Dr. Carien Coetzee
21 January 2020
Pathogenesis-related (PR) proteins are the proteins that are responsible for hazes and sediments in wine and are derived from the grape berry where the concentration can be affected by environmental factors such as fungal infection1,2 and ultraviolet radiation3. The two principal groups of PR proteins responsible for protein instability in wine are thaumatin-like proteins and chitinases with the thaumatin-like proteins contributing 70% of the total protein in wine4.
The concentration of the PR proteins in wine is largely determined by their concentrations in pre-fermentation juice which in turn is determined by the extraction of proteins from the grape berries. These proteins are highly resistant to low pH and proteolytic degradation5 and thus survive fermentation to potentially form commercially unacceptable hazes in the wine. Studies have shown that about 60% of the thaumatin-like proteins and chitinase survive during the fermentation of Sauvignon Blanc juice. Sauvignon Blanc is in general considered to be a variety with a high degree of protein instability.
PR proteins are involved in defence mechanisms against pathogen attack as well as resulting from wounding and certain abiotic stress.6–8 Fungal diseases such as downy mildew and powdery mildew would lead to increased levels of PR proteins in grape berries. Interestingly, grapes infected by Botrytis cinerea and the resulting juice contained less PR proteins compared to their uninfected counterparts. This phenomenon is possibly due to the secretion of protease by B. cinerea.2,9,10
Factors such as damage incurred during mechanical harvesting could possibly be one of the stress factors leading to increased concentrations of PR proteins in the grape berry and the resulting wine. Researchers tested this hypothesis by comparing the protein content of Sauvignon blanc grapes obtained by hand harvesting (HH) compared to grapes obtained from machine harvesting (MH) in a study titled:
Materials and Methods
Sauvignon Blanc from South Australia was used for the study.
Treatment 1: Hand-harvested (HH) fruit
- Whole bunches were harvested
- The bunches were transported 300 km (20 hours)
- Intact berries were sampled on arrival
Treatment 2: Machine harvested (MH) fruit
- Mainly broken berries without stalks were present in the harvester bin
- The berry/juice mixture was transported 500 km (20 hours)
- Intact berries were sampled on arrival
- Berry/juice mixture was sampled on arrival
Testing the stress response –
Protein content of intact berries from HH compared to intact berries from MH
After 20 hours of transport of the HH whole bunches and the MH berry/juice mixture, intact berries from each treatment were sampled to analyse the protein content in the free-run juice (manual pressing of the berries).
Results showed that HH berries contained more thaumatin-like proteins compared to the MH grapes, while the difference in chitinase concentration between HH and MH free run juice was negligible. If there were any stress responses due to the damaging action of MH, the protein content would have been higher in these intact berry MH samples, which is not the case. There was thus no response to the stress caused by machine harvesting in the form of increased protein. Over the centuries the grapevine has adapted to such an extent, that the PR proteins may be formed even if there is no stress factor stimulating it.
Testing the extraction –
Protein content of intact berries from HH compared to free-run juice obtained from the MH berry/juice mixture
The free-run juice obtained from the MH berry/juice mixture (also after 20 hours transport) contained a considerably higher concentration of proteins compared to the free-run juice obtained from intact HH berries. The physical damage caused by MH facilitates extraction of protein from the skins and other berry solids, resulting in a higher protein concentration in the MH free-run juice.
Testing the effect of skin contact and pressing
Another study tested the extraction of PR proteins in Sauvignon Blanc using different harvesting techniques, applying different skin contact times and evaluating the effect of pressing conditions.11
Results showed that both harvesting method and processing resulted in significant differences in the extraction of proteins:
Juices obtained from hand-harvested grapes, juices subjected to the least amount of skin contact and juices collected from the lowest press fraction (0-0.4 MPa) showed the lowest extraction of proteins compared to other treatments tested.
Results showed that the increase in the protein concentration from the grapes was not due to the physiological response of the berries to stress, but rather the extraction of unstable proteins from the berry solids and skins especially during lengthy transports. Other additives such as sulphur dioxide can also increase the permeability of the grape skin cells, allowing greater leaching of contents from the berry into the must.
The non-selective harvest of the mechanical harvester would result in the harvesting of all bunches. Thus, grapes infected with downy mildew and/or powdery mildew would be harvested, increasing the likelihood of elevated unstable proteins in the resulting juice and wine (even prior to maceration). Hand harvesting would thus be advised in blocks where fungal infection is prevalent.
A 20-hour transport would inadvertently stimulate the extraction of various compounds from the berry skins. In a scenario where the machine-harvested grapes can be transported in a relatively short period of time to the cellar, the extraction of unstable proteins can be minimised.Download a PDF version of this blog
- Monteiro, S.; Barakat, M.; Piçarra-Pereira, M. A.; Teixeira, A. R.; Ferreira, R. B. Osmotin and Thaumatin from Grape: A Putative General Defense Mechanism Against Pathogenic Fungi. Phytopathology 2003, 93 (12), 1505–1512. https://doi.org/10.1094/PHYTO.2003.93.12.1505.
- GIRBAU, T.; STUMMER, B. The Effect of Uncinula Necator (Powdery Mildew) and Botrytis Cinerea Infection of Grapes on the Levels of Hazeâ€forming Pathogenesisâ€related Proteins in Grape Juice. … Grape Wine … 2008, No. 1992, 14–22.
- Tian, B.; Harrison, R.; Jaspers, M.; Morton, J. Influence of Ultraviolet Exclusion and of Powdery Mildew Infection on Sauvignon Blanc Grape Composition and on Extraction of Pathogenesis-Related Proteins into Juice. Aust. J. Grape Wine Res. 2015, 21 (3), 417–424. https://doi.org/10.1111/ajgw.12135.
- Pocock, K. F.; Hayasaka, Y.; Peng, Z.; Williams, P. J.; Waters, E. J. The Effect of Mechanical Harvesting and Long-Distance Transport on the Concentration of Haze-Forming Proteins in Grape Juice. Aust. J. Grape Wine Res. 1998, 4 (1), 23–29. https://doi.org/10.1111/j.1755-0238.1998.tb00131.x.
- Linthorst, H. J. M.; Van Loon, L. C. Pathogenesis‐related Proteins of Plants. CRC. Crit. Rev. Plant Sci. 1991, 10 (2), 123–150. https://doi.org/10.1080/07352689109382309.
- Jellouli, N.; Ben Jouira, H.; Skouri, H.; Ghorbel, A.; Gourgouri, A.; Mliki, A. Proteomic Analysis of Tunisian Grapevine Cultivar Razegui under Salt Stress. J. Plant Physiol. 2008, 165 (5), 471–481. https://doi.org/10.1016/j.jplph.2007.02.009.
- Pocock, K. F.; Hayasaka, Y.; McCarthy, M. G.; Waters, E. J. Thaumatin-like Proteins and Chitinases, the Haze-Forming Proteins of Wine, Accumulate during Ripening of Grape (Vitis Vinifera) Berries and Drought Stress Does Not Affect the Final Levels per Berry at Maturity. J. Agric. Food Chem. 2000, 48 (5), 1637–1643. https://doi.org/10.1021/jf9905626.
- Ferreira, R. B.; Monteiro, S. S.; Piçarra-Pereira, M. A.; Teixeira, A. R. Engineering Grapevine for Increased Resistance to Fungal Pathogens without Compromising Wine Stability. Trends Biotechnol. 2004, 22 (4), 168–173. https://doi.org/10.1016/j.tibtech.2004.02.001.
- Marchal, R.; Berthier, L.; Legendre, L.; Marchal-Delahaut, L.; Jeandet, P.; Maujean, A. Effects of Botrytis Cinerea Infection on the Must Protein Electrophoretic Characteristics. J. Agric. Food Chem. 1998, 46 (12), 4945–4949. https://doi.org/10.1021/jf980453b.
- Waters, E. J.; Alexander, G.; Muhlack, R.; Pocock, K. F.; Colby, C.; O’Neill, B. K.; Høj, P. B.; Jones, P. Preventing Protein Haze in Bottled White Wine. Aust. J. Grape Wine Res. 2005, 11 (2), 215–225. https://doi.org/10.1111/j.1755-0238.2005.tb00289.x.
- Tian, B.; Harrison, R.; Morton, J.; Jaspers, M.; Hodge, S.; Grose, C.; Trought, M. Extraction of Pathogenesis-Related Proteins and Phenolics in Sauvignon Blanc as Affected by Grape Harvesting and Processing Conditions. Molecules 2017, 22 (7). https://doi.org/10.3390/molecules22071164.