Using Microwave Pretreatment in the Enzymatic Hydrolysis of Pumpkin Seed Protein (Cucurbita maxima L.) by Alcalase and Investigating Its Antioxidant Properties

• Published in: Majallah-i pizhūhishhā-yi ʻulūm va ṣanāyiʻ-i ghaz̲āyī-i Īrāni Vol. 20; no. 2; pp. 295 - 308
• Main Authors: Zeinab Nooshi Manjili, Alireza Sadeghi Mahoonak, Vahid Erfani Moghadam, Mohammad Ghorbani, Hoda Shahiri Tabarestani
• Format: Journal Article
• Language: English
• Published: Ferdowsi University of Mashhad 01.05.2024
• Subjects: antioxidant activity; microwave pretreatment; protein hydrolysates; pumpkin seeds
• ISSN: 1735-4161; 2228-5415
• DOI: 10.22067/ifstrj.2023.82861.1264

Introduction Seeds and nuts have received increasing attention due to their nutritional value and the high therapeutic properties of their bioactive compounds. Most of the seeds are used as nuts, and some of them are considered agricultural waste. Pumpkin seeds have a high content of protein (30–40% in terms of dry matter). Proteins are among the vital health-giving components that provide nitrogen, essential amino acids and energy necessary for normal cells. Pumpkin seeds are a good source of amino acids such as valine, histidine, isoleucine, leucine, threonine and methionine. Protein hydrolysate is a mixture of peptides and amino acids that can show antioxidant, antimicrobial, anticancer, antidiabetic and antihypertensive properties. During hydrolysis, proteins are broken into small peptides and amino acids. Since enzymatic hydrolysis is performed in relatively mild conditions and no amino acid damage occurs, this type of hydrolysis is preferred over acid and alkaline hydrolysis. Hydrolysates obtained from pumpkin seed protein have bioactive properties, especially antioxidant activity. Pretreatment of proteins before enzymatic hydrolysis acts to improve the release of bioactive peptides from different proteins. Pretreatment can facilitate the unfolding the structure of proteins and thus increase the access of enzymes to peptide bonds. The main properties of microwaves usually show three characteristics: penetration, reflection and absorption. Microwave assisted enzymatic hydrolysis can shorten the time  and improve the speed of the reaction. The purpose of this research was to investigate the antioxidant activity of pumpkin seed protein hydrolysates (Cucurbita maxima L.) by alcalase enzyme in two conditions: without pretreatment and using microwave pretreatment.   Material and Methods In this study, Pumpkin (Cucurbita maxima L.) was purchased from the local market of Astane Ashrafieh in Gilan province. The seeds were scooped manuallyand then dried in an oven at 50°C for 72 hours. After the production of protein concentrate from pumpkin seeds, the chemical properties of the concentrate, such as the amount of fat, protein, ash and moisture, were measured. The isolated pumpkin seed solution was exposed to microwave energy with a power of 450-900 watts for 30–90 seconds and was used as a substrate solution in enzymatic hydrolysis experiments. It should be noted that after measuring the total antioxidantactivityr for different powers and times of microwave pretreatment, the power of 600 watts for 60 seconds was selected  and applied before enzymatic hydrolysis. Enzymatic hydrolysis was done by alcalase enzyme with a concentration of 0.5 to 2.5% compared to the protein substrate during 20 to 190 minutes, and the optimum temperature and pH of alcalase were determined in order to produce hydrolysates with antioxidant activity. Antioxidant activity was measured by using DPPH free radical inhibition, total antioxidant activity and iron chelation activity methods.   Result and Discussion Bioactive peptides produced by the enzymatic hydrolysis of proteins have significant antioxidant properties. Pumpkin seeds can be used as a rich source of nutrients and bioactive compounds in various food industries. The results showed that the maximum amount of antioxidant activity without pre-treatment was achieved in 165 minutes with a 2.2% ratio of E/S by using DPPH free radical scavenging activity (40.5%), total antioxidant power (0.79), and iron chelation activity (96.2%) methods. By using microwave pre-treatment, the maximum amount of antioxidant activity was achieved in a shorter time and with less enzyme (105 minutes and E/S ratio 1.5%) using DPPH free radical scavenging (52%), total antioxidant power (0.711), and iron chelation activity (93%). Therefore, it can be concluded that using microwave assisted enzymatic hydrolysis , in addition to achieving hydrolysates with proper antioxidant activity, is a suitable method to save time and reduce enzyme concentrations used in enzymatic hydrolysis.