Proteins haze formation

The mechanism of protein haze formation in wines is not fully understood. Slow denaturation of wine proteins is thought to lead to protein aggregation, flocculation into a hazy suspension and, finally, formation of visual precipitates. The importance of non-proteinaceous factors in white wine protein haze formation such as proan-thocyanidins (Koch and Sajak 1959 Waters et al. 1995a Yokotsuka et al. 1991) have been suspected for some time. Other factors such as polysaccharides, alcohol levels and pH have also been implicated (Mesquita et al. 2001 Siebert et al. 1996a). It has been observed that grape protein added to model wine does not precipitate or produce haze when heated, whereas visually obvious hazes occur when the same protein is added to a commercial wine (Pocock 2006). 

Protein haze in white wine thus differs in several aspects from protein haze in beer. It is well established that beer protein haze is due to interactions between proteins, derived from the barley storage protein hordein and rich in proline, and hop polyphenolic compounds (Bamforth 1999 Miedl et al. 2005 Siebert 1999 Siebert and Lynn 2003). White wine proteins are not derived from storage proteins of grape seed nor are they as rich in proline as hordein. In addition, wine protein haze formation cannot be eliminated by removing polyphenolic compounds by PVPP (Pocock et al. 2006) while in beer this has been applied as a commercial strategy (Leiper et al. 2005 Madigan et al. 2000). 

Mannoproteins are complex hydrocolloids released from yeast cell walls during autolysis (Goncalves et al., 2002 Charpentier et al., 2004). According to Feuillat (2003), mannoproteins are important to wine quality as these contribute to protein and tartrate stability, interact with aroma compounds, decrease the astringency and bitterness of tannins, and increase the body of wine. For instance, Dupin et al. (2000) reported that mannoproteins prevent protein haze formation. Using a model wine. Lubbers et al. (1994) noted that yeast cell walls bound volatile aroma compounds, especially those more hydrophobic, and could potentially change the sensory characteristics of wines through losses of these aromas. 

Chemical analysis of haze materials isolated from a beverage must be interpreted with caution because composition is often not well-related to cause. For example, beer hazes typically contain a high proportion of carbohydrate, with a modest amount of protein, and little polyphenol (Belleau and Dadic, 1981 Siebert et al., 1981). In order to prevent or delay haze formation, however, it is not necessary or helpful to remove carbohydrate. Reducing the amount of either protein or polyphenol typically has that effect. As a result, it appears that the large amount of carbohydrate found in the haze was coagulated with or adhered in some way to the protein-polyphenol haze backbone. 

Because warming can often disperse protein-polyphenol hazes, it is clear that covalent bonding is not involved in their formation. Asano et al. demonstrated that protein-polyphenol haze formation is inhibited by the nonpolar solvent dioxane and the hydrogen bond acceptor dimethylforma-mide (DMF), but not by a solution of sodium chloride (Asano et al., 1982). 

The time course of protein-polyphenol haze development in many packaged clear beverages has a two-phase pattern (see, for example, Fig. 2.17). At first no observable change occurs for some time. After this, haze formation begins and follows an essentially linear development rate. This phenomenon has been reported in beer (McMurrough et al., 1992) as well as apple juice, grape juice, and cranberry juice cocktail (Siebert, 1999, 2006). 

VI. ANALYSES RELATED TO PROTEIN-POLYPHENOL HAZE FORMATION... 

The removal of macromolecules by ultrafiltration has often been used in the production of clear fruit juices and wine (Girard and Fukumoto, 2000). This treatment removes both proteins and polysaccharides. Ultrafiltration through a 10,000 Da cut-off membrane has been shown to stabilize wines against haze formation (Flores, 1990). 

Adsorbents that remove proteins or polyphenols are used to treat a number of beverages to delay the onset of haze formation. Protein adsorbents include bentonite and silica. Bentonite removes protein nonspecifically (see Fig. 2.19) and so is unsuitable for stabilizing beverages where foam is desirable (beer and champagne). Silica, on the other hand, has remarkable specificity for HA proteins while virtually sparing foam-active proteins in beer (Siebert and Lynn, 1997b) (see Fig. 2.20). Silica removes approximately 80% of the HA protein from unstabilized beer, while leaving foam-active protein nearly untouched at commercial treatment levels. 

At one time, broad spectrum proteolytic enzymes (mainly papain and bromelain) were widely used to delay or minimize haze formation in beer (de Clerck, 1969). The enzymes cleaved protein chains, that when... 

Asano, K., Shinagawa, K., and Hashimoto, N. (1982). Characterization of haze-forming proteins of beer and their roles in chill haze formation. /. Am. Soc. Brew. Chem. 40,147-154. 

Protein clouding in white wines seems to be a greater problem when the wine pH is close to the isoelectric point of the various protein fractions. This is due to the fact that bentonite will remove, preferentially, the most positively charged proteins. The electrostatic charge of various protein fractions explains the observable phenomena of not being able to stabilize certain wines with the use of bentonite alone, or only with excessive amounts that can strip the wine character. But the pi of proteins only partially explains wine haze formation. It is also important to note that other factors, as yet not clearly identified, can intervene. 

Research into the isoelectric point of wine proteins has often been concurrent with smdies of wine protein size. Proteins with low isoelectric points (pi) were found to be significant contributors to total wine protein (Moretti and Berg 1965) and to wine haze (Bayly and Berg 1967). Hsu and Heatherbell (1987a) confirmed this observation and suggested that the majority of wine proteins had a pi of 4.1-5.8, whilst Lee (1986) suggested the major protein fractions of wine had a pi of 4.8-5.7. Dawes et al. (1994) fractionated wine proteins on the basis of their pi and found that the five different fractions all produced haze after heat treatment. Haze particle formation was found to differ between the fractions however, leading to a statement that other wine components, such as phenolic compounds, need to be considered to understand fully protein haze. 

The size and amount of protein haze formed in a wine is strongly influenced by other wine components. Pocock (2006) has demonstrated that one wine component, the sulfate anion, previously referred to as factor X, is essential for haze formation. If the sulfate anion is not present, heating does not result in sufficient denaturation of the proteins to lead to their aggregation, thus a haze will not form. 

Whilst sulfate appears to be fundamental to haze formation, other wine components such as phenolic compounds remain as candidate haze modulators. One possibility is that white wine phenolic compounds affect the particle size of denatured aggregated proteins, possibly through crosslinking. Several researchers (Oh et al. 1980 Siebert et al. 1996b) have suggested a hydrophobic mechanism for the interaction between phenolic compounds and proteins, in which the protein has a fixed number of phenolic binding sites. More of these sites are exposed when the protein is denatured. 

Pocock, K.F., Waters, EJ. (2006). Protein haze in bottled white wines how well do stability tests and bentonite fining trials predict haze formation during storage and transport . Aust. J. Grape Wine Res., 12, 212-220... 

Pocock, K.F., H0j, 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. Aust. J. Grape Wine Res., 9, 56-63 Pocock, K.F., Alexander, G.M., Hayasaka, Y, Jones, P.R., Waters, E.J. (2006). Sulfate - a candidate for the missing essential factor that is required for the formation of protein haze in white wine. J. Agric. Food Chem., 55, 1799-1807... 

Interactions between tannins and proteins have been extensively studied (Hager-man 1989 Haslam and Lilley 1988 Haslam et al. 1992), owing to their role in haze formation, astringency perception, and nutritional and anti-nutritional effects resulting from inhibition of various enzymes and reduction of dietary protein digestion. Other effects include reduced adsorption of /3-casein at the air-liquid interface in the presence of epigallocatechin gallate with potential consequences on foam properties (Sausse et al. 2003). 

The majority of the nitrogen compounds in beer have molecular weights between 5 and 70 kDa. The protein components of this fraction are of particular importance in brewing, as some of them contribute to foam formation (positive effect) while others, in association with polyphenols, lead to haze formation (undesirable effect) [10]. 

Haze formation is mostly attributed to proteins, polyphenols, and their interactions. It is also possible that there are also other factors that inbuence haze formation in beer, but their effect has not been yet clearly debned [ 15]. The amount of haze formed depends both on the concentration of proteins and polyphenols, and on their ratio. Polyphenols can combine with proteins to form colloidal suspensions that scatter light, which creates the cloudy appearance of beer. Beer polyphenols originate partly from barley and partly from hops. The beer polyphenols most closely associated with haze formation are the proanthocyanidins, which are dimers and trimers of catechin, epicatechin, and gaUocatechin. These have been shown to interact strongly with haze-active proteins [13,15-17] and their concentration in beer was directly related to the rate of haze formation [18]. Ahrenst-Larsen and Erdal [19] have demonstrated that anthocyanogen-free barley produces beer that is extremely resistant to haze formation, without any stabilizing treatment, provided that hops do not contribute polyphenols either. Not all proteins are equally involved in haze formation. It has been shown that haze-active proteins contain signibcant amounts of proline and that proteins that lack proline form little or no haze in the presence of polyphenols [13,15-17]. In beer, the source of the haze-active protein has been shown to be the barley hordein, an alcohol-soluble protein rich in proUne [16]. 

At least initially, the protein-polyphenol complexes are held together by weak associations and haze can be dispersed by warming, which in brewing is commonly referred to as reversible haze or chill haze. The practical consequence of this phenomenon is that beer should be bltered at the lowest possible temperature. The mechanism of haze formation appears to be... 

Pocock, K.F., Alexander, G.M., Hayasaka, Y., Jones, P.R. and Waters, E.J. (2007) Sulfate - a candidate for the missing essential factor that is required for the formation of protein haze in white wine. J. Agric. Food Chem., 55, 1799-1807. 

With each species of flavanoid the free phenol is regarded as being in equilibrium with the flavanoid-protein complex. Much evidence has been cited, however, that simple polyphenols do not react with protein to form insoluble haze and that only polymerized polyphenols are immediate haze precursors [59]. The addition of polymeric polyphenols, from various sources, to beer resulted in immediate haze formation. 

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