**3. Protein haze formation in white wine**

Protein haze can appear in bottled white wine as shown in **Figure 1** if unstable proteins are not removed before the wine bottling. Although research studies have investigated the protein stabilisation in white wines [12, 13, 56–59], the precise mechanism of protein haze formation still remains incompletely understood. One hypothesis [60] is that the first step in protein haze formation in wines is protein denaturation, a process accelerated by heating, after which the denatured proteins aggregate into large enough particles to be visually detected as haze, a process that may be affected either positively or negatively by non-protein wine components. In a recent study [61], two different mechanisms were proposed responsible for the heat-induced precipitation of the Arinto wine proteins: (1) at the higher pH values, it appears to result from isoelectric precipitation of proteins; (2) at the lower pH values, it seems to be associated with the presence of the non-protein wine components.

As the main soluble proteins remain in the finished wine, the slow denaturation of PR proteins is thought to lead to protein aggregation, flocculation into a hazy suspension and formation of precipitates. A study using purified thaumatin-like proteins and chitinases from grape juice [62] suggested that chitinases are the primary cause of heat-induced haze formation and their concentration was directly correlated to the turbidity of heat-induced haze formation, but conversely, thaumatin-like proteins seemed to have no measurable impact on turbidity. This result was confirmed by a latter study [54] in which chitinases were found to have a good linear correlation with protein stabilisation in Sauvignon Blanc wine. Different protein haze formation behaviour between thaumatin-like proteins and chitinases could be due to the difference in the protein structure of these two types of proteins. The thaumatin-like proteins start unfolding (or denaturing) at 62°C, but most of the proteins will refold again when the temperature drops down [63]. In contrast, chitinases have a lower unfolding temperature and this denaturation is irreversible. Thus, once chitinases are unfolded, they may aggregate and precipitate out of the solution.

A study used a reconstitution method [64] to investigate the heat-induced aggregation behaviour of purified wine proteins and showed that the chitinases were the protein most prone to aggregate and the one that formed the largest particles. It is important to note that in the reconstitution experiment, four thaumatinlike protein isoforms, chitinases, phenolics and polysaccharides in a Chardonnay wine were isolated individually, and the wine stripped of these compounds was used as a base to reconstitute each of the proteins alone or in combination with the isolated phenolics and/or polysaccharides. Although phenolics and polysaccharides did not show a significant impact on aggregation behaviour of chitinases,

**209**

haze formation.

**3.1 Phenolics**

**Figure 1.**

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

the thaumatin-like protein isoforms varied in susceptibility to haze formation and interactions with phenolics and polysaccharides. These observations obtained in the model system indicated the importance of non-protein factors in affecting protein

*Protein haze formed in Sauvignon Blanc wine after heating at 80°C for 2 h.*

Phenolic compounds are important constituents in white wines since they contribute to many characteristics such as appearance, taste, style and quality [65]. A reduction in protein haze observed in commercial wine fined with the addition of polyvinylpolypyrrolidone (PVPP) suggested that phenolics may play a modulating role in haze formation [60]. The interaction between phenolics and proteins has been studied in relation to haze formation [59, 66]. Hydrophobic bonding was suggested as the major model of interaction in tannin-protein complexes [67]. A conceptual model for the protein-phenolics interaction is that a protein molecule has a fixed number of polyphenol binding sites and more sites were exposed when protein hydrogen bonds were broken [68]. Thus, the concentration of various

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

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

#### **Figure 1.**

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

**3. Protein haze formation in white wine**

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.

Protein haze can appear in bottled white wine as shown in **Figure 1** if unstable proteins are not removed before the wine bottling. Although research studies have investigated the protein stabilisation in white wines [12, 13, 56–59], the precise mechanism of protein haze formation still remains incompletely understood. One hypothesis [60] is that the first step in protein haze formation in wines is protein denaturation, a process accelerated by heating, after which the denatured proteins aggregate into large enough particles to be visually detected as haze, a process that may be affected either positively or negatively by non-protein wine components. In a recent study [61], two different mechanisms were proposed responsible for the heat-induced precipitation of the Arinto wine proteins: (1) at the higher pH values, it appears to result from isoelectric precipitation of proteins; (2) at the lower pH values, it seems to be associated with the presence of the non-protein wine components. As the main soluble proteins remain in the finished wine, the slow denaturation of PR proteins is thought to lead to protein aggregation, flocculation into a hazy suspension and formation of precipitates. A study using purified thaumatin-like proteins and chitinases from grape juice [62] suggested that chitinases are the primary cause of heat-induced haze formation and their concentration was directly correlated to the turbidity of heat-induced haze formation, but conversely, thaumatin-like proteins seemed to have no measurable impact on turbidity. This result was confirmed by a latter study [54] in which chitinases were found to have a good linear correlation with protein stabilisation in Sauvignon Blanc wine. Different protein haze formation behaviour between thaumatin-like proteins and chitinases could be due to the difference in the protein structure of these two types of proteins. The thaumatin-like proteins start unfolding (or denaturing) at 62°C, but most of the proteins will refold again when the temperature drops down [63]. In contrast, chitinases have a lower unfolding temperature and this denaturation is irreversible. Thus, once chitinases are unfolded, they may aggregate and precipitate out of the solution. A study used a reconstitution method [64] to investigate the heat-induced aggregation behaviour of purified wine proteins and showed that the chitinases were the protein most prone to aggregate and the one that formed the largest particles. It is important to note that in the reconstitution experiment, four thaumatinlike protein isoforms, chitinases, phenolics and polysaccharides in a Chardonnay wine were isolated individually, and the wine stripped of these compounds was used as a base to reconstitute each of the proteins alone or in combination with the isolated phenolics and/or polysaccharides. Although phenolics and polysaccharides did not show a significant impact on aggregation behaviour of chitinases,

**208**

*Protein haze formed in Sauvignon Blanc wine after heating at 80°C for 2 h.*

the thaumatin-like protein isoforms varied in susceptibility to haze formation and interactions with phenolics and polysaccharides. These observations obtained in the model system indicated the importance of non-protein factors in affecting protein haze formation.
