Haze-active polyphenols

Siebert, K. J., Lynn, P. Y., and Carrasco, A. (1996b). Analysis of haze-active polyphenols and proteins in grape juices and wines. In "4th International Symposium on Cool Climate Viticulture and Enology," pp. VII-18-VII-21. Rochester, New York. 

Beer contains less haze-active polyphenols than haze-active proteins. Derived from barley hordeins, haze-active proteins (10-30 kOa) are acidic hydrophilic polypeptides, rich in both proline and glutamic acid [26] and glycosylated [32]. Much more haze is produced near pH 4.0 than at pH 3.0 or above pH 4.2. At the beer pH, ethanol at low concentration causes a modest decline of haze, while strong haze is observed at higher concentrations [33]. 

To preserve beer colloidal stability, brewers usually remove haze-active materials [34]. To get rid of haze-active proteins, precipitation with tannic acid, hydrolysis with papain and adsorption to bentonite [35] or silica gel [36, 37] are very effective, but unfortunately in some cases, such procedures also remove foam proteins. To remove haze-active polyphenols, the most usual way is adsorption to polyvinylpolypyrrolidone-PVPP. Because of the structural analogy between these compounds and proline [38], pyrrolidone rings bind polymerized flavanoids through hydrogen and ionic bonds. 

Only proteins that contain proline bind polyphenols. Asano et al. (1982) demonstrated that the haze-forming activity of a protein is roughly proportional to the mole percentage of proline it contains (see Fig. 2.3). DNA has codes for exactly 20 amino acids. If each of these were equally present in a protein, there would be 5 mol% of each one. In fact, most proteins have much less proline than this. There are a few exceptions. Casein has about 8 mol% proline and the grain prolamins (proline-rich, alcohol-soluble proteins) are even higher. Hordein, the barley prolamin, contains about 20 mol% proline. As a result, it readily forms haze with polyphenols and is the main beer haze-active (HA) protein. Hordein contains even more glutamine (Q) than proline (P), and often these amino acids are adjacent in the protein (see Fig. 2.4). In fact, the sequence P-Q-Q-P occurs... 

Protein molecule with fixed number of polyphenol binding sites (i.e., haze-active)... 

Siebert, K. J. and Lynn, P. Y. (1998). Comparison of polyphenol interactions with PVPP and haze-active protein. ]. Am. Soc. Brew. Chem. 56, 24431. 

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]. 

Siebert, K.J. Lynn, P.Y. Comparison of polyphenol interactions with polyvinylpolypyr-rolidone and haze-active protein. J. Amer. Soc. Brewing Chem. 1998, 56, 24-31. 

FIGURE 2.18 Possible mechanisms of polyphenol polymerization or activation leading to haze development based on concepts from Gardner and McGuinness (1977). 

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. 

Recently, proteolytic enzymes that cleave peptide bonds only adjacent to proline were introduced (Lopez and Edens, 2005). Since proline is involved in the polyphenol-binding sites and there is little proline in the foam-active proteins, these enzymes are specific for haze proteins and do little damage to foam proteins. 

Chill haze (or reversible haze), defined by non-covalent bonds between polyphenols and active proteins, can eventually turn into permanent haze that no longer dissolves as the beer warms. 

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