Influence of Technological Factors on the Quality of Chitosan Microcapsules with Boswellia serata L. Essential Oil

Essential oils contain many volatile compounds that are not stable and lose their pharmacological effect when exposed to the environment. The aim of this study is to protect Boswellia serrata L. essential oil from environmental factors by encapsulation and determine the influence of chitosan concentration and types (2%, 4%; medium and high molecular weights), essential oil concentration, different emulsifiers (Tween and Span), and technological factors (stirring time, launch height, drip rate) on the physical parameters, morphology, texture, and other parameters of the generated gels, emulsions, and microcapsules. For the first time, Boswellia serrata L. essential oil microcapsules with chitosan were prepared by coacervation. Hardness, consistency, stickiness, viscosity, and pH of chitosan gels were tested. Freshly obtained microcapsules were examined for moisture, hardness, resistance to compression, size, and morphology. Results show that different molecular weights and concentrations of chitosan affected gel hardness, consistency, stickiness, viscosity, mobility, and adhesion. An increase in chitosan concentration from 2% to 4% significantly changed the appearance of the microcapsules. It was found that spherical microcapsules were formed when using MMW and HMW 80/1000 chitosan. Chitosan molecular weight, concentration, essential oil concentration, and stirring time all had an impact on the hardness of the microcapsules and their resistance to compression.


Introduction
Essential oils are a complex mixture of compounds that are sensitive to oxygen, light, and high temperatures [1,2]. These environmental factors contribute to the degradation of the active substances and a reduction in the biological effects of the essential oil [2]. Microencapsulation technology is used to preserve the properties of an essential oil, during which it is coated with a protective shell [3,4]. Microencapsulated essential oils have a wide variety of applications across a range of food, cosmetic, and pharmaceutical products [5]. Microencapsulation of essential oils ensures the stability of volatile compounds during thermal processing, as well as during conversion of liquids to powder, and results in a slow and controlled release of the active compounds [5,6]. Various materials, such as soluble polymers, micro/nanocapsules, liposomes, micelles, and emulsions, have been developed and formulated as effective microencapsulation carriers [7]. Coacervation is one of the most efficient and frequently used microencapsulation methods in the pharmaceutical industry [8]. Coacervation-based microencapsulation is suitable for lipophilic materials, such as essential oils, vegetable oils and resins, and vitamin E, however the process has

Determination of pH Values of Chitosan Gels
Aqueous gel solutions containing 5% chitosan were prepared from chitosan with different molecular weights, and the pH was measured by using a WinLab ® Data Line pH-Meter [27]. Initially, the pH-meter electrode was calibrated in a beaker with purified water until a constant pH value was reached, then the pH of a 5% aqueous chitosan gel solution was measured. Each solution was measured three times, and the pH meter electrode was calibrated in purified water before each new measurement.

Texture Analysis of Chitosan Gels
A texture analysis of the chitosan gels was prepared according Ferreira et al. [28] with some modifications. A texture analyzer TA.XTplus (Stable Micro Systems, Godalming, UK) was used. Each gel was poured into a container and placed on the stage of the texture analyzer. The required tap A/BE was attached to the rod of the appliance, the size of which corresponded to the size of the container. Initially, a backward extrusion test was performed. The required T.A. parameters-15 mm distance and 10 mm/s speed-were selected in the settings. The test measured the hardness (g), consistency (g*s), stickiness (g), and viscosity index (g*s) of the gel. Each sample was analyzed three times, and the program automatically created a graph of the results.
A spreadability test was also performed with the same T.A. settings (15 mm distance and 10 mm/s speed). In the data processing section of the program, the calculations for the peak negative force and negative area were selected. This test measured mobility (g*s) and adhesion (g*s). The sample was analyzed three times, and the program created a graph of the results.

Formation of Microcapsules from Chitosan Gels
Microcapsules from chitosan gels were formatted according to the method of Souza et al. [29] with some modifications. The reconstituted chitosan gel was placed into a 10 mL syringe with a needle attached. A 1% NaOH solution was poured into a beaker, which was placed on MSH-20A magnetic stirrer. The mixing speed of the magnetic stirrer was set at 130 rpm. Chitosan gel was slowly added to the 1% NaOH solution. The microcapsules were formed with previously prepared chitosan gels (2% MMW, 4% MMW, 2% HMW 80/1000, 2% HMW 80/3000) dripped from different launch heights-4 and 10 cm-from the point of droplet release to the surface of the 1% NaOH solution in the beaker using different stirring times:  Table 1). The microcapsules were rinsed several times with purified water, placed on filter paper, and left to dry for 24 h.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Boswellia serrata Essential Oil
This assay was performed using a headspace technique on a Shimadzu GC-MS-QP2010 gas chromatograph-mass spectrometer system (Shimadzu, Tokyo, Japan) equipped with a Shimadzu autoinjector AOG-5000 (Shimadzu, Tokyo, Japan) with an Rxi ® -5 ms capillary column (length 30 m, diameter 0.25 mm, stationary phase layer thickness 0.25 µm) according to Pudziuvelyte et al. [30]. The temperature in the column was set at 50 • C for 5 min, then raised 2 • C/min up to 200 • C, then 15 • C/min up to 315 • C, and this temperature was maintained constant for 15 min. Injector temperature was 260 • C. Helium gas was used for the analysis. The sample was injected using a flow ratio of 1:60. The injection volume was 1 µL. Mass range 29-500 amu, scan time 0.2 s, injection site temperature 280 • C.

Preparation of Emulsions
The selected amount of chitosan gel was placed in a beaker, the required amount of emulsifier was added dropwise, and everything was mixed. Then the required amount of Boswellia serrata essential oil was added and mixed (Table 2).

Microscopic Analysis of Emulsions
Microscopy was used to assess the size of the essential oil droplets in the emulsion and their distribution [27]. One drop of emulsion was placed on a slide and covered with a coverslip. A Motic ® BA310 microscope with a Nis-Elements program was used to evaluate the image. The images were magnified 100 times and the program calculated the size of selected oil droplets. For each emulsion, 10 random droplets of oil were selected, and their average diameter was determined.

Emulsions Stability Study by Centrifugation
The prepared emulsions were placed in tubes and centrifuged by using a Sigma 3-18KS centrifuge. The spin time was set for 5 min at 10,000 rpm [27]. After centrifugation, the oil-phase and aqueous phase of the emulsions were separated.

Formation of Microcapsules with a Syringe Pump
The selected chitosan gel or emulsion was drawn into a 10 mL medical syringe with a needle attached. The syringe was attached to a LA-120 syringe pump (Jiangsu Zhengkang Medical Apparatus, Shanghai-Nanjing Railway, Changzhou, China) [31]. The gel was dripped at a rate of 0.5 mL/min, and the emulsion at a rate of 0.4 mL/min or 0.8 mL/min, with a diameter of 14.43, depending on the volume of the syringe ( Figure 1).

Microscopic Analysis of Emulsions
Microscopy was used to assess the size of the essential oil droplets in the emulsion and their distribution [27]. One drop of emulsion was placed on a slide and covered with a coverslip. A Motic ® BA310 microscope with a Nis-Elements program was used to evaluate the image. The images were magnified 100 times and the program calculated the size of selected oil droplets. For each emulsion, 10 random droplets of oil were selected, and their average diameter was determined.

Emulsions Stability Study by Centrifugation
The prepared emulsions were placed in tubes and centrifuged by using a Sigma 3-18KS centrifuge. The spin time was set for 5 min at 10,000 rpm [27]. After centrifugation, the oil-phase and aqueous phase of the emulsions were separated.

Formation of Microcapsules with a Syringe Pump
The selected chitosan gel or emulsion was drawn into a 10 mL medical syringe with a needle attached. The syringe was attached to a LA-120 syringe pump (Jiangsu Zhengkang Medical Apparatus, Shanghai-Nanjing Railway, Changzhou, China) [31]. The gel was dripped at a rate of 0.5 mL/min, and the emulsion at a rate of 0.4 mL/min or 0.8 mL/min, with a diameter of 14.43, depending on the volume of the syringe ( Figure 1). A beaker with 1% NaOH solution was placed on a MSH-20A magnetic stirrer set at 130 rpm stirring speed. The chitosan gel or emulsion was added to the 1% NaOH solution from a height of 10 cm (from the starting point of the gel drop to the surface of 1% NaOH solution). The microcapsules were produced using different stirring times: 5, 15, and 30 min. (Table 3). Freshly made microcapsules were rinsed several times with purified water and placed on filter paper to dry for 24 h. A beaker with 1% NaOH solution was placed on a MSH-20A magnetic stirrer set at 130 rpm stirring speed. The chitosan gel or emulsion was added to the 1% NaOH solution from a height of 10 cm (from the starting point of the gel drop to the surface of 1% NaOH solution). The microcapsules were produced using different stirring times: 5, 15, and 30 min. (Table 3). Freshly made microcapsules were rinsed several times with purified water and placed on filter paper to dry for 24 h.

Moisture Content
A moisture analyzer (Kern MLS, Balingen, Germany) was used to measure moisture content. The sample (0.1-0.2 g) was dried at 100-105 • C until a constant weight was obtained [32]. Samples were tested 2, 4, 6, and 24 h after preparation of the microcapsules. The test was repeated three times and the average of the obtained results was derived.

Compression Analysis
A Stable Micro Systems manual TA.XTplus texture analyzer was used to measure the crush resistance of fresh microcapsule shells. A pre-programed gnocchi compression test was chosen for this purpose. Ten fresh microcapsules from each series were placed on the surface of the texture analyzer, a flat base was attached to the device, and the maximum force of the device was set to 6500 g [31]. Other parameters were selected in the program settings-the descent distance to microcapsules, 3 mm, and the speed of descent, 2 mm/s. The base descended, pressed the microcapsules, then returned to its starting position. The test was repeated three times for each series, and the program automatically created a graph of the results.

Microscopic Analysis of Microcapsules
To assess the appearance of the dried microcapsules and their size, a microscopic examination was performed using a Motic ® BA310 microscope with the Nis-Elements program [33]. The microcapsules in each series were placed on a slide and the image was magnified 100 times. Three microcapsules were randomly selected from each series.

Statistical Analysis
Data obtained during the research were processed by Microsoft Office Excel 2016 (Redmond, WA, USA). The results were summarized, and their means and standard deviations were calculated. Student's t-test was used to assess the differences in results. Differences were considered statistically significant when p < 0.05.

Influence of Chitosan Molecular Weight and Concentration on Gel pH Values
Gels were prepared from chitosan with different molecular weights and concentrations and their pH values were determined. The obtained results are shown in Table 4. We compared 2% and 4% chitosan gels with different molecular weights. The pH values ranged from 3.34 ± 0.05 to 3.65 ± 0.03. When comparing different molecular weight chitosan gels at the same concentration, the changes in pH were insignificant. The lowest pH value was observed for 2% HMW 80/1000 chitosan gel (3.34 ± 0.05), which was slightly higher for the 2% HMW 80/3000 (3.35 ± 0.04) and 2% MMW (3.37 ± 0.03) chitosan gels. A significant difference was found for the 4% MMW chitosan gel, which had a maximum pH of 3.65 ± 0.03.
MMW chitosan was used in previous study, where it was dissolved in a 1% aqueous solution of lactic acid. The pH values ranged from 4.8 to 5.27. Compared to the results of our study, these values are higher due to the lower concentration of acid. Since this study aimed to produce a gel for application on the skin, these pH values were close to the pH value of skin (about 5). An acidic environment of the gel prevents the multiplication of microorganisms [34].
Senyigit et al. [35] used chitosan with small, medium, and large molecular weights dissolved in a 1% lactic acid solution, and measured pH values ranging from 4.98 ± 0.03 to 5.34 ± 0.04. The molecular weight of chitosan had no significant effect on pH.
Summarizing the obtained results, it can be stated that the molecular weight of chitosan did not affect the pH value of the gels, but the concentration of chitosan did. A higher concentration of chitosan had a significantly higher pH value (p < 0.05).

Influence of Different Molecular Weights and Concentrations of Chitosan on Gel Texture
Hardness (g), consistency (g*s), stickiness (g) and viscosity index (g*s) results were obtained by analyzing the texture of chitosan gels with different molecular weights and concentrations during the backward extrusion test. The hardness (g) results of the gels are shown in Figure 2.
A significant difference was found for the hardness of chitosan gels with different molecular weights. The 2% MMW chitosan gel had the lowest hardness, 21.46 ± 0.03 g; the 2% HMW 80/1000 chitosan gel had a medium average hardness, 34.74 ± 0.21 g; and the 2% HMW 80/3000 chitosan gel had highest hardness, 65.73 ± 1.79 g. A significant difference was found when comparing MMW chitosan gels of different concentrations: the 4% MMW chitosan gel was 4.5 times harder than the 2% chitosan gel. Sezer et al. [36] found that the hardness of their gel formulations were significantly affected by the molecular weight and concentration of the polymer. The hardness value of their hydrogel increased significantly 5-fold when the chitosan concentration was increased in from 1.5 to 2% [36].
After determining the consistency (g*s) of chitosan with different molecular weights and concentrations, a significant difference was found. Figure 3 shows the consistency (g*s) results of the gels.
Results show the impact of chitosan concentration and molecular weight on gel consistency. When evaluating the consistency of chitosan gels with different molecular weights at the same concentration, the 2% MMW chitosan gel had the lowest value (90.11 ± 0.92 g*s), followed by the 2% HMW 80/1000 chitosan gel (149.17 ± 0.72 g*s), and the 2% HMW 80/3000 chitosan gel (274.32 ± 8.66 g*s). The 4% MMW chitosan gel had a significantly higher consistency (402.29 ± 0.62 g*s) than the 2% MMW chitosan gel. According to Szczesniak et al. [37], higher concentrations of chitosan increased the consistency of the gels, where the consistency of a 5% chitosan gel was higher than 1-4% chitosan gels. A significant difference was found for the hardness of chitosan gels with different molecular weights. The 2% MMW chitosan gel had the lowest hardness, 21.46 ± 0.03 g; the 2% HMW 80/1000 chitosan gel had a medium average hardness, 34.74 ± 0.21 g; and the 2% HMW 80/3000 chitosan gel had highest hardness, 65.73 ± 1.79 g. A significant difference was found when comparing MMW chitosan gels of different concentrations: the 4% MMW chitosan gel was 4.5 times harder than the 2% chitosan gel. Sezer et al. [36] found that the hardness of their gel formulations were significantly affected by the molecular weight and concentration of the polymer. The hardness value of their hydrogel increased significantly 5-fold when the chitosan concentration was increased in from 1.5 to 2% [36].
After determining the consistency (g*s) of chitosan with different molecular weights and concentrations, a significant difference was found. Figure 3 shows the consistency (g*s) results of the gels.  Results show the impact of chitosan concentration and molecular weight on gel consistency. When evaluating the consistency of chitosan gels with different molecular weights at the same concentration, the 2% MMW chitosan gel had the lowest value (90.11 ± 0.92 g*s), followed by the 2% HMW 80/1000 chitosan gel (149.17 ± 0.72 g*s), and the 2% HMW 80/3000 chitosan gel (274.32 ± 8.66 g*s). The 4% MMW chitosan gel had a significantly higher consistency (402.29 ± 0.62 g*s) than the 2% MMW chitosan gel. According to Szczesniak et al.
[37], higher concentrations of chitosan increased the consistency of the gels, where the consistency of a 5% chitosan gel was higher than 1-4% chitosan gels.
The stickiness (g) of the gels was found to vary from −13.02 ± 0.13 g to −65.29 ± 0.11 g with significant differences. The obtained results are shown in Figure 4. Among the chitosan gels with different molecular weights at the same concentration, the least sticky was The stickiness (g) of the gels was found to vary from −13.02 ± 0.13 g to −65.29 ± 0.11 g with significant differences. The obtained results are shown in Figure 4. Among the chitosan gels with different molecular weights at the same concentration, the least sticky was 2% MMW chitosan gel (−13.02 ± 0.13 g), and the most was 2% HMW 80/3000 chitosan gel (−41.10 ± 2.99 g), with the 2% HMW 80/1000 chitosan gel (−20.46 ± 0.66 g) in between. The 4% MMW chitosan gel had significantly higher stickiness (65.29 ± 0.11 g) than the 2% MMW chitosan gel. The viscosity indices ranged from −20.95 ± 0.39 g*s to −177.88 ± 5.79 g*s. Among the chitosan gels with the same concentration but different molecular weights, the 2% MMW chitosan gel had lowest viscosity index (−20.95 ± 0.39 g*s), followed by the 2% HMW 80/1000 chitosan gel (−50.77 ± 5, 74 g*s) and the 2% HMW 80/3000 chitosan gel (−177.88 ± 5.79 g*s). The difference between these results is significant. When evaluating gels with different concentrations, the 4% MMW chitosan gel had a significantly higher viscosity index (−135.51 ± 0.64 g*s) than the 2% MMW chitosan gel. The results are shown in Figure  5. The viscosity indices ranged from −20.95 ± 0.39 g*s to −177.88 ± 5.79 g*s. Among the chitosan gels with the same concentration but different molecular weights, the 2% MMW chitosan gel had lowest viscosity index (−20.95 ± 0.39 g*s), followed by the 2% HMW 80/1000 chitosan gel (−50.77 ± 5, 74 g*s) and the 2% HMW 80/3000 chitosan gel (−177.88 ± 5.79 g*s). The difference between these results is significant. When evaluating gels with different concentrations, the 4% MMW chitosan gel had a significantly higher viscosity index (−135.51 ± 0.64 g*s) than the 2% MMW chitosan gel. The results are shown in Figure 5. The spreadability test was performed to determine the mobility (g*s) and adhesion (g*s) of the chitosan gels. The mobility values for chitosan gels with different molecular weights were found to range from −0.39 ± 0.22 g*s to 2.99 ± 0.74 g*s. The mobility (g*s) The spreadability test was performed to determine the mobility (g*s) and adhesion (g*s) of the chitosan gels. The mobility values for chitosan gels with different molecular weights were found to range from −0.39 ± 0.22 g*s to 2.99 ± 0.74 g*s. The mobility (g*s) results for the gels are shown in Figure 6. The spreadability test was performed to determine the mobility (g*s) and adhesion (g*s) of the chitosan gels. The mobility values for chitosan gels with different molecular weights were found to range from −0.39 ± 0.22 g*s to 2.99 ± 0.74 g*s. The mobility (g*s) results for the gels are shown in Figure 6.  The lowest mobility was determined in 2% MMW chitosan gel (−0.39 ± 0.22 g*s), slightly higher in 2% HMW 80/1000 chitosan gel (−0.83 ± 0.09 g*s), but there was no significant difference between these two gels. The maximum mobility had 2% HMW 80/3000 chitosan gel (2.99 ± 0.74 g*s). The results of this gel are significantly higher than those of the previous samples. Comparing MMW chitosan gels of different concentrations, it was found that 4% gel had a significantly higher mobility (4.78 ± 0.1 g*s) than 2% MMW gel. Figure 7 shows adhesion (g*s) results of the gels.
When evaluating the adhesion (g*s) of chitosan gels with different molecular weights at the same concentration, the 2% MMW chitosan gel had the lowest adhesion (−1.76 ± 0.04 g*s), 2% HMW 80/1000 chitosan gel had higher adhesion (−2.57 ± 0.23 g*s), and 2% HMW 80/3000 chitosan gel had the highest adhesion (−4.68 ± 0.68 g*s). The differences between these results are significant. The 4% MMW chitosan gel had significantly higher adhesion (−7.69 ± 0.19 g*s) compared to 2% MMW chitosan gel. Another study also prepared gels containing chitosan with different molecular weights (low molecular weight (LMW), MMW, and HMW). After analysis of their texture, the parameters of hardness, consistency, and stickiness were evaluated. MMW chitosan gel was found to have the lowest values for all of the listed properties, HMW chitosan gel the highest, and LMW chitosan gel fell in between [38]. Comparing these properties to the MMW and HMW gels prepared in our study, the obtained results are similar. The scientific literature also contains a study in which chitosan gel hardness and stickiness increased with increasing chitosan concentrations [39]. According to the results of Sezer et al. [36], the molecular weight and concentration of the chitosan used in the gel can influence the adhesion of the formulation.
Summarizing the results for hardness, consistency, stickiness, viscosity index, mobility, and adhesion of chitosan gels of different molecular weights, it can be concluded that 2% MMW chitosan gel had the lowest texture properties, 2% HMW 80/1000 chitosan gel had higher texture properties, and the 2% HMW 80/3000 chitosan gel had the highest.
Evaluating the results for chitosan gels with different concentrations, it was found that a higher concentration of chitosan produced higher values for these texture parameters.
The lowest mobility was determined in 2% MMW chitosan gel (−0.39 ± 0.22 g*s), slightly higher in 2% HMW 80/1000 chitosan gel (−0.83 ± 0.09 g*s), but there was no significant difference between these two gels. The maximum mobility had 2% HMW 80/3000 chitosan gel (2.99 ± 0.74 g*s). The results of this gel are significantly higher than those of the previous samples. Comparing MMW chitosan gels of different concentrations, it was found that 4% gel had a significantly higher mobility (4.78 ± 0.1 g*s) than 2% MMW gel. Figure 7 shows adhesion (g*s) results of the gels. When evaluating the adhesion (g*s) of chitosan gels with different molecular weights at the same concentration, the 2% MMW chitosan gel had the lowest adhesion (−1.76 ± 0.04 g*s), 2% HMW 80/1000 chitosan gel had higher adhesion (−2.57 ± 0.23 g*s), and 2% HMW 80/3000 chitosan gel had the highest adhesion (−4.68 ± 0.68 g*s). The differences between these results are significant. The 4% MMW chitosan gel had significantly higher adhesion (−7.69 ± 0.19 g*s) compared to 2% MMW chitosan gel. Another study also prepared gels containing chitosan with different molecular weights (low molecular weight (LMW), MMW, and HMW). After analysis of their texture, the parameters of hardness, consistency, and stickiness were evaluated. MMW chitosan gel was found to have the lowest values for all of the listed properties, HMW chitosan gel the highest, and LMW chitosan gel fell in between [38]. Comparing these properties to the MMW and HMW gels prepared in our study, the obtained results are similar. The scientific literature also contains a study in which chitosan gel hardness and stickiness increased with increasing chitosan concentrations [39]. According to the results of Sezer et al. [36], the molecular weight and concentration of the chitosan used in the gel can influence the adhesion of the formulation.
Summarizing the results for hardness, consistency, stickiness, viscosity index, mobility, and adhesion of chitosan gels of different molecular weights, it can be concluded that 2% MMW chitosan gel had the lowest texture properties, 2% HMW 80/1000 chitosan gel had higher texture properties, and the 2% HMW 80/3000 chitosan gel had the highest.

Formation of Microcapsules
The ability to form a gel in contact with anionic groups is an interesting feature of chitosan. This gel formation process is due to the presence of anionic groups that allow the formation of intrachain and interchain cross-links [40]. Different technological factors were assessed to determine the optimal conditions for the formation of microcapsules. Initially, we chose to form microcapsules from 2% MMW and 2% HMW 80/1000 chitosan gels using different gel release heights (4 and 10 cm) into a 1% NaOH solution with different mixing times (5, 15, and 30 min). It was found that more regular microcapsules were formed by releasing the gel from a 10 cm height. When the gel was dripped from 4 cm, the microcapsules formed an elongated shape instead of a sphere. Figures 8 and 9 show microcapsules formed from 2% HMW 80/1000 chitosan gel released from 4 and 10 cm height into a NaOH solution. Evaluating the results for chitosan gels with different concentrations, it was found that a higher concentration of chitosan produced higher values for these texture parameters.

Formation of Microcapsules
The ability to form a gel in contact with anionic groups is an interesting feature of chitosan. This gel formation process is due to the presence of anionic groups that allow the formation of intrachain and interchain cross-links [40]. Different technological factors were assessed to determine the optimal conditions for the formation of microcapsules. Initially, we chose to form microcapsules from 2% MMW and 2% HMW 80/1000 chitosan gels using different gel release heights (4 and 10 cm) into a 1% NaOH solution with different mixing times (5, 15, and 30 min). It was found that more regular microcapsules were formed by releasing the gel from a 10 cm height. When the gel was dripped from 4 cm, the microcapsules formed an elongated shape instead of a sphere. Figures 8 and 9 show microcapsules formed from 2% HMW 80/1000 chitosan gel released from 4 and 10 cm height into a NaOH solution.     Chitosan gels with 4% MMW and 2% HMW 80/3000 did not form microcapsules. The main reason for this was the viscosity of these gels was too high to form droplets. Thus we produced microcapsules from a 2% MMW chitosan gel using a syringe pump with a dripping speeds and different stirring times.
Microcapsules were formed from emulsions consisting of 2% MMW chitosan gel, 0.5% Tween 20, and different concentrations of Boswellia serrata essential oil. Emulsions that dripped at 0.8 mL/min speed formed round-spherical (regular) shape microcapsules with 0.1, 0.2, and 0.3% essential oil, whereas irregular microcapsules formed with 0.4% essential oil. By reducing the dripping speed of the emulsion to 0.4 mL/min, regular microcapsules were formed with 0.4% essential oil, but the microcapsules did not form properly with higher oil concentrations. Microcapsules were found to be more regular when the emulsion was dripped at 0.4 mL/min speed, which was selected for further studies. A 15 min stirring time was chosen to produce microcapsules in the following experiments.
We tested different emulsifiers in the emulsion composition. Emulsions consisting of 2% MMW chitosan gel, 0.5% Tween 80 and different concentrations of Boswellia serrata essential oil only produced suitable microcapsules with 0.1% of the essential oil. Span 20 and Span 80 emulsifiers could be used to encapsulate 0.1% and 0.2% of the essential oil, and when MMW was replaced by HMW 80/1000 chitosan, Tween 20 was selected to encapsulate Boswellia serrata essential oil at a concentration from 0.1% to 0.4%. Microcapsules could not be formed from an emulsion containing 3% MMW chitosan, 0.1% Boswellia serrata essential oil, and 0.5% Tween 20 because the emulsion was too viscous.
The results show that the most regular microcapsules were formed from 2% chitosan emulsion containing MMW or HMW 80/1000 when the optimal drip rate was 0.4 mL/min and the concentration of Boswellia serrata essential oil was 0.4%.

Influence of Different Technological Factors on the Appearance and Size of Microcapsules
After drying the prepared microcapsules, the influence of different technological factors (concentration of Boswellia serrata essential oil, mixing time, emulsifiers, different molecular weight of chitosan) on the appearance and size of microcapsules was evaluated. The shape and appearance of microcapsules can affect the following properties: mechanical strength, degree of swelling, and the protection and release of the encapsulated bioactive compounds [41]. Microscopic examination of the appearance of dried microcapsules revealed that they all have a slightly rough surface, with slight irregularities, in the shape of an irregular circle. A microscopic image of microcapsules No. 16,No. 17,and No.18 is shown in Figure 10.
The shape and appearance of microcapsules can affect the following properties: mechanical strength, degree of swelling, and the protection and release of the encapsulated bioactive compounds [41]. Microscopic examination of the appearance of dried microcapsules revealed that they all have a slightly rough surface, with slight irregularities, in the shape of an irregular circle. A microscopic image of microcapsules No. 16,No. 17,and No.18 is shown in Figure 10.  Table 3.  Table 3.
In order to compare the size of the produced and dried microcapsules and its dependence on Boswellia serrata essential oil concentration, we used a single chitosan gel composition, emulsifier, and microcapsule mixing time. The sizes of microcapsules from different batches are given in Table 5. 1014.81 ± 11.94 ** †  Table 3.
When comparing microcapsule diameters, no significant difference was found, except for microcapsules No. 6 and No. 12 (p < 0.05). Microcapsule No. 6 and No. 12 contained 0.1% and 0.3% of Boswellia serrata essential oil, respectively. In this example, microcapsules containing less essential oil were found to have a larger diameter (974.09 ± 26.78 µm) than microcapsules containing a higher concentration of essential oil (943.82 ± 31.47 µm).
To evaluate the effect of stirring time on the diameter of the microcapsules, microcapsules with the same composition and technological factors except stirring time were compared. Microcapsules formed at three different stirring times (5, 15, and 30 min) were evaluated. This technological factor was found to cause significant differences in microcapsule diameters. To evaluate the influence of emulsifiers on the diameter of microcapsules, microcapsules were formed using different emulsifiers. The largest significant difference in the diameter was found to be between the microcapsules containing 0.5% Tween 20 and the other emulsifiers. Microcapsule No. 19, which contained 0.5% Tween 80, was significantly smaller than microcapsule No. 5 (containing 0.5% Tween 20  [6] produced microcapsules that were 1224 ± 6.56 µm in a diameter. The larger microcapsule size may have been due to slightly different study conditions, e.g., their microcapsules contained not only chitosan but also the polysaccharide carrageenan, and Tween 40 was used as an emulsifier. In addition, their microcapsules were stirred in ethanol instead of aqueous NaOH, and ethanol has been shown to reduce the solubility and increase the hardness of chitosan [6]. Hu et al. [23] analyzed microcapsules formed from a lower concentration of chitosan (0.5%). The microcapsules were stirred at 400, 800, and 1500 rpm, and their sizes were 225 ± 4 µm, 131 ± 20 µm, and 11 ± 3 µm, respectively. Thus, higher stirring speeds produces smaller microcapsules [42]. In our study, the stirring speed was fixed at 130 rpm. It has been determined that smaller size microcapsules may increase the release of essential oil components [43]. Emulsifiers in the emulsion system form a protective membrane between the aqueous and oil phases, thus making the system more stable. Yang et al. [25] found that a complex of two emulsifiers (Span 80 and Tween 60) enhanced the membrane by increasing the viscosity of the interface. This might account for a decrease in microcapsule diameter from 626.5 µm (using Span 80) to 31.8 µm (using Span 80 and Tween 60) [44].
Javid et al. [26] produced microcapsules containing chitosan and eucalyptus or sandalwood essential oils. It was found that as the concentration of eucalyptus essential oil increased, the size of the microcapsules also increased, but the size decreased when using higher concentrations of sandalwood essential oil [45]. In our study, different concentrations of Boswellia serrata essential oil did not significantly change microcapsule size.
Summarizing our results, we can state that significant changes in the size of microcapsules was not caused by a change in the concentration of Boswellia serrata essential oil or the molecular weight of chitosan. Emulsifiers did have an effect on the size of the microcapsules, where the largest microcapsules were formed with Tween 20, and the smallest ones with Span 80. The microcapsules containing Span 20 and Tween 80 were arranged in a descending order, respectively.

Influence of Boswellia serrata Essential Oil Concentration on the Hardness of Microcapsules
Mechanical compression is a good way to determine the quality of produced microcapsules [46]. The mechanical properties of microcapsules are highly dependent on the physical properties of their shell. Polymeric materials are often used to form microcapsules to increase their compressive strength [47]. To evaluate the mechanical properties of microcapsules, the methods can test a single microcapsule or a group of microcapsules. Examination of a single microcapsule gives more accurate results [48]. The literature also states that the compressive force on microcapsules between two parallel plates cannot predict their mechanical properties in another medium, such as when the microcapsules are suspended in a flowing liquid. The motion and deformation of the particles then depends not only on their physical properties (internal rheology, surface-volume ratio, mechanical properties of the shell) but also on the local field flow [49]. In our study, it was not possible to evaluate just one microcapsule in a compression test, so a group of 10 microcapsules from each batch was tested by repeating the test 3 times.

Influence of Stirring Time on the Hardness of Microcapsules
The hardness results for microcapsules formed from emulsions containing 0.1% Boswellia serrata essential oil, 2% MMW chitosan, 0.5% Tween 20 that were prepared with different stirring times are shown in Figure 13. Comparing the microcapsules No. 4 (stirring time 5 min), No. 5 (stirring time 15 min), and No. 6 (stirring time 30 min), it was found that as the stirring time lengthened, the microcapsules required more force to crush them, though the difference between No. 5 and No. 6 was not statistically significant.  Figure 12 which were prepar from 2% HMW 80/1000 chitosan gel without a syringe pump. Once again, as the stirr time for the microcapsules lengthened, their resistance to crushing increased. Microca sules No. 1 (stirring time 5 min), No. 2 (stirring time 15 min), and No. 3 (stirring time min) (red curve in Figure 12) were prepared from 2% MMW chitosan gel using a syrin pump and the obtained data were very similar to that for microcapsules No. I, No. II, a No. III.
The hardness results for microcapsules formed from emulsions containing 0.1% B wellia serrata essential oil, 2% MMW chitosan, 0.5% Tween 20 that were prepared w different stirring times are shown in Figure 13. Comparing the microcapsules No. 4 (s ring time 5 min), No. 5 (stirring time 15 min), and No. 6 (stirring time 30 min), it was fou that as the stirring time lengthened, the microcapsules required more force to crush the though the difference between No. 5 and No. 6 was not statistically significant.  Summarizing the obtained results, we can state that microcapsules that consisted only of chitosan gel were stronger when stirred for longer time. Microcapsules composed of emulsions did not always show significant differences in hardness with longer stirring times, but there was a tendency for it to increase as the stirring time increased.

Influence of Surfactants on the Hardness of Microcapsules
In our study, microcapsules were formed with four different emulsifiers: Tween 20, Tween 80, Span 20, and Span 80. The results for their effect on hardness are shown in Figure 14.
Summarizing the obtained results, we can state that microcapsules that consisted only of chitosan gel were stronger when stirred for longer time. Microcapsules composed of emulsions did not always show significant differences in hardness with longer stirring times, but there was a tendency for it to increase as the stirring time increased.

Influence of Surfactants on the Hardness of Microcapsules
In our study, microcapsules were formed with four different emulsifiers: Tween 20, Tween 80, Span 20, and Span 80. The results for their effect on hardness are shown in Figure 14.  Table 3.
Comparing microcapsules No. 5,No. 19,No. 20,and No. 22 by the shell hardness, the lowest compressive forces were required to crush microcapsules containing Span 20 (microcapsules No. 20). A statistically significant difference in the hardness of microcapsules was found between No. 20 and No. 5,No. 19,and No. 22  Summarizing the results, we can state that the softest microcapsules are formed containing Span 20. Microcapsules with a stronger coating can be formed using Tween 20, Tween 80 or Span 80, and the differences between the hardness with these emulsifiers are insignificant.  Table 3.
Comparing microcapsules No. 5,No. 19,No. 20,and No. 22 by the shell hardness, the lowest compressive forces were required to crush microcapsules containing Span 20 (microcapsules No. 20). A statistically significant difference in the hardness of microcapsules was found between No. 20 and No. 5,No. 19,and No. 22  Summarizing the results, we can state that the softest microcapsules are formed containing Span 20. Microcapsules with a stronger coating can be formed using Tween 20, Tween 80 or Span 80, and the differences between the hardness with these emulsifiers are insignificant. Figure 15 shows the crushing results for chitosan microcapsules prepared from different molecular weight chitosan gels.  Figure 15 shows the crushing results for chitosan microcapsules prepared from different molecular weight chitosan gels. In all cases, microcapsules containing HMW 80/1000 chitosan were harder than microcapsules containing MMW chitosan. This was due to the higher value of HMW 80/1000 gel hardness determined in the texture study.

Moisture Content in Microcapsules
Moisture content is an important factor when assessing the viability of microorganisms in a product [50]. The results are shown in Figure 16. In all cases, microcapsules containing HMW 80/1000 chitosan were harder than microcapsules containing MMW chitosan. This was due to the higher value of HMW 80/1000 gel hardness determined in the texture study.

Moisture Content in Microcapsules
Moisture content is an important factor when assessing the viability of microorganisms in a product [50]. The results are shown in Figure 16.    Table 1.
Freshly made microcapsules No. I, No. II, and No. III (containing MMW chitosan) and microcapsules No. IV, No. V, and No. VI (containing HMW 80/1000 chitosan) were compared at 2, 4, 6, and 24 h after production. A significant difference was observed in the moisture content of the microcapsules measured at different times. The moisture content of the microcapsules decreased with time. After 2 h the moisture content varied from 77.82% ± 0.99 to 87.54% ± 0.51; by 4 h, the moisture content ranged from 58.44% ± 0.72 to 70.10% ± 0.63; by 6 h, it was found to be 11.04% ± 0.34 to 14.80% ± 0.22; and after 24 h, the moisture content ranged from 4.27% ± 0.28 to 5.6% 2 ± 0.38. This means that the microcapsules lost the largest amount of moisture during the first 6 h of drying, whereas the loss over the next 18 h was much smaller. A significant difference was also found between the moisture content of microcapsules prepared by using chitosan gels with different molecular weights. Microcapsules containing HMW 80/1000 chitosan had a higher moisture content than microcapsules containing MMW chitosan. The moisture content at 24 h in microcapsules with MMW chitosan ranged from 4.27% ± 0.28% to 4.47% ± 0.19 compared to 5.65% ± 0.33 and 5.68% ± 0.04 for HMW 80/1000 chitosan.
The results for moisture content measured 24 h after microcapsule formation are shown in Table 6. moisture content of 4.38% ± 0.31. Inulin has been shown to absorb higher amounts of water [41].
Summarizing the results, we can say that microcapsules lost the largest amount of moisture between 4 and 6 h after preparation. After the final drying, the moisture content of microcapsules with MMW chitosan varied from 4.21% ± 0.36% to 4.66% ± 0.39 and that of microcapsules with HMW 80/1000 chitosan varied from 5.44% ± 0.36 to 5.69% ± 0.45, and this difference was statistically significant.

Emulsion Microscopy and Evaluation of Stability
After microscopic examination of the emulsion samples, the droplet sizes of Boswellia serrata essential oil were measured and their distribution in the emulsion was visually compared. The droplets were more densely distributed in emulsions with a higher concentration of the essential oil. A microscopic view of emulsion No. 1 (containing 2% MMW chitosan, 0.1% essential oil, and 0.5% Tween 20) is shown in Figure 17 and emulsion No. 4 (containing 2% MMW chitosan, 0.4% essential oil, and 0.5% Tween 20) is shown in Figure 18.

Emulsion Microscopy and Evaluation of Stability
After microscopic examination of the emulsion samples, the droplet sizes of Boswellia serrata essential oil were measured and their distribution in the emulsion was visually compared. The droplets were more densely distributed in emulsions with a higher concentration of the essential oil. A microscopic view of emulsion No. 1 (containing 2% MMW chitosan, 0.1% essential oil, and 0.5% Tween 20) is shown in Figure 17    The droplet size of the emulsion is an important factor influencing the stability of the emulsion and its texture [54]. Measurement of the droplet size in our emulsions showed that the mean droplet size increased as the concentration of the essential oil increased, but there was no significant difference between the results. Another study has shown that droplet size also increases as the concentration of essential oil increases in the emulsions [55]. In our study, no significant difference was found between the results when comparing emulsions prepared with different emulsifiers. The results are shown in Table 7.  The droplet size of the emulsion is an important factor influencing the stability of the emulsion and its texture [54]. Measurement of the droplet size in our emulsions showed that the mean droplet size increased as the concentration of the essential oil increased, but there was no significant difference between the results. Another study has shown that droplet size also increases as the concentration of essential oil increases in the emulsions [55]. In our study, no significant difference was found between the results when comparing emulsions prepared with different emulsifiers. The results are shown in Table 7.  8.44 ± 2.42 *** * p > 0.05 vs. emulsion containing 0.1% essential oil, ** p > 0.05 vs. emulsion containing 0.2% essential oil, *** p > 0.05 vs. emulsion, containing 0.3% essential oil; n = 3. Composition of emulsions according to their number is presented in the Table 2.
The stability of the emulsions was tested with centrifugation, which showed that all emulsions were stable. The oil-phase and aqueous phase did not separate when the emulsions were centrifuged for 5 min at 10,000 rpm. The emulsions were stable because the chitosan molecules were adsorbed at the oil-water interface, which improved emulsion stability [54].
To summarize the results, as concentration of the essential oil in the emulsions increased, the droplets were more densely distributed, and their size tended to increase without any significant differences. All of the examined emulsions were stable.

Conclusions
Our texture analysis of chitosan gels revealed that different molecular weights and concentrations of chitosan affect the values of gel hardness, consistency, stickiness, viscosity index, mobility, and adhesion. Compared to MMW and HMW chitosan gels, HMW 80/3000 chitosan gel showed significantly higher results for the texture properties. It was also found that increasing the concentration of chitosan from 2 to 4% significantly increased gel hardness, consistency, stickiness, viscosity index, mobility, and adhesion. It was found that microcapsules with a regular shape were formed with either Tween 20 and 0.1 to 0.4% Boswellia serrata essential oil, Tween 80 and up to 0.1% Boswellia serrata essential oil, or Span 20 and Span 80 and 0.1 to 0.2% Boswellia serrata essential oil. Spherical microcapsules were formed using MMW and HMW 80/1000 chitosan. Microcapsules with HMW 80/1000 chitosan were harder and contained more moisture than microcapsules with MMW chitosan (p < 0.05). When the stirring time for the microcapsules was increased to 30 min, their diameter decreased significantly and they retained a regular oval shape. The resistance of microcapsule shell to crushing increased significantly with the amount of stirring. The compression test also showed that increasing concentrations of essential oil increased the resistance of microcapsules containing 2% MMW chitosan and 0.5% Tween 20 (p < 0.05). Our study results will be important for other researchers that are working with natural active substances and microencapsulation methods. For future scope, it will be necessary to analyze the release of active ingredients (for example, Boswellia serrata essential oil), and perform a thermogravimetric analysis to obtain the best heat resistance with a constant rate of weight loss and the highest encapsulation efficiency for the potential control of the releasing property during processing. In addition, there is huge interest from industry companies that want to improve their products with active substances and apply new technologies to their manufacturing processes. Coacervation is an easily applicable encapsulation method for the food, cosmetic, and pharmaceutical industries. New active compounds, products, and innovative and simple technologies will help manufacturers and distributors become leaders in the global market.
Author Contributions: L.P. analyzed the data and wrote the manuscript. A.S. performed the experiments, analyzed the data. R.M. conceived, designed and supervised the study. R.L. conceived, designed and supervised the study. J.B. conceived, designed and supervised the study. All authors have read and agreed to the published version of the manuscript.