Sequential hydrolysis of commercial casein hydrolysate by immobilized trypsin and thermolysin to produce bioactive phosphopeptides

Most reports only focus on the preparation of one specific peptide, while simultaneous preparation of various peptides is rarely investigated. A commercial preparation of casein hydrolysate from bovine milk was used as a source to obtain highly valuable bioactive peptides. A tailor-made protocol to immobilize trypsin and thermolysin in highly activated glyoxyl agarose was used to obtain heterogeneous protease biocatalysts. The intense multipoint covalent immobilization and the subsequent improvement of the thermal-stabilization enabled the reuse of the catalyst for three reaction cycles. The sequential hydrolysis with immobilized trypsin and thermolysin leads to an increase of 12% in the degree of hydrolysis (DH) starting from the commercial hydrolysate. After sequential hydrolysis, casein phosphopeptides (CPPs) were obtained with a high purity by selective precipitation using Ca2+/ethanol at pH 8. Consequently, chemical analysis of these hydrolysates revealed an increase in the content of phosphorous and calcium by 39 and 30%, respectively, with regard to the original sample. Sequential hydrolysis and selective precipitation yielded interesting bioactive peptides with 29 phosphorylation sites. These peptides, identified by LC-MS/MS shared common sequences with known peptides that show biological activities such as opioid agonists, immunostimulants, mineral carriers, antioxidant, bifidogenic, antihypertensives, antithrombotic, as well as peptides that show gastrointestinal mucosal protection activity.