Haze, formation in wine

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). 

Therefore, an alternative test method, such as heating at 80 °C for 2 h, an alkali modified Coomassie-dye assay (e.g. Boyes et al. 1997), or the more recently available reagent based test kits such as Proteotest or Prostab, could present an opportunity to decrease bentonite dose and reduce volume of wine occluded in bentonite lees if confirmed to predict more accurately haze formation in wine. If these alternative test methods could replace the current heat test method of 80 °C for 6 h, this would also reduce the amount of time and/or effort presently required by a winery s laboratory to conduct heat stability testing. 

Waters, E.J., Hayasaka, Y, Tattersall, D.B., Adams, K.S., Williams, P.J. (1998). Sequence analysis of grape (Vitis viniferd) berry chitinases that cause haze formation in wines. J. Agric. Food Chem., 46, 4950-4957... 

Aldehyde-tannin and aldehyde-anthocyanin condensation reactions result in polymer formation (Figure 1). These polymers may be responsible for haze formation in wine and the polymers may eventually precipitate out of solution (26). The polymerized tannins have different flavor properties than the monomeric starting units (21-29) and formation of anthocyanin polymers affects wine color. In addition, these reactions may result in a reduction of aldehyde flavors in the wine. These condensation reactions are discussed more fully in other chapters of this volume. The formation of strong covalent bonds between the aldehyde and the tannin or anthocyanin makes recovery of the bound aldehydes difficult. 

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. 

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). 

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. 

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. 

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). 

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... 

Proteinaceous materials in wines will stain blue-black using Amido Black lOB and pink to red using Eosine Y as described in method A (Section 17.5.1.1). Method B (Section 17.5.1.2) rehes on the interaction of tannin and protein, which forms a visually apparent haze or precipitate. In method B, formation of pronounced haze in the treated sample as compared with a control is indicative of unstable protein (Fig. 17.4). 

In some cases, however, the formation of salts and complexes is an unwanted phenomenon, as protein salts and complexes with polyphenols form hazes and sediments in preserved fruit, fruit juices and beer. Protein hazes can also form in white wines after bottling, where the main factor is inorganic sulfate ions. Protein complexes with transition metals are often coloured, and their formation in processed foods is generally undesirable. An example is the protein conalbumin occurring in egg white, which readily forms coloured complexes with metal ions (pink with Fe +, yellow with Cu + and Mn +) in media of pH > 6 through tyrosine and histidine residues of the polypeptide chain. Complexes with iron ions often cause discoloration of egg products, but in media with pH < 4, these complexes dissociate to the original colourless compounds. 

Microorganisms can cause various degrees of haze, cloudiness, or sediment formation as well as changes in the composition of wine constituents by metabolizing components of the wine. 

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... 

---