Proteinase and exopeptidase hydrolysis of whey protein: Comparison of the TNBS, OPA and pH stat methods for quantification of degree of hydrolysis

Whey protein hydrolysates were generated with Alcalase 2.4L and Debitrase HYW20 which are proteinase and exopeptidase enriched enzyme preparations, respectively. Degree of hydrolysis (DH) values were quantified with the TNBS, OPA and pH stat methods. Poor correlation was observed between the three methods for DH values in Debitrase HYW20 hydrolysates. For Alcalase 2.4L hydrolysates, the OPA method gave DH values that were approximately 15% lower than the pH stat, whereas TNBS DH values were similar to the pH stat method. As whey proteins are relatively rich in cysteine, a weak and unstable reaction between OPA and cysteine was thought to contribute to the under-estimation of DH in whey protein hydrolysates. Since TNBS reacts strongly with cysteine and TNBS DH values were unaffected by the type of enzyme preparation used to generate the hydrolysate, the TNBS method was deemed most suitable for the quantification of DH in whey protein hydrolysates.