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

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.