Proving the synergistic effect of Alcalase, PepX and PepN during casein hydrolysis by complete degradation of the released opioid precursor peptide VYPFPGPIPN
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A casein solution was hydrolyzed with Alcalase 2.4 L (EC 126.96.36.199) and the recombinantly produced aminopeptidases PepX (EC 188.8.131.52) and PepN (EC 184.108.40.206) 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.
KeywordsPepX 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.
- 4.Manninen AH (2004) Protein hydrolysates in sports and exercise: a brief review. J Sport Sci Med 3:60–63Google Scholar
- 11.Tan PS, van Kessel TA, van de Veerdonk FL, Zuurendonk PF, Bruins AP, Konings WN (1993) Degradation and debittering of a tryptic digest from β-casein by aminopeptidase N from Lactococcus lactis subsp. cremoris WG2. Appl Environ Microbiol 59:1430–1436Google Scholar
- 28.Henschen A, Lottspeich F, Brantl V, Teschemacher H (1979) Novel opioid peptides derived from casein (β-casomorphins). II. Structure of active components from bovine casein peptone. Hoppe Seylers Z Physiol Chem 360:1217–1224Google Scholar
- 30.Laugesen M, Elliott R (2003) The influence of consumption of A1 beta-casein on heart disease and type 1 diabetes—the authors reply. N Z Med J 116:U367Google Scholar
- 31.Laugesen M, Elliott R (2003) Ischaemic heart disease, type 1 diabetes, and cow milk A1 beta-casein. N Z Med J 116:U295Google Scholar
- 33.Arentz-Hansen H, Körner R, Molberg Ø, Quarsten H, Vader W, Kooy YM, Lundin KE, Koning F, Roepstorff P, Sollid LM, McAdam SN (2000) The intestinal T cell response to α-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase. J Exp Med 191:603–612CrossRefGoogle Scholar
- 35.Aleanzi M, Demonte AM, Esper C, Garcilazo S, Waggener M (2001) Celiac disease: antibody recognition against native and selectively deamidated gliadin peptides. Clin Chem 47:2023–2028Google Scholar
- 36.De Angelis M, Cassone A, Rizzello CG, Gagliardi F, Minervivi F, Calasso M, Di Cagno R, Francavilla R, Gobbetti M (2010) Mechanism of degradation of immunogenic gluten epitopes from Triticum turgidum L. var. durum by sourdough lactobacilli and fungal proteases. Appl Environ Microbiol 76:508–518CrossRefGoogle Scholar