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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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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.

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References

  1. Clemente A (2000) Enzymatic protein hydrolysates in human nutrition. Trends Food Sci Technol 11:254–262

    Article  CAS  Google Scholar 

  2. Damle MV, Harikumar P, Jamdar SN (2010) Debittering of protein hydrolysates using immobilized chicken intestinal mucosa. Process Biochem 45:1030–1035

    Article  CAS  Google Scholar 

  3. Ewert J, Glück C, Zeeb B, Weiss J, Stressler T, Fischer L (2018) Modification of the interfacial properties of sodium caseinate using a commercial peptidase preparation from Geobacillus stearothermophilus. Food Hydrocoll 81:60–70

    Article  CAS  Google Scholar 

  4. Manninen AH (2004) Protein hydrolysates in sports and exercise: a brief review. J Sport Sci Med 3:60–63

    Google Scholar 

  5. Lehrer SB, Horner WE, Reese G (1996) Why are some proteins allergenic? Implications for biotechnology. Crit Rev Food Sci Nutr 36:553–564

    Article  CAS  PubMed  Google Scholar 

  6. Maldonado J, Gil A, Narbona E, Molina JA (1998) Special formulas in infant nutrition: a review. Early Hum Dev 53:S23–S32

    Article  CAS  PubMed  Google Scholar 

  7. Saha BC, Hayashi K (2001) Debittering of protein hydrolyzates. Biotechnol Adv 19:355–370

    Article  CAS  PubMed  Google Scholar 

  8. FitzGerald RJ, O’Cuinn G (2006) Enzymatic debittering of food protein hydrolysates. Biotechnol Adv 24:234–237

    Article  CAS  PubMed  Google Scholar 

  9. Nishiwaki T, Yoshimizu S, Furuta M, Hayashi K (2002) Debittering of enzymatic hydrolysates using an aminopeptidase from the edible basidiomycete Grifola frondosa. J Biosci Bioeng 93:60–63

    Article  CAS  PubMed  Google Scholar 

  10. Raksakulthai R, Haard NF (2003) Exopeptidases and their application to reduce bitterness in food: a review. Crit Rev Food Sci Nutr 43:401–445

    Article  CAS  PubMed  Google 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 59:1430–1436

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Lin S-B, Nelles LP, Cordle CT, Thomas RL (1997) Physical factors related to C18 adsorption columns for debittering protein hydrolysates. J Food Sci 62:946–948 + 1010

    Article  CAS  Google Scholar 

  13. Lin S-B, Nelles LP, Cordle CT, Thomas RL (1997) Debittering casein hydrolysates with octadecyl-siloxane (C18) columns. J Food Sci 62:665–670

    Article  CAS  Google Scholar 

  14. Stressler T, Eisele T, Schlayer M, Lutz-Wahl S, Fischer L (2013) Characterization of the recombinant exopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 important for food protein hydrolysis. PLoS One 8:e70055. https://doi.org/10.1371/journal.pone.0070055

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Schechter I, Berger A (1967) On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 27:157–162

    Article  CAS  PubMed  Google Scholar 

  16. Ewert J, Glück C, Strasdeit H, Fischer L, Stressler T (2018) Influence of the metal ion on the enzyme activity and kinetics of PepA from Lactobacillus delbrueckii. Enzyme Microb Technol 110:69–78

    Article  CAS  PubMed  Google Scholar 

  17. Stressler T, Eisele T, Schlayer M, Fischer L (2012) Production, active staining and gas chromatography assay analysis of recombinant aminopeptidase P from Lactococcus lactis ssp. lactis DSM 20481. AMB Express 2:39. https://doi.org/10.1186/2191-0855-2-39

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Byun T, Kofod L, Blinkovsky A (2001) Synergistic action of an X-prolyl dipeptidyl aminopeptidase and a non-specific aminopeptidase in protein hydrolysis. J Agric Food Chem 49:2061–2063

    Article  CAS  PubMed  Google Scholar 

  19. Barry CM, O’Cuinn G, Harrington D, O’Callaghan DM, Fitzgerald RJ (2000) Debittering of a tryptic digest of bovine β-casein using porcine kidney general aminopeptidase and X-prolydipeptidyl aminopeptidase from Lactococcus lactis subsp. cremoris AM2. J Food Sci 65:1145–1150

    Article  CAS  Google Scholar 

  20. Merz M, Ewert J, Baur C, Appel D, Blank I, Stressler T, Fischer L (2015) Wheat gluten hydrolysis using isolated flavourzyme peptidases: product inhibition and determination of synergistic effects using response surface methodology. J Mol Catal B Enzym 122:218–226

    Article  CAS  Google Scholar 

  21. Irvine GB (1997) Size-exclusion high-performance liquid chromatography of peptides: a review. Anal Chim Acta 352:387–397

    Article  CAS  Google Scholar 

  22. Kopaciewicz W, Regnier FE (1982) Nonideal size-exclusion chromatography of proteins: effects of pH at low ionic strength. Anal Biochem 126:8–16

    Article  CAS  PubMed  Google Scholar 

  23. Törnqvist M, Fred C, Haglund J, Helleberg H, Paulsson B, Rydberg P (2002) Protein adducts: quantitative and qualitative aspects of their formation, analysis and applications. J Chromatogr B Anal Technol Biomed Life Sci 778:279–308

    Article  Google Scholar 

  24. Birch GG, Kemp SE (1989) Apparent specific volumes and tastes of amino acids. Chem Senses 14:249–258. https://doi.org/10.1093/chemse/14.2.249

    Article  CAS  Google Scholar 

  25. Kirimura J, Shimizu A, Kimizuka A, Ninomiya T, Katsuya N (1969) Contribution of peptides and amino acids to the taste of foods. J Agric Food Chem 17:689–695

    Article  CAS  Google Scholar 

  26. Zhao CJ, Schieber A, Gänzle MG (2016) Formation of taste-active amino acids, amino acid derivatives and peptides in food fermentations—a review. Food Res Int 89:39–47

    Article  CAS  PubMed  Google Scholar 

  27. Brantl V, Teschemacher H, Henschen A, Lottspeich F (1979) Novel opioid peptides derived from casein (β-casomorphins). I. Isolation from bovine casein peptone. Hoppe Seylers Z Physiol Chem 360:1211–1216

    Article  CAS  PubMed  Google 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 360:1217–1224

    CAS  PubMed  Google Scholar 

  29. Elliott RB, Harris DP, Hill JP, Bibby NJ, Wasmuth HE (1999) Type I (insulin-dependent) diabetes mellitus and cow milk: casein variant consumption. Diabetologia 42:292–296

    Article  CAS  PubMed  Google 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:U367

    PubMed  Google Scholar 

  31. Laugesen M, Elliott R (2003) Ischaemic heart disease, type 1 diabetes, and cow milk A1 beta-casein. N Z Med J 116:U295

    PubMed  Google Scholar 

  32. McLachlan CNS (2001) β-casein A1, ischaemic heart disease mortality, and other illnesses. Med Hypotheses 56:262–272

    Article  CAS  PubMed  Google 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 191:603–612

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Shan L, Qiao S-W, Arentz-Hansen H, Molberg Ø, Gray GM, Sollid LM, Khosla C (2005) Identification and analysis of multivalent proteolytically resistant peptides from gluten: Implications for celiac sprue. J Proteome Res 4:1732–1741

    Article  CAS  PubMed  PubMed Central  Google 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 47:2023–2028

    CAS  PubMed  Google 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 76:508–518

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

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.

Funding

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.

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Authors and Affiliations

  1. Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany

    Timo Stressler, Thomas Eisele, Jacob Ewert, Bertolt Kranz & Lutz Fischer

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  1. Timo Stressler
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  2. Thomas Eisele
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Correspondence to Timo Stressler.

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Stressler, T., Eisele, T., Ewert, J. et al. Proving the synergistic effect of Alcalase, PepX and PepN during casein hydrolysis by complete degradation of the released opioid precursor peptide VYPFPGPIPN. Eur Food Res Technol 245, 61–71 (2019). https://doi.org/10.1007/s00217-018-3140-2

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  • DOI: https://doi.org/10.1007/s00217-018-3140-2

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