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Enzymatic hydrolysis of thermo-sensitive grape proteins by a yeast protease as revealed by a proteomic approach
FRIN-04523; No of Pages 4 Food Research International xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Food Research International journal homepage: www.elsevier.com/locate/foodres
Enzymatic hydrolysis of thermo-sensitive grape proteins by a yeast protease as revealed by a proteomic approach Buchra Younes a,⁎, Clara Cilindre a, b, Philippe Jeandet a, Yann Vasserot a a b
Université de Reims, Faculté des Sciences, URVVC EA 4707, Laboratoire d’Oenologie et de Chimie Appliquée, BP 1039, 51687 Reims Cedex 02, France Université de Reims, Equipe Effervescence (GSMA), UMR CNRS 7331, BP 1039, 51687 Reims Cedex 02, France
a r t i c l e
i n f o
Article history: Received 12 July 2012 Received in revised form 28 January 2013 Accepted 30 January 2013 Available online xxxx Keywords: Saccharomyces cerevisiae yeast Proteolytic activity Grape protein 2D-E Wine Haze
a b s t r a c t Grape juice protein hydrolysis can be proposed as one of the potential alternative methods to prevent haze wine formation. Saccharomyces cerevisiae PlR1 is a wild yeast strain of grapes able to secrete an acidic proteolytic activity during growth on a synthetic medium with proline. The aim of the present work was to confirm, by SDS-PAGE and two-dimensional electrophoresis (2D-E), the hydrolysis of some grape juice proteins after incubation with a S. cerevisiae PlR1 culture supernatant, and then to identify these hydrolyzed proteins by nano-LC–MS/MS analysis. Results obtained showed that hydrolyzed proteins correspond to pathogenesis-related (PR) proteins, in particular, thaumatin-like (TL) proteins and chitinases. Some of these PR proteins are likely to be implicated in the wine haze formation. These proteins will be isolated and characterized in order to explain why they are sensitive to enzymatic hydrolysis. Otherwise, the protease will be purified, sequenced and cloned in yeast in order to improve the wine stability with regard to proteic haze. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Protein haze is a problem which may occur in both white and rosé wines following the precipitation of heat-unstable soluble proteins that may become insoluble and precipitate in bottle causing the formation of undesirable haze or deposits. The mechanism of haze formation is not entirely understood but in general, it is thought that it is the result of the thermic denaturation of wine proteins due to unfavorable storage conditions (Dawes, Boyes, Keene, & Heatherbell, 1994). Otherwise, this problem can be induced by different physico-chemical factors such as ethanol and phenolic concentrations and pH (Sarmento, Oliveira, Slatner, & Boulton, 2000). It was also evidenced that wine proteins alone cannot form haze without the presence of other compounds of low molecular weight such as factor(s) X (Pocock, Alexander, Hayasaka, Jones, & Waters, 2007). Moreover, haze formation does not depend on the total protein concentration but on a specific grape protein fraction (Lamikanra & Inyang, 1988) with a molecular mass between 13 and 30 kDa and a low isoelectric point (4.1–5.8) (Hsu & Heatherbell, 1987; Waters, Wallace, & Williams, 1992). These fractions have been identified as pathogenesis related (PR) proteins, among which thaumatin-like (TL) proteins and chitinases, the major PR proteins responsible for haze formation in wine (Waters, Hayasaka, Tattersall, Adams, & Williams, 1998). This phenomenon of haze formation is commonly prevented using bentonite. Since this practice presents disadvantages (lack of specificity, deleterious ⁎ Corresponding author. Tel./fax: +33 3 26 91 33 40. E-mail address: [email protected] (B. Younes).
effect on the sensory properties, loss of wine volume, waste disposal problem) (Waters et al., 2005), alternative techniques have been proposed (Flores, Heatherbell, & McDaniel, 1990; Pachova, Ferrando, Guell, & Lopez, 2002; Pocock, HØj, Adams, Kwiatkowski, & Waters, 2003; Vincenzi, Polesani, & Curioni, 2005; Waters, Pellerin, & Brillouet, 1994; Weetall, Zelko, & Bailey, 1984). One of them is the addition of proteolytic enzymes (Waters et al., 1992). But little or no activity against wine proteins was detected at normal winemaking temperatures (15–18 °C) (Feuillat & Ferrari, 1982; Modra & Williams, 1988). According to Waters, Peng, Pocock, and Williams (1995), PR proteins are known to be naturally resistant to hydrolysis by exogenous proteases. Nevertheless, some proteases (trenolin and porcine pepsine) were active against PR proteins after incubation at optimal conditions (45 °C) (Pocock et al., 2003). On the other hand, proteins, identified by capillary zone electrophoresis, were found to be protease-sensitive following incubation at optimal conditions for the proteolytic activities tested (Dizy & Bisson, 1999), suggesting that PR wine proteins are not intrinsically resistant to protease degradation. It should be noted that the operating conditions (pH, temperature, enzyme doses, incubation time and nature of proteins) vary between studies and may account for these discrepancies. A possible source of proteases may be wine yeast. It is known that non-Saccharomyces yeast strains naturally associated with grapes can secrete acidic proteases (Strauss, Jolly, Lambrechts, & Van Rensburg, 2001). Some of these proteases were active against wine proteins (Lagace & Bisson, 1990; Rensburg & Pretorius, 2000), but none of them were specific of haze-forming protein hydrolysis. Contrary to non-Saccharomyces yeast strains, fermentative yeast of the
0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.01.063
Please cite this article as: Younes, B., et al., Enzymatic hydrolysis of thermo-sensitive grape proteins by a yeast protease as revealed by a proteomic approach, Food Research International (2013), http://dx.doi.org/10.1016/j.foodres.2013.01.063
2
B. Younes et al. / Food Research International xxx (2013) xxx–xxx
genus Saccharomyces do not secrete any acidic proteolytic activity (Ogrydziak, 1993). However, Saccharomyces cerevisiae PlR1, a wild yeast strain isolated from Pinot noir grapes, was recently shown to secrete an acidic proteolytic activity whose optimal pH and temperature conditions were 2.5 and 38 °C, respectively (Younes et al., 2011). The aim of this work was to study the impact of the extracellular proteolytic activity produced by S. cerevisiae PlR1 on grape juice proteins by getting rid of the matrix must or wine, and to identify precisely the proteins sensitive to this activity in order to know if some of them could be associated to protein haze. Protein identification was realized using 2D-E followed by nano-LC–MS/MS analysis. 2. Materials and methods
stained with silver nitrate (Rabilloud, 1990) and colloidal Coomassie Blue (CBB) G-250 (Candiano et al., 2004). 2.5. Image analysis Digitized images were obtained using the GS-800 scanner (Bio-Rad). Computerized SDS-PAGE analysis (band detection) was performed using Quantity One 4.6.2 software (Bio-Rad) and computerized 2D-E gel analysis, (spot detection and quantification) was performed using the PDQuest Basic 8.0.1 software (Bio-Rad). Selected proteins features were modeled as Gaussians, and the relative optical densities (OD) were computed. Differences in spots intensities in gels before and after incubation were calculated and reported as percent of intensity before incubation.
2.1. Yeast strain, culture conditions and supernatant preparation 2.6. In-gel digestion and nano-LC–MS/MS analysis S. cerevisiae PlR1 has been isolated following spontaneous fermentation of a Pinot noir grape juice. Culture conditions and supernatant preparation were as previously described (Younes et al., 2011). Proline was used as the only nitrogen source for yeast growth.
Spots of interest were excised manually. Nano-LC–MS/MS analysis was performed as described by Jégou et al. (2009). 3. Results and discussion
2.2. Protein preparation Ammonium sulfate (NH4)2SO4 (80% saturation) was added to a Chardonnay grape juice (Champagne area, France) and proteins were allowed to precipitate at 4 °C overnight under agitation. The precipitate was collected by centrifugation (9500 g, 15 min, 4 °C). The pellet was dissolved in a minimum volume of 30 mM acetic acid before dialyzing 3 times against Milli-Q water at 4 °C using regenerated cellulose dialysis membranes with a molecular mass cut off 10 kDa (spectra/por dialysis membrane, USA). The retentate was filtered through a 0.45 μm membrane (Millipore, USA). The final concentration of must proteins was estimated as 940 μg/ml equivalents of BSA (Bradford, 1976). 2.3. Proteolytic activity 80 μl of the grape juice protein extract was mixed with 400 μl of the culture supernatant in a final volume of 1 ml of McIlvaine's buffer (0.1 M citric acid, 0.2 M disodium phosphate), pH 3.5, and incubated at 38 °C. Samples were collected at 0, 24 and 48 h and the reaction was stopped by freezing at − 20 °C. Grape juice protein hydrolysis was followed by SDS-PAGE and 2D-E. Simultaneously, two blanks were mixed with McIlvaine's buffer, and incubated under the same conditions. The first one corresponded to grape juice proteins and the second one corresponded to the culture supernatant. Three independent experiments were done. 2.4. Electrophoretic analyses
To highlight the effect of S. cerevisiae PlR1 proteolytic activity, SDS-PAGE was first performed (Fig. 1). The profile of the first blank sample (Fig. 1, lane a) before incubation appears to be identical with that of the grape juice proteins before precipitation (Fig. 1, lane j). Therefore, 80% saturation of ammonium sulfate is efficient to precipitate all grape juice proteins. When grape juice proteins were incubated alone, some protein intensities, mainly those of 25 and 27 kDa, decreased, leading to the nearly complete disappearance of the protein at 27 kDa after 48 h (Fig. 1, lane g). When grape juice proteins were incubated with culture supernatant, the disappearance of the proteins at 25 and 27 kDa was accelerated (Fig. 1, lane e). Both of them completely disappeared, the first one after 48 h and the second one after 24 h. Moroever the protein of 30 kDa which was shown to be naturally thermo-resistant (Fig. 1, lane a, d, g) completely disappeared after 24 h (Fig. 1, lane e). The disappearance of these different bands seems to confirm the ability of the activity secreted by S. cerevisiae PlR1 to hydrolyze some grape juice proteins at optimal conditions. To further characterize these proteins, 2D-E analyses were performed. The profile of grape juice proteins mixed with the culture supernatant before incubation (Fig. 2C) was enriched with several kDa MW
a
b
c
d
e
f
g
h
i
j
150 75 50
2.4.1. SDS-PAGE Vertical SDS-PAGE was performed in a Mini-Protean 3 electrophoresis cell (Bio-Rad, USA) using 12% resolving gel (Laemmli, 1970). 6 μl of the reaction mixture was loaded at each lane and proteins were silver-stained according to Rabilloud (1990). 2.4.2. 2D-E Protein analysis with 2D-E was performed according to the method of Cilindre et al. (2008) with modifications. Precast dry polyacrylamide 7 cm length gels (ReadyStrip IPG, pH 3–6, Bio Rad, USA) were rehydrated with 125 μl of a mixture containing proteins diluted in a sample buffer. Isoelectric focusing (IEF) was conducted at 20 °C in an IPGphor unit (Amersham Pharmacia, Sweden) as follows: a linear increase from 50 to 4000 V to give a total of 10,000 Vh. Electrophoretic migration along the second dimension was performed using a Mini-Protean 3 electrophoresis cell (Bio-Rad, USA). Analytical and preparative gels were respectively
37 25 20 15
0h
24h
48h
Fig. 1. Evidence, by SDS-PAGE, for the proteolytic activity of the culture supernatant of S. cerevisiae PlR1 against grape juice proteins after 24 and 48 h of incubation at 38 °C and pH 3.5. Lanes a, d and g: grape juice proteins precipitated by ammonium sulfate; lanes c, f and i: culture supernatant of S. cerevisiae PlR1; lanes b, e and h: grape juice proteins mixed with the culture supernatant of S. cerevisiae PlR1; lane j, profile of grape juice proteins before precipitation. Molecular weight standard are given on the left side of the gel.
Please cite this article as: Younes, B., et al., Enzymatic hydrolysis of thermo-sensitive grape proteins by a yeast protease as revealed by a proteomic approach, Food Research International (2013), http://dx.doi.org/10.1016/j.foodres.2013.01.063
B. Younes et al. / Food Research International xxx (2013) xxx–xxx
A
3
IEF
6
B
SDS-PAGE
kDa
3
150 75 50
4
3
37 25 20
1
C
3
2
IEF
6
D
SDS-PAGE
kDa
5
150 75 50 37 25 20
Fig. 2. 2D-E analysis of grape juice proteins precipitated by ammonium sulfate without S. cerevisiae PlR1 culture supernatant before (A) and after (B) 48 h of incubation; or with S. cerevisiae PlR1 culture supernatant before (C) or after (D) 48 h on incubation at pH 3.5 and 38 °C. Spots circled correspond to yeast proteins. Molecular weight standard proteins are given on the left side of gels.
spots (circled) when compared with the profile of grape juice proteins alone (Fig. 2A). These supplemental spots correspond to proteins found in the supernatant of S. cerevisiae PlR1 (data not shown). After 48 h of incubation, five spots numbered from 1 to 5 (Fig. 2A) corresponding to grape juice proteins have lost differently their intensities (Fig. 2B and D). Differences in spot intensities strictly caused by thermo-sensitivity (i.e. differences in spot intensities between gels A and B) showed that spots 3 and 5 seem to be the most thermo-sensitive ones (they lose, respectively, 71 and 70 % of their relative intensity) (Table 1), while spots 1, 2 and 4 seem to be more thermo-stable. In the presence of the yeast supernatant (Fig. 2D), these spots have either completely disappeared such as spot numbers 3, 4 and 5, or were found with a lower intensity in the case of spot numbers 1 and 2 (71 and 26 % respectively). So, it is clear that the sensitivity of these spots to both temperature and enzymatic hydrolysis is different and is likely linked to the nature of the protein. Nano-LC–MS/MS analysis of these spots, excised from the preparative 2D-E gel, allows us to identify four proteins corresponding to Vitis vinifera grape proteins. Two proteins VVTL1 (gi|2213852) and
a class IV endochitinase (gi|2306811) which belong, respectively, to the PR-5 and the PR-3 families, have already been identified in our previous work (Younes et al., 2011) and were sensitive to hydrolysis by the S. cerevisiae PlR1 proteolytic activity secreted during alcoholic fermentation. Two new proteins: a putative thaumatin-like (gi|7406716), which belongs to the PR-5 family and a hypothetical protein (gi|225426795) are also characterized. A BLAST search showed a high homology of this last protein with a thaumatin-like protein from V. vinifera (gi|225426795). Those identified proteins, previously found in a healthy wine of Chardonnay (Cilindre et al., 2008), are heat-sensitive pathogenesis-related proteins. Marangon, Vincenzi, Lucchetta, and Curioni (2010), reported that TL proteins are more sensitive to temperature than chitinases, while Breiteneder (2004) indicated that they are generally resistant to proteases and to heat denaturation. Increasing precipitation of chitinases can be induced by an increasing temperature, while TL proteins are more stable (Sauvage, Bach, Moutounet, & Vernhet, 2010). In our study, both spot numbers 3 and 5, the most sensitive to temperature, contain a class IV endochitinase that supports the hypothesis that the chitinase is less heat-stable than the other PR proteins identified. At least five isoforms
Table 1 Proteins of grape juice identified by nano-LC–MS/MS after 2-DE. Spot number
Relative optical density (%) after incubationa
Exp mass (kDa)/pI
Protein name (organism)
Hyp mass (kDa)/pI
Accession number
Number of peptides matched
Sequence coverage (%)
Hypothetical protein (V. vinifera) VVTL1 (V. vinifera) Putative thaumatine-like (V. vinifera) Class IV endochitinase (V. vinifera) VVTL1 (V. vinifera) Class IV endochitinase (V. vinifera) Class IV endochitinase (V. vinifera)
23.9/4.56 23.9/5.09 24.0/4.94 27.3/5.01 23.9/5.09 27.3/5.01 27.3/5.01
gi|225426795 gi|2213852 gi|7406716 gi|2306811 gi|2213852 gi|2306811 gi|2306811
7 6 10 4 4 7 3
41 37 49 24 23 35 22
Without supernatant
With supernatant
1
100
71 ± 7
27/4.5
2 3
86 ± 4 28 ± 19
26 ± 4 0
28/4.8 34/4.8
4 5
90 ± 2 30 ± 1
0 0
34/4.95 34/5.2
a
Values are the mean of three determinations. Means and standard deviations were determined by Microsoft Excel.
Please cite this article as: Younes, B., et al., Enzymatic hydrolysis of thermo-sensitive grape proteins by a yeast protease as revealed by a proteomic approach, Food Research International (2013), http://dx.doi.org/10.1016/j.foodres.2013.01.063
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of chitinase were revealed in plant tissues (Derckel, Legendre, Audran, Haye, & Lambert, 1996) and according to Marangon et al. (2011) every isoform has a different thermal sensitivity which may account for the different behavior observed among the chitinases identified. In spite of the effect of temperature on PR grape juice protein stability, the supplemental disappearance of protein spots in the presence of the supernatant of S. cerevisiae PlR1 containing the proteolytic activity, confirms its capacity to hydrolyze some PR proteins implied in haze wine formation. Acknowledgments The authors would like to thank the Syrian government and the A.R.O.C.U. association for their support. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.foodres.2013.01.063. References Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254. Breiteneder, H. (2004). Thaumatin-like proteins — A new family of pollen and fruit allergens. Allergy, 59, 479–481. Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggeri, G. M., Carnemolla, B., et al. (2004). Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis, 25, 1327–1333. Cilindre, C., Jégou, S., Hovasse, A., Schaeffer, C., Castro, A. J., Clément, C., et al. (2008). Proteomic approach to identify champagne wine proteins as modified by Botrytis cinerea infection. Journal of Proteome Research, 7, 1199–1208. Dawes, H., Boyes, S., Keene, J., & Heatherbell, D. (1994). Protein instability of wines: Influence of protein isoelectric point. American Journal of Enology and Viticulture, 45, 319–326. Derckel, J. -P., Legendre, L., Audran, J. -C., Haye, B., & Lambert, B. (1996). Chitinases of the grapevine (Vitis vinifera L.): Five isoforms induced in leaves by salicylic acid are constitutively expressed in other tissues. Plant Science, 119, 31–37. Dizy, M., & Bisson, L. F. (1999). White wine protein analysis by capillary zone electrophoresis. American Journal of Enology and Viticulture, 50, 120–127. Feuillat, M., & Ferrari, G. (1982). Hydrolyse enzymatique des protéines du raisin en vinification. Comptes rendus des seances — Academie d'agriculture de France, 13, 1070–1075. Flores, J. H., Heatherbell, D. A., & McDaniel, M. R. (1990). Ultrafiltration of wine: Effect of ultrafiltration on white Riesling and Gewürztraminer wine composition and stability. American Journal of Enology and Viticulture, 41, 207–214. Hsu, J. C., & Heatherbell, D. A. (1987). Heat-unstable proteins in wine. I. Characterization and removal by bentonite fining and heat treatment. American Journal of Enology and Viticulture, 38, 11–16. Jégou, S., Conreux, A., Villaume, S., Hovasse, A. s., Schaeffer, C., Cilindre, C., et al. (2009). One step purification of the grape vacuolar invertase. Analytica Chimica Acta, 638, 75–78. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.
Lagace, L. S., & Bisson, L. F. (1990). Survey of yeast acid proteases for effectiveness of wine haze reduction. American Journal of Enology and Viticulture, 41, 147–155. Lamikanra, O., & Inyang, I. D. (1988). Temperature influence on muscadine wine protein characteristics. American Journal of Enology and Viticulture, 39, 113–116. Marangon, M., Van Sluyter, S. C., Neilson, K. A., Chan, C., Haynes, P. A., Waters, E. J., et al. (2011). Roles of grape thaumatin-like protein and chitinase in white wine haze formation. Journal of Agricultural and Food Chemistry, 59, 733–740. Marangon, M., Vincenzi, S., Lucchetta, M., & Curioni, A. (2010). Heating and reduction affect the reaction with tannins of wine protein fractions differing in hydrophobicity. Analytica Chimica Acta, 660, 110–118. Modra, E. J., & Williams, P. J. (1988). Are proteases active in wines and juices? The Australian Grapegrower & Winemaker, 292, 42–46. Ogrydziak, D. M. (1993). Yeast extracellular proteases. Critical Reviews in Biotechnology, 13, 1–55. Pachova, V., Ferrando, M., Guell, C., & Lopez, F. (2002). Protein adsorption onto metal oxide materials in white wine model systems. Journal of Food Science, 67, 2118–2121. Pocock, K. F., Alexander, G. M., Hayasaka, Y., Jones, P. R., & Waters, E. J. (2007). Sulfate — A candidate for the missing essential factor that is required for the formation of protein haze in white wine. Journal of Agricultural and Food Chemistry, 55, 1799–1807. Pocock, K. F., HØj, P. B., Adams, K. S., Kwiatkowski, M. J., & Waters, E. J. (2003). Combined heat and proteolytic enzyme treatment of white wines reduces haze forming protein content without detrimental effect. Australian Journal of Grape and Wine Research, 9, 56–63. Rabilloud, T. (1990). Mechanisms of protein silver staining in polyacrylamide gels: A 10-year synthesis. Electrophoresis, 11, 785–794. Rensburg, P., & Pretorius, I. S. (2000). Enzymes in winemaking: Harnessing natural catalysts for efficient biotransformations — A review. South African Journal of Enology and Viticulture, 21, 52–73. Sarmento, M. R., Oliveira, J. C., Slatner, M., & Boulton, R. B. (2000). Influence of intrinsic factors on conventional wine protein stability tests. Food Control, 11, 423–432. Sauvage, F. -X., Bach, B., Moutounet, M., & Vernhet, A. (2010). Proteins in white wines: Thermo-sensitivity and differential adsorbtion by bentonite. Food Chemistry, 118, 26–34. Strauss, M. L. A., Jolly, N. P., Lambrechts, M. G., & Van Rensburg, P. (2001). Screening for the production of extracellular hydrolytic enzymes by non-Saccharomyces wine yeasts. Journal of Applied Microbiology, 91, 182–190. Vincenzi, S., Polesani, M., & Curioni, A. (2005). Removal of specific protein components by chitin enhances protein stability in a white wine. American Journal of Enology and Viticulture, 56, 246–254. Waters, E. J., Alexander, G., Muhlack, R., Pocock, K. F., Colby, C., O'Neill, B. K., et al. (2005). Preventing protein haze in bottled white wine. Australian Journal of Grape and Wine Research, 11, 215–225. Waters, E. J., Hayasaka, Y., Tattersall, D. B., Adams, K. S., & Williams, P. J. (1998). Sequence analysis of grape (Vitis vinifera) berry chitinases that cause haze formation in wines. Journal of Agricultural and Food Chemistry, 46, 4950–4957. Waters, E. J., Pellerin, P., & Brillouet, J. -M. (1994). A Saccharomyces mannoprotein that protects wine from protein haze. Carbohydrate Polymers, 23, 185–191. Waters, E. J., Peng, Z., Pocock, K. F., & Williams, P. J. (1995). Proteins in white wine, II: Their resistance to proteolysis is not due to either phenolic association or glycosylation. Australian Journal of Grape and Wine Research, 1, 94–99. Waters, E. J., Wallace, W., & Williams, P. J. (1992). Identification of heat-unstable wine proteins and their resistance to peptidases. Journal of Agricultural and Food Chemistry, 40, 1514–1519. Weetall, H. H., Zelko, J. T., & Bailey, L. F. (1984). A new method for the stabilization of white wine. American Journal of Enology and Viticulture, 35, 212–215. Younes, B., Cilindre, C., Villaume, S., Parmentier, M., Jeandet, P., & Vasserot, Y. (2011). Evidence for an extracellular acid proteolytic activity secreted by living cells of Saccharomyces cerevisiae PlR1: Impact on grape proteins. Journal of Agricultural and Food Chemistry, 59, 6239–6246.
Please cite this article as: Younes, B., et al., Enzymatic hydrolysis of thermo-sensitive grape proteins by a yeast protease as revealed by a proteomic approach, Food Research International (2013), http://dx.doi.org/10.1016/j.foodres.2013.01.063
Contents lists available at SciVerse ScienceDirect
Food Research International journal homepage: www.elsevier.com/locate/foodres
Enzymatic hydrolysis of thermo-sensitive grape proteins by a yeast protease as revealed by a proteomic approach Buchra Younes a,⁎, Clara Cilindre a, b, Philippe Jeandet a, Yann Vasserot a a b
Université de Reims, Faculté des Sciences, URVVC EA 4707, Laboratoire d’Oenologie et de Chimie Appliquée, BP 1039, 51687 Reims Cedex 02, France Université de Reims, Equipe Effervescence (GSMA), UMR CNRS 7331, BP 1039, 51687 Reims Cedex 02, France
a r t i c l e
i n f o
Article history: Received 12 July 2012 Received in revised form 28 January 2013 Accepted 30 January 2013 Available online xxxx Keywords: Saccharomyces cerevisiae yeast Proteolytic activity Grape protein 2D-E Wine Haze
a b s t r a c t Grape juice protein hydrolysis can be proposed as one of the potential alternative methods to prevent haze wine formation. Saccharomyces cerevisiae PlR1 is a wild yeast strain of grapes able to secrete an acidic proteolytic activity during growth on a synthetic medium with proline. The aim of the present work was to confirm, by SDS-PAGE and two-dimensional electrophoresis (2D-E), the hydrolysis of some grape juice proteins after incubation with a S. cerevisiae PlR1 culture supernatant, and then to identify these hydrolyzed proteins by nano-LC–MS/MS analysis. Results obtained showed that hydrolyzed proteins correspond to pathogenesis-related (PR) proteins, in particular, thaumatin-like (TL) proteins and chitinases. Some of these PR proteins are likely to be implicated in the wine haze formation. These proteins will be isolated and characterized in order to explain why they are sensitive to enzymatic hydrolysis. Otherwise, the protease will be purified, sequenced and cloned in yeast in order to improve the wine stability with regard to proteic haze. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Protein haze is a problem which may occur in both white and rosé wines following the precipitation of heat-unstable soluble proteins that may become insoluble and precipitate in bottle causing the formation of undesirable haze or deposits. The mechanism of haze formation is not entirely understood but in general, it is thought that it is the result of the thermic denaturation of wine proteins due to unfavorable storage conditions (Dawes, Boyes, Keene, & Heatherbell, 1994). Otherwise, this problem can be induced by different physico-chemical factors such as ethanol and phenolic concentrations and pH (Sarmento, Oliveira, Slatner, & Boulton, 2000). It was also evidenced that wine proteins alone cannot form haze without the presence of other compounds of low molecular weight such as factor(s) X (Pocock, Alexander, Hayasaka, Jones, & Waters, 2007). Moreover, haze formation does not depend on the total protein concentration but on a specific grape protein fraction (Lamikanra & Inyang, 1988) with a molecular mass between 13 and 30 kDa and a low isoelectric point (4.1–5.8) (Hsu & Heatherbell, 1987; Waters, Wallace, & Williams, 1992). These fractions have been identified as pathogenesis related (PR) proteins, among which thaumatin-like (TL) proteins and chitinases, the major PR proteins responsible for haze formation in wine (Waters, Hayasaka, Tattersall, Adams, & Williams, 1998). This phenomenon of haze formation is commonly prevented using bentonite. Since this practice presents disadvantages (lack of specificity, deleterious ⁎ Corresponding author. Tel./fax: +33 3 26 91 33 40. E-mail address: [email protected] (B. Younes).
effect on the sensory properties, loss of wine volume, waste disposal problem) (Waters et al., 2005), alternative techniques have been proposed (Flores, Heatherbell, & McDaniel, 1990; Pachova, Ferrando, Guell, & Lopez, 2002; Pocock, HØj, Adams, Kwiatkowski, & Waters, 2003; Vincenzi, Polesani, & Curioni, 2005; Waters, Pellerin, & Brillouet, 1994; Weetall, Zelko, & Bailey, 1984). One of them is the addition of proteolytic enzymes (Waters et al., 1992). But little or no activity against wine proteins was detected at normal winemaking temperatures (15–18 °C) (Feuillat & Ferrari, 1982; Modra & Williams, 1988). According to Waters, Peng, Pocock, and Williams (1995), PR proteins are known to be naturally resistant to hydrolysis by exogenous proteases. Nevertheless, some proteases (trenolin and porcine pepsine) were active against PR proteins after incubation at optimal conditions (45 °C) (Pocock et al., 2003). On the other hand, proteins, identified by capillary zone electrophoresis, were found to be protease-sensitive following incubation at optimal conditions for the proteolytic activities tested (Dizy & Bisson, 1999), suggesting that PR wine proteins are not intrinsically resistant to protease degradation. It should be noted that the operating conditions (pH, temperature, enzyme doses, incubation time and nature of proteins) vary between studies and may account for these discrepancies. A possible source of proteases may be wine yeast. It is known that non-Saccharomyces yeast strains naturally associated with grapes can secrete acidic proteases (Strauss, Jolly, Lambrechts, & Van Rensburg, 2001). Some of these proteases were active against wine proteins (Lagace & Bisson, 1990; Rensburg & Pretorius, 2000), but none of them were specific of haze-forming protein hydrolysis. Contrary to non-Saccharomyces yeast strains, fermentative yeast of the
0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.01.063
Please cite this article as: Younes, B., et al., Enzymatic hydrolysis of thermo-sensitive grape proteins by a yeast protease as revealed by a proteomic approach, Food Research International (2013), http://dx.doi.org/10.1016/j.foodres.2013.01.063
2
B. Younes et al. / Food Research International xxx (2013) xxx–xxx
genus Saccharomyces do not secrete any acidic proteolytic activity (Ogrydziak, 1993). However, Saccharomyces cerevisiae PlR1, a wild yeast strain isolated from Pinot noir grapes, was recently shown to secrete an acidic proteolytic activity whose optimal pH and temperature conditions were 2.5 and 38 °C, respectively (Younes et al., 2011). The aim of this work was to study the impact of the extracellular proteolytic activity produced by S. cerevisiae PlR1 on grape juice proteins by getting rid of the matrix must or wine, and to identify precisely the proteins sensitive to this activity in order to know if some of them could be associated to protein haze. Protein identification was realized using 2D-E followed by nano-LC–MS/MS analysis. 2. Materials and methods
stained with silver nitrate (Rabilloud, 1990) and colloidal Coomassie Blue (CBB) G-250 (Candiano et al., 2004). 2.5. Image analysis Digitized images were obtained using the GS-800 scanner (Bio-Rad). Computerized SDS-PAGE analysis (band detection) was performed using Quantity One 4.6.2 software (Bio-Rad) and computerized 2D-E gel analysis, (spot detection and quantification) was performed using the PDQuest Basic 8.0.1 software (Bio-Rad). Selected proteins features were modeled as Gaussians, and the relative optical densities (OD) were computed. Differences in spots intensities in gels before and after incubation were calculated and reported as percent of intensity before incubation.
2.1. Yeast strain, culture conditions and supernatant preparation 2.6. In-gel digestion and nano-LC–MS/MS analysis S. cerevisiae PlR1 has been isolated following spontaneous fermentation of a Pinot noir grape juice. Culture conditions and supernatant preparation were as previously described (Younes et al., 2011). Proline was used as the only nitrogen source for yeast growth.
Spots of interest were excised manually. Nano-LC–MS/MS analysis was performed as described by Jégou et al. (2009). 3. Results and discussion
2.2. Protein preparation Ammonium sulfate (NH4)2SO4 (80% saturation) was added to a Chardonnay grape juice (Champagne area, France) and proteins were allowed to precipitate at 4 °C overnight under agitation. The precipitate was collected by centrifugation (9500 g, 15 min, 4 °C). The pellet was dissolved in a minimum volume of 30 mM acetic acid before dialyzing 3 times against Milli-Q water at 4 °C using regenerated cellulose dialysis membranes with a molecular mass cut off 10 kDa (spectra/por dialysis membrane, USA). The retentate was filtered through a 0.45 μm membrane (Millipore, USA). The final concentration of must proteins was estimated as 940 μg/ml equivalents of BSA (Bradford, 1976). 2.3. Proteolytic activity 80 μl of the grape juice protein extract was mixed with 400 μl of the culture supernatant in a final volume of 1 ml of McIlvaine's buffer (0.1 M citric acid, 0.2 M disodium phosphate), pH 3.5, and incubated at 38 °C. Samples were collected at 0, 24 and 48 h and the reaction was stopped by freezing at − 20 °C. Grape juice protein hydrolysis was followed by SDS-PAGE and 2D-E. Simultaneously, two blanks were mixed with McIlvaine's buffer, and incubated under the same conditions. The first one corresponded to grape juice proteins and the second one corresponded to the culture supernatant. Three independent experiments were done. 2.4. Electrophoretic analyses
To highlight the effect of S. cerevisiae PlR1 proteolytic activity, SDS-PAGE was first performed (Fig. 1). The profile of the first blank sample (Fig. 1, lane a) before incubation appears to be identical with that of the grape juice proteins before precipitation (Fig. 1, lane j). Therefore, 80% saturation of ammonium sulfate is efficient to precipitate all grape juice proteins. When grape juice proteins were incubated alone, some protein intensities, mainly those of 25 and 27 kDa, decreased, leading to the nearly complete disappearance of the protein at 27 kDa after 48 h (Fig. 1, lane g). When grape juice proteins were incubated with culture supernatant, the disappearance of the proteins at 25 and 27 kDa was accelerated (Fig. 1, lane e). Both of them completely disappeared, the first one after 48 h and the second one after 24 h. Moroever the protein of 30 kDa which was shown to be naturally thermo-resistant (Fig. 1, lane a, d, g) completely disappeared after 24 h (Fig. 1, lane e). The disappearance of these different bands seems to confirm the ability of the activity secreted by S. cerevisiae PlR1 to hydrolyze some grape juice proteins at optimal conditions. To further characterize these proteins, 2D-E analyses were performed. The profile of grape juice proteins mixed with the culture supernatant before incubation (Fig. 2C) was enriched with several kDa MW
a
b
c
d
e
f
g
h
i
j
150 75 50
2.4.1. SDS-PAGE Vertical SDS-PAGE was performed in a Mini-Protean 3 electrophoresis cell (Bio-Rad, USA) using 12% resolving gel (Laemmli, 1970). 6 μl of the reaction mixture was loaded at each lane and proteins were silver-stained according to Rabilloud (1990). 2.4.2. 2D-E Protein analysis with 2D-E was performed according to the method of Cilindre et al. (2008) with modifications. Precast dry polyacrylamide 7 cm length gels (ReadyStrip IPG, pH 3–6, Bio Rad, USA) were rehydrated with 125 μl of a mixture containing proteins diluted in a sample buffer. Isoelectric focusing (IEF) was conducted at 20 °C in an IPGphor unit (Amersham Pharmacia, Sweden) as follows: a linear increase from 50 to 4000 V to give a total of 10,000 Vh. Electrophoretic migration along the second dimension was performed using a Mini-Protean 3 electrophoresis cell (Bio-Rad, USA). Analytical and preparative gels were respectively
37 25 20 15
0h
24h
48h
Fig. 1. Evidence, by SDS-PAGE, for the proteolytic activity of the culture supernatant of S. cerevisiae PlR1 against grape juice proteins after 24 and 48 h of incubation at 38 °C and pH 3.5. Lanes a, d and g: grape juice proteins precipitated by ammonium sulfate; lanes c, f and i: culture supernatant of S. cerevisiae PlR1; lanes b, e and h: grape juice proteins mixed with the culture supernatant of S. cerevisiae PlR1; lane j, profile of grape juice proteins before precipitation. Molecular weight standard are given on the left side of the gel.
Please cite this article as: Younes, B., et al., Enzymatic hydrolysis of thermo-sensitive grape proteins by a yeast protease as revealed by a proteomic approach, Food Research International (2013), http://dx.doi.org/10.1016/j.foodres.2013.01.063
B. Younes et al. / Food Research International xxx (2013) xxx–xxx
A
3
IEF
6
B
SDS-PAGE
kDa
3
150 75 50
4
3
37 25 20
1
C
3
2
IEF
6
D
SDS-PAGE
kDa
5
150 75 50 37 25 20
Fig. 2. 2D-E analysis of grape juice proteins precipitated by ammonium sulfate without S. cerevisiae PlR1 culture supernatant before (A) and after (B) 48 h of incubation; or with S. cerevisiae PlR1 culture supernatant before (C) or after (D) 48 h on incubation at pH 3.5 and 38 °C. Spots circled correspond to yeast proteins. Molecular weight standard proteins are given on the left side of gels.
spots (circled) when compared with the profile of grape juice proteins alone (Fig. 2A). These supplemental spots correspond to proteins found in the supernatant of S. cerevisiae PlR1 (data not shown). After 48 h of incubation, five spots numbered from 1 to 5 (Fig. 2A) corresponding to grape juice proteins have lost differently their intensities (Fig. 2B and D). Differences in spot intensities strictly caused by thermo-sensitivity (i.e. differences in spot intensities between gels A and B) showed that spots 3 and 5 seem to be the most thermo-sensitive ones (they lose, respectively, 71 and 70 % of their relative intensity) (Table 1), while spots 1, 2 and 4 seem to be more thermo-stable. In the presence of the yeast supernatant (Fig. 2D), these spots have either completely disappeared such as spot numbers 3, 4 and 5, or were found with a lower intensity in the case of spot numbers 1 and 2 (71 and 26 % respectively). So, it is clear that the sensitivity of these spots to both temperature and enzymatic hydrolysis is different and is likely linked to the nature of the protein. Nano-LC–MS/MS analysis of these spots, excised from the preparative 2D-E gel, allows us to identify four proteins corresponding to Vitis vinifera grape proteins. Two proteins VVTL1 (gi|2213852) and
a class IV endochitinase (gi|2306811) which belong, respectively, to the PR-5 and the PR-3 families, have already been identified in our previous work (Younes et al., 2011) and were sensitive to hydrolysis by the S. cerevisiae PlR1 proteolytic activity secreted during alcoholic fermentation. Two new proteins: a putative thaumatin-like (gi|7406716), which belongs to the PR-5 family and a hypothetical protein (gi|225426795) are also characterized. A BLAST search showed a high homology of this last protein with a thaumatin-like protein from V. vinifera (gi|225426795). Those identified proteins, previously found in a healthy wine of Chardonnay (Cilindre et al., 2008), are heat-sensitive pathogenesis-related proteins. Marangon, Vincenzi, Lucchetta, and Curioni (2010), reported that TL proteins are more sensitive to temperature than chitinases, while Breiteneder (2004) indicated that they are generally resistant to proteases and to heat denaturation. Increasing precipitation of chitinases can be induced by an increasing temperature, while TL proteins are more stable (Sauvage, Bach, Moutounet, & Vernhet, 2010). In our study, both spot numbers 3 and 5, the most sensitive to temperature, contain a class IV endochitinase that supports the hypothesis that the chitinase is less heat-stable than the other PR proteins identified. At least five isoforms
Table 1 Proteins of grape juice identified by nano-LC–MS/MS after 2-DE. Spot number
Relative optical density (%) after incubationa
Exp mass (kDa)/pI
Protein name (organism)
Hyp mass (kDa)/pI
Accession number
Number of peptides matched
Sequence coverage (%)
Hypothetical protein (V. vinifera) VVTL1 (V. vinifera) Putative thaumatine-like (V. vinifera) Class IV endochitinase (V. vinifera) VVTL1 (V. vinifera) Class IV endochitinase (V. vinifera) Class IV endochitinase (V. vinifera)
23.9/4.56 23.9/5.09 24.0/4.94 27.3/5.01 23.9/5.09 27.3/5.01 27.3/5.01
gi|225426795 gi|2213852 gi|7406716 gi|2306811 gi|2213852 gi|2306811 gi|2306811
7 6 10 4 4 7 3
41 37 49 24 23 35 22
Without supernatant
With supernatant
1
100
71 ± 7
27/4.5
2 3
86 ± 4 28 ± 19
26 ± 4 0
28/4.8 34/4.8
4 5
90 ± 2 30 ± 1
0 0
34/4.95 34/5.2
a
Values are the mean of three determinations. Means and standard deviations were determined by Microsoft Excel.
Please cite this article as: Younes, B., et al., Enzymatic hydrolysis of thermo-sensitive grape proteins by a yeast protease as revealed by a proteomic approach, Food Research International (2013), http://dx.doi.org/10.1016/j.foodres.2013.01.063
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B. Younes et al. / Food Research International xxx (2013) xxx–xxx
of chitinase were revealed in plant tissues (Derckel, Legendre, Audran, Haye, & Lambert, 1996) and according to Marangon et al. (2011) every isoform has a different thermal sensitivity which may account for the different behavior observed among the chitinases identified. In spite of the effect of temperature on PR grape juice protein stability, the supplemental disappearance of protein spots in the presence of the supernatant of S. cerevisiae PlR1 containing the proteolytic activity, confirms its capacity to hydrolyze some PR proteins implied in haze wine formation. Acknowledgments The authors would like to thank the Syrian government and the A.R.O.C.U. association for their support. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.foodres.2013.01.063. References Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254. Breiteneder, H. (2004). Thaumatin-like proteins — A new family of pollen and fruit allergens. Allergy, 59, 479–481. Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggeri, G. M., Carnemolla, B., et al. (2004). Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis, 25, 1327–1333. Cilindre, C., Jégou, S., Hovasse, A., Schaeffer, C., Castro, A. J., Clément, C., et al. (2008). Proteomic approach to identify champagne wine proteins as modified by Botrytis cinerea infection. Journal of Proteome Research, 7, 1199–1208. Dawes, H., Boyes, S., Keene, J., & Heatherbell, D. (1994). Protein instability of wines: Influence of protein isoelectric point. American Journal of Enology and Viticulture, 45, 319–326. Derckel, J. -P., Legendre, L., Audran, J. -C., Haye, B., & Lambert, B. (1996). Chitinases of the grapevine (Vitis vinifera L.): Five isoforms induced in leaves by salicylic acid are constitutively expressed in other tissues. Plant Science, 119, 31–37. Dizy, M., & Bisson, L. F. (1999). White wine protein analysis by capillary zone electrophoresis. American Journal of Enology and Viticulture, 50, 120–127. Feuillat, M., & Ferrari, G. (1982). Hydrolyse enzymatique des protéines du raisin en vinification. Comptes rendus des seances — Academie d'agriculture de France, 13, 1070–1075. Flores, J. H., Heatherbell, D. A., & McDaniel, M. R. (1990). Ultrafiltration of wine: Effect of ultrafiltration on white Riesling and Gewürztraminer wine composition and stability. American Journal of Enology and Viticulture, 41, 207–214. Hsu, J. C., & Heatherbell, D. A. (1987). Heat-unstable proteins in wine. I. Characterization and removal by bentonite fining and heat treatment. American Journal of Enology and Viticulture, 38, 11–16. Jégou, S., Conreux, A., Villaume, S., Hovasse, A. s., Schaeffer, C., Cilindre, C., et al. (2009). One step purification of the grape vacuolar invertase. Analytica Chimica Acta, 638, 75–78. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.
Lagace, L. S., & Bisson, L. F. (1990). Survey of yeast acid proteases for effectiveness of wine haze reduction. American Journal of Enology and Viticulture, 41, 147–155. Lamikanra, O., & Inyang, I. D. (1988). Temperature influence on muscadine wine protein characteristics. American Journal of Enology and Viticulture, 39, 113–116. Marangon, M., Van Sluyter, S. C., Neilson, K. A., Chan, C., Haynes, P. A., Waters, E. J., et al. (2011). Roles of grape thaumatin-like protein and chitinase in white wine haze formation. Journal of Agricultural and Food Chemistry, 59, 733–740. Marangon, M., Vincenzi, S., Lucchetta, M., & Curioni, A. (2010). Heating and reduction affect the reaction with tannins of wine protein fractions differing in hydrophobicity. Analytica Chimica Acta, 660, 110–118. Modra, E. J., & Williams, P. J. (1988). Are proteases active in wines and juices? The Australian Grapegrower & Winemaker, 292, 42–46. Ogrydziak, D. M. (1993). Yeast extracellular proteases. Critical Reviews in Biotechnology, 13, 1–55. Pachova, V., Ferrando, M., Guell, C., & Lopez, F. (2002). Protein adsorption onto metal oxide materials in white wine model systems. Journal of Food Science, 67, 2118–2121. Pocock, K. F., Alexander, G. M., Hayasaka, Y., Jones, P. R., & Waters, E. J. (2007). Sulfate — A candidate for the missing essential factor that is required for the formation of protein haze in white wine. Journal of Agricultural and Food Chemistry, 55, 1799–1807. Pocock, K. F., HØj, P. B., Adams, K. S., Kwiatkowski, M. J., & Waters, E. J. (2003). Combined heat and proteolytic enzyme treatment of white wines reduces haze forming protein content without detrimental effect. Australian Journal of Grape and Wine Research, 9, 56–63. Rabilloud, T. (1990). Mechanisms of protein silver staining in polyacrylamide gels: A 10-year synthesis. Electrophoresis, 11, 785–794. Rensburg, P., & Pretorius, I. S. (2000). Enzymes in winemaking: Harnessing natural catalysts for efficient biotransformations — A review. South African Journal of Enology and Viticulture, 21, 52–73. Sarmento, M. R., Oliveira, J. C., Slatner, M., & Boulton, R. B. (2000). Influence of intrinsic factors on conventional wine protein stability tests. Food Control, 11, 423–432. Sauvage, F. -X., Bach, B., Moutounet, M., & Vernhet, A. (2010). Proteins in white wines: Thermo-sensitivity and differential adsorbtion by bentonite. Food Chemistry, 118, 26–34. Strauss, M. L. A., Jolly, N. P., Lambrechts, M. G., & Van Rensburg, P. (2001). Screening for the production of extracellular hydrolytic enzymes by non-Saccharomyces wine yeasts. Journal of Applied Microbiology, 91, 182–190. Vincenzi, S., Polesani, M., & Curioni, A. (2005). Removal of specific protein components by chitin enhances protein stability in a white wine. American Journal of Enology and Viticulture, 56, 246–254. Waters, E. J., Alexander, G., Muhlack, R., Pocock, K. F., Colby, C., O'Neill, B. K., et al. (2005). Preventing protein haze in bottled white wine. Australian Journal of Grape and Wine Research, 11, 215–225. Waters, E. J., Hayasaka, Y., Tattersall, D. B., Adams, K. S., & Williams, P. J. (1998). Sequence analysis of grape (Vitis vinifera) berry chitinases that cause haze formation in wines. Journal of Agricultural and Food Chemistry, 46, 4950–4957. Waters, E. J., Pellerin, P., & Brillouet, J. -M. (1994). A Saccharomyces mannoprotein that protects wine from protein haze. Carbohydrate Polymers, 23, 185–191. Waters, E. J., Peng, Z., Pocock, K. F., & Williams, P. J. (1995). Proteins in white wine, II: Their resistance to proteolysis is not due to either phenolic association or glycosylation. Australian Journal of Grape and Wine Research, 1, 94–99. Waters, E. J., Wallace, W., & Williams, P. J. (1992). Identification of heat-unstable wine proteins and their resistance to peptidases. Journal of Agricultural and Food Chemistry, 40, 1514–1519. Weetall, H. H., Zelko, J. T., & Bailey, L. F. (1984). A new method for the stabilization of white wine. American Journal of Enology and Viticulture, 35, 212–215. Younes, B., Cilindre, C., Villaume, S., Parmentier, M., Jeandet, P., & Vasserot, Y. (2011). Evidence for an extracellular acid proteolytic activity secreted by living cells of Saccharomyces cerevisiae PlR1: Impact on grape proteins. Journal of Agricultural and Food Chemistry, 59, 6239–6246.
Please cite this article as: Younes, B., et al., Enzymatic hydrolysis of thermo-sensitive grape proteins by a yeast protease as revealed by a proteomic approach, Food Research International (2013), http://dx.doi.org/10.1016/j.foodres.2013.01.063