Methods for Formulation and Preparation of Nanoemulsions

Properties and Composition of Essential Oils

The stable nanoemulsions are usually of the O/W type, w'hich consists of oil in the dispersed phase, with mean droplet diameter ranging from 30 to 250 mm, in a suitable dispersion medium, usually aqueous. This matrix is essentially stabilized by an emulsifying agent, which can be a food-grade surfactant like lecithins and polysor- bates, or it can be a biopolymer like starch, gum, and protein, depending on the application of nanoemulsion. These surfactants not only reduce the surface tension of the solution but also provide them with desired attributes like specific interfacial behaviour and load-bearing capacity (Donsi et al., 2011; Malathi et al., 2017; Kumar et al., 2020). Essential oils usually consist of 20 to 40 different types of compounds in different concentration but are dominated by unsaturated hydrocarbons (>70%). The major antimicrobial agents present in these O/W nanoemulsions are identified by their similarity to alcohols, terpenes, phenols, and aldehyde. For instance, in clove oil. eugenol is the main constituent, which is a phenol. The menthol and terpineol are present in sage and peppermint oils. There is a huge variation in structure and composition of these compounds, thus their antimicrobial activity solely depends upon the configuration of their structure (Pathania et al., 2018; Mwaurah et al., 2020). Sometimes antimicrobial agents’ activity is also affected by various unit operations performed especially in the food industry (Table 11.1).

Emulsions are generally thermodynamically unstable leading to their separation with time. Unlike them, the stability of an essential oil nanoemulsion (EON) is determined by the Brownian motion which dominates over gravitational force. Additionally, the EON is more stable when the net attractive forces between the droplets are more. As per a general trend, the strength of these forces reduces with

Packaging Unit Operation

Antimicrobial Agent

Target Microorganism

Results

References

Thermo-compression Extrusion molding

Chitosan

Escherichia coli Staphylococcus aureus Salmonella enteritidis Bacillus cereus

o Processing improved mechanical properties of packaging

о Antimicrobial activities of films were not affected by processing

о High-temperature processing inactivated lysozymes activities

Tome et al. (2012)

Thermoforming Extrusion molding

Lysozymes

Micrococcus lysodeikticus

о Increase in plasticizer favored lysozyme activity due to their increase in cross-linking

Mlalila et al. (2018): Jbilou et al. (2014)

Compression molding

Potassium sorbate (KS)

None (research was aimed to check the stability of KS under compression molding)

о Processing retained antimicrobial agent in films о Mechanical and barrier properties improved

Mlalila et al. (2018): Ortega-Toro et al. (2015): Kumar et al. (2020)

Extrusion molding

KS

Zygosaccharomyces bailii

о KS was not affected during processing

Flores et al. (2010)

the reduction in droplet size, thereby reducing the aggregation in an EON (Landry et al., 2014; Kumar et al., 2017). Furthermore, when the droplet diameter is made smaller than the wavelength of visible light (400-700 nm), the droplets are unable to scatter light and become transparent which makes them suitable for incorporating in shampoos and clear beverages. This phenomenon of optical transparency is achieved when the mean droplet diameter is less than 38 nm. Between 38 and 90 nm, an EON appears hazy and finally white when the mean droplet size crosses 90 nm due to induced reflection of light (Mason et ah, 2007). Generally, the reactivity of an EON increases with the decrease in droplet size due to increased surface area-to-volume ratio which increases the activity of lipophilic compounds present in EONs (Donsi et ah, 2012). The antimicrobial action and applications of essential oil nanoemulsions and their correlation with the formulation and mean droplet size are provided in Table 11.2.

Method of Fabrication of Essential Oil Nanoemulsions

The top-down and bottom-up approaches are used to fabricate the EON. In the former, the oil phase is disrupted into uniform, fine, and homogeneous droplets by mechanical means while the latter emphasizes gathering and assembling of molecules into a structure.

Top-Down Fabrication

This process employs a mechanical mean for reducing the size of droplet, thereby generating fluid mechanical stresses within EONs. The emulsification is achieved by reducing the size of droplets followed by absorption of emulsifying agent on these droplets to avoid their reaggregation and to provide stability to EONs. The amount of the emulsifying agent used, dispersed phase and dispersion medium concentration and processing conditions of temperature and pressure are the key factors in the fabrication of EONs (Silva and Cerqueira, 2015; Devgan et al., 2019). Some of the processes involved in top-down fabrication include ultrasonication, colloid milling, membrane emulsification, and high-pressure homogenization. The top-down fabrication processes are characterized by their high-energy input. Ultrasonication process is widely used in laboratories to fabricate EONs. It produces low-and high-pressure sound waves alternatively at high frequency (> 19kHz) which produces bubbles in the EON matrix. The bubbles then implode inside the surrounding liquid, thus releasing energy in the form of shock waves which locally increase the pressure in the liquid to as high as 1.3 MPa (Donsi et al., 2011). Ultrasonication can produce uber-fine nanoemulsions, but local hotspots produced during this process might affect the antimicrobial activity and reactivity of EONs (Salvia-Trujillo et al., 2015a; Kumar, 2018). High-pressure homogenization comprises compressing the fluid to high pressures (60-400 MPa) and forcing it through micrometric homogenization chamber. This results in the formation of acute turbulence, cavitation, and shear stress which disrupt the droplets into a fine and uniform size. To further maintain the uniformity in the size of droplets, multiple passes of EON fluid have been recommended through micrometric homogenization chamber. The size of the droplets has been found directly proportional to the pressure applied during this process (Ghosh et al.,

TABLE 11.2

Antimicrobial Action, Applications of Essential Oil Nanoemulsions and their Correlation with Formulation and Mean Droplet Size

Type of Essential Oil

Purpose

Formulae

Reference/Tested

Microorganism(s)

Surfactant

Mean Droplet Size (mm)

Nanoformulation Antimicrobial Effect

References

Eugenol/Clove

oil

Food (Fruit juice), pharma (antiinflammatory and antiseptic properties)

C10H12O2

Listeria

monocytogenes, Escherichia coli

Polysorbate

20.80.

maltodextrin

conjugates

130

Negative

Ghosh et al. (2014); Shah et al. (2012)

Mandarin oil

Pharma and cosmetic industries (aromatic and anti-depression properties)

^-10^16

L. innocua

Polysorbate 80

90

Slightly positive

Severino et al. (2014); Dons) and Ferrari (2016)

Oregano oil (carvacrol +thymol+ cymene)

Pharma industries (antiviral, antioxidant, antidiabetic, anti-inflammatory and cancer suppressant)

C,0H,4O

L. monocytogenes, Salmonella typhimurium, E. coli

Polysorbate 20

105

Slightly positive

Bhargava et al. (2015); Pathania et al. (2018)

Pure carvacrol/ cymophenol

Pharma industries (antimicrobial, antioxidant and antidiabetic.)

C,0H,4O

E. coli, S. enteric Enteritidis

Polysorbate 80. Lecithin

172

Positive

Landry et al. (2015)

Lemongrass oil

Food, cosmetic and pharma industries (treatment of high blood pressure and digestive problems

^-"51^84^5

S. typhimurium, E. coli

Polysorbate 80

15

Positive

Kim et al. (2013); Pathania et al. (2018)

Cinnamaldehyde

Food (flavouring agent)

C„HsO

Lactobacillus delbrueckii, E. coli, S. cerevisiae

Polysorbate 20

83

Slightly positive

Donsi and Ferrari (2016)

Type of Essential Oil

Purpose

Formulae

Reference/Tested

Microorganism(s)

Surfactant

Mean Droplet Size (mm)

Nanoformulation Antimicrobial Effect

References

Eucalyptus oil

Pharma industry (treatment of nasal and chest congestion, asthma, arthritis, and skin ulcers)

CigH180

Proteus mirabilis, E. coli

Polysorbate 20

110

Positive

Saranya et al. (2012)

Basil oil

Food- and cosmetic- based applications

C10H16O

E. coli

Polysorbate 80

140

Slightly positive

Ghosh et al. (2014); Pathania et al. (2018)

Palm oil

Food based applications (cooking, frying and as an emulsifying agent)

cl6H32o2

c18h402

L. delbrueckii,

E. coli, S. cerevisiae

Polysorbate 80, glycerol and polysorbate 20

97

Slightly positive to neutral

Baldissera et al. (2013)

Peppermint oil

Food, Pharma and cosmetic industries

^62^108^7

L. monocytogenes, Staphylococcus aureus

Starch and glycerol

149

Slightly negative

Donsi and Ferrari (2016)

Sage oil

Pharmaceuticals industry (therapeutic properties)

CI0H16O + C,0H,6

L. delbrueckii,

E. coli, S. cerevisiae

Polysorbate 80, Lecithin

110-170

Positive

Flores et al. (2013)

Limonene/ Lemon oil

Food (as flavouring agent),

pharmaceuticals (obesity, cancer, bronchitis)

C10H,6

Staphylococcus aureus, E. coli, L. delbrueckii

Polysorbate 80, starch

90-185

Positive

Saranya et al. (2012); Ghosh et al. (2014)

Sunflower oil

Food and cosmetic (frying, cooking, emollient)

c,8H32o2

L. delbrueckii, S. cerevisiae

Polysorbate 20

78

Neutral to slightly positive

Donsi et al. (2011)

Type of Essential Oil

Purpose

Formulae

Reference/Tested

Microorganism(s)

Surfactant

Mean Droplet Size (mm)

Nanoformulation Antimicrobial Effect

References

Thyme oil

Pharmaceuticals industry (antibiotic and antifungal properties)

с.ЛА

S. aureus, E. coli,

L. monocytogenes

Lecithin, sorbitan monolaurate, polysorbate 80

80-115

Slightly positive

Landry et al. (2015); Donsi et al. (2011)

Crabwood oil/ Andiroba oil

Pharmaceutical and chemical industries (treatment of arthritis, joint aches, anti-inflammatory properties, fuel, solvent for dissolving dyes, insect repellent)

Trypanosoma evansi

Polysorbate 80 and polysorbate 20

130

Slightly positive

Shah et al. (2012); Donsi and Ferrari (2016)

Canola oil

Food and industries (frying, baking, an ingredient in sauces and marinades, manufacturing of plastics, and adhesives and is used as a biofuel)

c,8H34o2

+

C18H30O2

Staphylococcus aureus, E. coli,

L. monocytogenes

Starch

155

Neutral

Majeed et al. (2016); Pathania et al. (2018)

Neem oil/ Azadirachtin

Agricultural, cosmetics, food, and pharmaceuticals industries (manure, soil conditioner, fumigant, urea coating agent, food additive, emollient and wound healing properties)

Сз5Н.мО,6

Vibrio vulnificus, E. coli, S. aureus

Polysorbate 20

110-140

Positive

Ghotbi et al. (2014); Flores et al. (2013)

Type of Essential Oil

Purpose

Formulae

Reference/Tested

Microorganism(s)

Surfactant

Mean Droplet Size (mm)

Nanoformulation Antimicrobial Effect

References

Anethole/Anise

oil

Food and pharmaceutical industries (flavouring agent in alcohols, meat and candies; manufacturing of soap, perfume, and sachets)

C,„H120

Pseudomonas

aeruginosa,

L. monocytogenes, E. coli

Triglycerides

120-145

Slightly positive

Orav et al. (2008); Donsi and Ferrari (2016)

Savory oil

Pharmaceutical and food industries (strong antiseptic,

antidiuretic, treatment of diarrhoea, nausea, loss of appetite)

C10H14O + C,0H,6

Bacillus cereus,

L. monocytogenes, S. aureus

Polysorbate 20

110-170

Slightly positive

Tozlu et al. (2011); Pathania et al. (2018)

2014; Donsi and Ferrari, 2016; Donsi et ah, 2012). Colloid milling essentially utilizes high-speed rotors to produce intense shear, vibration, and friction which collectively reduce the size of EONs. This type of system is scalable and cost-effective but unable to provide extremely fine droplet size, thereby limiting their applicability to the fabrication of only coarse nanoemulsions (Pan et ah, 2014). Membrane emulsification comparatively requires less energy and thus produces low shear. It is utilized to fabricate O/W nanoemulsions in a narrow size range. The dispersion phase is forced to pass through a micrometric membrane into an aqueous phase containing a hydrophilic emulsifying agent by applying either positive pressure on one side or negative pressure on the other (Liu et ah, 2011). Despite the simplicity and flexibility of this process, it is limited to the laboratories experimentation, and no industry application has been reported yet.

With respect to the industrial applicability, high-pressure homogenization offers major advantages in terms of simplicity of process, scalability, cost, and high throughput. Colloid milling has found its application in the food, pharmaceutical, and cosmetic industries, where the size of particles/droplets is kept comparatively large. The ultrasonication process confronts scalability issues when it comes to the industrial applicability, yet some of the paints, cosmetics, and food-processing industries have developed several prototypes as per their use (Salvia-Trujillo et ah, 2015b; Donsi and Ferrari 2016; Mlalila et ah, 2018).

Bottom-Up Fabrication

The bottom-up process for fabrication of essential oil-based nanoemulsions is a physicochemical process in which the structured oil molecules are encompassed by an emulsifying agent. This process is driven by the balanced repulsive and attractive forces making a thermodynamic equilibrium. The assembling of oil molecules in a structure depends on their intrinsic properties like geometry solubility and surface activity. Additionally, the environmental components like temperature and properties of the dispersed phase and dispersing medium such as ionic strength, pH. and concentration significantly affect the fabrication process (McClements and Rao, 2011; Sessa and Donsi, 2015; Kehinde et ah, 2020). Some mechanical energy is also required for the purpose of agitation.

The bottom-up fabrication process is characterized by its low-energy requirement for producing extremely small droplets, utilizing the simple and scalable equipment. Nevertheless, a strict protocol needs to be followed in the selection of the emulsifying agent and oil type for formulating stable EONs. Additionally, the surfactant-to- oil ratio has to be kept lower than in the top-down fabrication process. Some of the processes involved in bottom-up fabrication include solvent demixing, phase inversion, and spontaneous emulsification. In the solvent demixing technique, the oil phase is dissolved in a preselected organic solvent followed by its segregation in nano form by the addition of an aqueous anti-solvent containing an emulsifying agent for stabilization (Baldissera et ah, 2013). The organic solvent diffuses rapidly in the aqueous anti-solvent, leading to the formation of nanoemulsions with high degree of encapsulation. Precise intricacy is required in this process so some scientists have developed a comparatively more energy-intensive process like spray-drying of oil (thymol) in solvent (hexane) using maltodextrin, which results in a stable and transparent EON (Shah et al., 2012). Akin to solvent demixing, spontaneous emulsification includes mixing a preselected oil in a hydrophilic emulsifying agent to form stable O/W EONs. The mixing leads to interfacial turbulence which further gives rise to the formation of EONs (McClements and Rao, 2011). Phase inversion pertains to a process in which O/W emulsion phases back to W/O on agitation and vice versa depending on the environmental factors like temperature, pH, and concentration of surfactant. It produces extremely fine-sized droplets in a continuous phase. It has widespread application in pharmaceuticals, cosmetics, food processing, and detergent manufacturing (Liu et ah, 2011; Donsi et ah, 2011).

 
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