Development of a method for assessing haze -active protein in beer by dye binding

A new protein assay that involves the binding of Bromopyrogallol Red (BPR) to haze-active protein in beer was developed. When the BPR dye reagent binds to haze-active (HA) protein, there is an absorbance change at 600 nm that can be monitored by difference spectra for estimation of the amount of HA protein in samples.;The most frequently used method to measure the protein fraction that actually forms haze in beer is a nephelometric procedure. However, this has the disadvantage common to other nephelometric methods of a severely limited linear range of response due to variations in the quantity and nature of the haze produced. It would be particularly useful to have a rapid and simple method for estimating the amount of haze-active protein in beer as this could be used to compare the effectiveness of various stabilization approaches, evaluate different lots of chillproofing materials, and to estimate the extent of stabilization (and likely shelf-life) of a given beer.;Fifteen candidate dyes were each tested for response to the HA protein gliadin (a proline-rich, alcohol soluble protein) in pH 4 phosphate buffer containing 5% ethanol after 30 min incubation. The absorbance changes in the 200--800 nm range were recorded. Four dyes gave a good response to gliadin. Alizarin Red S (ARS) and Nuclear Fast Red (NFR) produced responses in the UV region while Bromopyrogallol Red (BPR), and Plasmocorinth B (PCB) responded in the visible range. Responses of each of the four dyes to a range of proteins were determined. The UV responding dyes were considered to be unsuitable for use in beer due its high UV absorbance background. Of the two visible range dyes, BPR showed the strongest affinity for the model haze active protein gliadin. The protein-dye binding conditions were optimized. The BPR dye produced a good linear response in the range 50--600 mg/L gliadin (r2 = 0.99). This assay is simple and rapid, with the dye binding process virtually complete in 25 min with good color stability for 1 hr.;Applying BPR reagent to beer samples revealed interference from some beer constituents. A procedure involving first passing beer through a C18 solid phase cartridge and then through a 10 kDa centrifugal membrane concentrator followed by dye addition and spectroscopy was developed. This assay demonstrated reasonable results for beer samples subjected to different stabilizing treatments.