Stability and Kinetic Study of Vitamin C containing Hydrogenated and Middle-Chain Triglyceride Coconut Oil-Based Double Emulsion

Vitamin C or ascorbic acid, is an organic compound that is highly required by human since it acts as antioxidant, help increase metabolism, and improves the immune system. Vitamin C is easily oxidized and damaged during storage due to several external factors such as light, metal, shear, etc. The encapsulation technique is able to improve the stability as well as the activity of Vitamin C in order to protect the bioactive compound from deleterious external factors. Coconut oil which is composed of about 50% lauric acid, has an antiviral property. This research aimed to obtain the stable water-in-oil-in-water (W₁/O/W₂) double emulsion using coconut oil upon the incorporation of Vitamin C.  Hydrogenated (HCNO) and Medium-Chain Triglycerides (MCT) coconut oil was used as the oil phase, and various emulsifiers such as Tween-20 0.5%, Tween-20 1%, and Tween-20 0.5%/ PgPr 0.5% were applied to strengthen the outer interfacial layer. The double emulsion stability was monitored macroscopically, and the destabilization kinetics was studied using the zero and first-order kinetic models. It turned out that the HCNO-based double emulsion demonstrated higher stability compared to the MCT-based double emulsion. The lowest destabilization rate constants of 4.5 x 10^-3 h^-1 and 6.8 x 10^-3 h^-1 were obtained for HCNO and MCT-based double emulsions both stabilized with Tween-20 0.5%, respectively. The coconut oil-based double emulsion enriched with Vitamin C would be potentially developed for various functional food applications.

Double emulsion; HCNO; Kinetics; Tween-20; Vitamin C

There are several techniques to encapsulate Vitamin C, such as spray drying, spray cooling/ chilling, fluidized bed coating, liposomes, and extrusion (Comunian et al., 2022). Spray drying has been used commercially because it is continued in operation and readily scalable. Encapsulation of water-soluble materials derived from the extraction process containing a polysaccharide matrix in solution has been widely carried out using the spray drying technique (Matsunaga et al., 2014). Other techniques except for liposomes also, were also applied to ascorbic acid in water-based solution. However, research has shown that ascorbic acid has a longer shelf-life when encapsulated within a lipophilic matrix, such as liposomes or in the form of an emulsion (Comunian et al., 2022). Additionally, the Vitamin C-containing emulsion can be spray dried to further improve its stability. Emulsion is a mixture containing at least two immiscible phases, mostly oil, and water, whereas one phase is dispersed in another phase in the form of small droplets (McClements, 2016). Double emulsion is a more complex emulsion system whereby two interfacial layers exist in the system, such as oil-in-water-in-oil (O/W/O) or water-in-oil-in-water (W/O/W). Both aqueous and oil dispersed phases of double emulsions could be potentially used as the encapsulation vehicle of water based and oil based bioactive materials. The presence of surfactants or emulsifiers was crucial in order to obtain stable emulsions. Emulsifiers are active ingredients that are responsible for reducing the surface tension between the immiscible phases, whereby the need of renewable and environmentally friendly emulsifiers to replace synthetic ones has been soaring (Qadariyah et al., 2022).

Coconut oil has been recognized as one of the healthiest oil despite its high saturated fatty acids, up to 90% (Lima and Block, 2019; Boateng et al., 2016). However, more than 50% of their fatty acid belongs to medium chain triglyceride (MCT), which is easily absorbed and digested in the body. MCT consists of caproic acid, caprylic acid, and capric acid. Since this oil is not accumulated in the body, it can be readily used as a source of energy (Boateng et al., 2016). It also possesses good oxidative stability and can extend the shelf life of end products. On the other hand, hydrogenated coconut oil (HCNO) primarily consists of long-chain saturated fatty acids resulting from the hydrogenation process. HCNO oil is highly stable and can also prolong the shelf life of products while maintaining their solid texture, even at high temperature.

Double emulsion has been attractive due to the increasing demand for nutritious food. It has been fascinating for the food industry due to its capability to encapsulate compounds, fabricate polymersomes, and act as fat replacers or sweetness enhancers in different foods (Loffredi and Alamprese, 2024; Kumar et al., 2022; Mudric et al., 2019). Recent studies demonstrated the use of W₁/O/W₂ double emulsion as a vehicle for the co-delivery of both hydrophilic and hydrophobic bioactive compounds such as curcumin/ catechin (Aditya et al., 2015), insulin/ quercetin (Han et al., 2022), and ascorbic acid/ tocopherol (Khan et al., 2023). Tania and Kuswahyuning (2020) investigated the stability of paraffin-based W/O/W double emulsion whereby Span-80 and sodium carboxymethyl cellulose were varied to strengthen the inner and outer interfacial layers. Ying et al. (2021) studied the encapsulation of soy peptide in W/O/W stabilized with PgPr and octenyl succinic anhydride (OSA) starch/ maltodextrin as lipophilic and hydrophilic emulsifiers, respectively. Sapei et al. (2022a; 2022b; 2018) improved the stability of W₁/O/W₂ double emulsion using rice husk biosilica and chitosan-modified rice husk ash on the outer interfacial layer. Molet-Rodriguez, Martín-Belloso, and Salvia-Trujillo (2021) used lipid gelling agent along with PgPr and sodium alginate/ Tween 80 to increase the stability of W/O/W emulsion. Chevalier, Gomes, and Cunha (2022) investigated different hydrophilic emulsifiers such as sodium caseinate, whey protein isolate, and Tween-80 on the stability of W₁/O/W₂ double emulsion. Sodium caseinate produced the most stable emulsion by favoring the formation of small droplets. Su et al. (2022) studied the encapsulation of amino acids in W₁/O/W₂ double emulsion stabilized with PgPr and gum arabic/ xanthan gum as lipophilic and hydrophilic emulsifiers. Snoussi et al. (2020) investigated the encapsulation of catechin in W₁/O/W₂ double emulsion with the addition of chitosan/ sodium caseinate and lactose/ sodium caseinate in W₁ and W₂, respectively.  Encapsulation of Vitamin C using W/O/W double emulsions as vehicles has been attempted by several investigations (Sapei et al., 2023; Khan et al., 2023; Dai et al., 2022; Hu et al., 2022; Kheynoor et al., 2022; Fraj et al., 2019). PgPr was extensively used as lipophilic emulsifier to form a highly stable W/O emulsion, whereas protein such as whey protein isolate, sodium caseinate, and soluble protein was mainly added in the outer continuous phase to stabilize the outer interfacial layer of W/O/W double emulsions containing Vitamin C (Khan et al., 2023; Dai et al., 2022; Hu et al., 2022; Fraj et al., 2019). Moreover, some previous investigations combined non-ionic emulsifiers such as PgPr and Tween to strengthen the inner and outer W/O/W interfacial layers, respectively (Sapei et al., 2023; Kheynoor et al., 2022). The use of non-ionic emulsifiers is preferred over proteins in food applications, as proteins are inherently complex and their behavior is highly sensitive to pH and ionic strength due to the presence of both positive and negative charges in their structure.

        However, there has been no publication yet investigating the effect of different concentration of external emulsifiers using Tween-20 and combined Tween-20/ PgPr on the stability of coconut oil-based W/O/W double emulsion containing Vitamin C in liquid form. There is also no study related to the double emulsion stability with time and the evaluation of its destabilization kinetics. The kinetics data could be useful for predicting the emulsion stability behavior in the long term. In this research, the double emulsion stability prepared with two different coconut oil derivatives, namely HCNO and MCT were compared. The product would be of importance, innovative functional food which helps boost the immunity and maintain overall health.

2.1. Materials

      Hydrogenated coconut oil (HCNO) comprised of 0.5% caproic acid, 5% caprylic acid, 6% capric acid, 45% lauric acid, 20% myristic acid, 11% palmitic acid, 12% stearic acid, and trace amounts of oleic, linoleic, and linoleic acids; middle chain triglycerides (MCT) comprised of 60% caprylic acid (C8) and 40% capric acid (C10); Vitamin C/ ascorbic acid (Sigma-Aldrich, UK); Tween-20 (Merck, Germany), Polyglycerol Polyricinoleate/ PgPr 4120 (Palsgaard, Denmark), and demineralized water.

2.2. Preparation of primary emulsion/ water-in-oil (W₁/O) emulsion

        The internal aqueous phase (W₁) was prepared by adding 25% (w/w) of ascorbic acid into the remaining water and then mixed at 100 rpm for 3 minutes using a magnetic stirrer until homogeneous. The oil phase was prepared by adding 6% (w/w) PgPr into the oil phase. The mixture was stirred using a magnetic stirrer at 800 rpm for 7 minutes. Afterward, the aqueous phase (W₁) with a fraction of 30% (w/w) was dispersed in the oil phase and homogenized using a rotor-stator (IKA T25 digital ULTRATURRAX, Germany) at 20,000 rpm for 6 minutes. To prepare the primary emulsion using HCNO, the aqueous phase and oil phase were heated to an elevated temperature of 60°C. This was necessary because HCNO has a higher melting point compared to MCT oil.

2.3.  Preparation of double emulsion/ water-in-oil-in-water (W₁/O/W₂) emulsion

         The external aqueous phase (W₂) was prepared by mixing emulsifiers at certain amounts, as depicted in Table 1. The concentrations of emulsifiers were in % (w/w) relative to the outer aqueous phase (W₂). The emulsifiers were added to water and mixed using a magnetic stirrer at 300 rpm for 7 minutes. The fractions of primary emulsion (W₁/O) dispersed into the outer aqueous phase were 30% (w/w) and 40% (w/w) for HCNO and MCT oil, respectively. The mixtures were homogenized using a rotor-stator (IKA T25 digital ULTRATURRAX, Germany) at 8,000 rpm for 3 minutes until homogeneous. The resulting double emulsion was poured into the vial (inner diameter = 25 mm and height = 95 mm) at room temperature (~28-30^?C). The stability of each double emulsion was continuously monitored for up to 4 days.

Table 1 Variation of emulsifiers used for the secondary emulsion

2.4.  Determination of the stability of double emulsion and its destabilization kinetics
     The stability of the double emulsion was evaluated macroscopically by measuring the ratio of the emulsion height after a certain time to its initial height immediately after secondary emulsification. The monitoring was conducted until 4 days, whereby a significant phase separation between the emulsion and aqueous phase was distinctly seen. Liquid with a milky and homogeneous appearance demonstrated a stable emulsion without any emulsion instabilities such as flocculation, sedimentation, or creaming. The emulsion stability (%S) was calculated according to equation (1).

whereby h_t = the height of the double emulsion at a particular time and h₀ = the height of the initial double emulsion right after the preparation.

         Kinetic models of zero order and first order, according to equations (2) and (3), respectively, were used to study the destabilization kinetics of the double emulsion quantitatively. The coefficients of determination (R²) of both models were also determined. The experimental data used for the kinetic study was within the range of 0 to 10 hours and 0 to 24 hours for HCNO and MCT-based double emulsion, respectively.

Whereby S is the percentage of emulsion stability at a certain time (t) in hours; S₀ is the initial stability of the double emulsion in percentage; k₀ and k₁ are the destabilization rate constants derived from the zero-order (% stability/hour) and first-order model (per hour), respectively.

3.1.  Stability of double emulsion prepared using HCNO and MCT coconut oil

    All double emulsions prepared according to Table 1 were completely stable right after the preparation. However, the stability of double emulsions decreased with time, as seen in Figure 1. The double emulsions stability was monitored for up to 4 days. Interestingly, the double emulsion prepared with HCNO demonstrated a much-delayed destabilization at the beginning and tended to be leveled off after approximately 24 hours with much higher emulsion stability percentages. In contrast, the double emulsion prepared with MCT demonstrated a continuous decrease in stability with time. 

Figure 1 Stability of the double emulsions with time monitored until 4 days. (A) HCNO; (B) MCT

Furthermore, the macroscopic stability of HCNO and MCT-based double emulsion after 4-day storage can be seen in Figure 2. The percentages of double emulsion stability prepared using HCNO and MCT observed on days 0, 1, and 4 were depicted in Table 2 and Table 3, respectively. 

Figure 2 Macroscopic stability of double emulsions prepared using various emulsifiers’ types and concentrations after 4-day storage. A) HCNO; B) MCT

Table 2 Stability of double emulsions prepared using HCNO

Table 3 Stability of double emulsions prepared using MCT

        As seen in Figure 2, it was obvious that double emulsions were subjected to instability indicated by the separation of the aqueous phase from the double emulsion phase. The inner aqueous phase droplets dispersed in the oil globules could agglomerate and coalesce and finally diffuse into the external aqueous phase (Hu et al., 2022; Leister and Karbstein, 2020; Schuch et al., 2013).  This was strongly influenced by differences in osmotic pressure and Laplace pressure between the aqueous phases (Heidari et al., 2022). The increased volume of the external aqueous phase (W₂) would, in turn, trigger the phase separation between the aqueous phase and emulsion phase due to the difference in densities. The water-rich phase underwent sedimentation out from the whole emulsion due to its higher density. The partial detachment of the hydrophilic emulsifiers initially present at the interface between oil and outer aqueous phase into the outer aqueous phase with time could be another factor of this instability. This would lead to flocculation and, thus, coalescences of oil globules in order to minimize their surface tension. However, the creaming of primary emulsions was hardly seen in all double emulsions. Furthermore, the separation of the external aqueous phase was remarkably seen in double emulsions prepared using MCT compared to those prepared using HCNO, albeit a much lower W₁/O fraction was used in MCT-based double emulsion. The W₁/O fraction used for the MCT-based double emulsion was 40%, while it was only 30% when HCNO was used. The increase in the dispersed phase fraction would definitely increase the stability of double emulsions due to an increase in the overall viscosity. However, the MCT-based double emulsions still demonstrated much lower stability compared to the HCNO-based double emulsions. The fatty acid profiles of both oils did make this difference. HCNO is composed of entirely saturated fatty acids due to hydrogenation ranging from short to long fatty acid chains, whereas MCT is comprised of middle chain fatty acids of C₈ and C_10, imparting a much higher melting point to HCNO with a melting point of about 31 - 33^?C. On the other hand, the melting point of MCT is relatively low, about 5 - 7^?C. The HCNO oil tended to turn into gel or solid upon storage reducing the destabilization rate of the double emulsion. The formed crystal network reduced the permeability of the oil phase due to increased tortuosity of the diffusive path between inner and outer aqueous phases (Nelis et al., 2019).  The solidified HCNO also increases the viscosity of the dispersed phase, resulting in higher viscosity of the double emulsion and thereby increasing its stability. Vice versa, the dispersed MCT oil remained liquid upon storage and thus facilitating the diffusion of the inner aqueous phase into the outer aqueous phase. Besides that, the oil globules were prone to flocculation and coalescence, leading to a higher rate of double emulsion destabilization. 

           Based on the microscopic structures of double emulsions prepared using HCNO and MCT, as depicted in Figure 3, it was confirmed that the double emulsion prepared using HCNO seemed to be very thick in contrast to the liquid MCT-based double emulsion. The oil globule sizes in HCNO-based double emulsion seemed bigger than those in MCT-based double emulsion. This again confirmed the higher release rate of the inner aqueous phase into the outer aqueous phase reducing the MCT oil globule sizes and inferring higher instability of MCT-based double emulsion (Schuch et al., 2013). This instability could be dominated by the coalescence of inner droplets followed by the coalescence of both inner and outer aqueous phase (Leister and Karbstein, 2020). However, the double emulsion formation was still partially retained after 4 days storage demonstrated by the presence of inner aqueous phase droplets inside the oil globules.

Figure 3 Microscopic structure of double emulsions after 4 days storage. (A) HCNO; (B) MCT

         The double emulsion with HCNO demonstrated quite high stability of above 85% after 4 days, whereas the double emulsion with MCT retained about 70% stability after 4 days (Table 2 vs. Table 3). This was in line with the previous investigation whereby the stability of MCT-based W/O/W emulsions were much lower than those prepared using HCNO upon the addition of thickeners such as isomalto-oligosaccharides (IMO) and inulin (Sapei et al., 2023). The stability of these double emulsions prepared with HCNO was a bit higher compared to the stability of Pickering palm oil-based W/O/W emulsions stabilized with rice husk silica/ chitosan particles (Sapei et al., 2022b). However, the stability of Pickering palm oil-based W/O/W double emulsions stabilized using Tween 20/ rice husk silica demonstrated a much superior stability of more than 96% after 4 days (Sapei et al., 2018). Moreover, the double emulsion (H2) prepared using Tween-20 0.5% showed the highest stability. In the case of MCT-based emulsion, sample M1 which was prepared using mixed emulsifiers PgPr/ Tween-20 (0,5%/0,5%), showed the highest stability In order to strengthen the interfacial layer between the oil and external aqueous phase and suppress the desorption rate of emulsifiers from the interface, it is generally desirable to use emulsifier mixtures with low and high HLB values, such as PgPr (HLB = 4) and Tween-20 (HLB = 16.7). However, the use of a single emulsifier in HCNO-based double emulsion seemed to be more efficient. The use of Tween-20 only could be superior to the use of mixed PgPr/ Tween-20 due to the interaction or competition amongst the emulsifiers adsorbed at the inner interfacial and outer interfacial layers (Schuch et al., 2013). Previous investigation, on the contrary, showed the lowest stability on palm oil-based W/O/W emulsion stabilized with Tween-20 only, which demonstrated synergism when being combined with rice husk silica (Sapei et al., 2018). This implied a different mechanism between mixed polymeric emulsifiers and polymeric emulsifiers/ particles in strengthening the interfacial layers, thus stabilizing the entire emulsion. The polymeric emulsifier was easily adsorbed but easily desorbed from the interfaces, whereas particles could form a multilayer barrier once adsorbed at the interfaces (Zheng et al., 2022; Sapei et al., 2018). Creaming has been a common instability phenomenon of oil in water emulsion whereby oil-rich phase moves upward due to its lower density (Sapei et al., 2022b; McClements, 2016). In all resulting double emulsions, the emulsions part appeared on the upper layer, with an increasing portion of the aqueous layer at the bottom during the double emulsion destabilization with time. One of the major causes of the distinction of these two layers between the double emulsion layer and water-rich layer was the release of the inner aqueous phase into the outer aqueous phase inducing water-rich phase sedimentation. Furthermore, increasing Tween 20 concentrations up to 1% tended to deteriorate the emulsion stability. The excess hydrophilic emulsifier would be, in turn, dispersed in the outer aqueous phase instead of being adsorbed at the interfacial layer attracting the emulsifiers attached to the interfacial layers into the outer aqueous phase, thus promoting the destabilization of the double emulsion. Moreover, the excess of Tween 20 could possibly modify the antioxidant efficacy by improving the antioxidant oxidative stability (Yamamoto and Misawa, 2018) and modifying the crystallization as well as melting behavior of coconut oil (Maruyama et al., 2014).

3.2. Kinetics of double emulsion destabilization according to zero and first-order models

It has been pronounced that the stability of HCNO-based double emulsion was higher than the MCT-based double emulsion with time. The short-term W/O/W double emulsions stability would be furthermore quantitatively proved through the determination of destabilization rate constants according to zero and first-order kinetic models, as could be seen in Tables 4 and 5. The first-order kinetic model appeared to be more suitable in determining the destabilization rate constants compared to the zero-order based on the R² values. The lowest destabilization rate constant of about 4.5 x 10^-3 h^-1 was obtained for HCNO-based double emulsion stabilized with Tween-20 0.5% (emulsion H2). The highest destabilization rate was observed when mixed emulsifiers Tween 20 0.5%/ PgPr 0.5% were used for HCNO-based double emulsion (emulsion H1). The destabilization rate constant became double when Tween-20 used was doubled, inferring that the excessive use of hydrophilic emulsifier did not increase the stability of the double emulsion. This short-term destabilization kinetic study within the first 24 hours was in line with the long-term stability data shown in Table 2.

 In the case of double emulsion prepared with MCT, the lowest destabilization constant of about 6.8 x 10^-3 h^-1 was obtained for that stabilized with Tween 20 0.5% (emulsion M2). Emulsion M1, which was stabilized with Tween-20 0.5%/ PgPr 0.5%, demonstrated a slightly higher destabilization rate constant than that of M2 even though the M1 sample appeared to be the most stable in the long-term, i.e., after 4 days as seen in Table 2 and Figure 3. This again implied that the use of mixed polymeric emulsifiers of different HLB values did not significantly improve the stability of the double emulsion due to the complexity of the presence of two interfacial layers whereby the diffusion and interaction of emulsifiers between the interfaces could possibly occur. Similarly with the HCNO-based double emulsion, the use of doubled Tween-20 of 1% also resulted in a doubled destabilization rate constant for the MCT-based double emulsion (M3), which seemed to be consistent.

Table 4 Destabilization rate constants and R² values of HCNO-based double emulsions according to zero and first-order kinetic models

Table 5 Destabilization rate constants and R² values of MCT-based double emulsions according to zero and first-order kinetic models

Double emulsions prepared with different coconut oil types, namely hydrogenated (HCNO) and MCT, were developed with the incorporation of Vitamin C. The inner aqueous phase was potentially developed for functional food products such as low-calorie creamers with high nutrients. Various emulsifiers of combined Tween 20/ PgPr were used to strengthen the outer interfacial layer. It turned out that the double emulsions stabilized with Tween-20 0.5% seemed to sufficiently stabilize the double emulsion, both derived from HCNO and MCT. The HCNO-based double emulsions demonstrated remarkable long-term stability of more than 85% owing to their higher melting point compared to MCT. The lowest destabilization rate constants according to the first-order model kinetics were 4.5 x 10-3 h-1 and 6.8 x 10-3 h-1 for HCNO and MCT-based double emulsions stabilized with Tween-20 0.5%, respectively. Both HCNO and MCT-based double emulsions have been promising to be developed due to the superior health benefits of coconut oil, which exceed those of other oils besides its great potential as an encapsulation vehicle for vitamins and other antioxidant compounds in the inner aqueous phase. However, designing a double emulsion with high kinetic stability has been a great challenge and needs further investigation to unravel the complex factors affecting its stability.

         We thank PT. Lautan Natural Krimerindo for providing the hydrogenated and MCT coconut oil. We wish to thank Ms. Dyah Ayu Ambarsari for the technical assistance during the experiments. The research was funded by Indonesia Endowment Fund for Education (Lembaga Pengelola Dana Pendidikan/ LPDP) in collaboration with Directorate General of Higher Education, Research, and Technology, Ministry of Education, Culture, Research and Technology of the Republic of Indonesia under the research grant scheme of “Hibah Riset Keilmuan” 2022 (Contract number: 159/E4.1/AK.04.RA/2021).

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