The wine proteins: origin, characteristics and functionality Andrea Curioni
Dipartimento di Biotecnologie Agrarie Centro interdipartimentale per la Ricerca in Viticoltura ed Enologia (CIRVE) Università di Padova
1 The CIRVE campus in Conegliano 2 Protein Structure / Functionality
Aminoacid sequence Protein
Protein structure • Size • Charge • Hydrophobicity Proprieties
Functionality Environment Detectable • pH • Solvent effects • Ionic strength • Temperature • Etc. 3 Proteins in wine
Implications in wine
–Hazing of white wines (negative) –“Mouthfeel” and aroma –Foam volume and stability
The wine proteins Tarragona 2011 4 Protein Haze in wine
Serious quality defect Prevention: Protein removal by bentonite treatments
Bottled wine Flocculation Coagulation Precipitation
Bentonite Other methods? several drawbacks: • Loss of aroma Knowledge is • Cost needed • Waste • …..
The wine proteins Tarragona 2011 5 Wine Proteins: Origin
Where do the wine proteins came from?
The wine proteins Tarragona 2011 6 Wine Proteins: Origin
• The wine proteins derive from Grape (mainly): involved in wine hazing Microorganisms
The wine proteins Tarragona 2011 7 Grape Proteins
• Accumulate after veraison – with sugars
• Low quantity – ≈ hundreds mg/Kg
• heterogeneous - > 300 components
• Few main components Pocock et al. (2000) JAFC 48, 1637
The wine proteins Tarragona 2011 8 The Grape Proteins
similar in all the varieties
Sarry et al., 2004 Proteomics, 4, 201
pH
The wine proteins Tarragona 2011 9 Grape Proteins: Identification by MS
PR-proteins
Sarry et al., 2004 Proteomics, 4, 201 10 The wine proteins Tarragona 2011 Grape Proteins: the main components
Pathogenesis related (PR)-Proteins
– THAUMATIN-LIKE PROTEINS (TLP, PR 5) • ≈ 24 kDa – CHITINASES (PR 3) • ≈ 30 kDa
– Osmotins
– Beta-(1,3)-glucanases
The wine proteins Tarragona 201111 Thaumatin-like Proteins (TLP)
• Antifungal activity
• Expressed mainly in the berry
• Several types – main: VvTL1 (constitutive) – minor : VvTL2 (less present in healthy grapes), . • Tattersall, et al. (1997). Plant Physiology 114, 759; Pocock et al. (2000) • No sweet taste • Peng, et al. (1997) J. Agric. Food Chem., 45, 4639
The wine proteins Tarragona 2011 12 Chitinases
• Up to 13 isoforms (4 basic and 9 acidic) Derckel et al.(1996), Plant Sci. 119, 31
• Chitinolitic activity
• Main: class IV chitinase Robinson et al., 1997 Plant Physiol. 114, 771
Catalytic domain
CHITIN- BINDING DOMAIN The wine proteins Tarragona 201113 Chitinases
reduced Chitinase activity Chitinolytic activity detection after SDS-PAGE of grape berries (1), wine (2) and pomegranate fruit (3) proteins under (A)–(C) reducing and (D)–(F) nonreducing conditions. Gels contained (A), (D) 0.01, (B), (E) 0.05, and (C), (F) 0.10% glycol chitin. In (D)–(E), the arrowheads indicate the chitinase isoform retarded in the presence of glycol chitin.
Vincenzi and Curioni (2005) Electrophoresis, 26, 60
Not reduced Chitinase is active in wine Chitinase can bind chitin
0.01 % 0.05 % 0.10 % Percent chitin in the gel
14 PR-Proteins
Pathogen defense
1. Inducibility by 2. Resistance . pathogens . acidic pH . abiotic stress . solvents . proteolysis
Grape: only in part .Stable PR-Proteins are Constitutive
The wine proteins Tarragona 2011 15 What happens to the grape proteins during winemaking?
The wine proteins Tarragona 2011 16 Proteins and winemaking
Proteins (but not PR-P) 1. Juice extraction •Denaturation (acidic pH) •Degradation (proteases) •Precipitation (tannins) • Low pH only PR-proteins • Grape Proteases resist 2. Fermentation
• Yeast Proteases • Alcohol WINE
The wine proteins Tarragona 2011 17 Proteins in wine: Quantity
Low concentration 10 – 250 mg/L
Large variability
(reported: < 1 mg/L - > 1 g/L).
Are proteins quantified correctly?
The wine proteins Tarragona 2011 18 Quantification by the Smith assay of the protein recovered by the KDS method from different Prosecco and Manzoni Bianco wine samples. Data are Vincenzi et al., AJEV 2005 expressed in BSA equivalents. N Protein concentration wine ° (mg/L) ± SD 1 Proseccoa 14.9 ± 1.9 2 Proseccoa 15.5 ± 1.5 3 Proseccoa 19.7 ± 0.5 4 Proseccoa 15.7 ± 1.6 5 Proseccoa 20.0 ± 0.5 6 Proseccoa 14.2 ± 0.7 7 Proseccoa 12.2 ± 2.6 8 Proseccoa 14.1 ± 1.7 9 Proseccoa 16.9 ± 1.3 10 Proseccoa 14.7 ± 1.8 11 Proseccob 121.5 ± 2.9 12 Incrocio Manzoni 6.0.13a 30.5 ± 3.6 A Commercial bottled wines; 13 Incrocio Manzoni 6.0.13a 30.5 ± 3.6 b wine samples taken before a ± 14 Incrocio Manzoni 6.0.13 26.5 1.9 bentonite fining. 15 Incrocio Manzoni 6.0.13a 176.1 ± 9.3 16 Incrocio Manzoni 6.0.13b 328.0 ± 40.5 The wine proteins Tarragona 2011 19 Grape/Juice vs Wine
Grape
WineWine
Marangon et al. (2009) JAFC, 57, 4415
2D-PAGEThe wine of proteinswine proteins (cv. Manzoni Bianco) (Polesani, 2004, unpublished). Tarragona 2011 20 Wine Proteins: Preparative Purification
1. Cation exchange chromatography 2. Hydrophobic interaction chromatography (SCX) for Chardonnay wine. (HIC, Phenyl Sepharose)
SDS-PAGE bands used for MS identification
SDS-PAGE and RP-HPLC profile of purified proteins Gazzola et al., unpublished21 Identification by Nano LC-MS/MS
band sequence protein
PREDICTED: Vitis vinifera class IV chitinase (CHI4D), C 1-2 Class IV chitinase [Vitis vinifera] mRNA
LOC100232841, PREDICTED: Vitis vinifera VVTL1 C 4 VVTL1 [Vitis vinifera] (LOC100232841), mRNA
LOC100256970, PREDICTED: Vitis vinifera hypothetical C 6-7 α Vacuolar invertase 1, [Vitis Vinifera]. protein LOC100256970 (LOC100256970), mRNA
LOC100256970, PREDICTED: Vitis vinifera hypothetical C 6-7 β Vacuolar invertase 1, [Vitis vinifera]. protein LOC100256970 (LOC100256970), mRNA
PREDICTED: Vitis vinifera thaumatin-like protein (TL3), C 6-7 γ Thaumatin-like protein [Vitis vinifera] mRNA
D 1-2-3- LOC100232841, PREDICTED: Vitis vinifera VVTL1 VVTL1 [Vitis vinifera]. 4 (LOC100232841), mRNA
E 1-2-3 Lipid transfer protein isoform 1 [Vitis vinifera] Lipid Transfer Protein 1 [Vitis vinifera].
LOC100232841, PREDICTED: Vitis vinifera VVTL1 H 4 VVTL1 [Vitis vinifera]. (LOC100232841), mRNA
LOC100232841, PREDICTED: Vitis vinifera VVTL1 I 1 VVTL1 [Vitis vinifera]. (LOC100232841), mRNA 22 Gazzola et al.2011, unpublished How do the wine protein behave to form haze?
The wine proteins Tarragona 201123 Proteins and Haze formation
• 1. Protein denaturation – Limiting step •2. Interactions (?)
• 3. Insoluble particles formation (invisible) • 4. Aggregation Visible HAZE
The wine proteins Tarragona 201124 Proteins and Haze formation
• 1. Wine Protein denaturation
– Temperature (!) Can be reversible – Other factors (?)
The wine proteins Tarragona 201125 Thermal stability of wine proteins
Chitinase TLP
Repeated DSC scans of chitinase from Repeated DSC scans of thaumatin-like Sauvignon blanc showing a melt protein from Semillon showing a melt temperature of 55 °C, no reversibility temperature of 61 °C and some of thermal unfolding reversibility of thermal unfolding.
Falconer et al.; J. Agric. Food Chem. 2010, 58, 975. Copyright © 2009 American Chemical Society
The wine proteins Tarragona 201126 Haze formation at 30°C
Effect of incubation at 30°C for 22 h on the protein composition of wine. (A) PAGE of proteins from Sauvignon blanc wine after 22 h at 30 °C. The wine was centrifuged and the obtained pellet washed with model wine. Chitinase Proteins from 100 μL for control (before heating, C) and supernatant (after heating, S) and from 500 μL of pellet (after heating, P) were loaded per TLP lane.
(B) Reverse phase (C8) HPLC chromatograms of unheated Sauvignon blanc wine (C) and supernatant after 22 h at 30 °C (S).
Chitinase is more sensitive than LTP
Marangon et al; J. Agric. Food Chem. 2011, 59, 733-740. Copyright © 2010 American Chemical Society
The wine proteins Tarragona 201127 Chitinases and haze
Wine Treatment with Chitin specific interaction with the chitin binding domain of Chitinases
Proteins: - 29% Haze : - 80% (Bentonite: -90%) (Bentonite: -100%)
Chitinase is strongly involved in wine hazing
Vincenzi et al. (2005) Am. J. Enol. Vitic. 6:3:246
The wine proteins Tarragona 201128 Proteins and Haze formation
The wine proteins do not form haze in model wine, but only in real wines!
model real 2. interactions with other compounds • the “factor(s) X”
– Sulfate
– Tannins
The wine proteins Tarragona 201129 Proteins and Haze formation
• Sulfate (Pocock et al. JAFC 2007, 55, 1799; Marangon et al. JAFC 2011, 59, 73)
2- 2 - - - - SO4 > HPO4 > acetate > Cl > NO3 Hofmeister series: • Remove water • Promote Hydrophobic interactions
Model wine Effect of increasing sulfate concentration on the haze produced by heating wine proteins (150 mg/L) in model wine.
Effect of protein concentration and composition on haze formed in model wine. 30 The wine proteins Tarragona 2011 Proteins and Haze formation
• Tannins Fixed number of tannin binding sites
0,4
0,35
0,3
0,25
0,2 Torbidity
0,15 nm) (Abs 540
0,1
0,05
0
300 15 0 75 37.5 Ta
The wine proteins Tarragona 201131 Proteins and Haze formation Model of interaction hydrophobic sites tannins not exposed
few sites exposed
hydrophobic sites exposed
native protein + tannins heat denatured protein + tannins soluble aggregation 32 The wine proteins Tarragona 2011 Hydrophobicity and Haze
HIC fractionation of wine proteins
Increasing Hydrophobicity Tot
Increasing Hydrophobicity
The wine proteins Tarragona 201133 Hydrophobicity and Haze
HIC fractions (protein: 75 mg/L) + wine tannins (50 mg/L)
Ultrafiltered wine Heated 0,2 2 Increasing Hydrophobicity R = 0.8868 0,16 r = - 0.942 ** ProteineProt. + Tannins + Tannini (RT) 0,12 0,08 0,40 R2 = ProteineProt. + Tannins + Tannini + heat riscaldati° 0,04 (80 C) 0.8136 0,35 0 0,30 ProteineHeated/cooled bollite + Tannini 200 150 100 50 Prot. + Tannins (RT) 0,25 Conductivity (mS/cm) 0,20
Turbidity0,15 (540 nm) 0,10 Haze formation 0,05 upon heating is 0,00 related to protein 1234567hydrophobicity HIC fraction
The wine proteins Tarragona 201134 Protein reduction and Haze
Wine Proteins (75 mg/l) heated and cooled + / - a reducing agent (DTT) + Wine Tannins (50 mg/l) (25°C)
Turbidity developed after the reaction at 25 ◦C of wine Haze at 25°C tannins (50mgL−1) added at room temperature to wine proteins dissolved at 75mgL−1 in model wine. Protein only in reducing samples were previously heated for 15 min at four different conditions temperatures in the absence (○) or presence (●) of 420mM DTT.
- Marangon et al. Anal. Chim. Acta 660 (2010) 110 35 The wine proteins Tarragona 2011 Effect of heating and reduction: Model
(a) when proteins and tannins are heated together haze develops due to the interactions with the new binding sites exposed on the heat denatured (unfolded) protein;
(b) when proteins are pre-heated in the absence of tannins and then reacted with tannins at 25°C, the effect on haze is minimal, because proteins refolds during cooling
(C) when proteins are pre-heated in the absence of tannins and in the presence of a reducing agent (DTT) protein can expose new binding sites; in this case refolding does not occur due to the DTT effect, this fact allowing the protein–tannins interaction Marangon et al. Anal. Chim. Acta 660 (2010) 110 and haze formation 36 The wine proteins Tarragona 2011 Conclusions
• Hazing proteins in wine can be related to grape Chitinase and TLPs.
• All these proteins are present in different forms which behave differently.
• Chitinases are the most heat-sensitive components, but also some forms of TLPs can produce haze.
• TLPs are present in in larger amounts compared to chitinases. Therefore they contribution to to wine hazing can be important.
• However, factors of non-protein nature are necessary to wine hazing.
• These factors are not fully identified, although tannins and sulfate seem to be involved.
• In order to find valuable methods to prevent wine protein hazing, other studies are necessary to elucidate its basic mechanism and the factors involved.
37 Acknowledgments
• Simone Vincenzi • Matteo Marangon • Marco Lucchetta • Diana Gazzola
CAMPUS DI CONEGLIANO Facoltà di Agraria - Università di Padova Centro Interdipartimentale per la Ricerca in Viticoltura ed Enologia CIRVE
The wine proteins Tarragona 201138