**3. Methodology for the formulation of nano-emulsions of citrus essential oils**

A reproducible process for the formulation of nano-emulsions of essential citrus oils is described. The initial process of preparing the nano-emulsions consists

*Nanoemulsions - Properties, Fabrications and Applications*

with methanolic KOH is performed and the amount of mL used is recorded to reach a pH of 3. In order to determine the concentration of aldehydes, the following math-

a = Volume of the potassium hydroxide solution used in the neutralization of the

meq = Milliequivalent corresponding to the carbonyl compound in which the

Aldehydes are a family of organic compounds (R▬CHO), which are indicative of the quality of essential oils, the higher the concentration of aldehydes, the higher the oil quality [29]. The released HCl is evaluated, which is related to the content of carbonyl groups in the sample and it can be calculated in grams of the aldehyde. The results are shown in **Table 1**, these are within the expected ranges (For Persian lemon is 3.5–7.5 and 0.8 1.5% for pink grapefruit) according to

**Citric essential oil %pH mL %Aldehydes** Persian lemon 3.89 9.96 3.67 Pink grapefruit 1.07 2.75 1.04

%*carbonyl compounds* = (*<sup>a</sup>* <sup>×</sup> *<sup>N</sup> meq* <sup>×</sup> 100)⁄*<sup>P</sup>*

N = Normality of the potassium hydroxide solution.

P = Weight of the sample, in grams.

**50**

**Table 1.**

ematical formula is used:

*IR spectra of the different gums.*

where

**Figure 5.**

sample in mL.

result is expressed.

literature [28].

*Concentration of Aldehydes.*

**Figure 7.**

*Process for the formulation of nano-emulsions (A) oil and water phase of nano-emulsions, (B) preparation of pre emulsion by high speed agitation, (C) high pressure process.*

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.


**53**

**Figure 8.**

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

necessary to evaluate each system previously described in **Table 2**.

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

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 distribu-

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

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;

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

**4.1 Dynamic light scattering**

tion than Control 2 (with Arabic gum).

citrus essential oil from oxidizing in the presence of light.

besides, its appearance including color did not change.

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

**4. Characterization of nano-emulsions**

#### **Table 2.** *Details of the formulation of nano-emulsions.*

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