Haze-active Proteins Research Articles

Characterization of haze-active proteins in apple juice.

The nature of the haze-active protein (HAP) in apple juice was investigated. Heat treatment removed protein indiscriminately while polyvinylpolypyrrolidone (PVPP) treatment was fairly specific for proteins of 15 and 28 kDa. Presumably, the PVPP bound to polyphenols, which in turn were complexed with protein. Three candidate apple HAPs were isolated. Two were extracted from juice with acetone and fractionated by hydrophobic interaction chromatography and solid phase extraction with C18 (HAP I) or SAX (HAP II) material. Hydroxyproline-rich protein was extracted from apple tissue (HAP III). The order of haze formation with tannic acid was gliadin > HAP III > HAP II > HAP I > bovine serum albumin, which shows increasing haze formation with increasing proline content. The sizes of HAP I, II, and III were 28, 15, and 12 kDa; the first two corresponded to the sizes of proteins removed by PVPP treatment and are involved in juice haze formation.

Effects of protein-polyphenol interactions on beverage haze, stabilization, and analysis.

The haze-forming activity of a polypeptide depends greatly on its proline content. Haze-forming polyphenols have at least two binding groups, each of which has at least two hydroxy groups on an aromatic ring. The protein/polyphenol ratio has a strong influence on the amount of haze formed; the largest amount occurs when the numbers of polyphenol binding ends and protein binding sites are nearly equal. This has important consequences for turbidimetric methods used to measure haze-active proteins and polyphenols in beverages. The ratio also influences the effectiveness of a number of stabilization procedures.

Development of Monoclonal Antibody Sandwich-ELISA for Determination of Beer Foam-Active Proteins

The development of a method to determine foam-active and haze-active proteins by enzyme-linked immunosorbent assays (ELISA) based on polyclonal antibodies was previously reported. Several different monoclonal antibodies against foam-active proteins in beer were developed. These monoclonal antibodies have enabled us to develop a sandwichELISA for the detection of foam-active proteins. Compared with our former ELISA, this new system showed the following advantages: The detection limits for the foam-active protein determined with the new ELISA system were greatly improved, and the contents of foam-active protein quantified by new ELISA system were highly correlated with the foam adhesion on beer. This system was applied to the evaluation of foam-active proteins during the brewing process.

Comparison of Polyphenol Interactions with Polyvinylpolypyrrolidone and Haze-Active Protein

Model system experiments were carried out to determine if the binding of haze-active (HA) polyphenols to polyvinylpolypyrrolidone (PVPP) resembles the binding of HA polyphenols to HA proteins. Gallic acid and methyl gallate were shown to be “single-ended” polyphenols; these compounds can bind to one HA protein molecule, but they cannot cross-link to another to develop haze. Single-ended polyphenols can interfere with haze formation under certain conditions, apparently by blocking the access of an HA polyphenol to sites of attachment on HA proteins. Single-ended polyphenols were bound by PVPP and removed from solution. Single-ended polyphenols produced chromic shifts when they bound to polyproline and to soluble polyvinylpyrrolidone (PVP). Both polyproline (a model of an HA protein) and soluble PVP formed much more haze with catechin than with epicatechin under some conditions. These findings suggest that the mechanisms by which HA polyphenols attach to PVPP and HA protein are similar, but not identical.