Comparison of a modified spectrophotometric and the pH-stat methods for determination of the degree of hydrolysis of whey proteins hydrolysed in a tangential-flow filter membrane reactor

A procedure was developed to determine the degree of hydrolysis (DH) of whey protein hydrolysates (WPH) during hydrolysis in either 3 kDa or 10 kDa tangential-flow filter (TFF) enzymatic membrane reactors (EMR). Protease N Amano G (IUB 3.4.24.28, Bacillus subtilis) was used to hydrolyse an initial 5% (w v−1) aqueous solution of whey protein isolate (86.98% protein) at pH 7.0 and 55 °C with continuous recirculation and simultaneous removal of hydrolysates through the TFF, in single- or two-stage operation. The DH in the permeate and the retentate were determined as the concentration of the free α-NH2 using 2,4,6-trinitrobenzene 1-sulphonic acid (TNBS) and compared to the pH-stat method. In the new method, the DH of the permeate, the retentate and for the total EMR process could be quantified together or independently. The pH-stat method exaggerated the DH in the EMR because of the leakage of the alkali. The TNBS method was more reliable for DH estimation in the EMR.