Document Type : Full Research Paper


Department of Food Science and Technology, Shahr-e-Qods Branch, Islamic Azad University, Tehran, Iran


Introduction: Bioactive peptides are protein fragments with 2 to 20 amino acids that have different biological properties depending on the type of amino acids and peptide sequences, including antioxidant, antihypertensive, and antidiabetic. These peptides are inactive in their parent protein sequences but are released during fermentation, enzymatic hydrolysis, or food processing, and exhibit a positive effect on body function and health being. Lentil protein hydrolysate containing antioxidant peptides can be considered as an ingredient of functional foods. One major challenge in using protein hydrolysate in the formulation of functional foods is their stability against the various processes applied to food such as heat and pH treatments.
Materials and Methods: In this study, Lentil protein (Lens esculinaris) was hydrolyzed by Alcalase enzyme under controlled conditions (enzyme/substrate ratio of 90 Anson unit (AU)/ kg protein, 55°C, one hour). The intensity of enzymatic hydrolysis was monitored by the OPA method and antioxidant activity was evaluated based on DPPH and ABTS radical scavenging activity. The heat stability of lentil protein hydrolysate was evaluated by heating samples at 37, 50, 75 (for 15 -60 min), and 90°C (for 5 minutes). The pH stability was investigated by exposing the sample at a pH of 2, 5, 7, 9, And 11 for 1 hr and then adjusting on 7. OPA method was also used to evaluate the possible effect of pH and heat treatments on the content of free amino groups.
Results and Discussion: The results showed that hydrolysis of Lentil protein by Alcalase under controlled conditions produced antioxidant peptides. Heating at 37, 50, and 75°C for 15 minutes reduced the DPPH radical scavenging activity by 1.25, 4.9, and 10.17% and ABTS radical scavenging activity by 3.8, 6.8, and 9%, respectively. The results of the OPA assay also showed a significant (P<0.05) decrease in the number of free amino groups in protein hydrolysate exposed to heat treatment. With increasing the time of treatment up to 60 minutes, the antioxidant activity decreased more significantly (P<0.05), simultaneously with a decrease in the content of free amino acid groups in the protein hydrolysate sample. So that, after heat treatment at 37, 50, and 75 ° C for 60 minutes, the free amino acid groups reached from 33/66 μM leucin /mg protein to 29.51, 27.59, and 25.68 μM leucin /mg protein  and the most decrease in antioxidant activity was measured for samples exposed to 75°C for 60 minutes. It caused a 27.2%, and 29.2% reduction in DPPH and ABTS radical scavenging activity, respectively. Also, exposure to heat treatment at 90°C for 5 minutes caused a 15% and 13% decrease in DPPH and ABTS radical scavenging activity. The results obtained from consideration the antioxidant  activity of samples exposed to pH treatment (2, 5, 7, 9, and 11 for 1 hour) showed the highest antioxidant activity of peptides at neutral pH and confirmed that acidic and alkaline conditions caused a significant decrease in antioxidant activity (P<0.05). As exposure to pHs 2 and 11 for one hour led to respectively 16.3 and a 29.2% decrease in DPPH radical scavenging activity and 16 and 18.2% decrease in ABTS radical scavenging activity. The results of the OPA assay also confirmed the role of acidic and basic pH on less exposure of free amino acid groups in protein structure.
The results showed the potential of using Alcalase enzyme to hydrolyze Lentil protein and produce antioxidant peptides and the Lentil protein hydrolysate with antioxidant activity exhibited relative stability toward different heat and pH treatments. It was concluded that peptides retained 88% and 76% of antioxidant activity at maximum heat (90 ° C for 5 minutes) and pH treatment ( pH=11, for 1 hour). According to the results of the OPA assay, the observed decrease in antioxidant activity  may be due to the changes that happen in protein and peptide structure when are exposed to heat and pH treatments. Altogether, our results showed that Lentil protein hydrolysate can be considered as a potential food ingredient with stable antioxidant activity.


Main Subjects

Abdul-Hamid, A., Bakar, J., Bee, G.H., 2002. Nutritional quality of spray dried protein hydrolysate from Black Tilapia (Oreochromis mossambicus). Food chemistry 78, 69-74.
Alemán, A., Giménez, B., Pérez-Santin, E., Gómez-Guillén, M., Montero, P., 2011. Contribution of Leu and Hyp residues to antioxidant and ACE-inhibitory activities of peptide sequences isolated from squid gelatin hydrolysate. Food Chemistry 125, 334-341.
Brand-Williams, W., Cuvelier, M.-E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. LWT-Food science and Technology 28, 25-30.
Bamdad, F., Wu, J., Chen, L., 2011. Effects of enzymatic hydrolysis on molecular structure and antioxidant activity of barley hordein. Journal of Cereal Science 54, 20-28.
Church, F.C., Swaisgood, H.E., Porter, D.H., Catignani, G.L., 1983. Spectrophotometric assay using o-phthaldialdehyde for determination of proteolysis in milk and isolated milk proteins. Journal of Dairy Science 66, 1219-1227.
de Castro, R.J.S., Sato, H.H., 2015. Biologically active peptides: Processes for their generation, purification and identification and applications as natural additives in the food and pharmaceutical industries. Food Research International 74, 185-198.
Escudero, E., Mora, L., Toldrá, F., 2014. Stability of ACE inhibitory ham peptides against heat treatment and in vitro digestion. Food chemistry 161, 305-311.
Jian, L., Lee, A., Binns, C., 2007. Tea and lycopene protect against prostate cancer. Asia Pacific journal of clinical nutrition 16, 453-457.
Kiokias, S., Dimakou, C., Oreopoulou, V., 2007. Effect of heat treatment and droplet size on the oxidative stability of whey protein emulsions. Food Chemistry 105, 94-100.
Ketnawa, S., Benjakul, S., Martínez-Alvarez, O., Rawdkuen, S., 2017. Fish skin gelatin hydrolysates produced by visceral peptidase and bovine trypsin: Bioactivity and stability. Food chemistry 215, 383-390.
Lowry, O., Rosebrough, N., Farr, A., Randall, R., 1951. Total protein estimation by Lowry's method. J. Biol. Chem 193, 265.
Lai, T., Lin, Z., Zhang, R., Guo, X., Ma, Z., Liao, W., Hu, X., 2016. Processing stability of antioxidant protein hydrolysates extracted from degreased walnut meal. International Journal of Food Engineering 2, 155-161.
Mendis, E., Rajapakse, N., Kim, S.-K., 2005. Antioxidant properties of a radical-scavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate. Journal of agricultural and food chemistry 53, 581-587.
Mine, Y., Li-Chan, E., Jiang, B., 2010. Bioactive proteins and peptides as functional foods and nutraceuticals. John Wiley & Sons.
Maqsoudlou, A., Mahoonak, A.S., Mora, L., Mohebodini, H., Toldrá, F., Ghorbani, M., 2019. Peptide identification in alcalase hydrolysated pollen and comparison of its bioactivity with royal jelly. Food research international 116, 905-915.
Nalinanon, S., Benjakul, S., Kishimura, H., Shahidi, F., 2011. Functionalities and antioxidant properties of protein hydrolysates from the muscle of ornate threadfin bream treated with pepsin from skipjack tuna. Food Chemistry 124, 1354-1362.
Ovissipour, M., Safari, R., Motamedzadegan, A., Rasco, B., Pourgholam, R., Mohagheghi, E., Molla, A.E., 2009. Use of hydrolysates from yellowfin tuna Thunnus albacares fisheries by-product as a nitrogen source for bacteria growth media. International Aquatic Research 1, 73-77.
Pokorný, J., Schmidt, S., 2001. Natural antioxidant functionality during food processing. Antioxidants in food, 331-350.
Rao, S., Sun, J., Liu, Y., Zeng, H., Su, Y., Yang, Y., 2012. ACE inhibitory peptides and antioxidant peptides derived from in vitro digestion hydrolysate of hen egg white lysozyme. Food chemistry 135, 1245-1252.
Rajabzadeh M, Pour-Ashuri P., Shabanpour B, Alishahi A. 1397. Evaluation of Applied and Antioxidant Properties of Hydrolyzed Sperm Protein Egg Protein. (Oncorhyynchus mykiss) Journal of Innovation in Food Science and Technology, 10: 35-23.
Sharma, N., Singh, N., Singh, O., Pandey, V., Verma, P., 2011. Oxidative stress and antioxidant status during transition period in dairy cows. Asian-Australasian Journal of Animal Sciences 24, 479-484.
Urbano, G., Porres, J.M., Frías, J., Vidal-Valverde, C., 2007. Nutritional value, Lentil. Springer, pp. 47-93.
Wong, F.-C., Xiao, J., Michelle, G., Ong, L., Pang, M.-J., Wong, S.-J., Teh, L.-K., Chai, T.-T., 2019. Identification and characterization of antioxidant peptides from hydrolysate of blue-spotted stingray and their stability against thermal, pH and simulated gastrointestinal digestion treatments. Food chemistry 271, 614-622.
Zhu, C.-Z., Zhang, W.-G., Kang, Z.-L., Zhou, G.-H., Xu, X.-L., 2014. Stability of an antioxidant peptide extracted from Jinhua ham. Meat science 96, 783-789.