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Identification of antioxidant peptides of hen egg-white lysozyme and evaluation of inhibition of lipid peroxidation and cytotoxicity in the Zebrafish model

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Abstract

Hen egg lysozyme was hydrolyzed with pepsin in situ on a cation-exchange column to isolate antioxidant peptides. The most cationic fraction was eluted with 1 M NaCl. Five positively charged peptides f(109–119) VAWRNRCKGTD, f(111–119) WRNRCKGTD, f(122–129) AWIRGCRL, f(123–129) WIRGCRL and f(124–129) IRGCRL were identified using tandem mass spectrometry. Using ORAC-FL , all five peptides presented antioxidant activity with values of (1970, 3123, 2743, 2393 and 0.313 µmol Trolox/µmol peptide), respectively. Using method TBARS in Zebrafish larvae, all five synthetic peptides were found to efficiently inhibit lipid peroxidation (36.8, 51.6, 55.56, 63.2, 61.0 % inhibition of lipid peroxidation), respectively. None of the five peptides were toxic in Zebrafish eggs and larvae at concentrations lower than 50 µg/ml. Concentrations higher than 50 µg/ml were toxic for both Zebrafish eggs and larvae.

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References

  1. Mine Y, Ma FP, Lauriau S (2004) Antimicrobial peptides released by enzymatic hydrolysis of hen egg white lysozyme. J Agric Food Chem 58:1088–1094

    Article  Google Scholar 

  2. Somboonpatarakun C, Shinya S, Kawaguchi Y, Araki T, Fukamizo T, Klaynongsruang S (2015) A goose-type lysozyme from ostrich (Struthio camelus) egg white: multiple roles of His101 in its enzymatic reaction. Biosci Biotechnol Biochem. doi:10.1080/09168451.2015.1091716

    Google Scholar 

  3. You SJ, Udenigwe CC, Aluko RE, Wu J (2010) Multifunctional peptides from egg white lysozyme. Food Res Int 43(3):848–855. doi:10.1016/j.foodres.2009.12.004

    Article  CAS  Google Scholar 

  4. Jiménez-Saiz R, Martos G, Carrillo W, López-Fandiño R, Molina E (2011) Susceptibility of lysozyme to in vitro digestion and immunoreactivity of its digests. Food Chem 127:1719–1726. doi:10.1016/j.foodchem.2011.02.047

    Article  Google Scholar 

  5. Dias R, Vilas-Boas E, Campos FM, Hogg T, Couto JA (2015) Activity of lysozyme on Lactobacillus hilgardii strains isolated from port wine. Food Microbiol 49:6–11

    Article  CAS  Google Scholar 

  6. Brand J, Dachmann E, Pichler M, Lotz S, Kulozik U (2016) A novel approach for lysozyme and ovotransferrin fractionation from egg white by radial flow membrane adsorption chromatography: impact of product and process. Sep Purif Technol. doi:10.1016/j.seppur.2016.01.0327

    Google Scholar 

  7. Brand J, Silberbauer A, Kulozik U (2016) Comparison of different mechanical methods for the modification of the egg white protein ovomucin, part A: physical effects. Food Bioprocess Technol 9(3):501–510

    Article  CAS  Google Scholar 

  8. Prosapio V, Reverchon E, De Marco I (2016) Production of lysozyme microparticles to be used in functional foods, using an expanded liquid antisolvent process. J Supercrit Fluid 107:106–113

    Article  CAS  Google Scholar 

  9. Hasselberg FX (1978) Uses of enzymes and inmobilized enzymes. Nelson-Hall Inc., Chicago, pp 117–131

    Google Scholar 

  10. Lee-Huang S, Huang PL, Sun Y, Huang PL, Kung HF, Blithe DL, Chen HC (1999) Lysozyme and RNases as anti-HIV components in beta-core preparations of human chorionic gonadotropin. Proc Natl Acad Sci USA 96:2678–2681

    Article  CAS  Google Scholar 

  11. Kokoshis PL, Williams DL, Cook JA, Di Luzio NR (1978) Increased resistance to Staphylococcus aureus infection and enhancement in serum lysozyme activity by glucan. Science 199:1340–1342

    Article  CAS  Google Scholar 

  12. Jolles P, Jolles J (1984) What’s new in lysozyme research? Always a model system, today as yesterday. Mol Cell Biochem 63:165–189

    Article  CAS  Google Scholar 

  13. Sava G, Ceschia V, Zabucchi G (1988) Evidence for host-mediated antitumor effects of lysozyme in mice bearing the MCa mammary carcinoma. Eur J Cancer Clin Oncol 24:1737–1743

    Article  CAS  Google Scholar 

  14. Recio I, Visser S (1999) Two ion-exchange chromatographic methods for the isolation of antibacterial peptides from lactoferrin. In situ enzymatic hydrolysis on an ion-exchange membrane. J Chromatogr A 831:191–201

    Article  CAS  Google Scholar 

  15. Elagamy EI, Ruppanner R, Ismail A, Champagne CP, Assaf R (1996) Purification and characterization of lactoferrin, lactoperoxidase, lysozyme and immunoglobulins from camel’s milk. Int Dairy J 6:129–145

    Article  CAS  Google Scholar 

  16. Chiu HC, Lin CW, Suen SY (2007) Isolation of lysozyme from hen egg albumen using glass fiber-based cation-exchange membranes. J Membr Sci 290:259–266

    Article  CAS  Google Scholar 

  17. Byun HG, Lee JK, Park HG, Jeon JK, Kim SK (2009) Antioxidant peptides isolated from the marine rotifer, Brachionus rotundiformis. Process Biochem 44:842–846

    Article  CAS  Google Scholar 

  18. Je JY, Park PJ, Kwon JY, Kim SK (2004) A novel angiotensin I converting enzyme inhibitory peptide from Alaska pollack (Theragra chalcogramma) frame protein hydrolysate. J Agric Food Chem 52:7842–7845

    Article  CAS  Google Scholar 

  19. Philanto-Leppala A (2000) Biocative peptides derived from bovine whey proteins: opiod and ace-inhibitory peptides. Trends Food Sci Technol 11:347–356

    Article  Google Scholar 

  20. Sarmadi BH, Ismail A (2010) Antioxidative peptides from food proteins: a review. Peptides 31:1949–1956

    Article  CAS  Google Scholar 

  21. Li H, Aluko RE (2006) Structural modulation of calmodulin and calmodulin-dependent protein kinase II by pea protein hydrolysates. Int J Food Sci Nutr 57:178–189

    Article  CAS  Google Scholar 

  22. Zhu L, Chen C, Tang X, Xiong Y (2008) Reducing, radical scavenging, and chelation properties of in vitro digests of Alcalase-treated zein hydrolysate. J Agric Food Chem 56:2714–2721

    Article  CAS  Google Scholar 

  23. Chen HM, Muramoto K, Yamauchi F (1995) Structural analysis of antioxidative peptides from soybean.beta.-conglycinin. J Agric Food Chem 43:574–578

    Article  CAS  Google Scholar 

  24. Meisel H, FitzGerald RJ (2003) Biofunctional peptides from milk proteins: mineral binding and cytomodulatory effects. Curr Pharm Des 9:1289–1295

    Article  CAS  Google Scholar 

  25. Thammasirirak S, Pukcothanung Y, Preecharram S, Daduang S, Patramanon R et al (2010) Antimicrobial peptides derived from goose egg white lysozyme. Comp Biochem Physiol C Toxicol Pharm 151:84–91

    Article  Google Scholar 

  26. Asoodeh A, Memarpoor Yazdi M, Chamani J (2012) Purification and characterisation of angiotensin I converting enzyme inhibitory peptides from lysozyme hydrolysates. Food Chem 131:291–295

    Article  CAS  Google Scholar 

  27. Abdou AM, Higashiguchi S, Aboueleinin AM, Kim M, Ibrahim HR (2007) Antimicrobial peptides derived from hen egg lysozyme with inhibitory effect against Bacillus species. Food Control 18:173–178

    Article  CAS  Google Scholar 

  28. Jeong JB, De Lumen BO, Jeong HJ (2010) Lunasin peptide purified from Solanum nigrum L. protects DNA from oxidative damage by suppressing the generation of hydroxyl radical via blocking fenton reaction. Cancer Lett 293:58–64

    Article  CAS  Google Scholar 

  29. Hoq MI, Ibrahim HR (2011) Potent antimicrobial action of triclosan–lysozyme complex against skin pathogens mediated through drug-targeted delivery mechanism. Eur J Pharmacol Sci 42:130–137

    Article  CAS  Google Scholar 

  30. Senger MR, Rico EP, de Bem Arizi M, Frazzon AP, Dias RD, Bogo MR et al (2006) Exposure to Hg2+ and Pb2+ changes NTPDase and ecto-50-nucleotidase activities in central nervous system of Zebrafish (Danio rerio). Toxicol 226:229–237

    Article  CAS  Google Scholar 

  31. Gerlai R, Lee V, Blaser R (2006) Effects of acute and chronic ethanol exposure on the behavior of adult Zebrafish (Danio rerio). Pharmacol Biochem Behav 85:752–761

    Article  CAS  Google Scholar 

  32. Gerlai R, Ahmad F, Prajapati S (2008) Differences in acute alcohol induced behavioral responses among Zebrafish populations. Alcohol Clin Exp Res 32:1763–1773

    Article  Google Scholar 

  33. Egan RJ, Bergner CL, Hart PC, Cachat JM, Canavello PR, Elegante MF et al (2009) Understanding behavioral and physiological phenotypes of stress and anxiety in Zebrafish. Behav Brain Res 205:38–44

    Article  CAS  Google Scholar 

  34. Barbazuk WB, Korf I, Kadavi C, Heyen J, Tate S, Wun E et al (2000) The syntenic relationship of the Zebrafish and human genomes. Genome Res 10:1351–1358

    Article  CAS  Google Scholar 

  35. Crosier PS, Kalev-Zylinska ML, Hall CJ, Flores MV, Horsfield JA, Crosier KE (2002) Pathways in blood and vessel development revealed through Zebrafish genetics. Int J Dev Biol 46:493–502

    CAS  Google Scholar 

  36. Mullins MC, Hammerschmidt M, Haffter P, Nusslein-Volhard C (1994) Large scale mutagenesis in the Zebrafish: in search of genes controlling development in a vertebrate. Curr Biol 4:189–202

    Article  CAS  Google Scholar 

  37. Reimers MJ, La Du JK, Periera CB, Giovanini J, Tanguay RL (2006) Ethanol-dependent toxicity in Zebrafish is partially attenuated by antioxidants. Neurotoxicol Teratol 28:497–508

    Article  CAS  Google Scholar 

  38. Carrillo W, García-Ruiz A, Recio I, Moreno-Arribas MV (2014) Antibacterial activity of hen egg white lysozyme modified by heat and enzymatic treatments against oenological lactic acid bacteria and acetic acid bacteria. J Food Prot 77:1732–1739

    Article  CAS  Google Scholar 

  39. López-Expósito I, Minervini F, Amigo L, Recio I (2006) Identification of antibacterial peptides from bovine kappa-casein. J Food Prot 69:2992–2997

    Google Scholar 

  40. Ou B, Hampsch-Woodill M, Prior RL (2001) Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. J Agric Food Chem 49:4619–4626

    Article  CAS  Google Scholar 

  41. Dávalos A, Gómez-Cordóves C, Bartolomé B (2004) Extending applicability of the oxygen radical absorbance capacity (ORAC-fluorescein) assay. J Agric Food Chem 52:48–54

    Article  Google Scholar 

  42. Hernández-Ledesma B, Dávalos A, Bartolomé B, Amigo L (2005) Preparation of antioxidant enzymatic hydrolysates from alpha-lactalbumin and beta-lactoglobulin. Identification of active peptides by HPLC-MS/MS. J Agric Food Chem 53:588–593

    Article  Google Scholar 

  43. Westerfield M (1995) The Zebrafish book: a guide for the laboratory use of Zebrafish (Danio rerio), 3rd edn. University of Oregon Press, Eugene, p 385

    Google Scholar 

  44. OECD. Test No. 236: Fish embryo acute toxicity (FET) test: OECD Publishing

  45. Domingues I, Oliveira R, Lourenco J, Grisolia CK, Mendo S et al (2010) Biomarkers as a tool to assess effects of chromium (VI): comparison of responses in Zebrafish early life stages and adults. Comp Biochem Physiol C Toxicol Pharmacol 152:338–345

    Article  Google Scholar 

  46. Nagel R (2002) DarT: the embryo test with the Zebrafish Danio rerio a general model in ecotoxicology and toxicology. Altex 19:38–48

    Google Scholar 

  47. Ibrahim HR, Thomas U, Pellegrini A (2001) A helix-loop-helix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeabilization action. J Biol Chem 276:43767–43774

    Article  CAS  Google Scholar 

  48. Thomas K, Aalbers M, Bannon GA, Bartels M, Dearman RJ, Esdaile DJ et al (2004) A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins. Regul Toxicol Pharmacol 39:87–98

    Article  CAS  Google Scholar 

  49. Polverino de Laureto P, Frare E, Gottardo R, Van Dael H, Fontana A (2002) Partly folded status of members of the lysozyme/lactalbumin superfamily: a comparative study by circular dichroism spectroscopy and limited proteolysis. Prot Sci 11:2932–2946

    Article  Google Scholar 

  50. Fu TJ, Abbott UR, Hatzos C (2002) Digestibility of food allergens and non allergenic proteins in simulated gastric fluid and simulated intestinal fluid—a comparative study. J Agric Food Chem 50:7154–7160

    Article  CAS  Google Scholar 

  51. Ibrahim HR, Inazaki D, Abdou A, Aoki T, Kim M (2005) Processing of lysozyme at distinct loops by pepsin: a novel action for generating multiple antimicrobial peptide motifs in the newborn stomach. Biochem Biophy Acta—Gen Subj 1726:102–114

    Article  CAS  Google Scholar 

  52. Epand RM, Vogel HJ (1999) Diversity of antimicrobial peptides and their mechanisms of action. Biochem Biophys Acta 1462:11–28

    Article  CAS  Google Scholar 

  53. Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55:27–55

    Article  CAS  Google Scholar 

  54. Bijelic A, Molitor C, Mauracher SG, Al-Oweini R, Kortz U, Rompel A (2015) Hen egg-white lysozyme crystallisation: protein stacking and structure stability enhanced by a tellurium(VI)-centred polyoxotungstate. Chem Bio Chem. doi:10.1002/cbic.201402597

    Google Scholar 

  55. Ibrahim HR, Matsuzaki T, Aoki T (2001) Genetic evidence that antibacterial activity of lysozyme is independent of its catalytic function. FEBS Lett 506:27–32

    Article  CAS  Google Scholar 

  56. Huang WY, Majumder K, Wu J (2010) Oxygen radical absorbance capacity of peptides from egg white protein ovotransferrin and their interaction with phytochemicals. Food Chem 123:635–641

    Article  CAS  Google Scholar 

  57. Li YW, Li B (2013) Characterization of structure–antioxidant activity relationship of peptides in free radical systems using QSAR models: key sequence positions and their amino acid properties. J Theor Biol 318:29–43

    Article  CAS  Google Scholar 

  58. Je JY, Park PJ, Kim SK (2005) Antioxidant activity of a peptide isolated from Alaska pollack (Theragra chalcogramma) frame protein hydrolysate. Food Res Int 38:45–50

    Article  CAS  Google Scholar 

  59. Elias RJ, Kellerby SS, Decker EA (2008) Antioxidant activity of proteins and peptides. Crit Rev Food Sci Nutr 48:430–441

    Article  CAS  Google Scholar 

  60. Wu SJ, Ng LT (2008) Antioxidant and free radical scavenging activities of wild bitter melon (Monordica charantia Linn. Var. abbreviate Ser.) in Taiwan. LWT 41:323–330

    Article  CAS  Google Scholar 

  61. Zou Y, Lu Y, Wei D (2004) Antioxidant activity of a flavonoid-rich extract of Hypericum perforatum L. in vitro. J Agric Food Chem 52:5032–5039

    Article  CAS  Google Scholar 

  62. Lin RJ, Yen CM, Chou TH (2013) Antioxidant, anti-adipocyte differentiation, antitumor activity and anthelmintic activities against Anisakis simplex and Hymenolepis nana of yakuchinone A from Alpinia oxyphylla. BMC Complement Altern Med 237:1–13

    Google Scholar 

  63. Charron RA, Fenwick JC, David RS, Lean DRS, Moon TW (2000) Ultraviolet-B radiation effects on antioxidant status and survival in the Zebrafish, Brachydanio rerio. Photochem Photobiol 72:327–333

    Article  CAS  Google Scholar 

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Acknowledgments

This study was supported with Project Number AGL2015-66886-R of Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM), Madrid, Spain, and project number CPU-1373-2014-UTA of Universidad Técnica de Ambato, Ecuador/Universidad Nacional Rio Negro, Argentina. This work has been reviewed in the English edition by Emilio Labrador. Carrillo W. thanks Comunidad Autónoma de Madrid for the research contract to complete his PhD. Daniel Alejandro Barrio is member of the Carrera del Investigador, CONICET, Argentina.

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Authors and Affiliations

  1. Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM), Nicolás Cabrera, 9, 28049, Madrid, Spain

    W. Carrillo, J. A. Gómez-Ruiz, B. Miralles, M. Ramos & I. Recio

  2. Facultad de Ciencia e Ingeniería en Alimentos, Universidad Técnica de Ambato, Av. Los Chasquis y Rio Payamino, Campus Huachi, CP 1801334, Ambato, Ecuador

    W. Carrillo

  3. Universidad Nacional de Rio Negro, Don Bosco y Leloir s/n (8500), Viedma, Argentina

    D. Barrio

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  1. W. Carrillo
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All animal work conformed to ethical guidelines and was approved by relevant local animal ethics committees.

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Carrillo, W., Gómez-Ruiz, J.A., Miralles, B. et al. Identification of antioxidant peptides of hen egg-white lysozyme and evaluation of inhibition of lipid peroxidation and cytotoxicity in the Zebrafish model. Eur Food Res Technol 242, 1777–1785 (2016). https://doi.org/10.1007/s00217-016-2677-1

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