Garlic essential oil‐based nanoemulsion carrier: Release and stability kinetics of volatile components

Abstract An O/W nanoemulsion of garlic essential oil (GEO) at different oil‐to‐emulsion (O/E) ratios (5%, 10%, 15%, and 25%) was formulated to protect the volatile components of GEO. The effects of O/E ratios on the encapsulation efficiency (EE%) of volatile compounds and droplet size of nanoemulsions were studied. The results showed that with increasing in E/O ratio, droplet size increased while EE% decreased so that the droplet size was below 100 nm for all samples and the EE% was almost above 80% for most samples. The effects of various factors such as temperature (5°C–45°C), pH values (3–7), ionic strength (0–500 mM), and O/E ratios (5%–25%) on kinetic of nanoemulsions stability were studied. Reducing pH values and raising the temperature, ionic strength, and O/E ratios intensified the instability process and constant rate of instability in all nanoemulsions. The effects of temperature and O/E ratios on the release kinetics of volatile components were evaluated over time, and kinetic parameters such as release rate constant (k), Q10, and activation energy (Ea) were calculated in which results showed a zero‐degree model to describe the release kinetic behavior of most nanoemulsions. Both temperature and O/E ratios factors as well as their interaction (which had a synergistic effect) had a significant effect on increasing the release rate of volatiles so that the degree of reaction rate was changed from zero to the first order at simultaneous high levels of both factors. FT‐IR spectroscopy was carried out to study interactions among nanoemulsion ingredients. The presence of sulfur‐containing functional groups of garlic oil (thiosulphate, diallyl trisulfide, etc.) in nanoemulsions was confirmed by FT‐IR.


| INTRODUC TI ON
Kinetic modeling is a very applicable technique for food processing and food quality. The area of kinetics in food systems has received a great deal of attention in past years, primarily due to efforts to optimize or at least maximize the quality of food products during processing and storage. Kinetic modeling enables us to describe chemical, biochemical, and physical reactions and their rates quantitatively (Heldman et al., 2006). Moreover, a good understanding of reaction kinetics can provide a better idea of how to formulate or fortify food products to preserve the existing nutrients or components in a food system. Unfortunately, limited kinetic information is available at present for food systems or ingredients that inhibit the rapid development of new functional food products with improved stability or the optimization of processing conditions (Heldman et al., 2006).
In recent years, nanoemulsions have shown great potential in the encapsulation of lipophilic compounds which can develop the application of essential oil in food formulation (Salvia-Trujillo et al., 2015).
Nanoemulsions are the fine droplet emulsions that have a droplet size below 200 nm and they have shown relatively higher kinetic stability against flocculation, coalescence, and creaming than macroemulsions due to their smaller droplet size (McClements, 2012;Saberi et al., 2013;Salvia-Trujillo et al., 2015). Fabrication of nanoemulsions for encapsulation and controlled delivery of functional ingredients is one of the functional areas of nanotechnology in the food industry (McClements, 2012;Velikov & Pelan, 2008;Yang et al., 2012).
Researchers reported that the nanoemulsions based on essential oil have antimicrobial activity against positive and negative bacteria, mold, yeast, fungi, viruses, and spores without adding any synthetic or natural antimicrobial additives (Weiss et al., 2009). Regarding the previous results, these nanoemulsions had an antimicrobial potential when they were formed only by water, oil phase, surfactant, and alcohol which is attributed to the electrostatic beyond between cationic charge of nanosize droplets of emulsions and anionic charge of the pathogen cell (Jumaa et al., 2002). Nanosize droplets bind to the outer membrane of the microbial cell by electrostatic attraction and transmit their energy to the outer membrane and consequently disrupt the cells (Donsì et al., 2011;Liang et al., 2012).

Artiga-Artigas et al. (2018) evaluated the effect of lecithin,
Tween 20, and SMP (Sorbitan Monopalmitate) on encapsulation and stabilization of curcumin-loaded nanoemulsions. They reported that nanoemulsions with 2.0% w/w lecithin did not suffer destabilization phenomena during almost 86 days of the experiment, whereas those containing Tween 20 or SMP at the same concentrations were destabilized after 5 days or along 24 hr, respectively. Indeed, Tween 20 concentrations of 0.5% w/w showed the fastest release of curcumin.
Md Saari et al. (2020) worked on the release kinetics of encapsulated curcumin based on the cumulative amount of curcumin released from nanoemulsion. The data were analyzed and fitted using five kinetic models such as zero, first, second, Higuchi, and Korsmeyer-Peppas equations. The results showed that both Higuchi and Korsmeyer-Peppas models could fit well into the data satisfactorily. The model achieves the highest correlation coefficient (>0.9) for both free curcumin and encapsulated curcumin. Doi et al. (2019) studied the impact of garlic oil droplet properties on flavor release from the heated emulsion and explained that higher oil levels increased flavor retention but decreased physical stability and low molecule surfactants had slow flavor release but decreased physical stability. Also, the emulsifier type impacted emulsion stability and flavor retention.
Fabrication of nanoemulsion based on GEO has a great advantage due to covering its unfavorable flavor, especially in a dairy beverage, such as buttermilk, kefir, etc., and maintaining its biological functions. This article aimed to study the release kinetics of volatile components from the nanoemulsion containing essential oil of garlic and to study different factors affecting release phenomena in this type of nanoemulsion.

| Materials
The essential oil of garlic was purchased from Tehran Zardband Co.
Tween 80 and Citric acid were purchased from Sigma-Aldrich Co.
The used materials were analytical grade.

| Nanoemulsion preparation
A combination of low energy and high energy methods was used for the production of nanoemulsions. At first, essential oil of garlic and tween 80 were mixed by magnetic stirrer for 30 min at 500 rpm and then the mixed oil phase (tween 80 and essential oil of garlic mixture) was added slowly to the aqueous phase while mixing by magnetic stirrer for 30 min at 700 rpm (25°C ± 3°C). Then, the produced premixed emulsions were transferred to the water bath ultrasonic with 100 w powers and 40 kHz frequency for 15 min (30°C ± 5°C).
The ratio of surfactant-to-oil was fixed for all of the nanoemulsions (1:1), while the aqueous phase content was varied (50, 70, 80, and 90%).

| Droplet size (Z average) and Polydispersity Index (PDI)
Dynamic light scattering (Malvern Instruments Ltd with precision (range) of measurement 0.6 nm to 6 microns) was used to determine droplet size. All of the nanoemulsions were diluted by distilled water with a ratio of 1:50 to prevent multi-scattering during droplet size measurement. Experiments were carried out at 25°C and with a 90 angle of refraction.

| Encapsulation efficiency
Gas chromatography (Agilent Technologies) equipped with a flame ionization detector (FID) was used to measure encapsulation efficiency. Type of used column was HP5, 50 m × 0.32 mm × 25 μm (19091J-413) Agilent technologies.INC. Nitrogen was used as carrier gas with a flow rate of 0.7 ml/min. The injection inlet temperature was 120°C and the temperature of the detector was 300°C.
Also, the initial temperature of the oven was 40°C and then the temperature increased with a rate of 5°C/min until reached 90°C and was maintained for 2 min at this temperature. Before injection, the samples were heated to 40°C, and then sampling was done from samples headspace (Karagoz et al., 2017). Then, encapsulation efficiency was determined by comparing the total area under the peaks of the nanoemulsions and the not emulsified mixture headspace (Hassanzadeh et al., 2018).

| Stability tests
The prepared nanoemulsions were subjected to various conditions to evaluate their stability against gastrointestinal digestion and various storage conditions. The pH value of nanoemulsions was adjusted by HCl (0.1 N) in different states (2, 3, 4, and 6), also NaCl was added to the nanoemulsions in different formulations (0, 250, and 500 mM) and finally, the nanoemulsions were stored at different storage temperatures (5, 25, and 45). The absorbance changes of nanoemulsions were recorded at 500 nm by spectrophotometer (Pharmacia Nova spec II) at 3-day intervals during storage time to study their stability.

Release kinetic of entrapped material
The release of volatile compounds of garlic oil was recorded during storage time by gas chromatography (it was carried out in the way described in the encapsulation efficiency part) (Kiralan et al., 2019).
Then, the recorded data were fitted to the zero (Equation 1), first (Equation 2), and second (Equation 3) order reaction, and one of them was selected based on the highest R 2 and the lowest sum of squared errors (SSE).
Where C is the concentration, t is the time, C 0 is the initial concentration, and k is the rate constant (Heldman et al., 2006).
For calculating activation energy (Ea), firstly arranged the calculated reaction rate constant (K) at different storage temperatures versus the temperature (in degrees Kelvin), then it was taken a natural logarithm of the reaction rate constant (Ln K) and inversed the temperature (1/T).
The sorted data are fitted with the Arrhenius model (Equation 4) and the slope (Ea/R) and the intercept (ln A) are calculated for the next steps.
Also, by substituting a certain constant global amount of gases (8.314472) in the calculated slope (Ea/R), the amount of activation energy (Ea) was determined for each nanoemulsion.

Kinetic stability
The absorbance changes of nanoemulsions were recorded during storage time. Then, the recorded data were fitted to the zero (Equation 1), first (Equation 2), and second (Equation 3) order reaction, and one of them was selected based on the highest R 2 and the lowest sum of squared errors (SSE), where c is the absorbance, t is the time, c 0 is the initial absorbance, and k is the rate constant (Heldman & Lund, 2007 were mixed with KBr (50 mg) to produce the pellet. The analysis was performed within the spectral region of 4000-500 cm −1 with 64 scans recorded at a 4 cm −1 resolution.

| Statistical analysis
All of the experiments were carried out in triplicate at least for mean and standard deviation calculation. Duncan's multiple range tests were used for mean treatments comparison.

| Droplet size and Polydispersity Index (PDI)
The results show that the droplet size is below 100 nm for all nanoemulsions and this may be due to equal content of surfactant and GEO so that the surfactant molecules were able to surround the encapsulated material.
Statistical analysis showed that the droplet size of nanoemulsions was significantly affected by O/E ratios in nanoemulsions formulation (p < .05). As shown in Table 1, by increasing the proportion EE( % ) = 1 − the total area under the peak of the emulsions headspace the total area under the peak of not emulsified mixture headspace × 100. (1) of O/E ratios in nanoemulsion formulations, the droplet size has also increased.
The increase in droplet size with the increase of GEO concentration could be attributed to the following mechanisms: (1) the increase in the number of GEO droplets can increase the probability of droplet collisions which, in turn, enhances flocculation and coalescence phenomena.
(2) Increase in GEO concentration can decrease homogenization efficiency for disruption of oil droplets.
Analysis of variance of data related to polydispersity index shows that except for nanoemulsion containing 25% garlic essential oil, there is no significant difference between the other samples. The polydispersity index for all samples is below 0.4, which is generally in the appropriate range, indicating a uniform particle size.
Maybe it can be noted that there was an equal content of es-

| Encapsulation efficiency
As mentioned in the 2.2.4 section, encapsulation efficiency was determined by comparing the total area under the peaks of the nanoemulsions and not emulsified mixture headspace ( Figure 1). Based on the obtained results, it was clear that O/E ratios were significantly affected encapsulation efficiency (p < .05). The encapsulation efficiency was decreased significantly from 92% to 77% with increasing O/E ratios from 10% to 25% ( Table 1) which shows that surfactant molecules were not well able to surround the oil molecules in the higher O/E ratios and especially in the 25%. The present study results are similar to Mazloom and Farhadyar (2014), which reported that encapsulation efficiency was decreased by raising blueberry essential oil content in nanoemulsion formulation. Also, Rachmadi et al. (2015) encapsulated silymarin and curcumin by ultrasonic emulsification method and they reported that the encapsulation efficiency decreased with increasing oil phase percent in nanoemulsion formulation so that increasing the content of silymarin and curcumin from 5 to 35 mg in oil phase (10% w/w) decreased the encapsulation efficiency from 99% to 75% for curcumin and 80% for silymarin. Reduction of turbidity of nanoemulsions during storage time at ambient temperature may be due to droplet coalescence and/ or Ostwald ripening processes and consequently instability of nanoemulsions. It seems that accelerating the instability of nanoemulsions by decreasing pH value is due to reducing the electrostatic repulsion between the oil phase droplets encircled by a surfactant (McClements, 2005). In this situation, the attractive colloidal interactions (van der Waals) overcome the repulsive interactions (steric repulsion generated by hydrophilic head groups of surfactant and electrostatic repulsion) and it leads to droplet coalescence and/or Ostwald ripening processes of the dispersed phase (Rao & McClements, 2013  β-carotene and declared that color fading due to degradation of β-carotene was accelerated by raising the temperature from 5 to 55°C. Also, Rao & McClements (2012) reported that microemulsions produced by lemon oil and sucrose monopalmitate formed gel when they were stored at 5°C, were relatively stable at 25°C, while growth, aggregation, and precipitation of droplets were observed at 45°C. In addition, they declared that produced nanoemulsions stored at 5°C and 25°C were relatively stable, but

F I G U R E 2
The effect of storage temperature on changes in turbidity of nanoemulsions in various formulations. Nanoemulsion containing 5% GEO (a), Nanoemulsion containing 15% GEO (b), Nanoemulsion containing 25% GEO (c) nanoemulsions stored at 45°C involved aggregations and precipitation of droplets.

| Kinetic stability of nanoemulsions
All of the studied factors (storage temperature, GEO percent, ionic strength, and pH value) significantly affected the changes in turbidity and instability process of nanoemulsions during storage time; therefore, more understanding on kinetic behavior instability of nanoemulsions under influence of studied factors can be considerable.
The recorded data of turbidity versus storage time were fitted with zero-, first-, and second-order models and kinetic order, and constant rate of instability process of nanoemulsions, associated with their coefficient of determination, was calculated.
Results obtained from fitting the recorded data of changes in turbidity of nanoemulsions versus storage time with kinetic order models showed that although changes in turbidity of nanoemulsions (instability mechanism of nanoemulsions) followed first and second order in some states, in general, in many cases, the recorded data were fitted with zero order in higher coefficient of determination. It is necessary to note that the negative value of the constant rate of nanoemulsions is indicative of a decline in turbidity of nanoemulsions during storage time, and it can be stated that the instability trend of nanoemulsions followed zero order at ambient temperature. As shown in Table 1, by comparing the obtained constant rates, it can be found that, generally, the constant rate values of changes in turbidity of changes in turbidity of nanoemulsions was increased during the storage time ( Table 2). By comparing the obtained constant rates and kinetic orders for the three mentioned factors (in studied ranges), it can be understood that the pH values factor (especially at pH 3) more affected the instability trend of nanoemulsions compared to the other two factors (storage temperature and ionic strength).

| Kinetic parameters for stability tests
As mentioned before, the storage temperature is another factor that affected the trend of the kinetic change of turbidity of nanoe- In this equation, ṁ is the mass flow rate (kg s −1 ) of each ingredient, C is the concentration of each ingredient (kg), D is the diffusion coefficient of each ingredient (m 2 s −1 ), A is the area (m 2 ), and x is the thickness (m) (Cussler, 2009 (Figure 4).

mulsions. As demonstrated in
Also, as shown in Figure 4, the volatile component release signal changed from 20,000 pA at first storage days to 140,000 pA (for nanoemulsions with 10% garlic essential oil), to 180,000 pA (for nanoemulsions with 15% GEO), and to 200,000 pA (for nanoemulsions with 25% garlic essential oil) at last storage day by increasing temperature from 24°C to 50°C. By comparing these changes, it can be found that the temperature effect on the release of the volatile components was more significant at a higher O/E ratio, and as it was discussed previously, this issue approved a typical synergetic relation between studied factors (temperature and O/E ratio). In general, intensifying release and migration of GEO volatile components from inner phase to nanoemulsions headspace by raising temperature can be due to several reasons; the first reason is Where NA is the Avogadro number, T is the temperature, and η is the viscosity of the solution or is the solute radius. Einstein assumed that the solute radius is larger than the solvent radius (Sharma & Yashonath, 2007). According to the above equation, temperature and diffusion coefficient have a direct relation and the relation between viscosity and diffusion coefficient is opposite. Also, it is necessary to clarify that the diffusion coefficient of droplets in liquids has a direct relation with volatile components migration from the inner phase to the outer phase of nanoemulsions.

| Release kinetics of volatile components
By fitting the obtained experimental data from gas chromatography to different kinetic order models (zero, first, and second), it was clear that the release kinetics of entrapped volatile components in the inner phase of nanoemulsions containing GEO followed zero order in different O/E ratios and temperatures (Table 4). Although experimental data were fitted with a first-order model with higher R 2 in higher O/E ratio (25%) and temperature (37°C and 50°C), generally, the obtained results indicated that release kinetic of volatile components from inner phase to outer phase of nanoemulsions followed zero order, and the release rate of volatile components was independent of the concentration. This may occur in two different situations: (a) when intrinsically the release rate is independent of the concentration of component and (b) when the concentration of the compound is so large that the overall release rate appears to be independent of its concentration (Heldman et al., 2006).

| FT-IR analysis
FT-IR spectroscopy has been used for the characterization of the samples because of the frequency at which a characteristic group of essential oil compounds is modified by surfactants interactions.
Moreover, this technique was used as a tool to investigate the interaction between essential oil, water, and surfactants by measuring the shown in Figure 5.  and 25% GEO) spectra and they have a similar spectrum, and the peak intensity in some regions is the only difference between them, which indicates ingredients change in their formulation. As shown in Figure 5c,d, the peak intensity of aromatic (-CH) vibrations was increased by increasing the GEO percentage in the nanoemulsion formulation. Also, where the GEO percent was lower, the water content would be high, which is verified by (-OH) group absorbance peak in the region of 3450 cm −1 . Moreover, the absorbance peaks of S = S and C-S groups and diallyl disulfide which are related to GEO components have more intensity in 25% nanoemulsions compared to the 5% nanoemulsions. new outlook to study more on the release phenomena of nanocarrier and connected kinetic parameters and to more focus on the gas chromatography method for the evaluation of encapsulation efficiency and release content of volatile components as a precise method.

ACK N OWLED G EM ENT
The authors are very grateful and appreciative of the Food Analysis Laboratory of the Urmia University, which technically and financially supported this study.

CO N FLI C T O F I NTE R E S T
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.