Thermal pre-treatment of β-Lactoglobulin as a tool to steer enzymatic hydrolysis and control the release of peptides

Abstract

Tryptic hydrolysis of β-Lactoglobulin (β-Lg) has been shown as an excellent way to produce several functional peptides. However enzymatic hydrolysis results in a mixture of numerous different peptides, which is problematic regarding the enrichment of one functional peptide in foods. Therefore, a new approach was investigated to control enzymatic hydrolysis by thermal pre-treatment of the substrate to produce specifically desired peptides. For this a 5% β-Lg solution (w/w) was denatured at 80°C and the influence of the surrounding pH (pH 4.6, 6.8 and 8) on the formed denatured particles was characterised by degree of denaturation (DD) and particle size. The native, as well as the denatured protein was then hydrolysed by trypsin (EC 3.4.21.4) at enzyme optimal conditions (pH 8 and 37°C). Hydrolysis was monitored by degree of hydrolysis (DH) using the pH-stat method and the hydrolysates were analysed by MALDI-TOF-MS/MS after several DHs. It could be demonstrated that the attacked cleavage sites during tryptic hydrolysis of native β-Lg followed no specific order. Denaturation independent of the environmental conditions led to a decrease of the DH during the total hydrolysis. Of special interest was the formation of non-native monomers by thermal denaturation at pH 8 and the subsequent decrease of attacked peptide bonds to only five different cleavage sites after a DH of 1%. This conditions were favorable, due to the release of a slightly heterogeneous hydrolysate, containing the functional peptide f(142-148), with reported ACE-inhibitory activity.