Search

Immobilised trypsin is an alternative to free trypsin for producing protein hydrolysates with increased functionalities. However, the influence of hydrolytic conditions on this process remains unclear. The influence of salts on β-lactoglobulin (β-Lg) hydrolysis by free and immobilised trypsin was compared. For both forms of trypsin, 0.1 m Tris accelerated the release of most final peptides except f (71–75), and had no significant effects on the hydrolysis of intact β-Lg. Increasing NaCl concentrations from 0 to 0.02 m increased the degree of hydrolysis (DH) by 22.4% for free trypsin versus 62.1% for immobilised trypsin. The presence of 0.1 or 0.5 m NaCl hindered the release of peptides associated with the breakdown of intact protein. This led to 2–4 fold decreases in depleting intact β-Lg and DH, except immobilised trypsin at 0.1 m NaCl (DH increased by 44.3% versus without NaCl). Potential mechanisms underlying the effects of salts are discussed.

Although immobilized trypsin is a viable alternative to the free one in solution for producing protein hydrolysates, the change of selectivity introduced by immobilization is unclear. In this study, we compared the selectivity of free and immobilized trypsin towards different cleavage sites of β-lactoglobulin (β-Lg) with a focus on the impact of environmental pH. Both free and immobilized trypsin exhibited greater accessibility to native β-Lg at elevated pH (from pH 7.2 to 8.7). Additionally, free trypsin preferred to attack cleavage sites located at the C-terminus at pH 7.8, whereas an opposite preference for the N-terminus was observed at pH 8.7. Regarding the immobilized trypsin, the pH did not significantly influence its preference for the C- or N-terminus. Generally, immobilization of trypsin resulted in more focused cleavage within its specificity during the initial stage of hydrolysis, and some peptides were formed more rapidly by the immobilized trypsin.