Estimation of Degree of Hydrolysis of Protein Hydrolysates by Size Exclusion Chromatography

Abstract

Estimating the degree of hydrolysis (DH) of commercial protein hydrolysates from their molecular weight (MW) median values generated by high performance size exclusion chromatography with UV detection (HPSEC/UV) was evaluated. DH is defined as the proportion (%) of cleaved peptide bonds in a protein hydrolysate. Several methods for DH determination are available, and protein hydrolysates are often tested both for DH by one of these methods and for MW profile by an HPSEC/UV method. We describe a means of estimating the DH of a protein hydrolysate from its MW profile, thereby eliminating the need for the performance of a second, separate test. In this study, a plot of average DH (%) vs. MW median (Da) was constructed from pancreatin digestion time data for seven protein substrates, determined by an HPSEC/UV system with a peptide gel filtration column. A general formula obtained from the plot enables DH estimation for a protein hydrolysate from its MW median value. By using this approach, the DH values for a total of thirteen commercial protein hydrolysates derived from whey, casein, and soy were estimated. Method accuracy was assessed by comparing the MW median-estimated DH to the DH determined by TNBS colorimetry, which generally agreed well, with R2 > 0.95. This study demonstrated that a reliable estimate of protein hydrolysate DH may be obtained from its MW median so that the performance of a separate DH test may not be necessary.