Pathogenesis-related (PR) proteins are the proteins responsible for hazes and sediments in wine. They are derived from the grape berry where the concentration can be affected by various factors such as cultivar, region and vintage, as well as viticultural and environmental factors.1-5 The two principal groups of PR proteins responsible for protein instability in wine are thaumatin-like proteins (TLP) and chitinases with the latter being mainly responsible for haze formation.6-9
The concentration of the PR proteins in wine is largely determined by their presence in pre-fermentation juice, which in turn is determined by the extraction of proteins from the grape berries. These proteins are resilient to low pH and proteolytic degradation and thus survive the winemaking process potentially forming commercially unacceptable hazes in the wine.4,5,10,11 Some cultivars, such as Sauvignon blanc, generally have a higher risk of protein instability and the effect of oenological processes (other than targeted fining treatments) on these haze-forming proteins needs investigation.
A recent study aimed to elucidate the fate of the PR proteins during alcoholic fermentation of Sauvignon blanc and Chardonnay musts, specifically reporting the change in protein quantity and quality over time.12 The study specifically monitored the impact of factors such as fermentation temperature, grape cultivar and yeast strain on the protein levels of the juice and resulting wines.
Materials and methods
The study was done using Sauvignon blanc and Chardonnay juice. Fermentation was conducted using two strains of Saccharomyces cerevisiae (commercial strains BM45 and EC1118) and two strains of Saccharomyces paradoxus (RO88 and P01 146, strains isolated from wine). Two fermentation temperatures were tested (15°C and 30°C). The total protein content was measured on day zero (two hours after inoculation), day seven and day 14 (fermentation completed). Reverse-phase chromatography was used to monitor the evolution of total grape proteins, as well as chitinases and TLP during alcoholic fermentation.
Total protein (PR proteins and non-PR proteins)
- Sauvignon blanc musts contained about half the amount of total proteins compared to the Chardonnay musts.
- During fermentation, the total protein content of both varieties decreased significantly and the levels dropped by 82% – 90% from the beginning to the end of fermentation. This decrease occurred at a similar rate for both juices and could be due to denaturation or proteolytic activity.6
- Fermentation temperatures showed a significant impact on the total protein content of both cultivars. The fermentations conducted at a higher temperature (30°C) resulted in wines with lower total protein content compared to wines fermented at a lower temperature (15°C).
PR proteins (chitinase and TLP)
- There were more TLP present than chitinase for both cultivars.
- The total TLP and chitinase content was three-fold higher in the Sauvignon blanc must when compared to the Chardonnay must (even though the total protein content was higher in the Chardonnay musts). This confirms the general tendency of cultivars, such as Sauvignon blanc, to contain higher concentrations of the haze-forming proteins, which, if not removed, may increase the risk of haze formation.
- Even though a decrease in total proteins was observed during fermentation, the proportion of PR proteins increased compared to the non-PR protein fraction. This increase was seen from day zero to day seven. For the Sauvignon blanc fermented at 15°C, the fraction of PR proteins remained high even at the end of fermentation. This confirms the relatively higher stability of the PR proteins during vinification.
Effect of yeast strain and fermentation temperature on chitinase and TLP
- Differences in the chitinase content: The paradoxus strains resulted in 75% – 100% less chitinase compared to the wines fermented with S. cerevisiae (for both cultivars and both fermentation temperatures).
- Differences in the TLP content: Sauvignon blanc must fermented with the paradoxus P01 146 also resulted in significantly lower TLP fraction compared to the wines fermented with other yeast strains (for both fermentation temperatures).
- Higher concentrations of chitinase were observed when fermentation was done at 15°C compared to 30°C (especially for the Sauvignon blanc musts fermented with cerevisiae). No distinguishable pattern or trend was observed for TLP.
Significant changes in the concentration of free chitinases were observed in a yeast strain-dependant manner. Yeasts differ in its ability to reduce haze-forming proteins during fermentation and certain yeasts are capable of specifically removing chitinases from fermenting musts. The primary mechanism involved is thought to be the binding of chitinases to chitin located in the yeast cell walls ultimately removing chitinase from the fermenting must. Therefore, yeast cells with a higher chitin content in their cell walls are capable of reducing the main haze-forming protein, reducing the risk of haze formation. Currently, common S. cerevisiae wine yeasts do not show significant variation in chitin content in its cell walls. This opens a window for yeast breeding programmes, which can target the production of yeasts with increased cell wall chitin for the production of wine with lower chitinase content.
The wines were subjected to standard heat test to determine whether the difference in protein composition and concentration had any effect on the stability of the wine. Significant differences were seen between the wines fermented with different strains. In agreement with the results obtained above, the wines fermented with S. paradoxus reduced the protein haze by 60 – 71% when compared to wines fermented with S. cerevisiae.
This study highlights that there is no correlation between total protein content and protein haze formation. A high total protein content in a juice does not translate to a higher risk of protein instability. It is the quantity of the PR proteins (especially grape chitinases)9 that are important and directly correlated to the protein haze formation.
Specific yeast strains hold significant potential to decrease the concentration of haze-forming proteins (particularly chitinase) in wines. The commercialisation of these types of yeast might significantly reduce the bentonite requirements thereby minimising the volume lost due to sedimentation. The development of these yeasts would be especially advantageous in the production of Sauvignon blanc wines where protein instabilities and haze formation are often a challenge.
- Monteiro, S., Barakat, M., Piçarra-Pereira, M.A., Teixeira, A.R. & Ferreira, R.B., 2003. Osmotin and thaumatin from grape: A putative general defense mechanism against pathogenic fungi. Phytopathology 93(12): 1505 – 1512. https://doi.org/10.1094/PHYTO.2003.93.12.1505.
- Girbau, T. & Stummer, B., 2008. The effect of Uncinula necator (powdery mildew) and Botrytis cinerea infection of grapes on the levels of haze-forming pathogenesis-related proteins in grape juice. Grape Wine (?) 1992: 14 – 22.
- Tian, B., Harrison, R., Jaspers, M. & Morton, J., 2015. Influence of ultraviolet exclusion and of powdery mildew infection on Sauvignon blanc grape composition and on extraction of pathogenesis-related proteins into juice. Australian Journal of Grape and Wine Research 21(3): 417 – 424. https://doi.org/10.1111/ajgw.12135.
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- Marangon, M., Van Sluyter, S.C., Neilson, K.A., Chan, C., Haynes, P.A., Waters, E.J. & Falconer, R.J., 2011. Roles of grape thaumatin-like protein and chitinase in white wine haze formation. Journal of Agricultural and Food Chemistry 59(2): 733 – 740. https://doi.org/10.1021/jf1038234.
- Sauvage, F.X., Bach, B., Moutounet, M. & Vernhet, A., 2010. Proteins in white wines: Thermo-sensitivity and differential adsorbtion by bentonite. Food Chemistry 118(1): 26 – 34. https://doi.org/10.1016/j.foodchem.2009.02.080.
- Tian, B., Harrison, R., Morton, J., Jaspers, M., Hodge, S., Grose, C. & Trought, M., 2017. Extraction of pathogenesis-related proteins and phenolics in Sauvignon blanc as affected by grape harvesting and processing conditions. Molecules 22(7). https://doi.org/10.3390/molecules22071164.
- Feuillat, M., 2003. Yeast macromolecules: Origin, composition and enological interest. American Journal of Enology and Viticulture 54(3): 211 LP – 213.
- Linthorst, H.J.M. & Van Loon, L.C., 1991. Pathogenesis‐related proteins of plants. CRC Critical Reviews in Plant Sciences 10(2): 123 – 150. https://doi.org/10.1080/07352689109382309.
- Ndlovu, T., Buica, A. & Bauer, F.F., 2019. Chitinases and thaumatin-like proteins in Sauvignon blanc and Chardonnay musts during alcoholic fermentation. Food Microbiology 78 (December): 201 – 210. https://doi.org/10.1016/j.fm.2018.10.018.
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