Proving the synergistic effect of Alcalase, PepX and PepN during casein hydrolysis by complete degradation of the released opioid precursor peptide VYPFPGPIPN

Abstract

A casein solution was hydrolyzed with Alcalase 2.4 L (EC 3.4.21.62) and the recombinantly produced aminopeptidases PepX (EC 3.4.14.11) and PepN (EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 in various combinations to analyze the synergistic effect of these peptidases during casein hydrolysis. The sequential application of PepX or PepN after prehydrolysis with Alcalase resulted in an relative degree of hydrolysis (rDH) increase of 1.12- or 2.00-fold, respectively, compared to only using Alcalase. By a combined application of PepX and PepN the rDH increased ~ 2.32-fold. Using Alcalase, PepX and PepN simultaneously from the beginning the rDH increased ~ 2.42-fold. Compared to the single application of PepX or PepN after an Alcalase treatment, the combined usage led to an increased amount of small peptides (< 1.1 kDa) and free amino acids. After the sequential application of first Alcalase and then PepX and PepN, only 14 peptides, which originated mainly from the C-terminal end of the β-casein chain remained. Even the opioid precursor peptide VYPFPGPIPN [β-casein, ƒ(59–68); V-β-casomorphine-9], generated by the Alcalase treatment was fully hydrolyzed after adding PepX and PepN. Therefore, the synergistic effect of PepX and PepN during casein hydrolysis was confirmed. The simultaneous application of Alcalase, PepX and PepN from the beginning showed similar results as the sequential application, but only three remaining peptides were observed by the mass spectrometric analysis. Additionally, the hydrolysis time was reduced from 16 h (sequential approach) to 6.5 h (simultaneous approach). This indicated a further synergism between Alcalase and the two aminopeptidases.