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Abstract

Objective: This study aimed to determine appropriate parameters for encapsulating pomelo peel essential oil (Citrus maxima) using the alginate/chitosan complex. Methods: The investigated parameters included the concentration of sodium alginate solution (2 ‒ 3.5% w/v based on the volume of mixture), the concentration of pomelo essential oil (20 ‒ 40% w/w based on dry matter of wall marerials), the concentration of Tween 80 (0 ‒ 20% w/w based on dry matter of wall marerials), the concentration of CaCl₂ solution (0.5 ‒ 3.5% w/v based on the volume of mixture), time (10 min – 20 min) and speed of emulsion homogenization (489-4402 × g), the concentration of chitosan solution (0.5 ‒ 2% w/v based on the volume of mixture), and pH of chitosan solution (4 ‒ 6). Results: The results showed encapsulation yield (EY%) and encapsulation efficiency (EE%) of 91.64% and 85.18%, respectively, when using the concentration of sodium alginate solution as 3% (w/v based on the volume of mixture), the concentration of essential oil as 30% (w/w based on dry matter of wall marerials), the concentration of Tween 80 as 15% (w/w based on dry matter of wall marerials), the concentration of CaCl₂ solution as 1.5% (w/v based on the volume of mixture), homogenization time as 10 min and homogenization speed as 4402 × g, the concentration of chitosan as 2% (w/v based on the volume of mixture) and pH of Chitosan solution as 5. Conclusion: The alginate/chitosan complex was proven effective in encapsulating pomelo essential oil (Citrus maxima) on a laboratory scale. The resulting encapsulated particles had a relatively uniform size and a high ability to retain essential oils in the core of the particles. Further studies should be conducted to elucidate the mechanism of the encapsulation process and to additionally evaluate the physical and chemical properties of the encapsulated particles.

Keywords

Encapsulation pomelo essential oils alginate/chitosan complex

Introduction

Pomelo (Citrus maxima) is a fruit tree belonging to the Rutaceae family. Pomelo originates from Southeast Asia and is mainly grown in Thailand and Malaysia.¹ Pomelo is spherical with an average diameter of about 15 cm, protected on the outside by a peel, followed by a flavedo layer with pigment, oil pockets, and an albedo layer containing flavonoid compounds.² Pomelo contains a lot of vitamin C, which helps increase iron absorption and prevent anemia.³ Pomelo peel contains large amounts of cellulose, pectin, vitamins, and other bitter substances.⁴ Cellulose is essential in improving body shape, reducing blood fat, and preventing the accumulation of fat that causes obesity.⁵ Furthermore, pomelo peel contains many bioflavonoid compounds, which have been shown to help prevent breast cancer cells.⁶ Pomelo essential oil is an aromatic liquid obtained by pressing or extracting with appropriate solvents.⁷ Organic compounds in pomelo essential oil include aliphatic hydrocarbons, alcohol (linalool), aldehyde (citral), acid, ester, and some aromatic compounds.⁸ Pomelo essential oil has been shown to inhibit the growth of food spoilage bacteria and pathogenic strains.⁹ The monoterpenes compounds in pomelo essential oil have analgesic, antibacterial, and expectorant effects.¹⁰ Limonene is the main ingredient in pomelo essential oil (accounting for 70-90%) with anti-inflammatory, antiseptic, antiviral, and bactericidal effects.⁹ The aldehyde compounds in pomelo essential oil have anti-fungal, anti-inflammatory, anti-infectious, antiviral, bactericidal, antiseptic, and sedative effects.¹¹ Currently, pomelo essential oil has many applications in the food and pharmaceutical fields thanks to its high-value biological compounds. However, the use of pomelo essential oil is limited due to its volatile properties and decomposition under adverse environmental impacts (temperature, light, pH). Encapsulation is wrapping one substance in another to create particles with sizes from nanometers (nm) to millimeters (mm).¹² The technique is often used to encapsulate biological compounds that have high value but are easily decomposed under the influence of the environment.¹³ Based on the technique used, the encapsulation technique is divided into two main methods: physical methods (extrusion, spray drying, freeze-drying, and so on) and chemical methods (ion-gel, coacervation, liposomes, and so on).¹⁴ Among the above methods, ion-gel is the most commonly used method because of its simplicity and ease of research on a laboratory scale.¹⁵ Choosing the suitable wall material helps increase the amount of bioactive compounds encapsulated inside and provides better protection for them.¹⁶ Therefore, this study aimed to apply the ion-gel method in encapsulating pomelo essential oil using the alginate/chitosan complex as a wall material. The novelty of this study was that it applied the ion-gel complex technique to encapsulate pomelo essential oil from Vietnam and investigated the effects of emulsion parameters on the retaining ability of pomelo essential oil inside.

Materials and Methods

Materials

Pomelo essential oil was provided by AOTA Ltd company. Chemicals used for this study were purchased at Xilong Chemical Company in China, including sodium alginate (NaC₆H₇O₆, purity as 99%), calcium chloride (CaCl₂, purity as 95%), trisodium citrate dihydrate (C₆H₅Na₃O₇.2H₂O, purity as 99%), tween 80 (C₆₄H₁₂₄O₂₆, purity as 98%), chitosan (purity as 98%), acetic acid (CH₃COOH, purity as 99%), hexane (C₆H₁₄, purity as 99%), sodium hydroxide (NaOH, purity as 98%).

Process of Encapsulation of Pomelo Essential Oil Using Alginate/Chitosan Complexes

Initially, the sodium alginate solution was diluted to a concentration of 2% ‒ 3.5% (w/v based on the volume of mixture), and the pH was adjusted to 4 with sodium hydroxide solution (with a concentration of 5 M) and acetic acid solution (with a concentration of 0.5 M). Next, the emulsifier Tween 80 (concentration 0% ‒ 20% w/w based on dry matter of wall marerials) was added to the sodium alginate solution and stirred well. Then, the mixture was homogenized with pomelo essential oil (a concentration of 20% ‒ 40% w/w based on dry matter of wall marerials) with different homogenization times (about 10, 15, and 20 min) and homogenization speeds from 489, 1957, and 4402 × g. The emulsion was formed into capsules using a 25G×1” syringe containing the sample to be dropped into a well-stirred calcium chloride bath (with a concentration of 0.5% ‒ 3.5%). After 30 min, the capsule was rinsed with distilled water, left to dry for about 5 min, then stirred in chitosan solution. In particular, this chitosan solution was prepared with a concentration of 0.5% ‒ 2% (w/v based on the volume of mixture) in the solution of acid acetic 1% (by volume), and the pH was adjusted similarly to the sodium alginate solution (with pH values of 4, 5, and 6) and remained stable for 12 h. The capsule was removed after 60 min, drained, and analyzed. All experiments were conducted at room temperature (25 °C ± 3 °C).

Determination of Encapsulation Yields

Encapsulation yield was determined similarly to the study by Soliman et al, 2013 using photometric methods.¹⁷ 0.5 gram of the capsules were put into a beaker, and 2.5 ml of acetic acid 1% along with 2.5 ml of trisodium citrate solution (0.055 M) were added. After that, the capsule was crushed, 15 ml of hexane was added, and the mixture was stirred. After 30 min, the mixture was centrifuged for 10 min at 1252 × g. The top solution layer was taken for photometric measurement and was calculated the encapsulation efficiency (EY%) according to the formula equation (1): $EY (%) = \frac{F \times V \times m_{1}}{1000 \times m_{o}}$ In there:

F: the concentration of pomelo essential oil (mg/mL) was calculated based on the standard curve of pomelo essential oil dissolved in hexane at different concentrations from 0 to 10 mg/ml. Absorbance was determined at 340 nm according to Soliman et al (2013).¹⁷ The standard curve equation of pomelo essential oil was y = 0.0035x + 0.0003, R²= 0.9984.

V: the volume of hexane (mL).

m1: the mass of microencapsulated particles obtained (g).

m0: the initial mass of essential oil added to the system (g)

Determination of Encapsulation Efficiency

According to the report of the authors Sutaphanit et al, 2014 the encapsulation efficiency index was determined by photometric methods.¹⁸ 0.5 gram of the capsules were placed in a beaker, and the surface oil was removed with 5 ml of distilled water and 5 ml of hexane. After that, 2.5 ml of acetic acid 1% and 2.5 ml of trisodium citrate solution 0.055 M were added and crushed. Next, 15 ml of hexane was added to the mixture and stirred for 30 min. This mixture was centrifuged for 10 min at 1252 × g. The top liquid was aspirated and optically measured to calculate the encapsulation efficiency (EE%) index according to the formula equation (2): $EE (%) = \frac{F^{'}}{F} * 100$ In there:

F': the essential oil concentration measured after washing the surface oil (mg/mL).

F: the total essential oil concentration (mg/mL) calculated from the standard curve equation of pomelo essential oil as y = 0.0035x + 0.0003, R²= 0.9984.

Statistical Analysis

Each experiment was repeated three times. Excel 2016 for Microsoft Office was used to process experimental data. Study results were shown as mean ± SD. ANOVA and LSD analysis for each experiment were performed through Statgraphics XV software with a 95% confidence level.

Results

Physical Properties of Pomelo Essential Oil

Essential oil color: colorless, transparent, shiny.

The smell of essential oil: fragrant mixed with the strong smell of the peel, light, cool.

Essential oil taste: pungent, astringent, and gradually turning bitter.

Density: 0.8396.

Refractive index: 1.469

Effect of Sodium Alginate and Pomelo Essential Oil Concentrations

The effects of sodium alginate solution and pomelo essential oil concentration on encapsulation yields and encapsulation efficiency are shown the result in Figure 1.

Figure 1.

The effect of sodium alginate and pomelo essential oil concentrations. Note: Figure 1A: Encapsulation yield; Figure 1B: Encapsulation efficiency.

The effects of sodium alginate and pomelo essential oil concentrations had a statistically significant on the EY and EE index at the 95% confidence level (p < 0.05) through ANOVA analysis. LSD analysis showed that the EY value at 20% essential oil concentration was different from the remaining treatments. Additionally, with alginate concentrations, there also were differences between the 2.5% (w/v based on the volume of mixture) treatment and the other treatments. Figure 1 shows that the EY and EE indexes reached their highest values when the concentration of pomelo essential oil was 30% (w/w based on dry matter of wall marerials), and the sodium alginate concentration was 3% (w/v based on the volume of mixture), with EY% as 67.01% and EE% as 90.97%.

Effect of Tween 80 Emulsifier Concentrations

Figure 2 shows the influence of a tween 80 concentration on EY and EE indexes.

Figure 2.

The effect of Tween 80 concentration on the EY and EE indexes.

The effect of tween 80 concentration was significant on EY and EE index at 95% confidence level (p < 0.05) through ANOVA analysis. Based on LSD analysis, The EE and EY% values at Tween 80 concentrations were all different from each other (p < 0.05). Figure 2 presents that when increasing the concentration of tween 80 from 0% to 15% (w/w based on dry matter of wall marerials), the EY and EE indexes increased. This could be explained by the fact that surface tension helped ensure the stability of the particles.

Effect of CaCl₂ Concentration

The effect of CaCl₂ concentration on EY and EE indexes was shown in Figure 3.

Figure 3.

The effect of CaCl₂ concentration on the indicators.

The CaCl₂ solution concentration was significant effects on EY and EE values at a 95% confidence level (p < 0.05) through ANOVA analysis. LSD analysis showed that EY and EE values at different CaCl₂ solution concentrations were different. According to Figure 3, when CaCl₂ concentration increased from 0.5% to 1.5% (w/v based on the volume of mixture), the EY value increased (from 36.22% to 86.39%) but decreased at CaCl₂ concentration of 3.5% (w/v based on the volume of mixture). The EE value did not change significantly when increasing the CaCl₂ concentration.

From the above experimental results, the encapsulation yield and encapsulation efficiency was highest at the CaCl₂ solution concentration of 1.5% (w/v based on the volume of mixture), with 86.39% and 80.79%, respectively.

Effects the Time and Speed of Emulsion Homogenization

The effect of time and speed of homogenization on EY and EE indexes was shown in Figure 4.

Figure 4.

The effect of time and speed homogenization on the indicators. Note: Figure 4A: Encapsulation yield; Figure 4B: Encapsulaation efficiency.

Through ANOVA analysis, emulsion homogenization time and speed significantly affected the EE and EY index at the 95% confidence level (p < 0.05). LSD analysis showed that EY and EE values at the 20-min homogenization time differed from the remaining times (p < 0.05). EY and EE indexes reached the highest value at a homogenization speed of 4402 × g and a homogenization time of 10 min (with EY as 88.06% and EE as 82.79%). Based on Figure 4, both EE and EY indexes tended to increase when increasing the homogenization time to 15 min but decreased in the homogenization time as 20 min.

Effect of Concentration and pH of Chitosan Solution

Figure 5 shows the effect of the concentration of chitosan and the pH of the chitosan solution on EY and EE indexes.

Figure 5.

The effect of concentration and pH of the chitosan solution. Note: Figure 5A: Encapsulation yield; Figure 5B: Encapsulaation efficiency.

ANOVA analysis showed that the pH of the chitosan solution has a statistically significant effect on the EY index at a 95% confidence level (p < 0.05), and the concentration of the chitosan solution has a statistically significant effect on the EE index at the 95% confidence level (p < 0.05). Additionally, LSD results showed that the EE (%) and EY (%) values at different chitosan concentrations and chitosan solution pHs were different (p < 0.05). Based on Figure 5, the EY and EE values reached the highest values of 91.64% and 85.18% when using a concentration of chitosan solution as 2% (w/v based on the volume of mixture) and pH of chitosan solution as 5.

The Visualization of Pomelo Essential Oil Encapsulated Capsules

Capsules used for visualization evaluation were synthesized with sodium alginate as 3% (w/v), chitosan as 2% (w/v) at pH 5, emulsifier tween 80 concentration as 15% (w/w), CaCl₂ concentration as 1.5% (w/w), and pomelo essential oil concentration as 30% (w/w). Homogenization process would be performed for 10 min at 4402 × g.

The capsules had the appearance as shown in Figure 6. They were milky white, round, or spherical, and the particles produced had a certain hardness.

Figure 6.

Visualization of pomelo essential oil encapsulated capsules.

Discussions

Effect of Sodium Alginate and Pomelo Essential Oil Concentrations

At the same sodium alginate concentration, EY and EE indexes tended to increase with increasing pomelo essential oil concentration. However, when sodium alginate concentration increased, these indicators tended to decrease. This was explained as increasing sodium alginate concentration also increases sodium concentration. It helped form a thick, stable network structure that covers all the essential oils added during the homogenization process. In addition, the decrease in the EE index was due to the increase in space occupied by alginate. This caused a decrease in free volume in the polymer matrix and led to a decrease in the amount of essential oil retained in the pores.^19,20 For high essential oil concentrations, the amount of sodium alginate was not enough to cover the oil droplets completely and prevent them from clumping together.²¹ Free oil droplets would stick to the particle surface, which led to a decrease in encapsulation efficiency.

The report of Chan et al (2011) also showed similar results to this study.²² Therefore, the following experiments were conducted with a sodium alginate concentration of 3% (w/v based on the volume of mixture) and a pomelo essential oil concentration of 30% (w/w based on dry matter of wall marerials).

Effect of Tween 80 Emulsifier Concentrations

As the Tween 80 concentration increased, the surface tension decreased, leading to a decrease in droplet sizes and an increase in EY and EE values.²³ On the other hand, when continuing to increase to 20% (w/w based on dry matter of wall marerials), the values of the EE and EY indexes decreased because the surfactants had absorbed enough onto the surface layer, and they tended to leave the surface into the liquid.²⁴ This resulted in decreased EY and EE indexes.

From the above experimental results, EY and EE indexes have the highest value (90.52%) at the Tween 80 concentration of 15% (w/w based on dry matter of wall marerials). Similar to the studies of Guttoff et al (2015), emulsifier Tween was a nonionic surfactant with an HLB higher than 10, with higher HLB numbers, lipophilic surfactants forming oil droplets smaller at the phase boundary.²⁵

Effect of CaCl₂ Concentration

When the CaCl₂ concentration was low, the emulsion after homogenization dropping into the CaCl₂ reservoir did not have enough conditions to create cross-linking between alginate ions and calcium in a matrix, and bioactive compounds would easily be lost.²⁶ On the contrary, increasing the concentration of CaCl₂ led to increase the amount of Ca²⁺ ions in the solution, creating more cross-links between alginate chains, helping the system have a thick, sturdy structure, and the resulting particles were better coated. This helped limit bioactive compound loss in the core of particles.²⁷ But when increasing the CaCl₂ concentration above the threshold, the system would reduce its mechanical strength due to loss of cross-linking and unstable structure. Essential oils would be lost from the capsules and mixed into the CaCl₂ solution tank, leading to a reduction in the formation of pores in the alginate matrix during granulation and reduced encapsulation efficiency.²⁸

This result was similar to the report of Yousefi et al (2020), in which sodium alginate at a concentration of 1.42% (w/v based on the volume of mixture) and CaCl₂ at a concentration of 1.28% (w/v based on the volume of mixture) had the highest EE% values. An increased CaCl₂ concentration that was too high would also lead to the diffusion of more Ca ^+ 2 ions into the space of the polymer matrix and displaced drug molecules, leading to a decrease in EE%.²⁹

Effects the Time and Speed of Emulsion Homogenization

This was explained by the fact that when the speed and time of homogenization increased, the emulsion became more stable by providing more energy to break the particles into smaller particles.³⁰ However, when the homogenization time lasted too long, it would lead to a decrease in the amount of essential oil entering the emulsion due to the evaporation of the essential oil.³¹ In addition, longer homogenization time also increased the collision of particles, leading to agglomeration to form larger-sized particles under the effect of Vander Walls's force.³² Therefore, the homogenization process should be carried out with a time and homogenization speed that should not exceed the critical value, but they must still be large enough to create a stable emulsion, avoiding phase separation and loss of essential oils.³³

The results obtained from this study were consistent with the research results of Gupta et al (2017).³⁴ Therefore, a homogenization time of 10 min and a homogenization speed of 4402 × g were chosen for the following experiment.

Effect of Concentration and pH of Chitosan Solution

This could be explained by the fact that the pH value of 5 was close to the pKa value of chitosan; in this value, the charge density of chitosan molecules decreases rapidly.³⁵ On the other hand, when the pH value of the chitosan solution was higher than its pKa value, it is difficult for them to dissolve in water and agglomeration. When the pH values of the chitosan solution were lower than its pKa value, the essential oil would be lost.³⁶

Research results on the effect of concentration and pH of chitosan solution were consistent with the report by Bastos et al (2018), with the appropriate pH range of chitosan used in the microencapsulation process being pH 4 ‒ 5.³⁷ Therefore, a chitosan solution with a concentration of 2% (w/v based on the volume of mixture) and a pH value of 5 was appropriate for creating capsules.

Conclusion

Appropriate parameters for the encapsulation process of pomelo peel essential oil (Citrus maxima) using the alginate/chitosan complex have been determined as follows: the concentration of sodium alginate solution and essential oil concentration as 3% (w/v based on the volume of mixture) and 30% (w/w based on dry matter of wall marerials), respectively, the concentration of tween 80 as 15% (w/w based on dry matter of wall marerials), the concentration of CaCl₂ as 1.5% (w/v based on the volume of mixture), chitosan solution concentration as 2% (w/v based on the volume of mixture), pH of chitosan solution as 6. The emulsion was stable when homogenizing at a speed of 4402 × g for 10 min. Depending on the conditions in the laboratory, further evaluation of the structure of pomelo essential oil encapsulated particles through modern techniques such as SEM, TEM, and TGA/DSC analysis is necessary for this study. Pomelo essential oil capsules using alginate/chitosan complex as a wall material showed good effectiveness in storing essential oil inside and opened up great application potential in food and cosmetics.

Footnotes

Acknowledgements

This study was supported by Nong Lam University,Ho Chi Minh City,Viet Nam.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research,authorship,and/or publication of this article.

Ethical Approval

Ethical Approval is not applicable for this article.

Funding

The authors received no financial support for the research,authorship,and/or publication of this article.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

ORCID iD

Thuong Nhan Phu Nguyen

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De Carvalho

CWP

Garcia-Rojas

. Formation and characterization of the complex coacervates obtained between lactoferrin and sodium alginate. Int J Biol Macromol. 2018;120:332-338. doi:10.1016/j.ijbiomac.2018.08.050

Encapsulation of Pomelo Peel Essential oil (Citrus maxima) Using the Alginate/Chitosan complex

Abstract

Keywords

Introduction

Materials and Methods

Materials

Process of Encapsulation of Pomelo Essential Oil Using Alginate/Chitosan Complexes

Determination of Encapsulation Yields

Determination of Encapsulation Efficiency

Statistical Analysis

Results

Physical Properties of Pomelo Essential Oil

Effect of Sodium Alginate and Pomelo Essential Oil Concentrations

Effect of Tween 80 Emulsifier Concentrations

Effect of CaCl2 Concentration

Effects the Time and Speed of Emulsion Homogenization

Effect of Concentration and pH of Chitosan Solution

The Visualization of Pomelo Essential Oil Encapsulated Capsules

Discussions

Effect of Sodium Alginate and Pomelo Essential Oil Concentrations

Effect of Tween 80 Emulsifier Concentrations

Effect of CaCl2 Concentration

Effects the Time and Speed of Emulsion Homogenization

Effect of Concentration and pH of Chitosan Solution

Conclusion

Footnotes

Acknowledgements

Declaration of Conflicting Interests

Ethical Approval

Funding

Statement of Human and Animal Rights

Statement of Informed Consent

ORCID iD

References

Effect of CaCl₂ Concentration

Effect of CaCl₂ Concentration