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Application of nano-encapsulated olive leaf extract in controlling the oxidative stability of soybean oil
Food Chemistry 190 (2016) 513–519
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Application of nano-encapsulated olive leaf extract in controlling the oxidative stability of soybean oil Adeleh Mohammadi, Seid Mahdi Jafari ⇑,1, Afshin Faridi Esfanjani, Sahar Akhavan Department of Food Materials and Process Design Engineering, Faculty of Food Science and Technology, University of Agricultural Sciences and Natural Resources, Gorgan, Iran
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Article history: Received 4 December 2014 Received in revised form 27 April 2015 Accepted 28 May 2015 Available online 29 May 2015 Keywords: Olive leaf extract Nano-emulsion Encapsulation Antioxidant activity
a b s t r a c t Our objective was to evaluate the antioxidant activity of olive leave extract (OLE) encapsulated by nano-emulsions in soybean oil. The average droplet size one day after production was 6.16 nm for primary W/O nano-emulsion and, 675 nm and 1443 nm for multiple emulsions stabilized by WPC alone and complex of WPC–pectin, respectively. The antioxidant activity of these emulsions containing three concentrations of 100, 200 and 300 mg OLE during storage was evaluated in soybean oil by peroxide value, TBA value and rancimat thermal stability test and was compared with blank (non-encapsulated) OLE and synthetic TBHQ antioxidant. Nano-encapsulated OLE was capable of controlling peroxide value better than unencapsulated OLE. But because of blocking phenolic compounds within dispersed emulsions droplets, thermal stability of encapsulated OLE was lower. To summarize, with increased solubility and controlled release of olive leaf phenolic compounds through their nano-encapsulation, a higher antioxidant activity was achieved. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Lipid oxidation is a complex series of reactions that occur during processing, storage, shipment, and final preparation of food stuffs containing lipids. Oxidation mechanism starts to take place immediately after oil extraction (Iqbal & Bhanger, 2007; Taghvaei & Jafari, 2015). Oxidation may lead to formation of toxic compounds, off-flavors and lower the quality and nutritional value of foods. This process may be inhibited by diverse procedures including prevention of oxygen access, inactivation of enzymes catalyzing oxidation, reduction of oxygen pressure, addition of chelating agents, use of lower temperature and the use of suitable packaging. Another method of protection against oxidation is to use specific additives called the antioxidants (Pokorny, Yanishlieva, & Gordon, 2001; Ramarathnam, Osawa, Ochi, & Kawakishi, 1995). Harmful effects of the synthetic antioxidants such as tert-butyl hydroquinone (TBHQ), butylated hydroxy toluene (BHT), and butylated hydroxy anisole (BHA), for health have been demonstrated previously. Therefore, there has been a recent attention on the use of natural phenolic compounds due to their antioxidant properties and potential health effects (Anagnostopoulou, Kefalas, Papageorgiou, Assimepoulou, & Boskou, 2006; Rahmanian, Jafari, & Calanagis, 2014; Taghvaei & Jafari, 2015). Several studies have ⇑ Corresponding author at: Pishro Food Technology Research Group, Gorgan, Iran. 1
E-mail address: [email protected] (S.M. Jafari). This author equally contributed as first author.
http://dx.doi.org/10.1016/j.foodchem.2015.05.115 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
been conducted with the target of protecting foods against oxidation by using natural antioxidants such as grape seed extract, chestnut, rosemary, green tea, etc (Gibis & Weiss, 2012; Lorenzo, González-Rodríguez, Sánchez, Amado, & Franco, 2013; Samotyja & Malecka, 2007). Olive leaf (Olea europaea) is one of the medicinal plants, which is considered as a good and cheap source of antioxidants. Among different parts of olive tree, the leaf is one of the richest sources of phenolic compounds (Rafiee, Jafari, Alami, & Khomeiri, 2011; Rahmanian, Jafari, & Wani, 2015; Proestos, Boziaris, Nychas, & Komaitis, 2006). Oleuropein and its derivatives such as hydroxytyrosol and tyrosol are the most abundant phenolic compounds in olive leaf. A linear relationship exists between the amount of oleuropein in leaves and their antioxidant property (Farag, El-Baroty, & Basuny, 2003). Nano-encapsulation of bioactive compounds by nonionic emulsifiers and through O/W or W/O nano-emulsions can protect and increase their properties (such as antioxidativity, stability and, solubility) (Lawrence & Rees, 2012). In this technology, nanoparticles of encapsulated compound (5–100 nm) are produced in a continuous phase (Flanagan & Singh, 2006). Also, it has been well documented that nano-emulsions have a pronounced effect on the antioxidant activity of different bioactive compounds such as green tea polyphenols, resveratrol, and flavanols (Fang & Bhandari, 2010; Zhou et al., 2000; Zhou, Miao, Yang, & Liu, 2005). On the other hand, emulsification of a W/O emulsion within an aqueous phase containing hydrophilic emulsifiers could lead to the production of W/O/W multiple emulsions. As a result, the release
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rate of encapsulated compounds within the inner aqueous phase is controlled (Choi, Decker, & McClements, 2009). Food biopolymers, such as proteins and polysaccharide are among the hydrophilic emulsifiers which may be applied in external aqueous phase of W/O/W double emulsions. Whey protein has excellent emulsifying properties and can be used alone or in a complex with polysaccharides such as pectin within the external aqueous phase of W/O/W emulsions (Bouyer, Mekhloufi, Rosilio, Grossiord, & Agnely, 2012). Besides, whey proteins are a rich of cysteine and this amino acid is a potent intracellular antioxidant which could impart with a good antioxidant capacity (Walzem, Dillard, & German, 2002). There have been some studies describing the antioxidant capacity of olive leaf extract (Rafiee, Jafari, Alami, & Khomeiri, 2012; Taghvaei et al., 2014) but, due to sensitivity of its natural phenolic compounds against pH, light, oxygen, etc., it is essential to protect them by a novel technology such as encapsulation. Antioxidant activity of encapsulated olive leaf extract within nano-emulsions and multiple emulsions could be a solution which has not been investigated yet. Therefore, objective of this study was to evaluate nano-encapsulation of olive leaf extract and its application in soybean oil. 2. Materials and methods 2.1. Materials Refined soybean oil without any antioxidant additives was purchased from a local oil refining factory (Alia Golestan Co., Iran). Olive leaves of Mission variety were collected from Gorgan, Iran, and citrus pectin (GA: > 65%) was purchased from MP biomedical (Netherland). Whey protein concentrate (80% protein) and sorbitan monooleate (span 80) was obtained from Sapoto cheese (USA) and Merck (Germany), respectively. Sodium azide was purchased from Sigma–Aldrich (St. Louis, USA). TBHQ were purchased from Nova international (India). Other chemical were of analytical grade. 2.2. Microwave assisted extraction of phenolic compounds from olive leaf For preparing phenolic compounds, microwave assisted extraction (MAE) was used according to the method of Rafiee et al. (2011). Briefly for each extraction, 12 g of dried leaves was blended with 120 ml methanol solvent in a 500 ml volumetric flask and were placed in a microwave oven. While magnetic stirring, 6 min irradiation was performed (8 s power on and 15 s power off in order to prevent super-boiling of solvent). Then, the methanol extract was filtered and solvent removed under reduced pressure at 40 °C by means of a rotary evaporator (IKA RV 10 basic, Germany) and remaining dried by a freeze-drier (Operon FDB-5503) at 20 °C. 2.3. Determination of total phenolic content Total phenolic content of each extract was determined by the Folin–Ciocalteu micro-method (Rafiee et al., 2012). Briefly, 20 ll of extract solution was mixed with 1.16 ml distilled water and 100 ll Folin–Ciocalteu reagent, followed by addition of 300 ll Na2CO3 solution (20%) before 8 min. Subsequently, the mixture was incubated in a shaking incubator at 40 °C for 30 min and its absorbance was measured at 760 nm (WPA S2000, UK). Gallic acid was used as a standard for calibration curve. 2.4. Biopolymer solution preparation Wall materials solutions as the outer queues phase of multiple emulsions were prepared by dispersing the powdered pectin in
distilled water and stirring at 50 °C for 30 min and then were kept at room temperature overnight. In the case of WPC, their solutions were kept refrigerated for 24 h for complete hydration. The pH of solution was afterwards adjusted on 6.0 with phosphate buffer. The solution was heat-treated at 70 °C for 20 min, and cooled down quickly. The solution of WPC–pectin was prepared by adding pectin solution into the solution of WPC, and stirring at room temperature for an hour. Then, the pH of solution was set on 6 by phosphate buffer and was kept in a refrigerator overnight. 0.004% sodium azide was included in the solutions as an antimicrobial substance. The final percentage of biopolymers in outer aqueous phase of multiple emulsions was 8% WPC for single layer and, 8%WPC–0.2% Pectin for double layer treatments (Lutz, Aserin, Wicker, & Garti, 2009). 2.5. Preparation of emulsions W/O/W multiple emulsions were prepared using a two-step emulsification procedure. 2.5.1. Preparation of the primary W/O micro-emulsion (nanoencapsulated particles) First, nanoparticles of olive leaf extract were prepared via a nonionic emulsifier. According to the method of Sadeghi, Madadlou, and Yarman (2013), W/O nanoemulsion was prepared by dropwise addition of 7% olive leaf extract into a continuous phase consisting 31% span 80 and 62% soybean oil while stirring (300 rpm) until the system became transparent. Before this step, span 80 and soybean oil as an organic phase should be mixed together at 300 rpm so that the span 80 is dissolve completely. 2.5.2. Preparation of W/O/W multiple emulsions In the second emulsification step, nanocapsules of olive leaf extract (initial W/O nano-emulsion) were coated by biopolymers (WPC, pectin) through W/O/W double emulsions. The pre-W/O/W emulsions were formed by gradually adding the primary W/O nano-emulsions into the continuous aqueous phase containing biopolymers at 10 °C during homogenization with a rotor–stator homogenizer (8000 rpm for 5 min, T25 IKA, Germany). These W/O/W emulsions were then further emulsified using the mentioned homogenizer (15000 rpm for 8 min). All multiple emulsions were composed of 30% primary nano-emulsion and 70% outer aqueous phase containing biopolymers (w/w) (Lutz et al., 2009). 2.6. Determining particle size of emulsions Size distribution of water droplets in W/O nano-emulsions and oil droplets in W/O/W multiple emulsions were determined by laser light scattering method using Zetasizer (Malvern Instruments, Worcestershire, UK). Before measuring the size of oil particles, W/O/W emulsions were diluted in deionized water by 1 to 5 ratios. Particle size was reported as volume-surface (Sauter) mean diameter, D32 = (Rnid3i /Rnid2i ), where ni is the number of particles with diameter di and volume-weighted (De-Brouker) mean diameter, D43 = (Rnid4i /Rnid3i ) (Jafari, Beheshti, & Assadpoor, 2012). 2.7. Sample preparation for oxidative stability determination Three olive leaf extract (OLE) concentrations of 100, 200 and 300 mg were dissolved in soybean oil in four different forms including blank (non-encapsulated) OLE, nano-encapsulated OLE in primary W/O nano-emulsions, in W/O/W emulsions stabilized by WPC alone and, in W/O/W emulsions stabilized by mixture of WPC–pectin. The samples were transferred into a series of glass
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bottles and placed in an oven at 60 °C for 20 days. Soybean oil without any additives and containing 100 and 200 mg/kg TBHQ were also selected as control samples. 2.7.1. Determination of the thermal oxidative stability index (Rancimat test) Oxidative stability analyses were made through the measurement by Rancimat (Metrohm, 743, Switzerland) test based on the AOCS (2007). The air flow rate and the temperature was set at 20 l/h and at 110 °C, respectively. 2.7.2. Peroxide value (PV) The Peroxide value was determined according to the official methods of AOCS (2007). Briefly, the oil sample (3 g) was dissolved in glacial acetic acid (30 ml) and chloroform (20 ml) (3:2 v/v). Then saturated KI solution (1 ml) was added. The mixture was kept in the dark for 1 min, after adding of distilled water (50 ml), mixture was titrated against sodium thiosulfate (0.01 N). The PV value (mEq of oxygen/kg) was calculated using the following equation:
PV value ¼ 1000ðS NÞ=W where S is the volume of sodium thiosulfate solution (blank corrected) in ml, N is the normality of sodium thiosulfate solution and W is the weight of oil sample (gram). 2.7.3. Thiobarbituric acid (TBA) value Thiobarbituric acid test to measure secondary oxidation products of malonaldehyde was performed according to AOCS (2007) procedure. Oil sample (200 mg) was dissolved in a small volume of 1-butanol and made up to volume with the same solvent (25 ml), then 5.0 ml of this solution mixed with 10 ml of TBA reagent (0.2%), incubated for 2 h at 95 °C water bath and cooled under running tap-water for about 10 min until it reaches room temperature. The absorbance was measured at 532 nm against a blank (reaction with all the reagents except the oil) and TBA value was calculated by the following equation:
TBA value ¼ 50 ðA BÞ=m where A is the absorbance of the test solution, B is the absorbance of the reagent blank and m is the weight of oil sample (mg). 2.8. Statistical analysis The experiments were all carried out in triplicate. The collected data were analyzed by ANOVA; the means were compared by the Duncan’s multiple range tests at the 5% level through SPSS version 21 (IBM, USA). 3. Results and discussion 3.1. Total phenolic content of olive leaf extract The phenolic compounds can effectively scavenge free radicals and chelate transition metals, thus stopping progressive oxidative damage (Brewer, 2011). The total phenolic compounds in olive leaf was 206.81 ± 0.02 mg GAE/g of sample. This result concurred with the reports of Rafiee et al. (2012). 3.2. Evaluation of nano-encapsulation by different emulsions At the first stage, nano-encapsulation of olive leaf extract was performed by preparation of water-in-oil (W/O) nano-emulsions. By measuring droplet size distribution in these primary W/O nano-emulsions through laser light scattering, it was observed that there are many small dispersed droplets with a size well below
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1 lm (<10 nm), as shown in Fig. 1A. In fact, these droplets are water droplets containing olive leaf extract dispersed within oil phase. Successful preparation of nano-encapsulated olive bioactive compounds could be verified by transparent appearance of these emulsions (Fig. 1A). Double layer encapsulation of initial W/O nanoemulsions with biopolymers (WPC–pectin) resulted in W/O/W double emulsions. The milky appearance of W/O/W emulsions can be an indication of increase in the size of dispersed droplets coated by biopolymers (Fig. 1B and C). This is predictable since initial dispersed water droplets in O/W emulsions are placed within bigger oil droplets that now are themselves as dispersed droplets in W/O/W double emulsions. Our analysis of droplet size distribution of W/O/W emulsions revealed that double emulsions prepared with a complex of WPC and pectin had larger droplets than their counterparts stabilized with WPC only (Fig. 1B and C). Also, existence of a higher peak in droplet size distribution of the W/O nano-emulsions (Fig. 1A) compared with double emulsions could be due to much lower droplet size in these emulsions which is appropriate to much higher number of droplets. But in W/O/W emulsions, addition of biopolymers increases the size of particles, and as a result, the number of droplets decreases (Jafari, He, & Bhandari, 2007). Given the same conditions and parameters involved in the production of double emulsions, it can be concluded that the simultaneous application of two biopolymers (WPC and pectin) increases the thickness of biopolymers layer around dispersed oil droplets and results in an increase in their size. Droplet size distribution of the W/O/W emulsions (WPC and pectin) was bimodal with a small peak around 141.8 nm (corresponding to the span 80 or WPC small droplets) and a large peak around 712.4 nm (corresponding to the double layer of WPC and pectin) (Fig. 1C). A bimodal emulsion can be regarded as a system consisting of two different size ranges as the larger particles are suspended in a continuous fluid containing the smaller particles. When comparing emulsion size results of W/O nano-emulsion and W/O/W double emulsions by single WPC and WPC–pectin, it can be seen that W/O samples had a much smaller D32 (0.831 nm) and D43 (1.089 nm) than W/O/W double emulsions (Table 2), and differences between these two means were smaller in W/O emulsions due to a uniform droplet size distribution (single peak). Also, Sauter mean size was proportional to the bimodal distribution in W/O/W double emulsions made with WPC–pectin (Table 2). 3.3. Thermal oxidative stability analysis In this stage, the samples containing different forms of nano-encapsulated olive leaf extract (as shown in Table 1) along with a sample with unencapsulated olive leaf extract and one with TBHQ as a synthetic antioxidant were analyzed for oxidative stability by Rancimat test. According to our results revealed in Fig. 2, the highest thermal stability was for the oil sample containing TBHQ with a concentration of 200 mg/kg. Also, increasing the concentration of TBHQ from 100 to 200 mg/kg resulted in a significant increase in thermal stability of soybean oil (P < 0.05). We found that thermal stability of oil samples enriched with unencapsulated olive leaf extract was higher than those samples with nano-encapsulated olive leaf extract. These phenomena could be explained by the fact that in oil samples containing encapsulated antioxidants, some layer forming biopolymers (pectin and WPC in our case) around dispersed emulsion droplets are covering the antioxidants within them and prevent their anti-oxidant activity in oil samples. In particular, a complex formation between pectin and WPC at high temperatures results in a strong barrier which does not allow permeation of phenolic compounds into oil medium around dispersed droplets. This was verified by observing the
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Fig. 1. Droplet size distribution of nano-encapsulated olive leaf extract in initial W/O nano-emulsion (A); and in W/O/W double emulsions stabilized by single WPC (B), and WPC–pectin (C).
Table 1 Experiment design of different treatments. Independent factor
Nano-encapsulation method Antioxidant concentration
Levels
Treatment codes
Within initial W/O nanoemulsion Within W/O/W double emulsions stabilized by single WPC Within W/O/W double emulsions stabilized by WPC – Pectin Un-encapsulated olive leaf extract 100, 200, 300 mg/kg for OLE containing samples 100 and 200 mg/kg for TBHQ containing samples
OLEN OLENW OLENWP OLE –
A. Mohammadi et al. / Food Chemistry 190 (2016) 513–519 Table 2 Mean droplet diameters (D43 and D32, nm) of W/O and W/O/W double emulsions. Samples
D43 (nm)
D32 (nm) c
W/O micro-emulsion W/O/W emulsions (single WPC) W/O/W emulsions (WPC–pectin)
1.089 ± 0.15 535.98 ± 0.24b 3821.99 ± 0.09a
0.831 ± 0.03c 353.76 ± 0.12b 2654.66 ± 0.43a
Results are shown as mean ± SD of three independent measurements. Different letters within the same column indicate significant differences (P < 0.05).
lowest thermal stability for the oil samples containing nano-encapsulated olive leaf extract with pectin–WPC (Fig. 2). Armando, Maythe, and Beatriz (1998) indicated that a higher oil oxidation at high temperatures could be due to an increases rate of complex formation between biopolymers; while at low temperatures, the effect of blocking does not exist or is negligible.
3.4. Storage oxidative stability results From the perspectives of food quality and safety, the control of oil oxidation in food is important to prevent foods from deterioration and protect human health, Therefore, determination of the peroxide value is one of the most important quality control measurements for food systems specially edible oils, because it is an indicator of the primary oxidation state of the oils. This indicator measures the concentration of hydroperoxides which are unstable and can easily break down to form low-molecular-weight oxygenated constituents such as alcohols, aldehydes, free fatty acids, and ketones, ultimately leading to rancidity (Akinoso, Aboaba, & Olayanju, 2010; Pizarro, Esteban-Díez, Rodríguez-Tecedor, & González-Sáiz, 2013). The peroxide value of blank soybean oil sample without any added antioxidants reached to a maximum value of 46 meq/kg after 20 days storage (Fig. 3). On the other hand, surprisingly we found that peroxide value for oil samples containing 100, 200 and 300 mg/kg unencapsulated olive leaf extract and 100, 200 and 300 mg/kg nano-encapsulated olive leaf extract were 37.4, 35.6, 28.2, 26.5, 22.3, and 20.6, respectively. Also, peroxide value for those samples consisting 100, 200 and 300 mg/kg nano-encapsulated olive leaf extract with WPC and 100, 200 and 300 mg/kg nano-encapsulated with pectin–WPC reached to a maximum of 22.6, 19, 14.4, 18.05, 14.6, and 12.2 (meq/kg oil), respectively after 20 days storage at 55° (Fig. 3).
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In general, our results revealed that an increase in the concentration of phenolic extracts of olive leaf, significantly decreases the peroxide value of soybean oil samples (P < 0.05). As it has been shown in Fig. 3, peroxide value of all samples had an increasing trend over storage time. But this increased trend for samples with encapsulated olive leaf extract and those with synthetic TBHQ antioxidant was slower (with a smaller slope) compared with blank oil sample (without any antioxidants) and the sample with unencapsulated olive leaf extract. Nano-encapsulation of olive leaf extract by W/O nano-emulsions and W/O/W double emulsions had a positive effect on retention of antioxidant activity of olive phenolic compounds during storage time, so that addition of encapsulated natural antioxidants to the soybean oil could diminish its oxidation successfully in comparison with non-encapsulated form. Another interesting result was that among nano-encapsulated forms, the lowest peroxide value over storage time was related to oil samples enriched with multiple emulsions stabilized by WPC–pectin complex. This was contrary to the results of Rancimat, possibly due to different mechanisms of oxidation reactions at the temperatures of 110 °C and 60 °C. These results were in agreement with Taghvaei et al. (2014). Nano-particulation of phenolic compounds could increase bioavailability, solubility, and antioxidant capacity of these bioactive compounds, compared with conventional dispersion methods (Flanagan & Singh, 2006). So, nano-capsules of olive leaf extract (dispersed droplets in W/O and W/O/W emulsions) can control peroxide value much better than simple olive leaf extract during storage of edible oils. On other hand, nano-encapsulation through formation of double emulsions stabilized with WPC and pectin can increase the inhibitory rate of natural antioxidants because of controlled release of phenolic compounds and a gradual availability to antioxidant activity of these antioxidants within external oil phase and a supplementary antioxidant activity by applied biopolymers (WPC and pectin). Several studies have shown the antioxidant properties of WPC (Bounous, 2000; El-Kady et al., 2010), pectin and pectin derivatives (Liu, Lin, Lee, & Hou, 2007; Yang, Cheng, Lin, Liu, & Hou, 2004). The TBA test determines the amount of malondialdehyde (MDA), a major secondary by-product of lipid oxidation in food samples. Based on our results revealed in Fig. 4, TBA values of different samples, as influenced by storage time were minimum and had less differences at the start of the study. However, these value increased significantly (P < 0.05) as the time of storage. TBA value showed an increasing trend until day 12 in all samples except
Rancimat induction period (h)
14 12 10 8 6 4 2 0
Fig. 2. Rancimat analysis of soybean oil samples containing un-encapsulated olive leaf extract (OLE), encapsulated in initial nano-emulsion (OLEN), or double emulsions with single WPC (OLENW), and a complex of WPC–pectin (OLENWP) compared with TBHQ at 110 °C (data are the average of three replicates).
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4. Conclusion Our results indicated that nano-encapsulation of olive leaf extract by W/O nano-emulsions and W/O/W double emulsions could be a suitable novel technique to improve the antioxidant capacity of natural ingredients. It was revealed that oxidation protection offered by encapsulated olive leaf extract with pectin–WPC is comparable with the capacity of a widely used synthetic antioxidant (TBHQ). We found that nano-encapsulation with WPC–pectin improved the antioxidant activity because of better controlled release of phenolic compounds. On the other hand, due to blockage of phenolic compounds within dispersed droplets of emulsions, the thermal stability of oil samples containing encapsulated forms of olive leaf extract was lower than unencapsulated form. Finally, we can conclude that it is possible to substitute synthetic antioxidants by natural ones like olive leaf phenolic compounds but we have to apply some forms of encapsulation techniques in order to protect their properties.
Fig. 3. The peroxide value (average of three replicates) of soybean oil samples containing OLE at 100 ( ), 200 (4) and 300 () mg/kg, OLEN at 100 ( ), 200 ( ) and 300 (+) mg/kg, OLENW at 100 ( ), at 200 ( ), at 300 (j) mg/kg, OLENWP at 100 ( ), at 200 (d), at 300 (s) mg/kg, TBHQ at 100 ( ) and 200 () mg/kg and soybean oil without any additive ( ) during 20 days storage at 60 °C. Abbreviations are explained in Fig. 2 and Table 1.
OLENW and OLENWP. The samples containing encapsulated olive leaf extract by WPC and pectin (OLENW and OLENWP) showed a peak in day 8, and then TBA values decreased. When measuring TBA value, malondialdehyde reacts with thiobarbituric acid. Thus, the rate of thiobarbituric acid increases during oil oxidation. In this process, aldehydes may be oxidized to carboxylic acids, so the amount of thiobarbituric acid will decrease. According to Taghvaei et al. (2014), this could be related to the oxidation of secondary autoxidation products and formation of carboxylic acids. Our result indicated that the highest and lowest TBA value was related to blank and TBHQ containing samples, respectively. In general, TBA value for oil samples containing encapsulated olive leaf extract with pectin–WPC had a similar behavior with TBHQ samples. This could indicate the high capability of this form of nano-encapsulation for natural olive leaf phenolic compounds which can compete with a synthetic antioxidant due to better controlled release, and a supplementary antioxidant activity of WPC and pectin.
Fig. 4. The TBA value (average of three replicates) of soybean oil samples containing OLE at 100 ( ), 200 (4) and 300 () mg/kg, OLEN at 100 ( ), 200 ( ) and 300 (+) mg/kg, OLENW at 100 ( ), at 200 ( ), at 300 (j) mg/kg, OLENWP at 100 ( ), at 200 (d), at 300 (s) mg/kg, TBHQ at 100 ( ) and 200 () mg/kg and soybean oil without any additive ( ) during 20 days storage at 60 °C. Abbreviations are explained in Fig. 2 and Table 1.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Application of nano-encapsulated olive leaf extract in controlling the oxidative stability of soybean oil Adeleh Mohammadi, Seid Mahdi Jafari ⇑,1, Afshin Faridi Esfanjani, Sahar Akhavan Department of Food Materials and Process Design Engineering, Faculty of Food Science and Technology, University of Agricultural Sciences and Natural Resources, Gorgan, Iran
a r t i c l e
i n f o
Article history: Received 4 December 2014 Received in revised form 27 April 2015 Accepted 28 May 2015 Available online 29 May 2015 Keywords: Olive leaf extract Nano-emulsion Encapsulation Antioxidant activity
a b s t r a c t Our objective was to evaluate the antioxidant activity of olive leave extract (OLE) encapsulated by nano-emulsions in soybean oil. The average droplet size one day after production was 6.16 nm for primary W/O nano-emulsion and, 675 nm and 1443 nm for multiple emulsions stabilized by WPC alone and complex of WPC–pectin, respectively. The antioxidant activity of these emulsions containing three concentrations of 100, 200 and 300 mg OLE during storage was evaluated in soybean oil by peroxide value, TBA value and rancimat thermal stability test and was compared with blank (non-encapsulated) OLE and synthetic TBHQ antioxidant. Nano-encapsulated OLE was capable of controlling peroxide value better than unencapsulated OLE. But because of blocking phenolic compounds within dispersed emulsions droplets, thermal stability of encapsulated OLE was lower. To summarize, with increased solubility and controlled release of olive leaf phenolic compounds through their nano-encapsulation, a higher antioxidant activity was achieved. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Lipid oxidation is a complex series of reactions that occur during processing, storage, shipment, and final preparation of food stuffs containing lipids. Oxidation mechanism starts to take place immediately after oil extraction (Iqbal & Bhanger, 2007; Taghvaei & Jafari, 2015). Oxidation may lead to formation of toxic compounds, off-flavors and lower the quality and nutritional value of foods. This process may be inhibited by diverse procedures including prevention of oxygen access, inactivation of enzymes catalyzing oxidation, reduction of oxygen pressure, addition of chelating agents, use of lower temperature and the use of suitable packaging. Another method of protection against oxidation is to use specific additives called the antioxidants (Pokorny, Yanishlieva, & Gordon, 2001; Ramarathnam, Osawa, Ochi, & Kawakishi, 1995). Harmful effects of the synthetic antioxidants such as tert-butyl hydroquinone (TBHQ), butylated hydroxy toluene (BHT), and butylated hydroxy anisole (BHA), for health have been demonstrated previously. Therefore, there has been a recent attention on the use of natural phenolic compounds due to their antioxidant properties and potential health effects (Anagnostopoulou, Kefalas, Papageorgiou, Assimepoulou, & Boskou, 2006; Rahmanian, Jafari, & Calanagis, 2014; Taghvaei & Jafari, 2015). Several studies have ⇑ Corresponding author at: Pishro Food Technology Research Group, Gorgan, Iran. 1
E-mail address: [email protected] (S.M. Jafari). This author equally contributed as first author.
http://dx.doi.org/10.1016/j.foodchem.2015.05.115 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
been conducted with the target of protecting foods against oxidation by using natural antioxidants such as grape seed extract, chestnut, rosemary, green tea, etc (Gibis & Weiss, 2012; Lorenzo, González-Rodríguez, Sánchez, Amado, & Franco, 2013; Samotyja & Malecka, 2007). Olive leaf (Olea europaea) is one of the medicinal plants, which is considered as a good and cheap source of antioxidants. Among different parts of olive tree, the leaf is one of the richest sources of phenolic compounds (Rafiee, Jafari, Alami, & Khomeiri, 2011; Rahmanian, Jafari, & Wani, 2015; Proestos, Boziaris, Nychas, & Komaitis, 2006). Oleuropein and its derivatives such as hydroxytyrosol and tyrosol are the most abundant phenolic compounds in olive leaf. A linear relationship exists between the amount of oleuropein in leaves and their antioxidant property (Farag, El-Baroty, & Basuny, 2003). Nano-encapsulation of bioactive compounds by nonionic emulsifiers and through O/W or W/O nano-emulsions can protect and increase their properties (such as antioxidativity, stability and, solubility) (Lawrence & Rees, 2012). In this technology, nanoparticles of encapsulated compound (5–100 nm) are produced in a continuous phase (Flanagan & Singh, 2006). Also, it has been well documented that nano-emulsions have a pronounced effect on the antioxidant activity of different bioactive compounds such as green tea polyphenols, resveratrol, and flavanols (Fang & Bhandari, 2010; Zhou et al., 2000; Zhou, Miao, Yang, & Liu, 2005). On the other hand, emulsification of a W/O emulsion within an aqueous phase containing hydrophilic emulsifiers could lead to the production of W/O/W multiple emulsions. As a result, the release
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rate of encapsulated compounds within the inner aqueous phase is controlled (Choi, Decker, & McClements, 2009). Food biopolymers, such as proteins and polysaccharide are among the hydrophilic emulsifiers which may be applied in external aqueous phase of W/O/W double emulsions. Whey protein has excellent emulsifying properties and can be used alone or in a complex with polysaccharides such as pectin within the external aqueous phase of W/O/W emulsions (Bouyer, Mekhloufi, Rosilio, Grossiord, & Agnely, 2012). Besides, whey proteins are a rich of cysteine and this amino acid is a potent intracellular antioxidant which could impart with a good antioxidant capacity (Walzem, Dillard, & German, 2002). There have been some studies describing the antioxidant capacity of olive leaf extract (Rafiee, Jafari, Alami, & Khomeiri, 2012; Taghvaei et al., 2014) but, due to sensitivity of its natural phenolic compounds against pH, light, oxygen, etc., it is essential to protect them by a novel technology such as encapsulation. Antioxidant activity of encapsulated olive leaf extract within nano-emulsions and multiple emulsions could be a solution which has not been investigated yet. Therefore, objective of this study was to evaluate nano-encapsulation of olive leaf extract and its application in soybean oil. 2. Materials and methods 2.1. Materials Refined soybean oil without any antioxidant additives was purchased from a local oil refining factory (Alia Golestan Co., Iran). Olive leaves of Mission variety were collected from Gorgan, Iran, and citrus pectin (GA: > 65%) was purchased from MP biomedical (Netherland). Whey protein concentrate (80% protein) and sorbitan monooleate (span 80) was obtained from Sapoto cheese (USA) and Merck (Germany), respectively. Sodium azide was purchased from Sigma–Aldrich (St. Louis, USA). TBHQ were purchased from Nova international (India). Other chemical were of analytical grade. 2.2. Microwave assisted extraction of phenolic compounds from olive leaf For preparing phenolic compounds, microwave assisted extraction (MAE) was used according to the method of Rafiee et al. (2011). Briefly for each extraction, 12 g of dried leaves was blended with 120 ml methanol solvent in a 500 ml volumetric flask and were placed in a microwave oven. While magnetic stirring, 6 min irradiation was performed (8 s power on and 15 s power off in order to prevent super-boiling of solvent). Then, the methanol extract was filtered and solvent removed under reduced pressure at 40 °C by means of a rotary evaporator (IKA RV 10 basic, Germany) and remaining dried by a freeze-drier (Operon FDB-5503) at 20 °C. 2.3. Determination of total phenolic content Total phenolic content of each extract was determined by the Folin–Ciocalteu micro-method (Rafiee et al., 2012). Briefly, 20 ll of extract solution was mixed with 1.16 ml distilled water and 100 ll Folin–Ciocalteu reagent, followed by addition of 300 ll Na2CO3 solution (20%) before 8 min. Subsequently, the mixture was incubated in a shaking incubator at 40 °C for 30 min and its absorbance was measured at 760 nm (WPA S2000, UK). Gallic acid was used as a standard for calibration curve. 2.4. Biopolymer solution preparation Wall materials solutions as the outer queues phase of multiple emulsions were prepared by dispersing the powdered pectin in
distilled water and stirring at 50 °C for 30 min and then were kept at room temperature overnight. In the case of WPC, their solutions were kept refrigerated for 24 h for complete hydration. The pH of solution was afterwards adjusted on 6.0 with phosphate buffer. The solution was heat-treated at 70 °C for 20 min, and cooled down quickly. The solution of WPC–pectin was prepared by adding pectin solution into the solution of WPC, and stirring at room temperature for an hour. Then, the pH of solution was set on 6 by phosphate buffer and was kept in a refrigerator overnight. 0.004% sodium azide was included in the solutions as an antimicrobial substance. The final percentage of biopolymers in outer aqueous phase of multiple emulsions was 8% WPC for single layer and, 8%WPC–0.2% Pectin for double layer treatments (Lutz, Aserin, Wicker, & Garti, 2009). 2.5. Preparation of emulsions W/O/W multiple emulsions were prepared using a two-step emulsification procedure. 2.5.1. Preparation of the primary W/O micro-emulsion (nanoencapsulated particles) First, nanoparticles of olive leaf extract were prepared via a nonionic emulsifier. According to the method of Sadeghi, Madadlou, and Yarman (2013), W/O nanoemulsion was prepared by dropwise addition of 7% olive leaf extract into a continuous phase consisting 31% span 80 and 62% soybean oil while stirring (300 rpm) until the system became transparent. Before this step, span 80 and soybean oil as an organic phase should be mixed together at 300 rpm so that the span 80 is dissolve completely. 2.5.2. Preparation of W/O/W multiple emulsions In the second emulsification step, nanocapsules of olive leaf extract (initial W/O nano-emulsion) were coated by biopolymers (WPC, pectin) through W/O/W double emulsions. The pre-W/O/W emulsions were formed by gradually adding the primary W/O nano-emulsions into the continuous aqueous phase containing biopolymers at 10 °C during homogenization with a rotor–stator homogenizer (8000 rpm for 5 min, T25 IKA, Germany). These W/O/W emulsions were then further emulsified using the mentioned homogenizer (15000 rpm for 8 min). All multiple emulsions were composed of 30% primary nano-emulsion and 70% outer aqueous phase containing biopolymers (w/w) (Lutz et al., 2009). 2.6. Determining particle size of emulsions Size distribution of water droplets in W/O nano-emulsions and oil droplets in W/O/W multiple emulsions were determined by laser light scattering method using Zetasizer (Malvern Instruments, Worcestershire, UK). Before measuring the size of oil particles, W/O/W emulsions were diluted in deionized water by 1 to 5 ratios. Particle size was reported as volume-surface (Sauter) mean diameter, D32 = (Rnid3i /Rnid2i ), where ni is the number of particles with diameter di and volume-weighted (De-Brouker) mean diameter, D43 = (Rnid4i /Rnid3i ) (Jafari, Beheshti, & Assadpoor, 2012). 2.7. Sample preparation for oxidative stability determination Three olive leaf extract (OLE) concentrations of 100, 200 and 300 mg were dissolved in soybean oil in four different forms including blank (non-encapsulated) OLE, nano-encapsulated OLE in primary W/O nano-emulsions, in W/O/W emulsions stabilized by WPC alone and, in W/O/W emulsions stabilized by mixture of WPC–pectin. The samples were transferred into a series of glass
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bottles and placed in an oven at 60 °C for 20 days. Soybean oil without any additives and containing 100 and 200 mg/kg TBHQ were also selected as control samples. 2.7.1. Determination of the thermal oxidative stability index (Rancimat test) Oxidative stability analyses were made through the measurement by Rancimat (Metrohm, 743, Switzerland) test based on the AOCS (2007). The air flow rate and the temperature was set at 20 l/h and at 110 °C, respectively. 2.7.2. Peroxide value (PV) The Peroxide value was determined according to the official methods of AOCS (2007). Briefly, the oil sample (3 g) was dissolved in glacial acetic acid (30 ml) and chloroform (20 ml) (3:2 v/v). Then saturated KI solution (1 ml) was added. The mixture was kept in the dark for 1 min, after adding of distilled water (50 ml), mixture was titrated against sodium thiosulfate (0.01 N). The PV value (mEq of oxygen/kg) was calculated using the following equation:
PV value ¼ 1000ðS NÞ=W where S is the volume of sodium thiosulfate solution (blank corrected) in ml, N is the normality of sodium thiosulfate solution and W is the weight of oil sample (gram). 2.7.3. Thiobarbituric acid (TBA) value Thiobarbituric acid test to measure secondary oxidation products of malonaldehyde was performed according to AOCS (2007) procedure. Oil sample (200 mg) was dissolved in a small volume of 1-butanol and made up to volume with the same solvent (25 ml), then 5.0 ml of this solution mixed with 10 ml of TBA reagent (0.2%), incubated for 2 h at 95 °C water bath and cooled under running tap-water for about 10 min until it reaches room temperature. The absorbance was measured at 532 nm against a blank (reaction with all the reagents except the oil) and TBA value was calculated by the following equation:
TBA value ¼ 50 ðA BÞ=m where A is the absorbance of the test solution, B is the absorbance of the reagent blank and m is the weight of oil sample (mg). 2.8. Statistical analysis The experiments were all carried out in triplicate. The collected data were analyzed by ANOVA; the means were compared by the Duncan’s multiple range tests at the 5% level through SPSS version 21 (IBM, USA). 3. Results and discussion 3.1. Total phenolic content of olive leaf extract The phenolic compounds can effectively scavenge free radicals and chelate transition metals, thus stopping progressive oxidative damage (Brewer, 2011). The total phenolic compounds in olive leaf was 206.81 ± 0.02 mg GAE/g of sample. This result concurred with the reports of Rafiee et al. (2012). 3.2. Evaluation of nano-encapsulation by different emulsions At the first stage, nano-encapsulation of olive leaf extract was performed by preparation of water-in-oil (W/O) nano-emulsions. By measuring droplet size distribution in these primary W/O nano-emulsions through laser light scattering, it was observed that there are many small dispersed droplets with a size well below
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1 lm (<10 nm), as shown in Fig. 1A. In fact, these droplets are water droplets containing olive leaf extract dispersed within oil phase. Successful preparation of nano-encapsulated olive bioactive compounds could be verified by transparent appearance of these emulsions (Fig. 1A). Double layer encapsulation of initial W/O nanoemulsions with biopolymers (WPC–pectin) resulted in W/O/W double emulsions. The milky appearance of W/O/W emulsions can be an indication of increase in the size of dispersed droplets coated by biopolymers (Fig. 1B and C). This is predictable since initial dispersed water droplets in O/W emulsions are placed within bigger oil droplets that now are themselves as dispersed droplets in W/O/W double emulsions. Our analysis of droplet size distribution of W/O/W emulsions revealed that double emulsions prepared with a complex of WPC and pectin had larger droplets than their counterparts stabilized with WPC only (Fig. 1B and C). Also, existence of a higher peak in droplet size distribution of the W/O nano-emulsions (Fig. 1A) compared with double emulsions could be due to much lower droplet size in these emulsions which is appropriate to much higher number of droplets. But in W/O/W emulsions, addition of biopolymers increases the size of particles, and as a result, the number of droplets decreases (Jafari, He, & Bhandari, 2007). Given the same conditions and parameters involved in the production of double emulsions, it can be concluded that the simultaneous application of two biopolymers (WPC and pectin) increases the thickness of biopolymers layer around dispersed oil droplets and results in an increase in their size. Droplet size distribution of the W/O/W emulsions (WPC and pectin) was bimodal with a small peak around 141.8 nm (corresponding to the span 80 or WPC small droplets) and a large peak around 712.4 nm (corresponding to the double layer of WPC and pectin) (Fig. 1C). A bimodal emulsion can be regarded as a system consisting of two different size ranges as the larger particles are suspended in a continuous fluid containing the smaller particles. When comparing emulsion size results of W/O nano-emulsion and W/O/W double emulsions by single WPC and WPC–pectin, it can be seen that W/O samples had a much smaller D32 (0.831 nm) and D43 (1.089 nm) than W/O/W double emulsions (Table 2), and differences between these two means were smaller in W/O emulsions due to a uniform droplet size distribution (single peak). Also, Sauter mean size was proportional to the bimodal distribution in W/O/W double emulsions made with WPC–pectin (Table 2). 3.3. Thermal oxidative stability analysis In this stage, the samples containing different forms of nano-encapsulated olive leaf extract (as shown in Table 1) along with a sample with unencapsulated olive leaf extract and one with TBHQ as a synthetic antioxidant were analyzed for oxidative stability by Rancimat test. According to our results revealed in Fig. 2, the highest thermal stability was for the oil sample containing TBHQ with a concentration of 200 mg/kg. Also, increasing the concentration of TBHQ from 100 to 200 mg/kg resulted in a significant increase in thermal stability of soybean oil (P < 0.05). We found that thermal stability of oil samples enriched with unencapsulated olive leaf extract was higher than those samples with nano-encapsulated olive leaf extract. These phenomena could be explained by the fact that in oil samples containing encapsulated antioxidants, some layer forming biopolymers (pectin and WPC in our case) around dispersed emulsion droplets are covering the antioxidants within them and prevent their anti-oxidant activity in oil samples. In particular, a complex formation between pectin and WPC at high temperatures results in a strong barrier which does not allow permeation of phenolic compounds into oil medium around dispersed droplets. This was verified by observing the
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Fig. 1. Droplet size distribution of nano-encapsulated olive leaf extract in initial W/O nano-emulsion (A); and in W/O/W double emulsions stabilized by single WPC (B), and WPC–pectin (C).
Table 1 Experiment design of different treatments. Independent factor
Nano-encapsulation method Antioxidant concentration
Levels
Treatment codes
Within initial W/O nanoemulsion Within W/O/W double emulsions stabilized by single WPC Within W/O/W double emulsions stabilized by WPC – Pectin Un-encapsulated olive leaf extract 100, 200, 300 mg/kg for OLE containing samples 100 and 200 mg/kg for TBHQ containing samples
OLEN OLENW OLENWP OLE –
A. Mohammadi et al. / Food Chemistry 190 (2016) 513–519 Table 2 Mean droplet diameters (D43 and D32, nm) of W/O and W/O/W double emulsions. Samples
D43 (nm)
D32 (nm) c
W/O micro-emulsion W/O/W emulsions (single WPC) W/O/W emulsions (WPC–pectin)
1.089 ± 0.15 535.98 ± 0.24b 3821.99 ± 0.09a
0.831 ± 0.03c 353.76 ± 0.12b 2654.66 ± 0.43a
Results are shown as mean ± SD of three independent measurements. Different letters within the same column indicate significant differences (P < 0.05).
lowest thermal stability for the oil samples containing nano-encapsulated olive leaf extract with pectin–WPC (Fig. 2). Armando, Maythe, and Beatriz (1998) indicated that a higher oil oxidation at high temperatures could be due to an increases rate of complex formation between biopolymers; while at low temperatures, the effect of blocking does not exist or is negligible.
3.4. Storage oxidative stability results From the perspectives of food quality and safety, the control of oil oxidation in food is important to prevent foods from deterioration and protect human health, Therefore, determination of the peroxide value is one of the most important quality control measurements for food systems specially edible oils, because it is an indicator of the primary oxidation state of the oils. This indicator measures the concentration of hydroperoxides which are unstable and can easily break down to form low-molecular-weight oxygenated constituents such as alcohols, aldehydes, free fatty acids, and ketones, ultimately leading to rancidity (Akinoso, Aboaba, & Olayanju, 2010; Pizarro, Esteban-Díez, Rodríguez-Tecedor, & González-Sáiz, 2013). The peroxide value of blank soybean oil sample without any added antioxidants reached to a maximum value of 46 meq/kg after 20 days storage (Fig. 3). On the other hand, surprisingly we found that peroxide value for oil samples containing 100, 200 and 300 mg/kg unencapsulated olive leaf extract and 100, 200 and 300 mg/kg nano-encapsulated olive leaf extract were 37.4, 35.6, 28.2, 26.5, 22.3, and 20.6, respectively. Also, peroxide value for those samples consisting 100, 200 and 300 mg/kg nano-encapsulated olive leaf extract with WPC and 100, 200 and 300 mg/kg nano-encapsulated with pectin–WPC reached to a maximum of 22.6, 19, 14.4, 18.05, 14.6, and 12.2 (meq/kg oil), respectively after 20 days storage at 55° (Fig. 3).
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In general, our results revealed that an increase in the concentration of phenolic extracts of olive leaf, significantly decreases the peroxide value of soybean oil samples (P < 0.05). As it has been shown in Fig. 3, peroxide value of all samples had an increasing trend over storage time. But this increased trend for samples with encapsulated olive leaf extract and those with synthetic TBHQ antioxidant was slower (with a smaller slope) compared with blank oil sample (without any antioxidants) and the sample with unencapsulated olive leaf extract. Nano-encapsulation of olive leaf extract by W/O nano-emulsions and W/O/W double emulsions had a positive effect on retention of antioxidant activity of olive phenolic compounds during storage time, so that addition of encapsulated natural antioxidants to the soybean oil could diminish its oxidation successfully in comparison with non-encapsulated form. Another interesting result was that among nano-encapsulated forms, the lowest peroxide value over storage time was related to oil samples enriched with multiple emulsions stabilized by WPC–pectin complex. This was contrary to the results of Rancimat, possibly due to different mechanisms of oxidation reactions at the temperatures of 110 °C and 60 °C. These results were in agreement with Taghvaei et al. (2014). Nano-particulation of phenolic compounds could increase bioavailability, solubility, and antioxidant capacity of these bioactive compounds, compared with conventional dispersion methods (Flanagan & Singh, 2006). So, nano-capsules of olive leaf extract (dispersed droplets in W/O and W/O/W emulsions) can control peroxide value much better than simple olive leaf extract during storage of edible oils. On other hand, nano-encapsulation through formation of double emulsions stabilized with WPC and pectin can increase the inhibitory rate of natural antioxidants because of controlled release of phenolic compounds and a gradual availability to antioxidant activity of these antioxidants within external oil phase and a supplementary antioxidant activity by applied biopolymers (WPC and pectin). Several studies have shown the antioxidant properties of WPC (Bounous, 2000; El-Kady et al., 2010), pectin and pectin derivatives (Liu, Lin, Lee, & Hou, 2007; Yang, Cheng, Lin, Liu, & Hou, 2004). The TBA test determines the amount of malondialdehyde (MDA), a major secondary by-product of lipid oxidation in food samples. Based on our results revealed in Fig. 4, TBA values of different samples, as influenced by storage time were minimum and had less differences at the start of the study. However, these value increased significantly (P < 0.05) as the time of storage. TBA value showed an increasing trend until day 12 in all samples except
Rancimat induction period (h)
14 12 10 8 6 4 2 0
Fig. 2. Rancimat analysis of soybean oil samples containing un-encapsulated olive leaf extract (OLE), encapsulated in initial nano-emulsion (OLEN), or double emulsions with single WPC (OLENW), and a complex of WPC–pectin (OLENWP) compared with TBHQ at 110 °C (data are the average of three replicates).
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4. Conclusion Our results indicated that nano-encapsulation of olive leaf extract by W/O nano-emulsions and W/O/W double emulsions could be a suitable novel technique to improve the antioxidant capacity of natural ingredients. It was revealed that oxidation protection offered by encapsulated olive leaf extract with pectin–WPC is comparable with the capacity of a widely used synthetic antioxidant (TBHQ). We found that nano-encapsulation with WPC–pectin improved the antioxidant activity because of better controlled release of phenolic compounds. On the other hand, due to blockage of phenolic compounds within dispersed droplets of emulsions, the thermal stability of oil samples containing encapsulated forms of olive leaf extract was lower than unencapsulated form. Finally, we can conclude that it is possible to substitute synthetic antioxidants by natural ones like olive leaf phenolic compounds but we have to apply some forms of encapsulation techniques in order to protect their properties.
Fig. 3. The peroxide value (average of three replicates) of soybean oil samples containing OLE at 100 ( ), 200 (4) and 300 () mg/kg, OLEN at 100 ( ), 200 ( ) and 300 (+) mg/kg, OLENW at 100 ( ), at 200 ( ), at 300 (j) mg/kg, OLENWP at 100 ( ), at 200 (d), at 300 (s) mg/kg, TBHQ at 100 ( ) and 200 () mg/kg and soybean oil without any additive ( ) during 20 days storage at 60 °C. Abbreviations are explained in Fig. 2 and Table 1.
OLENW and OLENWP. The samples containing encapsulated olive leaf extract by WPC and pectin (OLENW and OLENWP) showed a peak in day 8, and then TBA values decreased. When measuring TBA value, malondialdehyde reacts with thiobarbituric acid. Thus, the rate of thiobarbituric acid increases during oil oxidation. In this process, aldehydes may be oxidized to carboxylic acids, so the amount of thiobarbituric acid will decrease. According to Taghvaei et al. (2014), this could be related to the oxidation of secondary autoxidation products and formation of carboxylic acids. Our result indicated that the highest and lowest TBA value was related to blank and TBHQ containing samples, respectively. In general, TBA value for oil samples containing encapsulated olive leaf extract with pectin–WPC had a similar behavior with TBHQ samples. This could indicate the high capability of this form of nano-encapsulation for natural olive leaf phenolic compounds which can compete with a synthetic antioxidant due to better controlled release, and a supplementary antioxidant activity of WPC and pectin.
Fig. 4. The TBA value (average of three replicates) of soybean oil samples containing OLE at 100 ( ), 200 (4) and 300 () mg/kg, OLEN at 100 ( ), 200 ( ) and 300 (+) mg/kg, OLENW at 100 ( ), at 200 ( ), at 300 (j) mg/kg, OLENWP at 100 ( ), at 200 (d), at 300 (s) mg/kg, TBHQ at 100 ( ) and 200 () mg/kg and soybean oil without any additive ( ) during 20 days storage at 60 °C. Abbreviations are explained in Fig. 2 and Table 1.
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