The effect of molecular weight and amino acid composition on antioxidant properties of peptide components of pumpkin seed protein hydrolysate

Document Type : Complete scientific research article

Authors

1 Department of Food Science & Technology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran

2 Department of Food Science & Technology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran.

Abstract

Background and purpose: The formation of free radicals in aerobic organs is inevitable. These radicals cause cells and tissues damages. Oxidation of lipids by producing harmful compounds and as a result spoilage of food products, is one of the most important concerns of the food industry. The use of synthetic antioxidants is tightly controlled due to their harmful effects on human health. Therefore, scientists' research has turned to identification and extraction of natural antioxidants compounds. Bioactive peptides are specific protein components that are inactive at the primary protein source and produced by enzymatic hydrolysis, microbial fermentation, and chemical synthesis. These peptides have different health-promoting properties such as antihypertensive, anti-cholesterol, anti-cancer, strengthening the immune system and antioxidant properties that are influenced by their amino acid composition and sequence. The aim of this study was to investigate the effect of molecular weight and amino acid composition on the antioxidant properties (DPPH radical scavenging activity, Fe reducing power and ABTS radical scavenging activity) of peptide components of pumpkin seed protein hydrolysate.
Materials and Methods: Pumpkin seed protein hydrolysate was prepared under optimum conditions based on the results of previous research and then the effect of molecular weight distribution on the bioactive properties of the resulting fractions and also the relationship between amino acid composition of these peptides and their antioxidant properties after separation and purification by size exclusion chromatography (SEC) and reverse phase high pressure liquid chromatography (RP-HPLC) was examined.
Results: The results of size exclusion chromatography showed that fractions 49 to 56 and 49 to 63 with a molecular weight in range of 9000 to 13,000 daltons have highest DPPH and ABTA radical scavenging activity and after further purification of these fractions by RP-HPC, this ability in fractions with a holding time of 22 minutes showed the highest level. The results of amino acid analysis showed that fractions with high antioxidant capacity contain high amounts of hydrophobic amino acids (about 33.5%), which include non-polar aliphatic groups (such as valine, isoleucine, proline, leucine, tryptophan, and tyrosine). In other words, low molecular weight peptides, with aromatic and hydrophobic amino acids have higher antioxidant activity.
Conclusion: Pumpkin seed protein hydrolysate with peptide components with significant antioxidant properties, can compete with synthetic antioxidants used in the food industry, so it can be used as a natural preservative in food formulations to produce functional products with the aim of increasing public health level and also in medicinal purposes.

Keywords


  1. Blanca, H.L., Ana, Q., Lourdes, A., and Isidra, R. 2007. Identification of bioactive peptides after digestion of human milk and infant formula with pepsin and pancreatin. International Dairy J. 17:1. 42–49.

2.Chalamaiah, M., Jyothirmayi, T., Bhaskarachary, K., Vajreswari, A., Hemalatha, R., and Kumar, B.D. 2013. Chemical composition, molecular mass distribution and antioxidant capacity of rohu (Labeo rohita) roe (egg) protein hydrolysates prepared by gastrointestinal proteases. Food Research International. 52:1. 221–229.

  1. Chang, S.K., Ismail, A., Yanagita, T., Esa, N.M., and Baharuldin, M.T.H. 2015. Antioxidant peptides purified and identified from the oil palm (Elaeis guineensis Jacq.) kernel protein hydrolysate. J. Functional Foods. 14:1. 63-751.

4.Clement, A., Vioque, J., and Millan, F. 1999. Vegetable Protein Hydrolysate in Nutricion y Obesidad. Food Science. 2:1. 289- 296.

5.Collins, A.R. 2005. Antioxidant intervention as a route to cancer prevention. European J. of Cancer. 41:13.1923–30.

6.Contreras, M., Hernández-ledesma, B., Amigo, L., and Martín-Álvarez, P.J. 2011. Production of antioxidant hydrolyzates from a whey protein concentrate with thermolysin: Optimization by response surface methodology. LWT - Food Science and Technology. 44:1. 9-15.

7.Corêa, A.P.F., Daroit, D.J., Fontoura, R., Meira, S.M.M., Segalin, J., and Brandelli, A. 2014. Hydrolysates of sheep cheese whey as a source of bioactive peptides with antioxidant and angiotensin - converting enzyme inhibitory activities. Peptides. 61:1. 48-55.

8.Dey, S.S., and Dora, K.C. 2014. Antioxidative activity of protein hydrolysate produced by alcalase hydrolysis from shrimp waste (Penaeus monodon and Penaeus indicus). J of food science and technology. 51:3.449-457.

9.Escudero, E., Aristoy, M.C., Nishimura, H., Arihara, K., and Toldrá, F. 2012. Antihypertensive effect and antioxidant activity of peptide fractions extracted from Spanish dry-cured ham. Meat Science. 91:1. 306–311

10.Etemadi, M., Sadeghi mahoonak, A.R. Ghorbani, M. and Maqsoudlou, Y. 2016. Production and evaluation of chelating activity and reducing power of hydrolyzed proteins derived from soy protein. J of Food Science and Nutrition. 13: 1.65-74. (In Persian)

11.Gauthier, SF., Pouliot, Y., and Saint-Sauveur, D. 2006. Immuno modulatory peptides obtained by the enzymatic hydrolysis of whey proteins. International Dairy J. 16:11. 1315-1323.

12.Gibbs, B.F., Kermasha, S., Alli, I., and Mulligan, C.N. 1999. Encapsulation in the food industry. International J of Food Sciences and Nutrition. 50:3. 213–224.

13.Jemil, I., Jridi, M., Nasri, R., Ktari, N., Salem, R.B.S.-B., Mehiri, M., and Nasri, M. 2014. Functional, antioxidant and antibacterial properties of protein hydrolysates prepared from fish meat fermented by Bacillus subtilis A26. Process Biochemistry. 49: 6.963-972

14.Jia, J., Maa, H., Zhao, W., Wang, Z., Tian, W., and Luo, L. 2010. The use of ultrasound for enzymatic preparation of ACE-inhibitory peptides from wheat germ protein. Food Chemistry. 119:1. 336–42.

15.Kaveh, Sh., Sadeghi mahoonak, A.R., Ghorbani, M. and Sarabandi, Kh. 2019. Comparison of antioxidant properties of hydrolyzed fenugreek seed protein with alcalase and pancreatin. J. Innovation in Food Science and Technology. 11: 4.77-88. (In Persian)

16.Lassoued, I., Mora, L., Nasri, R., Jridi, M., Toldrá, F., Aristoy, M.-C., and Nasri, M. 2015. Characterization and comparative assessment of antioxidant and ACE inhibitory activities of thornback ray gelatin hydrolysates. J. Functional Foods. 13:1. 225-238.

17.Li, Y., Jiang, B., Zhang, T., Mu, W., and Liu, J. 2008. Antioxidant and free radical-scavenging activities of chickpea protein hydrolysate (CPH). Food Chemistry. 106:2. 444-450.

18.Liang, L., Cai, S., Gao, M., Chu, X., Pan, X., Gong, K., Xiao, C., Chen, Y., Zhao, Y., Wang, B., and Sun, k. 2019. Purification of antioxidant peptides of Moringa oleifera seeds and their protective effects on H2O2 oxidative damaged Chang liver cells. J. Functional Foods. 64:1. 103698.

19.Maqsoudlou, A., Sadeghi mahoonak, A.R., Mora, L., Mohebodini, H., Toldrá, F., and Ghorbani, M. 2019. Peptide identification in alcalase hydrolysated pollen and comparison of its bioactivity with royal jelly. Food ResearchInternational. 116:1. 905-915.

20.Matthäus, B. 2002. Antioxidant activity of extracts obtained from residues of different oilseeds. J of Agricultural and Food Chemistry. 50:1. 3444–3452.

21.Mazloomi, N., Mora, L., Aristoy, M., Sadeghi mahoonak. A.R., Ghorbani, M., Houshmand, Gh., and Toldrá, F. 2020. Impact of Simulated Gastrointestinal Digestion on the Biological Activity of an Alcalase Hydrolysate of Orange Seed (Siavaraze, Citrus sinensis) by-Products. Foods. 9: 9. 1217. 

22.Mazloomi, N., Sadeghi mahoonak, A.R., Ghorbani, M. and Hooshmand, Gh.R. 2019. Determination of optimal production conditions of antioxidant peptides resulting from hydrolysis of orange kernel protein with alcalase enzyme. Food science and Technology. 88: 16. 343-356. (In Persian)

23.Mccann, KB., Shiell, BJ., Michalski, WP., Lee, A., Wan, J., Roginski, H. 2006. Isolation and characterization of a novel antibacterial peptide from bovine As1-casein. International Dairy J. 16:1. 316–323.

24.Meisel, H., and Fitz-gerald, R.J. 2003. Biofunctional peptides from milk proteins, mineral binding and cytomodulatory effects. Current Pharmaceutical design. 9: 1. 1289–1295.

25.Mendis, E., Rajapakse, N., and Kim, S.K. 2005. Antioxidant properties of a radicals scavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate. Food Chemistry. 53: 3. 581-587.

26.Meshginfar, N., Sadeghi-mahoonak, A. R., Ghorbani, M. and Aalamai, M. 2016. Effects of protein hydrolysate from sheep visceral on oxidative stability of soybean oil and chicken sausage. J of Food Processing and Preservation. 41: 2.12875.

27.Moayedi, A., Mora, L., Aristoy, M., Hashemi, M., Safari, M., and Toldra, F. 2016. ACE-Inhibitory and Antioxidant Activities of Peptide Fragments Obtained from Tomato Processing By-Products Fermented Using Bacillus subtilis: Effect of Amino Acid Composition and Peptides Molecular Mass Distribution. Applied Biochemistry Biotechnology. 181: 1.48-64.

28.Mohamed, R.A., Ramadan, R.S., and Ahmed, L.A. 2009. Effect of substituting pumpkin seed protein isolate for casein on serum liver enzymes, lipid profile and antioxidant enzymes in CCl4-intoxicated rats. Advanced Biomedical Research. 3: 1. 9-15.

29.Nkosi, C.Z, Opoku A.R., and Terblanche S.E. 2006. Antioxidative effects of pumpkin seed (Cucurbita pepo) protein isolate in CCl4 Induced liver injury in low protein fed rats. Phytotherapy Research 20:11. 935-940

30.Normohamadi, E., Sadeghi-mahonak, A.R., Aalami, M., Ghorbani, M. and Sadeghi, M. 2018. Optimization of pumpkin seed meal protein hydrolysis with alcalase to achieve to maximum antioxidant properties. J. Food Processing and Preservation, 9: 2.11-17. (In Persian)

31.Ovissipour, M.R., Abedian Kenari, A., Motamedzadegan, A., and Nazari, R.M. 2010. Optimization of Enzymatic Hydrolysis of Visceral Waste Proteins of Yellowfin Tuna (Thunnus albacares). Food and Bioprocess Technology. 5: 2. 696-705.

32.Palmieri, V.O., Grattagliano, I., Portincasa, P., and Palasciano, G. 2006. Systemic oxidative alterations are associated with visceral adiposity and liver steatosis in patients with metabolic syndrome. J. Nutrition. Oxford University Press.136: 12.3022–3026.

33.Phongthai, S., and Rawdkuen, S. 2019. Fractionation and characterization of antioxidant peptides from rice bran protein hydrolysates stimulated by. Cereal Chemistry. 97:2. 316–325.

34.Power, O., Jakeman, P., and FitzGerald, R. 2013. Antioxidative peptides: enzymatic production, in vitro and in vivo antioxidant activity and potential applications of milk-derived antioxidative peptides. Amino Acids. 44: 3. 797-820.

35.Sadeghian, A., Sadeghi-mahoonak, A.R. Ghorbani, M., Aalami, M. and Joshghani, H. 2020. Effect of process time on functional and antioxidant properties of quinoa hydrolyzed protein with alcalase and pancreatin. Journal of Nutrition Science and Food Technology. 14: 4.89-102. (In Persian)

  1. Saiga, A.I., Tanabe, S., and Nishimura, T. 2003. Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment. J. Agriculture and Food Chemistry. 51: 12. 3661-3667.

37.Sakanaka, S., Tachibana, Y., Ishihara, N., and Juneja, L.R. 2004. Antioxidant activity of egg-yolk protein hydrolysates in a linoleic acid oxidation system. Food Chemistry. 86: 1.99-103.

38.Salwanee, S., Wan Aida, W.M., Mamot, S., and Maskat, M.Y. 2013. Effects of enzyme concentration, temperature, pH and time on the degree of hydrolysis of protein extract from viscera of tuna (Euthynnus affinis) by using alcalase. Sains Malaysiana. 42:3. 279-287.

39.Samaranayaka, G.P.A., and Li-Chan, C.Y.E. 2011. Food-derived peptidic antioxidants: A review of their production, assessment, and potential applications. J. Functional Foods. 3: 4. 229-254.

40.Shariat-alvai, M., Sadeghi mahoonak, A.R., Gorbani, M., Aalami, M. and Mohammadzadeh, J. 2019. Determining the optimal conditions for the production of hydrolyzed proteins with antioxidant capacity and reduction of nitric oxide from tomato waste by alcalase. Food science and Technology. 15: 84. 137-151. (In Persian)

41.Sun, J., He, H., and Xie, B.J. 2004. Novel antioxidant peptides from fermented mushroom Ganoderma lucidum. Food Chemistry. 52: 6646–52.

42.Tang, C.-H., Peng, J., Zhen, D.-W., and Chen, Z. 2009. Physicochemical and antioxidant properties of buckwheat (Fagopyrum esculentum Moench) protein hydrolysates. Food Chemistry. 115: 2.672-678.

43.Wiriyaphan, C., Chitsomboon, B., and Yongsawadigul, J. 2012. Antioxidant activity of protein hydrolysates derived from threadfin bream surimi byproducts. Food Chemistry. 132: 1.104-111.

44.Yildirim, A., Mavi, A., and Kara, A.A. 2001. Determination of antioxidant and antimicrobial activities of Rumex crispus L. extracts. J. Agricultural and Food Chemistry. 49: 8.4083-4089.

45.You, L., Zhao, M., Regenstein, J.M., and Ren, J. 2010. Changes in the antioxidant activity of loach (Misgurnus anguillicaudatus) protein hydrolysates during a simulated gastrointestinal digestion. Food Chemistry. 120: 3.810–6.

46.Zhang, S.B., Wang, Z., and Xu, S.Y.2008. Antioxidant and antithrombotic activities of rapeseed peptides. J of the American Oil Chemists' Society. 85: 6.521-527.

47.Zhao, Q., Xiong, H., Selomulya, C., Chen, D. X., Zhong, H., Wang, S., Sun, W., and Zhou, Q. 2012. Enzymatic hydrolysis of rice dreg protein: Effects of enzyme type on the functional properties and antioxidant activities of recovered proteins. Food Chemistry. 134: 1.1360-1367.

48.Zhu, K., Zhou, H., and Qian, H. 2006. Antioxidant and free radical-scavenging activities of wheat germ protein hydrolysates (WGPH) prepared with alcalase. Process Biochemistry. 41:6. 1296-1302.

49.Zhu, L., Chen, J., Tang, X., and Xiong, Y.L. 2008. Reducing, radical scavenging and chelation properties of in vitro digests of alcalase-treated zein hydrolysate. J. of Agricultural and Food Chemistry. 56: 8. 2714-2721.

50.Živanović, I., Vaštag, Z., Popović, S., Popović, L., and Peričin, D. 2011. Hydrolysis of hull-less pumpkin oil cake protein isolate by Pepsin. International J of Biological and Life Sciences. 7: 1. 30-34.