A kinetic study of bovine haemoglobin hydrolysis by pepsin immobilized on a functionalized alumina to prepare hydrolysates containing bioactive peptides

Abstract

The hydrolysis kinetics of native and denatured haemoglobin, using pepsin immobilized on aluminium oxide, was studied in order to produce hydrolysates containing bioactive peptides. Pepsin was immobilized on acidic alumina and on 2-ethanolamine- O -phosphate (2-EAOP)-modified acidic alumina. Surface charge of the two supports was determined as a function of pH. Kinetic studies were performed at 23 degrees C in 0.1 M acetate buffer, pH 4.5. At this pH, the surface charge of the two supports was almost the same. The coating of alumina by 2-EAOP only introduced a two carbon spacer between alumina surface and the reaction medium. Adsorption on the two supports of haemoglobin, haem and peptides produced in the course of hydrolyses were compared. Fixation of 2-EAOP on a pepsin-alumina complex gave hydrolysis kinetics of urea-denatured haemoglobin close to that obtained with the same amount of pepsin in solution, but with comparatively less adsorption of peptides and complete adsorption of haem. Heterogeneous hydrolyses of haemoglobin with pepsin, immobilized on functionalized alumina, resulted in the presence of VV-haemorphin-4, VV-haemorphin-7 and neokyotorphin in the supernatants without haem, the presence of which makes further purification difficult.

Artificial neural networks to model the production of blood protein hydrolysates for plant fertilisation

“…The influence of pH on the degree of hydrolysis is the consequence of the trade‐off between the denaturation of the substrate structure and the proteolytic activity of the subtilisin. As the pH becomes acid, the denaturation of the quaternary structure of haemoglobin (the major protein present in blood meal) increases the exposure of peptide bonds to enzyme attack, resulting in higher degrees of hydrolysis . In contrast, the proteolytic activity of subtilisin is favoured by alkaline conditions, with an optimal pH between 7 and 8 .…”

“…High degrees of hydrolysis ensure adequate levels of free amino acids and small peptides, as well as favouring the solubilisation of the final product. Phenomenological approaches to modelling the hydrolysis of blood protein are normally based on simple Michaelis–Menten mechanisms . Given the complexity of an enzymatic reaction, influenced by a range of processes such as enzyme thermal deactivation or substrate inhibition, empirical methods based on direct analysis of experimental data are preferred .…”

Food peptidomics of in vitro gastrointestinal digestions of partially purified bovine hemoglobin: low‐resolution versus high‐resolution LC‐MS/MS analyses

“…Nonetheless, the blood of slaughtered animals is a potential source of bioactive peptides. Indeed, few publications have reported that the blood proteins, especially HB, are potential sources of bioactive peptides . Bovine HB is a protein easily digestible by the human GI digestion process.…”

Enzyme engineering (immobilization) for food applications

“…However, both whole cells and proteolytic enzymes have poor stability. Few studies have also reported the immobilization of pepsin on aluminum oxide and its subsequent use in the release of BPs from hemoglobin (Ticu et al, 2004). The use of immobilized enzymes can, however, overcome these challenges (Walsh, 2007).…”