Identification and characterization of immunomodulatory peptides from pepsin–soy protein hydrolysates

Abstract

To obtain immunomodulatory peptides from isolated soy protein (ISP), pepsin was selected to prepare hydrolysates and 4-h treatment (Pepsin-ISPH4h) showed the highest yield and immunomodulatory activities. The Pepsin-ISPH4h was sequentially fractionated by 30, 10 and 1-kDa molecular weight cut-off (MWCO) membranes, in which 1-kDa MWCO permeate (1P) exhibited the most significant enhancement of phagocytosis activity without causing excessive inflammation as compared with Pepsin-ISPH4h. To further purify and enhance the immunomodulatory activity, 1P was distinct by high-performance liquid chromatography equipped with a reverse-phase column and in vivo immunomodulatory activity of fractions was examined in mice. Fraction 1 (F1) significantly elevated phagocytosis activity of mice spleen macrophages and neutrophils. However, increase of phagocytosis activity did not result from the induction of macrophages M1 or M2 polarization. The immunomodulatory peptide sequence, EKPQQQSSRRGS, from F1 was identified by LC–MS/MS. Phagocytosis activity and macrophage M1 polarization were elevated by synthetic peptide treatment. Hence, our results indicated that isolated soy protein hydrolysates prepared by pepsin could provide a source of peptides with immunomodulatory effects.

Keywords

Immunomodulatory peptide / Isolated soy protein (ISP) / Lipopolysaccharide (LPS) / Molecular weight cut-off (MWCO) / Pepsin / Soybean

References

[1] Adler-Nissen J. Some fundamental aspects of food protein hydrolysis. Enzymic hydrolysis of food proteins, 1986, London: Elsevier Applied Science Publishers, 9-24.

[2] Ahn C-B, Cho Y-S, Je J-Y. Purification and anti-inflammatory action of tripeptide from salmon pectoral fin byproduct protein hydrolysate. Food Chem, 2015, 168: 151-156.

[3] Akbarian M, Khani A, Eghbalpour S, Uversky VN. Bioactive peptides: synthesis, sources, applications, and proposed mechanisms. Int J Mol Sci, 2022, 23: 1445.

[4] Asadullah K, Sterry W, Volk HD. Interleukin-10 therapy—review of a new approach. Pharmacol Rev, 2003, 55: 241-269.

[5] Ashaolu TJ. Health applications of soy protein hydrolysis. Int J Pept Res Ther, 2020, 26: 2333-2343.

[6] Bayne CJ. Origins and evolutionary relationships between the innate and adaptive arms of immune systems. Integr Comp Biol, 2013, 43: 293-299.

[7] Bueno-Gavilá E, Abellán A, Girón-Rodríguez F, María Cayuela J, Tejada L. Bioactivity of hydrolysates obtained from chicken egg ovalbumin using artichoke (Cynarascolymus L.) proteases. Foods, 2021, 10: 246.

[8] Chalamaiah M, Hemalatha R, Jyothirmayi T, Diwan PV, Uday Kumar P, Nimgulkar C, Dinesh Kumar B. Immunomodulatory effects of protein hydrolysates from rohu (Labeo rohita) egg (roe) in BALB/c mice. Food Res Int, 2014, 62: 1051-1061.

[9] Chalamaiah M, Yu W, Wu J. Immunomodulatory and anticancer protein hydrolysates (peptides) from food proteins: a review. Food Chem, 2018, 245: 205-222.

[10] Coscueta ER, Campos DA, Osório H, Nerli BB, Pintado M. Enzymatic soy protein hydrolysis: a tool for biofunctional food ingredient production. Food Chem X, 2019, 1.

[11] Dia VP, Bringe NA, de Mejia EG. Peptides in pepsin-pancreatin hydrolysates from commercially available soy products that inhibit lipopolysaccharide-induced inflammation in macrophages. Food Chem, 2014, 152: 423-431.

[12] Egusa S, Otani H. Soybean protein fraction digested with neutral protease preparation, “Peptidase R”, produced by Rhizopusoryzae, stimulates innate cellular immune system in mouse. Int Immunopharmacol, 2009, 9: 931-936.

[13] Fang Y, Pan X, Zhao E, Shi Y, Shen X, Wu J, Pei F, Hu Q, Qiu W. Isolation and identification of immunomodulatory selenium-containing peptides from selenium-enriched rice protein hydrolysates. Food Chem, 2019, 275: 696-702.

[14] Gille C, Spring B, Tewes L, Poets CF, Orlikowsky T. A new method to quantify phagocytosis and intracellular degradation using green fluorescent protein-labeled Escherichia coli: comparison of cord blood macrophages and peripheral blood macrophages of healthy adults. Cytometry A, 2006, 69: 152-154.

[15] Gordon S. Phagocytosis: an immunological process. Immunity, 2016, 44: 463-475.

[16] Hamer M, Endrighi R, Poole L. Physical activity, stress reduction, and mood: insight into immunological mechanisms. Methods Mol Biol, 2012, 934: 89-102.

[17] He P, Wang Q, Zhan Q, Pan L, Xin X, Wu H, Zhang M. Purification and characterization of immunomodulatory peptides from enzymatic hydrolysates of duck egg ovalbumin. Food Funct, 2021, 12: 668-681.

[18] Hemshekhar M, Faiyae S, Faiyaz S, Choi K-YG, Krokhin OV, Mookherjee N. Immunomodulatory functions of the human cathelicidin LL-37 (aa 13–31)-derived peptides are associated with predicted α-helical propensity and hydrophobic index. Biomolecules, 2019, 9: 501.

[19] Hou H, Fan Y, Li B, Xue C, Yu G, Zhang Z, Zhao X. Purification and identification of immunomodulatory peptides from enzymatic hydrolysates of Alaska pollock frame. Food Chem, 2012, 134: 821-828.

[20] Iwasaki A, Medzhitov R. Control of adaptive immunity by the innate immune system. Nat Immunol, 2015, 16: 343-353.

[21] Jacquot A, Gauthier SF, Drouin R, Boutin Y. Proliferative effects of synthetic peptides from β-lactoglobulin and α-lactalbumin on murine splenocytes. Int Dairy J, 2010, 20: 514-521.

[22] Jamdar SN, Rajalakshmi V, Pednekar MD, Juan F, Yardi V, Sharma A. Influence of degree of hydrolysis on functional properties, antioxidant activity and ACE inhibitory activity of peanut protein hydrolysate. Food Chem, 2010, 121: 178-184.

[23] Jiang H, Zhang J, Shi B-Z, Xu Y-H, Li Z-H, Gu J-R. Application of EGFP-EGF fusions to explore mechanism of endocytosis of epidermal growth factor. Acta Pharmacol Sin, 2007, 28: 111-117.

[24] Kim JY, Kim TH, Kim SS. Anti-inflammatory effect of a human prothrombin fragment-2-derived peptide, NSA9, in EOC2 microglia. Biochem Biophys Res Commun, 2008, 368: 779-785.

[25] Kim H, Wang SY, Kwak G, Yang Y, Kwon IC, Kim SH. Exosome-guided phenotypic switch of M1 to M2 macrophages for cutaneous wound healing. Adv Sci, 2019, 6: 1900513.

[26] Kong X, Guo M, Hua Y, Cao D, Zhang C. Enzymatic preparation of immunomodulating hydrolysates from soy proteins. Bioresour Technol, 2008, 99: 8873-8879.

[27] Koyama T, Uchida K, Fukushima K, Ohashi Y, Uchiyama K, Inoue G, Takahira N, Takaso M. Elevated levels of TNF-α, IL-1β and IL-6 in the synovial tissue of patients with labral tear: a comparative study with hip osteoarthritis. BMC Musculoskelet Disord, 2021, 22: 33.

[28] Lee SM, Son K-N, Shah D, Ali M, Balasubramaniam A, Shukla D. Histatin-1 attenuates LPS-induced inflammatory signaling in RAW264.7 macrophages. Int J Mol Sci, 2021, 22: 7856.

[29] Li Z, An L, Zhang S, Shi Z, Bao J, Tuerhong M, Abudukeremu M, Xu J, Guo Y. Structural elucidation and immunomodulatory evaluation of a polysaccharide from Stevia rebaudiana leaves. Food Chem, 2021, 364.

[30] Liu Q, Kong B, Xiong YL, Xia X. Antioxidant activity and functional properties of porcine plasma protein hydrolysate as influenced by the degree of hydrolysis. Food Chem, 2010, 118: 403-410.

[31] Mallet J-F, Duarte J, Vinderola G, Anguenot R, Beaulieu M, Matar C. The immunopotentiating effects of shark-derived protein hydrolysate. Nutrition, 2014, 30: 706-712.

[32] Marques AH, Bjørke-Monsen A-L, Teixeira AL, Silverman MN. Maternal stress, nutrition and physical activity: impact on immune function, CNS development and psychopathology. Brain Res, 2015, 1617: 28-46.

[33] Mercier A, Gauthier SF, Fliss I. Immunomodulating effects of whey proteins and their enzymatic digests. Int Dairy J, 2004, 14: 175-183.

[34] Minato KI, Abe C. Preedy V. Chapter 17—immunomodulating effect of immodulating effect of polysaccharide. Bioactive food as dietary interventions for arthritis and related inflammatory diseases, 2013, Amsterdam: Elsevier, 241-250.

[35] Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity. J Immunol Methods, 1983, 65: 55-63.

[36] Murray PJ, Allen JE, Biswas SK, Fisher EA, Gilroy DW, Goerdt S, Gordon S, Hamilton JA, Ivashkiv LB, Lawrence T, Locati M, Mantovani A, Martinez FO, Mege J-L, Mosser DM, Natoli G, Saeij JP, Schultze JL, Shirey KA, Sica A, Suttles J, Udalova I, van Ginderachter JA, Vogel SN, Wynn TA. Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity, 2014, 41: 14-20.

[37] Nielson P, Petersen D, Dambmann C. Improved method for determining food protein degree of hydrolysis. J Food Sci, 2001, 66: 642-646.

[38] Polak E, Stępień AE, Gol O, Tabarkiewicz J. Potential immunomodulatory effects from consumption of nutrients in whole foods and supplements on the frequency and course of infection: preliminary results. Nutrients, 2021, 13: 1157.

[39] Ranamukhaarachchi S, Meissner L, Moresoli C. Production of antioxidant soy protein hydrolysates by sequential ultrafiltration and nanofiltration. J Membr Sci, 2013, 429: 81-87.

[40] Rho SJ, Lee JS, Chung YI, Kim YW, Lee HG. Purification and identification of an angiotensin I-converting enzyme inhibitory peptide from fermented soybean extract. Process Biochem, 2009, 44: 490-493.

[41] Ricker D, Johnson L, Murphy P. Functional properties of improved glycinin and β-nglycinin fractions. J Food Sci, 2004, 69: 303-311.

[42] Rőszer T. Understanding the mysterious M2 macrophage through activation markers and effector mechanisms. Mediat Inflamm, 2015, 2015.

[43] Santiago-López L, Hernández-Mendoza A, Vallejo-Cordoba B, Mata-Haro V, González-Córdova AF. Food-derived immunomodulatory peptides. J Sci Food Agric, 2016, 96: 3631-3641.

[44] Sapan HB, Paturusi I, Islam AA, Yusuf I, Patellongi I, Massi MN, Pusponegoro AD, Arief SK, Labeda I, Rendy L, Hatta M. Interleukin-6 and interleukin-10 plasma levels and mRNA expression in polytrauma patients. Chin J Traumatol, 2017, 20: 318-322.

[45] Siemensma AD, Weijer WJ, Bak HJ. The importance of peptide lengths in hypoallergenic infant formulae. Trends Food Sci Technol, 1993, 4: 16-21.

[46] Song Y, Zhang W, Zhang L, Wu W, Zhang Y, Han X, Yang C, Zhang L, Zhou D. Cerebrospinal fluid IL-10 and IL-10/IL-6 as accurate diagnostic biomarkers for primary central nervous system large B-cell lymphoma. Sci Rep, 2016, 6: 38671.

[47] Toopcham T, Mes JJ, Wicheers HJ, Yongsawatdigul J. Immunomodulatory activity of protein hydrolysates derived from Virgibacillus halodenitrificans SK1-3-7 proteinase. Food Chem, 2017, 224: 320-328.

[48] Tsou M-J, Lin S-B, Chao C-H, Chiang W-D. Enhancing the lipolysis-stimulating activity of soy protein using limited hydrolysis with Flavourzyme and ultrafiltration. Food Chem, 2012, 134: 1564-1570.

[49] Van Ginderachter JA, Movahedi K, Hassanzadeh Ghassabeh G, Meerschaut S, Beschin A, Raes G, De Baetselier P. Classical and alternative activation of mononuclear phagocytes: picking the best of both worlds for tumor promotion. Immunobiology, 2006, 211: 487-501.

[50] Wen L, Jiang Y, Zhou X, Bi H, Yang B. Structure identification of soybean peptides and their immunomodulatory activity. Food Chem, 2021, 359.

[51] Wu W, Zhang M, Ren Y, Cai X, Yin Z, Zhang X, Min T, Wu H. Characterization and immunomodulatory activity of a novel peptide, ECFSTA, from wheat germ globulin. J Agri Food Chem, 2017, 65: 5561-5569.

[52] Yang R, Zhang Z, Pei X, Han X, Wang J, Wang L, Long Z, Shen X, Li Y. Immunomodulatory effects of marine oligopeptide preparation from Chum Salmon (Oncorhynchus keta) in mice. Food Chem, 2009, 113: 464-470.

[53] Yang Q, Cai X, Huang M, Wang S. A specific peptide with immunomodulatory activity from Pseudostellaria heterophylla and the action mechanism. J Funct Foods, 2020, 68.

[54] Yu W, Field CJ, Wu J. Purification and identification of anti-inflammatory peptides from spent hen muscle proteins hydrolysate. Food Chem, 2018, 253: 101-107.

[55] Zaky AA, Simal-Gandara J, Eun J-B, Shim J-H, El-Aty AMA. Bioactivities, applications, safety, and health benefits of bioactive peptides from food and by-products: a review. Front Nutr, 2022, 8.

[56] Zhang J, Gao S, Li H, Cao M, Li W, Liu X. Immunomodulatory effects of selenium-enriched peptides from soybean in cyclophosphamide-induced immunosuppressed mice. Food Sci Nutr, 2021, 9: 6322-6334.

[57] Zheng W, Zhao T, Feng W, Wang W, Zou Y, Zheng D, Takase M, Li Q, Wu H, Yang L, Wu X. Purification, characterization and immunomodulating activity of a polysaccharide from flowers of Abelmoschus esculentus. Carbohydr Polym, 2014, 106: 335-342.

[58] Ziegler F, Ollivier J, Cynober L, Masini J, Coudray-Lucas C, Levy E, Giboudeau J. Efficiency of enteral nitrogen support in surgical patients: small peptides v non-degraded proteins. Gut, 1990, 31: 1277-1283.

Funding

Ministry of Science and Technology(NSC100-2221-E029-002)