Purification, Identification and Functional Analysis of a Novel Immunomodulatory Peptide from Silkworm Pupa Protein

  • Zhiyong Li
  • Shan Zhao
  • Xiangdong Xin
  • Bei Zhang
  • Attaribo Thomas
  • Asakiya Charles
  • Kwang Sik Lee
  • Byung Rae Jin
  • Zhongzheng GuiEmail author


In this study, we isolated and characterized an immunomodulatory peptide from silkworm (Bombyx mori) pupa protein hydrolysates. Ultrasound-pretreated hydrolysates were prepared by alcalase digestion and their pro-proliferative activity was assessed with the splenic lymphocyte proliferation assay. Peptide fractions exhibiting the highest activity were purified by Sephadex-G100 and -G15 gel filtration chromatography. The structure of the purified peptides was analyzed using Liquid Chromatography Electrospray Ionisation Tandem Mass Spectrometry (LC–ESI–MS/MS); their digestive stability and their effects of the expression on immune-related cytokines were also evaluated. We purified a novel immunomodulatory peptide with a molecular weight of about 441.06 Da. The amino acid sequence was Asp-His-Ala-Val (DHAV). The splenocyte proliferation rate was 91.1% (P < 0.05) in the presence of 100 µg/mL purified peptide. DHAV was stable in the presence of the gastrointestinal proteases pepsin and trypsin, and stimulated the expression of immune-related factors including interleukin-6 and -12, nuclear factor-κB, cyclin D1, and cyclin-dependent kinase 4. Thus, DHAV purified from silkworm pupa protein hydrolysate has immunomodulatory activity and potential therapeutic value.


Silkworm pupa protein Immunomodulatory peptide Purification Identification 



This study was funded by the Special Fund for Agro-scientific Research in the Public Interest of China (No. 201403064).

Compliance with Ethical Standards

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with animals performed by any of the authors.

Informed Consent

Informed consent was obtained from all individual participants included in the study.


  1. Ajaykrishna PGG, Sivakumar TR, Jin C, Li SH, Cheng HJ, Jia JQ, Gui ZZ (2016) Optimization of polyphenol extraction from mulberry fruit by response surface methodology with ultra-sonic assisted. Sericologia 56(3):74–83Google Scholar
  2. Alanyali S (2017) Expression of Cyclin D1 and its relationship to hormone receptors and survival in breast cancer. Turk J Oncol. Google Scholar
  3. Arzeni C, Perez OE, Pilosof AMR (2012) Functionality of egg white proteins as affected by high intensity ultrasound. Food Hydrocolloids 29(2):308–316CrossRefGoogle Scholar
  4. Chen J, Zheng MZ, Wang H (2009) Development of a sports beverage of silkworm pupa protein peptide. Food Sci 30(14):318–320Google Scholar
  5. Chen J, Liu S, Ye R, Cai G, Ji B, Wu Y (2013) Angiotensin-I converting enzyme (ACE) inhibitory tripeptides from rice protein hydrolysate: purification and characterization. J Funct Foods 5(4):1684–1692CrossRefGoogle Scholar
  6. Cian RE, Lópezposadas R, Drago SR, Sánchez DMF, Martínezaugustin O (2012) A Porphyra columbina hydrolysate upregulates IL-10 production in rat macrophages and lymphocytes through an NK-κB, and p38 and JNK dependent mechanism. Food Chem 134(4):1982–1990CrossRefGoogle Scholar
  7. Conlon JM, Mechkarska M, Lukic ML, Flatt PR (2014) Potential therapeutic applications of multifunctional host-defense peptides from frog skin as anti-cancer, anti-viral, immunomodulatory, and anti-diabetic agents. Peptides 57(7):67–77CrossRefGoogle Scholar
  8. Duarte J, Vinderola G, Ritz B, Perdigón G, Matar C (2006) Immunomodulating capacity of commercial fish protein hydrolysate for diet supplementation. Immunobiology 211(5):341–350CrossRefGoogle Scholar
  9. Ghassem M, Arihara K, Babji AS, Said M, Ibrahim S (2011) Purification and identification of ACE inhibitory peptides from Haruan (Channa striatus) myofibrillar protein hydrolysate using HPLC-ESI-TOF MS/MS. Food Chem 129:1770–1777CrossRefGoogle Scholar
  10. Gill HS, Doull F, Rutherfurd KJ, Cross ML (2000) Immunoregulatory peptides in bovine milk. Br J Nutr 84(S1):111–117Google Scholar
  11. Gui ZZ (2017) Study on development and utilization of silkworm pupa resources. Sci Seric 43(4):0537–0545Google Scholar
  12. Hara S, Yamakawa M (1996) Production in of Moricin, a novel type antibacterial peptide from the silkworm. Biochem Biophys Res Commun. 220:664–669. CrossRefGoogle Scholar
  13. Hartmann R, Meisel H (2007) Food-derived peptides with biological activity: from research to food applications. Curr Opin Biotechnol 18(2):163–169CrossRefGoogle Scholar
  14. Hu AB, Li Q, Shi HJ, Tai Q, Wu LW, Xiong J, Fu BM, He XS (2012) Donor-derived bone marrow transfusion produces mixed chimerism and promotes a Th2 shift in Th1/Th2 balance in rat heterotopic small bowel transplantation. Dig Liver Dis 44(12):988–994CrossRefGoogle Scholar
  15. Jacquot A, Gauthier SF, Drouin R, Boutin Y (2010) Proliferative effects of synthetic peptides from β-lactoglobulin and α-lactalbumin on murine splenocytes. Int Dairy J 20:514–521CrossRefGoogle Scholar
  16. Jia JQ, Wu QY, Yan H, Gui ZZ (2015) Purification and molecular docking study of a novel angiotensin-І converting enzyme (ACE) inhibitory peptide from alcalase hydrolysate of ultrasonic-pretreated silkworm pupa (Bombyx mori) protein. Process Biochem 50:876–883CrossRefGoogle Scholar
  17. Jolles P, Fiat AM, Migliore-Samour D, Drouet L, Caen J (1992) Peptides from milk proteins implicated in antithrombosis and immunomodulation. In: Renner B, Sawatzki G (eds), New perspectives in infant nutrition, symposium Antwerp. Thieme Medical Publications, New York, pp 160–172Google Scholar
  18. Léonil J, Gagnaire V, Mollé D, Pezennec S, Bouhallab S (2000) Application of chromatography and mass spectrometry to the characterization of food proteins and derived peptides. J Chromatogr A 881:1–21CrossRefGoogle Scholar
  19. Mercier A, Gauthier SF, Fliss I (2004) Immunomodulating effects of whey proteins and their enzymatic digests. Int Dairy J 14(3):175–183CrossRefGoogle Scholar
  20. O’Sullivan J, Arellano M, Pichot R, Norton I (2014) The effect of ultrasound treatment on the structural, physical and emulsifying properties of dairy proteins. Food Hydrocolloids 42:386–396CrossRefGoogle Scholar
  21. Ren ZX, Zhou ZF, Fang Y, Jia JQ, Gui ZZ (2015) Process optimization and property testing of silkworm pupa protein treated by ultrasonic. Sci Seric 41(3):548–554Google Scholar
  22. Sapan HB, Paturusi I, Islam AA, Yusuf I, Patellongi I, Massi MN, Pusponegoro AD, Arief SK, Labeda I, Rendy L, Hatta M (2017) Impact of interleukin-6 and interleukin-10 serum levels and mRNA expression level on polytrauma patients. Chin J Traumatol. Google Scholar
  23. Sütas Y, Soppi E, Korhonen H, Syväoja EL, Saxelin M, Rokka T, Isolauri E (1996) Suppression of lymphocyte proliferation in vitro by bovine caseins hydrolyzed with Lactobacillus casei GG-derived enzymes. J Allergy Clin Immunol 98(1):216–224CrossRefGoogle Scholar
  24. Wu FY, Yan H, Ma XN, Jia JQ, Zhang GZ, Guo XJ, Gui ZZ (2012) Comparison of the structural characterization and biological activity of acidic polysaccharides from Cordyceps militaris cultured with different media. World J Microbiol Biotechnol. Google Scholar
  25. Wu QY, Jia JQ, Yan H, Du JJ, Gui ZZ (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 68:17–24CrossRefGoogle Scholar
  26. Yoshikawa M, Kishi K, Takahashi M, Watanabe A, Miyamura T, Yamazaki M, Chiba H (1993) Immunostimulating peptide derived from soybean protein. Ann N Y Acad Sci 685(1):375–376CrossRefGoogle Scholar
  27. Zhao HH, Wang Jh, Liu YJ (2017) Digestive stability of CP4 -EPSPS protein in simulative gastric and intestinal fluid. Biotechnology 27(2):180–185Google Scholar
  28. Zhou J, Han D (2006) Safety evaluation of protein of silkworm (Antheraea pernyi) pupae. Food Chem Toxicol 44(7):1123–1130CrossRefGoogle Scholar
  29. Zhou ZF, Ren ZX, Yu HY, Jia JQ, Gui ZZ (2017) Effects of different modification techniques on molecular structure and bioactivity of Bombyx mori pupa protein. J Asia-Pacific Entomol 20:35–41CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Zhiyong Li
    • 1
  • Shan Zhao
    • 1
  • Xiangdong Xin
    • 1
  • Bei Zhang
    • 1
  • Attaribo Thomas
    • 1
  • Asakiya Charles
    • 1
  • Kwang Sik Lee
    • 2
  • Byung Rae Jin
    • 2
  • Zhongzheng Gui
    • 1
    • 3
    Email author
  1. 1.School of BiotechnologyJiangsu University of Science and TechnologyZhenjiangChina
  2. 2.College of Natural Resources and Life ScienceDong-A UniversityBusanSouth Korea
  3. 3.Sericultural Research InstituteChinese Academy of Agricultural ScienceZhenjiangChina

Personalised recommendations