Influence of temperature and degree of hydrolysis on the peptide composition of trypsin hydrolysates of β-lactoglobulin: Analysis by LC–ESI-TOF/MS

Enzymatic hydrolysis of proteins is influenced, either positively or negatively, by the hydrolysis conditions, temperature, enzyme concentration and pH, as well as substrate pre-treatments, e.g. heat-denaturing, glyco-conjugation and/or cross-linking. Purified bovine β-lactoglobulin (96.0% nitrogen) was hydrolysed using trypsin (EC 3.4.21.4, bovine pancrease) at between 30 and 50 °C to degrees of hydrolysis (DHs) between 1 and ∼9.0%. The time taken to reach the desired DH varied greatly, being shortest at 45 and 50 °C and longest at 30 °C. The hydrolysates were analysed by tandem liquid chromatography–electrospray ionisation time-of-flight mass spectra (LC–ESI-TOF/MS) and results showed that the detectable peptides, at both 30 °C and 35 °C, were similar at DH 1%. However, not only were the detectable peptides produced at 40–50 °C different from those produced at lower temperatures, but the trypsin released peptides due to non-specific hydrolysis of β-Lg. The pattern resembled a shift of trypsinolysis towards chymotrypsinolysis, probably due to steric ‘stretching’ and increase of the catalytic pocket, thus allowing bulky amino acids to be processed. Hydrolysis at 30 °C to DH 5% and 10% also led to the release of peptides due to non-specific cleavage by trypsin. These results indicate that trypsin could only release peptides in a predictable manner at temperatures near, but lower than, the declared optimum of 37 °C. Above this temperature and above DH 5–10% at 30 °C, hydrolysis followed a mixed trypsin- and chymotrypsin-like activity. Lys–Pro, Lys–Ile(–Pro) and Lys–Phe bonds remained stable to trypsin at all temperatures. Some peptides with a high content of hydrophobic amino acids were undetected by ESI-TOF/MS, probably due to their poor ionisation.