The Wine Proteins: Origin, Characteristics and Functionality Andrea Curioni

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 1The CIRVE campus in Conegliano 2Protein Structure / Functionality Aminoacid sequence  Protein • Size • Charge Protein structure • Hydrophobicity Proprieties Functionality  Environment • pH Detectable • Solvent effects • Ionic strength • Temperature • Etc. 3Proteins in wine Implications in wine –Hazing of white wines (negative) –“Mouthfeel” and aroma –Foam volume and stability The wine proteins Tarragona 2011 4Protein Haze in wine  Serious quality defect  Prevention: Protein removal by bentonite treatments Bottled wine Precipitation <ul style="display: flex;"><li style="flex:1">Flocculation </li><li style="flex:1">Coagulation </li></ul>Bentonite Other methods? several drawbacks: • Loss of aroma Knowledge is needed • Cost • Waste • ….. The wine proteins Tarragona 2011 5Wine Proteins: Origin Where do the wine proteins came from? The wine proteins Tarragona 2011 6Wine Proteins: Origin • The wine proteins derive from  Grape (mainly): involved in wine hazing  Microorganisms The wine proteins Tarragona 2011 7Grape 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 8The Grape Proteins similar in all the varieties Sarry et al., 2004 Proteomics, 4, 201 pH The wine proteins Tarragona 2011 9Grape 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 2011 11 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 2011 13 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 Percent chitin in the gel <ul style="display: flex;"><li style="flex:1">0.01 % </li><li style="flex:1">0.05 % </li><li style="flex:1">0.10 % </li></ul>14 PR-Proteins Pathogen defense <ul style="display: flex;"><li style="flex:1">1. Inducibility by </li><li style="flex:1">2. Resistance </li></ul>. pathogens . acidic pH . solvents . abiotic stress . proteolysis Grape: only in part PR-Proteins are Constitutive .Stable 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 WINE • Alcohol 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 expressed in BSA equivalents. Vincenzi et al., AJEV 2005 N°Protein concentration wine (mg/L) ± SD 123456789Proseccoa Proseccoa Proseccoa Proseccoa Proseccoa Proseccoa Proseccoa Proseccoa Proseccoa 14.9 ± 1.9 15.5 ± 1.5 19.7 ± 0.5 15.7 ± 1.6 20.0 ± 0.5 14.2 ± 0.7 12.2 ± 2.6 14.1 ± 1.7 16.9 ± 1.3 14.7 ± 1.8 121.5 ± 2.9 30.5 ± 3.6 30.5 ± 3.6 26.5 ± 1.9 176.1 ± 9.3 328.0 ± 40.5 10 Proseccoa 11 Proseccob 12 Incrocio Manzoni 6.0.13a 13 Incrocio Manzoni 6.0.13a 14 Incrocio Manzoni 6.0.13a 15 Incrocio Manzoni 6.0.13a ACommercial bottled wines; bwine samples taken before bentonite fining. 16 Incrocio Manzoni 6.0.13b The wine proteins Tarragona 2011 19 Grape/Juice vs Wine Grape WWiniene Marangon et al. (2009) JAFC, 57, 4415 The wine proteins 2D-PAGE of wine proteins (cv. Manzoni Bianco) (Polesani, 2004, unpublished). Tarragona 2011 20 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 Identification by Nano LC-MS/MS band C 1-2 C 4 <ul style="display: flex;"><li style="flex:1">sequence </li><li style="flex:1">protein </li></ul>PREDICTED: Vitis vinifera class IV chitinase (CHI4D), mRNA Class IV chitinase [Vitis vinifera] LOC100232841, PREDICTED: Vitis vinifera VVTL1 (LOC100232841), mRNA VVTL1 [Vitis vinifera] LOC100256970, PREDICTED: Vitis vinifera hypothetical protein LOC100256970 (LOC100256970), mRNA C 6-7 α Vacuolar invertase 1, [Vitis Vinifera]. LOC100256970, PREDICTED: Vitis vinifera hypothetical protein LOC100256970 (LOC100256970), mRNA C 6-7 β Vacuolar invertase 1, [Vitis vinifera]. PREDICTED: Vitis vinifera thaumatin-like protein (TL3), mRNA C 6-7 γ Thaumatin-like protein [Vitis vinifera] D 1-2-3- LOC100232841, PREDICTED: Vitis vinifera VVTL1 VVTL1 [Vitis vinifera]. <ul style="display: flex;"><li style="flex:1">4</li><li style="flex:1">(LOC100232841), mRNA </li></ul><ul style="display: flex;"><li style="flex:1">E 1-2-3 </li><li style="flex:1">Lipid transfer protein isoform 1 [Vitis vinifera] </li></ul>Lipid Transfer Protein 1 [Vitis vinifera]. VVTL1 [Vitis vinifera]. LOC100232841, PREDICTED: Vitis vinifera VVTL1 (LOC100232841), mRNA H 4 I 1 LOC100232841, PREDICTED: Vitis vinifera VVTL1 (LOC100232841), mRNA VVTL1 [Vitis vinifera]. 22 Gazzola et al.2011, unpublished How do the wine protein behave to form haze? The wine proteins Tarragona 2011 23 Proteins and Haze formation • 1. Protein denaturation – Limiting step • 2. Interactions (?) • 3. Insoluble particles formation (invisible) • 4. Aggregation Visible HAZE The wine proteins Tarragona 2011 24 Proteins and Haze formation • 1. Wine Protein denaturation Can be reversible – Temperature (!) – Other factors (?) The wine proteins Tarragona 2011 25 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 2011 26 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. Proteins from 100 μL for control (before heating, C) and supernatant (after heating, S) and from 500 μL Chitinase of pellet (after heating, P) were loaded per lane. TLP (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 2011 27 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 2011 28 Proteins and Haze formation The wine proteins do not form haze in model wine, but only in real wines! <ul style="display: flex;"><li style="flex:1">model </li><li style="flex:1">real </li></ul>2. interactions with other compounds • the “factor(s) X” – Sulfate – Tannins The wine proteins Tarragona 2011 29 Proteins and Haze formation • Sulfate (Pocock et al. JAFC 2007, 55, 1799; Marangon et al. JAFC 2011, 59, 73) SO4 > HPO4 > acetate- > Cl- > NO3 2- <ul style="display: flex;"><li style="flex:1">2 - </li><li style="flex:1">-</li></ul>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 30 formed in model wine. The wine proteins Tarragona 2011

The Wine Proteins: Origin, Characteristics and Functionality Andrea Curioni

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