Analytical and Bioanalytical Chemistry

, Volume 410, Issue 15, pp 3573–3586 | Cite as

Peptidomic strategy for purification and identification of potential ACE-inhibitory and antioxidant peptides in Tetradesmus obliquus microalgae

  • Carmela Maria Montone
  • Anna Laura Capriotti
  • Chiara Cavaliere
  • Giorgia La Barbera
  • Susy Piovesana
  • Riccardo Zenezini Chiozzi
  • Aldo Laganà
Research Paper
Part of the following topical collections:
  1. Discovery of Bioactive Compounds


Microalgae are unicellular marine organisms that have promoted complex biochemical pathways to survive in greatly competitive marine environments. They could contain significant amounts of high-quality proteins which, because of their structural diversity, contain a range of yet undiscovered novel bioactive peptides. In this work, a peptidomic platform was developed for the separation and identification of bioactive peptides in protein hydrolysates. In this work, a peptidomic platform was developed for the extraction, separation, and identification of bioactive peptides in protein hydrolysates. Indeed, extraction of proteins from recalcitrant tissues is still a challenge due to their strong cell walls and high levels of non-protein interfering compounds. Therefore, seven different protein extraction protocols, based on mechanical and chemical methods, were tested in order to produce high-quality protein extracts. Proteins obtained by means of the best protocol, consisting of milling the recalcitrant tissue with glass beads, were subjected to enzymatic digestion with Alcalase® and subsequently the hydrolysate was purified by two-dimensional semi-preparative reversed phase liquid chromatography. Fractions were assayed for antioxidant and antihypertensive activities and only the most active ones were finally analyzed by RP nanoHPLC-MS/MS. Around 500 peptide sequences were identified in these fractions. The identified peptides were subjected to an in silico analysis by PeptideRanker algorithm in order to assign a score of bioactivity probability. Twenty-five sequenced peptides were found with potential antioxidant and angiotensin-converting-enzyme-inhibitory activities. Four of these peptides, WPRGYFL, GPDRPKFLGPF, WYGPDRPKFL, SDWDRF, were selected for synthesis and in vitro tested for specific bioactivity, exhibiting good values of antioxidant and ACE-inhibitory activity.

Graphical abstract

Workflow showing the entire peptidomic approach developed for identification of bioactive peptides in microalgae


Protein extraction methods Peptidomics Microalgae Antioxidant peptides ACE-inhibitory peptides Off-line two-dimensional chromatography High resolution mass spectrometry 



This work has been carried out within the framework of the Research Project “Microalgae as a source of bioactive compounds: chromatographic fractionation of peptides and lipids and their mass spectrometric characterization,” supported by Sapienza, no. RM11715C82118E74.

Moreover, the authors wish to thank Prof. Francesca Pagnanelli for providing the microalgae samples.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest

Supplementary material

216_2018_925_MOESM1_ESM.xlsx (61 kb)
ESM 1 (XLSX 61 kb)
216_2018_925_MOESM2_ESM.pdf (832 kb)
ESM 2 (PDF 831 kb)


  1. 1.
    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;408:2677–85. Scholar
  2. 2.
    Piovesana S, Capriotti ALAL, Cavaliere C, La Barbera G, Samperi R, Zenezini Chiozzi R, et al. Peptidome characterization and bioactivity analysis of donkey milk. J Proteome. 2015;119:21–9. Scholar
  3. 3.
    Yu Z, Yin Y, Zhao W, Chen F, Liu J. Application and bioactive properties of proteins and peptides derived from hen eggs: opportunities and challenges. J Sci Food Agric. 2014;94:2839–45. Scholar
  4. 4.
    Lafarga T, Hayes M. Bioactive peptides from meat muscle and by-products: generation, functionality and application as functional ingredients. Meat Sci. 2014;98:227–39. Scholar
  5. 5.
    Capriotti AL, Cavaliere C, Foglia P, Piovesana S, Samperi R, Zenezini Chiozzi R, Laganà A. Development of an analytical strategy for the identification of potential bioactive peptides generated by in vitro tryptic digestion of fish muscle proteins. Anal Bioanal Chem 2015;407. doi:
  6. 6.
    Halim NRA, Yusof HM, Sarbon NM. Functional and bioactive properties of fish protein hydolysates and peptides: a comprehensive review. Trends Food Sci Technol. 2016;51:24–33. Scholar
  7. 7.
    Zenezini Chiozzi R, Capriotti AL, Cavaliere C, La Barbera G, Piovesana S, Samperi R, 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. 2016;408:5657–66. Scholar
  8. 8.
    Capriotti AL, Caruso G, Cavaliere C, Samperi R, Ventura S, Zenezini Chiozzi R, et al. Identification of potential bioactive peptides generated by simulated gastrointestinal digestion of soybean seeds and soy milk proteins. J Food Compos Anal. 2015;44:205–13. Scholar
  9. 9.
    Batista AP, Gouveia L, Bandarra NM, Franco JM, Raymundo A. Comparison of microalgal biomass profiles as novel functional ingredient for food products. Algal Res. 2013;2:164–73. Scholar
  10. 10.
    Ejike CECC, Collins SA, Balasuriya N, Swanson AK, Mason B, Udenigwe CC. Prospects of microalgae proteins in producing peptide-based functional foods for promoting cardiovascular health. Trends Food Sci Technol. 2017;59:30–6. Scholar
  11. 11.
    Hallenbeck PC, Grogger M, Mraz M, Veverka D. Solar biofuels production with microalgae. Appl Energy. 2016;179:136–45. Scholar
  12. 12.
    Faried M, Samer M, Abdelsalam E, Yousef RS, Attia YA, Ali AS. Biodiesel production from microalgae: processes, technologies and recent advancements. Renew Sust Energ Rev. 2017;79:893–913. Scholar
  13. 13.
    Duarte JH, Fanka LS, Costa JAV. Utilization of simulated flue gas containing CO2, SO2, NO and ash for Chlorella fusca cultivation. Bioresour Technol. 2016;214:159–65. Scholar
  14. 14.
    Kim S-K, Chojnacka K. Marine algae extracts. Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA; 2015.CrossRefGoogle Scholar
  15. 15.
    Admassu H, Gasmalla MAA, Yang R, Zhao W. Bioactive peptides derived from seaweed protein and their health benefits: antihypertensive, antioxidant, and antidiabetic properties. J Food Sci. 2018;83:6–16. Scholar
  16. 16.
    Alzahrani MAJ, Perera CO, Hemar Y. Production of bioactive proteins and peptides from the diatom Nitzschia laevis and comparison of their in vitro antioxidant activities with those from Spirulina platensis and Chlorella vulgaris. Int J Food Sci Technol. 2017;
  17. 17.
    Ochoa-Méndez CE, Lara-Hernández I, González LM, Aguirre-Bañuelos P, Ibarra-Barajas M, Castro-Moreno P, et al. Bioactivity of an antihypertensive peptide expressed in Chlamydomonas reinhardtii. J Biotechnol. 2016;240:76–84. Scholar
  18. 18.
    Piovesana S, Capriotti AL, Cavaliere C, La Barbera G, Montone CM, Zenezini Chiozzi R, Laganà A. Recent trends and analytical challenges in plant bioactive peptide separation, identification and validation. Anal Bioanal Chem 2018; 852.
  19. 19.
    Harnedy PA, O’Keeffe MB, FitzGerald RJ. Fractionation and identification of antioxidant peptides from an enzymatically hydrolysed Palmaria palmata protein isolate. Food Res Int. 2017;100:416–22. Scholar
  20. 20.
    Neves AC, Harnedy PA, O’Keeffe MB, FitzGerald RJ. Bioactive peptides from Atlantic salmon (Salmo salar) with angiotensin converting enzyme and dipeptidyl peptidase IV inhibitory, and antioxidant activities. Food Chem. 2017;218:396–405. Scholar
  21. 21.
    Zenezini Chiozzi R, Capriotti AL, Cavaliere C, La Barbera G, Piovesana S, Laganà A. Identification of three novel angiotensin-converting enzyme inhibitory peptides derived from cauliflower by-products by multidimensional liquid chromatography and bioinformatics. J Funct Foods. 2016;27:262–73. Scholar
  22. 22.
    Cuellar-Bermudez SP, Aguilar-Hernandez I, Cardenas-Chavez DL, Ornelas-Soto N, Romero-Ogawa MA, Parra-Saldivar R. Extraction and purification of high-value metabolites from microalgae: essential lipids, astaxanthin and phycobiliproteins. Microb Biotechnol. 2015;8:190–209. Scholar
  23. 23.
    Di Caprio F, Visca A, Altimari P, Toro L, Iaquaniello G, Pagnanelli F. Two stage process of microalgae cultivation for starch and carotenoid production. Chem Eng Trans. 2016;49:415–20. Scholar
  24. 24.
    Capriotti AL, Cavaliere C, Piovesana S, Stampachiacchiere S, Ventura S, Zenezini Chiozzi R, et al. Characterization of quinoa seed proteome combining different protein precipitation techniques: improvement of knowledge of nonmodel plant proteomics. J Sep Sci. 2015;38:1017–25. Scholar
  25. 25.
    Capriotti AL, Cavaliere C, Ferraris F, Gianotti V, Laus M, Piovesana S, et al. New Ti-IMAC magnetic polymeric nanoparticles for phosphopeptide enrichment from complex real samples. Talanta. 2018;178:274–81. Scholar
  26. 26.
    Muñoz R, Navia R, Ciudad G, Tessini C, Jeison D, Mella R, et al. Preliminary biorefinery process proposal for protein and biofuels recovery from microalgae. Fuel. 2015;150:425–33. Scholar
  27. 27.
    Jensen SK. Improved Bligh and Dyer extraction procedure. Lipid Technol. 2008;20:280–1. Scholar
  28. 28.
    Abedini Najafabadi H, Pazuki G, Vossoughi M. Experimental study and thermodynamic modeling for purification of extracted algal lipids using an organic/aqueous two-phase system. RSC Adv. 2015;5:1153–60. Scholar
  29. 29.
    Matyash V, Liebisch G, Kurzchalia TV, Shevchenko A, Schwudke D. Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J Lipid Res. 2008;49:1137–46. Scholar
  30. 30.
    Yoo G, Park W-K, Kim CW, Choi Y-E, Yang J-W. Direct lipid extraction from wet Chlamydomonas reinhardtii biomass using osmotic shock. Bioresour Technol. 2012;123:717–22. Scholar
  31. 31.
    Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med. 1999;26:1231–7. Scholar
  32. 32.
    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. Scholar
  33. 33.
    Klimek-Ochab M, Brzezińska-Rodak M, Zymańczyk-Duda E, Lejczak B, Kafarski P. Comparative study of fungal cell disruption—scope and limitations of the methods. Folia Microbiol (Praha). 2011;56:469–75. Scholar
  34. 34.
    Nagai K, Yotsukura N, Ikegami H, Kimura H, Morimoto K. Protein extraction for 2-DE from the lamina ofEcklonia kurome (laminariales): recalcitrant tissue containing high levels of viscous polysaccharides. Electrophoresis. 2008;29:672–81. Scholar
  35. 35.
    Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye binding. Anal Biochem. 1976;72:248–54. Scholar
  36. 36.
    Peterson GL. Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Anal Biochem. 1979;100:201–20. Scholar
  37. 37.
    González López CV, del Carmen Cerón García M, Fernández FGA, Bustos CS, Chisti Y, Sevilla JMF. Protein measurements of microalgal and cyanobacterial biomass. Bioresour Technol. 2010;101:7587–91. Scholar
  38. 38.
    Barka A, Blecker C. Microalgae as a potential source of single-cell proteins. A review. Biotechnol Agron Soc Environ. 2016;20:427–36.Google Scholar
  39. 39.
    González-García E, Marina ML, García MC. Plum (Prunus domestica L.) by-product as a new and cheap source of bioactive peptides: extraction method and peptides characterization. J Funct Foods. 2014;11:428–37. Scholar
  40. 40.
    Awuor OL, Edward Kirwa M, Betty M, Jackim MF (2017) Optimization of Alcalase hydrolysis conditions for production of Dagaa (Rastrineobola argentea) protein hydrolysate with antioxidative properties. Ind Chem; 3.
  41. 41.
    Jeong H-S, Kim H-Y, Ahn SH, Oh SC, Yang I, Choi I-G. Optimization of enzymatic hydrolysis conditions for extraction of pectin from rapeseed cake (Brassica napus L.) using commercial enzymes. Food Chem. 2014;157:332–8. Scholar
  42. 42.
    Kalli A, Smith GT, Sweredoski MJ, Hess S. Evaluation and optimization of mass spectrometric settings during data-dependent acquisition mode: focus on LTQ-Orbitrap mass analyzers. J Proteome Res. 2013;12:3071–86. Scholar
  43. 43.
    Gilar M, Olivova P, Daly AE, Gebler JC. Two-dimensional separation of peptides using RP-RP-HPLC system with different pH in first and second separation dimensions. J Sep Sci. 2005;28:1694–703. Scholar
  44. 44.
    Furuta T, Miyabe Y, Yasui H, Kinoshita Y, Kishimura H. Angiotensin I converting enzyme inhibitory peptides derived from phycobiliproteins of dulse Palmaria palmata. Mar Drugs. 2016;14:32. Scholar
  45. 45.
    Connolly A, O’Keeffe MB, Piggott CO, Nongonierma AB, FitzGerald RJ. Generation and identification of angiotensin converting enzyme (ACE) inhibitory peptides from a brewers’ spent grain protein isolate. Food Chem. 2015;176:64–71. Scholar
  46. 46.
    Sheih I-CC, Wu T-KK, Fang TJ. Antioxidant properties of a new antioxidative peptide from algae protein waste hydrolysate in different oxidation systems. Bioresour Technol. 2009;100:3419–25. Scholar
  47. 47.
    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.Google Scholar
  48. 48.
    Brahmachari G. Chemistry and pharmacology of naturally occurring bioactive compounds. 1st ed. Boca Raton: CRC Press; 2013.Google Scholar
  49. 49.
    Wang Q. Preparation of functional peanut oligopeptide and its biological activity. In: Peanut processing characteristics and quality evaluation. Singapore: Springer Singapore; 2018. p. 461–537.CrossRefGoogle Scholar
  50. 50.
    Tsai J-S, Chen T-J, Pan BS, Gong S-D, Chung M-Y. Antihypertensive effect of bioactive peptides produced by protease-facilitated lactic acid fermentation of milk. Food Chem. 2008;106:552–8. Scholar

Copyright information

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

Authors and Affiliations

  • Carmela Maria Montone
    • 1
  • Anna Laura Capriotti
    • 1
  • Chiara Cavaliere
    • 1
  • Giorgia La Barbera
    • 1
  • Susy Piovesana
    • 1
  • Riccardo Zenezini Chiozzi
    • 1
  • Aldo Laganà
    • 1
  1. 1.Department of ChemistrySapienza Università di RomaRomeItaly

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