**4. Characterization of nano-emulsions**

To demonstrate which formulation leads to an optimization of the use of the microfluidizer and natural gums in the formation of these nano-emulsions, it is necessary to evaluate each system previously described in **Table 2**.

### **4.1 Dynamic light scattering**

*Nanoemulsions - Properties, Fabrications and Applications*

*pre emulsion by high speed agitation, (C) high pressure process.*

**52**

**Table 2.**

**Figure 7.**

**Experiment Laps Oil** 

*Details of the formulation of nano-emulsions.*

**phase**

**Span 20**

Control 3 10 9.86% 1.45% 4.89% Span 20: 1.12% + Tween

**Tween 80**

of mixing the components in an Erlenmeyer flask using magnetic stirring in a water bath at 40°C for 10 minutes. Followed by high speed mechanical agitation (8000 rpm) for 5 minutes. This first step helps to dissolve the gums and/or surfactants in the aqueous phase as much as possible, and thus it eliminates any possible lumps that could lead to plugging in the microfluidizer interaction chamber. In addition, this step allows to form a pre-emulsion, that is, the oil phase is dispersed as droplets in the aqueous continuous phase; however, at this point, the droplets are micrometric in size and therefore the pre-emulsions have a milky appearance. This pre-emulsion is then introduced into the high pressure homogenizer (microfluidizer), and subjected to high pressure (the nano-emulsions were subjected to pressures ranging from 10,000 to 30,000 psi) collecting a sample every 1, 3, 5 and 10 laps. In this high pressure process, the droplet size of the nano-emulsion decreases as the number of times the nano-emulsion is introduced to the equipment increases (number of turns or laps or steps), although sometimes the droplet size increases again when the number of laps increases up to a certain value, due to degradation of gums or surfactants. Finally, the nano-emulsion sample is collected and prepared to be sterilized. In **Figure 7**, a summary of the process is presented. After a series of experiments varying concentrations, applied pressure and number of laps, it was possible to obtain a visually appropriate formulation with relative stability, verified by the characterizations. Therefore, comparison controls are generated, which are described in **Table 2**, replacing mesquite gum with Arabic gum, Tween 80 and Span 20 surfactants, and finally the substitution of mesquite gum for deionized water. These controls were defined in this way to investigate if mesquite gum would have an influence on the characteristics and kinetic stability of the nanoemulsion. Samples of these controls were taken at 1, 3, 5 and 10 steps, **Table 2** shows the samples that resulted in the best size distribution and best visual appearance.

*Process for the formulation of nano-emulsions (A) oil and water phase of nano-emulsions, (B) preparation of* 

Delta control 10 9.86% 1.45% 4.89% Mesquite gum: 4.93% 78.86% 20,000 Control 2 3 9.86% 1.45% 4.89% Arabic gum: 4.93% 78.86% 20,000

Control 4 5 9.86% 1.45% 4.89% Deionized water 83.76% 20,000

**Variable Deionized** 

80: 3.81%

**water**

8.86% 20,000

**PSI**

When comparing these controls against the Delta Control nano-emulsion in **Figure 8** it is observed that Control 3 with 10 turns in the homogenizer presents a smaller size, in this case a single population with a size of 19 nm, but it has a higher polydispersity index (PDI of 0.143). This is attributed to its composition based solely on surfactants Tween 80 and Span 20 with high HLB. On the other hand, Control 4 nano-emulsion, which does not contain gum, and only carries 5% of surfactants Tween 80/Span 20, had a droplet size of 25.3 nm, a lower size than Control Delta but larger than Control 3. With these experiments, it can be implied that the components responsible for the small drop size are the surfactants Tween 80 and Span 20, since greater interfacial activity with the presence of these surfactants was expected. In addition to increasing the concentration of these surfactants (Control 3) produces a greater interfacial area and a smaller drop size. Finally, it is observed that the Control 2 experiment (control using Arabic gum instead of mesquite gum), has a droplet size of 46.8 nm, very similar to Control Delta, which shows that the mesquite gum, in effect, has a very similar performance to that of Arabic gum (in terms of the droplet size). It should be noted that Control Delta nano-emulsion (with mesquite gum) has a narrower size distribution than Control 2 (with Arabic gum).

A small initial droplet size is not a guarantee that the kinetic stability will be better compared to nano-emulsions with a larger droplet size. For this reason, nanoemulsions were monitored in order to see if there was an increase in the droplet size or an increase in the number of populations, which would indicate instability or some other problem such as cremation, sedimentation, flocculation, or some change in coloration or appearance, which would reduce shell life. All the samples were refrigerated at 4°C and were wrapped in aluminum foil in order to prevent the citrus essential oil from oxidizing in the presence of light.

The first monitoring study to be discussed is Control Delta (**Figure 9**), which represents the best formulation that includes mesquite gum. It was observed to be stable for 4 months. The PDI varied between 0.071 and 0.091 and the sizes vary from 41 to 46 nm, therefore it is considered to be a very stable nano-emulsion; besides, its appearance including color did not change.

**Figure 8.** *Graph of volume size distribution in DLS of nano-emulsions.*

**Figure 9.** *DLS graph of the 4-month follow-up of Delta Control.*

Therefore, a certain percentage of the droplets increased slightly in size, however, there appeared to be no coalescence since the size remained almost constant. On the other hand, the fact that no additional larger populations were produced, unlike the other controls, as shown below, is indicative of the appropriate steric stabilization conferred by mesquite gum.

The next control that was evaluated is the one that incorporates Arabic gum instead of mesquite gum (control 2). From the first series evaluated on this research, we have seen that the nano-emulsions that incorporate Arabic gum developed during this research do not present a very good stability performance, this is confirmed with the results shown in **Figure 10**, where there appears to be an apparent gradual reduction of the droplet size (~25 nm) but with an increase of the PDI from 0.115 to 0.247, to finally increase again at the fourth month (droplet size of ~35 nm). The greatest sign of instability is the presence of other populations with a size around 3 μm that occurs in parallel to the apparent reduction in droplet by the second month, which is attributed to the phenomenon of Ostwald ripening [36], which is one of the main mechanisms of instability in nano-emulsions. This experiment confirms that mesquite gum has advantages over Arabic gum in the formulation of the nano-emulsions of Persian lemon oil. The mesquite gum confers a better steric stabilization as compared to Arabic gum, improving the kinetic stability of the nano-emulsion.

In the follow-up of Control 3 shown in **Figure 11** (nano-emulsion with excess of Tween 80 and Span 20, and without mesquite gum), it seems to be a very stable nano-emulsion, however with each measurement the droplet size, the presence of larger size populations and the PDI increase. For example, from the second month, it evolves from having a single population of 19.0 nm, to having the population of 20.7 nm in coexistence with populations of 467 and 5033 nm, which do not occur in Control Delta. Therefore, it is deduced that the presence of the mesquite gum helps to maintain the stability of the nano-emulsions, providing an additional steric stabilization against coalescence.

**55**

**Figure 12.**

*Development of Nano-Emulsions of Essential Citrus Oil Stabilized with Mesquite Gum*

Control 4 nano-emulsion incorporates only 5% of surfactant, a mixture of Tween 80/Span 20, and mesquite gum was not included, therefore its water content increases to 85% (as mesquite gum is replaced by water). This control was formulated in order to verify if there is any effect on the size of the nano-emulsion and its stability with the presence of the mesquite gum. This is observed in **Figure 12**, where first, apparent reductions followed by increases in the size of the droplets are seen (droplet size range from ~15 to ~20 nm with PDI range from 0.165 to 0.453), with the presence of large droplet populations, with size around 2000–4000 nm by the fourth month. This may be attributed to a combination of Ostwald ripening and

In general, the results from the experiments described above showed that nanoemulsions without mesquite gum result in droplet sizes which are smaller than those obtained with samples that include mesquite gum in their formulation, but better kinetic stability and smaller PDI are obtained with the nano-emulsions that contain mesquite gum. This is attributed to the additional steric stability conferred by mesquite gum, which results it a better kinetic stability, since there is virtually no droplet size growth. An arrangement of the different surfactants and stabilizers at the droplet interface is proposed in the scheme of **Figure 13**. It is proposed that the larger hydrodynamic size obtained with mesquite gum is due to its location at the surface of the droplet, where carbohydrate chains of the gum can interact with the sorbitan

Next, the results of the antibacterial activity tests of Control Delta, Control 2,

Control 3 and Control 4 nano-emulsions are shown (in this study, each

*DOI: http://dx.doi.org/10.5772/intechopen.84157*

coalescence destabilization phenomena.

*DLS graph of the 4-month follow-up of Control 3.*

**Figure 11.**

groups and EO groups of Tween 80.

**4.2 Minimum inhibitory concentration**

*DLS graph of the 4-month follow-up of Control 4.*

**Figure 10.** *DLS graph of the 4-month follow-up of Control 2.*

*Development of Nano-Emulsions of Essential Citrus Oil Stabilized with Mesquite Gum DOI: http://dx.doi.org/10.5772/intechopen.84157*

**Figure 11.** *DLS graph of the 4-month follow-up of Control 3.*

*Nanoemulsions - Properties, Fabrications and Applications*

stabilization conferred by mesquite gum.

*DLS graph of the 4-month follow-up of Delta Control.*

**Figure 9.**

Therefore, a certain percentage of the droplets increased slightly in size, however, there appeared to be no coalescence since the size remained almost constant. On the other hand, the fact that no additional larger populations were produced, unlike the other controls, as shown below, is indicative of the appropriate steric

The next control that was evaluated is the one that incorporates Arabic gum instead of mesquite gum (control 2). From the first series evaluated on this research, we have seen that the nano-emulsions that incorporate Arabic gum developed during this research do not present a very good stability performance, this is confirmed with the results shown in **Figure 10**, where there appears to be an apparent gradual reduction of the droplet size (~25 nm) but with an increase of the PDI from 0.115 to 0.247, to finally increase again at the fourth month (droplet size of ~35 nm). The greatest sign of instability is the presence of other populations with a size around 3 μm that occurs in parallel to the apparent reduction in droplet by the second month, which is attributed to the phenomenon of Ostwald ripening [36], which is one of the main mechanisms of instability in nano-emulsions. This experiment confirms that mesquite gum has advantages over Arabic gum in the formulation of the nano-emulsions of Persian lemon oil. The mesquite gum confers a better steric stabilization as compared to Arabic gum, improving the kinetic stability of the nano-emulsion. In the follow-up of Control 3 shown in **Figure 11** (nano-emulsion with excess of Tween 80 and Span 20, and without mesquite gum), it seems to be a very stable nano-emulsion, however with each measurement the droplet size, the presence of larger size populations and the PDI increase. For example, from the second month, it evolves from having a single population of 19.0 nm, to having the population of 20.7 nm in coexistence with populations of 467 and 5033 nm, which do not occur in Control Delta. Therefore, it is deduced that the presence of the mesquite gum helps to maintain the stability of the nano-emulsions, providing an additional steric

**54**

**Figure 10.**

*DLS graph of the 4-month follow-up of Control 2.*

stabilization against coalescence.

Control 4 nano-emulsion incorporates only 5% of surfactant, a mixture of Tween 80/Span 20, and mesquite gum was not included, therefore its water content increases to 85% (as mesquite gum is replaced by water). This control was formulated in order to verify if there is any effect on the size of the nano-emulsion and its stability with the presence of the mesquite gum. This is observed in **Figure 12**, where first, apparent reductions followed by increases in the size of the droplets are seen (droplet size range from ~15 to ~20 nm with PDI range from 0.165 to 0.453), with the presence of large droplet populations, with size around 2000–4000 nm by the fourth month. This may be attributed to a combination of Ostwald ripening and coalescence destabilization phenomena.

In general, the results from the experiments described above showed that nanoemulsions without mesquite gum result in droplet sizes which are smaller than those obtained with samples that include mesquite gum in their formulation, but better kinetic stability and smaller PDI are obtained with the nano-emulsions that contain mesquite gum. This is attributed to the additional steric stability conferred by mesquite gum, which results it a better kinetic stability, since there is virtually no droplet size growth. An arrangement of the different surfactants and stabilizers at the droplet interface is proposed in the scheme of **Figure 13**. It is proposed that the larger hydrodynamic size obtained with mesquite gum is due to its location at the surface of the droplet, where carbohydrate chains of the gum can interact with the sorbitan groups and EO groups of Tween 80.

#### **4.2 Minimum inhibitory concentration**

Next, the results of the antibacterial activity tests of Control Delta, Control 2, Control 3 and Control 4 nano-emulsions are shown (in this study, each

**Figure 12.** *DLS graph of the 4-month follow-up of Control 4.*

#### **Figure 13.**

*Schematic of the proposed arrangement of surfactants and stabilizers at the interface of the nano-emulsion droplets.*

nano-emulsion was evaluated at 1, 3, 5 and 10 steps into the microfluidizer). To determine the MIC (minimum inhibitory concentration) of the nano-emulsions against the test organisms *Escherichia coli* and *Staphylococcus aureus* the broth microdilution method was used as recommended by the National Committee for Clinical Laboratory standards. This test was performed in sterile 96-well microplates. The nano-emulsions were properly prepared and transferred to each microplate into two lines in order to verify reproducibility. The inoculate (10 μL) containing 5x105 CFU (colony-forming unit) of each microorganism was added to each well. A number of wells were reserved in each plate to test for sterility control (no inoculate added), inoculate viability (no nano-emulsion added), and the nano-emulsion inhibitory effect. Plates were aerobically incubated at 35°C. After incubation for 18–24 h, bacterial growth was evaluated by the presence of turbidity and a pellet formed at the bottom of the well. MIC was defined as the lowest concentration of nano-emulsions that had no macroscopically visible growth. A sterilization process was applied to the nano-emulsion samples prior to MIC studies, in order to ensure that no previous contamination was present in the samples.

**Table 3** shows the MIC results for nano-emulsions sterilized during 40 minutes under UV light corresponding to *Escherichia coli* and **Table 4** shows the MIC results corresponding to *Staphylococcus aureus*. Additionally, a nano-emulsion with the same composition and processing of Control Delta was prepared, but replacing the Persian lemon essential oil with industrial D-limonene, since this component could be the active bactericidal component.

In general, the best result of MIC was obtained with *Staphylococcus aureus*. The delta control nano-emulsion resulted in a MIC of 6.25% for both bacteria. Therefore, with these results it was confirmed that the nano-emulsions of Persian lemon oil developed under the method described in this research have an antibacterial effect against *Staphylococcus aureus* and *Escherichia coli.*


**57**

**Figure 14.**

*Development of Nano-Emulsions of Essential Citrus Oil Stabilized with Mesquite Gum*

Control Limonene All steps 25 Control Delta All steps 6.25 Control 2 All steps 25

> 3 5 10

5, 10

**Nano-emulsion Steps in Homogenizer MIC (% of concentration of the nano-emulsion)**

1.56 3.12 6.25 12.5

3.12 6.25

Considering that the nano-emulsions contain 10% Persian lemon oil, the MIC of this essential oil could be considered as 0.625% for both *Staphylococcus aureus* and *Escherichia coli* and (taking into account the composition of Control Delta samples). Considering the best results, we have a MIC of 0.625% for Control Delta, Control 3 and Control 4 for *Escherichia coli* and 0.156% for Control 3 and *Staphylococcus aureus*; it is inferred then, that these nano-emulsions (Control 2, 3 and 4), which present smaller droplet sizes, their antibacterial power can be attributed to a greater interfacial area, since there is a greater contact area between Persian lemon oil and bacteria. However, due to the better kinetic stability of Control Delta nano-emulsion, it is considered as more promising. Some adjustments could be made to improve these results, such as increasing the concentration of Tween 80/Span 20 in Control Delta in order to reduce the size, but maintaining the presence of mesquite gum in order to

*MIC results for nano-emulsions sterilized during 40 minutes. Bacteria used: Staphylococcus aureus.*

When carrying out the antibacterial activity tests, it was observed that when the nano-emulsions were subjected to treatment with UV light, they became slightly more transparent, so it was suspected that the UV light radiation can cause a reduction in the droplet size. In **Figures 14** and **15**, we observe the effect of the UV light treatment on the two nano-emulsions with better kinetic stability behavior and droplet size. In the case of Control 3 nano-emulsion at 10 steps (formulation without mesquite gum, but with additional Tween 80 and Span 20), there was no significant reduction in size (from 1 to 0.5 nm reduction), after sterilization treatment at different exposure times. However, for Control Delta nano-emulsion there was a reduction of approximately 10 nm after treatment with UV light, which may indicate that the mesquite gum is being broken into smaller carbohydrate chains, or

*DOI: http://dx.doi.org/10.5772/intechopen.84157*

Control 3 1

Control 4 1, 3

**Table 4.**

preserve the steric stability conferred by it.

*Size distribution chart by volume of control 3 10 steps with UV treatment.*

**Table 3.**

*MIC results for nano-emulsions sterilized during 40 minutes. Bacterium used: Escherichia coli.*


*Development of Nano-Emulsions of Essential Citrus Oil Stabilized with Mesquite Gum DOI: http://dx.doi.org/10.5772/intechopen.84157*

#### **Table 4.**

*Nanoemulsions - Properties, Fabrications and Applications*

contamination was present in the samples.

be the active bactericidal component.

Control 3 1, 3, 5

nano-emulsion was evaluated at 1, 3, 5 and 10 steps into the microfluidizer). To determine the MIC (minimum inhibitory concentration) of the nano-emulsions against the test organisms *Escherichia coli* and *Staphylococcus aureus* the broth microdilution method was used as recommended by the National Committee for Clinical Laboratory standards. This test was performed in sterile 96-well microplates. The nano-emulsions were properly prepared and transferred to each microplate into two lines in order to verify reproducibility. The inoculate (10 μL) containing 5x105 CFU (colony-forming unit) of each microorganism was added to each well. A number of wells were reserved in each plate to test for sterility control (no inoculate added), inoculate viability (no nano-emulsion added), and the nano-emulsion inhibitory effect. Plates were aerobically incubated at 35°C. After incubation for 18–24 h, bacterial growth was evaluated by the presence of turbidity and a pellet formed at the bottom of the well. MIC was defined as the lowest concentration of nano-emulsions that had no macroscopically visible growth. A sterilization process was applied to the nano-emulsion samples prior to MIC studies, in order to ensure that no previous

*Schematic of the proposed arrangement of surfactants and stabilizers at the interface of the nano-emulsion* 

**Table 3** shows the MIC results for nano-emulsions sterilized during 40 minutes under UV light corresponding to *Escherichia coli* and **Table 4** shows the MIC results corresponding to *Staphylococcus aureus*. Additionally, a nano-emulsion with the same composition and processing of Control Delta was prepared, but replacing the Persian lemon essential oil with industrial D-limonene, since this component could

In general, the best result of MIC was obtained with *Staphylococcus aureus*. The delta control nano-emulsion resulted in a MIC of 6.25% for both bacteria. Therefore, with these results it was confirmed that the nano-emulsions of Persian lemon oil developed under the method described in this research have an antibacte-

**Nano-emulsion Steps in Homogenizer MIC (% of concentration of the nano-emulsion)**

6.25 12.5

rial effect against *Staphylococcus aureus* and *Escherichia coli.*

Control Limonene All steps 25 Control Delta All steps 6.25 Control 2 All steps 25

10

Control 4 All steps 6.25

*MIC results for nano-emulsions sterilized during 40 minutes. Bacterium used: Escherichia coli.*

**56**

**Table 3.**

**Figure 13.**

*droplets.*

*MIC results for nano-emulsions sterilized during 40 minutes. Bacteria used: Staphylococcus aureus.*

Considering that the nano-emulsions contain 10% Persian lemon oil, the MIC of this essential oil could be considered as 0.625% for both *Staphylococcus aureus* and *Escherichia coli* and (taking into account the composition of Control Delta samples). Considering the best results, we have a MIC of 0.625% for Control Delta, Control 3 and Control 4 for *Escherichia coli* and 0.156% for Control 3 and *Staphylococcus aureus*; it is inferred then, that these nano-emulsions (Control 2, 3 and 4), which present smaller droplet sizes, their antibacterial power can be attributed to a greater interfacial area, since there is a greater contact area between Persian lemon oil and bacteria. However, due to the better kinetic stability of Control Delta nano-emulsion, it is considered as more promising. Some adjustments could be made to improve these results, such as increasing the concentration of Tween 80/Span 20 in Control Delta in order to reduce the size, but maintaining the presence of mesquite gum in order to preserve the steric stability conferred by it.

When carrying out the antibacterial activity tests, it was observed that when the nano-emulsions were subjected to treatment with UV light, they became slightly more transparent, so it was suspected that the UV light radiation can cause a reduction in the droplet size. In **Figures 14** and **15**, we observe the effect of the UV light treatment on the two nano-emulsions with better kinetic stability behavior and droplet size. In the case of Control 3 nano-emulsion at 10 steps (formulation without mesquite gum, but with additional Tween 80 and Span 20), there was no significant reduction in size (from 1 to 0.5 nm reduction), after sterilization treatment at different exposure times. However, for Control Delta nano-emulsion there was a reduction of approximately 10 nm after treatment with UV light, which may indicate that the mesquite gum is being broken into smaller carbohydrate chains, or

**Figure 14.** *Size distribution chart by volume of control 3 10 steps with UV treatment.*

**Figure 15.** *Size distribution graph by volume of control delta 10 steps with UV treatment.*

that rearrangement of the carbohydrate chains is taking place, thereby reducing the hydrodynamic droplet size.

On the other hand, although the literature indicates that it is not that clear which of the components of the citrus essential oils is the cause of the antibacterial effect, the nano-emulsion with industrial D-limonene results in a higher MIC than Control Delta which is prepared with Persian lemon oil; thus, it may be inferred that the aldehydes of the Persian lemon oil could be the components that are mainly responsible for this effect, as compared to the terpene components.
