European Food Research and Technology

, Volume 245, Issue 12, pp 2687–2695 | Cite as

Identification and in silico analysis of antithrombotic peptides from the enzymatic hydrolysates of Tenebrio molitor larvae

  • Fangyuan Chen
  • Han Jiang
  • Yongbo Lu
  • Wenwei Chen
  • Guangrong HuangEmail author
Original Paper


Tenebrio molitor is an excellent source of high-quality protein that produces various bioactive peptides. It is a traditional Chinese herbal medicine which has the effect of “activating blood and dissolving stasis”. It aimed to obtain antithrombotic peptides from the Tenebrio molitor larvae hydrolysate generated by treatment with pepsin and trypsin. The hydrolysate was subjected to ion exchange chromatography and gel filtration chromatography; the obtained antithrombotic activity values of the fractions were 40.87% and 65.61% at 8.0 mg/mL, respectively. After further preparation by reverse-phase liquid chromatography, the peptides with antithrombotic activity of 28.66% at 0.2 mg/mL were identified by liquid chromatography tandem mass spectrometry as SLVDAIGMGP and AGFAGDDAPR. Both of the peptides were shown to be nontoxic and could interact with thrombin exosite 1 by molecular docking. These results indicate that peptides from Tenebrio molitor might be used as potential antithrombotic components in the future.


Tenebrio molitor larvae Antithrombotic peptide Enzymatic hydrolysis Isolation Identification In silico analysis 



Reverse-phase liquid chromatography


Liquid chromatography tandem mass spectrometry


Degree of hydrolysis


Trifluoroacetic acid


Discovery Studio 2.5


Analysis of variance



This work was financially supported by the Science and Technology Project of Zhejiang Province, China [grant number LGN19C200018].

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Compliance with ethics requirements

This article does not contain any studies with human or animal subjects.


  1. 1.
    Wakefield TW, Caprini J, Comerota AJ (2008) Thromboembolic diseases. Curr Probl Surg 45(12):844–899PubMedCrossRefPubMedCentralGoogle Scholar
  2. 2.
    Luchtman-Jones L, Broze GJ (1995) The current status of coagulation. Ann Med 27(1):47–52PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Syed AA, Mehta A (2018) Target specific anticoagulant peptides: a review. Int J Pept Res Ther 24(1):1–12CrossRefGoogle Scholar
  4. 4.
    Hahn D, Bae JS (2019) Recent progress in the discovery of bioactive components from edible natural sources with antithrombotic activity. J Med Food 22(2):109–120PubMedCrossRefPubMedCentralGoogle Scholar
  5. 5.
    Craik DJ, Fairlie DP, Spiros L, David P (2013) The future of peptide-based drugs. Chem Biol Drug Des 81(1):136–147PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Qiao M, Tu M, Wang Z, Mao F, Chen H, Qin L, Du M (2018) Identification and antithrombotic activity of peptides from blue mussel (Mytilus edulis) protein. Int J Mol Sci 19(1):138PubMedCentralCrossRefGoogle Scholar
  7. 7.
    Tu M, Feng L, Wang Z, Qiao M, Shahidi F, Lu W, Du M (2017) Sequence analysis and molecular docking of antithrombotic peptides from casein hydrolysate by trypsin digestion. J Funct Foods 32:313–323CrossRefGoogle Scholar
  8. 8.
    Gou M, Wang L, Liu X (2017) Anticoagulant activity of a natural protein purified from Hypomesus olidus. Nat Prod Res 31(10):1168–1171PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Zhang SB (2016) In vitro antithrombotic activities of peanut protein hydrolysates. Food Chem 202:1–8PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Sabbione AC, Añón MC, Nardo AE, Scilingo A (2016) Amaranth peptides with antithrombotic activity released by simulated gastrointestinal digestion. J Funct Foods 20:204–214CrossRefGoogle Scholar
  11. 11.
    Yang WG, Wang Z, Xu SY (2007) A new method for determination of antithrombotic activity of egg white protein hydrolysate by microplate reader. Chinese Chem Lett 18(4):449–451CrossRefGoogle Scholar
  12. 12.
    Zhang SB, Wang Z, Xu SY (2008) Antioxidant and antithrombotic activities of rapeseed peptides. J Am Oil Chem Soc 85(6):521–527CrossRefGoogle Scholar
  13. 13.
    Cheng S, Tu M, Liu H, Zhao G, Du M (2019) Food-derived antithrombotic peptides: preparation, identification, and interactions with thrombin. Crit Rev Food Sci 59(sup1):S81–S95CrossRefGoogle Scholar
  14. 14.
    Siemianowska E, Kosewska A, Aljewicz M, Skibniewska KA, Polak-Juszczak L, Jarocki A, Jędras M (2013) Larvae of mealworm (Tenebrio molitor L.) as European novel food. Agr Sci 04(06):287–291Google Scholar
  15. 15.
    Tang Y, Debnath T, Choi EJ, Kim YW, Ryu JP, Jang S, Chung SU, Choi YJ, Kim EK (2018) Changes in the amino acid profiles and free radical scavenging activities of Tenebrio molitor larvae following enzymatic hydrolysis. PLoS One 13(5):e0196218PubMedPubMedCentralCrossRefGoogle Scholar
  16. 16.
    Schneppenheim R, Theede H (1980) Isolation and characterization of freezing-point depressing peptides from larvae of Tenebrio molitor. Comp Biochem Phys B 67(4):561–568CrossRefGoogle Scholar
  17. 17.
    Chae JH, Kurokawa K, So YI, Hwang HO, Kim MS, Park JW, Jo YH, Lee YS, Lee BL (2012) Purification and characterization of tenecin 4, a new anti-Gram-negative bacterial peptide, from the beetle Tenebrio molitor. Dev Comp Immunol 36(3):540–546PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Dai C, Ma H, Luo L, Yin X (2013) Angiotensin I-converting enzyme (ACE) inhibitory peptide derived from Tenebrio molitor (L.) larva protein hydrolysate. Eur Food Res Technol 236(4):681–689CrossRefGoogle Scholar
  19. 19.
    Li XD, Niu ZX, Zhang BL (2006) Various methods available for the determination of hydrolyzed degree of whey protein. China Dairy Ind 34:56–62Google Scholar
  20. 20.
    Lafarga T, Wilm M, Wynne K, Hayes M (2016) Bioactive hydrolysates from bovine blood globulins: generation, characterisation, and in silico prediction of toxicity and allergenicity. J Funct Foods 24:142–155CrossRefGoogle Scholar
  21. 21.
    Tu M, Liu H, Zhang R, Chen H, Fan F, Shi P, Xu X, Lu W, Du M (2018) Bioactive hydrolysates from casein: generation, identification, and in silico toxicity and allergenicity prediction of peptides. J Sci Food Agric 98(9):3416–3426PubMedCrossRefGoogle Scholar
  22. 22.
    Neumann T, Junker HD, Keil O, Burkert K, Ottleben H, Gamer J, Sekul R, Deppe H, Feurer A, Tomandl D (2005) Discovery of thrombin inhibitor fragments from chemical microarray screening. Lett Drug Des Discov 2(8):590–594CrossRefGoogle Scholar
  23. 23.
    Lin K, Zhang LW, Han X, Cheng DY (2017) Novel angiotensin I-converting enzyme inhibitory peptides from protease hydrolysates of Qula casein: quantitative structure-activity relationship modeling and molecular docking study. J Funct Foods 32:266–277CrossRefGoogle Scholar
  24. 24.
    Wu Q, Jia J, Yan H, Du J, Gui Z (2015) A novel angiotensin-I converting enzyme (ACE) inhibitory peptide from gastrointestinal protease hydrolysate of silkworm pupa (Bombyx mori) protein: biochemical characterization and molecular docking study. Peptides 68:17–24PubMedCrossRefGoogle Scholar
  25. 25.
    Qiao M, Tu M, Chen H, Mao F, Yu C, Du M (2018) Identification and in silico prediction of anticoagulant peptides from the enzymatic hydrolysates of Mytilus edulis proteins. Int J Mol Sci 19(7):2100–2112PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Huang NT, Wang YR, Xu WB, Zhao SH, Zhang YJ (2009) Technique of bionic enzymatic method for extracting Scolopendra. J Beijing Univ Tradit Chin Med 32:706–709Google Scholar
  27. 27.
    Qin Z, Li X, He W, Zhou Y, Zhao Y (2012) Preparation and anticoagulant activity of polypeptide in Eupolyphaga sinensis walker. J Anhui Agri Sci 40(8910–8911):8934Google Scholar
  28. 28.
    Sabbione AC, Ibañez SM, Martínez EN, Añón MC, Scilingo AA (2016) Antithrombotic and antioxidant activity of amaranth hydrolysate obtained by activation of an endogenous protease. Plant Foods Hum Nutr 71(2):174–182PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Sabbione AC, Scilingo A, Añón MC (2015) Potential antithrombotic activity detected in amaranth proteins and its hydrolysates. LWT Food Sci Technol 60(1):171–177CrossRefGoogle Scholar
  30. 30.
    Li XR, Chi CF, Li L, Wang B (2017) Purification and identification of antioxidant peptides from protein hydrolysate of scalloped hammerhead (Sphyrna lewini) cartilage. Mar Drugs 15(3):61–76PubMedCentralCrossRefGoogle Scholar
  31. 31.
    Ren Y, Wu H, Lai F, Yang M, Li X, Tang Y (2014) Isolation and identification of a novel anticoagulant peptide from enzymatic hydrolysates of scorpion (Buthus martensii Karsch) protein. Food Res Int 64:931–938PubMedCrossRefPubMedCentralGoogle Scholar
  32. 32.
    Maruyama S, Nonaka I, Tanaka H (1993) Inhibitory effects of enzymatic hydrolysates of collagen and collagen-related synthetic peptides on fibrinogen/thrombin clotting. BBA Protein Struct M 1164(2):215–218CrossRefGoogle Scholar
  33. 33.
    Nasri R, Amor IB, Bougatef A, Nedjar-Arroume N, Dhulster P, Gargouri J, Châabouni MK, Nasria M (2012) Anticoagulant activities of goby muscle protein hydrolysates. Food Chem 133(3):835–841CrossRefGoogle Scholar
  34. 34.
    Marlowe CK, Sinha U, Gunn AC, Scarborough RM (2000) Design, synthesis and structure–activity relationship of a series of arginine aldehyde factor Xa inhibitors. Part 1: structures based on the (d)-Arg-Gly-Arg tripeptide sequence. Bioorg Med Chem Lett 10(1):13–16PubMedCrossRefGoogle Scholar
  35. 35.
    Abdelhedi O, Nasri R, Mora L, Jridi M, Toldra F, Nasri M (2018) In silico analysis and molecular docking study of angiotensin I-converting enzyme inhibitory peptides from smooth-hound viscera protein hydrolysates fractionated by ultrafiltration. Food Chem 239:453–463PubMedCrossRefGoogle Scholar
  36. 36.
    Tanaka-Azevedo AM, Morais-Zani K, Torquato RJ, Tanaka AS (2010) Thrombin inhibitors from different animals. J Biomed Biotechnol 1:641025Google Scholar
  37. 37.
    Liu H, Tu M, Cheng S, Chen H, Wang Z, Du M (2019) An anticoagulant peptide from beta-casein: identification, structure and molecular mechanism. Food Funct 10(2):886–892PubMedCrossRefGoogle Scholar
  38. 38.
    Feng L, Tu M, Qiao M, Fan F, Chen H, Song W, Du M (2018) Thrombin inhibitory peptides derived from Mytilus edulis proteins: identification, molecular docking and in silico prediction of toxicity. Eur Food Res Technol 244(2):207–217CrossRefGoogle Scholar
  39. 39.
    Guo SW, Robertson DH, Brooks CL, Michal V (2003) Detailed analysis of grid-based molecular docking: a case study of CDOCKER-A CHARMm-based MD docking algorithm. J Comput Chem 24(13):1549–1562CrossRefGoogle Scholar
  40. 40.
    Clark AM, Labute P (2007) 2D depiction of protein–ligand complexes. J Chem Inf Model 47(5):1933–1944PubMedCrossRefPubMedCentralGoogle Scholar
  41. 41.
    Guinto ER, Vindigni A, Ayala YM, Cera DED (1995) Identification of residues linked to the slow fast transition of thrombin. Proc Natl Acad Sci USA 92:11185–11189PubMedCrossRefPubMedCentralGoogle Scholar
  42. 42.
    Neumann T, Junker HD, Keil O, Burkert K, Ottleben H, Gamer J, Sekul R, Deppe H, Feurer A, Tomandl D (2005) Discovery of thrombin inhibitor fragments from chemical microarray screening. Lett Drug Des Discov 2:590–594CrossRefGoogle Scholar
  43. 43.
    Bode W (2006) Structure and interaction modes of thrombin. Blood Cell Mol Dis 36(2):122–130CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Fangyuan Chen
    • 1
  • Han Jiang
    • 1
  • Yongbo Lu
    • 1
  • Wenwei Chen
    • 1
  • Guangrong Huang
    • 1
    Email author
  1. 1.Key Lab of Marine Food Quality and Hazard Controlling Technology of Zhejiang Province, College of Life SciencesChina Jiliang UniversityHangzhouChina

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