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
• Size • Charge
Protein structure
• Hydrophobicity
Proprieties
Functionality
Environment
• pH
Detectable
• Solvent
effects
• Ionic strength • Temperature • Etc.
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Proteins in wine
Implications in wine
–Hazing of white wines (negative)
–“Mouthfeel” and aroma –Foam volume and stability
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Protein Haze in wine
Serious quality defect Prevention: Protein removal by bentonite treatments
Bottled wine
Precipitation
- Flocculation
- Coagulation
Bentonite
Other methods?
several drawbacks: • Loss of aroma
Knowledge is needed
• Cost • Waste • …..
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Wine Proteins: Origin
Where do the wine proteins came from?
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Wine Proteins: Origin
• The wine proteins derive from
Grape (mainly):
involved in wine hazing
Microorganisms
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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
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The Grape Proteins similar in all the varieties
Sarry et al., 2004 Proteomics, 4, 201
pH
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Grape Proteins: Identification by MS
PR-proteins
Sarry et al., 2004 Proteomics, 4, 201
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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
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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
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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
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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
- 0.01 %
- 0.05 %
- 0.10 %
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PR-Proteins
Pathogen defense
- 1. Inducibility by
- 2. Resistance
. pathogens
. acidic pH
. solvents
. abiotic stress
. proteolysis
Grape: only in part
PR-Proteins are
Constitutive
.Stable
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What happens to the grape proteins during winemaking?
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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
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Proteins in wine: Quantity
Low concentration
10 – 250 mg/L
Large variability
(reported: < 1 mg/L - > 1 g/L).
Are proteins quantified correctly?
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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
123456789
Proseccoa 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
A
Commercial bottled wines;
b
wine samples taken before bentonite fining.
16 Incrocio Manzoni 6.0.13b
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Grape/Juice vs Wine
Grape
WWiniene
Marangon et al. (2009) JAFC, 57, 4415
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2D-PAGE of wine proteins (cv. Manzoni Bianco) (Polesani, 2004, unpublished).
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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
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Identification by Nano LC-MS/MS
band
C 1-2 C 4
- sequence
- protein
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].
- 4
- (LOC100232841), mRNA
- E 1-2-3
- Lipid transfer protein isoform 1 [Vitis vinifera]
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].
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Gazzola et al.2011, unpublished
How do the wine protein behave to form haze?
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Proteins and Haze formation
• 1. Protein denaturation
– Limiting step
• 2. Interactions (?) • 3. Insoluble particles
formation (invisible)
• 4. Aggregation
Visible HAZE
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Proteins and Haze formation
• 1. Wine Protein denaturation
Can be reversible
– Temperature (!)
– Other factors (?)
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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
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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
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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
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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
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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-
- 2 -
- -
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
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formed in model wine.
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