Food Chemistry 118 (2010) 2634

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Proteins in white wines: Thermo-sensitivity and differential adsorbtion by bentonite

Francois-Xavier Sauvage *, Benoit Bach, Michel Moutounet, Aude Vernhet
UMR 1083 Sciences for Enology, INRA/Montpellier SupAgro, 2 Place Viala, 34 060 Montpellier Cedex 01, France

a r t i c l e

i n f o

a b s t r a c t
Protein fractions in a Chardonnay wine (invertases, glucanases, chitinases and thaumatin-like proteins) were identied using 2D-electrophoresis and mass spectrometry. The sensitivity of these fractions to heat-induced denaturation and precipitation following heat-treatments at different temperatures was studied and compared to their afnity for bentonite, a clay used to adsorb proteins and stabilise wines with regards to protein hazes. The different proteins exhibited different sensitivity with regards to heat-induced precipitation, glucanases being the most sensitive and invertases the least. Thaumatines and chitinases were characterised by a wide range of behaviours attributed to structural micro-heterogeneities. Protein depletion upon the addition of increasing bentonite doses was also dependent on the considered fraction. A good correlation was shown between protein afnity for bentonite and their sensitivity to heat precipitation. Results were discussed considering the current winemaking practices used to assess white wine stability and dene bentonite doses needed to achieve their stabilisation. 2009 Published by Elsevier Ltd.

Article history: Received 21 November 2008 Received in revised form 6 January 2009 Accepted 24 February 2009

Keywords: White wine stability Wine proteins Thermo-sensitivity Bentonite adsorption

1. Introduction Protein synthesis in grape berries quickly increases after veraison, along with sugar accumulation (Giribaldi, Perugini, Sauvage, & Schubert, 2007; Murphey, Spayd, & Powers, 1989; Pocock, Hayasaka, Mc Carthy, & Waters, 2000). Protein diversity in musts and wines is much lower than in grapes since only soluble proteins are extracted during the winemaking process (Ferreira, Piarra-Pereira, Monteiro, Loureiro, & Teixeira, 2002; Murphey et al., 1989). Moreover, protein amounts in wines are always lower than in the corresponding musts. That decrease is attributed to proteolytic activity, to precipitation by polyphenols and to unfavourable conditions related to the low pHs and the increasing ethanol contents. Protein concentrations reported for white wines vary between 10 and about 500 mg l1 (Santoro, 1995; Vincenzi, Polesani, & Curioni, 2005). Grape variety, maturity and winemaking process are some factors that strongly affect the nal protein content. These proteins have molecular mass ranging from 9 to 66 kDa and isoelectric points ranging from 3 to 9 (Brissonnet & Maujean, 1993; Dawes, Boyes, Keene, & Heatherbell, 1994; Hsu & Heatherbell, 1987). Most of them, identied as being chitinases and thaumatin-like proteins, are found within the range 2030 kDa and are acidic (Dawes et al., 1994; Hsu & Heatherbell, 1987; Mesquita et al., 2001; Murphey et al., 1989; Pocock et al., 2000; Pocock, Hayasaka, Peng, Williams, & Waters, 1998). Though present in rather small amounts, proteins are of primary importance for the colloidal stability and clarity of white wines (Bayly & Berg, 1967; Hsu & Heatherbell, 1987; Waters, Wallace, & Williams, 1991). Haze or deposit formation in bottled
* Corresponding author. Fax: +33 (0) 4 99 61 28 57. E-mail address: sauvage@supagro.inra.fr (F.-X. Sauvage). 0308-8146/$ - see front matter 2009 Published by Elsevier Ltd. doi:10.1016/j.foodchem.2009.02.080

wines, due to protein aggregation during storage, is a common defect of commercial wines making them unacceptable for consumers. Numerous works have been devoted to the identication of protein structures implied in colloidal instabilities, as well as to the elucidation of the factors that trigger or prevent haze formation (Dawes et al., 1994; Hsu & Heatherbell, 1987; Pocock, Alexander, Hayasaka, Jones, & Waters, 2007; Waters et al., 1991; Waters, Dupin, & Stockdale, 2000; Waters, Peng, Pocock, & Williams, 1995; Waters, Wallace, & Williams, 1992). Aggregation is usually attributed to slow protein unfolding during storage, induced by exposure to high temperatures. As a consequence, most studies to explore the development of these hazes have been conducted using heat-induced precipitation. Chitinases and thaumatin-like proteins, which constitute the major part of white wine proteins, are also important contributors to heat-induced protein instability (Dawes et al., 1994; Hsu & Heatherbell, 1987; Waters et al., 1992). Attempts to correlate the total wine protein contents to their sensitivity to protein haze failed (Bayly & Berg, 1967); this can be related to a different sensitivity of the various protein fractions to heat-denaturation (Hsu & Heatherbell, 1987; Waters et al., 1991, 1992), and/or to the inuence of factors other than protein composition. Among others, haze formation was shown to be strongly affected by the presence of non-protein compounds such as polyphenols (Dawes et al., 1994; Waters et al., 1995), ions (Besse, Clarck, & Scollary; 2000; Pocock et al., 2007) and polysaccharides (Dupin et al., 2000; Mesquita et al., 2001). Different tests have been proposed to assess wine stability/ instability with regards to protein haze (Pocock & Waters, 2006). These tests are based upon different types of procedures, leading to protein aggregation and precipitation. Heat stability trials, based on heat-induced precipitation, are the most common. These tests

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are empirical and do not necessarily reect changes and destabilisation phenomena liable to occur in real wine storage conditions. If needed, stability is usually obtained by bentonite ning, due to protein adsorption by the clay particles. The level of bentonite addition required for stabilisation is also determined by stability tests. These levels have increased during the last 20 years, so that doses in the order of 100200 g hl1 are often employed (Dubourdieu & Canal-Llaubre, 1989). Though effective, bentonite ning generates different problems, especially when such high doses are needed. First, this treatment is not selective enough and may adversely affect wine quality by inducing signicant aroma losses and sometime colour alteration (Bayonove et al., 1995; Cabaroglu, Canbas, Lepoutre, & Gunata, 2002). Bentonite ning also causes substantial volume losses (between 3% and 10%) (Hoj et al., 2001) and the disposal of spent bentonites constitute a non negligible source of waste. Finally, bentonite handling is also of concern for occupational health and safety issues. For these reasons, increasing attention is given today to the development of alternative practices for protein stabilisation, that would maintain quality, reduce costs and be more sustainable (Waters et al., 2005). Thorough knowledge of the structures and mechanisms involved is needed to be in position to control and prevent protein hazes while avoiding excessive and detrimental treatments. In addition to the identication of the role played by the other wine compounds, additional information regarding: (i) the sensitivity of the different wine proteins to heat-induced precipitation and (ii) their adsorption by bentonites is necessary. It is clear from previous works that the wine protein fractions exhibit different behaviours with regards to heat-induced precipitation and adsorption (Dawes et al., 1994; Hsu & Heatherbell, 1987; Murphey et al., 1989). In the present study the major proteins in a Chardonnay wine were identied using 2D-electrophoresis and mass spectrometry. This identication was used to study the sensitivity of each protein fraction to heat-treatment and to correlate this sensitivity to their adsorption by bentonite. To this end, a methodology to analyse and quantify wine proteins using 1D-electrophoresis coupled with image analysis was used, which is described rst.

2. Materials and methods 2.1. Materials 2.1.1. Wine sample The Chardonnay white wine used in this study was elaborated in 2005 at the Pech Rouge Experimental Unit (INRA Gruissan, France). Following to harvest, grapes were pressed without skin maceration and the must settled down (one night, 15 C). Alcoholic fermentation was achieved at a temperature of 15 C using the yeast Firmivin (DSM). After fermentation, the wine was racked and the SO2 level was adjusted to prevent oxidation and malolactic fermentation. It was then stored during two months at 15 C and bottled after a second racking and a membrane ltration. 2.1.2. Chemicals The bentonite used was a commercial suspension of bentonite Electra (Martin Vialatte). Thio-urea, urea, CHAPS, Triton X-100, Dithiothreitol, bromophenol blue, Tris buffer, glycine and Sodium Docedyl Sulfate were purchased from SigmaAldrich. Ampholines were from GE Healthcare. 2.2. Protein quantication in white wines using 1D-electrophoresis coupled with image analysis Problems encountered to quantify proteins in wines using 1D electrophoresis and image analysis are related to their low concen-

trations and to the presence of non-protein compounds, especially polyphenols, that interfere with the analysis. To overcome these problems, a methodology was developed to obtain both protein concentration and separation. This was achieved by adsorption on bentonite of the proteins contained in a 1 ml wine aliquot, followed by their desorption using Laemmli buffer. Different bentonite doses, between 20 and 500 g hl1, were assayed. After bentonite addition and 30 min under soft shaking the mixture was centrifuged for 15 min at 20,000g and at 4 C. The supernatant was removed and 100 ll of Laemmli buffer were added to the pellet to allow protein desorption. The suspension was shaken for 15 min and the bentonite separated by centrifugation, as described before. Several successive desorption steps were performed (up to 5 steps) and the supernatants analysed. With the experimental wines (protein concentrations below 150 mg l1) it was veried that no protein could be detected in the treated wines at a bentonite dose of 200 g hl1 and higher. Up to 200 g hl1 bentonite, the rst washing step using 100 ll Laemmli buffer allowed the recovery of the whole protein fraction. Conditions for further experiments were then: wine sample, 1 ml; bentonite dose, 200 g hl1 and Leammli buffer volume for desorption, 100 ll. Proteins in the buffer were concentrated 10 fold by comparison to the original wine. They were separated by 1D SDSPAGE electrophoresis, performed in 14% acrylamide resolving gel (resolving gel length, 60 mm). As their initial concentration in wine was unknown, three different volumes (10, 20 and 40 ll) were deposited in the gel. Running was rst conducted at 20 mA per gel all along the stacking gel and 30 mA per gel until the run end. Gels were then stained with colloidal CBB G-250 (Biorad) (Neuhoff, Arold, Taube, & Ehrhadt, 1988) and scanned at 300 dpi with an Image scanner (GE Biosciences). A low molecular weight calibration kit (Pharmacia, Biotech), ranging from 14 400 to 97,000 Da, was included in each electrophoretic run. Image elaboration and analysis were carried out with the Totallab software (Nonlinear Dynamics Ltd.). Image analysis was used to calculate the volume of each staining band from sample. This volume (product of the band area by the intensity of each pixel within this area) is proportional to the protein amount in each band provided saturation of the coloration has not been reached. It can be compared to the volume of the band obtained with a calibration protein. The volume of the Bovine Serum Albumin band of the low molecular weight calibration kit was used and results expressed in lg equivalent BSA. Quantication was performed for the three different deposited volumes (between 10 and 40 ll) and results represent the linear regression of the three measures. 2.3. Separation and identication of wine proteins by 2Delectrophoresis and mass spectrometry 2.3.1. Protein extract preparation for 2DE separation To be in position to perform a 2D-electrophoresis separation of the wine proteins it was rst required to purify (partial purication) and concentrate the whole wine protein content. Proteins were then dissolved under their native form by means of a suitable tensioactive mixture. Polyphenols in the Chardonnay wine (750 ml aliquot) were rst removed by adsorption using Fractogel (50 ml, FractogelTSK, Toyopearl HW50F, TosoHaas, Allemagne). Adsorption was realised in batches for 30 min, under gentle stirring. Adsorbent was removed by centrifugation (14,000g, 15 min, 4 C) and the supernatant recovered. Protein concentration and purication from the dephenolysed wine was then achieved by successive ultraltration steps. The wine was rst ultraltered using a 150 kDa plane membrane (50 cm2, TAMI Industry, Nyons, France) using the spirelab device developed by TAMI (Nyons, France). This rst step allowed the separation between wine polysaccharides, concentrated in the

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retentate, and proteins (MW <150 kDa), recovered in the permeate. A second ultraltration step on a 5 kDa titanium/zirconium membrane (50 cm2, TAMI Industry, Nyons, France), along with extensive dialtration using deionised water (Milli-Q), allowed protein concentration and the removal of salts and small molecules. Dialtration was monitored via conductivity measurements. The 5 kDa membrane was washed twice in 2 ml of an isoelectrofocusing (IEF) rehydration solution to recover adsorbed proteins. Washing solutions were added to the ultraltration retentate. The composition of the rehydratation solution was: 2 M thiourea, 7 M urea, 4% CHAPS, 0.5% Triton X-100, 65 mM DTT, traces of bromophenol blue, 5% ampholines mix. Protein concentration in the nal extract (after 10 fold dilution) was assessed using CBB G-250 (Bio-Rad) with BSA as standard according to the Bradfords procedure (Bradford, 1976). It was adjusted to 1 lg ll1 for next experiments. 2.3.2. 2DE Separation Isoelectrofocusing (IEF) was carried out with 300 lg protein using 18 cm long Immobiline Dry-strips (pH interval 310, nonlinear, GE Healthcare). Sample loading was performed passively for 8 h at 20 C in an IPGphor system (GE Healthcare). IEF migration program was as follows: active rehydration at 50 V during 9 h, ramping until 300 V in 1 min, start focusing at 300 V for 30 min, ramping again until 8000 V in 3 h, focusing for 11 h at 8000 V, decreasing until 300 V in 3 h. For a complete focusing the total power reached 105,000 Vh. Strips were then equilibrated twice upon gentle agitation for 15 min in a 50 mM TrisHCl (pH 8.8) buffer containing 6 M urea, 10% glycerol and 2% SDS, added rst with 1% DTT, and in a second time with 2.5% iodoacetamine (Grg, Weiss, & Dunn, 2004). Second-dimension SDSPAGE was performed on a 12% acrylamide gel using an ISODALT apparatus (GE Healthcare). Separation was achieved at 15 C in a buffer (pH 8.3) containing 25 mM Tris, 0.192 M glycine, and 0.1% SDS. Running conditions were 1 h at 40 V and 30 mA per gel, 1 h at 70 V and 40 mA per gel, and then 11 h at 110 V per gel. Gels were stained and scanned as described before for 1D-electrophoresis. Image elaboration and 2D analysis were completed with the ImageMaster 2D-Platinium version 5 software (GE Healthcare). Molecular weights and isoelectric points of spots were determined according to migration of 2D standards (Bio-Rad). 2.3.3. In-gel digestion and mass spectrometry Gels spots from 2D-electrophoresis were excised and washed twice with deionised water. Destaining was realised by at least three successive washes using a 50% acetonitrile aqueous solution (ACN) and 50 mM NH4HCO3, as described before (Grg et al., 2004). Gel fragments were successively dehydrated by ACN, re-hydrated and dehydrated again by NH4HCO3/ACN washes and nally dried under vacuum on a centrifugal evaporator. Final rehydration was achieved in a digestion solution composed of 0.1 M NH4HCO3, 5 mM CaCl2 and 0.015 mg ml1 trypsin (GOLD, Promega). Samples were rst left on ice for 45 min to allow trypsin activation and then during one night at 37 C to complete the in-gel protein digestion. To collect the digested peptides, gels spots were dehydrated/re-hydrated/dehydrated twice with ACN/NH4HCO3 (100 mM ACN), and washed twice in 5% formic acid and ACN. Washing solutions were pooled and dried in a vacuum centrifuge. The pellets (containing digested products) were re-suspended in 10 ll of 2% formic acid and desalted using C18 Zip Tips (Millipore Inc., France) according to manufacturers instructions. The nal peptide solution ACN/ TFA/water (50%/0.1%/49.9%) was concentrated to a 2 ll volume by vacuum evaporation. MS analyses were performed using an UltraFlex matrix-assisted laser desorption ionisation time-of-ight (MALDI-TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany) in reecton mode

with an accelerating voltage of 19 kV. Different peptides ions generated by trypsin autolysis were used as internal standards for calibrating the mass spectra. Spots identied by MALDI-TOF were also analysed online using nanoow HPLC nanospray ionisation on a quadrupole time-of-ight (Q-TOF) mass spectrometer. Spectra were recorded using Analyst QS 1.1 software (Applied Biosystems). 2.3.4. Identication by database search The NCBI non-redundant database was explored with the MASCOT software (Matrix Science, London, UK). To conrm protein identities, the SWISS-PROT and Trembl databases were also explored with Peptident software. For identication by peptide mass ngerprinting (MALDI-TOF), the following parameters were used for database searches (Grg et al., 2004): (1) mass tolerance of 50 ppm, (2) a minimum of four peptide mass matching the protein, and (3) up to one missed cleavage was accepted only when two consecutive lysine or arginine residues were present or when residues were followed by a proline or acidic/basic residues. Sequence coverage was at least 15% for any identication. All MS/MS spectra were searched against the Viridiplantae entries of either the SwissProt or Trembl database or ESTs in Genbank databases by using the Mascot v 2.1 algorithm, by sequence query analysis. All signicant hits were manually inspected.
TM

2.4. Thermo-sensibility of white wine proteins Ten millilitre aliquots of the Chardonnay wine were heated to 30, 40, 50, 60, 70 or 80 C in a waterbath during 30 min. Samples were cooled down and left at room temperature for at least one night. Then, they were centrifuged at 20,000g and at 4 C during 20 min, to separate the precipitate. Proteins in the supernatants and in the pellets were identied and quantied by 1D-electrophoresis and image analysis, as described before. Experimental protocol for the supernatants was the same than that described for wine, except that in this case, a 12% acrylamide resolving gel was used. Precipitated proteins, recovered in the pellet, were solubilised in 1 ml Laemmli buffer before electrophoresis (Hsu & Heatherbell, 1987). 2.5. Protein adsorption on bentonite Increasing volumes of a 10 g l1 bentonite suspension were added to 1 ml Chardonnay wine aliquots to obtain nal bentonite concentrations between 0 and 150 g hl1. Suspensions were shaken during 30 min at ambient temperature before being centrifuged (20,000g, 4 C, 15 min). Proteins in the supernatants were analysed as described for wines. Adsorbed proteins on bentonites were recovered by washing the clay particles with 0.1 ml Laemmli buffer and directly analysed using electrophoresis (14% acrylamide resolving gel) and image analysis.

3. Results and discussion 3.1. Quantication of wine proteins by 1D-electrophoresis and image analysis In the present work, the opportunity of improving protein patterns obtained from 1D-electrophoresis of white wines was experienced. The aim was to obtain proles allowing protein quantication by image analysis. To this end, proteins have to be concentrated and separated rst. This was achieved by an adsorption/desorption step on bentonite, clay being added in excess to ensure the adsorption of all proteins. Protein proles obtained in this way for different white wines are shown in Fig. 1; pattern quality allowed the quantication of each protein band within

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MM ( Kda) Standard

29

94 66 43 30

20

14

Fig. 1. Protein proles obtained by 1D SDSPAGE electrophoresis for white wines of different varieties and using the described extraction and concentration procedure. 1: Muscat, 2: Garganega, 3: Chasselas, 4: Moscatel, 5: Sauvignon, 6: Ugni Blanc, 7: Chardonnay I5, 8: Chardonnay I19, 9: Chardonnay I41, 10: Chardonnay I10.

Fig. 3. Protein prole obtained by 2D-electrophoresis separation and staining using CBB-G-250. Proteins in the Chardonnay wine (I10) were identied by mass spectrometry and were: thaumatin-like proteins (spots a1, a2, a3, a5), chitinases (spots a4 and a6), glucanases (spots a7 and a8) and invertases (spots a9 and a10).

the whole pool. These patterns were typical of those reported in the literature for white wines (Dawes et al., 1994; Hsu & Heatherbell, 1987; Pocock et al., 2000), with protein fractions of molecular weight ranging from 14 to 66 kDa, the majority of them being comprised between 20 and 30 kDa. The reproducibility of the quantication obtained via image analysis was assayed on the Chardonnay I10 wine (Fig. 2a), selected for further experiments due to its protein composition. The I10 protein pattern exhibited nine major bands so that this wine was characterised by the variety of its protein content. Results of protein quantication (Fig. 2b) represent a mean of six separate experiments, including the initial separation and concentration step. The I10 wine contained 142 mg l1 protein in equivalent BSA, and the standard deviation between the six aliquots was 11%. It is important to note that when bands are quantied separately, the standard deviation may be different, depending on the concentration of each protein in the initial wine. For the I10 wine, these standard deviation uctuated from 10% (band corresponding to the most concentrated protein fraction)

to 25% (band corresponding to the less concentrated protein fraction). 3.2. Identication of white wine proteins Proteins in the I10 wine were extracted and separated by 2Delectrophoresis for further identication by mass spectrometry. The protein prole evidenced ten major spots (Fig. 3), the number of spots detected onto 2DE gels being in agreement with the number of bands detected on 1D SDSPAGE gels. MS analyses (Table 1) allowed identifying the different proteins in these spots: 4 thaumatin-like proteins (spots a1, a2, a3, a4), 2 chitinases (spots a5, a6), 2 glucanases (spots a7, a8) and 2 invertases (spots a9, a10). All these proteins, considered as pathogenesis-related proteins (PRPs) except for invertases, originated from grape and not from yeast. This specic grape origin of wine proteins is in accordance with previous data (Ferreira et al., 2000). However some proteins of fungal

MM kDa 94 66 43

B
120

Concentration in eq BSA (mg l-1)

S 1 2 3 4 5 6

100 80 60 40 20 0 1 2 3 4 5 6 M

30

20

14

Fig. 2. (A) Protein proles in the I10 Chardonnay wine obtained by 1D SDSPAGE electrophoresis (14% acrylamide gel) for a deposited volume equivalent to 400 ll of wine. (B) Protein quantication on 6 aliquots, M being the average of the six quantications. Under the experimental conditions this Chardonnay wine exhibited 9 different protein bands, corresponding to an average protein content of 142 mg l1 equivalent BSA, with an 11% standard deviation.

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Table 1 Protein identication by Maldi-TOF MS (results non shown) and peptide sequence determination using HPLC-ion trap MS/MS. Spot number a10 a9 a8, a7 a5, a6 a4 Score* (peptide number) 88(3) 144(5) 75(2) 45(4) 122(1) 56(1) a3 167(4) 52(2) a2 755(6) 527(6) 56(1) a1 852(2) 131(5) 49(1)
* **

Sequence SSLAVDDVDQR; VLVDHSIVEGFSQGGR; VYPTEAIYGAAR SSLAVDDVDQR; TFFCTDLSR; IYGSIVPVLDDEKPTMR; VLVDHSIVEGFSQGGR; VYPTEAIYGAAR NIFNAISAAGLGNQIF; NLFDAILDAVYSALER AAFLSALNSYSGFGNDGSTDANKR; AINGAVECNGGNTAAVNAR; AAFLSALNSYSGFGNDGSTDANK; DYCSQLGVSPGDNLTC RTNCNFDASGNGK RLDSGQSWTITVK LDSGQSWTITVNPGTTNAR; TSLFTCTSGTNYK ; TSCTFDANGR; DRCPDAYSYPQDDK; rldsgqswtitvnpgttnar; GIQCSADINGQCPSELK RTNCNFDASGNGK; GISCTADIVGECPAALK LDSGQSWTITVNPGTTNAR; TSLFTCPSGTNYK; TSCTFDANGR ; DRCPDAYSYPQDDK; rldsgqswtitvnpgttnar; GIQCSVDINGQCPSELK LDSGQSWTITVNPGTTNAR; TSLFTCTSGTNYK; TSCTFDANGR; DRCPDAYSYPQDDK; rldsgqswtitvnpgttnar; GIQCSADINGQCPSELK RTNCNFDASGNGK RTNCNFDASGNGK; VLVDHSIVEGFSQGGR LDSGQSWTITVNPGTTNAR; TSLFTTCSGTNYK ; TSCTFDANGR; DRCPDAYSYPQDDK; ldsgqswtitvnpgttnar SSLAVDDVDQR

Access number** (SP Trembl) Q9S944_VITVI Q9S944_VITVI Q9M3U4_VITVI Q7XAU6_VITVI Q7XAU7_VITVI Q9LLB7_VITVI Q9M4G6_VITVI Q7XAU7_VITVI O04708_VITVI Q9M4G6_VITVI Q7XAU7_VITVI Q7XAU7_VITVI O04708_VITVI Q9S944_VITVI

Molecular mass (Da) 71,501 71,501 37,489 28,366 24,835 25,269 24,947 24,835 24,866 24,947 24,835 24,835 24,866 71,501

Name Vacuolar invertase 1 Vacuolar invertase 1 b1-3 glucanase Class IV chitinase Thaumatin-like protein Thaumatin-like protein Thaumatin-like protein Thaumatin-like protein Thaumatin-like protein (VVTL1) Thaumatin-like protein Thaumatin-like protein Thaumatin-like protein Thaumatin-like protein (VVTL1) Vacuolar invertase 1

Identication score along with the number of peptides (into brackets) that matched with peptides of the SP Trembl database. Identication number in the SP Trembl database.

or bacterial origin may also be present, though rarely evidenced (Monteiro et al., 2001). I10 wine proteins were mainly acidic, with isoelectric points (IEP) below 5, except for glucanases, the IEPs of which were found to be around 7. This also is in agreement with previous works (Cilindre, Castro, Clment, Jeandet, & Marchal, 2007; Dawes et al., 1994). 3.3. Heat-induced precipitation of white wine proteins The conformational stability of proteins in a given solvent is weak and strongly affected by even small changes in the external variables (temperature, pH, salts, etc.). Changes from native to unfolded conformation, such as those induced by high temperature, are known to lead to protein aggregation (intermolecular interactions) and eventually precipitation (Chi, Krishnan, Randolph, & Carpenter, 2003; Murphy & Kendrick, 2007). However, complete unfolding is not required and also only partially unfolded structures may be prone to aggregate. In complex media such as wines, aggregation may involve proteins only, but can also result from attractive interactions between proteins and other non-protein compounds, as well-known for polyphenols. The presence of different protein structures, along with a large diversity in wine composition, makes the elucidation of mechanisms a very complex task and information is still required to rationalise both stability/instability trials and stabilization operations. Thanks to the identication of the I10 wine protein composition it was possible to evaluate here the sensitivity of each protein family to heat-induced precipitation. Haze-forming precipitates were removed by centrifugation, and proteins in the pellets (results non shown) and non-precipitated proteins in the heat-treated wine (Fig. 4) were analysed and quantied by 1D-electrophoresis and image analysis. Results showed differences between the various protein families. The most heat-unstable were found to be glucanases (band 2); most of them were precipitated by a 30 min heat-treatment at a temperature as low as 40 C. By contrast, temperatures higher than 60 C were needed to signicantly affect invertases (band 1). At 70 C an important reduction of the invert-

ase band was observed and most of them were removed when the temperature was raised to 80 C. Chitinases (bands 3 and 4) and thaumatin-like proteins (bands 5, 6) exhibited different behaviours, depending on the considered structures. Increasing the temperature between 40 and 80 C progressively induced an increasing precipitation of chitinases in band 4, most being eliminated after 30 min at 70 C. Two different behaviours were observed for chitinases in band 3: most of them were highly sensitive to heat-induced precipitation (85% were precipitated at 40 C, as glucanases) but others (15%) remained unaffected by temperatures below 80 C. Thaumatin-like proteins were broadly more stable than chitinases, in accordance with previous results (Waters et al., 1992). The disparities observed here for chitinases and thaumatins could be related to the strong micro-heterogeneities evidenced between structurally similar wine proteins (Monteiro et al., 2001). Such micro-heterogeneities, which cannot be resolved by techniques based on separation according to the mass or the charge, were attributed to the limited proteolysis of common precursors during the winemaking process, generating a large number of structurally related but different polypeptides. Such a limited proteolysis is sufcient to induce subtle changes in the balance of intramolecular forces that affect protein conformational stability (Murphy & Kendrick, 2007). In the present conditions a large part of the proteins in the band 7 (40%) did not precipitate, even for temperatures as high as 80 C. Proteins in band 7 were thaumatins (about 4 mg l1), along with low molecular weight (14 kDa) proteins (about 27 mg l1), not separated by the applied experimental conditions (12% acrylamide gel). In most cases, heat-induced precipitation of wine proteins is studied using heat-tests performed at high temperature. The most widely used is that proposed by Pocock and Rankine (1973), which consists in a 6 h 80 C treatment followed by cooling down during 12 h at 4 C. Present results evidence that heat-induced aggregation is prone to occur for much less drastic conditions than those usually applied in these tests. They also emphasised the high heatsensitivity of b-glucanases. This last protein class is not always found in white wines, where its presence was underlined only

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31

30C

60C

40C

50C

70C

80C

160 B 140 120 % of the initial content wine 100 80 60 40 20 0 1 2 3 4 band

Fig. 4. Protein patterns in the I10 Chardonnay wine following to 30 min heat-treatments at different temperatures. (A) SDSPAGE Electrophoresis (12% acrylamide resolving gel, Coomassie staining) performed on aliquots of heated-wine supernatants after cooling down and haze removal by centrifugation (molecular weight standard proteins on the right). (B) Protein contents in the supernatants as quantied by image analysis, in percentage of each protein band with regards to the initial wine. Band 1: invertases (initial content 12 mg l1); band 2: glucanases (initial content 9 mg l1); bands 3 and 4: chitinases (initial contents 8 and 11 mg l1, respectively); bands 5, 6: thaumatin-like proteins (initial contents 17 and 54 mg l1, respectively); band 7 (31 mg l1): low molecular weight unknown proteins (14 kDa, 87%) and thaumatins (13%), not separated here due to the experimental conditions (acrylamide percentage in gel of 12% and resolving gel length 60 mm).

wine

MW

30C 40C 50C 60C 70C 80C

three times (Cilindre et al., 2007; Esteruelas et al., 2009). It was recently found to be involved in naturally occurring hazes that had formed in a Sauvignon wine, along with VVTL1 thaumatin-like protein and ripening-related protein grip22 precursor (Esteruelas et al., 2009). Proteins in these spontaneous hazes were associated with phenolics and polysaccharides, present in quantitatively comparable levels. It is important to mention that b-glucanases in the I10 wine exhibited signicantly higher isoelectric point (IEP) values than the other protein fractions (Fig. 3), and were then more distant from their IEP in the wine pH range. Heat-induced precipitation of proteins involves two distinct phenomena, protein unfolding and colloidal aggregation, which both are strongly inuenced by protein charge (Chi et al., 2003). The inuence of that charge on both intramolecular and intermolecular interactions is however complex and cannot be discussed on the only basis of protein isoelectric point; it also depends on the overall charge density and on the charged group distribution within the structure (random or anisotropic). Such structural information, along with information related to the other wine macromolecules (Vernhet,

Pellerin, Prieur, Osmianski, & Moutounet, 1996), is needed to identify the exact physico-chemical mechanisms involved in protein instabilities in wines. 3.4. Protein adsorption on bentonite Bentonite ning is the most common treatment applied to achieve white wine stabilization with regards to protein haze. One millilitre aliquots of the Chardonnay wine were treated with increasing bentonite doses, up to 150 g hl1. Adsorption was studied by the analysis of residual proteins in the ned wine along with that of the adsorbed proteins. More than 50% of the whole protein content was removed with 50 g hl1 bentonite (Fig. 5). Increasing this dose up to 100 g hl1 resulted in the removal of 85% of the protein content. About 15% of this protein content remained non-adsorbed even for a bentonite concentration as high as 150 g hl1, indicating that the concerned structures only have low afnity for the clay particles. Residual proteins all belonged to the thaumatin-like family. Protein patterns clearly showed selectivity in pro-

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F.-X. Sauvage et al. / Food Chemistry 118 (2010) 2634

100

80 proteine distribution (%)

60

40

20

0 0 50 100
-1

150

bentonite dose (g.hL )

Fig. 5. Quantication of adsorbed (j) and non-adsorbed (s) proteins in the I10 wine after bentonite ning. Results are expressed in % of the whole protein content.

tein adsorption by bentonite (Fig. 6). Glucanases were adsorbed rst, along with part of the thaumatin-like and low MW (14 kDa) proteins (in the order of 20%). Doses as low as 6 g hl1 were enough to achieve glucanase depletion in the experimental wine. Chitinase adsorption started from 8 to 10 g hl1 bentonite, progressively increased with increasing clay doses and was complete at 60 g hl1. The adsorption of thaumatin-like and 14 kDa proteins continued in parallel. A plateau value (70% adsorption) was reached for thaumatins at bentonite doses between 60 and 80 g hl1, while at 80 g hl1 the depletion of 14 kDa proteins was achieved. Invertase adsorption only began at 20 g hl1 and was total at 100 g hl1. Differences between wine proteins with regards to their removal by

bentonite are in accordance with the earlier observations of Hsu and Heatherbell (1987). However in their work, the protein identities were still unknown. Depletion of the protein fractions in the I10 wine as a function of bentonite concentration has to be compared to their sensitivity towards heat-induced precipitation. Glucanases, that exhibited the highest afnity for the clay, were the most heat-sensitive. Invertases, that needed the highest bentonite doses to be removed, were shown to be the most resistant to heat-induced precipitation. Chitinases, low molecular weight proteins and thaumatins exhibited intermediate behaviours, as observed with heat trials, and a specic part of the thaumatin (%30%) remained unaffected for bentonite doses lower than 150 g hl1 (it must be noted that this adsorption is achieved at a dose of 200 g hl1). Protein adsorption at interfaces often involves structural changes (partial or total unfolding) due to the formation of physico-chemical bonds between protein amino-acids and surface interaction sites (Haynes & Norde, 1995). Such structural changes have been evidenced for protein adsorption on negatively charged montmorillonites (main component of bentonites) when adsorption occur at pH values lower than the protein isoelectric point (Gougeon et al., 2003; Servageant-Noinville, Revault, Quiquampoix, & Baron, 2000). The relationship between protein adsorption on bentonite and their sensitivity to heat-induced precipitation could then be linked to their inherent conformational stability. As already discussed, the different behaviours observed in a given protein family, especially important for chitinases and thaumatins, could be related to micro-heterogeneities generated between structurally similar proteins by grape processing (Monteiro et al., 2001). 3.5. Consequences for stability tests and bentonite ning trials Though the use of heat-stability tests to warrant white wine stability during transport and storage is a prevalent practice, the question of how they really predict situations that may occur during wine storage and transport is not yet solved. Indeed, only few works have been devoted to the study of the relationship between test procedures and long term stability of wines in real (long-time

120

100

adsorbed protein (%)

80

60 glucanases 40 chitinases thaumatins 20 invertases unknowns 0 0 20 40 60 80 100

-1

120

140

160

bentonite dose (g.hL )

Fig. 6. Percentage of adsorbed proteins as a function of the bentonite dose, represented for each different protein family. Results showed selectivity in protein removal by the clay particles.

F.-X. Sauvage et al. / Food Chemistry 118 (2010) 2634

33

low temperature) conditions (Pocock & Waters, 2006). The fact that the same tests, associated with bentonite ning trials, are used to determine the bentonite doses needed to stabilise wines raises the question of the relevance of these doses. Solving this question is a complex task due to wine variability and to incomplete knowledge concerning: (i) the precise structures and physico-chemical properties of wine proteins and, (ii) the mechanisms actually involved in their instability, including the inuence of non-protein compounds. Results of the present work provide however some information to direct further studies devoted to the rationalisation of heat-test use. With the experimental Chardonnay wine, a correlation was found between protein sensitivity to heat-induced precipitation and their removal by bentonite; the most heat-sensitive fractions were those that were also adsorbed rst. These fractions are also those most likely to precipitate under excessive temperature exposure during practical storage and transport conditions. It could then be concluded that usual heat-tests, which induce the precipitation of almost all wine proteins including invertases, lead to an overestimation of the bentonite doses required for stabilization. These doses should be reasoned considering the different sensitivity of wine proteins and the conditions actually met in bottled wines. Additional information is needed to be in position to propose such less drastic heat-stability test conditions. First, heattests in the present study were performed at different temperatures but for a given incubation duration of 30 min. As temperature is known to affect protein unfolding rates, kinetics of heat-induced precipitation of the different protein classes have to be determined for different temperatures. Moreover, results must be validated using a large selection of wines because: (i) the wine composition is known to inuence protein heat-stability and aggregation; (ii) if most wines were shown to contain the same set of structurally related proteins, the micro-heterogeneities evidenced within a same protein family (Monteiro et al., 2001) may be wine-dependent and induce conicting results. Finally, all these results will have to be correlated with realistic storage conditions to validate the new test. Acknowledgements The authors thank VINIFLHOR for their nancial support. References
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