Optimization of Antioxidant Peptides Production from Tryptic Hydrolysis of Pomegranate Seed Protein

Document Type : Research Article

Authors

Maryam Rahimipanah ¹

Alireza Sadeghi Mahoonak ¹

Mohammad Ghorbani ¹

Hoda Shahiri Tabarestani ¹

Mohsen Nabimeybodi ²

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

² Traditional Pharmacy and Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Shahid Sadooghi University of Medical Sciences, Yazd, Iran

https://doi.org/10.22067/ifstrj.2022.76797.1174

Abstract

Introduction
 High levels of free radicals can damage biomolecules and eventually cause oxidative stress. Bioactive peptides produced during enzymatic hydrolysis keep high health properties, such as antioxidant properties. The production of antioxidant peptides has received much attention as a new generation of natural antioxidants. Plants are one of the most abundant sources of biopolymers, especially protein. As long as the protein structure is intact, its amino acid sequence is inactive; however, during proteolysis, fermentation, and gastrointestinal digestion, these amino acids are released as oligopeptides ordinally with less than 20 amino acids and below 10 kDa in molecular weight. These peptides are more digestible and can exhibit specific bioactive properties such antioxidant properties. In this regard, the use of food waste containing protein to produce bioactive peptides and increase their value has received increasing attention. Enzymatic hydrolysis can increase their functional properties by converting proteins into peptides without affecting their nutritional value. Pomegranate seed protein is a by-product of the pomegranate seed oil industry and can be a good source of bioactive peptides with antioxidant properties. According to our knowledge, there isn’t any data about the enzymatic hydrolysis of pomegranate seed protein for antioxidant peptides production. In this study, the optimal conditions for enzymatic hydrolysis of pomegranate seed protein with trypsin using the responses surface method and the effect of hydrolysis on protein structure were investigated.
Materials and Methods
 In this study, the protein was extracted from pomegranate seed, and using trypsin the optimization of enzymatic hydrolysis conditions of protein was determined by Face-Centered Central Composite design, which is one of the responses surface design methods. The effect of independent variables including temperature (30 to 45 °C), time (30 to 180 minutes), and enzyme to substrate ratio (1 to 3 w/w) on DPPH free radical scavenging activity and Fe⁺³ reducing power as responses, was evaluated. Validation tests were performed for confirmation of the proposed values by software and the degree of hydrolysis of the samples was determined. In the next step, the unhydrolyzed and hydrolyzed protein was evaluated for molecular weight distribution and their surface hydrophobicity was compared. Finally, scanning electron microscopy images were used to confirm the hydrolysis process.
Results and Discussion
 Under optimal conditions obtained from the response surface method (temperature: 37.6 °C, time: 136.55 minutes, and enzyme to substrate ratio: 2.2%), trypsin-derived hydrolyzate, showed DPPH free radical scavenging power: 87±0.89% and Fe⁺³ reduction power: 0.293±0.44. Under these conditions, the degree of hydrolysis was equal to 30.1±1%. The optimum conditions of hydrolysis were validated by RSM. The increase in the surface hydrophobicity of the protein after the hydrolysis process indicated the unfolding of the pomegranate seed protein chain and the exposure of its structure during the reaction. The electrophoretic profile of denatured pomegranate seed protein showed smaller peptide bands and lower band intensity, along with losing some of the peptide fractions after hydrolysis. so the efficacy of trypsin at cleaving the protein was confirmed. Evaluation of images obtained by scanning electron microscopy showed that unhydrolyzed protein had complex structures comprised of random sheets of different sizes and shapes and the protein degraded into small fragments and looser structure with many folds after enzyme hydrolysis, resulting in smaller particles compared with untreated samples with the same SEM parameters
Conclusion
Considering the consumer’s tendency toward functional foods and present concerns about the application of synthetic additives and according to the results, the hydrolyzed pomegranate seed protein prepared by trypsin shows good antioxidant capacity. In addition, there will be a reduction in waste generated by the pomegranate processing industry. Further studies will need for the isolation and identification of the specific peptides and amino acid sequences and the evaluation of their possible incorporation in food matrices.

Keywords

Enzymatic hydrolysis

Pomegranate seed

Scanning electron microscopy (SEM)

SDS-PAGE electrophoresis

Surface hydrophobicity

Subjects

• Food Chemistry

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References

1. Akbarbaglu, Z., Mahdi Jafari, S., Sarabandi, K., Mohammadi, M., Khakbaz Heshmati, M., & Pezeshki, A. (2019). Influence of spray drying encapsulation on the retention of antioxidant properties and microstructure of flaxseed protein hydrolysates. Colloids and Surfaces B: Biointerfaces 178: 421–429. https://doi.org/10.1016/j.colsurfb.2019.03.038.
2. Aluko, R.E. (2015). Structure and function of plant protein-derived antihypertensive peptides. Current Opinion in Food Science 4: 44–50. https://doi.org/10.1016/j.cofs.2015.05.002.
3. Chalamaiah, M., Yu, W., & Wu, J. (2017). Immunomodulatory and anticancer protein hydrolysates (peptides) from food proteins : a review. Food Chemistry 245: 205–222. https://doi.org/10.1016/j.foodchem.2017.10.087.
4. Cotabarren, J., Rosso, A.M., Tellechea, M., García-, J., Rivera, J.L., & Obregón, W.D. (2019). Adding value to the chia (Salvia hispanica) expeller: Production of bioactive peptides with antioxidant properties by enzymatic hydrolysis with Papain. Food Chemistry 274: 848–856. https://doi.org/10.1016/j.foodchem.2018.09.061.
5. Derakhshan, Z., Ferrante, M., Tadi, M., Ansari, F., Heydari, A., & Hosseini, M.S. (2018). Antioxidant activity and total phenolic content of ethanolic extract of pomegranate peels, juice and seeds. Food and Chemical Toxicology. https://doi.org/10.1016/j.fct.2018.02.023.
6. Fathi, M., Hosseini, F.S., & Rashidi, L. (2022). Optimized enzymatic hydrolysis of olive pomace proteins using response surface methodology. Applied Food Biotechnology 9(2): 79–90. https://doi.org/10.22037/afb.v9i2.36192.
7. Gerzhova, A., Mondor, M., Benali, M., & Aider, M. (2016). Study of total dry matter and protein extraction from canola meal as affected by the pH, salt addition and use of zeta-potential/turbidimetry analysis to optimize the extraction conditions. Food Chemistry 201: 243–252. https://doi.org/10.1016/j.foodchem.2016.01.074.
8. Hall, F., Johnson, P.E., Liceaga, A., & Johnson, P.E. (2018). Effect of enzymatic hydrolysis on bioactive properties and allergenicity of cricket (Gryllodes sigillatus) protein. Food Chemistry. https://doi.org/10.1016/j.foodchem.2018.04.058.
9. Hamzeh, A., Rezaei, M., Khodabandeh, S., Motamedzadegan, A., Noruzinia, M., & Regenstein, J. Mac. (2019). Optimization of antioxidant peptides production from the mantle of cuttlefish (Sepia pharaonis) using RSM and fractionation. Journal of Aquatic Food Product Technology 28(4): 392–401. https://doi.org/10.1080/10498850.2019.1594480.
10. Houde, M., Khodaei, N., Benkerroum, N., & Karboune, S. (2018). Barley protein concentrates: Extraction, structural and functional properties. Food Chemistry 254: 367–376. https://doi.org/10.1016/j.foodchem.2018.01.156.
11. Islam, M.S., Hongxin, W., Admassu, H., Noman, A., Ma, C., & An wei, F. (2021). Degree of hydrolysis, functional and antioxidant properties of protein hydrolysates from Grass Turtle (Chinemys reevesii) as influenced by enzymatic hydrolysis conditions. Food Science and Nutrition 9(8): 4031–4047. https://doi.org/10.1002/fsn3.1903.
12. Jahanbani, R., Ghaffari, S.M., Salami, M., Vahdati, K., Sepehri, H., & Sarvestani, N.N. (2016). Antioxidant and anticancer activities of walnut (Juglans regia) protein hydrolysates using different proteases. Plant Foods for Human Nutrition 71(4): 402–409. https://doi.org/10.1007/s11130-016-0576-z.
13. Jin, F., Wang, Y., Tang, H., Regenstein, J.M., & Wang, F. (2020). Limited hydrolysis of dehulled walnut (Juglans regia) proteins using trypsin: Functional properties and structural characteristics. Lwt 133(35): 110035. https://doi.org/10.1016/j.lwt.2020.110035.
14. Kato, A., & Nakai, S. (1980). Hydrophobicity determined by a fluorescence probe. Biochimica et Biophysica Acta 624: 13–20.
15. Kim, S.S., Ahn, C.B., Moon, S.W., & Je, J.Y. (2018). Purification and antioxidant activities of peptides from sea squirt (Halocynthia roretzi) protein hydrolysates using pepsin hydrolysis. Food Bioscience 25: 128–133. https://doi.org/10.1016/j.fbio.2018.08.010.
16. Kurozawa, L.E., Park, K.J., & Hubinger, M.D. (2008). Optimization of the enzymatic hydrolysis of chicken meat using response surface methodology. Journal of Food Science 73(5). https://doi.org/10.1111/j.1750-3841.2008.00765.x.
17. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259), 680–685. https://doi.org/10.1038/227680a0.
18. Maluf, J.U., Fiorese, M.L., Maestre, K.L., Dos Passos, F.R., Finkler, J.K., Fleck, J.F., & Borba, C.E. (2020). Optimization of the porcine liver enzymatic hydrolysis conditions. Journal of Food Process Engineering 43(4). https://doi.org/10.1111/jfpe.13370.
19. Meshginfar, N., Sadeghi, A., Farah, M., Tsopmo, A., Mahoonak, A.S., Hosseinian, F., & Tsopmo, A. (2019). Physicochemical, antioxidant, calcium binding, and angiotensin converting enzyme inhibitory properties of hydrolyzed tomato seed proteins. Journal of Food Biochemistry 43(2): 1–10. https://doi.org/10.1111/jfbc.12721.
20. Mirzapour, M., Rezaei, K., Sentandreu, M.A., & Moosavi-movahedi, A.A. (2016). In vitro antioxidant activities of hydrolysates obtained from Iranian wild almond (Amygdalus scoparia) protein by several enzymes. International Journal of Food Sciences and Technology 51: 609–616. https://doi.org/10.1111/ijfs.12996.
21. Nioi, C., Kapel, R., Rondags, E., & Marc, I. (2012). Selective extraction, structural characterisation and antifungal activity assessment of napins from an industrial rapeseed meal. Food Chemistry 134(4): 2149–2155. https://doi.org/10.1016/j.foodchem.2012.04.017.
22. Nourmohammadi, E., Sadeghimahoonak, A., Ghorbani, M., Alami, M., & Sadeghi, M. (2017). The optimization of the production of anti-oxidative peptides from enzymatic hydrolysis of Pumpkin seed protein. Iranian Food Science and Technology Research Journal 13(1): 14–26. https://doi.org/10.22067/ifstrj.v1395i0.45423.
23. Olivares-Galván, S., Marina, M.L., & García, M.C. (2020). Extraction and characterization of antioxidant peptides from fruit residues. Foods 9(8): 1018. https://doi.org/10.3390/FOODS9081018.
24. Ranjbar Nadamani, E., Sadeghi Mahoonak, A., Ghorbani, M., Jakobson, S., & Khori, V. (2020). Optimization of Cajanus Cajan’s antioxidant and nutritional characteristics during hydrolysis process by pepsin using response surface method. Iranian Food Science and Technology Research Journal 15(5): 583–596. https://doi.org/10.22067/ifstrj.v15i4.76968.
25. Singh, T.P., Siddiqi, R.A., & Sogi, D.S. (2018). Statistical optimization of enzymatic hydrolysis of rice bran protein concentrate for enhanced hydrolysate production by papain. LWT - Food Science and Technology. https://doi.org/10.1016/j.lwt.2018.09.014.
26. Talekar, S., Patti, A.F., Singh, R., Vijayraghavan, R., & Arora, A. (2018). From waste to wealth : High recovery of nutraceuticals from pomegranate seed waste using a green extraction process. Industrial Crops & Products 112: 790–802. https://doi.org/10.1016/j.indcrop.2017.12.023.
27. Tehranifar, A., Zarei, M., Nemati, Z., Esfandiyari, B., & Reza, M. (2010). Investigation of physico-chemical properties and antioxidant activity of twenty Iranian pomegranate (Punica granatum) cultivars. Scientia Horticulturae 126(2): 180–185. https://doi.org/10.1016/j.scienta.2010.07.001.
28. Wang, D., & Shahidi, F. (2018). Protein hydrolysate from turkey meat and optimization of its antioxidant potential by response surface methodology. Poultry Science 97(5): 1824–1831. https://doi.org/10.3382/ps/pex457.
29. Xie, J., Du, M., Shen, M., Wu, T., & Lin, L. (2019). Physico-chemical properties, antioxidant activities and angiotensin-I converting enzyme inhibitory of protein hydrolysates from Mung bean (Vigna radiate). Food Chemistry 270(235): 243–250. https://doi.org/10.1016/j.foodchem.2018.07.103.
30. Zang, X., Yue, C., Wang, Y., Shao, M., & Yu, G. (2019). Effect of limited enzymatic hydrolysis on the structure and emulsifying properties of rice bran protein. Journal of Cereal Science 85: 168–174. https://doi.org/10.1016/j.jcs.2018.09.001.