Chymotrypsin selectively digests β-lactoglobulin in whey protein isolate away from enzyme optimal conditions: potential for native α-lactalbumin purification.

The present study examines the resistance of the alpha-lactalbumin to alpha-chymotrypsin (EC 3.4.21.1) digestion under various experimental conditions. Whey protein isolate (WPI) was hydrolysed using randomised hydrolysis conditions (5 and 10% of WPI; pH 7.0, 7.8 and 8.5; temperature 25, 37 and 50 degrees C; enzyme-to-substrate ratio, E/S, of 0.1%, 0.5 and 1%). Reversed-phase high performance liquid chromatography (RP-HPLC) was used to analyse residual proteins. Heat, pH adjustment and two inhibitors (Bowman-Birk inhibitor and trypsin inhibitor from chicken egg white) were used to stop the enzyme reaction. While operating outside of the enzyme optimum it was observed that at pH 8.5 selective hydrolysis of beta-lactoglobulin was improved because of a dimer-to-monomer transition while alpha-la remained relatively resistant. The best conditions for the recovery of native and pure alpha-la were at 25 degrees C, pH 8.5, 1% E/S ratio, 5% WPI (w/v) while the enzyme was inhibited using Bowman-Birk inhibitor with around 81% of original alpha-la in WPI was recovered with no more beta-lg. Operating conditions for hydrolysis away from the chymotrypsin optimum conditions offers a great potential for selective WPI hydrolysis, and removal, of beta-lg with production of whey protein concentrates containing low or no beta-lg and pure native alpha-la. This method also offers the possibility for production of beta-lg-depleted milk products for sensitive populations.