The wine proteins: origin, characteristics and functionality

Transcription

1 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

2 The CIRVE campus in Conegliano 2

3 Protein Structure / Functionality Aminoacid sequence Protein structure Proprieties Protein Size Charge Hydrophobicity Functionality Detectable effects Environment ph Solvent Ionic strength Temperature Etc. 3

4 Proteins in wine Implications in wine Hazing of white wines (negative) Mouthfeel and aroma Foam volume and stability The wine proteins Tarragona

5 Protein Haze in wine Bottled wine Flocculation Coagulation Precipitation Serious quality defect Prevention: Protein removal by bentonite treatments Bentonite several drawbacks: Loss of aroma Cost Waste.. Other methods? Knowledge is needed The wine proteins Tarragona

6 Wine Proteins: Origin Where do the wine proteins came from? The wine proteins Tarragona

7 Wine Proteins: Origin The wine proteins derive from Grape (mainly): involved in wine hazing Microorganisms The wine proteins Tarragona

8 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

9 The Grape Proteins similar in all the varieties Sarry et al., 2004 Proteomics, 4, 201 ph The wine proteins Tarragona

10 Grape Proteins: Identification by MS PR-proteins Sarry et al., 2004 Proteomics, 4, 201 The wine proteins Tarragona

11 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

12 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

13 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 The wine proteins DOMAIN Tarragona

14 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

15 PR-Proteins Pathogen defense 1. Inducibility by pathogens abiotic stress Grape: only in part PR-Proteins are Constitutive 2. Resistance acidic ph solvents proteolysis Stable The wine proteins Tarragona

16 What happens to the grape proteins during winemaking? The wine proteins Tarragona

17 Proteins and winemaking 1. Juice extraction Low ph Grape Proteases Proteins (but not PR-P) Denaturation (acidic ph) Degradation (proteases) Precipitation (tannins) only PR-proteins resist 2. Fermentation Yeast Proteases Alcohol WINE The wine proteins Tarragona

18 Proteins in wine: Quantity Low concentration mg/l Large variability (reported: < 1 mg/l - > 1 g/l). Are proteins quantified correctly? The wine proteins Tarragona

19 Quantification by the Smith assay of the protein recovered by the KDS method from different Prosecco and Manzoni Bianco wine samples. Data are expressed in BSA equivalents. N wine Protein concentration (mg/l) ± SD 1 Prosecco a 14.9 ± Prosecco a 15.5 ± Prosecco a 19.7 ± Prosecco a 15.7 ± Prosecco a 20.0 ± Prosecco a 14.2 ± Prosecco a 12.2 ± Prosecco a 14.1 ± Prosecco a 16.9 ± Prosecco a 14.7 ± Prosecco b ± Incrocio Manzoni a 30.5 ± Incrocio Manzoni a 30.5 ± Incrocio Manzoni a 26.5 ± Incrocio Manzoni a ± Incrocio Manzoni b ± 40.5 The wine proteins Tarragona 2011 Vincenzi et al., AJEV 2005 A Commercial bottled wines; b wine samples taken before bentonite fining. 19

20 Grape/Juice vs Wine Grape Wine Marangon et al. (2009) JAFC, 57, 4415 The wine proteins Tarragona D-PAGE of wine proteins (cv. Manzoni Bianco) (Polesani, 2004, unpublished). 20

21 Wine Proteins: Preparative Purification 1. Cation exchange chromatography (SCX) for Chardonnay wine. 2. Hydrophobic interaction chromatography (HIC, Phenyl Sepharose) SDS-PAGE bands used for MS identification SDS-PAGE and RP-HPLC profile of purified proteins Gazzola et al., unpublished 21

22 Identification by Nano LC-MS/MS band sequence protein C 1-2 C 4 PREDICTED: Vitis vinifera class IV chitinase (CHI4D), mrna LOC , PREDICTED: Vitis vinifera VVTL1 (LOC ), mrna Class IV chitinase [Vitis vinifera] VVTL1 [Vitis vinifera] C 6-7 α LOC , PREDICTED: Vitis vinifera hypothetical protein LOC (LOC ), mrna Vacuolar invertase 1, [Vitis Vinifera]. C 6-7 β LOC , PREDICTED: Vitis vinifera hypothetical protein LOC (LOC ), mrna Vacuolar invertase 1, [Vitis vinifera]. C 6-7 γ D PREDICTED: Vitis vinifera thaumatin-like protein (TL3), mrna LOC , PREDICTED: Vitis vinifera VVTL1 (LOC ), mrna Thaumatin-like protein [Vitis vinifera] VVTL1 [Vitis vinifera]. E Lipid transfer protein isoform 1 [Vitis vinifera] Lipid Transfer Protein 1 [Vitis vinifera]. H 4 I 1 LOC , PREDICTED: Vitis vinifera VVTL1 (LOC ), mrna LOC , PREDICTED: Vitis vinifera VVTL1 (LOC ), mrna VVTL1 [Vitis vinifera]. VVTL1 [Vitis vinifera]. 22 Gazzola et al.2011, unpublished

23 How do the wine protein behave to form haze? The wine proteins Tarragona

24 Proteins and Haze formation 1. Protein denaturation Limiting step 2. Interactions (?) 3. Insoluble particles formation (invisible) 4. Aggregation Visible HAZE The wine proteins Tarragona

25 Proteins and Haze formation 1. Wine Protein denaturation Temperature (!) Other factors (?) Can be reversible The wine proteins Tarragona

26 Thermal stability of wine proteins Chitinase TLP Repeated DSC scans of chitinase from Sauvignon blanc showing a melt temperature of 55 C, no reversibility of thermal unfolding Repeated DSC scans of thaumatin-like protein from Semillon showing a melt temperature of 61 C and some reversibility of thermal unfolding. Falconer et al.; J. Agric. Food Chem. 2010, 58, 975. Copyright 2009 American Chemical Society The wine proteins Tarragona

27 Haze formation at 30 C Chitinase TLP 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. 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 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, Copyright 2010 American Chemical Society The wine proteins Tarragona

28 Chitinases and haze Wine Treatment with Chitin specific interaction with the chitin binding domain of Chitinases Proteins: - 29% (Bentonite: -90%) Haze : - 80% (Bentonite: -100%) Chitinase is strongly involved in wine hazing Vincenzi et al. (2005) Am. J. Enol. Vitic. 6:3:246 The wine proteins Tarragona

29 Proteins and Haze formation The wine proteins do not form haze in model wine, but only in real wines! 2. interactions with other compounds the factor(s) X model real Sulfate Tannins The wine proteins Tarragona

30 Proteins and Haze formation Sulfate (Pocock et al. JAFC 2007, 55, 1799; Marangon et al. JAFC 2011, 59, 73) SO 4 2- > HPO > acetate - > Cl - > NO 3 - 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

31 Proteins and Haze formation 0,4 Tannins Fixed number of tannin binding sites 0,35 0,3 Torbidity (Abs 540 nm) 0,25 0,2 0,15 0,1 0, Ta The wine proteins Tarragona

32 Proteins and Haze formation Model of interaction hydrophobic sites not exposed tannins few sites exposed native protein + tannins soluble heat hydrophobic sites exposed denatured protein + tannins aggregation 32 The wine proteins Tarragona 2011

33 Hydrophobicity and Haze HIC fractionation of wine proteins Increasing Hydrophobicity Tot Increasing Hydrophobicity The wine proteins Tarragona

34 Hydrophobicity and Haze T urbidity (540 nm) 0,40 0,35 0,30 0,25 0,20 0,15 0,10 0,05 0,00 HIC fractions (protein: 75 mg/l) + wine tannins (50 mg/l) Increasing Hydrophobicity Proteine + Tannini Prot. + Tannins (RT) Proteine Prot. + Tannins Tannini riscaldati + heat Proteine Heated/cooled bollite + Tannini Prot. + Tannins (RT) (80 C) Ultrafiltered wine HIC fraction Heated R 2 = r = ** R 2 = Conductivity (ms/cm) The wine proteins Tarragona Haze formation upon heating is related to protein hydrophobicity 0,2 0,16 0,12 0,08 0,04 0

35 Protein reduction and Haze Wine Proteins (75 mg/l) heated and cooled + / - a reducing agent (DTT) + Wine Tannins (50 mg/l) (25 C) Haze at 25 C only in reducing conditions Turbidity developed after the reaction at 25 C of wine tannins (50mgL 1 ) added at room temperature to wine proteins dissolved at 75mgL 1 in model wine. Protein samples were previously heated for 15 min at four different temperatures in the absence ( ) or presence ( ) of 420mM DTT. - Marangon et al. Anal. Chim. Acta 660 (2010) The wine proteins Tarragona 2011

36 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

37 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

38 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