Investigating the Effect of Utilizing Bioactive Compounds Extracted from Pumpkin Peel Using Supercritical Fluid and Subcritical Water Extraction Methods on Canola Oil Stability
Subject Areas :Azadeh Salami 1 , Narmela Asefi 2 * , Reza Esmailzadeh Kanari 3 , Mahdi Gharah Khani 4
1 - Ph.D Graduated of Food Science and Technology, Tabriz Branch, Islamic Azad University, Tabriz, Iran.
2 - Associate Professor, Department of Food Science and Engineering, Tabriz Branch, Islamic Azad University, Tabriz, Iran.
3 - Professor, Department of Food Science and Industry, Sari Agricultural Sciences and Natural Resources University, Sari, Iran.
4 - Assistant Professor, Department of Food Science and Engineering, Tabriz Branch, Islamic Azad University, Tabriz, Iran.
Keywords: Antioxidant, Canola Oil, Pumpkin, Subcritical water, Supercritical Fluid.,
Abstract :
Oxidation of oils and fats is one of the major problems facing the food industry, leading to a reduction in nutritional value and shelf life. Pumpkin peel has been considered for its antioxidant properties due to phenolic and carotenoid compounds. In this research, phenolic and carotenoid extracts of pumpkin peel were obtained using sub-critical water extraction and supercritical fluid extraction methods. Extracts were added separately and in combination to canola oil with 400 ppm concentration. The changes in the physicochemical properties of the samples (peroxide index, carbonyl number, acidy number, polar compounds, conjugate di-en number, color index, and oxidative stability) were evaluated during two situations: stored at 30° C for 60 days and frying heat treatment conditions at 180˚C for 24 hours. Furthermore, the obtained results were compared with canola oil containing 100 ppm of TBHQ as a synthetic antioxidant. According to the results, the lowest level of oil oxidation stability was observed in the control samples (1 hour after 60 days of storage), then in the samples containing TBHQ for 3 hours, then in the oil containing the combined extract obtained by the supercritical method, and finally in the sub-critical water. The most resistant sample in terms of oxidative stability is related to the sample containing phenolic and carotenoid extract obtained with sub-critical water (after 60 days for 5.5 hours). The results of this study suggest that phenolic-carotenoid extract of pumpkin peel in canola oil can be utilized as a natural antioxidant and as an alternative to synthetic antioxidants
Introduction
Today, vegetable oils have received more attention due to their beneficial effects such as cholesterol reduction and have entered the diet of people in various forms such as salad, cooking and frying oils (2). In addition to changing the organoleptic properties of the food, oxidation of oils leads to an increase in free radicals by increasing peroxide and reducing the shelf life of the oils and due to the loss of sulfur compounds with natural antioxidant properties, and ultimately causing cardiovascular diseases, mutagenesis and cancer. Therefore, stabilization of oils under thermal and storage conditions is inevitable (10). There are various methods for stabilizing oils, one of the most important methods is the use of antioxidants.Although synthetic antioxidants are effective during thermal processing and storage conditions, their use is controversial in terms of toxicity and food safety. The most potent synthetic antioxidant (TBHQ) is not permitted in Japan, Canada, and Europe, and BHA has been removed from the GRAS list (1). Phenolic compounds are a group of aromatic secondary plant metabolites that are widely distributed throughout the plant and have numerous biological effects such as antioxidant activity and antibacterial activity. Today, edible food wastes have an important and valuable place and can be considered as a rich source of polyphenols or antioxidants.Using these wastes as a source of polyphenols can have significant economic benefits for food producers (7). Pumpkin, scientifically known as Cucurbita pepo, belongs to the Cucurbitaceae family. The nutritional value of pumpkin is high, containing 2-10 mg of vitamin C and 9-10 mg of vitamin E per 100 g of pumpkin. This fruit also contains high amounts of vitamins B6, K, thiamine, riboflavin, and minerals such as potassium, phosphorus, magnesium, iron, and selenium, and is an excellent source of carotenoids and phenolic compounds for human consumption (6, 16). In addition to lycopene, beta-carotene, and alpha-carotene, this fruit also contains lutein, which is abundantly found in the skin of this fruit and has unique properties of provitamin, antioxidant, and health-improving properties (4, 5).Extraction is a sensitive process in obtaining valuable compounds such as phenolic and carotenoid compounds from plant tissues. Modern methods of extracting antioxidants currently include the use of supercritical fluid (SFE) and subcritical water (SWE). Supercritical fluid extraction has many advantages, the most important of which are reduced extraction time and no environmental pollution. SFE has properties between a gas and a liquid, and its high solubility increases mass transfer. Supercritical fluid has high permeability and is less viscous than liquid solvents. The best solvent for extracting natural plant compounds is carbon dioxide. Because carbon dioxide is a cheap, neutral, readily available, tasteless, and GRAS-approved compound (15).According to the studies conducted, no research has been reported so far on the extraction of bioactive compounds from pumpkin skin using modern methods and the addition of these compounds as a natural antioxidant to stabilize canola oil. In the present study, the use of supercritical fluid and critical temperature water as two methods was used to extract bioactive compounds (phenolics and carotenoids) from pumpkin skin without thermal damage, and by comparing both methods, the best efficiency of the extracted extract in increasing the shelf life of canola oil was investigated.
Materials and Methods
Materials Used
Pumpkin species Cucurbitapepo, variety Styarica, which have a more orange color than others, were prepared and after thorough washing, their skin was separated and dried at 40 ° C and ground into a powder with a particle size of 2 mm (8).Canola oil used in this study was purchased from Behshahr Agricultural and Industrial Complex (Mazandaran, Iran) and other required chemicals were purchased with analytical grade (Merck, Germany).
Extraction of the extract
Extraction using supercritical fluid
12 g of pumpkin peel along with glass beads were placed in the extractor (model ES080, Kimia Tajhiz- Iran). Carbon dioxide fluid as solvent was pumped at a flow rate of 15 ml/min using an HPLC pump (model 1100Agilent Technologies) to reach the desired pressure. The temperature, time and pressure mixture used were 60°C, 3 h and 25 MPa, respectively (23).
Extraction using subcritical water fluid
12 g of pumpkin peel along with glass beads were placed in the extractor (model ES080, Kimia Tajhiz- Iran).Water was used as the solvent at a flow rate of 1 mL/min using an HPLC pump (Agilent Technologies Model 1100) to achieve the desired pressure. The temperature, time, and pressure used were 120°C, 3 h, and 5 MPa, respectively (23).
Use of extracts in oil
Freshly refined canola oil, without antioxidants, was used for oil tests. The concentrations of phenolic and carotenoid extracts were selected between 100-400 ppm.According to the results of the preliminary tests, the optimal concentration of the extract was determined to be 400 ppm, and the mixture of these two compounds was added in equal proportions to canola oil without antioxidants. The stability of canola oil was measured during storage at 30°C for 60 days (times 0, 15, 30, 45, 60) and during heating at 180°C for 0, 4, 8, 12, 16, 20, 24 hours, and compared with a canola oil sample containing 100 ppm of synthetic antioxidant TBHQ.
Peroxide value measurement
The AOCS method (Cd 8-53) was used to measure the peroxide value. The peroxide value was calculated using the following formula and expressed in milliequivalents of peroxide per 1000 grams of oil (4).
The absorbance of the samples was read at a wavelength of 420 nm against the control sample and the carbonyl number was obtained based on the carbonyl standard curve using the equation (13).2-5- Measurement of Acid Number
In order to measure the acid number, 10 grams of oil sample was weighed in an Erlenmeyer flask and dissolved in 50 ml of chloroform:ethanol solvent in a ratio of (50:50). Then, a few drops of phenolphthalein were added as a reagent and titrated with 0.1 normal potassium hydroxide. The acid number was obtained according to the following equation.
In this equation, m is the weight of the oil in grams, V is the volume of potassium hydroxide consumed in milliliters, and C is the concentration of potassium hydroxide in moles per liter (4).
Measurement of Oxidative Stability Index (OSI)
To determine the efficiency of the antioxidant added to the oil, a Rancimet device (Metrohem Model 743, Switzerland) was used. At a temperature of 120 ° C, 2.5 grams of oil sample was tested with different concentrations of 100, 200, 300, 400 ppm of the extract and 100 ppm of synthetic antioxidant TBHQ. The synthetic antioxidant TBHQ was used as a control (1).
Measurement of conjugated dianhydride
For this purpose, oil samples were dissolved in analytical grade hexane at a ratio of 600:1 and the absorbance at a wavelength of 234 nm was read with a spectrophotometer (Jenova-2100, England) and the amount of conjugated dianhydride was calculated using the relevant formula. The extinction coefficient was 29,000 mol/liter and the results were expressed in mmol/liter (21).
A = Sample absorption - Hexane control absorption
Total amount of polar compounds
The measurement of total polar compounds was carried out by thin layer chromatography (Perkinalmer, USA, model AP1165) in such a way that silica gel was dried for 24 hours at 160°C and mixed with water in a ratio of (5.5:5.9) and transferred to the desired column. The amount of polar compounds was measured gravimetrically and calculated according to the following formula
where Cp, Ws and Wn are the percentage of polar compounds, the weight of the sample and the weight of non-polar compounds, respectively (9).9-2- Color Index Test
The color index of the oil samples was determined by spectrophotometry (Jenova-2100 model) by measuring the absorbance of the oil samples at a wavelength of 420 nm against distilled water as a control sample (21).
Statistical Analysis
To statistically analyze the data obtained from the oil tests, a completely randomized design and two-way ANOVA were used to compare the means. Statistically significant differences between the means at the 95% probability level were determined using Duncan's multiple range test. The software used was SPSS version 20. In order to reduce error, all experiments were performed in triplicate
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