The aplication of Pistacia khinjuk extract nanoemulsion in a biopolymeric coating to improve the shelf life extension of sunflower oil

Abstract In the present study, a hydroalcoholic extract of P. khinjuk was obtained by sonication method at 60°C for 50 min. The measurement revealed that the total phenolic content of the extract was 46.0 mg/g. The results showed that the extract has an antioxidant activity of 73.5% and 8.3 (µmol TE/g DW) in DPPH radical scavenging method and FRAP assay, respectively. Also, Balango (Lallemantia royleana) and Fenugreek (Trigonella foenum‐graecum) seed gum and their composition (1:1) were used to prepare the nanoemulsion with P. khinjuk extract. The droplet mean size of nanoemulsions was ranged from 310.34 to 354.19 nm. The highest encapsulation efficiency was observed in Balango nanoemulsion. P. khinjuk extract nanoemulsion coating with Balango and TBHQ was added to sunflower oil at 200 and 100 ppm, respectively. During 24‐day storage at 60°C, samples were investigated for peroxide, acid, and p‐anisidine values at 4‐day intervals. The results showed that oils containing nanoemulsion had the highest stability during storage. However, in all samples peroxide, acid and p‐anisidine values increased but the rate of oxidation in samples containing both synthetic and natural antioxidants was slower than the control sample.

Recently, great attention has been directed to the field of natural antioxidants in order to find natural compounds with various health-promoting effects (Chong et al., 2015;Kazemi et al., 2020;Tavakoli et al., 2017). Some investigations demonstrated the antioxidant effects of Pistacia fruit family. It has been shown that the presence of phenolic compounds is responsible for high antioxidant capacities of fruit extracts (Pedraza-Chaverri et al., 2008).
Pistacia khinjuk (Anacardiaceae) is one of the Pistacia species widely distributed in Iran and is named Kolkhoung in the Persian language. This plant has various biological activities and is used for different pharmacological activities for instant antidiabetic, antitumor, anti-cholinesterase, antimicrobial, and antifungal activities (Taran et al., 2010). The application of P. khinjuk as a natural compound in traditional Persian medicine has been reported in several studies. This applications include the prevention and treatment of motion sickness, stomach discomfort, nausea, and vomiting such as motion sickness, stomach discomfort, nausea, and vomiting, (Dob et al., 2006).
The P. khinjuk fruit extract is also used in pharmaceutical industry.
Kolkhoung extract majorly contains flavonoids and phenolic compounds. It is reported that its fruits and resins have strong antioxidant activity due to being rich in secondary compounds (Hatamnia et al., 2016).
Moreover, direct use of phenolic compounds is not applicable due to unpleasant taste, interaction with food components, and potential decomposition reactions These decomposition reactions take place during the processing of food and their storage due to the different conditions including light and oxygen as well as temperature and enzymes. The encapsulation of extract is a successful strategy to overcome the concerns by improving the physicochemical stability of extract and protecting it from interactions with food ingredients (Fang & Bhandari, 2010). Encapsulation technology could be defined as the process of packaging materials in solid or liquid state in the small capsules enabling their release in a controlled rate manner. Various different materials could be used as encapsulating compounds including proteins and hydrocolloids. in both food and pharmaceutical industries (Anbinder et al., 2011;Esfahani et al., 2020;Mousavian et al., ). Due to the popular demand of natural biopolymers, the incorporation of phenolic compounds in natural wall materials as a value-added component is a growing area of research. Balango seed (Lallemantia royleana) is a plant with mucilaginous property which could be found in Asia, Europe, and the Middle East. The extracted Balango seed gum (BSG) is a high molecular weight gum with a slightly flexible chain (Salehi et al., 2014). Fenugreek (Trigonella foenum-graecum ) is a leguminous plant grown in the Mediterranean, northern Africa, western Asia (Brummer et al., 2003), and Iran. Fenugreek seed gum (FSG) has been used medicinally and as a condiment for many years.
Recently, research interest has increased about seed components (Roberts et al., 2012). The choice of appropriate material for encapsulation has shown crucial role in the process of encapsulation.
Bioactive materials could be encapsulated using various synthetic polymers with the food-grade quality. However, investigators are always seeking for cheap, abundant, natural, and popular biopolymers. Some studies have applied seed gums to encapsulate food ingredient for instant beet dye, in the combination of chia seed mucilage, maltodextrin, and Arabic gum (Antigo et al., 2020), bergamot essential oil in Balango hydrocolloids (Rezaeinia et al., 2019), and anthocyanins of black raspberry encapsulated based on fenugreek gum (Yousefi et al., 2015).
Up to date, there is no study indicating the antioxidant capacity of P. khinjuk extract in the nanoemulsion form for improving the storage time of sunflower oil. The aim of the present investigation was the evaluation of antioxidant activity of P. khinjuk extract nanoemulsion on the oxidative stability of sunflower oil. Therefore, first, the effect of type of seed gum, Balango and Fenugreek seed gum, on the characterization of nanoemulsion was studied, and second, the antioxidant activity of the free and encapsulated extract in sunflower oil was measured.

Determination of DPPH radical scavenging activity and FRAP test
Evaluation of DPPH radical scavenging assay and ferric reducing antioxidant power (FRAP) test was done using by method described by Tavakoli et al. (2019).

Seed gum extraction
Seed gums of Balango and Fenugreek were extracted using reported method by Salehi et al. (2014). The cleaned Balangu and Fenugreek seeds were soaked in distilled water (water/seed ratio 20:1) at 50°C, pH = 7, for 20 min. Separation of the hydrocolloid from the swollen seeds was achieved by passing the seeds through an extractor equipped with a rotating plate that scraped the gum layer on the seed surface. The extracted solution was then filtered and dried in an air forced oven at 50°C (convection oven, Memmert Universal, Schwabach, Germany), and finally, the powder was milled, packed, and kept at cool and dry condition.

Preparation of biopolymer solutions and Nanoemulsions
Biopolymer solutions of Balango and Fenugreek gums and complex of Balango gum and Fenugreek gum (1:1) were prepared using the explained method by Estakhr et al. (2020) and Delfanian et al. (2018).
Balango and Fenugreek seed gums were mixed in deionized water to achieve a total solids content of 0.5% (w/w). A magnetic mixer was used to the better dissolution of the compounds for 15 min at 20 Cº, and then, the solutions were kept in the refrigerator for 24 hr. The complex solutions of gums were prepared by adding a Balango gum solution into the solution of Fenugreek gum and stirring at 20 Cº.
Tween 80 as emulsifier was added to the gum solution at 2% w/w concentration. The P. khinjuk extract (200 ppm) was added to the mixture at a ratio of 1:5, and after half an hour stirring, the ultraturrax homogenizer (Ultraturrax T25, Janke & Kunkel, Germany) at 12,000 rpm and 21,000 rpm was used for homogenization during 5 min. Then, to further reduce the particle size, the probe type ultrasonic (PRO-250, mLabs, USA) with six cycles and 30 s was used.
Nanoemulsions were frozen for 24 hr at −20°C and then by using a freeze-drying machine at 0.017 mPa and −57°C for 48 hr were dried (Chranioti et al., 2016).

Encapsulation efficiency and total phenolic content
Encapsulation efficiency of nanoencapsulated powder was done using method described by Robert et al. (2010). In order to measuring the total phenolic content of different extract and oil samples was used the described method by Delfanian et al. (2018).

Characterization of nanoemulsion
Droplet mean size, polydispersity index (PDI), and zeta potential of the P.khinjuk extract-containing nanoemulsion were analyzed using the described method by Harris et al. (2011).

Surface Electronic Morphology of Nanoemulsion
The shape and surface of different nanoemulsions were observed by scanning electron microscopy (SEM). The samples were examined using a scanning electron microscope (JEOL JSM-6400, JEOL, Tokyo, Japan).

DSC
To evaluate the thermal properties of nanocapsules, the thermal analysis was performed by differential scanning calorimeter device (DSC 821e, Mettler Toledo, Germany). 5 mg of each nanocapsule was accurately sealed in a DSC aluminum pan and heated from 10 to 500°C with a constant rate of 10°C/min under the nitrogen flow rate of 20 ml/min. The energy (mW) given to the sample was determined and further evaluations were performed (Amiri et al., 2020).

F TIR
To confirm the formation of the nanoencapsulated P. khinjuk extract, the Fourier transformation infrared (FTIR) spectrum was tested using a spectrometer (Equinox 55, Bruker, Germany) at 25°C. The measurements were performed with a resolution of 4 cm −1 at the range of 1000-4000 cm −1 (Zou et al., 2006).

Release properties
The release properties were determined according to Mohammadi et al. (2016). The stability of nano-coatings was measured based on the release of phenolic compounds from nanoemulsions.

Acid value, peroxide value and p-anisidine value
The determination of Acid Value, peroxide value and p-anisidine value of the different oil samples was done based on the method ascribed by Tavakoli et al. (2017), Kazemi et al. (2020) andEstakhr et al. (2020), respectively.

Peroxide value and p-anisidine value
The determination of peroxide value and p-anisidine value of the different oil samples was done based on the method ascribed by Kazemi et al. (2020) and Estakhr et al. (2020), respectively.

Statistical analysis
The mean ± SD values obtained from experiments were subjected to analysis of variance by performing one-way ANOVA test. Duncan test was performed to determine the significant differences between means and the p values <.05 were considered as significant.

RE SULTS AND DISCUSS ION Total Phenolic Content and Antioxidant Activity
Medicinal plants that contain secondary metabolites have been known for antioxidant activities (Adrar et al., 2016), and they can be used as food preservatives (Bakry et al., 2016). In the present study, P. khinjuk extract was investigated for total phenolic content and antioxidant activity and data are depicted in Table 1. The total phenolic content of P. khinjuk was 46.0 mg/g DW. Antioxidant activity of extracts is one of the biological properties of great interest because they may preserve food from oxidation and spoilage reactions (Miguel, 2010).

Encapsulation efficiency and nanoemulsion characteristics
Encapsulation efficiency is an important parameter to be taken into attention when developing an encapsulation process. The nanocapsules obtained showed a relatively high value of encapsulation efficiency (over 50%). The nanocapsules with the Balango seed gum wall showed the highest encapsulation efficiency. It seems that interaction between gums caused a decrease in phenol: gum bound encapsulation efficiency.
Droplet size could be considered as a determining parameter in stability and encapsulation efficiency. Table 2 demonstrates that the size of all the samples is in the nanometer range which is a promising feature for their further applications. No difference (p > .05) in particle size was observed between nanocapsules with a single wall meaning the type of gum did not affect this parameter. The difference of the zeta potential among the samples was statistically significant (p < .05). The net charge of anionic groups in gums and carboxyl groups of P. khinjuk extract involves in the negative zeta potential of nanocapsules. The best PDI was achieved at nanocapsules with a composite wall. In other words, the lower PDI represents the smaller size of nanocapsules. Presumably, when the particle distribution index increased, the nanocapsules are more likely to aggregate together, so the particle size incremented. All nanoemulsions prepared were spherical in shape. Nanoemulsion with a composite coating showed uniform and round structure and it would be attributed to the potential interactions between polysaccharide groups of Fenugreek and Balango gum. Two influential factors on the morphology of capsules are bioactive components and wall materials. Nanocapsules with a single-layer wall showed the larger size and the results of particle size confirmed this observation.  (Musuc et al. 2013). This resulted in the improvement of its thermal resistance which is a determining parameter for the application of encapsulated extract in food industry.

F TIR
FTIR spectra of P. khinjuk extract, seed gums powder, and CSO nanoparticle have been presented in Figure 3. FTIR analysis is performed to investigate the interaction between gums as a wall material and the extract. Note: Mean ± SD within a column with the same lowercase letters are not significantly different at p < .05.

TA B L E 1
The antioxidant activity of P. khinjuk extract and its phenolic content shape changes, chemical structure, maximum IR absorption intensity, and sample peaks. As can be seen, the study area in this study is in the range of 1,000 to 4,000 (cm -1 ). Generally, the specific peaks

Release properties
The encapsulation of bioactive materials is carried out in order to improve their release profile in a controlled manner. In this investigation, the impact of wall materials on the release of P. khinjuk phenolic compounds from nanocapsules during 24 days storage at 60 •C was evaluated by measuring the total phenolic content (Figure 4).

Peroxide value
The degree of primary oxidation of sunflower oil (SFO) was evaluated using the measurement of PV of oils containing nanoemulsion and TBHQ and oil without antioxidants at 60°C for 24 days. As demonstrated in Figure 5, all treated samples showed an increase in their PVs during the storage. This could be the effect of the formation of lipid hydroperoxides. (Iqbal & Bhanger, 2007).

Acid value
The acid value could be considered as an index showing the quality of oil during the storage at accelerated condition.

p-Anisidine Value
Once the hydroperoxide breaks down to aldehydes, carbonyl, and ketones, the secondary lipid oxidation product is produced. This conver-  Table 4).
The pattern of the increase of p-AnV under the acceleration condition after 24 days of storage showed that the highest p-AnV value belongs to SFO while the TBHQ and nanoemulsions showed lower values.
The results also showed that all p-AnVs for each treatment were significantly different at different storage times. This difference was not observed between the days 0 and 4. The p-AnV value of SFO containing TBHQ was consistently higher than SFO with nanoemulsion.
This reveals that nanoemulsion extract has shown promising results for the retardation of secondary oxidation products formation in sunflower oil while it is stored at 60°C compared to TBHQ, a synthetic

CON CLUS IONS
The studies to date on P.khinjuk extract nanoemulsion suggested that because of the presence of phenolics compounds, it can be used as an important natural antioxidant source. P. khinjuk extract has higher antioxidant activity than TBHQ in free and encapsulated form. It is important to consider that the P. khinjuk extract nanoemulsion demonstrated sufficiently high antioxidant activity in sunflower oil. In conclusion, the oil-containing P. khinjuk extract nanoemulsion was found to possess good oxidation stability, and based on our findings, it seems that it could be considered as a promising alternative for synthetic antioxidants in the food industry.

ACK N OWLED G M ENT
Authors would like to express their sincere gratitude to Islamic Azad University, Sarvestan Branch.