**Abstract**

The microencapsulation technology consists of a trap of a compound inside a tiny sphere known as microsphere. The microencapsulation concerns many different active materials such as bioactive compounds, drugs, vitamins, enzymes, flavors, and pesticides. This technology has gained real interest in numerous fields such as agriculture, cosmetic, pharmaceutical, textile, and food. This chapter highlights the encapsulation of essential oils into nanoemulsion-based delivery system as a model for the encapsulation of natural bioactive compounds. Moreover, an investigation of different parameters affecting the stability of produced nanoemulsion was conducted, in addition to the study of the effect of the nanoencapsulation of essential oils on their antibacterial activity. Finally, an enumeration of the advantages of encapsulating essential oils into nanoemulsion-based delivery systems in order to provide a natural food preservatives has been provided.

**Keywords:** encapsulation, nanoemulsion, essential oil, formulation, stability, antibacterial activity, food preservation

## **1. Introduction**

In recent years, natural antimicrobials attracted consumer attention due to the increased awareness regarding food safety. In this context, new approaches have been adopted in the food preservation field. This includes the use of natural compounds with proven antibacterial activities, like essential oils, as safe preservatives. However, the incorporation of essential oils in foods is not economically and practically ideal. As a matter of fact, essential oils are not only volatile and chemically unstable in the presence of air, light, moisture and high temperatures, but also present hydrophobic properties*.*

With this respect, the nanoencapsulation of essential oils seems to be an attractive new approach to overcome these impediments.

Since the design of essential-oil-loaded particles is a complex process with interrelated steps [1], choosing encapsulating material and the encapsulation method should be in agreement with the intended matrix in which essential oils are to be introduced [2]. Basically, the nanoencapsulation of essential oils may imply coat, polymeric material, etc. to trap the core material in order to fix listed limitations of using essential oils as natural food preservative. Accordingly, different methods

could be adopted for the nanoencapsulation of essential oils, such as nanoemulsion, liposomes, cyclodextrin, etc. In the specific case of essential oil nanoemulsion, the preparation consists on a biphasic liquid system of one liquid solution dispersed in a continuous medium and no polymer shells are used. The immobilization of essential oils in nanoemulsions contributes efficiently to enhance their dispersibility in aqueous solutions, to protect them from interaction with food ingredients, to minimize their impact on the organoleptic properties, as well as to improve their absorption and bioavailability. In this context, numerous researches have been conducted on the nanoencapsulation of essential oils on the purpose of producing a natural powerful food conservator. Gathered data demonstrated that inappropriate formulation, due to a misunderstanding of the process of essential oil encapsulation, can lead to the instability and/or the inefficiency of the produced emulsion.

In this context, the purpose of this chapter is to better understand the phenomenon of encapsulating essential oils into nanoemulsion-based delivery systems. This would widen the knowledge of possible alternatives to consider while designing green food preservatives for future research. Accordingly, this chapter covers firstly a general description of the nanoemulsion delivery systems. Then, an enumeration of common parameters, often used in essential oil nanoemulsion characterization, was conducted. The third part of this chapter involves different adopted methods for the preparation of essential oil nanoemulsion. Moreover, the most relevant parameters affecting the nanoemulsion quality and stability were investigated. Also a special emphasis to the effect of the nanoencapsulation of essential oils on their antibacterial activity was provided. Finally, data on the efficiency of encapsulated essential oils into nanoemulsion-based delivery systems as natural food preservatives have been provided.

### **2. Nanoemulsion-based delivery system as an example of bioactive compound encapsulation**

According to the theory of emulsification, an emulsion is a thermodynamically unstable system consisting of at least two immiscible liquid phases, one of which is dispersed as globules in the other liquid phase [3]. Emulsions can be stabilized by increasing the repulsion between the dispersed and the continuous phases. As a matter of fact, the emulsion formation is a nonspontaneous phenomenon, which requires energy along with the use of emulsifiers. As a matter of fact, emulsifiers are amphiphile molecules that reduce the interfacial tension between the two phases and contribute to the stabilization of dispersed droplets with electrostatic or steric effects [4].

According to the proportion of each used liquid, an emulsion can be considered either as oil in water (O/W) or as water in oil (W/O) emulsion [2]. Indeed, if the oil droplets are dispersed throughout the aqueous phase, the emulsion is called oil-inwater (O/W). In the opposite case, where the water is dispersed as globules in the oil continuous phase, the emulsion is called water-in-oil emulsion (W/O).

It is worthy to mention the increasing interest accorded to multiple emulsions [1]. In this complex type of emulsion system, the W/O or O/W emulsions are dispersed in another immiscible liquid.

Accordingly, O/W/O emulsion is formed by very small oil droplets dispersed in water globules of a W/O emulsion, and W/O/W emulsion is formed by water droplets dispersed in the oil phase of an O/W emulsion (**Figure 1**). Multiple emulsion can be formed by a multistep mechanism. Actually, the deepest drop is formed in the first drop maker and then encapsulated in the next drop maker. In general, multiple emulsions present many advantages such as (1) a good ability to carry both hydrophilic and hydrophobic bioactive ingredients simultaneously, (2) high

**47**

*Encapsulation of Natural Bioactive Compounds: Nanoemulsion Formulation to Enhance…*

protection of sensitive bioactive molecules from gastrointestinal harsh conditions, and (3) sheltering essential oil's strong taste and smell [1]. Besides their importance, multiple emulsions present some limitations due to their complex structure and

*Schematic representation of the architectures of O/W/O (a) and W/O/W (b) multiple emulsions.*

Another special case of emulsion is nanoemulsion. Actually, nanoemulsions are isotropic, clear, and kinetically stable with droplet size inferior to 200 nm [4]. Two types of techniques could be adopted for nanoemulsion preparation: high-energy methods and low-energy methods [6]. This encapsulation method is gaining more and more interest in the scientific community due to its high stability, as compared to emulsions of larger droplet size [7]. Actually, nanoemulsion stability results of its nanoscale droplet size and its large surface area and free energy. With this respect, essential oil nanoemulsion can be formed by the encapsulation of essential oil as the dispersed phase at a nanoscale level.

• solubilize hydrophobic bioactive molecules and enhance their bioavailability;

In the last few decades, essential oil nanoemulsions have found enormous applications in the field of healthcare, cosmetics, food, agrochemicals, pharmaceuticals,

The encapsulation of essential oils in nanoemulsion-based delivery system

i.*Oils*: they are used to solubilize the lipophilic bioactive compound and to modulate the viscosity ratio between the dispersed and the continuous phases [8]. The commonly used oils in formulating essential oil food grade nanoemulsions are soyabean oil, ethyl oleate, sesame oil, castor oil, arachis oil, and

The main advantages of essential oil nanoemulsions are:

*DOI: http://dx.doi.org/10.5772/intechopen.84183*

their thermodynamic instability [5].

**Figure 1.**

• it possess high kinetic stability;

• can be used for taste masking;

• nontoxic and nonirritant; and

and biotechnology.

corn oil.

• suitable for human and veterinary use.

**2.1 Essential oil nanoemulsion components**

requires basically oil, emulsifier, and aqueous phase:

*Encapsulation of Natural Bioactive Compounds: Nanoemulsion Formulation to Enhance… DOI: http://dx.doi.org/10.5772/intechopen.84183*

**Figure 1.**

*Microencapsulation - Processes, Technologies and Industrial Applications*

could be adopted for the nanoencapsulation of essential oils, such as nanoemulsion, liposomes, cyclodextrin, etc. In the specific case of essential oil nanoemulsion, the preparation consists on a biphasic liquid system of one liquid solution dispersed in a continuous medium and no polymer shells are used. The immobilization of essential oils in nanoemulsions contributes efficiently to enhance their dispersibility in aqueous solutions, to protect them from interaction with food ingredients, to minimize their impact on the organoleptic properties, as well as to improve their absorption and bioavailability. In this context, numerous researches have been conducted on the nanoencapsulation of essential oils on the purpose of producing a natural powerful food conservator. Gathered data demonstrated that inappropriate formulation, due to a misunderstanding of the process of essential oil encapsulation, can lead to the instability and/or the inefficiency of the produced emulsion. In this context, the purpose of this chapter is to better understand the phenomenon of encapsulating essential oils into nanoemulsion-based delivery systems. This would widen the knowledge of possible alternatives to consider while designing green food preservatives for future research. Accordingly, this chapter covers firstly a general description of the nanoemulsion delivery systems. Then, an enumeration of common parameters, often used in essential oil nanoemulsion characterization, was conducted. The third part of this chapter involves different adopted methods for the preparation of essential oil nanoemulsion. Moreover, the most relevant parameters affecting the nanoemulsion quality and stability were investigated. Also a special emphasis to the effect of the nanoencapsulation of essential oils on their antibacterial activity was provided. Finally, data on the efficiency of encapsulated essential oils into nanoemulsion-based delivery systems as natural food preserva-

**2. Nanoemulsion-based delivery system as an example of bioactive** 

the stabilization of dispersed droplets with electrostatic or steric effects [4].

oil continuous phase, the emulsion is called water-in-oil emulsion (W/O).

According to the theory of emulsification, an emulsion is a thermodynamically unstable system consisting of at least two immiscible liquid phases, one of which is dispersed as globules in the other liquid phase [3]. Emulsions can be stabilized by increasing the repulsion between the dispersed and the continuous phases. As a matter of fact, the emulsion formation is a nonspontaneous phenomenon, which requires energy along with the use of emulsifiers. As a matter of fact, emulsifiers are amphiphile molecules that reduce the interfacial tension between the two phases and contribute to

According to the proportion of each used liquid, an emulsion can be considered either as oil in water (O/W) or as water in oil (W/O) emulsion [2]. Indeed, if the oil droplets are dispersed throughout the aqueous phase, the emulsion is called oil-inwater (O/W). In the opposite case, where the water is dispersed as globules in the

It is worthy to mention the increasing interest accorded to multiple emulsions [1]. In this complex type of emulsion system, the W/O or O/W emulsions are

Accordingly, O/W/O emulsion is formed by very small oil droplets dispersed in water globules of a W/O emulsion, and W/O/W emulsion is formed by water droplets dispersed in the oil phase of an O/W emulsion (**Figure 1**). Multiple emulsion can be formed by a multistep mechanism. Actually, the deepest drop is formed in the first drop maker and then encapsulated in the next drop maker. In general, multiple emulsions present many advantages such as (1) a good ability to carry both hydrophilic and hydrophobic bioactive ingredients simultaneously, (2) high

**46**

tives have been provided.

**compound encapsulation**

dispersed in another immiscible liquid.

*Schematic representation of the architectures of O/W/O (a) and W/O/W (b) multiple emulsions.*

protection of sensitive bioactive molecules from gastrointestinal harsh conditions, and (3) sheltering essential oil's strong taste and smell [1]. Besides their importance, multiple emulsions present some limitations due to their complex structure and their thermodynamic instability [5].

Another special case of emulsion is nanoemulsion. Actually, nanoemulsions are isotropic, clear, and kinetically stable with droplet size inferior to 200 nm [4]. Two types of techniques could be adopted for nanoemulsion preparation: high-energy methods and low-energy methods [6]. This encapsulation method is gaining more and more interest in the scientific community due to its high stability, as compared to emulsions of larger droplet size [7]. Actually, nanoemulsion stability results of its nanoscale droplet size and its large surface area and free energy. With this respect, essential oil nanoemulsion can be formed by the encapsulation of essential oil as the dispersed phase at a nanoscale level.

The main advantages of essential oil nanoemulsions are:


In the last few decades, essential oil nanoemulsions have found enormous applications in the field of healthcare, cosmetics, food, agrochemicals, pharmaceuticals, and biotechnology.

#### **2.1 Essential oil nanoemulsion components**

The encapsulation of essential oils in nanoemulsion-based delivery system requires basically oil, emulsifier, and aqueous phase:

i.*Oils*: they are used to solubilize the lipophilic bioactive compound and to modulate the viscosity ratio between the dispersed and the continuous phases [8]. The commonly used oils in formulating essential oil food grade nanoemulsions are soyabean oil, ethyl oleate, sesame oil, castor oil, arachis oil, and corn oil.


#### **2.2 Nanoemulsion characterization**

As detailed in the literature, the majority of the researches, dealing with the essential oil encapsulation into nanoemulsion-based delivery system, have considered that the droplet size and distribution measurements as the most important parameters for their nanoemulsion characterization [4, 6, 11]. Measurements could be determined by laser light scattering, and obtained results are expressed in terms of mean particle size, which is usually represented [11] with Sauter mean diameter, *d*3,2 (expressed in nm), calculated using the following equation:

$$\mathbf{d}\_{3,2} \text{= (Volume/Surface Area)} = (\Sigma \{ \mathbf{n}\_i^\* \, ^\circ \mathbf{d}\_i \}^3) / (\Sigma \{ \mathbf{n}\_i^\* \, ^\circ \mathbf{d}\_i \}^2) \tag{1}$$

where ni is the number of droplets and di is the droplet diameter.

In addition to these two listed parameters, the stability of essential oil nanoemulsion, which means its ability to resist physicotemporal changes, could be investigated by storing nanoemulsions at different temperatures and periods and measuring at each time point the droplet size variation [12]. Also, some researches have measured the viscosity, the density, the color, the turbidity as physical characterization of their nanoemulsion [13, 14], while others have focused their researches on the investigation of the biological activities of produced nanoemulsions [11, 15].

#### **2.3 Methods of nanoemulsion preparation**

The laborious step of formulation aims to produce stable nanoemulsion. Indeed, any emulsion system, if inappropriately formulated, may be subject to a variety of physicochemical phenomena, which can seriously affect the stability and the biological efficiency of the produced nanoemulsion [16].

A well-homogenized and stable emulsion whose droplet diameters figure in the nanoscale level can only be obtained under a complex alliance between physical and chemical forces [17]. As a matter of fact, the exclusive use of physical forces remains often insufficient to obtain stable nanoemulsions. The aim role of physical forces is to reduce the size of the dispersed phase droplets at a certain level, while chemical interactions between different medium components interfere to maintain newly formed droplets from fusing together.

**49**

**Table 1.**

*Encapsulation of Natural Bioactive Compounds: Nanoemulsion Formulation to Enhance…*

From a chemical point of view, the interfacial tension remains a critical parameter in the process of essential oil nanoemulsification. As a matter of fact, interfacial tension is known as the inward attraction of molecules at the surface of immiscible liquids due to the imbalance of their attractive forces [18]*.* With this respect, the nanoemulsion formation can occur only if the interfacial tension between the two immiscible phases decreased sufficiently to assure their mixture [11]. To reach such change in interfacial tension, an appropriate amount of an appropriate emulsifier should be used to surround and stabilize all neo-formed nanodroplets. Indeed, an emulsifier is not only able to reduce the interfacial tension of the two immiscible phases, but also it presents an effective stabilizer for the newly formed droplets [19]. In this way, the surfactants convert large globules into small ones and avoid small globules from coalescing into large ones, by reducing the repellent force between the liquids and withdrawing the attraction of liquids for their own molecules [20]. It is worthy to mention that to ensure its fundamental role in the nanoemulsification process, surfactants should be used at a higher concentration than its critical micellar concentration "CMC." As a matter of fact, the increase of surfactant concentrations on oil-water interface increases the adsorption of surfactant molecules, leading to the improvement of their ability to reduce the interfacial tension. Once the adsorption saturation is reached at the oil-water interface, the adsorption of surfactant molecules would stop increasing; thus, the interfacial tension remains

Physical emulsification is one of the most crucial steps in nanoencapsulation process since it affects deeply the quality of the final emulsion (encapsulation efficiency, nanoemulsion stability, or biological efficacy). Different homogenization methods, as detailed in **Table 1**, can be used such as high-pressure homogenization,

> **Obtained particle size (nm)**

**Disadvantages References**

[23]

[24]

110 - High production costs [11]

175 [2]


97 [15]

during the process due to high shear forces and

121 [25]

21 - High production costs

187 - Heat generation

cavitations

ultrasonic homogenization, and microfluidization [22].

*Thymus capitatus* essential oil

**bioactive compound**

*Melaleuca alternifolia* essential

*caryophyllata* essential oil

Lemon myrtle essential oil

*Thymus vulgaris* essential oil

*The different devices often used for the nanoencapsulation of essential oils.*

oil

Sonication Rosemary essential oil

**Devices Encapsulated** 

Microfluidization *Eugenia* 

*DOI: http://dx.doi.org/10.5772/intechopen.84183*

*2.3.1 Chemical forces*

constant [21].

*2.3.2 Physical forces*

High-pressure homogenization *Encapsulation of Natural Bioactive Compounds: Nanoemulsion Formulation to Enhance… DOI: http://dx.doi.org/10.5772/intechopen.84183*

### *2.3.1 Chemical forces*

*Microencapsulation - Processes, Technologies and Industrial Applications*

of newly formed drops.

**2.2 Nanoemulsion characterization**

**2.3 Methods of nanoemulsion preparation**

formed droplets from fusing together.

biological efficiency of the produced nanoemulsion [16].

ii.*Emulsifiers*: emulsifiers are amphiphilic molecules composed of two parts, polar and nonpolar regions [9]. According to their polar group nature, emulsifiers can also be classified into: anionic, cationic, nonionic, and zwitterionic emulsifiers. By lowering the interfacial tension between the two immiscible liquids, emulsifiers contribute significantly in the formulation of essential oil nanoemulsions [9]. Furthermore, they prevent coalescence

iii.*Aqueous phase*: the nanoemulsion stability is affected by the nature of the aqueous phase [4]. Particularly, consideration should be given to the aqueous phase pH and presence of electrolytes during nanoemulsion preparation. Theoretically, in essential oil nanoemulsion, continuous phase viscosity could influence droplet size through different mechanisms. It is worthy to mention that the relative importance of these mechanisms depends mainly on

the homogenizer design and the used operating conditions [10].

As detailed in the literature, the majority of the researches, dealing with the essential oil encapsulation into nanoemulsion-based delivery system, have considered that the droplet size and distribution measurements as the most important parameters for their nanoemulsion characterization [4, 6, 11]. Measurements could be determined by laser light scattering, and obtained results are expressed in terms of mean particle size, which is usually represented [11] with Sauter mean diameter,

d3,2= (Volume/Surface Area) = (∑(ni\*di)<sup>3</sup> )/(∑(ni\*di)<sup>2</sup> ) (1)

In addition to these two listed parameters, the stability of essential oil nanoemulsion, which means its ability to resist physicotemporal changes, could be investigated by storing nanoemulsions at different temperatures and periods and measuring at each time point the droplet size variation [12]. Also, some researches have measured the viscosity, the density, the color, the turbidity as physical characterization of their nanoemulsion [13, 14], while others have focused their researches on the investigation of the biological activities of produced nanoemul-

The laborious step of formulation aims to produce stable nanoemulsion. Indeed, any emulsion system, if inappropriately formulated, may be subject to a variety of physicochemical phenomena, which can seriously affect the stability and the

A well-homogenized and stable emulsion whose droplet diameters figure in the nanoscale level can only be obtained under a complex alliance between physical and chemical forces [17]. As a matter of fact, the exclusive use of physical forces remains often insufficient to obtain stable nanoemulsions. The aim role of physical forces is to reduce the size of the dispersed phase droplets at a certain level, while chemical interactions between different medium components interfere to maintain newly

*d*3,2 (expressed in nm), calculated using the following equation:

where ni is the number of droplets and di is the droplet diameter.

**48**

sions [11, 15].

From a chemical point of view, the interfacial tension remains a critical parameter in the process of essential oil nanoemulsification. As a matter of fact, interfacial tension is known as the inward attraction of molecules at the surface of immiscible liquids due to the imbalance of their attractive forces [18]*.* With this respect, the nanoemulsion formation can occur only if the interfacial tension between the two immiscible phases decreased sufficiently to assure their mixture [11]. To reach such change in interfacial tension, an appropriate amount of an appropriate emulsifier should be used to surround and stabilize all neo-formed nanodroplets. Indeed, an emulsifier is not only able to reduce the interfacial tension of the two immiscible phases, but also it presents an effective stabilizer for the newly formed droplets [19]. In this way, the surfactants convert large globules into small ones and avoid small globules from coalescing into large ones, by reducing the repellent force between the liquids and withdrawing the attraction of liquids for their own molecules [20]. It is worthy to mention that to ensure its fundamental role in the nanoemulsification process, surfactants should be used at a higher concentration than its critical micellar concentration "CMC." As a matter of fact, the increase of surfactant concentrations on oil-water interface increases the adsorption of surfactant molecules, leading to the improvement of their ability to reduce the interfacial tension. Once the adsorption saturation is reached at the oil-water interface, the adsorption of surfactant molecules would stop increasing; thus, the interfacial tension remains constant [21].
