European Food Research and Technology

, Volume 245, Issue 1, pp 61–71 | Cite as

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

  • Timo StresslerEmail author
  • Thomas Eisele
  • Jacob Ewert
  • Bertolt Kranz
  • Lutz Fischer
Original Paper


A casein solution was hydrolyzed with Alcalase 2.4 L (EC and the recombinantly produced aminopeptidases PepX (EC and PepN (EC 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.


PepX PepN Alcalase Casein hydrolysis Analytical methods 



Many thanks to Iris Klaiber and Berit Würtz (Core Facility for Mass Spectrometry) from the University of Hohenheim, for their support in the nano-LC-ESI-MS/MS measurements. Also many thanks to Julia Mangold (trainee from Staatsschule für Gartenbau und Landwirtschaft Hohenheim) for her support during the casein hydrolyses.


We express our gratitude to the German Federal Ministry of Economics and Technology (AIF/FEI Project No. 16541 N) for partial financial support of this research.

Compliance with ethics standards

Conflict of interest

The authors declare that there are no conflicts of interest regarding the publication of this article.

Compliance with ethics requirements

This article does not contain any studies with human or animal subjects.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Timo Stressler
    • 1
    Email author
  • Thomas Eisele
    • 1
  • Jacob Ewert
    • 1
  • Bertolt Kranz
    • 1
  • Lutz Fischer
    • 1
  1. 1.Department of Biotechnology and Enzyme Science, Institute of Food Science and BiotechnologyUniversity of HohenheimStuttgartGermany

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