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Purification and identification of endogenous antioxidant and ACE-inhibitory peptides from donkey milk by multidimensional liquid chromatography and nanoHPLC-high resolution mass spectrometry

Abstract

Donkey milk is a valuable product for the food industry due to its nutraceutical, nutritional, and functional properties. In this work, the endogenous peptides from donkey milk were investigated for their antioxidant and ACE-inhibitory activities, combining a two-dimensional peptide fractionation strategy with high-resolution mass spectrometry, bioinformatics analysis, and in vitro assays. After extraction, the endogenous peptides were fractionated twice, first by polymeric reversed phase and then by hydrophilic interaction chromatography. Fractions were screened for the investigated bioactivities and only the most active ones were finally analyzed by nanoRP-HPLC-MS/MS; this approach allowed to reduce the total number of possible bioactive sequences. Results were further mined by in silico analysis using PeptideRanker, BioPep, and PepBank, which provided a bioactivity score to the identified peptides and matched sequences to known bioactive peptides, in order to select candidates for chemical synthesis. Thus, five peptides were prepared and then compared to the natural occurring ones, checking their retention times and fragmentation patterns in donkey milk alone and in spiked donkey milk samples. Pure peptide standards were finally in vitro tested for the specific bioactivity. In this way, two novel endogenous antioxidant peptides, namely EWFTFLKEAGQGAKDMWR and GQGAKDMWR, and two ACE-inhibitory peptides, namely REWFTFLK and MPFLKSPIVPF, were successfully validated from donkey milk.

Analytical workflow for purification and identification of bioactive peptides from donkey milk sample

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References

  1. 1.

    Perna A, Intaglietta I, Simonetti A, Gambacorta E. Donkey milk for manufacture of novel functional fermented beverages. J Food Sci. 2015;80:S1352–9.

    CAS  Article  Google Scholar 

  2. 2.

    Polidori P, Vincenzetti S. Effects of thermal treatments on donkey milk nutritional characteristics. Recent Pat Food Nutr Agric. 2013;5:182–7.

    CAS  Article  Google Scholar 

  3. 3.

    Medhammar E, Wijesinha-Bettoni R, Stadlmayr B, Nilsson E, Charrondiere UR, Burlingame B. Composition of milk from minor dairy animals and buffalo breeds: a biodiversity perspective. J Sci Food Agric. 2012;92:445–74.

    CAS  Article  Google Scholar 

  4. 4.

    Jirillo F, Magrone T. Anti-inflammatory and anti-allergic properties of donkey’s and goat’s milk. Endocr Metab Immune Disord Drug Targets. 2014;14:27–37.

    CAS  Article  Google Scholar 

  5. 5.

    Tidona F, Sekse C, Criscione A, Jacobsen M, Bordonaro S, Marletta D. Antimicrobial effect of donkeys’ milk digested in vitro with human gastrointestinal enzymes. Int Dairy J. 2011;21:158–65.

    CAS  Article  Google Scholar 

  6. 6.

    Brumini D, Criscione A, Bordonaro S, Vegarud GE, Marletta D. Whey proteins and their antimicrobial properties in donkey milk: a brief review. Dairy Sci Technol. 2016;96:1–14.

    CAS  Article  Google Scholar 

  7. 7.

    Bučević-Popović V, Delaš I, Međugorac S, Pavela-Vrančić M, Kulišić-Bilušić T. Oxidative stability and antioxidant activity of bovine, caprine, ovine and asinine milk. Int J Dairy Technol. 2014;67:394–401.

    Article  Google Scholar 

  8. 8.

    Tafaro A, Magrone T, Jirillo F, Martemucci G, D’Alessandro AG, Amati L, et al. Immunological properties of donkey’s milk: its potential use in the prevention of atherosclerosis. Curr Pharm Des. 2007;13:3711–7.

    CAS  Article  Google Scholar 

  9. 9.

    Bruminia D, Bø Furlund C, Comi I, Gulbrandsen Devold T, Marletta D, Vegarud GE, et al. Antiviral activity of donkey milk protein fractions on echovirus type 5. Int Dairy J. 2013;28:109–11.

    Article  Google Scholar 

  10. 10.

    Mao X, Gu J, Sun Y, Xu S, Zhang X, Yang H, et al. Anti-proliferative and anti-tumour effect of active components in donkey milk on A549 human lung cancer cells. Int Dairy J. 2009;19:703–8.

    CAS  Article  Google Scholar 

  11. 11.

    Rutherfurd-Markwick KJ. Food proteins as a source of bioactive peptides with diverse functions. Br J Nutr. 2012;108 Suppl 2:S149–57.

    CAS  Article  Google Scholar 

  12. 12.

    Nagpal R, Behare P, Rana R, Kumar A, Kumar M, Arora S, et al. Bioactive peptides derived from milk proteins and their health beneficial potentials: an update. Food Funct. 2011;2:18–27.

    CAS  Article  Google Scholar 

  13. 13.

    Mohanty DP, Mohapatra S, Misra S, Sahu PS. Milk derived bioactive peptides and their impact on human health—a review. Saudi J Biol Sci. 2015. doi:10.1016/j.sjbs.2015.06.005.

    Google Scholar 

  14. 14.

    Capriotti AL, Cavaliere C, Piovesana S, Samperi R, Laganà A. Recent trends in the analysis of bioactive peptides in milk and dairy products. Anal Bioanal Chem. 2016;408:2670–85.

    Google Scholar 

  15. 15.

    Chianese L, Calabrese MG, Ferranti P, Mauriello R, Garro G, De Simone C, et al. Proteomic characterization of donkey milk “caseome”. J Chromatogr A. 2010;1217:4834–40.

    CAS  Article  Google Scholar 

  16. 16.

    Cunsolo V, Muccilli V, Fasoli E, Saletti R, Righetti PG, Foti S. Poppea’s bath liquor: the secret proteome of she-donkey’s milk. J Proteomics. 2011;74:2083–99.

    CAS  Article  Google Scholar 

  17. 17.

    Polidori P, Vincenzetti S. Protein profile characterization of donkey milk. In: Hurley W, editor. Milk protein. Rijeka: InTech. 2012; doi: 10.5772/45982.

  18. 18.

    Vincenzetti S, Amici A, Pucciarelli S, Vita A, Micozzi D, Carpi FM, et al. A proteomic study on donkey milk. Biochem Anal Biochem. 2012;1:109.

    CAS  Article  Google Scholar 

  19. 19.

    Bidasolo IB, Ramos M, Gomez-Ruiz JA. In vitro simulated gastrointestinal digestion of donkeys’ milk. Peptide characterization by high performance liquid chromatography–tandem mass spectrometry. Int Dairy J. 2012;24:146–52.

    CAS  Article  Google Scholar 

  20. 20.

    Piovesana S, Capriotti AL, Cavaliere C, La Barbera G, Samperi R, Zenezini Chiozzi R, et al. Peptidome characterization and bioactivity analysis of donkey milk. J Proteomics. 2015;119:21–9.

    CAS  Article  Google Scholar 

  21. 21.

    Guerrero A, Dallas DC, Contreras S, Bhandari A, Cánovas A, Islas-Trejo A, et al. Peptidomic analysis of healthy and subclinically mastitic bovine milk. Int Dairy J. 2015;46:46–52.

    CAS  Article  Google Scholar 

  22. 22.

    Sagar BK, Singh RP. Genesis and development of DPPH method of antioxidant assay. J Food Sci Technol. 2011;48:412–22.

    Article  Google Scholar 

  23. 23.

    Cushman DW, Cheung HS. Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochem Pharmacol. 1971;20:1637–48.

    CAS  Article  Google Scholar 

  24. 24.

    Mooney C, Haslam NJ, Pollastri G, Shields DC. Towards the improved discovery and design of functional peptides: common features of diverse classes permit generalized prediction of bioactivity. PLoS ONE. 2012;7:e45012.

    CAS  Article  Google Scholar 

  25. 25.

    D’Attoma A, Grivel C, Heinisch S. On-line comprehensive two-dimensional separations of charged compounds using reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography. Part I: orthogonality and practical peak capacity considerations. J Chromatogr A. 2012;1262:148–59.

    Article  Google Scholar 

  26. 26.

    Sànchez-Rivera L, Martìnez-Maqueda D, Cruz-Huerta E, Miralles B, Recio I. Peptidomics for discovery, bioavailability and monitoring of dairy bioactive peptides. Food Res Int. 2014;63:170–81.

    Article  Google Scholar 

  27. 27.

    Li Z, Jiang A, Yue T, Wang J, Wang Y, Su J. Purification and identification of five novel antioxidant peptides from goat milk casein hydrolysates. J Dairy Sci. 2013;96:4242–51.

    CAS  Article  Google Scholar 

  28. 28.

    Tenore GC, Ritieni A, Campiglia P, Stiuso P, Di Maro S, Sommella E, et al. Antioxidant peptides from “Mozzarella di Bufala Campana DOP” after simulated gastrointestinal digestion: in vitro intestinal protection, bioavailability, and anti-haemolytic capacity. J Funct Foods. 2015;15:365–75.

    CAS  Article  Google Scholar 

  29. 29.

    Sommella E, Pepe G, Ventre G, Pagano F, Manfra M, Pierri G, et al. Evaluation of two sub-2 μm stationary phases, core-shell and totally porous monodisperse, in the second dimension of on-line comprehensive two dimensional liquid chromatography, a case study: separation of milk peptides after expiration date. J Chromatogr A. 2015;1375:54–61.

    CAS  Article  Google Scholar 

  30. 30.

    Power O, Jakeman P, FitzGerald RJ. Antioxidative peptides: enzymatic production, in vitro and in vivo antioxidant activity and potential application of milk-derived antioxidative peptides. Amino Acids. 2013;44:797–820.

    CAS  Article  Google Scholar 

  31. 31.

    Lassoued I, Mora L, Barkia A, Aristoy MC, Nasri M, Toldrá F. Bioactive peptides identified in thornback ray skin’s gelatin hydrolysates by proteases from Bacillus subtilis and Bacillus amyloliquefaciens. J Proteomics. 2015;14(128):8–17.

    Article  Google Scholar 

  32. 32.

    Saito T, Nakamura T, Kitazawa H, Kawai Y, Itoh T. Isolation and structural analysis of antihypertensive peptides that exist naturally in Gouda cheese. J Dairy Sci. 2000;83:1434–40.

    CAS  Article  Google Scholar 

  33. 33.

    Liu Q, Cobb JS, Johnson JL, Wang Q, Agar JN. Performance comparisons of nano-LC systems, electrospray sources and LC-MS-MS platforms. J Chromatogr Sci. 2014;52:120–27.

    CAS  Article  Google Scholar 

  34. 34.

    Smith R, Ventura D, Prince JT. LC-MS alignment in theory and practice: a comprehensive algorithmic review. Brief Bioinform. 2013;16:104–17.

    Article  Google Scholar 

  35. 35.

    Wandy J, Daly R, Breitling R, Rogers S. Incorporating peak grouping information for alignment of multiple liquid chromatography mass spectrometry datasets. Bioinformatics. 2015;31:1999–06.

    Article  Google Scholar 

  36. 36.

    Nimmi OS, George P. Evaluation of the antioxidant potential of a newly developed polyherbal formulation for antiobesity. Int J Pharm Pharm Sci. 2012;4:505–10.

    CAS  Google Scholar 

  37. 37.

    Zhang QX, Wu H, Ling YF, Lu RR. Isolation and identification of antioxidant peptides derived from whey protein enzymatic hydrolysate by consecutive chromatography and Q-TOF MS. J Dairy Res. 2013;80:367–73.

    CAS  Article  Google Scholar 

  38. 38.

    Uenishi H, Kabuki T, Seto Y, Serizawa A, Nakajima H. Isolation and identification of casein-derived dipeptydyl-peptidase 4 (DPP-4)-inhibitory peptide LPQNIPPL from gouda-type cheese and its effect on plasma glucose in rats. Int Dairy J. 2012;22:24–30.

    CAS  Article  Google Scholar 

  39. 39.

    Kohmura M, Nio N, Ariyoshi Y. Inhibition of angiotensin-converting enzyme by synthetic peptide fragments of various β-caseins. Agric Biol Chem. 1990;54:1101–2.

    CAS  Google Scholar 

  40. 40.

    Jäkälä P, Vapaatalo H. Antihypertensive peptides from milk proteins. Pharmaceuticals. 2010;3:251–72.

    Article  Google Scholar 

  41. 41.

    Vizcaíno JA, Deutsch EW, Wang R, Csordas A, Reisinger F, Ríos D, et al. ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nat Biotechnol. 2014;30:223–6.

    Article  Google Scholar 

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Acknowledgments

This work has been carried out within the framework of the Research Project “Identification and characterization of new bioactive peptides in milk and dairy products,” supported by Sapienza Università di Roma, nr. C26H15WEZH.

The MS proteomics data have been deposited to the ProteomeXchange Consortium [41] via the PRIDE partner repository with the dataset identifier “PXD004035.”

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Correspondence to Anna Laura Capriotti.

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Zenezini Chiozzi, R., Capriotti, A.L., Cavaliere, C. et al. Purification and identification of endogenous antioxidant and ACE-inhibitory peptides from donkey milk by multidimensional liquid chromatography and nanoHPLC-high resolution mass spectrometry. Anal Bioanal Chem 408, 5657–5666 (2016). https://doi.org/10.1007/s00216-016-9672-z

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Keywords

  • Peptidomics
  • Donkey milk
  • Antioxidant peptides
  • ACE-inhibitory peptides
  • Off-line two-dimensional chromatography
  • High-resolution mass spectrometry