Antioxidant stability of vitamin C in double Pickering emulsion W/O/W with microcrystalline cellulose

ABSTRACT Vitamin C is a compound that is widely used in food processing as a preservative and antioxidant. The weakness of vitamin C is that it is unstable and very reactive to environmental conditions. To maintain the stability of vitamin C during processing and storage, emulsification was carried out with a double emulsion Pickering water in oil in water (W/O/W) system. The research method used was descriptive analysis method which consists of two stages. The first stage was to determine the right amount of emulsifier in the manufacture of W/O emulsion and the second stage is the manufacture of W/O/W emulsion which consists of four different treatments with microcrystalline cellulose (MCC) as the Pickering component. The results showed that the use of 5% sorbitan tristearate produced a stable W/O vitamin C emulsion at 14 days of storage with a coalescence index of 3.2% and a droplet size of 0.698 μm. The combination of polysorbate 80 and MCC produced a W/O/W emulsion with a droplet size of 0.952 μm, creaming index 2.615%, serum index 2.899%, and a lowest increase in IC50 of 0.32 ppm/day.


Introduction
Vitamin C is a common descriptor for all compounds that show qualitative biological activity of ascorbic acid, and in low doses, vitamin C is often used in food processing to maintain shelf life and improve taste. [1]The metabolic functions of vitamin C can be categorized based on its properties as an antioxidant and as an enzyme co-substrate.Vitamin C could lose electrons easily due to its reversible monovalent oxidation to ascorbyl radicals; this functions as a biochemical redox system.Properties of vitamin C are unstable and reactive to environmental conditions. [2]Therefore, the food manufacturing industry must find the right strategy to increase the shelf-life stability of Vitamin C-fortified functional foods and beverages . [3]he water-in-oil-in-water (W 1 /O/W 2 ) double emulsion encapsulation system is a method for coating a hydrophilic bioactive compound such as vitamin C. W 1 /O/W 2 consists of an internal aqueous phase (W 1 ), an intermediate phase (O), and an external aqueous phase (W 2 ).The right emulsifier needs to be considered in the formation of W 1 /O/W 2 double emulsions.Sorbitan tristearate as an emulsifier in the W 1 /O phase has good potential because it shows a strong viscoelastic effect when spread on the surface. [4]The use of the right amount of sorbitan tristearate emulsifier in the W 1 /O phase is important to know how to produce a stable double emulsion during processing and storage of vitamin C products.
Biopolymers such as proteins and polysaccharides are high molecular weight hydrophilic emulsifiers which can be applied in the external aqueous phase in W 1 /O/W 2 double emulsions.Whey protein has excellent emulsifying properties and can be used alone or as a complex with polysaccharides such as pectin in the external aqueous phase of W 1 /O/W 2 emulsions.Low molecular weight emulsifiers such as polysorbate 80 are generally used as suitable synthetic emulsifiers in multiple emulsions. [5]ne method to produce emulsions with high stability is through the use of Pickering-type emulsions. [6]Microcrystalline cellulose (MCC) can be used in beverage products as a thickener because of its high viscosity, so it can also be used to stabilize Pickering emulsions. [7]The use of MCC as a Pickering component in the vitamin C double emulsion was studied in this research to determine the stability of the antioxidant activity of the vitamin C double emulsion during storage.

Preparation of W 1 /O Emulsion
Phase 1 research aimed to determine the use of sorbitan tristearate as an emulsifier on the stability of the W 1 /O vitamin C emulsion.The aqueous phase 1 (W 1 ) was prepared with reference to Ref. [ 8 ], where 10% (w/v) of vitamin C was dissolved and 2% (w/v) of MgSO 4 was added to maintain osmotic pressure.A W 1 /O emulsion was prepared using the aqueous phase 1 and an oil (O) phase containing the emulsifier Sorbitan tristearate in five concentrations 1%, 2%, 3%, 4%, and 5% (w/v).Demineralized water was used as a medium for all aqueous phases.A 30% aqueous phase was added to 70% oil phase at room temperature (about 25°C), followed by first-stage emulsification at 10,000 rpm for 5 min using a homogenizer (DragonLab D500, China).

W 2 phase preparation
Phase 2 of the study aimed to determine the effect of the addition of MCC on the stability and antioxidant activity of the W 1 /O/W 2 double Pickering emulsion during storage.The DPPH analysis was performed to measure the peroxyl radical scavenging activity of the samples.Whey protein concentrate (WPC)-pectin and WPC-MCC solutions were prepared with reference to Ref. [ 9 ], WPC and pectin were dissolved in aqua demineral at 50°C for 30 min, then allowed to stand for 24 h at room temperature for complete hydration.The final concentration of the biopolymer solution consisted of 2.5% WPC and 1.25% pectin [w/v].The preparation of polysorbate 80 and polysorbate-MCC solutions refers to Ref. [ 10 ], with minor modifications in MCC addition.A 3.5% (w/v) polysorbate 80 and 1.25% MCC were dissolved into demineralized water at room temperature, 3.75% (w/v) NaCl was added to maintain the osmotic pressure of the emulsion system to be formed.The solution was then stored at 4°C 12 h before being used.

Preparation of W 1 /O/W 2 emulsion
The W 1 /O/W 2 emulsion was obtained by dispersing the best W 1 /O emulsion from the first phase into the outer phase (W 2 ).MCC was added as much as 1% w/v of the total volume of the W 1 /O/W 2 emulsion.The volume of the W 1 /O emulsion fraction was set at 20%.The W 1 /O emulsion phase was added to the aqueous phase at a total volume of 100 mL at room temperature, followed by emulsification at 1200 rpm for 5 min followed by sonication (QSonica 500, Newton, USA) with a frequency of 20 kHz and an amplitude of 70%, pulse 3 s on, 3 s off for 15 min.The W 1 /O/W 2 emulsion formed was stored in a dark glass bottle at 4°C to be analyzed for stability in 28 days.Detailed material for every treatment are showed in Table 1.

Analysis
Analysis was made on the W 1 /O emulsion to determine the stability of the emulsion during storage and the W 1 /O/W 2 double Pickering emulsion to determine the stability and antioxidant activity during storage.Particle size was measured using the particle size analyzer (Beckman Coulter LS 13 320).The measurements were done on the same day with emulsion preparation.One milliliter emulsion sample was diluted in the glass cell filled with distilled water (50 mL).Creaming index measured according to the W 1 /O/W 2 vitamin C emulsion was transferred in a 50-mL test tube to a height of 6 cm approximately and stored at 4°C.A weekly height of the cream at the upper emulsion was measured (h t ) and compared to the initial emulsion height (h 0 ) to determine the creaming index (CI) with equation.
Creaming index (%) = (h 0 − h 1 ) × 100 Flow behavior index was evaluated using viscometer (RAYPA Viscometer RP1) at 50, 100, and 200 rpm.The n value was calculated using equation: μApp = (4πN/n) × n − 1 where μApp is the apparent viscosity, N is the rpm data from viscometer, and n is the flow behavior index.Emulsion structure image was captured using transmission electron microscopy (TEM, JEOL JEM 1010) with uranyl acetate staining according to method.[ 11 ] Five microliter of sample was placed into a 400-mesh grid for 10 min before uranyl acetate staining.The grid was then loaded into the instrument to observe at 20,000-30,000× of magnification.Zeta potential of the emulsion was measured using a zeta potential measurement [Horiba SZ 100] according to Ref. [12] , samples were diluted 1:100 using distilled water 1 day after emulsion preparation.
Antioxidant activity was analyzed using 1,1-diphenyl-2-picrylhydrazyl [DPPH] assay according to the method of, 13 A reference cuvette was prepared by mixing 2 mL methanol and 0.5 mL DPPH stock solution (160 ppm) and the measuring cuvettes (five dilution solutions, e.g., 40, 20, 10, 5, 2.5 ppm) were prepared by mixing the sample, DPPH stock solution (160 ppm), and methanol with a total volume 2.5 mL.Measurements using spectrophotometer UV-9200 (Beijing Rayleigh Analytical Instrument Co., Ltd., China) with wavelength 517 nm were performed after the reference and measuring cuvettes were kept in the dark at room temperature.

W 1 /O/W 2 emulsion structure
The structure image of the W 1 /O/W 2 vitamin C emulsion was taken using.Observations were made at a magnification of 20,000-30,000× to capture the emulsion structure clearly.Figure 1a is an image of the emulsion structure with Polysorbate 80 emulsifier, the large black area is a W 1 /O emulsion system, the white droplets contained in the W 1 /O system are probably vitamin C solution which is still stable in system.Around the droplet there are also oil droplets which are separated from the system and form an O/W 2 system.A similar structure is also shown in Figure 1b where there is a W 1 /O system, the difference is the presence of black spots around the droplets which are suspected to be MCC.MCC appears to fill the space between droplets and strengthens the emulsion structure formed.Figure 1c showed the WPC-MCC emulsifier appears to form a large biopolymer complex in the system and protects the trapped droplets.The formation of the biopolymer complex causes the particle measurement results to produce high numbers.Emulsions with WPC-pectin in Figure 1d were also seen to form biopolymer complexes with less irregular shapes such as WPC-MCC.The mass thickness contrast method is the principle of taking image using TEM, where the higher the molecular mass of a compound, the darker the visible color will be. [14]The image shows that the W 1 /O system in the emulsion has a higher mass than the W 2 phase making a difference in the color of the dark and light colors.The same results were also found by Ref. [ 15 ], where the results of the TEM test showed large dark-colored droplets with clear droplets in a W 1 /O/W 2 emulsion system containing arbutin and emulsified using WPC-pectin, dark-colored droplets indicating the oil phase and clear droplets in it indicate the aqueous phase containing arbutin.
The W 1 /O/W 2 system also showed the presence of clear droplets scattered among the W 1 /O droplets, these droplets were most likely oil released from the W 1 /O phase due to the use of energy from ultrasound during emulsion formation.Ref. [ 16 ] stated that the greater the amplitude used, the ultrasonic waves will change the protein to be more polar and the fat-binding strength of the protein will decrease.Proteins can also be damaged during sonication due to large amplitudes producing large amounts of heat, thereby accelerating the denaturation process of the protein and causing the protein to be unable to bind to the W 1 /O emulsion formed. [17]W 1 /O/W 2 emulsion using polysorbate 80-MCC and polysorbate 80 had a clearer emulsion system than WPC-MCC and WPC-pectin based on TEM observations.

Stability of W 1 /O emulsion
The use of sorbitan tristearate at five different concentrations resulted in emulsions with different stabilities.The 5% concentration of sorbitan tristearate showed a stable W 1 /O emulsion at room temperature storage for 14 days.Figure 2b shows the oil separation (coalescence) occurring on day 7 for the concentration of sorbitan tristearate less than 4%, while at a concentration of 5% oil separation began to occur on day 14.Separation of oil from the emulsion is called coalescence as shown in Table 2. Coalescence is also called a separation process where the emulsion droplets combine to form larger droplets and can separate from the external phase of the system. [18]The coalescence process in emulsions is an irreversible process where droplets join where the droplets are close to each other, the use of a low emulsifier can also cause high intersurface tension so that it reduces the stability of the emulsion. [19]he increase in the concentration of sorbitan tristearate is directly proportional to the viscosity of the emulsion produced; this caused the emulsion to be more stable with Brownian movement due to gravity that can be minimized. [20]Another factor that increases the stability of the emulsion is the droplet size of the emulsion.Small droplet size can increase the stability of the emulsion against separation by gravity (Stoke's law) and prevent coalescence. [12]Sufficient amount of emulsifier combined with the right emulsification technique can produce smaller droplet sizes so that it has an impact on preventing creaming and coalescence. [21]ased on Table 3, the results showed that the use of an 5% emulsifier resulted in the lowest emulsion particle size of 0.698 μm.The particle size of the emulsion tends to decrease with the addition of the emulsifier sorbitan tristearate used.Increasing the concentration of the emulsifier can decrease the particle size along with the increase in the surface area that captures the dispersed phase. [22]In the emulsification process using a homogenizer, the water-phase droplets break down into smaller sizes and are distributed into the oil phase.

Stability of W 1 /O/W 2 emulsion
Stability of the W 1 /O/W 2 emulsion was seen from the creaming index and serum index values.Creaming index can be calculated by comparing the height of the cream layer formed with the overall emulsion height and becomes an indicator of the stability of the emulsion during storage. [12]Figure 3 showed W 1 /O/W 2 vitamin C emulsion made with nonionic polysorbate 80-MCC emulsifier had the lowest creaming index value after 28 days of storage at 2.615%, followed by WPC-MCC emulsifier 4.131%, WPC-pectin 5.256%, and polysorbate 80 8,519%.
The formation of a cream layer began to form since the seventh day of storage for all types of emulsifiers which indicated the incorporation of W 1 /O emulsion droplets in the system which formed a new emulsion with a higher density.The particle droplet size also affects the cream layer formed; from observations, it can be seen that the addition of MCC in the W 2 phase can produce smaller droplet sizes in both polysorbate 80 and WPC emulsifiers, implicating slower creaming process that occurs during storage and affected the value of creaming index.MCC plays a role in forming a matrix in the emulsion system so it can prevent droplets from moving freely and joining other droplets. [23]he increase in the concentration of MCC as a Pickering component can be evaluated to adjust the droplet size to produce and it has a direct impact on the stability of the emulsion. [24]igh value of creaming index in WPC-pectin emulsifier can be caused by flocculation in the components of the emulsion system formed.Ref. [ 8 ] stated that the speed of creaming is influenced by the flocculation that occurs in the components of the W 1 /O emulsion system.Flocculation happened due to the droplet aggregation process, thus accelerating the process of creaming. [25]The flocculation process is the process that most influences the occurrence of creaming in an emulsion system, it has a big role because the flocculated droplets move faster than individual droplets in the emulsion solution. [26]a: P80-MCC, b: P80, c: WPC-MCC, d: WPC-pectin) Figure 4 showed the layer formed on the W 1 /O/W 2 emulsion is divided into three, namely the cream layer at the top, the emulsion in the second layer, and the serum in the base layer.Emulsions with polysorbate 80 have a lower serum layer and cloudiness when compared to WPC emulsifiers.This is another indication of the instability of the emulsion during storage.The serum index value in Figure 5 also showed the emulsion with MCC added was lower when compared to the emulsion without the addition of either the polysorbate 80 or WPC emulsifier.
The increase in the serum index value was also influenced by the droplet particle size factor which caused coalescence and separation during emulsion storage.According to Ref. [27] , the phenomenon of serum formation is due to the emulsifier with hydrocolloid (pectin/MCC) which is sensitive to gravity Emulsion instability is influenced by the amount of surfactant used.The low concentration of WPC cannot bind the two components as a whole, resulting in separation and flocculation which eventually results in the creaming and seruming process. [10]The lack of WPC to meet the formed W 1 /O emulsion droplet surface causes pectin and MCC as polysaccharides or hydrocolloids to be unable to stabilize electrostatically. [26]Due to the lack of protein concentration, protein can act as a bridge for oil droplets to flocculate.

Flow behavior index and zeta potential
Measurement of flow behavior index was carried out on the sample on day 0 to determine the flow properties of the emulsion formed.Table 4 shows the results of the n-value calculation which were tested at a speed of 50 rpm using an L1 spindle.W 1 /O/W 2 emulsion with polysorbate 80-MCC had the highest n value followed by WPC-MCC, WPC-P, and polysorbate 80 treatments, respectively.The higher the viscosity of an emulsion system, the emulsion formed has a more stable potential. [28]he fusion of droplets can be inhibited due to slow droplet movement in high viscosity emulsion. [29]Emulsion systems are fragile, when the viscosity is high, the droplet will change slower (flocculation, coalescence) correlating the emulsion becomes more stable. [26WPC-MCC and WPCpectin emulsifiers should produce a higher viscosity emulsion system than polysorbate 80 emulsifiers because the hydrocolloids can increase the viscosity and stabilize the emulsion system. [30]The low viscosity of WPC can be caused by the lack of WPC concentration impacting poor stability of the emulsion completely and the viscosity becomes low. [28]able 4 showed samples polysorbate 80 has n value less than 1 indicating that the W 1 /O/W 2 vitamin C emulsion formed is included in a pseudoplastic non-Newtonian solution based on the "Power Law" theory. [31]Pseudoplastic solutions are non-Newtonian solutions which have a lower viscosity as the force applied to the solution. [28]This means that a pseudoplastic non-Newtonian solution will become dilute the longer it is stirred.Sample polysorbate 80-MCC, WPC-MCC, and WPC-P were on opposite with n value more than 1 indicate the solution is dilatant, dilatant solutions have a higher viscosity when force applied and impact on the stability of the emulsion during storage Zeta potential is the electrical surface property of a colloidal solid suspended in a liquid and indicates the criteria for electric attraction and repulsion at the interface between solids.A large zeta potential value indicates a stable solid without agglomeration, when coagulation between solids occurs due to gravity, the liquid stability decreases and the repulsion between solids decreases impacting zeta potential value decreases. [32]In general, the higher the absolute value of the zeta potential (±30 mV) will increase the repulsion between surfaces and have a direct impact on better emulsion stability. [21]able 5 showed W 1 /O/W 2 vitamin C emulsions with WPC-MCC and WPC-pectin have zeta potential values closer to absolute values, followed by polysorbate 80 and polysorbate 80-MCC treatments.All treatments experienced a change in the zeta potential value toward its absolute value at 28 days of storage.The zeta potential value getting closer to the absolute value indicates that the repulsion between droplets is getting higher with increasing stability. [9]WPC-based emulsifiers have a good zeta potential value caused by the protein isoelectric point being higher at pH 5.2, [33] in contrast to polysorbate 80 which is classified as a nonionic emulsifier does not have a positive/negative effect on the value of zeta potential. [10]The isoelectric point of MCC is in the pH range of 2.8, where at a pH below that MCC is negatively charged and has better repulsion. [34]The zeta potential value that is not close to the absolute value was also obtained in the research of Ref. [ 23 ], which used MCC in the manufacture of Pickering emulsions, the use of higher MCC compared to CMC increased the emulsion repulsion and stability during storage but did not reach the absolute zeta potential value.

Antioxidant activity of W 1 /O/W 2 emulsion
The antioxidant activity analysis was carried out on the W 1 /O/W 2 vitamin C emulsion during storage for 35 days at 4°C, as a control was also carried out on vitamin C solution with the same concentration for further testing of the IC 50 value.Figure 6 showed the control treatment had the lowest IC 50 value on day 0 followed by polysorbate 80-MCC, WPC-pectin, polysorbate 80, and WPC-MCC treatments.The control treatment had the lowest IC 50 value on day 0 compared to other treatments due to the presence of energy in the form of homogenization and ultrasound during the emulsification process, causing some damaged vitamin C in the W 1 /O/W 2 emulsion system. [35]Emulsion with polysorbate 80-MCC had the lowest IC 50 value on day 0 compared to other treatments, which was 30,886 ppm, followed by WPC-pectin, polysorbate 80, and WPC-MCC treatments.The use of hydrocolloid components such as pectin and MCC can protect the active components in the emulsion system, also reducing damage during the energizing process during emulsion formation. [36,37]The selection of low energy emulsification techniques such as membranes can be used to prevent damage to active components, increase droplet uniformity, and prolong emulsion stability. [38]he use of emulsifier polysorbate 80 and MCC showed the best stability compared to other treatments.This can be seen from the increase in IC 50 value after 35 days of storage in Figure 7. Emulsion with polysorbate 80-MCC increased IC 50 value by 0.32 ppm/day then WPC-MCC by 0.42 ppm/day, WPC-pectin 0.63 ppm/day, and polysorbate 80 1.85 ppm/day.The increase in the low IC 50 value indicates that the emulsifier polysorbate 80 and MCC have the best ability in terms of maintaining the active components contained in the W 1 /O/W 2 emulsion system.The ability of MCC to resist damage to the active components in the system due to the addition of hydrocolloids has protective properties against oxidation during storage. [33]Pickering components can slow down the transfer of active components from the W 1 /O system to the outer phase, this property being a good potential to regulate the release of active components compared to using an emulsifier without the addition of MCC. [10,24,39]The combination of polysorbate 80 and MCC was able to inhibit the increase in IC 50 values better.MCC produces emulsions with lower droplet sizes, better creaming, and serum index, so that it has a direct impact on emulsion stability and protection of the active components in it. [7,40]onclusion Based on the results of the study, it can be concluded that the use of 5% sorbitan tristearate can produce a W

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Table 1 .
Emulsion material for each treatment.

Table 3 .
Particle size of W 1 /O emulsion.
separation after flocculation occurs, the cloudy layer at the bottom (serum) is water which has a heavier molecular weight than oil.The flocculation impacts the emulsifier bonds within oil droplets causing the formation of serum.
IC 50 value of W 1 /O/W 2 emulsion during storage.
Figure 7. Increase of IC 50 value per day.
1 /O vitamin C emulsion which is stable during 14 days of storage with low particle size and coalescence index.W 1 /O/W 2 vitamin C emulsion with polysorbate 80-MCC emulsifier had the best stability, in terms of the emulsion structure formed, creaming index, low serum index values, and highest zeta potential value.The use of polysorbate 80-MCC as an emulsifier can protect the antioxidant activity of the W 1 /O/W 2 vitamin C emulsion after storage for 28 days, with an increase in IC 50 value of 0.3216 ppm/day.