Evaluation of antioxidant properties of nanoencapsulated sage (Salvia officinalis L.) extract in biopolymer coating based on whey protein isolate and Qodumeh Shahri (Lepidium perfoliatum) seed gum to increase the oxidative stability of sunflower oil

Abstract Sage leaf extract (SLE) is considered an excellent source of bioactive compounds mainly because of its high content of phenolics, widely known as natural antioxidants. This study aimed to compare the performance of free/encapsulated SLE by different coatings in protecting sunflower oil against oxidative deterioration. The coating materials were whey protein isolate and qodumeh seed gum at different ratios (1:0, 1:1, and 0:1). Each nanocapsule was analyzed for particle size, zeta potential, encapsulation efficiency, phenolics release, and SEM images. The total phenolic compounds of SLE were 31.12 mg GA/g. The antioxidant activity of SLE was increased in both DPPH and FRAP assays by increasing extract concentration from 50 to 250 ppm. All nanoparticles exhibited nanometric size, negative zeta potential, encapsulation efficiency higher than 60%, and gradual release during storage. The oxidative stability of sunflower oil with or without the incorporation of 250 ppm of free/encapsulated SLE was evaluated during 24 days of storage at 60°C. Peroxide value (PV), thiobarbituric acid value (TBA), oxidative stability index (OSI), color index (CI), and conjugated dienes (CD) were determined. COPM nanoparticles showed the lowest PV, TBA, CI, and CD but both SGUM and WHEY were more effective in delaying oil oxidation than TBHQ and free extract. Higher OSI was observed in oil‐containing nanoparticles with composite coating. Results obtained reinforce the use of whey protein isolate and qodumeh seed gum as a coating for encapsulating SLE to increase the shelf life of sunflower oil as a natural antioxidant.


| INTRODUC TI ON
Sunflower (Helianthus annus) is one of the most important oil crops grown worldwide due to high-yield oil, and lack of antinutritional factors (Aly et al., 2021;Jafari et al., 2022). Sunflower oil (SFO) is a kind of nutritious vegetable oil that contains more than 85% polyunsaturated fatty acids (PUFA), especially linoleic acid which is used for medical treatment (Meng et al., 2021;Sayyari & Farahmandfar, 2017). The ratio of omega-3 and omega-6 fatty acids is prominent for providing cardiovascular and heart health benefits (Aly et al., 2021).
However, due to its fatty acid composition with high PUFA, it is one of the most susceptible to suffering rancidity and oxidation progress. Fat oxidation results in unpleasant flavors, discoloration, changes in texture, nutritional value, shelf life, and appearance of SFO, so synthetic antioxidants such as tert-butyl hydroquinone (TBHQ), propyl gallate (PG), butylated hydroxytoluene (BHT), and butylated hydroxy anisole (BHA) were used .
Although synthetic antioxidants are attractive due to their low cost, wide availability, great stability, and effectiveness, their use is limited as they may generate health risks, gastrointestinal tract problem, and cancer risk (Xu et al., 2021). Today, there is growing interest to explore natural antioxidants like plant extracts which provide higher antioxidant activity, and improved sensory properties Wang et al., 2020).
Antioxidant properties of plants are effective in delaying oxidation and rancidity in fats and oils and they have similar activity as chemically synthetic antioxidants (Aly et al., 2021;Wang et al., 2020).
Natural extracts from different herbs, such as Heracleum persicum , Fumaria parviflora L. , sesame (Esmaeilzadeh , and Rosmarinus officinalis L. (Jafari et al., 2022), are stable for oxidation which is related to the presence of natural phenolic compounds.
Sage (Salvia officinalis L.), an evergreen shrub, belongs to the mint family (Labiatae). It is known for its aroma, flavor, and taste. Sage contains a wide array of bioactive compounds like phenolics, terpenoids, and organic acids that have shown antioxidant, antimicrobial, anticancer, and anti-inflammatory activities (El-Sayed & Youssef, 2019;Naziruddin et al., 2022). The extraction of bioactive compounds from plant materials with conventional methods such as maceration, shaker, and hydro-distillation is laborious due to long extraction time, low efficiency, and hazardous solvents (Wrona et al., 2017).
Ultrasound-assisted extraction (UAE) process is a potentially useful technique for the purification and isolation of bioactive compounds.
The high-intensity and high-frequency sound waves and also their interaction with plant materials distinguish UAE from the conventional methods (Sadat et al., 2021).
The efficiency of plant extracts pertains to biological activities and physicochemical properties. Low stability and water solubility, and the unpleasant taste of plant extract limit their application in food formulation. Encapsulation is a technology for maintaining the biological activities, control release, and bioavailability of bioactive compounds from plant materials which allow their application in different food formulations and preserving their functional properties (Reddy et al., 2022). It also enclosed bioactive compounds from light, oxygen, pH, water, and other adverse conditions (Jamshidi et al., 2020). A range of food-grade biopolymers is used to create nanoparticles such as polysaccharides, proteins, and a combination of them . Seed gums are new and plentiful polysaccharides. The Lepidium perfoliatum seed, which is known as Qodumeh Shahri in Iran, produces a high amount of mucilage. It can immobilize and bind a lot of water, and increase the viscosity of foods (Jamshidi et al., 2020). Whey protein isolate is obtained during the production of cheese or casein and it is a by-product of the dairy industry which is widely used in the food industry because of its functional properties, emulsification, gelatinization, film formation, and solubility in water (Tavares & Noreña, 2019).
Considering that sunflower oil is sensitive to oxidation like other vegetable oils, it is necessary to increase its shelf life by adding natural antioxidants as safe preservatives. The use of extract encapsulation controls the release of antioxidant compounds from the extract during the storage. To the best of our knowledge, studies carried out so far have predominantly focused on using free extracts to increase the shelf life of vegetable oils. Also, no research has been published about the antioxidant activity of the encapsulated sage extract in whey protein isolate and Qodumeh Shahri (Lepidium perfoliatum) seed gum in sunflower oil. Therefore, the present study aimed to evaluate (1) the antioxidant activity of the sage extract, (2) the effect of coating material on the properties of nanocapsules, and (3) the effect of free and nanoencapsulated extract on the extension of oxidative stability of sunflower oil during the accelerated thermal condition.

| Material
The common sage was collected from the local field area near Sari (Mazandaran, Iran) in the summer of 2021. Sunflower oil without antioxidant was purchased from North Agro-industrial Oil Company.
All solvents and chemicals were purchased from Sigma-Aldrich Company (Sigma). Qodumeh shahri seed gum was purchased from Reyhan gum parsian.

| Preparation of sage leaf extract
The leaves of sage were dried immediately after harvesting in a shady place for 1 week and the moisture content was below 10%.
The dried sage leaves were ground into powder using a mechanical grinder (Habi, Pars-Khazar). The powder was sieved using a 200μm sieve to remove any large pieces. To prepare sage leaf extract, 50 g of sage leaves was mixed with 250 ml of ethanol: water (70:30) solvent. The extraction was done using a ultrasonic bath (6.5l200 H, Dakshin, India) at 35°C for 30 min at a frequency of 35 kHz. The mixture was filtered using Whatman paper No. 1. Then, the solvent was evaporated using a rotary evaporator (RE 120) at 35°C and the final extract was kept at −18°C ).

| Total phenolic content of sage leaf extract
The total phenolic content (TPC) of sage leaf extract was calculated according to the method reported by Doymaz and Karasu (2018).
Initially, 2.5 ml of Folin-Ciocalteu phenol reagent (0.2 N) was added to 0.5 ml of extract and mixed with 2 ml of Na 2 CO 3 (7.5%). This mixture was kept for 20 min at room temperature in a dark place.
After incubation, the absorbance was recorded at 760 nm using a ultraviolet-vis spectrophotometer (Cintra 6, GBS Scientific). The total phenolic content was expressed as a gallic acid calibration curve (Doymaz & Karasu, 2018).

| Determination of antioxidant activity
The antioxidant activity of the extract was determined using 2,2-diphenyl-1-picrylhydrazyl radical scavenging method (DPPH) and ferric reduction antioxidant power (FRAP). Briefly, 0.1 ml of extract and 4.9 ml of DPPH solution (0.1 mM in ethanol) were mixed toughly and held at 25°C for 30 min. Then, the absorbance was
Initially, 0.05 g of coating powders was dispersed in deionized water at 30°C and after cooling, mixed overnight to enhance hydration.
Then, 10 ml of sage extract was combined with 40 ml of tween 80 and 50 ml of sunflower oil during homogenizing with a magnetic stirrer at 100 rpm for 15 min. After that, the formed emulsion was homogenized again using Ultra-Turrax homogenizer (IKA Labortechnik) at 15,000 rpm for 10 min followed by adding coating solution to nanoemulsion at a 5:1 ratio (Jafari et al., 2022).

| Properties of encapsulated sage extract
Nanoemulsions were dried using a freeze dryer (SP Scientific) at −50°C and 0.017 mPa for 48 h. The particle size, polydispersity index, and zeta potential of nanoemulsions were measured using a master-sizer light scattering (Malvern Instrument Ltd.

| Release rate of phenolic compounds
The release rate of phenolic compounds was measured according to the method described by Esmaeilzadeh Kenari et al. (2020). Initially, 20 g of different nanoparticles was poured into separate bottles and kept in an incubator at 60°C for 24 days. Then, 5 ml of phosphate buffer was mixed with 5 g of nanoparticles and centrifuged for 90 min at 1500 g and room temperature. The TPC of the lower phase was determined. The release rate was calculated using Equation 2  (Farahmandfar et al., 2018), and color index (CI) were determined  every 4 days.

| Statistical analysis
All experiments were performed in triplicate. Experimental data were analyzed using SPSS software (Statistical Program for Social Sciences) version 22. Significant differences (p < .05) were calculated using Duncan's multiple tests. (1) Release rate ( % ) = 100 − 100 × Encapsulated TPC in the outer phase Encapsulated TPC in the inner phase

| Total phenolic content of sage extract
The total phenolic content (TPC) of sage extract was 31.12 mg GA/g.

| Antioxidant activity of sage extract
The antioxidant activity of sage extract was determined by the DPPH radical scavenging and FRAP assay. Figure 1a

| Properties of nanocapsules
The results of the particle size of different nanocapsules are shown in Table 1. All nanocapsules showed a size below 270 nm and a statistically significant difference was observed. The pressure of ultra-turrax beside sonication energy caused nanosize of particles (Razavi et al., 2020). PDI is among the most important characteristic of nanocarrier systems. PDI of all samples was below 0.300 which indicates the normal distribution of particle size. The zeta potential is helpful to determine the net charge of nanocapsules.

| Morphology of nanocapsules
The morphological structure of nanocapsules depends on the interactions between the coating components, which affect the final physiochemical properties. The surface morphology of nanocapsules is presented in Figure 2. The surface of all nanocapsules was smooth and did not show cracks, pores, and bubbles. Figure 2c indicates the formation of high compatibility between gum and protein to form wall coating. These surface morphology images confirmed that the sage extract was well encapsulated into the polymer matrix (Esmaeilzadeh . A similar result was observed in a nanocapsule of Iranian golpar , Fumaria parviflora , rosemary leave (Jafari et al., 2022), and sesame seed extract (Esmaeilzadeh .

| Release rate of phenolic compounds
The gradual release rate of phenolic compounds was observed in all samples (Table 2) and differences were significant. There is a positive correlation between size diameter and release rate of phenolic compounds from nanoparticles. This result is in line with the reports of other researchers on the gradual release of phenolic compounds from extracts of Iranian golpar , rosemary leaf (Jafari et al., 2022), olive leaf (Mohammadi et al., 2016), and Ferula persica into soybean oil (Estakhr et al., 2020).

| Oil oxidation
Oils with high degree of unsaturation are prone to autooxidation.
The simplest test for evaluating the oil autooxidation is PV and TBA. Figure 3a shows     (Sayyari & Farahmandfar, 2017).
Control sample showed a lower OSI. Also, oil samples containing encapsulated sage extract exhibited higher OSI, which is related to the antioxidant activity of sage extract. In a study conducted by Upadhyay and Mishra (2015), the sage extract was found to have protective effect on oxidative stability of sunflower oil (Upadhyay & Mishra, 2015). Taghvaei et al. (2014) found that the thermal stability of soybean oil containing olive leaf extract in both free and encapsulated forms is higher than blank oil. However, oil containing encapsulated extract showed higher OSI (Taghvaei et al., 2014) which is in accordance with the results of the present study.
Color is considered a vital indicator contributing to the quality of edible oils and consumer preference. The results of color index of different oil samples are presented in Figure 5.    Kenari et al. (2020), who reported higher color index in soybean oil without antioxidant followed by oil containing TBHQ, nanoencapsulated Iranian golpar extract, and free extract . Similar results were reported by Salami et al. (2020) for lower color index of canola oil containing TBHQ than oil with pumpkin peel extract (Salami et al., 2020).

F I G U R E 3
Another indicator for evaluating oil oxidation is conjugated dienes An increasing trend in CD value of plant oil during thermal processing and storage time, and also low CD value of oil containing plant extracts previously reported by other publications Maghsoudlou et al., 2017;Salami et al., 2020;Talón et al., 2019).

| CON CLUS ION
In this study, the antioxidant effect of free and nanoencapsulated sage extract was compared to TBHQ synthetic antioxidant. All coating materials could extend the antioxidant properties of sage extract by controlling gradual release of phenolic compounds, and protecting phenolic compounds from environmental stresses. According to the results of oxidation parameters of oil, the use of complex coating of whey protein isolate and qodumeh shahri seed gum was suggested to encapsulate sage seed gum as a natural antioxidant to extend the shelf life and oxidative stability of sunflower oil.

ACK N OWLED G M ENTS
The authors of this article are extremely grateful to Sari Agricultural

Sciences and Natural Resources University and Vasteryoosh
Specialized Laboratory for their cooperation in this research.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.

E TH I C S S TATEM ENT
The study does not involve any human or animal testing.