**2. Pathogenesis-related proteins in grapes and juice**

Pathogenesis-related proteins are a group of plant proteins induced in pathological or related situations [15]. They were first discovered in tobacco as a result of a hypersensitive reaction to tobacco mosaic virus (TMV) [16]. PR proteins are typically acidic, of low molecular weight and highly resistant to proteolytic degradation and to low pH values. On the basis of similarities in amino acid sequences, serological relationship and/or enzymatic or biological activity, 11 families have been recognised and classified for tobacco and tomato [17]. Some of these PR protein

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progression.

*Pathogenesis-Related Proteins in Wine and White Wine Protein Stabilization*

found present in both grape skin and pulp but not in grape seed [29].

Protein content in grape berries generally increases during ripening [30–33]. The accumulation of PR proteins in grape berries during ripening has been observed [33, 34], with *véraison* being the trigger for gene expression. The expression of PR genes in grapes can also be modulated by the classical PR protein inducers such as wounding, chemical elicitors, pathogen attack and abiotic stress [18, 35]. Although the level of PR proteins in grape berries increases, the diversity of PR proteins decreases during grape ripening [36]. In addition, the level and proportion of PR proteins in grapes are dependent on the cultivar, region, climate and viticultural practices [36–41]. Therefore, the actual protein composition in ripe grape berries is a result of the interactions between environmental conditions and intrinsic factors. Sunlight-exposed fruits presented generally higher total soluble solids, anthocyanins and phenolic compounds and lower titratable acidity, malate and berry weight than non-exposed or canopy-shaded fruits [42–46]. One study on Riesling must show that the total amino acid concentration was significantly lower for fruits exposed to ambient UV-B levels than the low UV-B treatment and reduced UV-B affected amino acid composition, causing higher levels of arginine and glutamine, the main sources of amino acid for yeast metabolism [47]. In a later study [48], UV exclusion resulted in a lower concentration of not only phenolic compounds such as tannins but also PR proteins in grape skin. Interestingly, UV exclusion showed no

Fungal infection can significantly influence the concentration of PR proteins in grapes. Grey mould caused by *Botrytis cinerea* is one of the main fungal diseases found in grapevines. A study that compared the juice from healthy grapes against *Botrytis* [49] showed that most proteins normally present in the healthy juice, namely, those between 20 and 30 kDa and a major glycoprotein at 62/64 kDa disappeared in the *Botrytis* infected juice. These results suggested that some proteinases secreted by *Botrytis cinerea* could degrade grape proteins. Another study on *Botrytis cinerea* infection on Chardonnay and Semillon grapes has also revealed that the concentrations of both PR proteins and total proteins in botrytised grape juice decreased compared to the juice from healthy grapes [50]. Conversely, powdery mildew infection on grape berries has been documented as increasing levels of PR proteins [48, 50, 51]. The strongly induced expression of some PR genes such as Vv*Chi*3 (coding for an acidic class III chitinase), Vv*Glu*b (coding for a basic class I glucanase) and Vv*Tl*2 (coding for a thaumatin-like protein) has been reported in powdery mildew infected grape berries [35]. A recent study [38] also showed that a number of proteins were induced in leaf tissues of Cabernet Sauvignon in response to powdery mildew infection, suggesting that Cabernet Sauvignon is able to initiate a basal defence but is unable to restrict fungal growth or slow down disease

family members have also been found in grapevine. The two prominent soluble proteins accumulated in grapes during ripening have been identified as chitinases (PR-3 family) and thaumatin-like proteins (PR-5 family) [18, 19]. However, in early studies, the β-1, 3-glucanases (PR-2 family), a potential indicator of pathogen attack, were not found in grape juice and/or berry extracts [19–22]. With the accomplishment of grapevine genome sequencing programs in 2007 [23, 24] and the development of technology in protein analysis, proteomic analysis of grapevine has significantly improved knowledge of grape proteins and produced a better understanding of their characteristics [25]. These have consequently shown that there are more PR protein family members found in grapevines, such as osmotins (PR-5 family), β-1, 3-glucanases (PR-2 family) and the PR-10 proteins [26–28]. The two major PR proteins in wine, thaumatin-like proteins and chitinases, have been

*DOI: http://dx.doi.org/10.5772/intechopen.92445*

effect on the PR proteins in the grape pulp.

## *Pathogenesis-Related Proteins in Wine and White Wine Protein Stabilization DOI: http://dx.doi.org/10.5772/intechopen.92445*

*Chemistry and Biochemistry of Winemaking, Wine Stabilization and Aging*

the detected polypeptides by limited proteolysis.

**2. Pathogenesis-related proteins in grapes and juice**

proteins derived from grapes.

In previous studies, proteins from grapes and wines have been reported with molecular weight (MW) in the range 6–200 kDa and isoelectric points (pI) in a range 3–9 kDa, as shown in **Table 1** [4–9]. However, the majority of wine proteins have MW and pI in a low range (20–30 and 4.1–5.8 kDa, respectively) and possess a net positive charge at the pH of the wine [2, 5, 10]. Studies on fractions of wine proteins using denaturing polyacrylamide gel electrophoresis have shown that the wine protein fraction is mainly composed of only a few polypeptides with MW ranging from 15 to 30 kDa, but a more detailed examination of whole protein fraction indicates a very large number of distinct polypeptides, exhibiting similar MW but subtle differences in electrical charges [11]. In that study, the authors also revealed via highly specific antibodies and N-terminal sequencing analysis that most wine polypeptides were structurally similar, suggesting the existence of a common precursor to most or all of the wine proteins which could generate all of

*Proteins reported in grapes and wines from different grape varieties: molecular weight (MW) and isoelectric* 

**Grape variety Sample type MW pI Reference** Sauvignon Blanc Wine 14.6–77.1 kDa [4] Riesling and Gewürztraminer Grape 11.2–190 kDa [5]

Chardonnay, Verdeca and Pinot Noir Wine 6–200 kDa 3.6–9.0 [6] Macabeo, Xarel-lo, Parellada and Malvar Wine 14–94 kDa 3.0–5.6 [7] Muscadine Grape 19–100 kDa 5.6–7.6 [8]

Wine 11.2–65 kDa 4.1–8.0 [5]

Wine 12–50 kDa 4.6–8.8 [9]

In a study on Muscat of Alexandria wine [12], two major proteins with MW of 24 and 32 kDa, respectively, by SDS-PAGE are found with significant contribution to protein haze, and the 24 kDa protein produced about 50% more haze than the 32 kDa protein. The N-terminal sequence of the protein with MW of 24 kDa showed homology to thaumatin and to a number of plant thaumatin-like proteins, and the N-terminal sequence of enzyme digested peptides of the protein with MW of 32 kDa showed homology to plant chitinases [13]. Another study analysed the two main wine proteins present in sodium dodecyl sulphate capillary gel electrophoresis (SDS-CGE), which were determined with MW at 22 and 26 kDa, respectively, being concluded as corresponding to thaumatin-like proteins (TLPs) and chitinases [14]. Both thaumatin-like proteins and chitinases in wine are pathogenesis-related (PR)

Pathogenesis-related proteins are a group of plant proteins induced in pathological or related situations [15]. They were first discovered in tobacco as a result of a hypersensitive reaction to tobacco mosaic virus (TMV) [16]. PR proteins are typically acidic, of low molecular weight and highly resistant to proteolytic degradation and to low pH values. On the basis of similarities in amino acid sequences, serological relationship and/or enzymatic or biological activity, 11 families have been recognised and classified for tobacco and tomato [17]. Some of these PR protein

**206**

**Table 1.**

*point (pI).*

family members have also been found in grapevine. The two prominent soluble proteins accumulated in grapes during ripening have been identified as chitinases (PR-3 family) and thaumatin-like proteins (PR-5 family) [18, 19]. However, in early studies, the β-1, 3-glucanases (PR-2 family), a potential indicator of pathogen attack, were not found in grape juice and/or berry extracts [19–22]. With the accomplishment of grapevine genome sequencing programs in 2007 [23, 24] and the development of technology in protein analysis, proteomic analysis of grapevine has significantly improved knowledge of grape proteins and produced a better understanding of their characteristics [25]. These have consequently shown that there are more PR protein family members found in grapevines, such as osmotins (PR-5 family), β-1, 3-glucanases (PR-2 family) and the PR-10 proteins [26–28]. The two major PR proteins in wine, thaumatin-like proteins and chitinases, have been found present in both grape skin and pulp but not in grape seed [29].

Protein content in grape berries generally increases during ripening [30–33]. The accumulation of PR proteins in grape berries during ripening has been observed [33, 34], with *véraison* being the trigger for gene expression. The expression of PR genes in grapes can also be modulated by the classical PR protein inducers such as wounding, chemical elicitors, pathogen attack and abiotic stress [18, 35]. Although the level of PR proteins in grape berries increases, the diversity of PR proteins decreases during grape ripening [36]. In addition, the level and proportion of PR proteins in grapes are dependent on the cultivar, region, climate and viticultural practices [36–41]. Therefore, the actual protein composition in ripe grape berries is a result of the interactions between environmental conditions and intrinsic factors.

Sunlight-exposed fruits presented generally higher total soluble solids, anthocyanins and phenolic compounds and lower titratable acidity, malate and berry weight than non-exposed or canopy-shaded fruits [42–46]. One study on Riesling must show that the total amino acid concentration was significantly lower for fruits exposed to ambient UV-B levels than the low UV-B treatment and reduced UV-B affected amino acid composition, causing higher levels of arginine and glutamine, the main sources of amino acid for yeast metabolism [47]. In a later study [48], UV exclusion resulted in a lower concentration of not only phenolic compounds such as tannins but also PR proteins in grape skin. Interestingly, UV exclusion showed no effect on the PR proteins in the grape pulp.

Fungal infection can significantly influence the concentration of PR proteins in grapes. Grey mould caused by *Botrytis cinerea* is one of the main fungal diseases found in grapevines. A study that compared the juice from healthy grapes against *Botrytis* [49] showed that most proteins normally present in the healthy juice, namely, those between 20 and 30 kDa and a major glycoprotein at 62/64 kDa disappeared in the *Botrytis* infected juice. These results suggested that some proteinases secreted by *Botrytis cinerea* could degrade grape proteins. Another study on *Botrytis cinerea* infection on Chardonnay and Semillon grapes has also revealed that the concentrations of both PR proteins and total proteins in botrytised grape juice decreased compared to the juice from healthy grapes [50]. Conversely, powdery mildew infection on grape berries has been documented as increasing levels of PR proteins [48, 50, 51]. The strongly induced expression of some PR genes such as Vv*Chi*3 (coding for an acidic class III chitinase), Vv*Glu*b (coding for a basic class I glucanase) and Vv*Tl*2 (coding for a thaumatin-like protein) has been reported in powdery mildew infected grape berries [35]. A recent study [38] also showed that a number of proteins were induced in leaf tissues of Cabernet Sauvignon in response to powdery mildew infection, suggesting that Cabernet Sauvignon is able to initiate a basal defence but is unable to restrict fungal growth or slow down disease progression.

Extraction of PR proteins from grapes into juice can be greatly influenced by harvesting and grape processing conditions. Studies carried out in Australia [52, 53] showed that the juice obtained from mechanical harvesting coupled with long-distance transport had a higher concentration of PR proteins than juice obtained from hand harvesting fruit, which is likely due to the long skin contact during transport. A more recent study [54] conducted in New Zealand showed that Sauvignon Blanc juices from machine harvesting followed by 3 h skin contact had a significantly lower concentration of proteins, including PR proteins, than those from hand harvesting followed by 3 h skin contact. It was likely due to the greater juice yield in machine harvesting treatment and the interactions between proteins and phenolic compounds. In the following study [55], the authors confirmed that longer skin contact can increase the extraction of PR protein but the final concentration of PR proteins in juice can be modulated by the co-extracted phenolic compounds.
