Optimization of the Stability of Nano-emulsion Medium Chain Triglycerides (MCT) using α-Cyclodextrin

This study aims to determine the stability of nano-emulsion synthesized from virgin coconut oil (VCO) using α-cyclodextrin


INTRODUCTION
Indonesia is the largest coconut-producing country in the world, with a substantial 31.2% share of the global plantation area of the crop, which is equivalent to 3.86 million ha (Suratinojo, 2014).According to data obtained from the Directorate General of Plantations, Ministry of Agriculture, Indonesia in 2019, several provinces contributed to the large production volume in the country, including Riau, North Celebes, East Java, and Southeast Celebes (DGEC, 2021).Indonesia's vast expanse of coconut plantations contributes significantly to an annual yield of approximately 18.3 million tons (Mesu and Fangohoi, 2018).However, this is not in accordance with the sales of processed coconut products, such as virgin coconut oil (VCO).This is primarily due to the limited awareness among Indonesians concerning the numerous benefits of VCO, leading to low purchase interest.
In comparison to other vegetable oils, virgin coconut oil is unique due to its distinct properties as it contains approximately 44-52% medium chain triglycerides (MCT) (Ardianto and Mutiah, 2018).This sets it apart from the long-chain triglycerides (LCT) that are commonly found in vegetable oils.Several studies have shown that MCTs possess a smaller molecular size, lower melting point, and reduced energy content (8.4 compared to 9.2 kcal/g) (Silalahi, 2020).Consequently, MCTS are readily absorbed and digested by the body, leading to their rapid usage as an energy source, without being stored as body fat (Arpi, 2013).These triglycerides also exhibit remarkable stability compared to LCTs, making them an ideal choice for making nanoemulsions (Fitriani et al., 2016).
Nano-emulsion is an imbalance system that cannot be formed spontaneously, thereby necessitating a surfactant to lower the interfacial tension and facilitate the formation of new drops or droplet emulsion (Wilson et al., 2022).One of the surfactants that is often used in the production of nano-emulsions is tween 80.This is primarily due to its non-toxic properties and nonirritating attributes on the skin (Cheng et al., 2017).The natural surfactant commonly used in the manufacture of nano-emulsions is soy lecithin.In the manufacture of emulsions, combination of surfactants is recommended due to its ability to produce a more stable emulsion.The stability of the product can be further enhanced through the addition of a stabilizer, such as α-cyclodextrin (Sliwa and Girek, 2017).Furthermore, α-cyclodextrin is a nonreducing cyclic saccharide, consisting of 6 glucose units linked through 1-4-glycosidic bonds, which are produced by cyclodextrin glucopyranosyl transferase (Nopiasari et al., 2019).
Cyclodextrins are categorized into three types, namely α, β, and γ.The α-cyclodextrin variant, distinguished by its comparatively smaller size in relation to the β and γ types, exhibits a unique capacity to bind higher amounts of fat in serum (de Miranda et al., 2011, Nopiasari et al., 2019).According to a previous study, its resistance to fat hydrolysis plays a role in preventing damage to the nano-emulsion (Li et al., 2014).Despite its advantageous properties, the use of α-cyclodextrin in the production of nano-emulsions remains relatively uncommon.Therefore, this study aims to optimize the stability of nano-emulsion using α-cyclodextrin as a co-surfactant.The results are expected to serve as the basis for further studies on the production of nano-emulsions using cyclodextrins.

Materials
The materials used in this study included medium chain triglyceride (MCT) in VCO, α-cyclodextrin, tween 80, lecithin, and distilled water.Furthermore, VCO containing MCT was purchased from a marketplace in CV Ajisaka Kelapa, Malang, Indonesia.α-Cyclodextrin was purchased from Cyclochem Co. Ltd., Japan, Tween 80 was acquired from Kimia Indrasari Semarang, and Lecithin was obtained at Multi Kimia Raya Nusantara.

Nano-emulsion Production
The nano-emulsion was prepared by dissolving alpha-cyclodextrin with a magnetic stirrer at a speed of 200 rpm for 10 minutes.The alpha-cyclodextrin solution was then homogenized with a surfactant and MCT oil using an ultra turrax homogenizer (IKA T10 basic Ultra Turrax, China) at a speed of 18,000 rpm for 3 minutes.

Density Analysis
Density analysis was measured using a pycnometer and calculated based on Equation (1).

Emulsion Type and particle size Analysis
The type of the emulsion was determined by placing the nano-emulsion on an object glass, and adding the methylene blue.Particle size analysis was carried out to determine the type of emulsion based on its droplet size.It was also used to evaluate stability by observing changes in droplet size after being stored in varying temperatures (4 °C and room temperature).The particle size was measured using Horiba Partica LA-960, Japan.The emulsion is classified as nano, micro, and emulsion for the particle size of 1-100 nm, 100-400 nm, and ≥400 nm (Souto et al., 2022).

Zeta Potential Analysis
Zeta potential analysis was carried out to determine the stability of nano-emulsion after being stored at varying temperatures.Emulsions with high zeta potential tended to be more stable compared to those with lower levels.The accumulation of coagulate or flocculate could occur in samples with low zeta potential, leading to poor stability (Lu and Gao, 2010).The zeta potential was measured using the Horiba nano partica SZ-100, Japan.

Density Analysis from Nano-emulsion
The results of the density and viscosity analysis of the nano-emulsions is presented in Table 2. Table 2 showed the density and viscosity of nano-emulsion made from VCO, which was analyzed for 3 weeks with a weekly testing time after storage at 4 °C.The nano-emulsions were analyzed using 2 different types of emulsifiers.Furthermore, soy lecithin and tween Where is, ρ = density (g/mL); m2 = weight of the filled pycnometer (g); m1 = weight of empty pycnometer (g); and V = pycnometer volume (mL).

Emulsion Type and particle size Analysis
The type of the emulsion was determined by placing the nano-emulsion on an object glass, and adding the methylene blue.Particle size analysis was carried out to determine the type of emulsion based on its droplet size.It was also used to evaluate stability by observing changes in droplet size after being stored in varying temperatures (4 C and room temperature).The particle size was measured using Horiba Partica LA-960, Japan.The emulsion is classified as nano, micro, and emulsion for the particle size of 1-100 nm, 100-400 nm, and ≥400 nm (Souto et al., 2022).

Zeta Potential Analysis
Zeta potential analysis was carried out to determine the stability of nano-emulsion after being stored at varying temperatures.Emulsions with high zeta potential tended to be more stable compared to those with lower levels.The accumulation of coagulate or flocculate could occur in samples with low zeta potential, leading to poor stability (Lu and Gao, 2010).The zeta potential was measured using the Horiba nano partica SZ-100, Japan.

Density Analysis from Nano-emulsion
The results of the density and viscosity analysis of the nano-emulsions is presented in Table 2. Table 2 showed the density and viscosity of nano-emulsion made from VCO, which was analyzed for 3 weeks with a weekly testing time after storage at 4 °C.The nano-emulsions were analyzed using 2 different types of emulsifiers.Furthermore, soy lecithin and tween 80 served as emulsifiers in formulas 1-5 and 6-10, respectively.The nano-emulsion with soy lecithin and tween 80 produced a density range of 1.192-1.254g/mL and 1.178-1.233g/mL, respectively.80 served as emulsifiers in formulas 1-5 and 6-10, respectively.The nano-emulsion with soy lecithin and tween 80 produced a density range of 1.192-1.254g/ mL and 1.178-1.233g/mL, respectively.The results showed that there was no significant change in the density of each formula.However, the density obtained after storage at 4 °C increased due to the low storage temperature, leading to a decrease in the distance between the particles.At the same volume, the number of particles increased along with the density of the nano-emulsion (Wildan, 2013).

Emulsion Type Test
The emulsion type test was used to determine whether the nano-emulsion produced was o/w or w/o.The determination of the type of emulsion was shown by the distribution of methylene blue.The o/w emulsion is characterized by evenly spread of methylene blue in the nano emulsion.On the other hand, the w/o type of emulsion is described by the formation of blue spots (Rahmawanty and Sari, 2021).The results of the emulsion type test are presented in Figure 2. The result showed that addition of methylene blue to the nano-emulsion resulted in evenly dispersed of nano emulsion, indicating that the emulsion was the oil in water (o/w) type.This was because the emulsifiers in the form of tween 80 and soy lecithin had a high HLB value of 15 and 8, respectively.Emulsifiers with high (hydrophilic) and low HLB were likely to produce o/w and w/o emulsion type, respectively.Furthermore, the value required to form an o/w emulsion was 8-16 and w/o was 4-6 (Syahputri & Patricia, 2019).Soy lecithin was an amphoteric surfactant, with both negative and positive charges (Shamsuri & Jamil, 2020).The results of this study obtained 10 nano-emulsion formulas of the o/w type.

ANALYSIS OF THE EFFECT OF INDEPENDENT VARIABLES ON PH USING THE RESPONSE SURFACE METHOD (RSM)
In this study, the analysis was carried out using RSM to determine the effect of independent variables in the form of solid content and the ratio of MCT-wall to pH on the MCT nano-emulsion produced.It was also performed to achieve optimization, leading to the production of the best response.In this case, the wall was a combination of surfactants, namely tween 80 or lecithin, and alpha-cyclodextrin.The pH results from the 10 formulas are presented in Table 3.The equations obtained are Y = 4.4952380952381 + 0.027380952380951 X1 -0.00035714285714285 X1X1 + 0.52857142857141 X2 -0.14285714285714 X2X2 + 0.000000000000000399 X1X2 From the equation, X1 is the concentration of SC, X2 is the ratio, and Y is the pH.The equation stated that the pH response increased and was directly proportional to the SC concentration, ratio, and the interaction between SC concentration and ratio, as indicated by a positive constant value.Meanwhile, the pH response decreased along with increasing interaction between SC concentrations, as well as ratios, as indicated by a negative constant value.The results showed that there was no significant change in the density of each formula.However, the density obtained after storage at 4 °C increased due to the low storage temperature, leading to a decrease in the distance between the particles.At the same volume, the number of particles increased along with the density of the nano-emulsion (Wildan, 2013).

Emulsion Type Test
The emulsion type test was used to determine whether the nano-emulsion produced was o/w or w/o.The determination of the type of emulsion was shown by the distribution of methylene blue.The o/w emulsion is characterized by evenly spread of methylene blue in the nano emulsion.On the other hand, the w/o type of emulsion is described by the formation of blue spots (Rahmawanty and Sari, 2021).The results of the emulsion type test are presented in Figure 2. The result showed that addition of methylene blue to the nano-emulsion resulted in evenly dispersed of nano emulsion, indicating that the emulsion was the oil in water (o/w) type.This was because the emulsifiers in the form of tween 80 and soy lecithin had a high HLB value of 15 and 8, respectively.Emulsifiers with high (hydrophilic) and low HLB were likely to produce o/w and w/o emulsion type, respectively.Furthermore, the value required to form an o/w emulsion was 8-16 and w/o was 4-6 (Syahputri & Patricia, 2019).Soy lecithin was an amphoteric surfactant, with both negative and positive charges (Shamsuri & Jamil, 2020).The results of this study obtained 10 nano-emulsion formulas of the o/w type.

Analysis of the Effect of Independent Variables on pH using the Response Surface Method (RSM)
In this study, the analysis was carried out using RSM to determine the effect of independent variables in the form of solid content and the ratio of MCT-wall to pH on the MCT nano-emulsion produced.It was also performed to achieve optimization, leading to the production of the best response.In this case, the wall was a combination of surfactants, namely tween 80 or lecithin, and alpha-cyclodextrin.The pH results from the 10 formulas are presented in Table 3.The accuracy of the analysis could be seen from the value of the coefficient of determination (R2).The R2 represented the level of influence the independent variable had on the dependent variable, and the value often ranged from 0-1.Values closer to 1 indicated that the model could be considered good at predicting the response.In this analysis, the R2 value was 0.79, From the equation, X1 is the concentration of SC, X2 is the ratio, and Y is the pH.The equation state that the pH response increased and was directly proportional to the SC concentration, ratio, and th interaction between SC concentration and ratio, as indicated by a positive constant value.Meanwhile the pH response decreased along with increasing interaction between SC concentrations, as well a ratios, as indicated by a negative constant value.

Figure 3. Fitted response surface variable solid content and MCT-wall ratio to pH nano-emulsion
The accuracy of the analysis could be seen from the value of the coefficient of determinatio (R2).The R2 represented the level of influence the independent variable had on the dependent variable and the value often ranged from 0-1.Values closer to 1 indicated that the model could be considere good at predicting the response.In this analysis, the R2 value was 0.79, indicating that 79% of th independent variables affected the dependent variable.The other 21% were influenced by other factors which were not included in this study (Nduru et al., 2014).The value of R2 obtained is presented i Table 4.The ratio variable obtained a p-value of 0. indicating that it had no significant effect on the pH o the Pareto diagram that was obtained.In a case whe (α=0.05), it could be concluded that the ratio did no emulsion.The Pareto diagram obtained is presented  indicating that 79% of the independent variables affected the dependent variable.The other 21% were influenced by other factors, which were not included in this study (Nduru et al., 2014).The value of R2 obtained is presented in Table 4.
The next analysis involved evaluating the diagnostic plot (graph plot), which aimed to provide information on whether the model (response surface method) gave poor results, as shown in Figure 4.In Figure 3, the plot data points were distributed around the diagonal line and it could be concluded that the data were normally distributed.This indicated that the model could identify the effect of solid content and ratio on the pH of the nano-emulsion.Furthermore, the analysis of variance was carried out and the results are presented in Table 5.The results obtained were in the form of the P-value of the solid content variable (SC), namely 0.033 < α=0.05.Based on these findings, SC had a significant effect on the pH of the nano-emulsion.
The ratio variable obtained a p-value of 0.12 and was greater than the value of α=0.05, indicating that it had no significant effect on the pH of the nanoemulsion.This could also be proven in the Pareto diagram that was obtained.In a case where the ratio value did not cross the reference line (α=0.05), it could be concluded that the ratio did not have a significant effect on the pH of the nano-emulsion.The Pareto diagram obtained is presented in Figure 5.
Based on Figure 5, SC had the highest level of influence on the pH of the nano-emulsion.SC or solid content consisted of materials other than water, such as VCO, tween 80 or lecithin, and alpha-cyclodextrin.According to Mu'awanah et al., the amount of VCO used could affect the pH of the nano-emulsion produced.This was because the VCO contained fatty acids that could affect the pH of the product.An increase in the VCO concentration was expected to cause an increment in the acidity of the nano-emulsion (Mu'awanah et al., 2014).The surfactants used in this study were lecithin and tween 80 with HLB of 8 and 15 respectively.The lower the value, the higher the lipophilicity, thereby leading to lower pH.This could be proven based on the results obtained, where samples with lecithin produced nano-emulsions with a pH range of 5.2-5.5, while those with tween 80 ranged from 5.4-5.5.The optimum yield parameters of the nanoemulsion pH were obtained with variations of SC and MCT-wall ratio based on the critical value.Therefore, the critical value for optimizing the pH of the nano-emulsion was reached at an SC concentration of 38.33333% with Based on Figure 5, SC had the highest level of influence on the pH of the nano-emulsion.SC or solid content consisted of materials other than water, such as VCO, tween 80 or lecithin, and alphacyclodextrin.According to Mu'awanah et al., the amount of VCO used could affect the pH of the nanoemulsion produced.This was because the VCO contained fatty acids that could affect the pH of the product.An increase in the VCO concentration was expected to cause an increment in the acidity of the nano-emulsion (Mu'awanah et al., 2014).The surfactants used in this study were lecithin and tween 80 with HLB of 8 and 15 respectively.The lower the HLB value, the higher the lipophilicity, thereby leading to lower pH.This could be proven based on the results obtained, where samples with lecithin produced nano-emulsions with a pH range of 5.2-5.5, while those with tween 80 ranged from 5.4-5.5.The optimum yield parameters of the nano-emulsion pH were obtained with variations of SC and MCT-wall ratio based on the critical value.Therefore, the critical value for optimizing the pH of the nano-emulsion was reached at an SC concentration of 38.33333% with an MCT-wall ratio of 1.85000, which had a predicted pH value of 5.508968.When viewed from the predicted pH value obtained, it could be concluded that the best nano-emulsion formulas produced were 2, 6, and 9 with tween 80 or lecithin surfactant.

Particle Size and Zeta Potential Analysis
Particle size and zeta potential analysis was conducted after storage at 4 °C and room temperature for seven days, respectively, and the results are presented in Figure 6.  5, SC had the highest level of influence on the pH of the nano-emulsion.SC or solid content consisted of materials other than water, such as VCO, tween 80 or lecithin, and alphacyclodextrin.According to Mu'awanah et al., the amount of VCO used could affect the pH of the nanoemulsion produced.This was because the VCO contained fatty acids that could affect the pH of the product.An increase in the VCO concentration was expected to cause an increment in the acidity of the nano-emulsion (Mu'awanah et al., 2014).The surfactants used in this study were lecithin and tween 80 with HLB of 8 and 15 respectively.The lower the HLB value, the higher the lipophilicity, thereby leading to lower pH.This could be proven based on the results obtained, where samples with lecithin produced nano-emulsions with a pH range of 5.2-5.5, while those with tween 80 ranged from 5.4-5.5.The optimum yield parameters of the nano-emulsion pH were obtained with variations of SC and MCT-wall ratio based on the critical value.Therefore, the critical value for optimizing the pH of the nano-emulsion was reached at an SC concentration of 38.33333% with an MCT-wall ratio of 1.85000, which had a predicted pH value of 5.508968.When viewed from the predicted pH value obtained, it could be concluded that the best nano-emulsion formulas produced were 2, 6, and 9 with tween 80 or lecithin surfactant.

Particle Size and Zeta Potential Analysis
Particle size and zeta potential analysis was conducted after storage at 4 °C and room temperature for seven days, respectively, and the results are presented in Figure 6. an MCT-wall ratio of 1.85000, which had a predicted pH value of 5.508968.When viewed from the predicted pH value obtained, it could be concluded that the best nano-emulsion formulas produced were 2, 6, and 9 with tween 80 or lecithin surfactant.

Particle Size and Zeta Potential Analysis
Particle size and zeta potential analysis was conducted after storage at 4 °C and room temperature for seven days, respectively, and the results are presented in Figure 6.
Figure 6 (a) illustrated the progression of particle size for formulas 1, 2, 3, and 6 after being stored for 14 days.The particle size values decreased in formulas 1 and 2, and there were no significant changes in 3 and 6.Based on the results, formula 6 had the smallest particle size of 5.495 µm.Figure 6 (b) showed that all formulas had good stability with zeta potential values of more than ±30 mV.The highest zeta potential was obtained in formula 3 (-94,233 to -104,433 mV) with the highest amount of surfactant, which was adequate for making it stable (Eid et al., 2013).The negative zeta potential in the results showed that the emulsion had an electrode potential at zero.This was because there was no charge and flocculation in the globules, indicating that the emulsion system was stable.A similar trend was observed in a study by Östbring et al. (2021), who reported that the lowest zeta potential was found at the highest emulsion pH investigation.

CONCLUSION
In conclusion, the current study showed that 10 formulas of nano-emulsion were o/w type emulsion and had a density range of 1.178-1.254g/mL.The optimization of pH using RSM analysis revealed a value of 5.5, which was obtained in samples 2, 6, and 9 with tween 80 or lecithin as a surfactant.Based on the particle size analysis, formula 6 had the smallest particle size of 5.495 µm, while formula 3 had the highest zeta potential (-94,233 to -104,433 mV).The findings of this study showed that formula 6 had the best characteristics, with an optimum pH of 5.5, small particle size, and good stability, as indicated by the zeta potential value (-45.500 to -89.567 mV).

Figure 1 .
Figure 1.The result of GC MS for identification of triglycerides content in virgin coconut oil

Figure 2 .
Figure 2. The result of the emulsion type test

Figure 3 .
Figure 3. Fitted response surface variable solid content and MCT-wall ratio to pH nano-emulsion

Figure 4 .
Figure 4. Comparison of experimental data and estimated nano-emulsion pH

Figure 5 .
Figure 5. Pareto diagram from the standard effect of independent variables to the dependent variable

Figure 6 .Figure 5 .
Figure 6.The result of analysis after being stored for 14 days (a) particle size values (b) zeta potential

Figure 6 .
Figure 6.The result of analysis after being stored for 14 days (a) particle size values (b) zeta potential values Figure 6.The result of analysis after being stored for 14 days (a) particle size values (b) zeta potential values

Table 1 .
The composition of nano-emulsion medium chain triglycerides (MCT)

Table 2 .
The result of density and viscosity of nanoemulsions

Table 3 .
The result of pH nano-emulsion

Table 4
out and the results are presented in Table5.The re the solid content variable (SC), namely 0.033 < α=0 effect on the pH of the nano-emulsion.Figure 4. Comparison of experimental data and estim

Table 5 .
Analysis of variants of response surface regr

Table 4 .
Estimation effect data

Table 6 .
Prediction value of optimum pH in SC and ratio critical value

Table 6 .
Prediction value of optimum pH in SC and ratio critical value

Table 6 .
Prediction value of optimum pH in SC and ratio critical value