Formulation and characterization of micro-emulsions of peppermint and coriander oils extracted by using a supercritical fluid system

ABSTRACT The present study was designed to formulate and characterize the micro-emulsions of peppermint and coriander oil that extracted by using supercritical fluid extraction system. Moreover, Guar gum and maltodextrin were used as coating materials while preparing encapsulates of peppermint and coriander through freeze-drying. The moisture contents of mint leaves and coriander were 81.28 ± 7.12 and 84.62 ± 6.46% respectively whereas the recorded protein content in mint and coriander leaves were 1.456 ± 0.13 and 5.06 ± 0.03% respectively. The crude fat was high in mint leaves as compared to coriander. However, the ash content was 2.98 ± 0.278 in mint and 2.8 ± 0.12 in coriander leaves. The total phenolic contents (TPC) and antioxidant activity coriander essential oil was high as compared to peppermint oil. The results showed the better antioxidant activity of coriander than peppermint. Encapsulation efficiency of powder showed significant results of wall material. T3 (maltodextrin and coriander oil) exhibited better binding capability of bioactive components as compared to guar gum and mint. T3 was coriander and maltodextrin better ability to inhibit the oxidation process and had good DPPH assays as compared to other treatments. This study showed that the T3 (maltodextrin and coriander oil) exhibited better binding capability of bioactive components as compared to guar gum and mint. T3 was (coriander and maltodextrin) better ability to inhibit the oxidation process and had good DPPH assays to other treatments.


Introduction
Microencapsulation is a packaging technology used for solid, liquid, or gaseous materials with a thin layer of the polymer coating.This technology has been used in various industries including agriculture, pharmaceutical, medical, and food. [1]Encapsulation of oils is becoming more popular as a potential preservation technique.Encapsulation technique can be protected oil from oxidation.There are some popular methods of encapsulating including freeze-drying, coacervation, and emulsification.Different coating materials are being used for microencapsulation (such as lipids, proteins, cellulose, and polysaccharide gums). [2]The plants sources of essential oils are complex mixtures of natural volatile compounds.The essential oils have specific odors and are common sources of bioactive compounds. [3]The plant essential oils can be employed to extending the oxidative and microbiological shelf life.Natural bioactive compounds can saved food products from oxidative reactions. [4]Essential oils are immiscible in water, but it is also susceptible to environmental stimuli including temperature, light, oxygen, and others chemical.Therefore, it can be used as encapsulated to reduce the problems. [5]The effect of encapsulation on native oils due to components like bioactive compounds and unsaturated fatty acids. [6]The recent extraction methods are being used to achieve better yields of active compounds from natural sources.The major extraction methods are decoction, infusion, digestion, percolation maceration, and supercritical. [7]Supercritical fluid extraction (SFE) is recent method of extraction of oils. [8]SFE technique gives pure solvent-free extracts with high biological potential, and works at low temperatures that prevent the degradation of biological components.The extracted oil can further be processed in microencapsulation. [9]Peppermint oil is widely used in pharmaceutical, flavoring, and food products.It is one of the most common essential oils. [10]Among essential oil, Coriander sativum L. is considered one of the most beneficial medicinal herbs.The essential oil extracts from C. sativum have promising antibacterial, antifungal, and antioxidants properties, and play a significant role in enhance the shelflife of food products [10] The aim of current study was extracted the peppermint and coriander oil by using supercritical fluid system.Furthermore, formulation and characterization of micro-emulsions were measure.Different parameters were performed to compared the different treatment including proximate analysis, TPC, antioxidants, encapsulation efficiency, wettability and solubility.

Collection of raw material and sample preparation
The peppermint and coriander were obtained from the local market of Faisalabad, Pakistan.After that, peppermint and coriander were cleaned, and inedible particles removed.Furthermore, the fresh mint and coriander leaves were washed by using fresh water.Then, the leaves were dried in oven (35°C) and grounded prior to further analysis.The dried peppermint and coriander leaves were grounded and converted to powder form.The resultant powder was enclosed in a plastic bag and kept at room temperature for further analyses.

Proximate analysis
The proximate composition of peppermint and coriander were measured by using the respected method of AOAC. [11]The moisture content, crude protein, crude fat, crude fiber, and nitrogen-free extract were assessed.

Supercritical fluid extraction (SFE)
The 50 mL sample was loaded into high-pressure vessel.The oven temperature (35-558°C) was set.After that, high-pressure pump was used to pressurize the CO 2 and then charged into the vessel at the rate of 2 liter/min maintaining pressure of (100-300 bar) during the cycle.The supercritical CO 2 was passed to the temperature-controlled micrometer vale, and was exposed to the atmosphere which was containing the extract.A glass vial was used to collect extract after each run.After that the extract was stored in the refrigerator before analysis. [12]

Microencapsulation of peppermint and coriander oil
The mixtures of oils were stirred electromagnetically at 55°C for 4 hours without heating.All samples were incubated at 4°C for 12 hours.Vacuum filtration was used to take precipitates and then it was dried at 50°C for 24 hours.The powder was dried at room temperature for 24 hours to make it an equilibrium state for humidity content.The encapsulated peppermint and coriander oil powders were preserved in closed plastic bags at room temperature. [5]

Preparation of emulsion
The emulsion was prepared using Maltodextrin and Guar gum.The emulsion composition was similar for all samples 19.8% o(w/w) of wall materials (guar gum 0.5% and maltodextrin 15.4%), 70% (w/w) of water, and 10.2% (w/w) of oil.The wall material proportion and oil loading were established as per the work of Ogrodowska et al. [13] Thermomix was used to blend oil with aqueous solutions of wall materials at 40°C for 2 minutes and 9000 rpm (Vorwerk, Wuppertal, Germany).A high-pressure laboratory valve homogenizer was used for homogenizing emulsions at 240 bar (Panda 2K, GEA Niro Soavi, Parma, Italy). [6]

Emulsion stability
The emulsion sample was placed in a 50 mL graduated cylinder, sealed it, and stored at 25°C for 24 hours for evaluating emulsion stability.The separation phase of the emulsion was observed for whole 24 hours and expressed as % age of phase separation. [6]

Emulsion stability index (ESI)
For ESI, the liquid emulsion was transferred to a 10 mL measuring cylinder which was then capped and stored for 24 hours as outlined by Sarkar et al. [14] The volume of oil separated from the emulsion was measured for each sample and the emulsion stability index (ESI) was expressed within the range from 0 to 1 using the following equation.

Emulsion pH
The pH of the prepared emulsion was measured using a digital pH meter following the protocol of AOAC. [11]

Preparation of freeze-dried powder
Lyophilizer was used to prepare freeze-dried powders.The aluminum pans were frozen at 20°C for 24 hours where freeze-dried powders were placed.After that, the emulsions were lyophilized using an ice condenser at −50°C with a pressure of 0.12 mbar for freeze-drying for 72 hours. [6]

Microencapsulated powder tests
Total phenolic contents (TPC).TPC was determined using the Folin-Ciocalteau method.Sodium carbonate reduced the Folin-Ciocalteau reagent in the presence of phenolic substances by changing the color.A solution was prepared by mixing 3 mg of encapsulated phenolic powder with 10 mL of methanol after stirring it up for 5 minutes with a vortex.A microplate was used to mix 25 μL of the diluted sample with 125 μL Folin-Ciocalteu reagent.100 μL sodium carbonate was added to the microplate after keeping it for 5 min in a dark environment.The absorbance was measured at 745 nm after 2 hours of reaction time in a dark room. [15]ncapsulation efficiency.The encapsulation efficiency (EE) was determined by the following equations In equation 1 m 1 = the amount of essential oil contained in microencapsulates m 2 = the total amount of essential oil used.The total amount of microcapsules and the amount of essential oil contained in the microcapsules (m1) was determined using equation (2).In equation 2, m 3 = the amount of the essential oil from the aqueous phase collected after microcapsules filtration, by solvent extraction Enascuta et al. [16] All experiments were carried out in triplicate and the results presented are the average values.
Free radical-scavenging ability.DDPH radical scavenging activity was used to determine the radical scavenging activity of encapsulated oils.Different quantities of encapsulated oils were added up in a hydroalcoholic solution of water.After that, the ethanol was homogenized by ultrasonication for 1 minute.Concentrated solutions among 1 to 50 µg/mL of coriander essential oil were obtained.The retention degree of essential oils helped quantify the essential oils.A volume of 1 mL sample EO or 1 g complex in different concentrations was added to 2 mL DPPH ethanolic solution (5 mg/mL) and was strongly stirred.The mixtures were kept in darkness for 60 minutes, after that the absorbance was measured at a wavelength of 515 nm was calculated by Dima et al. [5] Wettability.The wettability was measured by following the protocols of Barros Fernandes et al. [17] One gram of powder was sprinkled over the surface of 100 mL of distilled water at room temperature without agitation.The extent of wettability of the samples was compared by recording the time of the powder particles to sediment from the water surface.
Solubility.Three grams of powder were dissolved in 10 mL solutions on a dry basis for checking solubility.Then, the solution was placed in the tube and centrifuged for 5 minutes at 1200 rpm.The solution was filtered using filter paper then the paper was dried in an oven for 3 hours at 105°C.The percentage of solubility was calculated by the difference in weight on the filter paper Mangiring et al. [15] Statistical analysis.Data obtained from each parameter were analyzed statistically using the respected method of Steel and Torrie. [18,19]sults and discussion Proximate analysis.The nutritional characteristics of raw materials are determined by proximate analysis.
The proximate composition of peppermint and coriander leaves was analyzed.The chemical composition of the fresh peppermint and coriander leaves has been shown in Table 1.The moisture contents in peppermint and coriander were 81.28 ± 7.12% and 84.62 ± 6.46% respectively, whereas the protein of peppermint and coriander were 1.456 ± 0.13 and 5.06 ± 0.03 respectively.However, the crude fat in peppermint leaves was 1.95 ± 0.182, and in coriander was 0.62 ± 0.056.The crude fiber was 6.90 ± 0.54 in peppermint, whereas coriander leaves was contained 4.56 ± 0.34 crude fiber.The ash content was 2.98 ± 0.278 in peppermint while coriander leaves was contained 2.8 ± 0.12 ash contents.Current result is similarly to Bukar and Saeed, (2014) who determined the nutrient composition (NFE, ash, crude fiber, crude fat, crude protein, and moisture) of peppermint.Another study showed the similar results of moisture to current findings (Moura et al. [20] ).Previous research is conducted by Dyab et al. [21] who determined the moisture of mint leaves.Javid et al. [22] was performed a proximate analysis of peppermint and found that moisture to be 6.5 ± 1.0% in the stem, fat 2.4 ± 0.5% in leaves, protein 5.5 ± 2.0% in leaves, and ash 10 ± 2.0% in leaves.Nehul et al. [23] performed proximate analysis on menta spicata and found moisture protein crude fat carbohydrates crude fiber and ash.
Emulsion pH.The pH of the emulsion is an important quality parameter.The pH of T 1 , T 2 , T3 and T4 was 4.71 ± 0.34, 48 ± 0.32, 4.92 ± 0.372 and 3.42 ± 0.23 respectively.According to the results of the current experiment, lower pH levels was categorically linked to a slower rate of respiration and efficient quality preservation.The pH was shown to the inverse to calculate the acidity index.Naeem et al. [24] demonstrated that coating vegetable fruits with guar gum+ethanolic isolates resulted in lower rates of changes in total soluble solid, total acidity, and pH when contrasted both to controls (treated control and uncoated).However, coatings enhanced with methanolic extracts.During storage, the pH values of coated and uncoated fruits both rose, and a substantial statistical variation was seen in the measurements.The conversion of organic acids into neutral molecules caused the pH to rise.

Emulsion Stability (ES).
The kinetic stability of emulsion formulations was shown by stability tests.Table 2 showed variable results with respect to the treatments.The stability of T 1, T 2, T 3, and T 4 was 2.80 ± 0.28, 2.37 ± 0.33, 2.92 ± 0.27 and 2.67 ± 0.25, respectively.The results depicted that the stability of T 3 (coriander: maltodextrin) was better as compared to T 1 (mint: maltodextrin).The encapsulated powder which is prepared from T3 presented high particle size, viscosity, and stability as compared to T1 and T2.The results were depicted lower value and less stable as compared to others.The better results was attained from T3 which shows maltodextrin is more stable as compared to other coating materials.In the case of peppermint encapsulates gum arabic acted as a thickening agent showing emulsion stability of 1.73 ± 0.02.Carneiro et al. [25,26] The stability of gum Arabic was not higher due to its lower viscosity.Its performance was increased when mixed with maltodextrin exhibiting good stability. [14]According to the findings by Shen and Li, [27] who acylated pea proteins were made by treating them with acetic anhydride (AA) or succinic anhydride (SA).Guar gum-pea protein compounds were made by incubating the combination at 60°C for 24 hours at mass ratios of 1:20 and 1:40, respectively.To examine the synergistic effects, acylated-guar gum-conjugated pea proteins were also synthesized.When contrasted to the unmodified protein (1.03 0.02 g oil/g), both connected and acylated pea proteins demonstrated the considerably enhanced oil-holding ability of up to 2.20 0.05 and 2.09 0.03 g oil/g protein.When contrasted to the unmodified protein (3.570.05 g water/g), the acylated pea protein showed a higher water retention capacity of up to 7.01 0.31 g water/g protein.The changed proteins enhanced emulsion stability and capacity by up to 96-100% and 95-100%, respectively.The 9% acetylated pea protein solutions created solid gels.The effects of sequential acylation and conjugation of pea proteins on the retention of water and emulsification capabilities were more favorable and synergistic.Emulsion Stability Index (ESI) was 1 for all treatments (Table 2) depicting emulsions stability.The 0 value showed a poor stability index and 1 shows higher stability.Similar results also showed that emulsion stability as 1. [14] According to Santos et al. [28] who measured the initial droplet size of microfluidized emulsions was optimized, yielding diameters of less than one micron independent of the processing parameters.The emulsion produced at 25,000 pressure and one pass, on the other hand, had the smallest polydispersity and mean droplet sizes.As stabilizers, various Span 80 and Tween 80 T 1 : Peppermint: Maltodextrin, T2 = Peppermint: GuarGum, T3 = Corinder : Maltodextrin, T4 = coriander: Guar Gum ratios were evaluated.The results showed that the surfactant ratio had a substantial influence on droplet size, and rheological, and physical stability characteristics.

Total phenolic content (TPC).
During the characterization of essential oils, TPC is an important indicator that shows the antioxidant activity.TPC of peppermint and coriander essential oil was 2.47 ± 0.19 and 6.9 ± 0.59 mg GAE/100 g respectively.The results showed that the TPC activity of coriander was better than peppermint.The TPC results of T1, T2, T3, and T4 4.44 ± 0.33, 2.47 ± 0.19, 6.9 ± 0.59 and 3.47 ± 0.23.T3 (coriander with maltodextrin) showed better antioxidant activity as compared to others treatments.However, outcomes showed that maltodextrin had better phenolic contents as compared to guar gum.According to Naeem et al., [25] the phenolic concentration of tomato homogenate derived from processed tomatoes is virtually identical to the control sets.It cannot include the measurement of phenols that have dispersed from the coating to the pulp of the fruits.On the contrary, both treated control and untreated revealed higher levels of TPC after just 15 and 30 days.
The fruits treated with ENEO exhibited a modest rise in TPC during 60 days in storage.

Encapsulation Efficiency (EE).
The process of encapsulation is described by encapsulation efficiency.It is the capability of the material to encapsulated the bioactive ingredient.Its depend upon the temperature, emulsion characteristics, and coating material.Table 3 show the encapsulation efficiency of powder.
The encapsulation efficiency of T 1 , T 2 , T 3 and T 4 were 58.81 ± 4.65%, 55 ± 5.23%, 91 ± 6.24%, 49 ± 3.54% respectively.T 3 (maltodextrin and coriander oil) showed the better binding capability of bioactive components as compared to other treatments.The range (62.3% to 95.7%) of encapsulation efficiency of maltodextrin was reported. [26]TPC of alginate micro-beads loaded with basil essential oil and free basil was 4.53 and 7.54 respectively.Sarkar et al. [14] determined the TPC of free basil essential oil and determined encapsulation efficiency of grape oil with gum Arabic and maltodextrin.The encapsulation efficiency of mint oil with gum arabic was up to 72-85% and for starch-based peppermint oil 79%.Ramdhan et al. [29] revealed that encapsulation efficiency is declined with increasing pectin.So it is deduced that the proportion or type of material is the influential factor affecting the stability of emulsions.Previously outcomes showed the encapsulation efficiencies rage 87.0% to 92.1%. [30]It was reported that tiny oil droplets were entrapped more efficiently with the wall matrix. [31]With the increase in hydrolyzate, encapsulation efficiency decreased from 98.28% to 88.37%.A similar trend was observed for the oil release rate. [5]When maltodextrin was used in the spray drying process, the microparticle exhibited heterogeneous and spherical structures and good encapsulation efficiency. [32]The encapsulation efficiency among different coating materials was partially varied.The lowest efficiency was obtained with capsules coated by Gum arabic, while the highest with capsules were coated in guar gum. [33]ee radical-scavenging ability.DPPH assay presents antioxidant activity of powder and inhibits oxidation.Table 3 showed significant differences in the different treatments (T 1 , T 2 , T 3 , and T 4 ).The results showed that T 3 was better ability to inhibit the oxidation process and had good DPPH assays as compared to other treatments.Earlier (DPPH assay) of free basil essential oil and alginate micro-beads loaded with basil essential oil was done.Kizil et al. [33] estimated the DPPH value up to 60.41 ± 0.60 in peppermint.Dyab et al. [22] determined the antioxidant activity of peppermint leaves.Wettability.The penetration of water due to capillary forces in encapsulated powder is wettability.This is important physically factor for ascertaining the reconstitution capability of microencapsulated powder.Wettability time increases due to a decrease in drying temperature.The relationship between wettability and particle size is inverse as more porosity is present in large particle sizes which require less penetration time.Due to less porosity in smaller particles of powder, it is very difficult for liquid to penetrate.is the data showed that the mean square for wettability provides a significant effect on different treatments.The yield of spray-dried powder, powder wettability, and bulk density were 69.8 ± 2.5, 92.5 ± 3.5, and 0.37 ± 0.01 g/cc respectively.The dependence on wettability is due to coating material and molecular collaboration among solid and liquid phases.It is affected by carbohydrates, temperature, particle size, protein concentration, and fat on the surface (Fernandes et al.) [32] Solubility.The capability of encapsulated ingredients to dissolve in water is known as solubility.
In food industries, solubility is considered an important physical characteristic for ensuring the quality of ingredients.Drying temperature and coating material affect the solubility directly.Mean square values of solubility give significant results on treatments.The results showed that carrier materials have good solubility (Table 3).The results of T 1 , T

Conclusion
It is concluded that microencapsulation is prepared by peppermint and coriander oils.Encapsulation of oils is becoming more popular as a potential preservation.It is mostly used in the encapsulation of bioactive compounds in aqueous solutions.T3 is composed of coriander and maltodextrin.T3 is showed high antioxidants and phenolic contents as compared to other treatments.However, the emulsion of coriander and maltodextrin is more wettability and solubility.Furthermore, overall composition of T3 was better as compared to other treatment.

Table 1 .
Proximate values of peppermint and coriander leaf.
T1 :Mint :MD, T2 = Mint: GG, T3 = Corinder :MD, T4 = coriander: GG 2 , T 3 and T 4 42.19 ± 3.15%, 40 ± 4.01%, 50 ± 4.21% and 50 ± 4.21%.Contrarily, multiple studies show that the formation of agglomerates increases the solubility of powder in water.It has a direct relationship with the water content of the capsule.More water in the coating increase solubility and less water decreases the capability.