques : 0, 1, 3 et 9 g/L ; et temps de

macération : 0, 4 et 11 jours) sur la teneur en tanins, protéines et pigments des vins a été étudié en

utilisant le cépage hybride interspécifique Frontenac. La concentration et le profil en flavan-3-ols ont

été analysés par chromatographie liquide à haute performance couplée à un détecteur de

fluorescence. Les concentrations en protéines et en pigments ont respectivement été mesurées par

spectrophotométrie-UV-visible par le test BCA et par la méthode de Boulton. Les résultats ont montré

que les traitements à la bentonite et à la chaleur réduisaient significativement la teneur en protéines

des vins en fin de fermentation alcoolique. Néanmoins le traitement à la chaleur était moins efficace

que le traitement à la bentonite (1,8 à 5,0 fois moins). Éliminer les protéines du vin n’a pas permis

une meilleure rétention des tanins dans le vin (p > 0.1032) mais fermenter le moût sans marc de

raisin a amélioré significativement leur rétention (jusqu’à 2,4 fois), et plus spécifiquement celle des

flavan-3-ols polymériques (jusqu’à 27,8%). Une addition de 3 g/L de tanins œnologiques dans les

vins, fermentés en présence et absence de marc, a été nécessaire pour augmenter significativement

la concentration en tanins des vins de Frontenac.

56

3.3. Abstract

The impact of different factors (must protein treatment: bentonite and heat; pomace: fermented with

and without; tannin addition: 0–9 g/L; and time of maceration: 0–11 days) on tannin, pigment, and

protein extraction/retention in Frontenac wines was investigated. Wine tannin concentration and

composition were determined by HPLC-fluorescence. Protein and pigment parameters were

analysed by BCA assay and Boulton’s method, respectively, using UV-spectrophotometry. Results:

Bentonite and heat significantly reduced wine protein concentration at the end of alcoholic

fermentation but heat was less efficient than bentonite (1.8–5.0 times less). Removing wine proteins

did not improve tannin retention in wines (p > 0.1032) but fermenting without pomace significantly

improved their retention (up to 2.4 times), especially that of polymeric flavan-3-ols (up to 27.8%). An

addition of 3 g/L of enological tannins in wines, fermented with or without pomace, was necessary to

increase wine tannin concentration significantly.

3.4. Introduction

Interspecific hybrid cultivars are crosses between Vitis vinifera and North American Vitis species,

such as Vitis labrusca and Vitis riparia (Pedneault & Provost, 2016). Hybrid cultivars were initially

developed to overcome the phylloxera crisis that appeared at the end of the 19th century in Europe.

Owing to their high tolerance to fungal diseases and frost, they are now increasingly used in cold and

humid areas where traditional wine cultivars from the Vitis vinifera species are challenging to grow

(Pedneault & Provost, 2016). In the past few decades, hybrid cultivars have contributed extensively

to the expansion of northern wine production in areas known for their harsh winters and short growing

seasons such as the Province of Quebec (Canada), Midwestern and Northeastern United States, and

Northern Europe (Pedneault et al., 2013). Despite significant advantages from the viticulture

standpoint, quality issues are known to affect wines produced from hybrid Vitis sp. varieties,

especially reds. Hybrid red grapes typically produce wines with atypical herbaceous aromas, unstable

colour, high acidity, and low tannin concentration (<100 mg/L catechin equivalent) (Manns et al.,

2013; Pedneault et al., 2013; Sun, Gates, et al., 2011).

57

Phenolic compounds, especially tannins (flavan-3-ol oligomers and polymers), play a key role in

colour stability and mouthfeel of red wine (Moreno-Arribas & Polo, 2009; Ribéreau-Gayon, Glories, et

al., 2006). Depending on their concentration and their structure, wine tannins have variable impacts

on wine astringency. Wine astringency correlates positively with the concentration of tannins and

their degree of polymerisation and galloylation (W. Ma et al., 2014). Young red wines made from

hybrids typically have a low mean degree of polymerisation (mDP ≤ 4) (Manns et al., 2013; Springer

& Sacks, 2014). From a sensory standpoint, this translates into bitterness rather than astringency

(Peleg, Gacon, Schlich, & Noble, 1999). Also, in cold climate, high concentrations of organic acids

are typical in hybrid red wines and may act as astringent at the low pH of red wine, and thus possibly

hamper the sensorial acceptability by negatively impacting the astringency perception of wines

(Gawel, 1998).

Low tannin concentration in hybrid red wines is a critical issue for winemakers in cold-climate regions.

Kassara & Kennedy (2011) highlighted an increasing preference of consumers for red wines with

high tannin concentration, while another study showed that high tannin wines are typically priced

higher than other red wines (Mercurio et al., 2010). Various winemaking processes have been

investigated to increase tannin concentration in red wines, including extended maceration,

exogenous tannin addition, thermovinification (pre-fermentative heat treatment), must freezing, and

clarifying/fining with bentonite (Sacchi et al., 2005; Springer, Chen, et al., 2016).

Extended maceration is a technique that involves extending skin contact after alcoholic fermentation.

This technique has been reviewed by several authors including Casassa & Harbertson (2014);

Sacchi et al. (2005); Smith et al. (2015). A key observation from these reviews is that extended

maceration can improve extraction of phenolic compounds (polymeric pigments and tannins) in Vitis

vinifera wines but grape composition may significantly impact their extractability. Thus, conflicting

results have been observed in some cases as highlighted by Smith et al. (2015). In hybrid cultivars

such as Chambourcin and Noble, Auw, Blanco, O'keefe, & Sims (1996) showed that the levels of

gallic acid, epicatechin, and procyanidins B3 and B4 increased with longer skin fermentation times.

Exogenous tannin addition is another widely used method in red winemaking, either to compensate

for natural tannin deficiency in berries, or to correct deficient tannin-anthocyanin ratio that may affect

colour stabilisation in red wine. Exogenous tannins can either be added to wine in the form of natural

grape additives, e.g. pomace, seed, and stalk (Nicolle, Marcotte, Angers, & Pedneault, 2018) or as

58

commercial enological tannins (Kyraleou et al., 2016). Enological tannins are typically classified into

two categories depending on their origin: (i) hydrolysable tannins, which are traditionally derived from

wood or chestnut and (ii) condensed tannins, which come mainly from grape skins and/or seeds

(Versari et al., 2013). Although enological tannin addition may improve tannin concentration, colour

stabilisation, and sensory properties of Vitis vinifera red wines (Kyraleou et al., 2016), this technique

showed little to no effect on both tannin concentration and mouthfeel of cold-climate hybrid red wines

made from Marechal Foch, Corot noir, and Marquette (Manns et al., 2013).

Commercial tannins are pricey, and thus add significant costs to wine production. Therefore, their

utilisation has to be carefully planned and perfectly timed. For instance, recent research has shown

that larger (endogenous or exogenous) tannins notably interact with cell wall material from grape skin

and pulp, largely eliminating them from must through adsorption and phase separation during final

winemaking steps (Bindon, Kassara, & Smith, 2017; Bindon, Li, Kassara, & Smith, 2016); (Springer &

Sacks, 2014). In hybrid red varieties, tannins are extracted and retained to a limited extent in wine

due to their strong interactions with proteins, mainly pathogenesis-related proteins (Springer,

Sherwood, et al., 2016). These proteins are present in higher concentration in hybrid red wines when

compared to European Vitis vinifera varieties (Springer, Sherwood, et al., 2016); (Springer & Sacks,

2014).

Processes such as heat and bentonite addition have been shown to decrease the protein

concentration in must and wine. Bentonite fining is commonly used in the wine industry to remove

unstable proteins from white wine to avoid haze. Bentonite is negatively charged at wine pH and

interacts electrostatically with the positively charged wine proteins, causing them to flocculate

(Ferreira et al., 2001). Thermovinification involves heating grapes and must from 60 to 80°C before

fermentation. Heating leads to conformational changes of proteins by eliminating water, resulting in

their denaturation and aggregation (Dufrechou et al., 2012). Springer, Chen, et al. (2016) have

explored bentonite addition, freezing, and heating to reduce the protein concentration of must before

fermentation and improve tannin retention in hybrid red wines. Although treatments such as bentonite

efficiently removed proteins (85% protein removal) in this study, authors had only considered juice

rather than proteins from grape solids, which significantly hampered the effect of the treatments.

In the current study, we proposed to investigate the impact of different combinations of treatments on

tannin, pigment, and protein concentration of wines made with a hybrid grape variety, during the

59

winemaking process, using a factorial experimental design. The treatments investigated were must

protein treatment (untreated, bentonite and heat-treated), pomace (fermentation with and without

pomace), tannin addition (0, 1, 3, and 9 g/L), and time of maceration (0, 4, and 11 days after the end

of alcoholic fermentation). The overall objective of the present study was to (i) understand the factors

involved in tannin retention in cold-hardy red wines (Frontenac) and (ii) propose potential winemaking

processes which may help cold-climate winemakers modulate the concentration of tannins (initially

very low) in their wines.


3.5.1. Chemicals

Acetone (HPLC grade), acetic acid (HPLC grade), hydrochloric acid (37% solution in water),

acetaldehyde (HPLC grade), and bicinchoninic acid (BCA) protein assay kit were purchased from

Fisher Scientific (Ottawa, ON, Canada). Methanol (HPLC grade) and acetonitrile (HPLC grade) were

purchased from EMD Millipore (Toronto, ON, Canada). Catechin and (−)-epicatechin standards,

sugar standards (glucose and fructose), albumin from bovine serum (BSA), iron (III) chloride, sodium

dodecyl sulphate (SDS), triethanolamine, trichloroacetic acid (TCA), sodium metabisulphite, sodium

hydroxide, and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich

(Oakville, ON, Canada). L-tartaric acid and sodium chloride were purchased from Fisher Scientific

(Fair Lawn, NJ, USA). Absolute ethanol was purchased from Commercial Alcohols (Brampton, ON,

Canada). Purified water was obtained from a MiliQ filtration system.

3.5.2. Experimental design

A factorial experimental design (3 must protein treatments*2 pomace treatments*4 tannin addition

doses*3 times) was performed in triplicate as follows:

- Factor 1: must protein treatment: untreated (control), bentonite-treated, and heat-treated must;

- Factor 2: pomace: must fermented with pomace (WP) and without pomace (WOP), with the term

pomace referring to the pulpy residue from berries after pressing for juice which contains skins and

seeds;

60

- Factor 3: tannin addition: 0, 1, 3, and 9 g/L of enological tannins (ET; 0, 5, 15, and 45 times the

recommended dose, respectively) in must;

- Factor 4: time of maceration: 0, 4, and 11 days after the end of alcoholic fermentation (e-AF;

corresponding to days 4, 8, and 15 of the winemaking process, respectively). The alcoholic

fermentation was completed in 4 days.

This design resulted in a total of 72 fermentations as described in Appendix C.


3.5.3.1. Grape material

The hybrid grape variety, red Frontenac (Landot (L. 4511) X Vitis riparia 89), used in this study were

obtained from a commercial grower located in Saint-Rémi (QC, Canada) (45° 16′ 0″ N, 73° 37′ 0″ W)

in 2015. The grapes were packed in hermetically sealed bags under argon and stored at –30°C until

the experiment was done as carried out by Springer, Chen, et al. (2016).

3.5.3.2. Winemaking

Red Frontenac grapes (approx. 120 kg) were thawed at 4°C and then manually destemmed and

pressed (1.8 bar) using a hydraulic press (Elnova, Quebec, CA). The must (total soluble solids at

23.4°Brix; titratable acidity at 14.1 g/L as tartaric acid equivalent; pH 3.1) and pomace (yield ~ 44%,

w/w) were equally divided into 25 L fermenter buckets, treated with 30 mg/L of sulphur dioxide (SO2,

using potassium metabisulfite), and cold-soaked at 4°C for 4 days. The pre-fermentative cold

maceration was used to enhance the extraction of phenolic compounds from grape berries. The must

and pomace were then recovered by pressing the grape mash using a hydraulic press (1.8 bar) and

kept separately (yield ~ 58%, w/w) in three batches until later recombination. Each batch was divided

again into three parts, for each must protein treatment.

Must protein treatments were conducted as follows:

- The control remained untreated and set for fermentation;

- Bentonite treatment: 30 g of a bentonite slurry (10%, w/w in water; KWK Krystal Klear; American

Colloid Company, Arlington Heights, IL, USA) was added to the must and left in the must during the

whole fermentation to avoid multiple filtration steps that might favour tannin oxidation reactions;

61

- Heat treatment: must and pomace (yield ~ 58%, w/w) were assembled, heated over a water-bath,

and maintained between 60°C and 80°C for 20 min. Must and pomace were then recovered by

pressing the grape mash through synthetic cheesecloth (yield ~ 52%, w/w).

Fermentations were carried out in 1 L glass jars equipped with lids drilled and fitted with fermentation

locks. After homogenisation, 500 mL of untreated and treated must were placed in the jars to receive

the pomace and tannin treatments. Pomace and tannin treatments were conducted as follows: In the

treatments having pomace, the initial proportion of pomace to juice was respected. Enological tannins

(TANIN VR GRAPE®; Laffort, Bordeaux, France), derived entirely from grape skins and seeds, were

also added at this moment, according to the experimental design described in Section 3.5.2.

Alcoholic fermentation was induced by a commercial dry yeast Saccharomyces cerevisiae (Lalvin BM

4X4; Lallemand Inc., Montreal, Canada) at 250 mg/L and carried out at 24°C until dryness. In order

to limit oxidation, the cap was punched once a day for the first two days. Alcoholic fermentation was

monitored daily by measuring the total soluble solid level (Brix) of the fermented must and the

concentration of glucose and fructose by high-performance liquid chromatography as described in

Appendix C. Musts were sampled at days 4 (e-AF), 8, and 15 of the winemaking process. All

samplings were followed by mixing the pomace and grape mash and purging with argon. Samples

were stored at –30°C until analyses. Final wines were pressed manually after 15 days using a French

press, packed in hermetically sealed bags under argon, and stored at 4°C.

3.5.4. Tannin analysis

3.5.4.1. HPLC-fluorescence

Flavan-3-ol concentration and composition analyses were measured as described by Nicolle et al.

(2018). The analysis was carried out on an Agilent 1260 infinity HPLC system (Agilent Technologies,

Santa Clara, CA, USA) equipped with a fluorescence detector (G1321C, Agilent, Santa Clara, CA,

USA). Briefly, samples were filtered through 0.45 μm PTFE syringe filters (25 mm diam., Silicycle,

Quebec City, QC, Canada) before HPLC analysis. Separation was performed on a Develosil Diol

column (250 mm × 4.6 mm; 5 μm particle size) fitted with a Cyano SecurityGuard column

(Phenomenex, Torrance, CA, USA) and maintained at 35°C. A gradient of acetonitrile:acetic acid

(98:2, v/v; solvent A) and methanol:water:acetic acid (95:3:2, v/v; solvent B) was used with the

62

following gradient: 0–40% B, from 0 to 35 min; 40–100% B, from 35 to 40 min; isocratic at 100% B,

from 40 to 45 min; and 100–0% B, from 45 to 50 min. The flow rate was maintained at 0.8 mL/min.

Compounds were separated according to their degree of polymerisation (1–9 units of flavan-3-ols,

and polymers (>10 units)) and detected using a fluorescence detector set at 230 and 321 nm for

excitation and emission wavelengths, respectively. Compounds were quantified based on an external

calibration curve of (−)-epicatechin (0–100 ppm). Correction factors were used to correct for the

respective responses of flavan-3-ols from various molecular weights in fluorescence (Prior & Gu,

2005).

3.5.4.2. Protein precipitation

In order to assess the performance of the HPLC-fluorescence method for tannin measurement

(section 3.5.4.1), wine tannins from days 4 and 15 of the winemaking process were analysed using

the traditional protein precipitation method as described by Harbertson et al. (2002) with some

modifications. Analyses were carried out in triplicate on a Shimadzu UV-Vis spectrophotometer UV-

2700 equipped with a high-absorbance kit including a partition plate and a dark filter when required

(Shimadzu, Quebec, Canada). Briefly, wine samples expected to carry tannin concentration inferior to

the limit of quantification of the method (< 100 mg/L or < 0.3 absorbance units (Jensen, Werge,

Egebo, & Meyer, 2008)) were concentrated using a Speed-vac concentrator (SAVANT SPD131DDA,

Thermo Scientific, Waltham, MA, USA) by ca. factor 2. Wine samples expected to have tannin

concentration superior to the upper limit of quantification (˃ 300 mg/L) were diluted in a model wine

solution consisting of 12% (v/v) ethanol in water and 5 g/L tartaric acid, with pH adjusted to 3.3. Then,

500 μL of prepared sample was added to 1 mL BSA solution (1 mg BSA/mL of buffer solution

containing 200 mM acetic acid and 170 mM sodium chloride in water, with pH adjusted to 4.9). The

mixture was incubated for 30 minutes at room temperature with occasional stirring and then

centrifuged at 10,000 rpm for 10 min at room temperature. The supernatant was discarded, the pellet

was washed with 500 μL of the same buffer, and the mixture was again centrifuged at 10,000 rpm for

5 min at room temperature. A total of three washes was completed. The pellet was then resuspended

in 875 μL of a buffer composed of 5% triethanolamine (v/v) and 10% SDS (w/v) in water. The mixture

was incubated 20 min at room temperature. After the incubation, the mixture was vortexed to dissolve

the pellet, transferred to a cuvette, and incubated for an additional 10 min at room temperature. The

background reading was recorded at 510 nm in a 10-mm semi microcuvette. After the reading, a

63

ferric chloride solution (125 μL; 10 mM ferric chloride in 10 mN aqueous hydrochloric acid) was

added, and the final absorbance was read after 10 min at 510 nm. The tannin response was

determined as the final absorbance minus the background absorbance. Accounting for dilution,

tannin concentration was calculated and expressed as mg/L catechin equivalent from catechin

standard curve.

3.5.5. Pigment analysis

Co-pigmented, monomeric, and polymeric anthocyanins were estimated using the method proposed

by Boulton (2001). Analyses were carried out in duplicate using a Shimadzu UV-Vis

spectrophotometer UV-2700. Briefly, wine samples were adjusted to pH 3.6 with sodium hydroxide

and centrifuged at 10,000 rpm for 15 min at room temperature. 20 μL of a 10% acetaldehyde (v/v)

solution and 160 μL of a 5% SO2 solution (using potassium metabisulphite) were successively added

to 2 mL of wine sample at room temperature in a 10-mm cuvette and set aside for 45 min. After 45

min, the wine samples with acetaldehyde were placed in a 2-mm cuvette, and then the absorbance at

520 nm read (Aacet). The reading at 520 nm of wine samples with SO2 could be taken directly (ASO2).

Besides, 50 μL of wine was diluted 40-fold in a model wine solution (12% ethanol, 5 g/L tartaric acid,

adjusted to pH 3.6) and the absorbance was measured at 520 nm (Awine). All the absorbance

measurements were converted to a 10-mm path length and a dilution of 1 before calculating. The

following equations were used to estimate the co-pigmented, monomeric, and polymeric

anthocyanins (CA, MA, and PP, respectively):

- CA = Aacet – Awine;

- MA = Awine – ASO2;

- PP = ASO2.

Total wine pigment at pH < 1 (Tpg) was estimated by measuring the absorbance at 520 nm (Boulton,

1998). The analysis was carried out in duplicate. Briefly, wine samples were dissolved 100-fold in 1 M

solution of hydrochloric acid and left at room temperature for 4 hours before reading. A quartz cuvette

was used for analyses and the readings corrected by multiplying by 100.

64

3.5.6. Protein analysis

Proteins from wine samples were precipitated by TCA precipitation as proposed by the International

Organization of Vine and Wine (OIV) in the resolution oeno 24/2004 (OIV, 2004) with few

modifications. Precipitation was done in triplicate. Briefly, proteins from centrifuged samples (10,000

rpm for 30 min at 4°C) were precipitated with freshly prepared ice-cold TCA at a final concentration of

10% (w/v). After 2-hours incubation on ice, the precipitated proteins were collected by centrifugation

at 10,000 rpm for 30 min at 4°C. The resulting pellet was washed four times with ice-cold acetone.

After brief drying, the protein pellet was resuspended in 0.1 M sodium hydroxide solution and stored

at –30°C until analysis. Protein concentration was determined on a Shimadzu UV-Vis

spectrophotometer UV-2700, based on the BCA protein assay kit (Pierce Laboratories, Rockford, IL,

USA) following the manufacturer’s instructions. Analyses were done in triplicate. Bovine serum

albumin was used as a standard.

3.5.7.Statistical analysis

Protein and tannin data were analysed with the SAS software (version 3.5 Basic Edition; SAS

Institute Inc., Cary, NC, USA) using analysis of variance (ANOVA) methods with PROC MIXED

statement, analysing the main and interaction effects of the factors with a block: must protein

treatments, pomace, tannin addition, and time of maceration. Since each wine was measured at the

end of alcoholic fermentation and 2-time intervals, a repeated-measures model was used with time

as repeated factor and batch as a random factor. The symmetric-composite variance/covariance

matrix was used to model the correlation between time-related measures for the same unit. The

Kenward-Roger method was used to calculate degrees of freedom. Residual analysis was carried out

to check the postulates of normality and homogeneity of the model. Multiple comparisons were made

using the Protected Fisher's Least Significant Difference (LSD) method (at the 0.05 probability level).

ANOVA analysis of pigment data, at day 15 of the winemaking process, was analysed using the

PROC MIXED procedure of the SAS software, analysing the main and interaction effects of the 3

factors with a block: must protein treatments, pomace, and tannin addition. Means were compared as

described earlier.

65

3.6. Results and discussion

3.6.1.Relevance of the method used for tannin quantification

The intrinsic heterogeneity of tannins as a group of molecules considerably challenges their

quantification in red wine (Kennedy, Ferrier, Harbertson, & des Gachons, 2006). Different

approaches have thus been proposed, either involving simple quantification methods for total tannin

analysis or extensive methods for detailed molecular characterisation. Spectrophotometric methods

have been developed for the global quantification of tannins based on their precipitation with proteins

(PrP) such as the bovine serum albumin or other polymers like methyl cellulose (Sarneckis et al.,

2006). The PrP method is described as time-consuming, and its precision is often questioned by

users (Brooks, McCloskey, Mckesson, & Sylvan, 2008), yet its utilisation for routine tannin analysis

has spread in the wine industry due to its high degree of correlation with wine perceived astringency

(r2 = 0.82) that provides hints on the sensory perception of wine (Kennedy, Ferrier, et al., 2006).

Recently, the PrP method was found to be less reliable in low-tannin cultivars such as interspecific

hybrids, due to its high limit of quantification (LOQ = 100 mg/L), which often results in non-

quantifiable tannin concentration, even after a pre-concentration stage (Springer & Sacks, 2014).

Pre-concentration of samples significantly modifies the matrix and may lead to inaccurate results,

thus hampering the validity of the precipitation method for the hybrid wine industry. Characterisation

of condensed tannins through depolymerisation using phloroglucinolysis (PG) or thiolysis followed by

high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) has also been

correlated with wine perceived astringency (r2 = 0.73) in addition to be highly correlated with the PrP

method as well (r2 = 0.91) (Kennedy, Ferrier, et al., 2006). The PG method is largely used to

determine tannin structure and mean degree of polymerisation (mDP) in wine research. However, it

has some limitations: This method is complex to implement, it presents issues with incomplete

depolymerisation of phenolic compounds (Kennedy, Ferrier, et al., 2006), and gives limited

information about the overall distribution of tannins based on their molecular weight or size.

The normal-phase HPLC coupled with fluorescence detection (HPLC-FLD) method used in the

current article might be a relatively affordable and time-effective alternative to the PrP and PG

methods to quantify and partly characterise tannins, especially in low-tannins cultivars (Lazarus,

66

Hammerstone, Adamson, & Schmitz, 2001). Though less conventional in oenology, HPLC-FLD

allows separating condensed tannins according to their degree of polymerisation along with high

sensitivity and a reduction of interfering signals from other UV-absorbing compounds (Lazarus et al.,

2001). To the best of our knowledge, no direct comparison has been reported yet between the tannin

concentration quantified by PrP and that obtained by HPLC-FLD. In the present article, the difference

in tannin level as determined by PrP and by HPLC-FLD was compared using samples from days 4

and 15 of the winemaking process. Results showed similar ranges of tannin concentration for both

methods (<100–2199 mg/L catechin equivalent and 71–2021 mg/L epicatechin equivalent, for PrP

and HPLC-FLD, respectively). A high correlation was found between both methods (r2 = 0.8579; Fig.

3.1). In contrast, Kyraleou et al. (2015) obtained a smaller tannin concentration using PrP rather than

HPLC-FLD (149.2–217.7 mg/L catechin equivalent and 233.9–533.4 mg/L catechin equivalent,

respectively). Such variations between studies could be attributed to the range of concentrations

considered to correlate methods. Indeed, in the current study, the dispersion could have been higher

if a narrower range would have been considered. Our results still suggest that HPLC-FLD could be a

suitable technique to quantify the tannin concentration in wines made from low-tannin cultivars such

as hybrids. In Quebec, most wineries send their samples out to commercial labs for analyses. In this

model, the use of HPLC for tannin analysis is viable since these labs get to run the analyses for the

industry. Besides, HPLC-FLD also provides valuable information about the relative distribution of

tannins of different degree of polymerisation. Such information could eventually be related to the

potential astringency or bitterness of red wines.

67

Figure 3.1. Regression of tannin concentration of experimental Frontenac wines as measured by protein

precipitation and HPLC-fluorescence. Wine samples from the end of alcoholic fermentation (e-AF) and day 11

after the e-AF (corresponding to days 0 and 15 of the winemaking process, respectively) were included in the

analysis.

3.6.2. Impact of winemaking processes on protein concentration

Based on the hypothesis that proteins extracted in juice during the winemaking process limit the

retention of tannins in hybrid red wines, the impact of two pre-fermentative treatments (bentonite and

heat) on protein removal from juice was evaluated. The potential of these treatments to remove

proteins was assessed in WP- and WOP-wines supplemented with various doses of ET (0, 1, 3, and

9 g/L), with measurements at the e-AF and 4 and 11 days after the e-AF. An overview of the impact

of treatments on protein and tannin concentration of Frontenac wines over time is represented using

a heat map (Fig. 3.2). The statistical analysis of the protein concentration of the experimental wines

showed that all factors, including must protein treatment, pomace, tannin addition, and time of

maceration were highly significant (Table 3.1, p < 0.0001). The analysis also revealed that most

factors interacted significantly with each other (p < 0.05).

68

Figure 3.2. Heat map of the protein and tannin concentration of experimental Frontenac wines made with

untreated (control, CT), bentonite-treated (BE), and heat-treated (HT) must, fermented with (WP) and without

(WOP) pomace, and with different doses of tannin addition (0, 1, 3, and 9 g/L) at 0, 4, and 11 days after the

end of alcoholic fermentation (e-AF). Red and blue colours represent the highest and the smallest

concentration of proteins or tannins in wines. Data are ranked from the lowest to the highest protein

concentration, 11 days after the e-AF (a), and from the highest to lowest tannin concentration, 11 days after

the e-AF (b).

Control wines mimicked the traditional on-skin fermentation. In these wines, the protein concentration

was found to decrease significantly (up to 4.9 times less) between the e-AF and the 11th day after the

e-AF (Table 3.2). This may be attributed to some of the grape proteins becoming insoluble or getting

hydrolysed by the proteolytic action of extracellular protease enzymes secreted by yeasts (Ferreira et

al., 2000). Adding bentonite to the must before AF reduced the protein concentration by 2.2–9.8

times at the e-AF when compared to the control wines, and the protein concentration remained stable

until the 11th day after the e-AF. The control wines supplemented with ≥3 g/L of ET resulted in a

similar or lower wine protein concentration than the bentonite-treated wines supplemented with

≥3 g/L of ET by the 11th day after the e-AF.

69

Heating the must before AF (heat treatment) removed wine proteins at the e-AF by 1.3–2.6 times

when compared to the control wines (Table 3.2). Still, this process was 2–5 times less efficient than

the bentonite treatment. In addition, at the e-AF, the heat-treated wines supplemented with 9 g/L of

ET showed a higher protein concentration than the control wines supplemented with 9 g/L of ET,

which might indicate better retention of the proteins in the wine matrix. The heat-treated wines

supplemented with ET ended-up having 2.0–4.1 times more proteins than the control wines

supplemented with ET on the 11th day after the e-AF. In V. vinifera cultivars, heating the must is

expected to precipitate almost all the proteins, as it denatures proteins by eliminating water, allowing

them to flocculate on contact with tannins and cations (Dufrechou et al., 2012). In the current study,

three phenomena might have contributed to the observed results: 1) proteins might have been

denaturated or underwent conformational changes without further precipitation, 2) protein extraction

may have been enhanced in the WP-wines, and 3) heat treatment may have caused other

macromolecules such as polysaccharides to be extracted that may have further stabilised the wine

proteins in solution.

Besides adding-on tannin concentration in wine, addition of exogenous tannins to must or wine is

also known to contribute in protein precipitation. Doses as high as 2 g/L are usually necessary to

eliminate them almost completely in V. vinifera cultivar (Ribéreau-Gayon, Glories, et al., 2006). In

Frontenac, supplementing the control wines with ET had a minor impact on the protein concentration

of the control WP-wines at the e-AF and 11 days after the e-AF. However, ET addition reduced the

protein concentration of the control WOP-wines up to 2.8 times at the e-AF, and up to 2.9 times by

the 11th day after the e-AF. Fermentations conducted in the presence of pomace (WP-wines) resulted

in an increased protein concentration in the bentonite-treated wines at the e-AF (up to 2.8 times

more). On the 11th day after the e-AF, both the bentonite- and heat-treated WP-wines exhibited a

significantly higher protein concentration than the WOP-wines, suggesting that the presence of grape

solid (e.g., pomace) mainly contributes to the protein load in Frontenac wines.

70

Table 3.1. Repeated measures analysis of variance (ANOVA) for protein, tannin, total pigment (Tpg), and co-pigmented, monomeric, and polymeric anthocyanin

(CA, MA, and PP, respectively). Main effects are: must protein treatment (MT; untreated, bentonite-treated, and heat-treated must); pomace (P; must fermented

with and without pomace); tannin addition (TA; 0, 1, 3, and 9 g/L); and time of maceration (TM; 0, 4, and 11 days after the end of alcoholic fermentation).

Effect Protein Tannin Tpg CA MA PP

F

Value

Pr > F F Pr > F F Value Pr > F F

Value

Pr > F F

Value

Pr > F F

Value

Pr > F

Value

MT 280.32 < 0.0001 2.48 0.1032 117.69 0.0003 134.05 < 0.0001 236.81 < 0.0001 156.33 < 0.0001

P 231.91 < 0.0001 1 411.91 < 0.0001 18.13 0.0003 9.01 0.0062 38.96 < 0.0001 6.82 0.0124

TA 17.13 < 0.0001 4 556.99 < 0.0001 2.95 0.0604 1.19 0.3426 13.48 < 0.0001 201.91 < 0.0001

EM 142.43 < 0.0001 659.07 < 0.0001

MT*EM 104.29 < 0.0001 15.37 < 0.0001

MT*P 13.91 < 0.0001 51.15 < 0.0001 42.05 < 0.0001 15.55 < 0.0001 20.40 < 0.0001 70.71 < 0.0001

MT*TA 56.84 < 0.0001 3.56 0.0102 7.07 0.0005 1.96 0.1258 1.05 0.4290 4.08 0.0026

P*EM 20.93 < 0.0001 125.07 < 0.0001

TA*P 20.34 < 0.0001 1 152.41 < 0.0001 1.38 0.2723 1.68 0.1971 1.35 0.2821 54.17 < 0.0001

TA*EM 0.93 0.4798 185.55 < 0.0001

MT*P*EM 21.42 < 0.0001 5.41 0.0007

MT*TA*EM 3.24 0.0008 4.19 < 0.0001

MT*TA*P 3.47 0.0071 7.64 < 0.0001 8.21 < 0.0001 5.14 0.0016 0.43 0.8514 2.32 0.0505

TA*P*EM 0.32 0.9265 16.51 < 0.0001

MT*TA*P*EM 1.57 0.1194 3.09 0.0012

71

Table 3.2. Protein concentration (mean ± standard deviation (SD), mg/L BSA equivalent) in experimental Frontenac wines made with untreated (control),

bentonite-treated, and heat-treated must, fermented with (WP) or without (WOP) pomace, and with different doses of tannin addition (0, 1, 3, and 9 g/L) at the end

of alcoholic fermentation (e-AF) and on the 4th and the 11th day following the e-AF (corresponding to days 4, 8, and 15 of the winemaking process, respectively).

Factor

Day after the e-AF stage (Time)

e-AF stage (day 0) 4 11

Pomace Tannin (g/L) Must treatment Mean ± SD Mean ± SD Mean ± SD

WP 0 Control 129.43 ± 7.98 a A x * a 101.79 ± 10.35 B a x 42.70 ± 6.90 a C y *

Bentonite 30.08 ± 3.50 b AB z 41.75 ± 7.95 b A y * 26.83 ± 4.22 b B z *

Heat 58.50 ± 3.78 c B y 58.70 ± 11.58 d B x 93.64 ± 1.22 b A x *

1 Control 115.59 ± 3.14 ab A x 87.52 ± 9.38 ab B x * 45.03 ± 1.98 a C y

Bentonite 35.36 ± 4.69 ab A y * 36.54 ± 6.37 b A z * 29.14 ± 1.83 b A z *

Heat 62.88 ± 5.17 c B z 74.66 ± 12.23 c AB x * 87.43 ± 17.39 b A x *

3 Control 117.38 ± 6.55 ab A x 87.17 ± 8.11 ab B x * 39.33 ± 4.24 a C y *

Bentonite 36.30 ± 6.80 ab A z * 50.70 ± 5.35 ab A y * 38.69 ± 4.54 b A y *

Heat 92.59 ± 1.55 b A y 96.37 ± 10.94 b A x * 91.87 ± 9.85 b A x *

9 Control 109.19 ± 12.44 b A x * 73.86 ± 5.91 b B y * 35.84 ± 1.97 a C z

Bentonite 49.86 ± 10.35 a A z* 61.20 ± 5.77 a A y * 53.89 ± 11.50 a A y *

Heat 133.55 ± 7.64 a A y * 129.16 ± 43.50 a A x * 145.78 ± 23.73 a A x *

WOP 0 Control 161.11 ± 15.75 a A x 87.35 ± 11.43 a B x 69.45 ± 9.18 a C y

Bentonite 20.10 ± 7.51 a A z 15.04 ± 3.44 a A y 9.55 ± 0.67 b A z

Heat 61.27 ± 8.89 c A y 50.84 ± 3.44 b A x 45.91 ± 21.18 a A x

1 Control 125.73 ± 10.20 b A x 48.31 ± 3.11 b B x 42.53 ± 4.21 b B x

Bentonite 12.82 ± 1.85 a A z 15.26 ± 2.51 a A y 10.61 ± 1.77 b A y

Heat 55.45 ± 6.50 c A y 45.16 ± 4.76 b A x 40.24 ± 5.48 a A x

72

3 Control 117.77 ± 36.49 b A x 36.88 ± 4.04 bc B y 24.11 ± 5.45 c B y

Bentonite 16.26 ± 3.72 a A z 18.48 ± 0.57 a A z 13.42 ± 4.19 ab A z

Heat 81.60 ± 20.38 b A y 72.81 ± 9.71 a A x 53.60 ± 7.37 a B x

9 Control 69.06 ± 0.47 c A y 26.84 ± 5.28 c B y 26.64 ± 7.04 c B y

Bentonite 27.83 ± 1.81 a A z 22.84 ± 2.88 a B y 25.92 ± 4.43 a C y

Heat 106.17 ± 21.29 a A x 82.20 ± 7.95 a B x 53.95 ± 13.18 a C x

a Values in the same row and in the same column followed by different letters are significantly different according to the Protected Fisher's Least Significant Difference method at

the 0.05 probability level: a, b, c, and d letters compare the dose of tannin addition for a given Must treatment*Pomace*Time; x, y, and z letters compare the must treatment for a

given Tannin*Pomace*Time; A, B, and C letters compare the day after the e-AF (time) for a given Must treatment*Pomace*Tannin; and asterisk (*) compare the pomace treatment

for a given Must treatment*Time*Tannin.

73

3.6.3. Impact of winemaking processes on wine polyphenol concentration

3.6.3.1.Impact on pigment concentration

The relative proportion of anthocyanin to tannin during the winemaking process may impact their own

stability and the resulting formation of polymeric pigments (Casassa & Harbertson, 2014). Young red

wine colour mainly depends on anthocyanins, but beyond that, polymeric pigments become

increasingly determinant of wine colour and long-term colour stability, while possibly reducing wine

perceived astringency (Casassa & Harbertson, 2014; Weber, Greve, Durner, Fischer, & Winterhalter,

2012). Therefore, the impact of must protein treatment, pomace, and tannin addition was evaluated

on wine parameters such as total pigment (Tpg) and co-pigmented, monomeric, and polymeric

anthocyanins (CA, MA, and PP, respectively; Fig. 3.3; see Appendix C for values and statistics).

The statistical analysis of colour-related variables showed that all factors (must protein treatment,

pomace, and tannin addition) were significant (p < 0.05), whereas the dose of tannin addition was not

significant (p > 0.05) on CA and Tpg (Table 3.1). The analysis also revealed that the interactions

between each factor including tannin addition were mostly not significant, except on PP.

The heat-treated wines had the highest amount of pigments based on the Tpg value (Fig. 3.3a).

Comparison of the bentonite- and heat-treated wines with the control wines showed that the heat

treatment favoured the extraction/retention of monomeric anthocyanins in wines as well as the

formation of co-pigmented and polymeric anthocyanins. As expected, the bentonite treatment tended

to limit both.

The presence of pomace during the winemaking process of the heat-treated wines neither impacted

the Tpg value, nor the monomeric and co-pigmented anthocyanin concentration of the wines but

reduced the formation of polymeric anthocyanins in wines (Fig. 3.3). This suggests that monomeric

anthocyanin and copigment extraction peaked early in the winemaking process, before AF. It is

possible that heating the pomace and the juice as well as carrying a cold pre-fermentative maceration

caused a cellular disruption that increased pigment extraction before AF. Moreover, the extraction

was also likely enhanced by the fact that frozen grapes were used for this study as also evidence by

Springer, Chen, et al. (2016). Indeed, freezing and unfreezing berries increases the extraction of cell

wall components including anthocyanins and condensed tannins (Garcia, Santesteban, Miranda, &

74

Royo, 2011; Sacchi et al., 2005). In contrast with heat-treated WP-wines, the presence of pomace in

bentonite-treated wines improved the formation of co-pigmented and polymeric anthocyanins as well

as the extraction/retention of monomeric anthocyanins.

75

Figure 3.3. Total pigment (a) and co-pigmented (b), monomeric (c), and polymeric anthocyanin (d) estimation

(in absorbance unit), in experimental Frontenac wines made with untreated (control), bentonite-treated, and

heat-treated must, fermented with and without pomace, and with different doses of tannin addition (0, 1, 3, and

9 g/L) at 11 days after the end of alcoholic fermentation.

76

3.6.3.2. Impact on tannin concentration

Must protein treatment, pomace, tannin addition, and time of maceration had a significant impact on

the anthocyanin and protein concentration of the experimental wines, and, therefore, their effect on

tannin concentration in Frontenac wines was studied (Fig. 3.4; see Appendix C for values and

statistics).

The statistical analysis of the wine tannin concentration was achieved through repeated measures

ANOVA (p ≤ 0.05; Table 3.1). The analysis revealed that the factors pomace, tannin addition, and

time of maceration had a significant impact on the tannin concentration (p ≤ 0.0001), whereas the

factor, must protein treatment, was non-significant (p = 0.1032). The interactions between all factors

were significant (p ≤ 0.05).

The HPLC-FLD analysis of the commercial tannins extract used in this assay showed a tannin

concentration of 14.9%, w/w dry weight (149.2 mg/g of tannin powder, in epicatechin equivalent). Its

composition mostly included oligomeric tannins composed of 2–5 units of flavan-3-ols (84%, w/w).

Increasing tannin addition in must (0, 1, 3, and 9 g/L of ET; calculated tannin concentration

corresponding to 0, 0.149, 0.448, and 1.3 g/L of tannins, respectively) linearly increased the tannin

concentration of the experimental wines (regression coefficients between 0.9667 and 0.9992).

However, the tannin concentration of both non-supplemented and supplemented WP-wines at a rate

of 1 g/L of ET was similar. In contrast, the tannin concentration of wines supplemented with 9 g/L of

ET ranged from 852 to 2 021 mg/L of epicatechin equivalent at the e-AF. Such concentration is not

excessively high when compared to those observed by Harbertson et al. (2008) in various red wines

(30–1900 mg/L for Pinot noir and Cabernet Sauvignon, respectively). Moreover, the addition of 9 g/L

enhanced the proportion of polymeric flavan-3-ols in the wines (up to 17.5%, on average). However,

the impact of high rate of tannin addition (3 to 9 g/L) on the sensory properties of the Frontenac wines

would need to be evaluated as tannin addition exceeding manufacturers recommendation was

previously found to have adverse impacts on the sensory characteristics of certain wines

(Harbertson, Parpinello, Heymann, & Downey, 2012).

78

Figure 3.4. Oligomeric (2–5 units of flavan-3-ols) and polymeric (>5 units of flavan-3-ols) flavan-3-ol

concentration (mean, mg/L epicatechin equivalent) in experimental Frontenac wines made with untreated

(control, CT), bentonite-treated (BE), and heat-treated (HT) must, fermented with (WP) and without (WOP)

pomace, and with different doses of tannin addition (0, 1, 3, and 9 g/L) at the end of alcoholic fermentation (e-

AF; a) and on the 4th (b) and the 11th (c) day following the e-AF (corresponding to days 4, 8, and 15 of the

winemaking process, respectively). For a given combination of Dose*Treatment*Day, small letters compare

the oligomeric flavan-3-ol concentration and capital letters compare the polymeric flavan-3-ol concentration of

pomace treatment at the 0.05 probability level. The statistics for the other combinations are available in

Appendix B.

The presence of pomace had a strong impact on tannin extraction and/or retention in the

experimental Frontenac wines. The WOP-wines supplemented with 3 and 9 g/L of ET showed a

significantly higher tannin concentration than the WP-wines at the e-AF (up to 2.4 times higher than

the control wines). Their composition majorly included oligomeric flavan-3-ols, but the proportion of

polymeric flavan-3-ols was also considerably improved (up to 27.8% increase in polymeric flavan-3-ol

concentration at the e-AF). Interestingly, among the WOP-wines, the bentonite-treated wines were

the most positively impacted by pomace removal. The bentonite-treated WOP-wines supplemented

with 3 and 9 g/L of ET had slightly higher tannin concentration than the control and heat-treated

79

wines by the 11th day after the e-AF, whereas the bentonite-treated WP-wines supplemented with 3

and 9 g/L of ET showed lower tannin concentration. As mentioned earlier (sections 3.4.2 and 3.4.3.1),

bentonite was shown to quickly remove proteins and some positively-charged anthocyanins during

the winemaking process, whereas pomace tended to release extra proteins and pigments in juice.

Thus, on the one hand, proteins limited the retention of tannins and, on the other hand, anthocyanins

contributed to tannin retention as anthocyanins and tannins compete for the binding sites during the

winemaking process (Bautista-Ortín et al., 2016; Kilmister, Mazza, Baker, Faulkner, & Downey,

2014). In addition, once denatured by the heat, the proteins can be less efficient at binding

condensed tannins (Sauvage, Bach, Moutounet, & Vernhet, 2010). All these phenomena could thus

explain why the WP-wines produced from heat-treated and control musts had better tannin retention

than the WP-wines produced from the bentonite-treated must.

Extending the maceration after the e-AF did not result in any noticeable improvement of the tannin

concentration, in any of the WP-wines that were non-supplemented with tannins, no matter the must

protein treatment (Fig. 3.4). This shows that the tannin concentration of Frontenac wines cannot be

improved from the use of Frontenac berries alone, even when proteins are previously removed from

the must.

The wines (WP- and WOP-wines) supplemented with at least 3 g/L of ET showed a significant

increase in tannin concentration. This effect was improved when pomace was removed, as the WOP-

wines resulted in a higher tannin concentration than the WP-wines. Yet most of the ET supplemented

wines also showed a progressive decrease in tannin concentration by the 11th day after the e-AF.

This effect was even more dramatic in the WOP-wines (1.5–2.5 times less). Such a decrease could

be related to the occurrence of polymerisation reactions between wine tannins and exogenous

tannins followed by precipitation (Gambuti, Capuano, Lisanti, Strollo, & Moio, 2010). It could also

relate to the formation of polymeric pigments during the extended maceration through direct and

acetaldehyde-mediated condensation reactions between anthocyanins and procyanidins, as

observed earlier (section 3.4.3.1) (Cheynier et al., 1998).

80

3.6.3.3. Relevant observations

Several relevant observations were made through the course of this study:

- Bentonite addition before AF significantly removed most of the proteins by the e-AF. Heating the

must also significantly reduced the protein concentration at the e-AF, although less efficiently than

bentonite. Nevertheless, supplementing those treated wines with ≥3 g/L of ET favoured protein

retention in wines.

- Heating must and pomace before AF favoured the extraction of monomeric, copigmented, and

polymeric anthocyanins, whereas bentonite addition limited their retention in wines, although less in

wines fermented with pomace.

- Surprisingly, removing proteins by applying a treatment before AF (bentonite or heat) did not

improve tannin retention in wines. On the contrary, fermenting in the presence of pomace

dramatically limited the retention of tannins in wines. Thus, extending the pomace maceration up to

11 days after the e-AF did not improve tannin retention. In contrast, fermenting without pomace

improved the proportion of polymeric flavan-3-ols in wines.

- WP- and WOP-wines supplemented with at least 3 g/L of ET showed a significant increase in tannin

concentration.

3.7. Conclusion

Red wines produced from Frontenac berries typically show low tannin concentration (<100 mg/L

catechin equivalent), even after maceration. Therefore, prefermentative treatments such as

thermovinification and tannin addition are necessary to increase wine tannin concentration. Our

results showed that a pre-fermentative cold maceration followed by pomace removal prior to

fermentation is an effective approach to significantly improve tannin retention in Frontenac wines,

especially the concentration in polymeric flavan-3-ols. Since wine proteins do not appear as the only

limiting factor in wine tannin retention, thermovinification (heating must and pomace before AF

between 60 and 80 °C) can be considered as an alternative to increasing tannin retention in wine.

Indeed, this winemaking process favours monomeric anthocyanin (colour) and copigment (colour

stabilisation) extraction before alcoholic fermentation, thus making fermentation possible in the

absence of pomace. Finally, an addition of at least 3 g/L of enological tannins in heat-treated must

fermented without pomace is necessary to increase the tannin concentration of Frontenac wines up

81

to a level that could modulate their astringency. However, the impact of such approach on wine

colour and stability as well as on wine astringency and aroma profile must be evaluated.

82

Chapitre 4. Evaluation of flavan-3-ols and

polysaccharides in musts and wines from Vitis

vinifera Cabernet Sauvignon and cold-hardy Vitis

sp. Frontenac

4.1. Avant-propos

Ce dernier chapitre répond à l’objectif 3 qui vise à explorer la teneur et la nature des polysaccharides

des vins des cépages hybrides interspécifiques Frontenac et Frontenac blanc et de les comparer au

cépage Vitis vinifera Cabernet Sauvignon afin d’anticiper un impact potentiel des polysaccharides sur

la rétention des tanins dans les vins de CHI rouges. Ces travaux seront soumis au journal Food

Chemistry sous la forme d’une communication courte, faisant l’objet de résultats préliminaires

nécessitant un approfondissement sur la thématique.

4.2. Résumé

La teneur et le profil en flavan-3-ols et en polysaccharides des moûts et des vins des cépages

hybrides interspécifiques Frontenac et Frontenac blanc et de Vitis vinifera Cabernet Sauvignon ont

été comparés. Les polysaccharides des moûts et des vins ont été précipités à l’éthanol, quantifiés à

l’aide de la méthode phénol-sulfurique de Dubois puis caractérisés par chromatographie par

perméation de gel/chromatographie d’exclusion stérique. La concentration et le profil en flavan-3-ols

ont été analysés par chromatographie liquide à haute performance couplée à un détecteur de

fluorescence. Résultats : La concentration totale en polysaccharides a augmenté pendant la

fermentation alcoolique et a diminué, ou s'est stabilisée, à la fin de la fermentation alcoolique. Les

vins issus du cépage hybride interspécifique Frontenac présentaient une concentration plus élevée

en polysaccharides totaux. Les polysaccharides des vins Frontenac et Frontenac blanc semblaient

également plus ramifiés que ceux de Cabernet Sauvignon. Il est connu que les polysaccharides

peuvent affecter la perception d’astringence des vins. Ces résultats suggèrent donc qu'une attention

particulière devrait être accordée à la composition en polysaccharides des cépages hybrides

interspécifiques dans le contexte de la production vinicole en climat froid.

83

4.3. Abstract

Changes in flavan-3-ol and polysaccharide content and profile of musts and wines from cold-hardy

Vitis sp. Frontenac and Frontenac blanc and V. vinifera Cabernet Sauvignon were compared. Must

and wine polysaccharides were precipitated by ethanol, quantified using the phenol-sulfuric method

of Dubois and characterised by GPC/SEC. The flavan-3-ol concentration and profile were analysed

by HPLC-FLD. Results: The total polysaccharide concentration increased during the alcoholic

fermentation and decreased or stabilized by the end of the fermentation. The wines made from the

cold-hardy hybrid cultivar Frontenac had a higher concentration in total polysaccharide when

compared to other studied varieties. Polysaccharides from Vitis sp. Frontenac and Frontenac blanc

wines also appeared to be more branched than those from V. vinifera Cabernet Sauvignon. As

polysaccharides are known to negatively impact the perceived astringency in wine, these results

suggest that significant attention should be given to the polysaccharide composition of cold-hardy

cultivars in the context of cold climate wine production.

4.4. Introduction

Cold-hardy Vitis cultivars issues from crosses between V. vinifera and native American species have

been largely implemented for wine production in northern areas such as Eastern Canada, Eastern

Europe, and Midwestern United States (Ehrhardt et al., 2014; J. Liu et al., 2015; Y. Ma, Tang, Xu, &

Li, 2017; Manns et al., 2013; K. Pedneault, M. Dorais, & P. Angers, 2013; Zhang et al., 2015). Cold-

hardy grapevines present certain specifications making them well suited for northern climates,

including a high resistance to very cold winter temperature lower than –30°C and a generally short

growing season (Fennell, 2004; Londo & Kovaleski, 2017). Along with cold resistance, most of them

also show a high degree of resistance to fungal diseases (Pedneault & Provost, 2016). In most

cases, the specific genetic of cold-hardy cultivars translates into certain berry characteristics such as

thick skins and high firmness in berries, even at full ripeness (Pedneault & Provost, 2016). These

characteristics highly contrasts with traditional V. vinifera berries that usually soften significantly along

the ripening process (Maury et al., 2009; Robin et al., 1997).

Changes in berry firmness are mainly attributable to modifications in mechanical properties of cell

walls occurring during ripening. Those involve berry cell wall components such as hemicellulose,

84

pectin, and cellulose that undergo solubilisation and depolymerisation processes but also

rearrangements of their associations (Goulao & Oliveira, 2008). Both the nature and the extent of

these changes are influenced by the grapevine’s genotype as well as its interactions with the

environment (Rihan, Al-Issawi, & Fuller, 2017). Berries from interspecific hybrid cultivars that present

cold-hardy and fungus-resistance properties have long been known for the particularly high pectin

content of their skin cell walls when compared to V. vinifera (Apolinar-Valiente et al., 2017; Lee et al.,

1975); (Springer & Sacks, 2014).

The winemaking process partly aims at extracting grape berry components such as tannins and

aroma. But along the process, macromolecules such as polysaccharides and proteins are also

extracted from berries as well as from fermenting microorganisms (yeast, bacteria). Pectic

polysaccharides mostly originate from grape berry cell wall, whereas microorganisms provide wine

with glycoproteins such as mannoproteins (Dols-Lafargue et al., 2007; Z. Guadalupe & Ayestarán,

2007; Vidal et al., 2003). The structure, concentration, and interactions between proteins, tannins,

and polysaccharides play a crucial role in the sensory properties of wine, especially regarding the

mouthfeel and taste of red wine. The role of tannins in the sensory properties of wine has been

largely studied over the years and extensively reviewed (Bajec & Pickering, 2008; W. Ma et al., 2014;

McRae & Kennedy, 2011; Scollary et al., 2012; Soares et al., 2017). In contrast, knowledge on wine

polysaccharides and proteins remains scarce. Recently, polysaccharides have been shown to inhibit

polyphenol-protein aggregation (including tannin-protein aggregation) and hence authors suggested

that polysaccharides can modulate wine astringency (Brandão et al., 2017; Lankhorst et al., 2017;

Watrelot, Schulz, & Kennedy, 2017).

Poor astringency is the main issue in red wine production from cold-hardy and fungus-resistant

cultivars (Nicolle et al., 2018; Nicolle, Marcotte, Angers, & Pedneault, 2019; Springer, Chen, et al.,

2016). Recent progress has highlighted the impact of proteins on tannin retention in hybrid red wine

(Nicolle et al., 2019; Springer, Sherwood, et al., 2016), but, thus far, little attention has been given to

polysaccharides in this context. Differences between the respective cell wall composition of cold-

hardy and V. vinifera cultivars suggest that polysaccharide content and composition of cold-hardy

berries might contribute to the poor astringency of the resulting wines. In this study, we compared the

composition of ethanol-precipitated polysaccharides and tannins of V. vinifera Cabernet Sauvignon

with that of two cold-hardy cultivars (Vitis sp. Frontenac and Frontenac blanc) using gel

85

permeation/size exclusion chromatography (GPC/SEC), and high-performance liquid

chromatography-fluorescence (HPLC-fluorescence), respectively.


4.5.1. Grape material

The cold-hardy hybrid grape varieties, Frontenac (FR) and Frontenac blanc (FB) (both issued from

Landot (L. 4511) X Vitis riparia 89) were harvested in a commercial vineyard located in Saint-Rémi

(QC, Canada) (45° 16′ 0″ N, 73° 37′ 0″ W). Berries from Vitis vinifera Cabernet Sauvignon were

imported from California (CA, USA) through a local dealer. All berries were harvested in 2015 and

stored at –30°C under control atmosphere until the experiment, as outlined by Springer, Chen, et al.

(2016).


The grapes were thawed at 4°C and then manually destemmed and pressed. The must and pomace

were placed in a 10 L fermenter bucket, treated with SO2 (30 mg/L, sulphur dioxide as potassium

metabisulfite), and cold-soaked (4°C, overnight). The must and pomace were transferred in a 10 L

fermentation unit equipped with a removable head plate fitted with two ports, one for sampling and

the other one for carbon dioxide discharge. Temperature regulation in the fermentation unit was

carried out by circulating water through two hoses connected to a temperature-controlled water bath.

Fermentations were performed as follows: Alcoholic fermentation (AF) was induced by a commercial

dry yeast Saccharomyces cerevisiae (Lalvin BM 4X4; Lallemand Inc., Montreal, Canada) at 250 mg/L

and carried out at 24°C until dryness. The cap was punched twice a day for the first two days and

then once a day. Alcoholic fermentation level was checked daily by measuring the concentration in

total soluble solid (°Brix). Fermenting must was sampled daily and stored at –30°C for future

analyses. At the end of alcoholic fermentation, wines were pressed manually using cotton

cheesecloth, packed in hermetically sealed bags under argon, and stored at 4°C. Fermentations

were performed in triplicates for each variety. The composition of musts and final wines (alcohol

concentration, % v/v; titratable acidity, g tartaric ac. eq./L; pH; primary amino nitrogen, mg/L; and

ammonia, mg/L) is provided in Table 4.1.

86

Table 4.1. Composition of musts and wines made from the cold-hardy Vitis sp. Frontenac blanc, Frontenac,

and V. vinifera Cabernet Sauvignon (Primary fermentable sugars, g/L; alcohol concentration, % v/v; titratable

acidity, g tartaric acid eq./L; pH; primary amino nitrogen, mg/L; and ammonia, mg/L).

Parameter Variety Must Wine

Primary fermentable sugars (g/L) Frontenac blanc 237.44 ± 1.85 a1 1.28 ± 0.38 a

Frontenac 227.85 ± 16.62 a 0.99 ± 0.01 a

Cabernet Sauvignon 256.35 ± 39.95 a 1.68 ± 0.38 a

Alcohol (v/v, %)

Frontenac blanc 0.00 a 15.11 ± 0.49 a

Frontenac 0.00 a 13.99 ± 0.00 b

Cabernet Sauvignon 0.00 a 15.56 ± 0.04 a

Titrable acidity (g/L tartaric acid eq.)

Frontenac blanc 13.24 ± 0.19 a 11.41 ± 0.04 b

Frontenac 14.14 ± 1.49 a 14.97 ± 0.95 a

Cabernet Sauvignon 4.25 ± 0.24 b 8.66 ± 0.88 c

pH Frontenac blanc 3.09 ± 0.06 b 3.18 ± 0.09 b

Frontenac 3.08 ± 0.12 b 3.25 ± 0.05 b

Cabernet Sauvignon 3.65 ± 0.08 a 3.86 ± 0.05 a

Primary amino nitrogen (mg/L) Frontenac blanc 272.33 ± 27.65 a n.a.

Frontenac 206.67 ± 5.86 b n.a.

Cabernet Sauvignon 118.00 ± 14.73 c n.a.

Ammonia (mg/L)

Frontenac blanc 11.67 ± 3.21 c n.a.

Frontenac 31.12 ± 6.87 b n.a.

Cabernet Sauvignon 49.00 ± 2.83 a n.a.

1 For a given matrix (must, wine) and parameter, values in the same column followed by different letters are

significantly different according to Tuckey’s honest significance test at the 0.05 probability level. n=3 samples

per variety X matrix (must, wine).

4.5.3. Sugars analysis

Ethanol, glucose, and fructose contents were quantified as described by Nicolle et al. (2019). Briefly,

analyses were performed on an HPLC system (Waters, Millipore Corp., Milford, Mass. USA)

equipped with a refractive index detector (Hitachi model L-7490 (Foster City California, USA), using a

Waters Sugar Pack-I column (6.5 mm x 300 mm) from Waters (Millipore Corp., Milford, Mass. USA).

Analyses were performed in duplicate.

87

4.5.4. Flavan-3-ol analysis

Flavan-3-ol content and composition were measured as described by Nicolle et al. (2018). The

analysis was carried out on an Agilent 1260 infinity HPLC system (Agilent Technologies, Santa Clara,

CA, USA) equipped with a fluorescence detector (G1321C, Agilent, Santa Clara, CA, USA).

Separation was performed on a Develosil Diol column (250 mm × 4.6 mm; 5 μm particle size) fitted

with a Cyano SecurityGuard column (Phenomenex, Torrance, CA, USA). Analyses were performed in

duplicate.

4.5.5. Polysaccharide analysis

4.5.5.1. Total polysaccharide precipitation and quantification

Total polysaccharides were precipitated as described by Segarra, Lao, López-Tamames, & De La

Torre-Boronat (1995) and quantified by UV-Vis spectroscopy (UV-Vis spectrophotometer UV-2700;

Shimadzu, Quebec, Canada) using the phenol-sulfuric method of Dubois, Gilles, Hamilton, Rebers,

& Smith (1956). Galactose was used as a standard for quantification. Total polysaccharide

precipitation and quantification were carried out in triplicate.

4.5.5.2. Total polysaccharide characterisation

One replicate of the ethanol-precipitated polysaccharide samples was characterised by gel

permeation/size exclusion chromatography (GPC/SEC) using the Malvern Panalytical OMNISEC

GPC/SEC system (Malvern Panalytical Ltd, Malvern, UK). The OMNISEC GPC/SEC system

combines multiple detectors (differential refractive index, diode-array-based UV\Vis

spectrophotometer, right angle and low angle light scattering, and four-capillary differential

viscometer) to quantify polysaccharides and measure their intrinsic viscosity (representative of

molecular structure, density and branching) and absolute molecular weight. Sample solutions were

filtered through 0.2 μm Nylon syringe filters (30 mm diam., Lab Products, Inc., Houston, TX, USA)

prior analysis. Polysaccharides were separated on an A2500 column (300 x 8 mm, Part Number

CLM3016, Malvern Panalytical, UK) maintained at 30°C, using an isocratic mobile phase composed

of H2O containing sodium sulfate (Na2SO4, 0.05 M) at a flow rate of 1.0 mL/min. The autosampler

chamber was maintained at 4°C. The injection volume was 100 L. The detectors were maintained

88

at 30C. Each sample was analyzed in triplicate. Polyethylene glycol was used as the calibration

standard and dextran was used as the verification standard. The molecular data for the

polysaccharide samples were calculated using the OMNISEC v10 software (Malvern Panalytical,

UK).

4.5.6. Statistical analysis

ANOVA analysis of the must and wine basic parameters (primary fermentable sugars, alcohol

concentration, titratable acidity, pH, primary amino nitrogen, and ammonia) were analysed using the

MIXED procedure of the SAS software (version 3.5 Basic Edition; SAS Institute Inc., Cary, NC, USA).

The DIFF option in a LSMEANS (least-squares means) statement was used, and means were

compared using the Tukey HSD ("Honestly Significant Difference") post-hoc test.

Flavan-3-ol and polysaccharide concentrations were analysed with the SAS software (version 3.5

Basic Edition; SAS Institute Inc., Cary, NC, USA) using ANOVA methods with te PROC MIXED

statement, analysing the main and interaction effects of the two following factors: cultivar and day of

fermentation. Since each wine was sampled during AF, a repeated-measures model was used, along

with the DIFF option in a LSMEANS (least-squares means) statement. Multiple comparisons were

made using the Tukey HSD ("Honestly Significant Difference") post-hoc test.

4.6. Results and discussion

4.6.1. Flavan-3-ols

The concentration in polymeric flavan-3-ols was significantly higher in FB musts when compared to

CS and FR musts (Fig. 4.1). At the end of AF, wines from all three varieties showed a similar

concentration in polymeric flavan-3-ol but the concentration in monomeric flavan-3-ols was

significantly higher in FB wines compared to CS and FR wines. CS wines showed a significantly

higher concentration in oligomeric flavan-3-ols compared to FB and FR wines.

89

(a)

0

500

1 000

1 500

2 000

2 500

0

50

100

150

200

250

300

350

400

0 1 2 3 4 5 6 7 8 9 10 11

Poly

sacc

har

ide

(mg/

L g

alac

tose

equ

ival

ent)

Fla

van

-3-o

l (m

g/L

EC

equ

ival

ent)

Alcoholic fermentation (day)

b) Frontenac rouge

0

500

1 000

1 500

2 000

2 500

0

50

100

150

200

250

300

350

400

0 1 2 3 4 5 6 7 8 9 10 11

Poly

sacc

har

ide

(mg/

L g

alac

tose

equ

ival

ent)

Fla

van

-3-o

l (m

g/L

EC

equ

ival

ent)


b) Frontenac rouge

0

500

1 000

1 500

2 000

2 500

0

50

100

150

200

250

300

350

400

0 1 2 3 4 5 6 7 8 9 10 11

Poly

sacc

har

ide

(mg/

L g

alac

tose

equiv

alen

t)

Fla

van

-3-o

l (m

g/L

EC

equiv

alen

t)


b) Frontenac rouge

(b)

0

500

1 000

1 500

2 000

2 500

0

50

100

150

200

250

300

350

400

0 1 2 3 4 5 6 7 8 9 10 11

Poly

sacc

har

ide

(mg/

L g

alac

tose

equiv

alen

t)

Fla

van

-3-o

l (m

g/L

EC

equiv

alen

t)


b) Frontenac rouge

90

0

500

1 000

1 500

2 000

2 500

0

50

100

150

200

250

300

350

400

450

0 1 2 3 4 5 6 7 8 9 10 11

Po

lysa

cch

ari

de (

mg/L

gala

cto

se e

qu

ivale

nt)

Fla

van

-3-o

l (m

g/L

EC

eq

uiv

ale

nt)


a) Cabernet Sauvignon

Polymeric flavan-3-ol

Oligomeric flavan-3-ol

Monomeric flavan-3-ol

Polysaccharide

0

500

1 000

1 500

2 000

2 500

0

50

100

150

200

250

300

350

400

450

0 1 2 3 4 5 6 7 8 9 10 11P

oly

sacch

ari

de (

mg/L

gala

cto

se e

qu

ivale

nt)

Fla

van

-3-o

l (m

g/L

EC

eq

uiv

ale

nt)



Polymeric flavan-3-ol

Oligomeric flavan-3-ol

Monomeric flavan-3-ol

Polysaccharide

0

500

1 000

1 500

2 000

2 500

0

50

100

150

200

250

300

350

400

0 1 2 3 4 5 6 7 8 9 10 11

Poly

sacchar

ide

(mg/L

gala

cto

se e

quiv

alent)

Fla

van

-3-o

l (m

g/L

EC

equiv

ale

nt)



(c)

Figure 4.1. Changes in the concentration of monomeric, oligomeric (2-5 flavan-3-ol units), and polymeric (≥ 6

flavan-3-ol units) flavan-3-ols (mean ± standard deviation, mg/L, epicatechin equivalent) and polysaccharides

(mean ± standard deviation, mg/L, galactose equivalent) during the alcoholic fermentation of the cold-hardy

Vitis sp. Frontenac blanc (a), Frontenac (b), and V. vinifera Cabernet Sauvignon (c). Mean comparison using

Tukey’s honest significant difference test at the 0.05 probability level is shown in Appendix D.

The kinetics of flavan-3-ol extraction during the fermentation varied between cultivars but some

similarities were also observed. For instance, the concentration of monomeric flavan-3-ols tripled in

wines from all three varieties when compared to musts and the concentration in oligomeric flavan-3-

ols increased by six to eight times during fermentation. Previous studies on V. vinifera cultivars

showed that small flavan-3-ol oligomers (1-3 flavan-3-ol units) are primarily extracted at the end of

the cold prefermentative maceration whereas larger oligomers (4-5 flavan-3-ols units) are mostly

extracted during further winemaking stages (González-Manzano et al., 2006).

In contrast with mono- and oligomers, the concentration in polymeric flavan-3-ols doubled in CS

wines during fermentation, whereas both FB and FR wines showed little to no significant difference in

this aspect (Fig. 4.1). Our results show a dramatic fall in polymeric flavan-3-ol concentration in both

FB and FR wines (up 2.5 and 1.9 times less, for FR and FB wines, respectively) two days after the

91

beginning of fermentation, suggesting that a particular physicochemical phenomenon occurred.

Previous studies showed that rise in ethanol concentration during AF weakens the hydrophobic

interactions between cell wall components and flavan-3-ols, thereby facilitating their extraction

(Casassa & Harbertson, 2014). However, based on the kinetics of fermentable sugars consumption

by yeast, both FR and FB ended their fermentation faster than CS (5 days versus 9 days) but yet

showed a dramatic decrease in their polymeric flavan-3-ol content (Figs. 4.2 and 4.3c). Cell wall

components from berries of cold-hardy cultivars have been shown to bind tannins at a higher rate

than those from V. vinifera berries (Springer, Sherwood, et al., 2016). Results on the negative impact

of pomace on tannin retention in Frontenac wines recently suggested that skin cell wall components,

including polysaccharides, could have a larger role than initially anticipated on tannin retention in

cold-hardy wines (Nicolle et al., 2019).

Cabernet Sauvignon Frontenac Frontenac blanc

ferm

enta

ble

sugars

ferm

enta

ble

sugars

, in

itia

l1 -

0.00

0.25

0.50

0.75

1.00

0 2 4 6 8 10

Fer

men

tatio

n p

rogr

ess


Figure 4.2. Kinetic of fermentable sugar consumption during the fermentation of the cold-hardy Vitis sp.

Frontenac blanc (a), Frontenac (b), and V. vinifera Cabernet Sauvignon (c).

92

(a)

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14 16 18

Mo

no

meri

c f

lav

an-3

-ol

(mg/L

eq

. E

C)

Ethanol (%, v/v)

(b)

0

20

40

60

80

100

120

140

160

180

200

0 2 4 6 8 10 12 14 16 18

Oli

go

meri

c f

lav

an

-3-o

l (m

g/L

eq

. E

C)

Ethanol (%, v/v)

93

(c)


0

20

40

60

80

100

120

0 2 4 6 8 10 12 14 16 18

Po

lym

eric

fla

van

-3-o

l (m

g/L

eq

. E

C)

Ethanol (%, v/v)

Figure 4.3. Monomeric (a), oligomeric (b; 2-5 flavan-3-ol units), and polymeric (c; ≥ 6 flavan-3-ol units) flavan-

3-ols (mean ± standard deviation, mg/L epicatechin equivalent) extraction as a function of increasing ethanol

concentration (mean ± standard deviation; %, v/v) during the alcoholic fermentation of the cold-hardy Vitis sp.

Frontenac blanc, Frontenac, and V. vinifera Cabernet Sauvignon. Mean comparison using Tukey’s honest

significant difference test at the 0.05 probability level is shown in Appendix D.

4.6.2. Total polysaccharides

On-skin fermentation performed in red winemaking strongly favours the extraction of polysaccharides

in wine. In our study, musts from FB, FR, and CS showed no significant difference in total

polysaccharide concentration, but significant differences were observed between wines. Indeed, FR

wines had a significantly higher concentration in polysaccharides than CS wines (1321.4 mg/L

compared to 921.7 mg/L galactose equivalent; Fig. 4.1).

The kinetic of polysaccharide extraction during the winemaking process showed that the

concentration of polysaccharides increased progressively up to 4.2 times for FB and 3.5 for FR

between 0 and 7 days of AF, reaching more than 1000 mg/L galactose equivalent, whereas it only

increased by 2.3 times in CS during the same period (Fig. 4.1). In the second half of the winemaking

process, the concentration in polysaccharides slightly decreased or stabilised. As previously

suggested by Guadalupe & Ayestaran (2007), such decrease might indicate that the precipitation rate

94

of polysaccharide compounds was higher than their solubilization, and that this process was

somewhat affected by the dynamic conditions provided by the winemaking process.

4.6.3. Polysaccharide profile

The parameters measured by GPC/SEC analyses provide significant information regarding the

molecular weight of the polysaccharides extracted from the studied varieties, including the number

average molecular weight (Mn), the weight average molecular weight (Mw), the Z average molecular

weight (Mz) and the molecular weight polydispersity ratio (Mw/Mn). These parameters are correlated

with key physical properties of polysaccharides such as viscosity and toughness that may affect wine

mouthfeel. Additional parameters such as intrinsic viscosity (IV), hydrodynamic radius (Rh), and

Mark-Houwink-Sakurada data were also measured (Table 4.2 and Fig. 4.4).

FR wine polysaccharide samples showed a smaller Mn value than those from CS (14 043 Da versus

39 403 Da, respectively; Table 4.2), but FB wine polysaccharide samples had a much higher Mn

value than both FR and CS (97 463 Da). These results suggest that FR wines could contain a

significant proportion of low-molecular weight polysaccharides such as rhamnogalacturonan-2 when

compared to CS and FB wines. Rhamnogalacturonan-2 have the lowest molecular weigth (10 000-

50 000 g/mol) among the polysaccharides presents in grape berries ; they are typically released from

cell wall pectins during skin contact in fermenting must (Gawel et al., 2018).

FB and FR wine polysaccharide samples showed the highest Mw and Mz values (137 933 and 445

533 Da, respectively; Table 4.2), whereas much lower values were observed for both these variables

in CS wine polysaccharide samples (56 617 and 101 467 Da, respectively; Table 4.2). In contrast

with Mn, Mw and Mz consider the molecular weight distribution of the polymers. Wine polysaccharides

from FR and FB samples showed high Mz values, suggesting that polysaccharides from these

cultivars had more massive chains than those from CS.

The ratio Mw/Mn, formerly known as dispersity, showed that the FR wine polysaccharide samples had

a broader molecular weight distribution than wine polysaccharide samples from FB and CS, including

larger polysaccharides as suggested by the Mz value of FR wine polysaccharide samples (Table 4.2).

Mannoproteins are the largest polysaccharides in wine (molecular weight > 50 000 units) (Gawel et

al., 2018). They are extracted from yeast cell walls during fermentation and later during yeast lee

95

contact. Our data suggest that FR wines might have higher content in mannoprotein when compared

to CS wines, but further characterisation is needed to fully explain these results.

Table 4.2. Average molecular weight values (number average molecular weight, Mn; weight average molecular

weight, Mw; Z average molecular weight, Mz; and molecular weight polydispersity ratio, Mw/Mn), and intrinsic

viscosity (IV) for the ethanol-precipitated polysaccharide of must, fermented must (middle of alcoholic

fermentation, mid-AF), and wine made from the cold-hardy Vitis sp. Frontenac blanc, Frontenac, and V.

vinifera Cabernet Sauvignon.

Matrix Variety Mn Mw Mz

Mw/Mn IV

(Da) (Da) (Da) (dL/g)

Must Frontenac

blanc

50,083 ± 2,4511 71,803 ± 1,314 96,673 ± 1,917 1.266 ± 0.060 0.117 ± 0.05

Frontenac 39,243 ± 432 63,743 ± 443 102,523 ± 2,846 1.625 ± 0.028 0.140 ± 0.001

Cabernet

Sauvignon

42,860 ± 870 54,240 ± 506 81,743 ± 3,334 1.266 ± 0.016 0.129 ± 0.005

Mid-AF Frontenac

blanc

105,500 ± 721 157,633 ± 1,159 467,100 ± 2,272 1.494 ± 0.020 0.274 ± 0.008

Frontenac 13,497 ± 462 66,183 ± 727 415,433 ± 12,458 4.907 ± 0.121 0.138 ± 0.004

Cabernet

Sauvignon

53,697 ± 570 76,777 ± 826 137,300 ± 3,857 1.43 ± 0.002 0.167 ± 0.02

Wine Frontenac

blanc

97,463 ± 355 137,933 ± 643 303,000 ± 954 1.437 ± 0.009 0.190 ± 0.003

Frontenac 14,043 ± 126 70,640 ± 404 445,533 ± 4,750 5.031 ± 0.045 0.185 ± 0.004

Cabernet

Sauvignon

39,403 ± 544 56,617 ± 219 101,467 ± 1,617 1.437 ± 0.015 0.309 ± 0.005

1 Standard deviations are representative of three analyses per sample; n=1 per matrix X variety.

The Mark-Houwink-Sakurada (MHS) plots are the most accurate method to visualise structural

differences in materials, as data from three detectors (light scattering, refractive index, and

viscometer) are combined to produce a plot of IV vs. molecular weight on log scales. The MHS plot of

the must polysaccharide samples shows that CS, FR, and FB presents narrows plots, which indicates

a combination of relatively low molecular weight and low dispersity (Fig. 4.4a). Also, the multiple

inflection points in the MHS plots in Fig. 4.4a indicate the presence of multiple species, as each

differently sloped segment is the result of a different component or a differently shaped (e.g.

branched) version. By the end of AF, higher molecular weight polysaccharides were extracted and

could be attributed to yeast cell walls. The MHS plot of the FR wine polysaccharide samples appears

as a combination of CS and FB wine samples (Fig. 4b). The FR wine samples overlap with CS wine

samples in the low molecular weight region, but after the intersection of the CS wine samples with the

96

FB wine samples, the MHS plot of FR wine samples follows that of FB wine samples strictly for a bit,

until it diverges in the highest molecular weight region. A downward curvature moving toward the

higher molecular weight at the end of the plot was observed for both FB and FR wine polysaccharide

samples. This indicates that molecular weight increased faster than the intrinsic viscosity. This is a

typical profile of a branched sample, as the presence of branches leads to an increase in molecular

weight but not necessarily an increase in the amount of volume the sample inhabits. The CS

polysaccharide samples exhibited a straight line which indicates a linear molecular structure, as an

increase in molecular weight corresponds to a proportional increase in IV.

0.01

0.1

1

10

1 000 10 000 100 000 1 000 000 10 000 000 100 000 000

Intr

insi

c vis

cosi

ty (

dL

/g)

Molecular weight (Da)

(a)

97

(b)

0.01

0.1

1

10

1 000 10 000 100 000 1 000 000 10 000 000 100 000 000

Intr

insi

c v

isco

sity

(d

L/g

)

Molecular weight (Da)

Figure 4.4. Mark–Houwink–Sakurada plot for must (a) and wine (b) polysaccharide samples of the cold-hardy

Vitis sp. Frontenac blanc (must, green; wine, maroon), Frontenac (must, grey; wine, blue), and V. vinifera

Cabernet Sauvignon (must, orange; wine, yellow).

Wine polysaccharides influence astringency perception of red wines. They are known to inhibit the

interactions and aggregations between salivary proteins and oligomeric and polymeric flavan-3-ols

(Carvalho et al., 2006). In red wine-like media, Vidal, Courcoux, et al. (2004) observed that increasing

tannin concentration also increased astringency intensity but this effect was reduced by the addition

of rhamnogalacturonan-2 polysaccharides. Similarly, Quijada-Morín, Williams, Rivas-Gonzalo, Doco,

& Escribano-Bailón (2014) showed that both mannoproteins and rhamnogalacturonan-2

polysaccharides strongly reduced astringency perception in Tempranillo red wines.

4.7. Conclusion

Our results showed that wines made from the cold-hardy cultivars Frontenac and Frontenac blanc

had less oligomeric and polymeric flavan-3-ols than those from Vitis vinifera Cabernet Sauvignon.

The total polysaccharide concentration of these wines clearly increased during the alcoholic

fermentation before decreasing or becoming stable by the end of the fermentation. The wines made

from the cold-hardy hybrid Frontenac showed a higher concentration in total polysaccharide, and

98

possibly had a higher content in mannoproteins and rhamnogalacturonan-2 polysaccharides

compared to Frontenac blanc and Cabernet Sauvignon wines. In addition, the wine polysaccharides

from Frontenac and Frontenac blanc seemed to be more branched than those from Cabernet

Sauvignon wines. This suggest that specific attention should be brought to the impact of the

polysaccharide composition of cold-hardy cultivars, such as Frontenac, as these polysaccharides

could strongly contribute to lowering the astringency of hybrid wines.

99

Conclusion & perspectives

Parfois décrits comme trop acides et sans astringence par certains consommateurs, les vins rouges

québécois représentent encore moins de 3% des ventes de vins au Québec. L’astringence est un

critère important pour la qualité d’un vin rouge. Elle est intimement liée à la concentration et structure

des tanins présents dans le vin. Diversifier les caractéristiques des vins produits à partir de cépages

rouges rustiques, plus spécifiquement leur astringence, pourrait améliorer leur qualité et permettre de

mieux rejoindre les attentes des consommateurs québécois.

Cette thèse avait pour but (1) de développer un procédé de vinification adapté à la composition

physico-chimique atypique des cépages hybrides interspécifique (CHI) rouges afin de produire des

vins plus riches en tanins, ayant un potentiel accru de satisfaire les goûts des consommateurs et,

conjointement, (2) de clarifier le rôle des constituants de la paroi cellulaire du raisin (polysaccharides

et protéines) et des anthocyanes sur la rétention des tanins dans les vins rouges de CHI. Elle devait

également valider ou réfuter l’hypothèse initiale, basée sur la revue de littérature, qui était que le

développement de procédés de vinification adaptés à la composition chimique des baies des

cépages hybrides, incluant notamment l’ajout de tanins exogènes en quantité suffisante et la

réduction des interactions avec les constituants de la paroi cellulaire du raisin (protéines et

polysaccharides) durant la vinification, permettra d’augmenter significativement la teneur en tanins

des vins rouges de CHI.

Plusieurs objectifs avaient été définis afin d’enrichir les connaissances actuellement peu

développées, sur ces cépages présentant des caractéristiques agronomiques importantes pour

l’implantation de pratiques durables en viticulture, non seulement en climat froid, mais aussi dans les

climats tempérés. En effet, l’optimisation de la protection du vignoble et le développement de la

viticulture biologique par l’utilisation de cépages tolérants ou résistants à différents stress biotiques et

abiotiques remet au premier plan l’utilisation des CHI au niveau mondial pour diminuer durablement

les pesticides et adapter la production vinicole aux changements climatiques. Ainsi, l’apport de

connaissance sur ces cépages a une retombée pour l’ensemble de la filière vitivinicole.

100

► Approche écologique et économique : co-fermentation de marc de raisin rouge

(Frontenac) et marc de raisin (Vidal)

La substitution partielle du marc rouge par du marc blanc, en début de fermentation alcoolique, s’est

avérée être une pratique œnologique intéressante pour améliorer le potentiel phénolique et la

stabilité de la couleur des vins de Frontenac. L'efficacité de l'addition se traduit par une augmentation

de la concentration de plusieurs composés phénoliques incolores (flavan-3-ols oligomériques et

copigments tels que flavan-3-ols monomériques) et de réactions de copigmentation intermoléculaires

dans les vins conduisant à une meilleure stabilisation de la couleur des vins rouges de Frontenac.

Cependant, l'effet global dépend clairement des proportions de marc rouge et de marc blanc

choisies. Une proportion trop élevée de marc blanc ajoutée au moût en début de fermentation

alcoolique (≥ 18% dans l’étude) peut nuire à la qualité du vin en raison d'une adsorption plus

importante des composés phénoliques tels que les flavan-3-ols polymériques et possiblement

certains copigments autres que les flavan-3-ols monomériques au cours de la macération, entraînant

une modification de couleur des vins finaux et une stabilisation moins efficace de la couleur. D’autre

part, l’ajout de marc blanc Vidal n’est pas sans impact sur le profil aromatique des vins puisqu’il libère

au cours de la fermentation des composés volatils typiques de son cépage. Ainsi, toutes les

proportions ne sont pas acceptables pour obtenir des effets bénéfiques sur le potentiel tanique d’un

vin et l'amélioration de sa couleur, sans pour autant entraver la typicité du cépage. Des analyses

sensorielles seraient pertinentes pour vérifier (1) l’impact de la modification de la composition

phénolique et volatile des vins sur ses caractéristiques organoleptiques telles que l’astringence et les

arômes et (2) l’acceptation de ces vins par les consommateurs. L’utilisation de marc de raisin, résidu

majoritaire peu transformé de la vinification en blanc, s’inscrit dans une démarche de développement

durable de plus en plus sollicitée par les consommateurs, ce qui pourraient apporter une valeur

ajoutée au produit.

Sur le plan scientifique, il a été montré qu’une proportion élevée de marc de raisin blanc (≥18%) –et

indirectement les composants de la paroi cellulaire du marc de Vidal, diminue la rétention des tanins

de masse moléculaire élevée dans le vin. Sur le plan technique, les parois cellulaires du marc de

Vidal pourraient donc être utilisées pour réduire l'astringence de certains vins rouges trop astringents.

101

► Maintien de la typicité des vins rouges Frontenac : ajout pré-fermentaire de tanins

œnologiques et fermentation sans marc de raisin

La fermentation sans marc de raisin après macération à froid a été une approche efficace pour

améliorer de manière significative la rétention des tanins dans les vins rouges de Frontenac, en

particulier la concentration en flavan-3-ols polymériques. En revanche, l’élimination des protéines du

vin par chauffage ou addition de bentonite, en amont de la fermentation alcoolique, n’a pas permis

une meilleure rétention des tanins dans ces vins. La thermovinification, qui correspond à un

traitement thermique des raisins, suivi du pressurage et de la fermentation en phase liquide, apparait

donc comme une bonne alternative pour les CHI. Elle permet l’obtention de vins plus riches en

pigments simples et en pigments polymériques. Cependant, l’ajout de tanins à une dose minimum de

3 g/L de tanins œnologiques (soit 5 fois la dose recommandée) dans le moût reste nécessaire pour

augmenter de façon significative la concentration en tanins des vins de Frontenac par rapport à celle

obtenue par vinification traditionnelle en rouge. À cet effet, du marc de raisin blanc riche en tanins

pourrait être ajouté lors de l’étape de chauffage pour augmenter le potentiel phénolique des vins si la

recherche de typicité n’est pas indispensable. L’ajout de tanins exogènes post-fermentation, après

retrait du marc de raisin et élimination partielle et naturelle des protéines, pourrait également être une

option à étudier.

Les moûts élaborés par thermovinification présentent généralement une turbidité élevée et sont

difficiles à clarifier. En vinification en blanc, il est reconnu que le degré de clarification du moût influe

sur la cinétique de fermentation de la levure, la viabilité cellulaire et les caractéristiques aromatiques

des vins finis. En vinification en rouge, peu de connaissances sur la teneur, la nature et les

caractéristiques physico-chimiques des particules responsables de la turbidité des moûts sont

disponibles. Il en est de même sur l’impact de ces particules sur les caractéristiques organoleptiques

des vins. De nouveaux essais incluant une étape de clarification du moût (absente de nos essais)

pourrait donc s’avérer intéressants afin d’avoir une meilleure compréhension de l’impact de la

turbidité des moûts issus de CHI et traités par thermovinification.

La thermovinification est une pratique croissante dans la vinification en rouge pour obtenir des vins

légers et fruités. Au Québec, ce traitement n’est pas ou peu utilisée. Le coût d’une telle installation

ainsi que le coût des dépenses énergétiques doivent donc être pris en considération. L’ajout de dose

importante de tanins, d’autant plus dans le cas d’une fermentation en présence de marc, nécessite

102

impérativement la conduite d’analyses sensorielles afin de valider l’impact d’une dose importante de

tanins sur les caractéristiques organoleptiques du vin (saveur, goût et couleur).

Sur le plan scientifique, nous avons démontré que le marc de raisin joue un rôle prépondérant sur la

rétention des tanins dans le vin et que les protéines solubles avaient un rôle négligeable sur ce

même aspect. Les constituants non solubles de la pellicule de raisin et/ou les polysaccharides

solubles apparaissent donc comme le principal facteur limitant la rétention des tanins dans les vins

de CHI.

► Les polysaccharides des cépages hybrides interspécifiques : différence structurale ?

Les vins élaborés à partir du CHI Frontenac ont montré une concentration plus élevée en

polysaccharides totaux et possiblement une teneur plus élevée en mannoprotéines et en

polysaccharides rhamnogalacturonanes-2 par rapport au cépage V. vinifera Cabernet Sauvignon.

Dans le cadre de notre étude, ces polysaccharides apparaissent également comme plus ramifiés que

ceux des vins Cabernet Sauvignon. Des études complémentaires devraient être conduites sur cette

thématique afin de confirmer ces résultats préliminaires et d’évaluer l’incidence d’une modification

structurale sur la perception d’astringence des vins rouges de CHI. Enfin, il pourrait être intéressant

de faire des essais de procédés de vinification permettant de contrôler la teneur et la nature des

polysaccharides du vin (ex. : enzymage, filtration du moût en amont de la fermentation) afin d’avoir

un impact potentiellement décisif sur la structure des vins rouges issus de CHI.

Sur le plan scientifique, les essais ont permis d’apporter des connaissances nouvelles sur la teneur

et la structure des polysaccharides des vins de CHI.

La Figure 5.1 propose deux itinéraires technologiques adaptée aux CHI cultivés en climat froid au vu

des résultats des études réalisées. L’ajout de tanins post-fermentation alcoolique, l’ajout d’enzymes

pectolytiques pré- ou post-fermentation alcoolique ainsi qu’une clarification du moût pré-fermentation

alcoolique en phase liquide restent néanmoins à être étudiés pour affiner le travail. Aussi, des

concentrations élevées d'acides organiques sont typiques dans les vins rouges issus de CHI ; ils

peuvent agir comme astringents au bas pH du vin rouge et donc éventuellement entraver

l'acceptabilité sensorielle en impactant négativement la perception d'astringence des vins. Il pourrait

être intéressant d’inclure un volet désacidification dans de futurs essais.

103

Figure 4.5.1. Proposition d’itinéraires technologiques adaptés à la vinification en rouge des cépages hybrides

interspécifiques cultivés en climat froid.

104

Annexes

Appendix A. Étapes de la vinification en rouge traditionnelle

1) Sélection des raisins : Dès leur réception, les grappes de raisin doivent subir un tri afin de séparer

les grappes saines des grappes non mûres, avariées ou pourries et ne conserver que les fruits de

qualité pour la production du vin.

2) Égrappage-foulage : Les grappes sélectionnées sont égrappées pour séparer les baies de raisin

de leurs rafles afin de réduire l'apport en tanins liés aux rafles et diminuer le caractère herbacé des

rafles non lignifiées. Les baies sont ensuite foulées pour déchirer et écraser la pellicule des baies afin

d'assurer une bonne diffusion des éléments solubles du marc vers le moût.

3) Macération pré-fermentaire : Cette étape optionnelle peut être réalisée afin de maximiser

l’extraction des constituants du raisin tels que les composés phénoliques en déstructurant la paroi

cellulaire. Elle peut être réalisée à chaud (e.g. 20 min à 60-80°C) ou à froid (e.g. 24 heures à 10°C).

Cette étape maximise principalement l’extraction des anthocyanes puisqu’elles s’extraient

préférentiellement en phase aqueuse.

4) Fermentation et macération alcoolique : En vinification en rouge traditionnelle, la macération et la

fermentation s'accomplissent simultanément. Elle s’initie par levurage, généralement au moyen de

levures sèches actives de Saccharomyces cerevisae. La fermentation alcoolique est une étape

importante du processus de vinification, elle contribue à l'optimisation de l'extraction des composés

du raisin en initiant la transformation des sucres du raisin en éthanol, en dioxyde de carbone et

produits secondaires. Pour renforcer l'extraction, divers procédés mécaniques peuvent être utilisés,

parmi eux le remontage et le lessivage du chapeau de marc.

5) Macération post-fermentaire : Cette étape optionnelle est souvent réalisée dans le but de

maximiser l’extraction des tanins de pépins, qui s’extraient une fois la cuticule dissoute par l’éthanol

dans le temps.

6) Soutirage et pressurage : En fin de fermentation alcoolique et après écoulage du vin (vin de

goutte), le marc est pressé afin d'extraire le liquide encore présent (vin de presse).

7) Fermentation malolactique : Le vin de presse et le vin de goutte subissent, séparément ou

ensemble, une fermentation malolactique en présence de bactéries Oenococcus oeni. Durant cette

étape, l'acide malique est transformé en acide lactique, rendant le vin moins acide et plus rond.

105

8) Élevage et assemblage : Le vin, selon la qualité visée, peut être élevé en fût ou être assemblé

avec d’autres vins.

9) Stabilisation et filtration : Une stabilisation à basse température est généralement réalisée afin

d’éviter que des cristaux d'acide tartrique ne se forment après embouteillage du vin. Une filtration est

également réalisée afin de clarifier le vin (limpidité et brillance) et d'éliminer toute bactérie du vin

pouvant entraver la stabilité à long terme.

10) Embouteillage : La dernière étape consiste à mettre en bouteille le vin.

Des actions correctives sur le moût ou le vin peuvent également être apportées en fonction de sa

composition chimique et du profil de vin recherché : acidification/désacidification (acidité du vin),

tannisage/collage protéique (amertume, astringence et structure en bouche du vin), enzymage

(astringence, couleur, filtrabilité du vin).

106

Appendix B. Supplementary material - Chapter 2

Table S2.1. Composition (mean ± standard deviation) of the must and control wines and RP/WP- (30%

RP/6% WP, 30% RP/12% WP, and 30% RP/18% WP) and WP-treated wines (23% WP) after 395 days of

bottling.

1 Abbreviations for parameters: TA, titratable acidity (g tartaric ac. eq./L); free SO2, free sulphur dioxide (mg/L); TSS, total

soluble solids (°Brix); PAN, primary amino nitrogen (mg/L); N, ammonia (mg/L). 2 Values in the same row followed by different letters are significantly different according to Tuckey’s honest significance


107

Table S2.2. Monomeric, oligomeric (2 to 5 flavan-3-ol units), and polymeric (≥ 6 flavan-3-ol units) flavan-3-ol compound concentration (mean ± standard deviation,

in mg/L epicatechin equivalent) in control (50% RP), RP/WP-treated (30% RP/6% WP, 30% RP/12% WP, and 30% RP/18% WP), and WP-treated (23% WP)

wines at different winemaking stages.

108

a Abbreviations for stages: PFM, after the pre-fermentative cold maceration; 1-FAM, m-FAM, and e-FAM, during the fermentative alcoholic maceration (days 1, 4, and 8); MLF,

after the malolactic fermentation (day 45); and BW, after bottling (day 395). b Values in the same row (lower-case letters) and the same column (capital letters) followed by different letters are significantly different according to Tuckey’s honest significance


109

Table S2.3. Phenol estimation (mean ± standard deviation, in absorbance unit) in control (50% RP), RP/WP-treated (30% RP/6% WP, 30% RP/12% WP, and 30%

RP/18% WP), and WP-treated (23% WP) wines at different winemaking stages.

110

a Abbreviations for parameters: TPI, Total Polyphenols Index (Abs 280 nm); P pH<1, pigments at acidic pH (Abs 520 nm in HCl); WP, wine pigment (Abs 520 nm); WPcor, wine

pigments corrected (Abs 520 nm with acetaldehyde); PRSO2, pigments resisting to sulphite bleaching (Abs 520 nm with SO2); CIcor, colour intensity corrected (Abs 420 nm + Abs

520 nm + Abs 620 nm with acetaldehyde); H, hue (Abs 420 nm/Abs 520 nm without acetaldehyde). b Abbreviations for stages: PFM, after the pre-fermentative cold maceration; 1-FAM, m-FAM, and e-FAM, during the fermentative alcoholic maceration (days 1, 4, and 8); MLF,

after the malolactic fermentation (day 45); and BW, after bottling (day 395). c Values in the same row (lower-case letters) and the same column (capital letters) followed by different letters are significantly different according to Tuckey’s honest significance


111

Table S2.4. Volatile compound parameters for GC-MS-SPME analysis.

112

a)

b)

Figure S2.1. Anthocyanin (a) (mg/L cyanidin-3-glucoside, delphinidin-3-glucoside, malvidin-3-glucoside,

pelargonidin-3-glucoside, and peonidin-3-glucoside equivalent depending of the aglycone) and flavan-3-ol

compound (b) (mg/L epicatechin equivalent) profiles in control wines and RP/WP- (30% RP/6% WP, 30%

RP/12% WP, and 30% RP/18% WP) and WP-treated wines (23% WP) after 395 days of bottling.

113

a)

b)

c)

114

d)

e)

Figure S2.2. CIELab parameters including L, lightness (a); a, red-green (b); b, blue-yellow (c); C, chroma (d);

H, hue (e) (mean ± standard deviation) for control wines and RP/WP- (30% RP/6% WP, 30% RP/12% WP,

and 30% RP/18% WP) and WP-treated wines (23% WP) at different winemaking stages: 1-FAM, m-FAM, and

e-FAM, during the fermentative alcoholic maceration (day 1, 4, and 8); MLF, after the malolactic fermentation

(day 45); and BW, after bottling (day 395).

115

Figure S2.3. Colour representation of control wines and RP/WP- (30% RP/6% WP, 30% RP/12% WP, and

30% RP/18% WP) and WP-treated wines (23% WP) from the CIELab data at different winemaking stages: 1-

FAM, m-FAM, and e-FAM, during the fermentative alcoholic maceration (day 1, 4, and 8); MLF, after the

malolactic fermentation (day 45); and BW, after bottling (day 395). The colour representation was obtained

from the website: http://colorizer.org/.

116

Appendix C. Supplementary material - Chapter 3

Figure S3.1. Factorial experimental design used to produce experimental Frontenac wines, including the

following factors: must protein treatment (untreated, bentonite-treated, and heat-treated must), pomace (must

fermented with and without pomace), tannin addition (0, 1, 3, and 9 g/L), and time of maceration (0, 4, and 11

days after the end of alcoholic fermentation, corresponding to days 4, 8, and 15 of the winemaking process,

respectively).

117

Material and Method: Sugars analysis

Glucose and fructose content was determined using an HPLC system (Waters, Millipore Corp.,

Milford, Mass. USA) equipped with a refractive index detector (Hitachi model L-7490 (Foster City

California, USA).

Briefly, samples were centrifuged at 10 000 rpm at 4°C for 15 min, diluted 10-fold, and filtered

through 0.45 μm PTFE syringe filters (25 mm diam., Silicycle, Quebec, Canada) prior HPLC analysis.

The separation was achieved on a Waters Sugar Pack-I column (6.5 mm x 300 mm) from Waters

(Millipore Corp., Milford, Mass. USA) maintained at 90ºC, using an isocratic mobile phase composed

of a solution of EDTA (50 mg/L) circulating at a flow rate of 0.5 mL/min. The injection volume was

50 L.

Calibration curves were prepared using authentic standards of glucose and fructose in a range of 5 to

20% (w/v) concentration in water. Peaks were identified by comparing retention times with standards.

118

Table S3.1. Total pigment (Tpg) and co-pigmented, monomeric, and polymeric anthocyanin (CA, MA, and PP, respectively) estimation (mean ± standard

deviation (SD)) in experimental Frontenac wines made with untreated (control), bentonite-treated, and heat-treated must, fermented with (WP) and without (WOP)

pomace, and with different doses of tannin addition (0, 1, 3, and 9 g/L) at 11 days after the end of alcoholic fermentation (corresponding to day 15 of the

winemaking process).

119


the 0.05 probability level: a, b, c, and d letters compare the dose of tannin addition for a given Must treatment*Pomace; A, B, and C letters compare the must treatment for a given

Tannin*Pomace; and asterisk (*) compare the pomace treatment for a given Must treatment*Tannin.

120

Table S3.2. Kinetic of tannin concentration (mean ± standard deviation (SD), mg/L epicatechin equivalent) in experimental Frontenac wines made with untreated

(control), bentonite-treated, and heat-treated must, fermented with (WP) and without (WOP) pomace, and with different doses of tannin addition (0, 1, 3, and 9 g/L)

at the end of alcoholic fermentation (e-AF) and on the 4th and the 11th day after the e-AF (corresponding to days 4, 8, and 15 of the winemaking process,

respectively).

121


the 0.05 probability level: a, b, c, and d letters compare the dose of tannin addition for a given Must treatment*Pomace*Time; x, y, and z letters compare the must treatment for a

given Tannin*Pomace*Time; A, B, and C letters compare the day after the e-AF (time) for a given Must treatment*Pomace*Tannin; and asterisk (*) compare the pomace treatment

for a given Must treatment*Time*Tannin.

122

Appendix D. Supplementary material - Chapter 4

Table S4.1. Monomeric, oligomeric (2-5 flavan-3-ol units), and polymeric (≥ 6 flavan-3-ol units) flavan-3-ol (mean ± standard deviation (SD), mg/L epicatechin

equivalent), polysaccharide (mean ± SD, mg/L galactose equivalent), and ethanol (%, v/v) concentration during the alcoholic fermentation of V. vinifera Cabernet

Sauvignon and cold-hardy Vitis sp. cultivars Frontenac and Frontenac blanc.

Cultivar

Parameter Day of

AF


Mean ± SD Mean ± SD Mean ± SD

Monomeric

flavan-3-ol

(mg/L EC eq.)

0 15.29 ± 0.76 G 1 b 17.93 ± 2.16 FG ab 25.88 ± 4.97 H a

1 16.99 ± 0.26 G b 14.04 ± 1.00 G b 27.92 ± 3.42 GH a

2 28.98 ± 0.32 F a 14.79 ± 0.42 G b 32.67 ± 4.87 G a

3 30.17 ± 0.02 F b 22.14 ± 3.68 F b 46.46 ± 2.52 F a

4 36.39 ± 0.79 E b 34.97 ± 4.52 E b 59.20 ± 4.54 E a

5 41.69 ± 1.23 D b 49.99 ± 4.76 D b 78.30 ± 8.20 D a

6 47.37 ± 2.65 C b 55.77 ± 6.20 BC b 87.05 ± 16.33 BC a

7 48.65 ± 4.50 BC b 56.67 ± 3.22 ABC b 89.34 ± 9.23 AB a

8 51.23 ± 2.22 ABC b 58.45 ± 3.45 ABC b 82.45 ± 6.45 CD a

9 48.40 ± 5.30 C b 53.45 ± 4.64 CD b 89.45 ± 8.37 AB a

10 53.64 ± 2.45 AB b 59.45 ± 2.45 AB b 91.23 ± 9.34 AB a

11 55.83 ± 7.99 A b 60.98 ± 12.34 A b 94.34 ± 4.56 A a

Oligomeric

flavan-3-ol

(mg/L EC eq.)

0 27.99 ± 5.81 I a 17.41 ± 4.47 F a 15.26 ± 9.87 G a

1 30.85 ± 2.93 I a 11.28 ± 1.14 F b 11.18 ± 2.29 G b

2 49.22 ± 2.72 H a 13.49 ± 0.73 F b 28.85 ± 6.99 F b

3 61.31 ± 0.75 G a 20.05 ± 11.16 F b 45.03 ± 1.32 E a

4 77.58 ± 1.10 F a 39.22 ± 6.85 E b 52.02 ± 3.56 E b

5 96.16 ± 3.34 E a 45.05 ± 6.12 E c 75.50 ± 7.22 D b

6 123.38 ± 2.55 D a 63.35 ± 6.18 D c 82.65 ± 13.44 D b

7 128.42 ± 15.60 D a 77.31 ± 4.23 C c 96.45 ± 11.20 C b

123

8 139.99 ± 13.67 C a 87.34 ± 2.34 BC b 98.39 ± 4.34 C b

9 144.27 ± 22.24 C a 93.45 ± 5.34 B b 109.34 ± 9.39 B b

10 165.77 ± 22.76 B a 109.34 ± 2.34 A b 119.98 ± 2.39 AB b

11 189.50 ± 27.35 A a 112.88 ± 5.39 A b 125.90 ± 19.30 A b

Polymeric

flavan-3-ol

(mg/L EC eq.)

0 54.25 ± 13.04 E b 68.74 ± 6.47 B b 101.74 ± 7.27 AB a

1 37.37 ± 9.52 F c 61.92 ± 10.86 BC b 84.48 ± 24.21 DE a

2 39.72 ± 5.98 F ab 28.06 ± 4.12 F b 53.41 ± 13.40 G a

3 42.69 ± 6.66 F ab 29.62 ± 5.78 EF b 54.35 ± 9.32 G a

4 60.39 ± 9.32 E ab 36.74 ± 0.82 E b 58.89 ± 8.72 G a

5 55.99 ± 0.00 E a 49.67 ± 9.90 D a 61.34 ± 12.90 G a

6 95.66 ± 0.00 C a 54.83 ± 5.58 CD b 60.50 ± 19.67 G b

7 86.64 ± 11.33 D a 65.60 ± 0.10 B b 71.12 ± 3.24 F ab

8 103.76 ± 12.22 BC a 69.09 ± 2.46 B b 79.34 ± 4.34 EF b

9 79.78 ± 14.65 D a 78.90 ± 6.78 A a 88.34 ± 8.45 CD a

10 113.94 ± 0.00 A a 82.34 ± 9.23 A b 95.43 ± 10.58 BC b

11 105.85 ± 17.56 AB a 84.34 ± 5.90 A b 109.12 ± 7.77 A a

Polysaccharide

(mg/L

galactose eq.)

0 439.81 ± 193.58 FG a 476.99 ± 59.75 G a 279.95 ± 85.20 F a

1 364.41 ± 90.58 G b 582.51 ± 79.74 G ab 698.31 ± 130.93 E a

2 567.43 ± 98.98 E b 956.24 ± 227.77 F a 974.35 ± 190.97 DE a

3 831.20 ± 97.53 BC b 1200.11 ± 204.38 E a 1095.49 ± 215.73 CDE ab

4 603.54 ± 17.94 E b 1201.87 ± 171.89 E a 1056.72 ± 198.98 BCD a

5 555.09 ± 121.23 EF c 1432.90 ± 123.34 A a 1160.70 ± 123.37 AB b

6 626.73 ± 17.47 DE b 1672.93 ± 249.02 CD a 1077.73 ± 137.21 ABCD a

7 1009.34 ± 107.47 A b 1411.50 ± 232.90 A a 1110.13 ± 156.40 ABC b

8 745.67 ± 76.54 CD c 1567.90 ± 301.87 AB a 1190.34 ± 130.44 A b

9 751.49 ± 111.09 C b 1459.98 ± 274.98 BC a 990.45 ± 120.40 CDE b

10 921.65 ± 116.29 AB b 1321.37 ± 201.85 DE a 989.45 ± 198.40 CDE ab

124

Ethanol

(%, v/v)

0 0.31 ± 0.53 A a 0.00 ± 0.00 A a 0.00 ± 0.00 A a

1 1.52 ± 0.64 B a 0.98 ± 0.36 A a 2.67 ± 1.00 B a

2 5.11 ± 0.69 C c 7.30 ± 0.97 B b 9.09 ± 0.45 C a

3 8.17 ± 0.80 D c 11.13 ± 0.53 C b 13.31 ± 0.51 D a

4 10.56 ± 0.55 E b 13.57 ± 0.72 D a 14.22 ± 0.72 E a

5 13.39 ± 0.00 F b 13.89 ± 0.54 D b 15.11 ± 0.49 EF a

6 13.59 ± 0.45 FG b 14.07 ± 0.82 D ab 15.22 ± 0.38 EF a

7 14.35 ± 0.68 G b 13.80 ± 0.00 D b 15.33 ± 0.23 F a

8 15.10 ± 0.44 G a 13.89 ± 0.10 D b 15.18 ± 0.34 EF a

9 15.57 ± 0.00 H a 13.72 ± 0.11 D b 15.34 ± 0.45 EF a

10 15.64 ± 0.00 I a 13.92 ± 0.00 D b 15.64 ± 0.12 F a

11 15.56 ± 0.04 G a 13.99 ± 0.00 D c 15.22 ± 0.22 EF b

1 Values on the same row (lower-case letters) and the same column (capital letters) followed by different letters are significantly different according to Tuckey’s honest significance


125

Références bibliographiques

Agarwal, P., & Agarwal, P. K. (2014). Pathogenesis related-10 proteins are small, structurally similar but with diverse role in stress signaling. Molecular biology reports, 41(2), 599-611. doi:10.1007/s11033-013-2897-4

Alcalde-Eon, C., Escribano-Bailón, M. T., Santos-Buelga, C., & Rivas-Gonzalo, J. C. (2006). Changes in the detailed pigment composition of red wine during maturity and ageing: a comprehensive study. Analytica Chimica Acta, 563(1), 238-254.

Amrani, J., & Mercierz, M. (1994). Localisation des tanins dans la pellicule de baie de raisin. Vitis, 33, 133-138.

Amrani Joutei, K., & Glories, Y. (1995). Tanins et anthocyanes: localisation dans la baie de raisin et mode d'extraction. Revue française d'oenologie, 35(153), 28-31.

Apolinar-Valiente, R., Gomez-Plaza, E., Terrier, N., Doco, T., & Ros-Garcia, J. M. (2017). The composition of cell walls from grape skin in Vitis vinifera intraspecific hybrids. Journal of the Science of Food and Agriculture, 97(12), 4029-4035. doi:10.1002/jsfa.8270

Auw, J., Blanco, V., O'keefe, S., & Sims, C. A. (1996). Effect of processing on the phenolics and color of Cabernet Sauvignon, Chambourcin, and Noble wines and juices. American Journal of Enology and Viticulture, 47(3), 279-286.

Bajec, M. R., & Pickering, G. J. (2008). Astringency: mechanisms and perception. Critical Reviews in Food Science and Nutrition, 48(9), 858-875. doi:10.1080/10408390701724223

Balík, J., Kumšta, M., & Rop, O. (2013). Comparison of anthocyanins present in grapes of Vitis vinifera L. varieties and interspecific hybrids grown in the Czech Republic. Chemical Papers, 67(10), 1285-1292.

Bate Smith, E., & Swain, T. (1965). Recent developments in the chemotaxonomy of flavonoid compounds. Lloydia, 28, 313-331.

Bautista-Ortín, A. B., Martínez-Hernández, A., Ruiz-García, Y., Gil-Muñoz, R., & Gómez-Plaza, E. (2016). Anthocyanins influence tannin–cell wall interactions. Food Chemistry, 206, 239-248.

Bindon, K., Kassara, S., & Smith, P. (2017). Towards a model of grape tannin extraction under wine‐like conditions: The role of suspended mesocarp material and anthocyanin concentration. Australian Journal of Grape and Wine Research, 23(1), 22-32.

Bindon, K., Li, S., Kassara, S., & Smith, P. (2016). Retention of proanthocyanidin in wine-like solution is conferred by a dynamic interaction between soluble and insoluble grape cell wall components. Journal of Agricultural and Food Chemistry, 64(44), 8406-8419.

Blouin, J., & Cruège, J. (2013). Analyse et composition des vins: comprendre le vin: La Vigne. Boulton, R. (1998). Estimation of copigmented anthocyanin content in red wines. Personal

correspondence. Boulton, R. (2001). The copigmentation of anthocyanins and its role in the color of red wine: A critical

review. American Journal of Enology and Viticulture, 52(2), 67-87. Brandão, E., Silva, M. S., García-Estévez, I., Williams, P., Mateus, N., Doco, T., . . . Soares, S.

(2017). The role of wine polysaccharides on salivary protein-tannin interaction: A molecular approach. Carbohydrate Polymers, 177, 77-85.

Brooks, L., McCloskey, L., Mckesson, D., & Sylvan, M. (2008). Adams-Harbertson protein precipitation-based wine tannin method found invalid. Journal of AOAC International, 91(5), 1090-1094.

Brossaud, F., Cheynier, V., & Noble, A. (2001). Bitterness and astringency of grape and wine polyphenols. Australian Journal of Grape and Wine Research, 7(1), 33-39.

126

Burtch, C. E., Mansfield, A. K., & Manns, D. C. (2017). Reaction kinetics of monomeric anthocyanin conversion to polymeric pigment and significance to color in interspecific hybrid wines. Journal of Agricultural and Food Chemistry.

Carbonneau, A., & Escudier, J. (2017). De l'œnologie à la viticulture: Quae. Carpita, N. C., & Gibeaut, D. M. (1993). Structural models of primary cell walls in flowering plants:

consistency of molecular structure with the physical properties of the walls during growth. The Plant Journal, 3(1), 1-30.

Carrau, F. M., Medina, K., Farina, L., Boido, E., Henschke, P. A., & Dellacassa, E. (2008). Production of fermentation aroma compounds by Saccharomyces cerevisiae wine yeasts: effects of yeast assimilable nitrogen on two model strains. FEMS yeast research, 8(7), 1196-1207. doi:10.1111/j.1567-1364.2008.00412.x

Carvalho, E., Mateus, N., Plet, B., Pianet, I., Dufourc, E., & De Freitas, V. (2006). Influence of wine pectic polysaccharides on the interactions between condensed tannins and salivary proteins. Journal of Agricultural and Food Chemistry, 54(23), 8936-8944.

Casassa, L. F., & Harbertson, J. F. (2014). Extraction, evolution, and sensory impact of phenolic compounds during red wine maceration. Annual Review of Food Science and Technology, 5, 83-109.

Cejudo-Bastante, M. J., Rodriguez-Morgado, B., Jara-Palacios, M. J., Rivas-Gonzalo, J. C., Parrado, J., & Heredia, F. J. (2016). Pre-fermentative addition of an enzymatic grape seed hydrolysate in warm climate winemaking. Effect on the differential colorimetry, copigmentation and polyphenolic profiles. Food Chemistry, 209, 348-357. doi:10.1016/j.foodchem.2016.04.092

Chen, K., Escott, C., Loira, I., Del Fresno, J. M., Morata, A., Tesfaye, W., . . . Suarez-Lepe, J. A. (2016). The effects of pre-fermentative addition of oenological tannins on wine components and sensorial qualities of red wine. Molecules, 21(11), 1445. doi:10.3390/molecules21111445

Cheynier, V., Souquet, J., Fulcrand, H., Sarni, P., & Moutounet, M. (1998). Stabilisation tanins-anthocyanes, données générales. Paper presented at the Workshop ITV.

Chira, K., Suh, J., Saucier, C., & Teissèdre, P. (2008). Les polyphénols du raisin. Phytothérapie, 6(2), 75-82.

Chisholm, M. G., Guiher, L. A., Vonah, T. M., & Beaumont, J. L. (1994). Comparison of some French-American hybrid wines with White Riesling using gas chromatography-olfactometry. American Journal of Enology and Viticulture, 45(2), 201-212.

Del Rio, J., & Kennedy, J. (2006). Development of proanthocyanidins in Vitis vinifera L. cv. Pinot noir grapes and extraction into wine. American Journal of Enology and Viticulture, 57(2), 125-132.

Diakou, P., & Carde, J. P. (2001). In situ fixation of grape berries. Protoplasma, 218(3-4), 225-235. Dols-Lafargue, M., Gindreau, E., Le Marrec, C., Chambat, G., Heyraud, A., & Lonvaud-Funel, A.

(2007). Changes in red wine soluble polysaccharide composition induced by malolactic fermentation. Journal of Agriculture and Food Chemistry, 55(23), 9592-9599. doi:10.1021/jf071677+

Dubé, G., & Pedneault, K. (2014). Le Québec en mode vinicole. Fruits Oubliés, 61, 9-18. Dubé, G., & Turcotte, I. (2011). Guide d'identification des cépages cultivés en climat froid: cépages

de cuve: Richard Grenier. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. t., & Smith, F. (1956). Colorimetric method for

determination of sugars and related substances. Analytical Chemistry, 28(3), 350-356. Ducasse, M.-A., Canal-Llauberes, R.-M., de Lumley, M., Williams, P., Souquet, J.-M., Fulcrand, H., . .

. Cheynier, V. (2010). Effect of macerating enzyme treatment on the polyphenol and polysaccharide composition of red wines. Food Chemistry, 118(2), 369-376.

127

Dufrechou, M., Poncet-Legrand, C. l., Sauvage, F. o.-X., & Vernhet, A. (2012). Stability of white wine proteins: combined effect of pH, ionic strength, and temperature on their aggregation. Journal of Agricultural and Food Chemistry, 60(5), 1308-1319.

Ehrhardt, C., Arapitsas, P., Stefanini, M., Flick, G., & Mattivi, F. (2014). Analysis of the phenolic composition of fungus‐resistant grape varieties cultivated in Italy and Germany using UHPLC‐MS/MS. Journal of Mass Spectrometry, 49(9), 860-869.

Fennell, A. (2004). Freezing tolerance and injury in grapevines. Journal of Crop Improvement, 10(1-2), 201-235.

Ferreira, R. B., Monteiro, S., Piçarra-Pereira, M. A., Tanganho, M. C., Loureiro, V. B., & Teixeira, A. R. (2000). Characterization of the proteins from grapes and wines by immunological methods. American Journal of Enology and Viticulture, 51(1), 22-28.

Ferreira, R. B., Piçarra-Pereira, M. A., Monteiro, S., Loureiro, V. l. B., & Teixeira, A. R. (2001). The wine proteins. Trends in Food Science & Technology, 12(7), 230-239.

Flancy, E. (1998). Oenologie, Fondements scientifiques et technologiques, Ed. Lavoisier, París, 134-145.

Fougère-Rifot, M., & Cholet, C. (1996). Evolution des parois des cellules de l'hypoderme de la baie de raisin lors de leur transformation en cellules de pulpe. Journal International des Sciences de la Vigne et du Vin, 30, 47-51.

Gagné. (2016). Étude des composés phénoliques chez les cépages hybrides interspécifiques cultivés au Québec pour la production vinicole.

Gagné, Saucier, C., & Gény, L. (2006). Composition and cellular localization of tannins in Cabernet Sauvignon skins during growth. Journal of Agricultural and Food Chemistry, 54(25), 9465-9471.

Gambuti, A., Capuano, R., Lisanti, M. T., Strollo, D., & Moio, L. (2010). Effect of aging in new oak, one-year-used oak, chestnut barrels and bottle on color, phenolics and gustative profile of three monovarietal red wines. European Food Research and Technology, 231(3), 455-465.

García‐Lomillo, J., & González‐SanJosé, M. L. (2017). Applications of wine pomace in the food industry: Approaches and functions. Comprehensive Reviews in Food Science and Food Safety, 16(1), 3-22.

Garcia, S., Santesteban, L., Miranda, C., & Royo, J. (2011). Variety and storage time affect the compositional changes that occur in grape samples after frozen storage. Australian Journal of Grape and Wine Research, 17(2), 162-168.

Gawel, R. (1998). Red wine astringency: A review. Australian Journal of Grape and Wine Research, 4(2), 74-95.

Gawel, R., Schulkin, A., Smith, P., Kassara, S., Francis, L., Herderich, M., & Johnson, D. (2018). Influence of wine polysaccharides on white and red wine mouthfeel. Wine & Viticulture Journal, 33(1), 34.

González-Manzano, S., Santos-Buelga, C., Pérez-Alonso, J., Rivas-Gonzalo, J., & Escribano-Bailón, M. (2006). Characterization of the mean degree of polymerization of proanthocyanidins in red wines using liquid chromatography-mass spectrometry (LC-MS). Journal of Agricultural and Food Chemistry, 54(12), 4326-4332.

Gordillo, B., Cejudo-Bastante, M. J., Rodriguez-Pulido, F. J., Jara-Palacios, M. J., Ramirez-Perez, P., Gonzalez-Miret, M. L., & Heredia, F. J. (2014). Impact of adding white pomace to red grapes on the phenolic composition and color stability of Syrah wines from a warm climate. Journal of Agricultural and Food Chemistry, 62(12), 2663-2671. doi:10.1021/jf405574x

Goulao, L. F., & Oliveira, C. M. (2008). Cell wall modifications during fruit ripening: when a fruit is not the fruit. Trends in Food Science & Technology, 19(1), 4-25.

128

Gros, C., & Yerle, S. (2014). Guide pratique de la vinification en rouge-2e éd: Dunod. Guadalupe, & Ayestaran, B. (2007). Polysaccharide profile and content during the vinification and

aging of Tempranillo red wines. Journal of Agricultural and Food Chemistry, 55(26), 10720-10728. doi:10.1021/jf0716782

Guadalupe, Z., & Ayestarán, B. (2007). Polysaccharide profile and content during the vinification and aging of Tempranillo red wines. Journal of Agricultural and Food Chemistry, 55(26), 10720-10728.

Habekost, M. (2013). Which color differencing equation should be used. International Circular of Graphic Education and Research, 6, 20-33.

Hanlin, R., Hrmova, M., Harbertson, J., & Downey, M. (2010). Review: Condensed tannin and grape cell wall interactions and their impact on tannin extractability into wine. Australian Journal of Grape and Wine Research, 16(1), 173-188.

Harbertson, J. F., Hodgins, R. E., Thurston, L. N., Schaffer, L. J., Reid, M. S., Landon, J. L., . . . Adams, D. O. (2008). Variability of tannin concentration in red wines. American Journal of Enology and Viticulture, 59(2), 210-214.

Harbertson, J. F., Kennedy, J. A., & Adams, D. O. (2002). Tannin in skins and seeds of Cabernet Sauvignon, Syrah, and Pinot noir berries during ripening. American Journal of Enology and Viticulture, 53(1), 54-59.

Harbertson, J. F., Kilmister, R. L., Kelm, M. A., & Downey, M. O. (2014). Impact of condensed tannin size as individual and mixed polymers on bovine serum albumin precipitation. Food Chemistry, 160, 16-21.

Harbertson, J. F., Parpinello, G. P., Heymann, H., & Downey, M. O. (2012). Impact of exogenous tannin additions on wine chemistry and wine sensory character. Food Chemistry, 131(3), 999-1008.

He, F., Liang, N.-N., Mu, L., Pan, Q.-H., Wang, J., Reeves, M. J., & Duan, C.-Q. (2012a). Anthocyanins and their variation in red wines I. Monomeric anthocyanins and their color expression. Molecules, 17(2), 1571-1601.

He, F., Liang, N.-N., Mu, L., Pan, Q.-H., Wang, J., Reeves, M. J., & Duan, C.-Q. (2012b). Anthocyanins and their variation in red wines II. Anthocyanin derived pigments and their color evolution. Molecules, 17(2), 1483-1519.

Hemingway, R. W., Foo, L. Y., & Porter, L. J. (1982). Linkage isomerism in trimeric and polymeric 2,3-cis-procyanidins. Journal of the Chemical Society, Perkin Transactions 1, 1209-1216.

Herderich, M., & Smith, P. (2005). Analysis of grape and wine tannins: Methods, applications and challenges. Australian Journal of Grape and Wine Research, 11(2), 205-214.

Jackson, R. S. (2008). Wine science: Principles and applications: Academic press. Jensen, J. S., Werge, H. H. M., Egebo, M., & Meyer, A. S. (2008). Effect of wine dilution on the

reliability of tannin analysis by protein precipitation. American Journal of Enology and Viticulture, 59(1), 103-105.

Kallithraka, S., Kim, D., Tsakiris, A., Paraskevopoulos, I., & Soleas, G. (2011). Sensory assessment and chemical measurement of astringency of Greek wines: Correlations with analytical polyphenolic composition. Food Chemistry, 126(4), 1953-1958. doi:10.1016/j.foodchem.2010.12.045

Kamas, J., Stein, L., & Nesbitt, M. (2010). Pierce’s Disease-Tolerant Grapes. Texas Fruit and Nut. Kassara, S., & Kennedy, J. A. (2011). Relationship between red wine grade and phenolics. 2. Tannin

composition and size. Journal of Agricultural and Food Chemistry, 59(15), 8409-8412. Keller, M. (2015). The science of grapevines: Anatomy and physiology: Academic Press. Kennedy, J. A. (2002). Understanding grape berry development. Practical Winery & Vineyard, 4, 1-5.

129

Kennedy, J. A., Ferrier, J., Harbertson, J. F., & des Gachons, C. P. (2006). Analysis of tannins in red wine using multiple methods: Correlation with perceived astringency. American Journal of Enology and Viticulture, 57(4), 481-485.

Kennedy, J. A., Matthews, M. A., & Waterhouse, A. L. (2000). Changes in grape seed polyphenols during fruit ripening. Phytochemistry, 55(1), 77-85.

Kennedy, J. A., Saucier, C., & Glories, Y. (2006). Grape and wine phenolics: history and perspective. American Journal of Enology and Viticulture, 57(3), 239-248.

Kilmister, R. L., Mazza, M., Baker, N. K., Faulkner, P., & Downey, M. O. (2014). A role for anthocyanin in determining wine tannin concentration in Shiraz. Food Chemistry, 152, 475-482. doi:10.1016/j.foodchem.2013.12.007

Koolman, J., Röhm, K.-H., Wirth, J., & Robertson, M. (2005). Color atlas of biochemistry (Vol. 2): Thieme Stuttgart.

Kyraleou, M., Kallithraka, S., Chira, K., Tzanakouli, E., Ligas, I., & Kotseridis, Y. (2015). Differentiation of wines treated with wood chips based on their phenolic content, volatile composition, and sensory parameters. Journal of Food Science, 80(12), C2701-C2710.

Kyraleou, M., Tzanakouli, E., Kotseridis, Y., Chira, K., Ligas, I., Kallithraka, S., & Teissedre, P.-L. (2016). Addition of wood chips in red wine during and after alcoholic fermentation: Differences in color parameters, phenolic content and volatile composition. OENO One, 50(4).

Lankhorst, P. P., Voogt, B., Tuinier, R., Lefol, B., Pellerin, P., & Virone, C. (2017). Prevention of Tartrate Crystallization in Wine by Hydrocolloids: The Mechanism Studied by Dynamic Light Scattering. Journal of Agricultural and Food Chemistry, 65(40), 8923-8929. doi:10.1021/acs.jafc.7b01854

Lazarus, S. A., Hammerstone, J. F., Adamson, G. E., & Schmitz, H. H. (2001). High-performance liquid chromatography/mass spectrometry analysis of proanthocyanidins in food and beverages. Methods in Enzymology, 335, 46-57.

Le Bourvellec, C., & Renard, C. (2012). Interactions between polyphenols and macromolecules: Quantification methods and mechanisms. Critical Reviews in Food Science and Nutrition, 52(3), 213-248.

Lee, C., Robinson, W., Van Buren, J., Acree, T., & Stoewsand, G. (1975). Methanol in wines in relation to processing and variety. American Journal of Enology and Viticulture, 26(4), 184-187.

Li, J. C., Li, S. Y., He, F., Yuan, Z. Y., Liu, T., Reeves, M. J., & Duan, C. Q. (2016). Phenolic and chromatic properties of beibinghong red ice wine during and after vinification. Molecules, 21(4), 431. doi:10.3390/molecules21040431

Liu, J.-J., & Ekramoddoullah, A. K. (2006). The family 10 of plant pathogenesis-related proteins: their structure, regulation, and function in response to biotic and abiotic stresses. Physiological and Molecular Plant Pathology, 68(1-3), 3-13.

Liu, J., Toldam-Andersen, T. B., Petersen, M. A., Zhang, S., Arneborg, N., & Bredie, W. L. (2015). Instrumental and sensory characterisation of Solaris white wines in Denmark. Food Chem, 166, 133-142. doi:10.1016/j.foodchem.2014.05.148

Liu, L., & Li, H. (2013). Research progress in amur grape, Vitis amurensis Rupr. Canadian Journal of Plant Science, 93(4), 565-575.

Londo, J. P., & Kovaleski, A. P. (2017). Characterization of wild North American grapevine cold hardiness using differential thermal analysis. American Journal of Enology and Viticulture, 68(2), 203-212.

130

Lorrain, B., Ky, I., Pechamat, L., & Teissedre, P. (2013). Evolution of analysis of polyhenols from grapes, wines, and extracts. Molecules, 18(1), 1076-1100.

Ma, W., Guo, A., Zhang, Y., Wang, H., Liu, Y., & Li, H. (2014). A review on astringency and bitterness perception of tannins in wine. Trends in Food Science & Technology, 40(1), 6-19.

Ma, Y., Tang, K., Xu, Y., & Li, J.-m. (2017). Characterization of the key aroma compounds in chinese Vidal icewine by gas chromatography–olfactometry, quantitative measurements, aroma recombination and omission tests. Journal of agricultural and food chemistry.

Manns, D. C., Coquard Lenerz, C., & Mansfield, A. K. (2013). Impact of processing parameters on the phenolic profile of wines produced from hybrid red grapes Maréchal Foch, Corot noir, and Marquette. Journal of Food Science, 78(5), C696-C702.

Martí, M. P., Pantaleón, A., Rozek, A., Soler, A., Valls, J., Macià, A., . . . Motilva, M. J. (2010). Rapid analysis of procyanidins and anthocyanins in plasma by microelution SPE and ultra‐HPLC. Journal of Separation Science, 33(17‐18), 2841-2853.

Martínez, J., Melgosa, M., Pérez, M., Hita, E., & Negueruela, A. (2001). Note. Visual and instrumental color evaluation in red wines. Food Science and Technology International, 7(5), 439-444.

Maury, C., Madieta, E., Le Moigne, M., Mehinagic, E., Siret, R., & Jourjon, F. (2009). Development of a mechanical texture test to evaluate the ripening process of Cabernet Franc grapes. Journal of Texture Studies, 40(5), 511-535.

McRae, J. M., & Kennedy, J. A. (2011). Wine and grape tannin interactions with salivary proteins and their impact on astringency: A review of current research. Molecules, 16(3), 2348-2364.

McRae, J. M., Schulkin, A., Kassara, S., Holt, H. E., & Smith, P. A. (2013). Sensory properties of wine tannin fractions: Implications for in-mouth sensory properties. Journal of Agricultural and Food Chemistry, 61(3), 719-727.

Mercurio, M. D., Dambergs, R. G., Cozzolino, D., Herderich, M. J., & Smith, P. A. (2010). Relationship between red wine grades and phenolics. 1. Tannin and total phenolics concentrations. Journal of Agricultural and Food Chemistry, 58(23), 12313-12319.

Monagas, M., Gomez-Cordoves, C., Bartolome, B., Laureano, O., & Ricardo da Silva, J. M. (2003). Monomeric, oligomeric, and polymeric flavan-3-ol composition of wines and grapes from Vitis vinifera L. Cv. Graciano, Tempranillo, and Cabernet Sauvignon. Journal of Agricultural and Food Chemistry, 51(22), 6475-6481. doi:10.1021/jf030325+

Moreno-Arribas, M. V., & Polo, M. C. (2009). Wine chemistry and biochemistry (Vol. 233): Springer. Mullins, M., Bouquet, A., & Williams, L. (1992). Biology of the grapevine: Cambridge University Press. Nicolle, P., Marcotte, C., Angers, P., & Pedneault, K. (2018). Co-fermentation of red grapes and white

pomace: A natural and economical process to modulate hybrid wine composition. Food Chemistry, 242, 481-490. doi:10.1016/j.foodchem.2017.09.053

Nicolle, P., Marcotte, C., Angers, P., & Pedneault, K. (2019). Pomace limits tannin retention in Frontenac wines. Food Chemistry, 277, 438-447. doi:10.1016/j.foodchem.2018.10.116

Ortega-Regules, A., Romero-Cascales, I., Ros-García, J., López-Roca, J., & Gómez-Plaza, E. (2006). A first approach towards the relationship between grape skin cell-wall composition and anthocyanin extractability. Analytica Chimica Acta, 563(1-2), 26-32.

Pedneault, Dorais, & Angers. (2013). Flavor of cold-hardy grapes: Impact of berry maturity and environmental conditions. Journal of Agricultural and Food Chemistry, 61(44), 10418-10438.

Pedneault, Dubé, G., & Turcotte, I. (2011). L'évaluation de la maturité du raisin par analyse sensorielle : un outil d'aide à la décision.

Pedneault, & Provost, C. (2016). Fungus resistant grape varieties as a suitable alternative for organic wine production: Benefits, limits, and challenges. Scientia Horticulturae.

131

Pedneault, K., Dorais, M., & Angers, P. (2013). Flavor of Cold-Hardy Grapes: Impact of Berry Maturity and Environmental Conditions. Journal of Agricultural and Food Chemistry, 61(44), 10418-10438. doi:10.1021/jf402473u

Pedroza, M. A., Carmona, M., Alonso, G. L., Salinas, M. R., & Zalacain, A. (2013). Pre-bottling use of dehydrated waste grape skins to improve colour, phenolic and aroma composition of red wines. Food Chemistry, 136(1), 224-236.

Peleg, H., Gacon, K., Schlich, P., & Noble, A. C. (1999). Bitterness and astringency of flavan-3-ol monomers, dimers and trimers. Journal of the Science of Food and Agriculture, 79(8), 1123-1128.

Pinelo, M., Arnous, A., & Meyer, A. (2006). Upgrading of grape skins: Significance of plant cell-wall structural components and extraction techniques for phenol release. Trends in Food Science & Technology, 17(11), 579-590.

Poncet-Legrand, C., Cartalade, D., Putaux, J.-L., Cheynier, V., & Vernhet, A. (2003). Flavan-3-ol aggregation in model ethanolic solutions: Incidence of polyphenol structure, concentration, ethanol content, and ionic strength. Langmuir, 19(25), 10563-10572.

Prior, R. L., & Gu, L. (2005). Occurrence and biological significance of proanthocyanidins in the American diet. Phytochemistry, 66(18), 2264-2280. doi:10.1016/j.phytochem.2005.03.025

Quijada-Morín, N., Williams, P., Rivas-Gonzalo, J. C., Doco, T., & Escribano-Bailón, M. T. (2014). Polyphenolic, polysaccharide and oligosaccharide composition of Tempranillo red wines and their relationship with the perceived astringency. Food Chemistry, 154, 44-51.

Ribéreau-Gayon, Dubourdieu, Donèche, & Lonvaud. (2006). Handbook of enology. The microbiology of wine and vinifications (Vol. 1): John Wiley & Sons.

Ribéreau-Gayon, Glories, Maujean, & Dubordieu. (2006). Handbook of enology. The chemistry of wine: Stabilization and treatments (Volume 2): John Wiley & Sons Ltd., West Sussex, UK.

Rihan, H. Z., Al-Issawi, M., & Fuller, M. P. (2017). Advances in physiological and molecular aspects of plant cold tolerance. Journal of Plant Interactions, 12(1), 143-157.

Riou, V., Vernhet, A., Doco, T., & Moutounet, M. (2002). Aggregation of grape seed tannins in model wine effect of wine polysaccharides. Food Hydrocolloids, 16(1), 17-23.

Robin, J., Abbal, P., & Salmon, J. (1997). Firmness and grape berry maturation. Definition of different rheological parameters during the ripening. Journal International des Sciences de la Vigne et du Vin (France).

Sacchi, K. L., Bisson, L. F., & Adams, D. O. (2005). A review of the effect of winemaking techniques on phenolic extraction in red wines. American Journal of Enology and Viticulture, 56(3), 197-206.

Sáenz-Navajas, M.-P., Avizcuri, J.-M., Ballester, J., Fernández-Zurbano, P., Ferreira, V., Peyron, D., & Valentin, D. (2015). Sensory-active compounds influencing wine experts' and consumers' perception of red wine intrinsic quality. LWT-Food Science and Technology, 60(1), 400-411.

Sánchez-Gómez, R., Zalacain, A., Pardo, F., Alonso, G., & Salinas, M. (2017). Moscatel vine-shoot extracts as a grapevine biostimulant to enhance wine quality. Food Research International, 98, 40-49.

Sarneckis, C. J., Dambergs, R., Jones, P., Mercurio, M., Herderich, M. J., & Smith, P. (2006). Quantification of condensed tannins by precipitation with methyl cellulose: development and validation of an optimised tool for grape and wine analysis. Australian Journal of Grape and Wine Research, 12(1), 39-49.

Sarni-Manchado, P., Cheynier, V., & Moutounet, M. (1999). Interactions of grape seed tannins with salivary proteins. Journal of Agricultural and Food Chemistry, 47(1), 42-47.

132

Sauvage, F.-X., Bach, B., Moutounet, M., & Vernhet, A. (2010). Proteins in white wines: Thermo-sensitivity and differential adsorbtion by bentonite. Food Chemistry, 118(1), 26-34.

Saxton, A. (1998). A macro for converting mean separation output to letter groupings in Proc Mixed. Proceedings of the 23rd SAS Users Group International, 22-25 Mar 1998, Nashville, 1243-1246.

Scheller, H. V., Jensen, J. K., Sørensen, S. O., Harholt, J., & Geshi, N. (2007). Biosynthesis of pectin. Physiologia Plantarum, 129(2), 283-295.

Scollary, G. R., Pásti, G., Kállay, M., Blackman, J., & Clark, A. C. (2012). Astringency response of red wines: Potential role of molecular assembly. Trends in Food Science & Technology, 27(1), 25-36.

Segarra, I., Lao, C., López-Tamames, E., & De La Torre-Boronat, M. C. (1995). Spectrophotometric methods for the analysis of polysaccharide levels in winemaking products. American Journal of Enology and Viticulture, 46(4), 564-570.

Singh, R., Tiwari, J., Sharma, V., Singh, B., Rawat, S., & Singh, R. (2014). Role of pathogen related protein families in defence mechanism with potential role in applied biotechnology. International Journal of Advanced Research, 2(8), 210-226.

Slegers, Angers, P., Ouellet, É., Truchon, T., & Pedneault, K. (2015). Volatile compounds from grape skin, juice and wine from five interspecific hybrid grape cultivars grown in Québec (Canada) for wine production. Molecules, 20(6), 10980-11016.

Slegers, Angers , P., & Pedneault, K. (2017). Volatile Compounds from Must and Wines from Five White Grape Varieties. Journal of Food Chemistry & Nanotechnology, 3(1), 8 - 18.

Smith, P., McRae, J., & Bindon, K. (2015). Impact of winemaking practices on the concentration and composition of tannins in red wine. Australian Journal of Grape and Wine Research, 21(S1), 601-614.

Soares, S., Brandão, E., Mateus, N., & De Freitas, V. (2017). Sensorial properties of red wine polyphenols: astringency and bitterness. Critical Reviews in Food Science and Nutrition, 57(5), 937-948.

Souquet, J., Veran, F., Mané, C., & Cheynier, V. (2006). Optimization of extraction conditions on phenolic yields from the different parts of grape clusters. Quantitative distribution of their proanthocyanidins. Paper presented at the XXIII International Conference on Polyphenols, Winnipeg, Manitoba, Canada. Groupe Polyphénols, Bordeaux, France.

Springer, Chen, L. A., Stahlecker, A. C., Cousins, P., & Sacks, G. L. (2016). Relationship of soluble grape-derived proteins to condensed tannin extractability during red wine fermentation. Journal of Agriculture and Food Chemistry, 64(43), 8191-8199. doi:10.1021/acs.jafc.6b02891

Springer, & Sacks. (2014). Protein-precipitable tannin in wines from Vitis vinifera and interspecific hybrid grapes (Vitis ssp.): differences in concentration, extractability, and cell wall binding. Journal of Agricultural and Food Chemistry, 62(30), 7515-7523.

Springer, Sherwood, R. W., & Sacks, G. L. (2016). Pathogenesis-related rroteins limit the retention of condensed tannin additions to red wines. Journal of Agricultural and Food Chemistry, 64(6), 1309-1317. doi:10.1021/acs.jafc.5b04906

Stafne, E. T. (2007). Factors affecting cold hardiness in grapevines. Res. Ext. Bull., Department of Horticulture and Landscape Architecture, Oklahoma State University, Stillwater, OK.

Sun, Gates, M. J., Lavin, E. H., Acree, T. E., & Sacks, G. L. (2011). Comparison of odor-active compounds in grapes and wines from Vitis vinifera and non-foxy American grape species. Journal of Agricultural and Food Chemistry, 59(19), 10657-10664. doi:10.1021/jf2026204

133

Sun, Sa, M. d., Leandro, C. a. o., Caldeira, I., Duarte, F. L., & Spranger, I. (2013). Reactivity of polymeric proanthocyanidins toward salivary proteins and their contribution to young red wine astringency. Journal of Agricultural and Food Chemistry, 61(4), 939-946.

Sun, Sacks, G., Lerch, S., & Heuvel, J. (2011). Impact of shoot thinning and harvest date on yield components, fruit composition, and wine quality of Marechal Foch. Journal of Agricultural and Food Chemistry, 62(1), 32-41.

Sun, Sacks, G., Lerch, S., & Heuvel, J. (2012). Impact of shoot and cluster thinning on yield, fruit composition, and wine quality of Corot noir. Journal of Agricultural and Food Chemistry, 63(1), 49-56.

Testing, A. S. f., & Materials. (1978). Standard definitions of terms relating to sensory evaluation of materials and products.

This, P., Lacombe, T., & Thomas, M. R. (2006). Historical origins and genetic diversity of wine grapes. TRENDS in Genetics, 22(9), 511-519. doi:10.1016/j.tig.2006.07.008

Trotignon, Y. (2015). La France au XXe siècle (Vol. 51): Walter de Gruyter GmbH & Co KG. Van Buren, J., Bertino, J., Einset, J., Remaily, G., & Robinson, W. (1970). A comparative study of the

anthocyanin pigment composition in wines derived from hybrid grapes. American Journal of Enology and Viticulture, 21(3), 117-130.

Versari, A., du Toit, W., & Parpinello, G. (2013). Oenological tannins: A review. Australian Journal of Grape and Wine Research, 19(1), 1-10.

Vidal, S., Courcoux, P., Francis, L., Kwiatkowski, M., Gawel, R., Williams, P., . . . Cheynier, V. (2004). Use of an experimental design approach for evaluation of key wine components on mouth-feel perception. Food Quality and Preference, 15(3), 209-217.

Vidal, S., Francis, L., Noble, A., Kwiatkowski, M., Cheynier, V., & Waters, E. (2004). Taste and mouth-feel properties of different types of tannin-like polyphenolic compounds and anthocyanins in wine. Analytica Chimica Acta, 513(1), 57-65.

Vidal, S., Williams, P., Doco, T., Moutounet, M., & Pellerin, P. (2003). The polysaccharides of red wine: Total fractionation and characterization. Carbohydrate Polymers, 54(4), 439-447.

Wallace, T. C., & Giusti, M. M. (2010). Extraction and normal-phase HPLC-fluorescence-electrospray MS characterization and quantification of procyanidins in cranberry extracts. Journal of Food Science, 75(8), C690-696. doi:10.1111/j.1750-3841.2010.01799.x

Wan, Y., Schwaninger, H., Li, D., Simon, C., Wang, Y., & He, P. (2008). The eco-geographic distribution of wild grape germplasm in China. VITIS-GEILWEILERHOF-, 47(2), 77.

Watrelot, A. A., Schulz, D. L., & Kennedy, J. A. (2017). Wine polysaccharides influence tannin-protein interactions. Food Hydrocolloids, 63, 571-579.

Weber, F., Greve, K., Durner, D., Fischer, U., & Winterhalter, P. (2012). Sensory and chemical characterization of phenolic polymers from red wine obtained by gel permeation chromatography. American Journal of Enology and Viticulture, ajev. 2012.12074.

Winkler, A. (1974). Development and composition of grapes. General viticulture, 138-196. Wolf, T. (2008). Wine grape production guide for eastern North America. Zabadal, T., Dami, I., Goffinet, M., Martinson, T., & Chien, M. (2007). Winter injury to grapevines and

methods of protection. Chien ML: Michigan State University Extension. Zhang, S., Petersen, M. A., Liu, J., & Toldam-Andersen, T. B. (2015). Influence of Pre-Fermentation

Treatments on Wine Volatile and Sensory Profile of the New Disease Tolerant Cultivar Solaris. Molecules, 20(12), 21609-21625. doi:10.3390/molecules201219791

Zoecklein, B. W., Fugelsang, K. C., Gump, B. H., & Nury, F. S. (1990). Phenolic compounds and wine color Production wine analysis (pp. 129-168): Springer.

134

Références internet :

AVQ (Association des Vignerons du Québec). Bilan de l’industrie 2017. URL <

http://vinsduquebec.com/bilan-de-lindustrie/> Accessed 18.10.01.

IFV (Institut Français de la Vigne et du Vin). Suivi de la Fermentation Malolactique ou FML dans les

vins par chromatographie. URL <https://www.vignevinsudouest.com/services-professionnels/

methode-analyse/chromatographie-FML.php> Accessed 17.09.06.

OIV (Organisation Internationale de la Vigne et du Vin). Resolution oeno 24/2004. Determination of

plant proteins in wines and musts. URL <http://www.oiv.int/public/medias/649/oeno-24-2004-

en.pdf> Accessed 18.02.01.

OIV (Organisation Internationale de la Vigne et du Vin). 2017 World Vitiviniculture Situation : OIV

Statiscal Report on World Vitiviniculture. URL < http://www.oiv.int/js/lib/pdfjs/web/viewer.

html?file=/public/medias/5479/oiv-en-bilan-2017.pdf> Accessed 18.10.01.

OIV (Organisation Internationale de la Vigne et du Vin). Conference de presse : Conjoncture

vitivinicole mondiale 2017. URL <http://www.oiv.int/public/medias/6018/conf-rence-de-

presse-oiv-avril-2018-fr.pdf> Accessed 18.10.01.

MAPAQ. (2016). Sommet sur l'alimentation - Cahier thématique.

URL<https://www.mapaq.gouv.qc.ca/fr/Publications/Cahier1_Sommet_Alimentation.pdf>

Accessed 18.11.28.

SAQ. (2016). Rapport annuel 2016.

URL <http://s7d9.scene7.com/is/content/SAQ/rapport-annuel-2015-16-fr> Accessed

17.09.06.

SAQ. (2017). Rapport annuel 2017.

URL <https://s7d9.scene7.com/is/content/SAQ/rapport-annuel-2017-fr> Accessed 18.11.28.

Jean Aubry. (2018). Vins du Québec : des pas de géant en moins de 20 ans ! (1). Journal Le Devoir.

URL <https://www.ledevoir.com/vivre/vin/537253/billet-vins-du-quebec-des-pas-de-geant-en-

moins-de-20-ans-1> Accessed 18.11.28.

Étude des composés impliqués dans la rétention des tanins ...

Documents

Transcript of Étude des composés impliqués dans la rétention des tanins ...