Methodology for Determining Degree of Hydrolysis of Proteins in Hydrolysates: A Review

Degree of hydrolysis (DH) is defined as the proportion of cleaved peptide bonds in a protein hydrolysate. Several methods exist for determining DH; the most commonly used of these include the pH-stat, trinitrobenzenesulfonic acid (TNBS), o-phthaldialdehyde (OPA), trichloroacetic acid soluble nitrogen (SN-TCA), and formol titration methods. The pH-stat method is based on the number of protons released during hydrolysis; the TNBS, OPA, and formol titration methods are based on the measurement of amino groups generated from hydrolysis. The SN-TCA method measures the amount of TCA-soluble nitrogen, rather than DH. The pH-stat is the simplest and most commonly used method, but does not determine peptide bonds directly. In addition, the accuracy of the method depends on the type of hydrolytic enzymes used, the size of the hydrolyzed peptides, and the reaction temperature. Generally, the TNBS and OPA methods compare well and do directly determine DH. However, the assumption that the response factor for all derivatized N-terminal amino acids is similar may lead to inaccuracies. In conclusion, there is no consensus as to the best method for determining the DH of protein hydrolysates; consequently, there is a need for a standardized approach if interstudy comparisons are to be made.