Food Chemistry
Zeinab Nooshi Manjili; Alireza Sadeghi Mahoonak; Vahid Erfani Moghadam; Mohammad Ghorbani; Hoda Shahiri Tabarestani
Abstract
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% ...
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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.
Elham Nourmohammadi; Alireza Sadeghi Mahoonak; Mohammad Ghorbani; Mehran Alami; Masoumeh Sadeghi
Abstract
Introduction: Proteins are vital substances for health since they provide nitrogen, amino acids and the energy required for normal body performance. However, the applications of proteins are limited due to their certain properties, such as their low solubility. The enzymatic hydrolysis of proteins is ...
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Introduction: Proteins are vital substances for health since they provide nitrogen, amino acids and the energy required for normal body performance. However, the applications of proteins are limited due to their certain properties, such as their low solubility. The enzymatic hydrolysis of proteins is an extensively used approach to produce bioactive peptides and promote the chemical, functional and nutritional properties of proteins. These compounds have interesting biological properties such as anti-oxidative, anti-hypertensive, anti-bacterial, anti-cancer and anti-thrombotic activities. Lipid peroxidation is one of the main reasons behind the deterioration of foodstuffs during processing and storage. In this case, the addition of anti-oxidative compounds is considered as an effective way to improve the shelf-life of lipid containing foods. Due to carcinogenic effect of synthetic anti-oxidative compounds, extensive efforts have been done to find natural anti-oxidative compounds with plant origin during recent years. Pumpkin (Cucurbitapepo) seeds are rich of proteins, unsaturated fatty acids, phytosterols and essential minerals like Zn, K, Ca, Mg, Fe, Cu and P. Oil content of pumpkin seeds is about 40-60%, and mostly consisted of oleic, palmitic and stearic acids. On the other hand, its protein content is about 45-46%, and this amount will reach to 55-56% after defatting. To date, pumpkin seeds have been mainly used for pumpkin oil production. After the oil extraction, a protein-rich by-product (pumpkin oil cake) remains, which is often used for animal feeding. In this study, the enzymatic hydrolysis of pumpkin oil cake protein isolate by a food-grade protease named trypsin was attempted and the optimum treatment was selected based on the DPPH radical scavenging and ferrous ion chelating activities Materials and Methods: In this study, the optimization of the hydrolysis of pumpkin (Cucurbitapepo) oil cake protein was investigated using response surface methodology (RSM) and central composite design (CCD) in order to achieve the maximum DPPH radicals scavenging and metal ion chelating activities. For this purpose, trypsin concentrations of 1-2% and hydrolysis temperatures and times of 35-45 ċ and 2-5 hours were examinedas independent variables. Preparations of pumpkin oil cake protein isolate (POCPI) Defatted pumpkin oil cake was dispersed in distilled water (1:10 w/v). The pH was adjusted to 10 with 1N NaOH, mixed for 1 hour at room temperature and centrifuged at 5000g for 20 minutes (Combi514R, South Korea). The supernatant was collected, pH was adjusted to 5 with 1N HCl and centrifugation was performed at the same condition. Supernatant was discarded and precipitate was collected as pumpkin oil cake protein isolate. Enzymatic hydrolysis Pumpkin oil cake protein isolate was dispersed in tris-HCl at pH= 8 for trypsin enzymatic treatment (5% w/v). After that, trypsin was added at 1%, 1.5% and 2% and hydrolysis was carried out for 2, 3.5 and 5h at 200 rpm in shaker incubator (8480-VS, South Korea). Hydrolysis temperatures were 35, 40 and 45˚C. At the end of hydrolysis, the enzyme was inactivated for 15 minutes at 85˚C; dispersion was centrifuged at 4000g for 30 minutes, the supernatant was collected and freeze dried. DPPH radical scavenging activity An aliquot of 1000 microliterpumpkin oil cake proteinhydrolysate was mixed with 1000 microliter of 0.1mM DPPH solution prepared in 96% ethanol. The mixture was allowed to stand for 60 minutes in the dark and the absorbance was read at 517 nm. The blank was prepared with the same manner except that 1000 microliter water was used instead of 1000 microliter pumpkin oil cake proteinhydrolysate. Ferrous ion chelating activity Pumpkin oil cake protein hydrolysate(4.7 ml) was mixed with 0.1 ml 2mM FeCl2 and 0.2 ml 5 mM ferrozine and was kept at room temperature for 20 min. The absorbance was read at 562 nm and the blank sample was prepared with the same manner except that 4.7 ml distilled water was used instead of sample. Results & Discussions: The results of this study, showed that the optimum conditions to reach the maximum DPPH radicals scavenging and metal ion chelating activities were 35 ċ, 5h, 1.1% enzyme concentration and 45 ċ, 2.05h and 2% enzyme concentration that showed DPPH radicals scavenging and metal ion chelating activities of 76.28% and 49.61% respectively. These results were to large extent similar to those suggested by Design Expert software (75.89% and 50.84%). The R2 was 0.9184% and 0.9761% for DPPH radicals scavenging and metal ion chelating activities respectively. Moreover the adjusted R2 was estimated to be 0.1333 and 0.1827 for DPPH radicals scavenging and metal ion chelating activities respectively, which suggested the suitability and fitness of proposed model for the considered responses. Conclusions: Based on the results, pumpkin oil cake protein hydrolysate demonstrated appropriate anti-oxidative and metal ion chelating abilities. The results of this study indicated that pumpkin oil cake protein hydrolysate had the ability to be used as an effective and natural anti-oxidative compound in lipid containing foods.
