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The Wine Proteins: Origin, Characteristics and Functionality Andrea Curioni

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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.

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
Precipitation

  • Flocculation
  • Coagulation

Bentonite

Other methods?

several drawbacks: • Loss of aroma

Knowledge is needed

• Cost • Waste • …..

The wine proteins

Tarragona 2011

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Wine Proteins: Origin

Where do the wine proteins came from?

The wine proteins

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Wine Proteins: Origin

• The wine proteins derive from

Grape (mainly):

involved in wine hazing

 Microorganisms

The wine proteins

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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

The wine proteins

Tarragona 2011

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The Grape Proteins similar in all the varieties

Sarry et al., 2004 Proteomics, 4, 201

pH

The wine proteins

Tarragona 2011

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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 2011

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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

The wine proteins

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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

The wine proteins

Tarragona 2011

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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 %

14

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

The wine proteins

Tarragona 2011

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What happens to the grape proteins during winemaking?

The wine proteins

Tarragona 2011

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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

The wine proteins

Tarragona 2011

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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?

The wine proteins

Tarragona 2011

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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

The wine proteins

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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

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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

21

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].

22

Gazzola et al.2011, unpublished

How do the wine protein behave to form haze?

The wine proteins

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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

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Proteins and Haze formation

• 1. Wine Protein denaturation

Can be reversible

– Temperature (!)

– Other factors (?)

The wine proteins

Tarragona 2011

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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

The wine proteins

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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

The wine proteins

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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

The wine proteins

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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

The wine proteins

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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

30

formed in model wine.

The wine proteins

Tarragona 2011

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    WINES . AFTER REPEAL t Copyright New York World-Tele;ram October, 1933 OUTLINE Foreword 1. Definition of Wine 2. Classifications of Wlinos a. By Color b. By Activity C. By Taste d. By Content e. By Alcoholic Percentage 3. Styles or Kinds of Wines 4. Origin of Wine Names 5. Uses of Wines 6. Wine Consumption in The United States 7. Imports of Wines 8. Sources of Supplies 9. Supplies from Abroad 10. The Wines of France French Bordeaux Wines French Burgundy Wines French Champagne Wines French Hermitage Wines 11. The Wines of Italy 12. The Wines of Spain 13. The Wines of Germany 14. The Wines of Portugal 15. The Wines of Hungary and taustria 16. The Wines of Switzerland 17. The Wines of Greece 18. The Wines of the United States Foreword Before prohibition, in the best years of wine production and importation, the United States was not a wine-drinking nation. In 1914, the last normal year, before the war and prohibition, while France was consuming 32 gallons per person, Italy 25, and Spain 18.5, the per capita wine consumption of the United States was less than one-half gallon. It has been said that many more people in the United States are wine-drinkers now because prohibition did not forbid wine making in homes and, that given ten more years of prohibition enforcement we would have become prolific users of wine. If this is only partially true, United States wineries and importers will find our people increasingly good customers when prohibition is repealed, and it seems probable that a sizable volume of newspaper advertising will gradually deve lop.
  • Science Sixtyfourth64 National Conference a Platform for Progress

    Science Sixtyfourth64 National Conference a Platform for Progress

    science sixtyfourth64 national conference a platform for progress 64th ASEV National Conference June 24–28, 2013 Portola Hotel and Monterey Conference Center Monterey, California USA TECHNICAL ABSTRACTS american society for enology and viticulture 2013 National Conference Technical Abstracts Oral Presentation Abstracts Wednesday, June 26 Enology — Flavor/Analysis ....................................................................59–62 Viticulture — Rootstocks .......................................................................63–66 Enology — Micro/Molecular Biology ....................................................67–70 Viticulture — Pests & Diseases ..............................................................71–74 Enology — Sensory/Sensory Impacts .....................................................75–78 Viticulture — Environmental Impacts ..................................................79–82 Enology — Flavor: Impact of Yeast and Bacteria ....................................83–86 Viticulture — General ...........................................................................87–90 Thursday, June 27 Enology — Wine Stability and Oxidation ..............................................91–93 Viticulture — Cultural Practices ............................................................94–96 Enology — Tannins (Part I) .................................................................97–100 Viticulture — Water Relations ...........................................................101–105 Enology — Tannins (Part II) ..............................................................106–108
  • Terra Alta Greenway (Tarragona)

    Terra Alta Greenway (Tarragona)

    Terra Alta Greenway Terra Alta is a rural area of Cataluña dotted with almond and pine groves through which a few trains used to run up until 1973. Now we can make use of the disused rail bed of this forgotten railway to travel through the spectacular countryside around the Sierra de Pandols ridge and the Beceite heights. A journey through tunnels and over viaducts takes us from Aragon to the Ebro. TECHNICAL DATA CONDITIONED GREENWAY Spectacular viaducts and tunnels between the ravines of the Canaletas river. LOCATION Between the stations of Arnes-Lledó and El Pinell de Brai TARRAGONA Length: 23 km Users: * * Punctual limitations due to steep slopes in la Fontcalda Type of surface: Asphalt Natural landscape: Forests of pine trees. Karst landscape of great beauty: cannons, cavities Cultural Heritage: Sanctuary of Fontcalda (S. XVI). Convent of Sant Salvador d'Horta in Horta. Church of Sant Josep en Bot Infrastructure: Condidtioned Greenway. 20 tunnels. 5 viaducts How to get there: To all the towns: HIFE Bus Company Connections: Tarragona: 118 Km. to Arnés-Lledó Barcelona: 208 Km. to Arnés-Lledó Castellón de la Plana: 169 Km. to Arnés-Lledó Maps: Military map of spain. 1:50.000 scale 470, 495, 496 and 497 sheets Official road map of the Ministry of Public Works Ministerio de Fomento More information on the Greenways guide Volume 1 Attention: Lack of lighting in some tunnels by vandalism. It is recommended to use a torch DESCRIPTION Km. 0 / Km. 13 / Km. 17,5 / Km. 23,7 Km 0 The Greenway begins at the Arnes-Lledó station alongside the river Algars which at this point forms the border between the autonomous communities of Aragon and Cataluña.