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Nanoemulsions and Their Potential Applications in Food Industry

Nanoemulsions and Their Potential Applications in Food Industry REVIEW published: 13 November 2019 doi: 10.3389/fsufs.2019.00095 Nanoemulsions and Their Potential Applications in Food Industry Jamuna Bai Aswathanarayan and Ravishankar Rai Vittal* Department of Studies in Microbiology, University of Mysore, Mysore, India Nanoemulsions have small droplet size and are kinetically stable colloidal systems. They have enhanced functional properties in comparison to conventional emulsions. The composition and structure of the nanoemulsions can be controlled for the encapsulation and effective delivery of bioactive lipophilic compounds. Nanoemulsions have potential application in the food industry for the delivery of nutraceuticals, coloring and flavoring agents, and antimicrobials. The nanoemulsion formulations of active ingredients can be used for developing biodegradable coating and packaging films to enhance the quality, functional properties, nutritional value, and shelf life of foods. This review focuses on preparation of food grade nanoemulsions using high-energy methods and low-energy approaches and their characterization for physical properties, stability, and microstructure. The application of nanoemulsion formulations for sustainable food processing and improving the delivery of functional compounds, such as colorants, flavoring agents, nutraceuticals, and preservatives or antimicrobial agents in foods has Edited by: José Antonio Teixeira, been discussed. University of Minho, Portugal Keywords: nanoemulsions, encapsulation, bioactive compounds, functional foods, nutraceuticals Reviewed by: Ana C. Pinheiro, Center for Biological Engineering, INTRODUCTION School of Engineering, University of Minho, Portugal Emulsions are defined as the dispersion of two immiscible liquids, with the spherical droplets Alejandra Acevedo-Fani, forming the dispersed phase, whereas the liquid surrounding it forms the continuous phase (Tadros Riddet Institute, New Zealand et al., 2004; McClements et al., 2007; Acosta, 2009). The commonly used liquids to form emulsion *Correspondence: are water and oil. The oil droplets dispersed in an aqueous phase are known as oil-in-water (o/w) Ravishankar Rai Vittal emulsions. These emulsion systems can be used for the delivery of hydrophobic active substances. raivittal@gmail.com The water droplets dispersed in oil are called the water-in-oil (w/o) emulsions and are used for the delivery of hydrophilic compounds. Multiple emulsion systems can also be developed such as Specialty section: This article was submitted to the water-in-oil-in-water (w/o/w) and oil-in-water-in-oil (o/w/o) emulsions. The w/o/w emulsions Sustainable Food Processing, are made of large oil droplets, containing water droplets dispersed in an aqueous phase. Whereas, a section of the journal in o/w/o emulsion system, water droplets containing oil droplets are dispersed in an oil phase. Frontiers in Sustainable Food Systems Bicontinuous nanoemulsion contains microdomains of oil and water—interdispersed within the Received: 23 November 2018 system (Garti and Benichou, 2004; Weiss et al., 2006). Accepted: 07 October 2019 Emulsions are categorized as coarse emulsions, microemulsions and nanoemulsions based Published: 13 November 2019 on their droplet size and stability (Komaiko and McClements, 2016). In Table 1, the various Citation: types of emulsion systems have been mentioned. However, there is some ambiguity regarding Aswathanarayan JB and Vittal RR their description based on size. The coarse emulsions are also known as conventional (2019) Nanoemulsions and Their emulsions or macroemulsions. They have particle size of diameter >200 nm range and are Potential Applications in Food thermodynamically metastable. They break down over time due to various destabilizing factors. Industry. Conventional emulsions are optically turbid as the dimension of the droplets is similar to Front. Sustain. Food Syst. 3:95. doi: 10.3389/fsufs.2019.00095 that of the wavelength of light and hence, scatters the incident light and appears opaque. The Frontiers in Sustainable Food Systems | www.frontiersin.org 1 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications TABLE 1 | Emulsion types: physicochemical properties, stability, and preparation methods. Emulsion/coarse Microemulsion Nanoemulsion emulsion/macroemulsion Size 1–100 μM 10–100 nm <200 nm Thermodynamic Stability Metastable Stable Metastable Kinetic stability Stable Unstable Stable Optical property Turbid Transparent Transparent/slightly translucent Polydispersity High (>40%) Low (<10%) Low (<10–20%) Preparation method High and low energy methods Low energy methods High and Low energy methods Effect of temperature and pH Stable to temperature and pH Effected by changes in Stable to temperature and pH changes composition, temperature and pH changes droplets in microemulsions are <100 nm in size and are COMPOSITION OF NANOEMULSIONS thermodynamically stable. However, their stability is affected by even slight variations in environmental conditions such Nanoemulsion formulation requires the use of two immiscible as composition and temperatures. Microemulsion forms liquids and an emulsifier. One of the immiscible liquids must spontaneously as its free energy is lower than its phase-separated be oleaginous and the other aqueous in nature, and they components. They are optically transparent as the particle make up the dispersed and aqueous phase. The o/w and w/o size is lesser than the wavelength and weakly scatters light nanoemulsion consists of a core–shell structure. For example, (Anton and Vandamme, 2011). The nanoemulsions have droplet in an o/w nanoemulsion system the amphiphillic shell is dimensions similar to the microemulsions ranging from <200 made of surface-active molecules, whereas the lipophilic core and in some cases <100 nm (Saifullah et al., 2016). Similar to contains non-polar molecules. The oil phase in a nanoemulsion conventional emulsions, nanoemulsions are thermodynamically is made of triacylglycerols, diacylgycerols, monoacylglycerols, metastable as phase separation occurs over time. However, and free fatty acids. Non polar essential oils, mineral oils, nanoemulsions are conferred with kinetic stability as there lipid substitutes, waxes, weighting agents, oil-soluble vitamins, is no gravitational separation and droplet aggregation due to and various lipophilic components are also used as oil phase. the reduced attractive force between the small sized droplets The viscosity, refractive index, density, phase behavior, and (McClements and Rao, 2011). The nanoemulsions unlike the interfacial tension of the oil phase components influences the thermodynamically stable microemulsions are not affected by formation, stability, and functional properties of nanoemulsion physical and chemical variations including temperature and pH. (Tadros et al., 2004; Wooster et al., 2008; McClements and Rao, They require less amount of surfactants for their preparation. 2011). However, the long-chain triacylglycerols are preferred for The droplet size of nanoemulsion apart from determining its nanoemulsion formulation due to their low cost, availability, optical property and stability, also influences its rheological and functional, and nutritional attributes (Witthayapanyanon et al., release behavior. Hence, the nanoemulsions are more suitable 2006). The aqueous phase of a nanoemulsion is made of than microemulsions for various applications. polar solvent and a cosolvent. It determines the polarity, The present review focuses on the increased application rheology, phase behavior, interfacial tension, and ionic strength of nanoemulsions in the food industries for sustainable food of nanoemulsion. The polar solvent generally used is water, processing and packaging. The nanoemulsions have the ability whereas carbohydrates, protein, alcohol, and polyols are used to encapsulate functional compounds and active ingredients as cosolvents (Saxena et al., 2017). The aqueous phase and including antioxidants and nutraceuticals. They are also useful oil phase can breakdown due to Ostwald ripening (increase in the controlled release of flavor compounds in foods (Velikov in mean droplet size over time), flocculation, coalescence, and and Pelan, 2008; McClements and Rao, 2011; Ines et al., gravitational separation (Kabalnov, 2001; McClements and Rao, 2015). Nanoemulsion encapsulation of bioactive compounds 2011). This can be prevented by adding a stabilizer agent increase its solubility, controlled release and absorption in to nanoemulsion. The stabilizers distribute on a particle and the gastrointestinal tract, and absorption through cells (Chen can form either a monolayer, multilayer, and solid particulate et al., 2006; McClements and Rao, 2011). Nanoemulsion based nanoemulsions. Some of the stabilizers used are emulsifiers, edible nanocoatings containing flavor and coloring ingredients, weighting agents, ripening retarders, and texture modifiers. antioxidants, enzymes, antimicrobials, and antibrowning agents Emulsifiers are surface active molecules and commonly used can be used to coat foods such as meats, dairy products stabilizers in nanoemulsion preparation to protect small droplets. such as cheese, fresh produce, and fresh cuts including fruit They reduce the interfacial tension resulting in formation of and vegetables and confectionaries to improve their shelf life. small and stable nanoemulsions. The emulsifiers also prevent The nanoemulsion coatings can also prevent moisture and collision and coalescence between the droplets and increases gas exchange, minimize moisture loss and oxidation of foods the kinetic stability of the nanoemulsions (Mason et al., (Azeredo et al., 2009; Rojas-Graü et al., 2009; Salvia-Trujillo et al., 2006). The emulsifiers can be cationic, anionic, nonionic, 2015; Donsi, 2018). and zwitterionic in nature. Some examples of emulsifiers Frontiers in Sustainable Food Systems | www.frontiersin.org 2 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications are small-molecule surfactants, phospholipids, proteins, and of food grade particles including proteins, polysaccharides, and polysaccharides (McClements, 2004; Kralova and Sjöblom, 2009). flavonoids have been investigated for pickering nanoemulsion Even polymers like polyvinyl alcohol are used as emulsifiers. preparation. However, colloidal lipid particles have shown to be The surfactants can be either ionic or non-ionic in nature. the most promising pickering stabilizers in O/W emulsions as Ionic surfactant prevents droplets aggregation by electrostatic they are able to impart good physical stability. These are simple to repulsion, whereas non-ionic surfactants reduce aggregation by prepare with tunable microstructure, and can be used to prepare steric hindrances, thermal fluctuation interactions, and hydration nanoemulsions using high pressure homogenization method and (McClements, 2004; Silva et al., 2015). For o/w emulsion cross flow microfluidic device (Schroder et al., 2017, 2018). preparations, hydrophile–lipophile balance values >10 are used as a criterion for selecting emulsifiers. For example, proteins insoluble in oils are used as emulsifiers for o/w nanoemulsion PROPERTIES OF NANOEMULSIONS AND preparation. Two or more emulsifiers have also been used DESTABILIZING MECHANISM together in nanoemulsion formulation for their synergistic effect. On using both hydrophilic and lipophilic (or cosurfactant) The optical properties of nanoemulsion are important for emulsifiers, a significant decrease in surface tension has been their application in food industry. Based on their droplet size observed (Shakeel et al., 2009; Qadir et al., 2016). Similarly, nanoemulsions are optically transparent or faintly turbid. Their block copolymers that are soluble only in the dispersed phase opacity is expressed in terms of turbidity (τ) and characterized can decrease surface tension (Tadros et al., 2004). However, by transmission measurements (McClements and Rao, 2011). some surfactants have found to be irritants, which limit their The mean particle size and narrow particle-size distribution application in foods. This has resulted in the formulation of influences the opacity of nanoemulsions (Wooster et al., 2008). w/o emulsions without surfactants (Glatter and Glatter, 2013). The rheological properties of nanoemulsions modify the texture Such surfactant-free nanoemulsions can be prepared by cooling of foods (Quemeda and Berli, 2002; Walstra, 2003; Genovese the continuous phase below its melting temperature, which et al., 2007). The relative droplet size is known to influence leads to formation of kinetically stable emulsion made up of the rheological properties of nanoemulsions. For example, lyotropic liquid crystalline nanostructure (Ridel et al., 2015; beverages have low viscosity and the nanoemulsions used for Duffus et al., 2016; Chen et al., 2018). Weighting agents are their preparation should have droplets which do not increase the used in nanoemulsion preparation to impede the gravitational overall viscosity (McClements and Rao, 2011). forces and reduce sedimentation and creaming. Weighting agents The physicochemical stability of nanoemulsions is such as ester and damar gum are used in o/w emulsion as their characterized as kinetically stable systems as these breaks density matches with that of the oil phase surrounding aqueous down over time due to destabilizing physical mechanisms phase. Ripening retarders have hydrophobic action which helps (gravitational separation, flocculation, coalescence) and chemical in retarding the ripening (Schuch et al., 2014). The generally instability (Figure 1). Gravitational separation is due to different used ripening retards such as mineral oils and long chain triacyl relative densities between the dispersed and continuous phases glycerols prevent diffusion of smaller oil molecules through the and results in creaming or sedimentation. Formation of aqueous phase to form larger molecules (Sonneville-Aubrun crystalline lipids or small oil droplets causes sedimentation in et al., 2004). The ripening inhibitors have low water solubility an o/w nanoemulsions. Similarly, creaming in nanoemulsions and cause entropy of mixing for balancing the curvature effects occurs due to large particle size as the movement of droplets is (Wooster et al., 2008; Li et al., 2009). The texture modifiers used influenced by gravity. In nanoemulsions with particle diameter in the nanoemulsions interact only with the aqueous phase and <70 nm, the Brownian motion effects movement of smaller increase its viscosity by thickening it or turning it into a gel. They particles and prevents creaming (Walstra, 2003; McClements prevent the movement of oil droplets and impart creaminess and and Rao, 2011). Droplet aggregation such as flocculation or thickness to aqueous phase. Some of the commonly used texture coalescence is less in nanoemulsions due to their relatively small modifiers are biopolymers including gums, vegetable proteins, particle size (Tadros et al., 2004). In nanoemulsions, colloidal and polysaccharides (Imeson, 2011). In Table 2, the commonly interactions is related to their droplet size and occurs due to the used food grade emulsifiers and various stabilizing agents have attractive interactions (van der Waals and hydrophobic) and been mentioned. repulsive interactions (electrostatic and stearic) between two In the recent years, there is an increased interest in the use adjacent droplets (McClements, 2005; McClements and Rao, of food-grade stabilizers for the preparation of nanoemulsions 2011). Ostwald ripening occurs as the droplet size increases over (Rayner et al., 2014). Pickering stabilization has superior time due to diffusion or movement of solubilized oil molecules stability in comparison to conventional surfactant-stabilized from small droplets to large droplets through dispersed phase nanoemulsions. The pickering particles form a dense layer at (Kabalnov, 2001; McClements and Rao, 2011). The decrease the oil and water interface and by steric mechanism prevent in droplet size causes an increase in aqueous-solubility of oil droplet flocculation and coalescence (Duffus et al., 2016). The present within a spherical droplet resulting in large number of three main prerequisites for efficient pickering stabilization have solubilized oil molecules. This solubilized oil molecules move to been identified. The particle should be in 200 nm−1 μm size larger droplets leading to a gradual increase in the droplet size. range with sufficient particle charge and should have affinity The aqueous solubility of oil phase determines the stability of to emulsion continuous phase (Duffus et al., 2016). A number a nanoemulsion to Ostwald ripening (Kabalnov and Shchukin, Frontiers in Sustainable Food Systems | www.frontiersin.org 3 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications TABLE 2 | Food grade stabilizing agents used in nanoemulsions preparations for food related applications. Stabilizing agent Compound Concentration References Emulsifier (Non-ionic Lecithin (Phospholipid) and Lecithin at 1 wt.% with oil phase and Tween 80 at 2 Tan et al., 2016 surfactant) Tween 80 wt.% with aqueous phase Lecithin and Tween 80 Lecithin/Tween 80 at 0.3 molar ratio Kumar et al., 2017 PEG 40 hydrogenated castor oil; Oil:surfactant at 2:1 Nantarat et al., 2015 Sorbitan monoleate Tween 20 With aqueous phase at 0.75 wt.% Severino et al., 2015 With both phases at 1 wt.% Artiga-Artigas et al., 2017 Tween 20 and Span 80 Tween 20 and aqueous phase at 1wt.% Alexandre et al., 2016 Span 80 and oil phase at 4 wt.% Tween 80 Mixing with both phases at 3 wt.% Acevedo Fani et al., 2015 Mixing with organic phase at 7.5 wt.% Chen et al., 2017 Mixing with oil phase at 4.5 wt.% Gahruie et al., 2017 Tween 80 and Span 80 Tween 80 with aqueous phase at 1.25 wt.% and Dammak et al., 2017 Span 80 with oil phase at 3.75 wt.% Sucrose monosterate Mixing with aqueous phase at 0.5 wt.% Ruiz-Montañez et al., 2017 Emulsifier (Anionic) Sodium dodecyl sulfate with aqueous phase at 2.5 wt.% Qian and McClements, with aqueous phase at 3 wt.% Lee et al., 2013 Emulsifier (Amphiphilic) Whey protein isolate With aqueous phase at 4 wt.% Hebishy et al., 2015 Egg yolk powder Mixing with water as continuous phase at 3 wt.% Schuch et al., 2014 for W/O/W emulsion formation Mixed with aqueous phase at 3 wt.% Lee et al., 2013 Plasticizer Glycerol Mixing with nanoemulsion and biopolymer at Alexandre et al., 2016 glycerol/biopolymer ratio of 0.3 Mixing with oil phase at 2% (v/v) Alexandre et al., 2016 Mixing with biopolymer at 30 wt.% and then with Dammak et al., 2017 emulsion solutions Poly(ethylene glycol) Mixing with nanoemulsion at 0.2% (w/v) Otoni et al., 2014b Ripening retardant Sodium chloride Mixing with aqueous phase at 0.5 wt.% for W/O/W Schuch et al., 2014 inner emulsion Texture modifier Sodium alginate Addition at 1% before coarse emulsion formation by Artiga-Artigas et al., 2017 homogenizer (Ultra-Turrax) at 11,000 rpm for 2 minutes at room temperature 1992). Chemical degradation of nanoemulsions occurs due constituents, operating conditions, and preparation methods. to oxidation and hydrolysis. The large specific surface area of The emulsification process involves break up of droplets nanoemulsions makes it prone to chemical degradation. The into smaller ones, adsorption of surfactants, and collision opacity of nanoemulsions also play a role in chemical stability. of droplets. The adsorption kinetics also affects the stability The clear nanoemulsions with small droplets get easily degraded and droplet size of nanoemulsions (Silva et al., 2015). The by UV or visible light due to transparency (Dickinson, 1992; high energy methods, involve the use of mechanical devices Friberg et al., 2004; McClements, 2005). which disrupts oil phase for it to interact with water phase and form smaller oil droplets. The excessive stress generated by the mechanical device disrupts oil phase. Most of the PREPARATION OF NANOEMULSIONS food industries use high energy methods to prepare oil- in-water nanoemulsions (Gutiérrez et al., 2008). In the low The nanoemulsions have numerous droplets which increases energy methods, nanoemulsions are prepared by altering the the surface area. Therefore, large amount of energy is required temperature or composition of the oil-water system and the to create extra surface. Thus, nanoemulsion formation is not energy input is from the chemical potential of the constituents spontaneous and requires energy input. The energy required (Bouchemal et al., 2004). for the formation of nanoemulsions (1G) is estimated by the Major factors involved in nanoemulsion preparation is to expression, 1G= 1Aγ-T1S, where 1A is increase in interfacial achieve significantly low interfacial tension (<10–3 mN/m) at area, γ is surface tension and T1S is the entropy of dispersion the oil/water interface which requires the use of appropriate (Tadros et al., 2004; Schramm, 2006). surfactant. The surfactant also helps in stabilizing droplets Nanoemulsions can be prepared by either high energy produced at low interfacial tension. Fluidity at the interface or low energy methods. Its size is dependent on the Frontiers in Sustainable Food Systems | www.frontiersin.org 4 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications FIGURE 1 | Nanoemulsion formation and destabilizing mechanisms. Coalescence involves droplets combining; flocculation involves droplet aggregating but do not combine; droplets grow in size through Ostwald ripening. This destabilizing mechanism results in creaming and phase separation. is another important factor for stimulating nanoemulsion addition of emulsifier molecules and its adsorption at the formation (Bhosale et al., 2014). newly formed interfaces causes stabilization of droplets in the interaction chamber. Repeated disruption and stabilization results in production of higher number of tiny droplets. The High Energy Methods reduction in interfacial tension and maintaining low viscosity The high energy methods involve the use of a mechanical device ratio of dispersed and continuous phase decreases droplet size such as high pressure valve homogenizers, microfluidizers and (Tadros et al., 2004; Lee and McClements, 2010). An increase ultrasonicators (Figure 2). These devices are used to apply highly in concentration of emulsifier in nanoemulsions has also disruptive force for disrupting dispersed phase into tiny droplets shown to decrease droplet diameter (Windhab et al., 2005). of nanoemulsions (Maali and Hamed Mosavian, 2013). The emulsifiers can also reduce instability of re-coalescence of droplets after disruption. This is by adsorbing at the interface High Pressure Valve Homogenization Method of the droplets to form a protective layer thereby, preventing The device used in the high pressure valve homogenization the coalescence. However, the adsorption process should occur (HPVH) method, includes a positive displacement pump, at a faster rate than coalescence (Jafari et al., 2008). But the pressure valve, and chambers for homogenization and organoleptic constraints and regulations, limits the use of higher interaction. The coarse emulsion is sucked into the concentrations of emulsifier in food emulsion. Studies have homogenization chamber by the suction stroke of the pump. shown that small-molecule emulsifiers such as Tween 20 and The homogenization chamber can be a simple orifice plate, sodium dodecyl sulfate (SDS), produce smaller droplet sizes than colliding jet or radial diffuser assemblies (Stang et al., 2001; larger molecules emulsifiers such as proteins (Anton et al., 2008). Donsì et al., 2009). High pressure of upto 300 MPa generated in High pressure homogenization cannot be applied for viscous the chamber during delivery stroke causes the coarse emulsion lipids (Witthayapanyanon et al., 2006). to be pushed out through small orifice of micrometric size by the Thus, high-pressure homogenization efficiently breaks down homogenizer valve. At this stage, the factors such as turbulence, droplets and increases stability (Schultz et al., 2004). The ease shear stress, and cavitation disrupts coarse emulsions into very of applicaion, scalability, reproducibility, and high throughput finer droplets (Tesch et al., 2003; Schultz et al., 2004). Further, makes high pressure valve homogenization technique, highly the fine droplets get stabilized in the interaction chamber. suitable for producing nanoemulsions in food industries Thus, in the high pressure homogenization, emulsification (Schubert and Engel, 2004). The high-pressure homogenization occurs in two stages. Firstly, disruption of dispersed phase preparation of the nanoemulsion is more efficient and gives better results in tiny droplet formation with increased surface area quality (Liu et al., 2019). High pressure homogenization method in the homogenization chamber. In the second stage, the Frontiers in Sustainable Food Systems | www.frontiersin.org 5 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications FIGURE 2 | High energy methods such as high pressure homogenization (HPH), microfluidizer, and ultrasonication break macroemulsions into smaller droplets. was used to prepare nanoemulsions of carvacrol, mandarin, for fat globule disruption in dairy applications. However, now bergamot, and lemon. Primary emulsions of essential oils they are extensively used for food processing applications were subjected to five cycles of high pressure homogenization as nanoemulsion for delivering supplemented nutrients and at 200 MPa to obtain nanoemulsions of 133–200 nm. The as a preservation technique. It was assumed that friction nanoemulsion of carvacrol showed significant growth inhibitory heat generated in the homogenizer may cause degradation of activity against Escherichia coli O157:H7 and Salmonella temperature sensitive nutrients. Recent studies have shown that Typhimurium. Carvacrol nanoemulsion was incorporated into no thermal degradation occurs in valve due to the short residence modified chitosan to form a bioactive coating. It increased the time (3–40 ms) (Håkansson, 2019). shelf life of green beans stored at 4 C for upto 13 days (Severino et al., 2015). The o/w nanoemulsions of jackfruit pulp extract rich in carotenoids was obtained by high-pressure homogenization Microfluidization Method (400–800 bar). The nanoemulsion was stabilized by sucrose The basic principle of emulsification by microfluidization is monostearate emulsifier and exhibited a longer stability in its similar to the high pressure valve homogenizer, except for the use antioxidant activity during storage at 4 C. Nanoemulsions with of a special microchannel having dimensions in the range of 50– a high stability were produced at 800 bar. The antioxidant 300 μm to form droplets in the microfluidization method. In this activity of a jackfruit pulp extract was protected by nanoemulsion technique, the coarse emulsion is pumped with high pressure (up (Ruiz-Montañez et al., 2017). Lycopene nanodispersions with a to 270 MPa) at the inlet through a microchannel at velocities of narrow polydispersity index and good stability for application 400 m/s. The channels at the downstream gets split into two small in beverages were developed using homogenization process. branches to form Y or T junction. These branches reconnect Homogenization pressure of 500 bar reduced the particle size at far downstream to an interaction channel. In the interaction and lycopene concentration significantly, while homogenization chamber, the coarse emulsions from two steams impinges on pressure of 700–900 bar resulted in large particle sizes with each other with very high velocity. Thus, the impulsive forces −1 high dispersibility. Homogenization pressure also effected zeta generated in two streams and shear rate as high as 107 s potential and turbidity of the lycopene nanodispersion (Shariffa are sufficient for disrupting and forming fine emulsions in et al., 2017). High pressure homogenization of 50–300 MPa the interaction channel (Souto et al., 2005). The process is was used to obtain stable lentil nanoemulsions. Stable lentil repeated for more than two cycles to increase emulsification nanoemulsions was obtained with homogenizer pressure above time and pressure. Thus, the size of nanoemulsion depends 200 MPa and the best stability was achieved at 300 MPa. High- on the disruption of droplets and its recoalescence. However, pressure homogenization decreased nanoemulsion viscosity the addition of a fast-adsorbing emulsifier and increasing the under all conditions (Tabilo-Munizaga et al., 2019). High- viscosity of continuous phases can reduce the recoalescence rate pressure homogenizers were initially used in dairy industry (Jafari et al., 2007; Bae et al., 2009; Mao et al., 2010). Frontiers in Sustainable Food Systems | www.frontiersin.org 6 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications Microfluidization was used to obtain nanoemulsions of 163 nm drop size and zeta potential of –29.7 were obtained thyme, lemongrass, and sage oil dispersed in sodium alginate (Ricaurte et al., 2016). solution. The technique reduced the average droplet size of The limitations of the microfluidization techniques are nanoemulsions and resulted in ζ-potentials between −41 and increased droplet size as a result of coalescence due to longer time −70 mV. Sage essential oil nanoemulsions showed good film involved in emulsification and the use of high pressure increases property such as higher transparency, water vapor resistance, the temperature of nanoemulsions. However, in comparison and flexibility. Edible films of thyme essential oil had strong to other homogenization techniques, the efficiency of droplet antimicrobial effect on E. coli. Microfluidizers could be used to disruption by microfluidization technique is high and results in prepare nanoemulsions with active ingredients for the formation formation of fine droplets with uniform size. of edible films, with different physical and functional properties (Acevedo Fani et al., 2015). Nanoemulsion of ginger essential Ultrasonication Method oil was produced by microfluidization. Edible films reinforced In this technique, the ultrasonic agitation by sound waves with montmorillonite was activated with ginger essential oil with more than 20 kHz frequency breaks coarse droplets into nanoemulsion. The incorporation of nanoemulsions improved nanoemulsions. Sound waves applied by sonotrode produce characteristics and antioxidant activity of montmorillonite mechanical vibration and acoustic cavitation, and with the films. The incorporation of montmorillonite and nanoemulsion collapse of cavitations, strong shock waves generated break the into gelatin-based films increased the thickness and decreased coarse droplets (Behrend et al., 2000). The acoustic and shock the solubility in water, moisture content and superficial waves create high pressure and turbulence which collapse the hydrophobicity of films (Alexandre et al., 2016). Dual-channel droplets. At high frequency (mega Hz), nanoemulsions can even microfluidization have also been used for efficiently producing be prepared without using an emulsifier (Kamogawa et al., 2004; label-friendly nanoemulsions from natural emulsifiers. The Jafari et al., 2007). emulsifiers either amphiphilic biopolymers (whey protein and The device consists of an ultrasonic chamber having an gum arabic) or biosurfactants (quillaja saponinand soy lecithin) ultrasonic probe. The disruptive forces created by the ultrasonic were used to prepare corn oil-in-water nanoemulsions. The probe in combination with cavitation, turbulence, and interfacial use of dual-channel microfluidization resulted in production waves breaks the coarse emulsions flowing in the ultrasonic of nanoemulsions with the mean particle diameter decreasing chamber to fine nanoemulsions (Kentish et al., 2008). Similarly, with increasing emulsifier concentration and homogenization bench-top sonicator is used for the production of nanoemulsions pressure. Using this technique, whey protein isolate and at small scale. The piezoelectric crystal probe in the sonicator quillaja saponin were more effective at forming nanoemulsions generates intense pressure waves. An optimum level of input of fine droplets than gum arabic and soy lecithin. Low energy is required for sonification to achieve smallest droplet amount of emulsifier was required and smaller droplets were diameter. On increasing the sonication time, there is also produced (Bai et al., 2016). Dual-channel microfluidization an increase in input energy which tends to disrupt higher proved to be an efficient method for continuously producing number of droplets and decrease their size (Jafari et al., 2007). carotenoid-loaded nanoemulsions from natural emulsifiers. The other factors which influence nanoemulsion formation It was used to prepare o/w nanoemulsions of β-carotene. are concentration of emulsifier, viscosity ratio of dispersed Two types of natural emulsifier, quillaja saponins and whey and continuous phase and the amplitudes of applied waves protein isolate were used for nanoemulsions preparation by (Nakabayashi et al., 2011). It was observed that nanoemulsions this novel homogenization method. At 4 and 25 C, the prepared by high intensity ultrasonication from flax seed oil and nanoemulsions remained physically stable throughout 14 days nonionic surfactant (Tween 40) had droplet radius below 70 nm storage. At 55 C, small amount of droplet aggregation occurred (Kentish et al., 2008). Similarly, high-intensity ultrasound has in saponin nanoemulsions. Thus, microfluidization technique also been used to obtain nanoemulsions with droplet radius of can be used to encapsulate β-carotene and improve its water 20 nm. These were prepared using grade emulsifiers including dispersibility and chemical stability in foods (Luo et al., 2017). sunflower oil, Tween 80, and Span 80 (Leong et al., 2009). Microfluidization was used to produce small sized fish oil The increased ultrasonification time and decreased surfactant nanoemulsion as fish oil is rich in polyunsaturated fatty acids concentration resulted in nanoemulsions with droplet diameter (PUFAs). Coarse emulsion had a droplet size of 1.5 μm, while of 29.3 nm. The nanoemulsions were prepared with basil oil microfluidization produced smaller droplet size (≥ 200 nm). Zeta and nonionic emulsifier (Tween 80 and water) and had a high potential values increased around –30 ± 2 mV. Nanoemulsions intrinsic stability with ultrasonication time of 15 min (Ghosh with an average droplet size around 200 nm were prepared et al., 2013). with 144 MPa and two passes of microfluidization. Thus, High intensity ultrasound (150 W) has been used to prepare microfluidization could be used to develop nanoemulsions with nanoemulsions of essential oils of Zataria multiflora. The better absorption in the digestive tract (García-Márquez et al., bioactivity of the essential oil was increased by nanoemulsion. 2017). Microfluidization has also been used to encapsulate high Further, increase in the antibacterial activity could be observed nutritional value oils, such as high-oleic palm oil (Ricaurte by decreasing the nanoemulsion droplet size. The small sized et al., 2016). o/w nanoemulsions of high-oleic palm oil was nanoemulsions could be easily incorporated in the basil seed obtained by microfluidization wherein, 1–20% w/w of the gum films. Increased nanoemulsion concentration in the film oil could be easily encapsulated. Stable nanoemulsions of matrix effected the microstructure of the film and improved Frontiers in Sustainable Food Systems | www.frontiersin.org 7 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications its mechanical properties. The films also showed significant surfactant in oil is more (lipophilic) than water phase and thus antimicrobial activity against potential foodborne pathogens water-in-oil nanoemulsion is formed. Temperature at which (Gahruie et al., 2017). Nanoemulsions of resveratrol and there is a conversion of oil-in-water to water-in-oil emulsion is resveratrol-cyclodextrin inclusion complex in a phospholipid termed as phase inversion temperature (PIT) (Pathak, 2017). stabilized nanoemulsion has been prepared by ultrasonic It is also to be noted that surface tension decreases with emulsification. The resveratrol nanoemulsion and inclusion increase in temperature and fine emulsions with small sized complex nanoemulsion had an average size of 20 and 24 nm, droplets are formed. At PIT, these small droplets are not stable respectively. The nanoemulsions developed by ultrasonication as they try to coalesce and form macroemulsions. If a large had a good loading and release efficiency. The nanoemulsion amount of emulsifier is used to reduce instability, droplets do prevented degradation of resveratrol on exposure to UV not coalesce immediately, and some liquid crystal structure forms irradiation (365 nm) (Kumar et al., 2017). at PI temperature (Salager et al., 2004). Thus, although it is Studies have been carried out to compare the efficiency of possible to form emulsions near PIT, they are very unstable. To high pressure homogenization and ultrasonication to develop produce stable and fine oil-in-water nanoemulsions, a cooling the nanoemulsions of capsaicin (oleoresin capsicum). Capsaicin process is required and final nanoemulsions are required to be nanoemulsions obtained by high pressure homogenization preserved at a temperature far below PIT (Liew et al., 2010). were optically translucent with 79% efficiency and significant In addition, if emulsions formed near PIT are rapidly cooled antimicrobial activity. However, nanoemulsions prepared by or heated, kinetically stable emulsions with small droplet size ultrasonication had good physical properties and smaller droplet and narrow size distribution can be produced. PIT can be size of 65 nm (Akbas et al., 2018). determined by noting down the change in different properties of nanoemulsions such as conductivity, viscosity, and turbidity (Rao and McClements, 2010). Low Energy Methods Phase inversion temperature method was used to prepare The low-energy methods, use the energy input from chemical cinnamon oil nanoemulsions. Cinnamon oil, non-ionic potential of the components to from nanoemulsions. The surfactant, and water were heated above the phase inversion nanoemulsions form spontaneously at oil and water phase temperature of the system. It was rapidly cooled with continuous interface by gentle mixing of the components. The spontaneous stirring resulting in spontaneous formation of small oil droplets emulsification can be controlled by two methods. One of the with mean droplet diameter of 101 nm. The cooling-dilution methods is to change temperature without altering composition. method increased stability of the nanoemulsions for 31 The other method is to keep the temperature constant and vary ◦ ◦ days at 4 C or 25 C (Chuesiang et al., 2018). The effect of the compositions and interfacial properties. Thus, nanoemulsion phase inversion temperature method on biological activity of formation by low energy methods depends on physicochemical cinnamon oil nanoemulsions has been studied. The surfactant properties such as temperature, composition, and solubility concentration effected the antimicrobial activity of the cinnamon (Anton and Vandamme, 2009). The low energy methods involved oil nanoemulsions. The increase in surfactant concentration in the nanoemulsion production are phase inversion temperature from 15 to 20 wt% enhanced the antimicrobial activity of (PIT), phase inversion composition (PIC), and solvent diffusion nanoemulsion in comparison to nanoemulsions with lower method. These methods involve minimal energy generation, and surfactant concentration (10 wt%) or with bulk cinnamon oil thereby prevent the degradation of heat labile compounds. (Chuesiang et al., 2019). Phase Inversion Temperature Method The method uses phase invasion property of the molecules, Phase Inversion Composition Method wherein the emulsifiers change their hydrophilicity or In this technique, varying the composition of constituents lipophilicity as a function of temperature at fixed composition changes the hydrophilic–lipophilic behavior of emulsifier (Figure 3). Depending on the hydration of the polar heads of the (Figure 3). On adding salt to an oil-in-water nanoemulsion non-ionic surfactants, they change their spontaneous curvature with ionic emulsifier, the electric charge of surfactant changes (Anton and Vandamme, 2009). At low temperature, oil-in-water and it turns to water-in-oil emulsion system (Maestro et al., emulsion is formed. Whereas, at high temperature, water-in-oil 2008). Similarly, a water-in-oil emulsion having high salt emulsion is formed as the solubility of emulsifier in water content can be converted to oil-in-water by diluting with decreases with increase in temperature. The phase inversion water (Liew et al., 2010). This technique has low cost, does temperature is the temperature at which there is transition not require the use of organic solvents and it has high from oil-in-water to water-in-oil emulsion (McClements and thermodynamic stability (Shakeel et al., 2009). It is difficult to Rao, 2011). At a particular temperature, the curvature of use phase inversion method for highly hydrophobic compounds emulsifier layer becomes zero and solubility of emulsifier (Witthayapanyanon et al., 2006). becomes approximately equal in water and oil phase. At this Phase inversion composition method has been used to prepare stage, there is no tendency to form either oil-in-water or water- food-grade nanoemulsions enriched with vitamin E acetate in-oil nanoemulsion and constituents form a bicontinuous or with a mean particle diameter of 40 nm. This method was lamellar liquid crystalline system. At higher temperature, the more effective at producing nanoemulsions at high surfactant surfactant layer becomes concave with negative curvature due to concentration than microfluidization technique. However, the dehydration of hydrophilic nonionic surfactant. The solubility of technique was not favorable for nanoemulsions preparations with Frontiers in Sustainable Food Systems | www.frontiersin.org 8 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications FIGURE 3 | Low energy methods involves breakdown of coarse w/o macroemulsions into nanoemulsions during phase inversion. In the Phase Inversion Composition technique, dilution by water induces a phase inversion while in the Phase Inversion Temperature method, cooling or decrease in temperature induces a phase inversion. label-friendly surfactants, such as Quillaja saponin, whey protein, fine droplets. Particle size could also be reduced by increasing casein, and sucrose monoesters (Mayer et al., 2013). the temperature and stirring speed used when the oil/surfactant mixture was added to water (Saberi et al., 2013). Similarly, vitamin D nanoemulsions have been prepared by spontaneous Spontaneous Emulsification Method emulsification. The nanoemulsions with small droplet diameters In this technique, spontaneous emulsions are formed on mixing <200 nm that were stable to droplet growth at ambient water and oil together with an emulsifier by gentle stirring temperatures but unstable at high temperatures (>80 C) were at a particular temperature (Figure 4). The mixing of phases obtained. The thermal stability of the nanoemulsions was by gentle magnetic stirring causes the emulsifier to enter the increased by using a cosurfactant during the formulation (Guttoff aqueous phase leading to increase of oil-water interfacial area et al., 2015). Spontaneous emulsification has been used to prepare resulting in oil droplet formation (Anton et al., 2008). The excess oil phase is removed by evaporation under reduced pressure fish oil nanoemulsions as these are rich in omega-3 fatty acids. Transparent nanoemulsions with physical stability at 37 C and (Bouchemal et al., 2004). The drawback of this method is the oxidative stabilities at 55 C for 14 days could be prepared use of high synthetic surfactants at large scale which is not (Walker et al., 2015). Cinnamaldehyde has been encapsulated feasible economically and has regulatory and sensory issues in in self-emulsifying emulsion systems. Stable cinnamaldehyde food industry. nanoemulsions were achieved only with the addition of medium- Thus, in the low energy method of nanoemulsion formation, chain triglyceride. The encapsulation efficiency of nanoemulsions the intrinsic physicochemical properties of surfactants and the was 80% for 1 week and slow release of cinnamaldehyde could oily phase plays a major role and can be easily scaled up. In the be expected. Phase separation occurred after 12 days of storage high-energy techniques, the size distribution and composition under 37 C. Encapsulation efficiency of cinnamaldehyde in of nanoemulsion can be controlled using mechanical devices, nanoemulsions was maintained around 80% within 1 week (Tian however, there could be degradation of constituents and the production processes cannot be scaled up (Date et al., 2010). et al., 2016). The effect of high and low energy approaches on Spontaneous emulsification method has been used to obtain the physicochemical properties of nanodispersions vitamin E acetate nanoemulsions of droplet diameters <50 nm has been evaluated. Solvent displacement and high- and low polydispersity indexes. Oil phase composition and pressure valve homogenization was used to prepare lutein surfactant-to-emulsion ratio had to be optimized to produce Frontiers in Sustainable Food Systems | www.frontiersin.org 9 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications FIGURE 4 | Spontaneous emulsification is a low energy method. In this method, nanoemulsions are formed on mixing water and oil together with an emulsifier by gentle stirring at a particular temperature. nanodispersions. The particle size and particle size distribution cannot be used to produce pickering nanoemulsions. of nanodispersions prepared prepared by both methods The vapor condensation method used for pickering were not significantly different. They also had very high nanoemulsion preparation is a single step process and lutein retentions (>90%). Therefore, solvent displacement has several advantages over the conventional techniques can also be a good alternative to high-pressure valve such as use of low concentration of nanoparticles homogenization in lutein nanodispersions preparation (Kang et al., 2018). (Tan et al., 2016). Novel Nanoemulsion Preparation CHARACTERIZATIONS OF Techniques NANOEMULSIONS The conventional nanoemulsification formulation involves the break drown of larger droplets or inversion of solvents. New The physiochemical properties of nanoemulsions such as emulsification approaches are being increasingly developed to physical properties, stabilities, rheological property, and increase the range of materials formulations and operating microstructure have to be characterized for its application in conditions, and to simultaneously lower the production costs. foods as these properties are known to influence the final texture, A bottom-up approach method based on condensation taste, flavor, and stability of foods. The basic physical properties has been developed to create nanoscale emulsions. Water-in- of nanoemulsion are particle size, size distribution, charge, and oil nanoemulsions are prepared by condensing water vapor lipid crystallinity. on subcooled oil-surfactant solution. The water droplets Particle structure and size distribution of nanoemulsions is nucleate at the oil and air interface to spontaneously measured by dynamic light scattering (DLS) and small-angle X- disperse within the oil. This occurs as a result of the ray scattering (SAXS), non-invasive techniques. DLS technique spreading dynamics of oil on water which depends on the measures size distribution of small particles or droplets in oil-surfactant concentration. The nanoemulsions formed suspensions in the range of 3 nm−5 μm. DLS records the using condensation approach have a peak radii of 100 nm. intensity fluctuations that occur as a result of change of relative An oil bath is placed in a humid environment with spatial location due to Brownian motion of small particles over appropriate concentration of surfactant, and on decreasing time when light is scattered by particles (Mason et al., 2006; Fryd the temperature below the dew point, water condensation and Mason, 2012). In the DLS technique, the size distribution is induced on the oil surface resulting in nanoemulsion profiles of nanoemulsions is observed as a single and narrow formation. The process is simple, rapid, scalable, and peak as it is monodisperse (Yun Zhang, 2003). DLS measurement energy efficient with potential application in processed foods utilizes the “Mie theory” mathematical model to determine the (Guha et al., 2017). scattering pattern of the droplets contained in the emulsion. DLS Pickering nanoemulsions have been developed using data is observed as plot of particle concentration and its size the vapor condensation strategy. The use of pickering (Mcclements, 2007). DLS can also be used to evaluate the stability nanoemulsion overcomes the problems associated with of nanoemulsions in various external conditions. surfactant desorption and Ostwald ripening. The high Small-angle X-ray scattering (SAXS)/small-angle neutron energy approach prevents adsorption of the particles scattering (SANS) is used to characterize the colloidal particles on the droplets whereas, the low energy approach for its shape, size, and nanostructure (Gradzielski, 2008). The Frontiers in Sustainable Food Systems | www.frontiersin.org 10 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications SAXS pattern is a function between intensity and scattering the microstructure of nanoemulsions at a resolution of <5 nm vector and obtained at low angles (typically 0.1–10 ), whereas (Acharya and Hartley, 2012; Silva et al., 2012). Transmission SANS uses short wavelength (λ < 10Å) to measure neutron electron microscopy (TEM) which has a resolution of (0.2 nm) scattering length density of nanoemulsions (Mason et al., 2006). has been used to image materials and biological samples Particle charge or the electrical surface charge of emulsions (Luykx et al., 2008; Silva et al., 2012). It has also been used is determined by measuring the zeta potential (ζ-potential). ζ- to study the morphology and structure of the nanoemulsions potential is the electro-kinetic potential in nanoemulsions. The (Bouchemal et al., 2004; Inugala et al., 2015). However, particles with surface charge on dispersion in liquid phase attract sample preparation for TEM uses high-energy electron beam the ions of opposite charges which form a firm attachment which can cause structural damage to the materials (Luykx known as stern layer. The electrical charge on the droplet surface et al., 2008). This problem can be overcome by the use of affects the interactions between emulsion droplets and in turn cryogenic preparation (cryo-TEM) and freeze-fracture (FFTEM) the stability of nanoemulsions. The nanoemulsions having high techniques. Cryo-TEM is effective in characterizing the structure absolute value of zeta potential (negative or positive, commonly of microemulsions (Spernath et al., 2009). The FFTEM provides above 30) are electrically stabilized, and those with low zeta detailed structure of bicontinuous nanoemulsions and droplets potential coagulate (Mcclements, 2007; Mohanraj and Chen, within discontinuous nanoemulsions (Krauel et al., 2007; 2007). Acharya and Hartley, 2012). Scanning electron microscope Lipid crystallinity of nanoemulsions is measured using a (SEM) is used to characterize materials in the nanometer thermo-analytical technique, differential scanning calorimetry to micrometer scale for microstructure morphology and (DSC). The technique measures the crystalline temperature chemical composition (Goldstein et al., 2003; Ferreira et al., differences of pure oil and oil in emulsion and thus assesses 2015). SEM uses narrow electron beam to provide surface the influence of oil crystallization on the stability of the structures. Hence, SEM is used to obtain surface structure nanoemulsion (Thanasukarn et al., 2004; Rafanan, 2013). The of oil droplets of nanoemulsion (Silva et al., 2012). SEM impurities present in the oil used for emulsion causes crystal can be used to process large amounts of materials at low formation and nucleation, which in turn affects the stability magnification, and high-resolution images can be obtained of nanoemulsion. The percentage of destabilized fat in the at higher magnification (Luykx et al., 2008). The drawbacks emulsions can be calculated using the DSC instrument. It associated with SEM are high cost, use of high vacuum measures the area under the non-emulsified enthalpy peaks and high sample conductivity. Surfactants present in the during a cooling cycle, and divides it by the area under all of sample can also interfere with SEM imaging as it causes the enthalpy peaks. However, as the oil is dispersed into small coating on the particle surfaces (Luykx et al., 2008). Atomic droplets, the interfacial layer reduces the nucleation growth of oil force microscopy (AFM) microscopy is used for structural crystal (Mcclements, 2007). characterization of nanoemulsion. It detects the changes of Nuclear magnetic resonance (NMR)-based techniques have force between a shape probe and an immobilized sample and also been used to study the types, structure, and diffusion produces a high-resolution 3-D profile of the sample surface properties of components in nanoemulsions (Acharya and (Preetz et al., 2010). Hartley, 2012; Hathout and Woodman, 2012). One of the NMR techniques, the Fourier transform pulsed gradient spin echo (FT-PGSE) technique measures self-diffusion coefficients of oil, APPLICATION OF NANOEMULSIONS IN water, and surfactant molecules at a time. It is also useful in FOOD determining the connectivity in emulsions and the PGSE helps to know the phase transient between the droplet emulsion phase The technological limitations of developing functional foods and bicontinuous emulsion (Gradzielski, 2008). is the low solubility, stability, and bioavailability of the Rheology of nanoemulsions is measured using shear device bioactive compounds. Most of the bioactive food ingredients are which gives the value of apparent viscosity and the dependent susceptible to degradation during food processing and oxidative relationship between apparent viscosity and shear stress. The deterioration during storage (Xianquan et al., 2005; Shahidi and dynamic oscillation methods are used to evaluate the viscoelastic Zhong, 2010). Certain bioactives have low solubility but rapid properties of emulsions. The stable emulsions flow curves have a metabolism which reduces its bioavailability, whereas some are −1 constant value of apparent viscosity at low shear rates (∼0.01s ) volatile and sensitive to processing conditions (McClements and and a strong shear thinning follows at high shear rates (Sharma Li, 2010; Jin et al., 2016). These challenges can be overcome by et al., 2010). The rheological properties of nanoemulsions are the use of nanoemulsions to encapsulate bioactive compounds affected by the surfactants used, shape, and number density of for their use in food matrix. The encapsulation of bioactive the droplets, and interactions between the constituent droplets compounds in an oil phase or emulsifier ensures it stability, (Acharya and Hartley, 2012). Use of cosurfactants has shown bioavailability, and controlled rate of release (McClements et al., to weaken the interactions between emulsifier and the anionic 2007). The nanoemulsion based delivery system should have surfactant, resulting in reduced viscosity (Howe and Pitt, 2008). compatibility with food matrix and minimal effect on the Microstructure characterization of nanoemulsions is organoleptic properties of the food such as its flavor, appearance, carried out using microscopy imaging techniques. Electron and texture. The encapsulation of bioactive compound can microscopy (EM) techniques is used for the visualization of protect it from processing conditions and prevent its degradation Frontiers in Sustainable Food Systems | www.frontiersin.org 11 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications for long duration from temperature, light, pH, and oxidative reduced particle size and improved the polydispersity of the conditions during storage. The application of nanoemulsion nanodispersions. The β-carotene nanodispersions had high ζ- based delivery system for foods requires that the technique is potential of −27 mV at pH 7 and thus were stable against particle economically feasible for industrial scale production (Borthakur aggregation (Chu et al., 2007). O/W β-carotene nanoemulsions et al., 2016; Pathak, 2017). Table 3 provides an overview of prepared using Tween 20 as emulsifier by high pressure nanoemulsions of bioactive compounds for food applications. homogenization had mean diameters from 132 to 184 nm. Only 25% of β-carotene in the nanoemulsions degraded after 4 weeks of storage at 4 and 25 C (Yuan et al., 2008). Solvent displacement Encapsulation of Flavor and Coloring technique was used to prepare β-carotene nanodispersions of 30– Agents 206 nm using emulsifiers such as sodium caseinate, Tween 20, The flavors and coloring compounds used in food have decaglycerol monolaurate, and sucrose fatty acid ester. Though, aldehyde, ketone, and esters as functional groups which make starch casein stabilized nanodispersions had large particles size them susceptible to oxidative and photolytic degradation. but they were most stable against oxidation. This might be due Encapsulation within nanoemulsions of these ingredients can to physical barrier and the antioxidant activity of caseins (Yin prevent these deleterious effects and enhance its shelf life et al., 2009). β -carotene nanoemulsions stabilized by modified (Goindi et al., 2016). Citral, a α,β-unsaturated aldehyde with starch and spray-dried to powders after the emulsification one additional double bond is an aromatic compound and process had better storage stability. Modified starches having used as flavors in food and cosmetics. However, its degradation low film oxygen permeability had a high retention of beta- produces off-flavor compounds. Nanoemulsions of citral have carotene during storage (Liang et al., 2013). Physicochemical been prepared to increase its stability. O/W nanoemulsion of properties of β -carotene nanoemulsions have been improved citral combined with natural antioxidants such as β-carotene, by coating emulsions with starch caseinate and chitosan- tanshinone, and black tea extract had a high chemical stability epigallocatechin-3-gallate conjugates (Wei and Gao, 2016). during storage. The nanoemulsions were prepared with lecithin- Sonication-assisted method with freeze drying has been used to stabilized palm kernel lipid in pH 3 buffer 1:1 ratio of citral and prepare β-carotene nanoemulsions with high water dispersibility antioxidants. Encapsulation with antioxidants led to decreased and chemical stability (Chen et al., 2017). Carotenoids were formation of off-flavor compounds, such as α, p-dimethylstyrene extracted from Cantaloupe melon. The nanoemulsions prepared and p-methylacetophenone (Yang et al., 2011). Similarly, O/W from these carotenoids had improved water solubility and nanoemulsion system of citral and ubiquinol-10 (Q H ) had color stability. The o/w carotenoid nanoemulsions encapsulated 10 2 good chemical stability. The Q H had a protective effect on in porcine gelatin and whey protein isolate had an average 10 2 citral and prevented its chemical degradation and oxidation. particle size of 70–160 nm. Gelatin was able to increase It reduced generation of off-flavor compounds (p-cresol, α,p- water solubility of carotenoids. The yogurt containing these dimethylstyrene, p-methylacetophenone) and lipid degradation nanoemulsions as natural coloring agent was stable for 60 days products. At Q H /citral ratio of 1:1, the ubisemiquinone (Q (Medeiros et al., 2019). 10 2 10 •− )/ubiquinone (Q ) redox transition was induced, leading to pro-oxidant behavior of Q H and its ability to maintain citral’s Encapsulation of Nutraceuticals 10 2 stability (Zhao et al., 2013). Gelatin and Tween 20 have been used Resveratrol, a natural polyphenol found in grape skins, as emulsifiers to increase the stability of citral from degradation blueberries, raspberries has many functional properties such as under acidic conditions in the food industry (Tian et al., 2017). antioxidant, anticancer, and antiobesity. Nanoemulsions based β-Carotene is a precursor of vitamin A and is used as a natural delivery systems have been used to encapsulate resveratrol. colorant, and an antioxidant in the food industry. However, Encapsulation of resveratrol by spontaneous emulsification it is easily degraded by heat, light, and oxygen. Attempts are using 10% oil phase (grape seed oil and orange oil), 10% being made to increase the stability of β-carotene toward various surfactant (Tween 80) and 80% aqueous phase had 100 nm processing conditions. β-carotene nanodispersions prepared by droplet size and could carry 120 ± 10 μg/ml of resveratrol. The emulsification-evaporation technique have shown an increased encapsulated resveratrol had improved chemical stability against stability. Nanoemulsion was prepared by emulsifying organic UV-light degradation (Davidov-Pardo and McClements, 2015). solution of β-carotene in aqueous phase containing emulsifier. O/W edible nanoemulsions of vitamin D (cholecalciferol) have Stable β-carotene nanodispersions were produced with weighted been used for the fortification of dairy emulsions. Emulsifiers mean diameter ranging from 60 to 140 nm (Tan and Nakajima, polysorbate 20, soybean lecithin and their mixtures and dispersed 2005a). O/W β-carotene nanodispersions prepared using non- oil phase of soybean oil or mixtures of the oil with cocoa butter ionic emulsifier polyglycerol esters of fatty acids (PGEs) having were used to prepare nanoemulsions of mean diameters <200 nm mean diameter of 85–132 nm showed improved physicochemical with high pressure homogenizer. Vitamin D3 (0.1–0.5 μg/mL) properties and physical stability. PGE decaglycerol monolaurate were encapsulated in the oil cores of stable nanoemulsions. −1 (10 g kg ) having high glycerol polymerization could be used Whole-fat milk was fortified with vitamin D3 nanoemulsions and for highly stable β-carotene nanodispersions (Tan and Nakajima, were stable for 10 days against particle growth and gravitational 2005b). Protein-stabilized β-carotene nanodispersions prepared separation (Golfomitsou et al., 2018). O/W nanoemulsions of using emulsification-evaporation had a mean particle size of kenaf seed oil stabilized with emulsifiers sodium caseinate, beta- 17 nm. The emulsifier sodium caseinate, at a high concentration cyclodextrin, and Tween 20 had improved in vitro bioaccessibility Frontiers in Sustainable Food Systems | www.frontiersin.org 12 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications TABLE 3 | Functional compounds encapsulated into nanoemulsions for food applications. Bioactive Function Method Particle size Constituents Reference compounds Citral Flavoring agent HPH (6 cycles for 150 MPa) 109-129 nm o/w : palm kernet fat/ citral/ undecane/ lecithin Yang et al., 2011, HPH (6 cycles/ 150 MPa) 98-120 nm aqueous buffer solution; Zhao et al., 2013 HPH (3 cycles/1000 bar) 467 nm o/w: MCT/ buffer solution (citric acid/sodium Tian et al., 2017 hydroxide/sodium chloride)/ soy lecithin/ citral/ undecane o/w: medium chain triacylglycerl/ buffer solution (citric acid/sodium hydroxide/sodium chloride)/ gelatin/ Tween 20/ citral/ undecane β-Carotene Coloring agent Sequential HPH (3 cycles/ 60-140 nm o/w: Hexane/β-carotene/ Tween 20 Tan and Nakajima, 60-140 MPa), MF and 85-132 nm o/w: Hexane/β-carotene/ Polyglycerol 2005a Solvent-evaporation 17-110 nm esters of monolaurate and monooleate; Tan and Nakajima, Sequential HPH (140 MPa), 132-184 nm o/w: Hexane/β-carotene/ sodium caseinate/ 2005b MF and Solvent-evaporation 30-206 nm whey protein isolate/soy protein isolate Chu et al., 2007 MF (1-3 cycles/ 140 MPa) 114-159 nm o/w: Hexane/β-carotene/Tween 20 Yuan et al., 2008 HPH 284 nm o/w: Hexane/β-carotene/starch/ Yin et al., 2009 SD 230 nm sodium caseinate/Tween 20/decaglycerol Liang et al., 2013 HPH (10 cycles/ 150 MPa) 70-160 nm monolaurate/sucrose fatty acid ester Wei and Gao, HPH (3 cycles/ 60 MPa) o/w: β-carotene/ 2016 Ultrasonication and MF Starch/ medium chain triacylglycerl Chen et al., 2017 HPH o/w: β-carotene/ Medeiros et al., sunflower oil/ starch 2019 caseinate/chitosan-epigallocatechin-3-gallate o/w: β-carotene/ vegetable oil/ casein o/w: Caotenoid extract/ soybean oil/gelatin/ whey protein isolate Resveratrol Nutraceutical Spontaneous emulsification 100 nm o/w: resveratrol/ grape seed oil/ Orange oil/ Davidov-Pardo Tween 80 and McClements, Vitamin D Nutraceutical HPH <200 nm o/w: Vitamin D/ polysorbate 20/ soybean Golfomitsou et al., lecithin/ cocoa butter 2018 Vitamin E and Nutraceutical HPH (4 cycles/ 28000 psi) 100 nm o/w: Vitamin E/ phytosterol/ kenaf seed oil/ Cheong et al., phytosterol sodium caseinate/ beta-cyclodextrin/ Tween 20 Astaxanthin Nutraceutical Spontaneous emulsification 150–160 nm o/w: Astaxanthin/ lecithin/ caprylic-capric Alarcon-Alarcon triglyceride/ chitosan et al., 2018 Curcumin Nutraceutical HPH (20 cycles/ 103 MPa) 80 nm o/w: curcumin/ Silva et al., 2018 MCT/SDS Lecithin Nutraceutical MF (5 cycles/ 150 MPa) <400 nm o/w: curcumin/ corn oil/ sodium alginate/ Artiga-Artigas Tween 20/ lecithin et al., 2018 Oregano EO Preservative Ultrasonication (750 W) 148 nm o/w: Oregano oil/Tween 80 Bhargava et al., 2015, Ultrasonication (20KHz/400 180-250 nm o/w: Oregano oil/ clove bud oil/Tween 80/ Otoni et al., 2014b W/10 min) distilled water Orange EO Preservative Ultrasonication (20KHz/750 20-30 nm o/w: orange oil/ Tween 80/ distilled water Sugumar et al., W/10 min) 2015 Cinnamaldehyde Preservative Spontaneous emulsification 20-500 nm o/w: Cinnamaldehyde/ Tween 80/ distilled Otoni et al., 2014a water Ginger EO Preservative MF (10 cycles/1000 psi) 133 nm o/w: Ginger EO/ Tween 20/ Span 80/ Canola oil Acevedo Fani et al., 2015 Ultrasonication (20 KHz/200 57 nm o/w: Ginger EO/ Tween 80 Noori et al., 2018 W/ 5 min) Curcumin Preservative Spontaneous emulsification 40-130 nm o/w: Curcumin/ Abdou et al., 2018 Tween 80/ Glycerol/ Water Capsaicin Preservative HPH (5 cycles/140 MPa) 65 nm o/w: Capsicum oleoresin/ Tween 80/ Water Akbas et al., 2018 (Capsicum Ultrasonication oleoresin) (75% amplitude/5 min) HPH, High Pressure homogenization; MF, Microfluidization; MCT, medium chain triacylglycerl. Frontiers in Sustainable Food Systems | www.frontiersin.org 13 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications and physicochemical stability of bioactive compounds and life of chicken breast filet. The nanoemulsion based active coating antioxidants. It was observed that after 8 weeks of storage at had higher antibacterial activity than antioxidant activity. It 4 C, the nanoemulsions were stable and maintained antioxidant reduced growth of psychrophilic bacteria in refrigerated chicken activities with high percentage retention of vitamin E and filets for 12 days (Noori et al., 2018). Nanoemulsions have been phytosterols (Cheong et al., 2018). Astaxanthin, a dietary used to increase the bioavailability of lipophilic compounds. supplement is a light sensitive molecule. Nanoemulsions have High pressure homogenization and ultrasoniction were used been formulated to photostabilize the pigment for its use in foods. to prepare capsaicin (oleoresin capsicum) nanoemulsions with The chitosan-coated and carrageenan-coated nanoemulsions Tween 80 as the aqueous phase. Ultrasonication improved of astaxanthin provided it protection from UV light and physical properties as nanoemulsions with particle size below photodegradation (Alarcon-Alarcon et al., 2018). 65 nm were obtained, whereas high pressure homogenization Nanoemulsions have been used to improve the bioaccessibility resulted in preparation of nanoemulsions with good inhibitory of lipophilic bioactive compounds. Curcumin nanoemulsions activity against S. aureus and E. coli (Akbas et al., 2018). prepared with chitosan and alginate had improved antioxidant capacity during in vitro digestion and a better control over lipid digestibility by decreasing free fatty acids. Thus, these NANOEMULSION BASED FOOD nanoemulsions can be used to fortify functional foods for PACKAGING MATERIALS targeting obesity (Silva et al., 2018). Similarly, nanoemulsions of curcumin have been prepared with improved antioxidant The nanoemulsions can be incorporated into films and capacity. Lecithin-stabilized nanoemulsions of curcumin had an coatings for potential food packaging applications. The films encapsulation efficiency of 75% and were stable for 86 days in and coatings made up of biopolymer matrix constitute comparison to other surfactants such as Tween 20 stabilized the continuous phase as they provide monodispersity and curcumin nanoemulsions (Bhosale et al., 2014). stability to nanoemulsion droplets. The increase in viscosity of the continuous phase reduces the coalescence of droplets Natural Preservatives (Artiga-Artigas et al., 2017). The preparation of nanoemulsion Plant essential oils including thyme, oregano, clove and orange bioedible films includes dispersing the bioactive ingredients and their components such as thymol, carvacrol, eugenol, in a continuous phase which makes up the matrix of limonene, and cinnamon have strong antimicrobial activity the packaging films. Food grade emulsions are added and against food borne pathogens. But their application in food appropriate high or low energy techniques are used for matrix is limited due to their hydrophobicity. Nanoemulsion homogenization. The homogenized formulation is cast into formulations of essential oils can be used to overcome this films with controlled thickness and dried. Further, these problem and they can be used in foods as natural preservatives films are characterized for the structural, morphological, and in food packaging (Alexandre et al., 2016). thermal, mechanical, and barrier properties (Otoni et al., Oregano oil nanoemulsions inhibited growth of foodborne 2014a). The biopolymers used for film preparation also bacteria Listeria monocytogenes, Salmonella Typhimurium and play a role in maintaining the functional properties of E. coli O157:H7 on fresh lettuce. It was observed that oregano dispersed nanoemulsions. oil nanoemulsions disrupted bacterial membranes (Bhargava The biopolymer pectin has been used to prepare edible et al., 2015). Orange oil nanoemulsions inhibited spoilage films of cinnamaldehyde and clove essential oil nanoemulsions of apple juice by Saccharomyces cerevisiae (Sugumar et al., with antimicrobial properties. The pectin films had low water 2015). Similarly, cinnamaldehyde nanoemulsions incorporated and vapor permeability due to the decrease in ratio of in pectin edible films inhibited growth of E. coli, Salmonella hydrophilic/hydrophobic (Otoni et al., 2014a; Sasaki et al., enterica, Listeria monocytogenes, and Staphylococcus aureus 2016). Cellulose and its derivatives have been used to prepare (Otoni et al., 2014a). Nanoemulsions of clove bud and oregano edible films containing nanoemulsions of clove and oregano essential oil with 180–250 nm droplet size incorporated into essential oils (Otoni et al., 2014b). Chitosan has been used for edible methylcellulose films had improved antimicrobial activity. preparation of nanoemulsion coatings and films of essential They prevented growth of yeasts and molds and improved shelf oils with antimicrobial activity such as carvacrol, mandarin oil, life of sliced bread (Otoni et al., 2014b). Nanoemulsions of ginger bergamot oil, and lemon oil (Severino et al., 2015). Sodium essential oil incorporated into gelatin-based films have shown alginate was used to formulate films of nanoemulsion containing to improve the physical properties of the active food packaging thyme, lemongrass, and sage essential oil (Acevedo Fani et al., films (Alexandre et al., 2016). Curcumin nanoemulsions with 2015). It was also useful in preparing films containing small, mean droplet size of 40 nm pectin edible coatings increased stable and less size-dispersed nanoemulsions of corn oil (Artiga- the shelf life of chilled chicken at 4 C for 12 days. The Artigas et al., 2017). Porcine gelatin has been used in preparing nanoemulsion reduced microbial spoilage by inhibiting growth films with antioxidant activity from canola oil and ginger of psychrophiles, yeast and mold growth. Further, it showed essential oil nanoemulsions (Alexandre et al., 2016). It has reduced values of total volatile nitrogen and thiobarbituric acid, also been used to prepare films of rutin-encapsulated soybean water holding capacity and texture and higher sensory scores in oil. However, the nanoemulsified essential oil increased the comparison to the control (Abdou et al., 2018). Nanoemulsions of water vapor permeability values resulting in poor water barrier ginger essential oil with sodium caseinate coating extended shelf properties (Dammak et al., 2017). Basil seed gums have been Frontiers in Sustainable Food Systems | www.frontiersin.org 14 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications used to prepare antimicrobial films of nano-emulsified Zataria structured liquids (NSSL) have been developed by NutraLease, multiflora essential oil (Gahruie et al., 2017). a technology start-up company. Small compressed micelles, The foods have also been immersed in nanoemulsions called nanodrops are developed and these micelles serve as a formulations to test their efficacy as coating agents without carrier for fat soluble bioactives. The micelles incorporated in the aid of polymer matrix. Green beans have been coated food products pass through the digestive system effectively and with chitosan solution containing nanoemulsions of mandarin to the absorption sites without undergoing degradation. These essential oil. And further irradiated with γ-irradiation, UV- nanoemulsions have been used for developing beverages with C, and treated with ozonized water. The synergistic effect of functional compounds. NutreLease with NSSL technology has the combined treatment of nanoemulsion coating and UV-C fortified beverages with nanoemulsions of lipophilic compounds irradiation inhibited the growth of Listeria innocua in green such as β-carotene, omega-3, vitamins, phytosterols, and beans (Severino et al., 2014). Sliced breads were immersed isoflavones. It claimed that the food products had improved in methylcellulose-and clove bud or oregano essential oil bioavailability and good shelf life (NutraLease, 2011). NSSL have nanoemulsions. There was a reduction in spoilage molds been used by Shemen industries to develop Canola Active oil resulting in increase of shelf life of sliced bread (Otoni which is fortified with non-esterified phytosterols. The minute et al., 2014b). Fresh-cut Fuji apples have been immersed in micelles carry the phytosterols to the large micelles that the nanoemulsion formulation of alginate-based edible coatings of body produces from the bile acid, where they compete with lemongrass essential oil for 2 min and stored at 4 C for 14 cholesterol for entry into the micelle. The phytosterols enter days. The nanoemulsion coating had an inhibitory effect on the micelle, thereby inhibiting transportation of cholesterol from E. coli and the growth of natural flora of apple was inhibited the digestive system into the bloodstream. Similarly, Aquanova for 2 weeks (Salvia-Trujillo et al., 2015). Fresh-water rainbow has developed NovaSol beverages fortified with nanoemulsions trout was immersed for 15 min in the nanoemulsion formulation of functional compounds and natural colorants (β-carotene, containing essential oils of Zataria multiflora Boiss and packaged apocarotenal, chlorophyll, curcumin, lutein, and sweet pepper within polyethylene bags and stored at 4 C. Its shelf-life increased extract). It claimed that encapsulated compounds had enhanced and sensory attributes were preserved during the storage time stability and standardized additive concentrations (AquaNova, (Shadman et al., 2017). Similarly, rainbow trout filets immersed 2011). The product NovaSOL sustain contains nanocarrier for 3 min in nanoemulsions of rosemary, laurel, thyme, and that introduces coenzyme Q1O and alpha-lipoic acid. The sage nanoemulsions were packed in stretch films and stored at micelle (30 nm) is stable to pH and temperature variations 2 C for 24 days. The nanoemulsions were effective in inhibiting and is completely water soluble. The micelle is assumed to growth of psychrotrophs and and Enterobacteriaceae members be optimum carrier system of lipophiles and can be used for (Ozogul et al., 2017). Spoilage of cherry tomatoes by molds such efficient intestinal and dermal resorption and penetration of as Botritis cinerea has been prevented by coating it with thymol active ingredients (AquaNova, 2011). RBC Life sciences has nanoemulsions. Quinoa protein and chitosan edible coating developed NanoceuticalsTM Slim Shake Chocolate. It employs TM containing thymol nanoemulsion significantly inhibited growth NanoCluster delivery system, a nanosize powder of nutritional of fungi on cherry tomatoes after 7 days when stored at 5 C supplements. CocoaClusters developed by this method is a (Robledo et al., 2018). Similarly, anise oil loaded nanoemulsions technologically advanced form of cocoa and does not require in comparison to coarse oil and bulk oil, significantly inhibited sugar. During the process of creating NanoClusters, pure Cocoa the growth of foodborne pathogens including E. coli O157:H7 is added to the “Cluster” formation. When consumed, it reduces and L. monocytogenes (Topuz et al., 2016). the surface tension of foods and supplements to increase wetness and absorption of nutrients. Another nutritional supplement R TM Nanoemulsions in Food Industry developed by RBC Life Sciences , is called NanoCeuticals . TM A number of in vitro and in vivo food challenge studies The product NanoCeuticals , with nanoscale ingredients is have shown the advantages of using nanoemulsions in foods is claimed to have antioxidant properties. A product Nanotea as delivery systems for bioactive compounds. However, there developed by Shenzhen Become Industry & Trade Co., Ltd. are very limited examples of nanoemulsions of bioactive contains nanofine powder produced. The product is claimed to compounds incorporated into commercial food products or have antimicrobial activity and high selenium supplementation used as packaging materials. Nanoemulsions can be used for by 10 times. LivOn Labs has developed a product Lypo- TM R sustainable food processing. They can prevent the functional Spheric Vitamin C which uses smart liposomal Nanospheres ingredients from temperature, oxidation, enzymatic reactions, to encapsulate Vitamin C. The product has increased the and pH variations. Food industries such as Nestle and Unilever bioavailability of all nonliposome encapsulated forms of vitamin and a few Start-ups have used nanoemulsions in their food C (http://www.nanotechproject.org). Bottled waters with oil- products (Silva et al., 2012; Salvia-Trujillo et al., 2017). Nestle soluble flavors and enriched with electrolytes, vitamins, and have developed w/o nanoemulsions and patented polysorbates nutraceuticals with nanoemulsion technology have also been and micelle-forming emulsions for rapid and uniform thawing developed (Piorkowski and McClements, 2014). of frozen foods in the microwave (Möller et al., 2009). Unilever Granalix BioTechnologies has commercially launched TM has used nanoemulsions in ice creams for reducing fat content GranaGard a food supplement based on pomegranate oil from 16 to 1% (Martins et al., 2007; Unilever, 2011). Novel which has shown to prevent neurodegeneration diseases in nanoencapsulation method known as Nano-sized self assembled animal models. GranaGard, is a submicron pomegranate seed oil Frontiers in Sustainable Food Systems | www.frontiersin.org 15 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications emulsion, and is an innovative formulation containing natural known to protect and increase the bioavailability of bioactive antioxidants, Punicic acid (an Omega 5 lipid), which constitutes compounds as shown by in vitro studies. But there are 80% of the nanoemuslion. The novel patented formulation was limited studies which show the actual health benefits of shown to delay disease onset and prevent neuronal death in including nanoemulsions in foods, their consumption, labeling, animal models (Binyamin et al., 2015). Therapeutics Solutions and public perception. Similarly, studies have been carried International, Inc., has produced nutraceuticals prepared from out to evaluate the use of high or low energy approach TM NanoStilbene , stable nanoemulsion of pterostilbene (75– to formulate nanoemulsions and the focus is on optimizing 90 nm) from GRAS ingredients by low energy emulsification processing parameters and ingredients used in nanoemulsion methods. The nanoemulsion of pterostilbene containing preparation. But there is not much research on reducing nanoparticles has been patented. Pterostilbene, the active the cost of production of nanoemulsions as its preparation ingredient in the Company’s patented ProJuvenol product is a and application for fortifying and packaging in foods require more potent analog of resveratrol. The nanoemulsions have a higher energy input and equipment investment. Similarly, the better solubility and stability than the parent compound (Dixon risks associated with the use of engineered nanoemulsions et al., 2017). in foods is not known. The potential toxicological effects A flavor nanoemulsion containing a hydrophobic oil droplets and biological fate of nanoparticles after digestion has not containing flavors, an aqueous phase, and a surfactant system been elucidated. including a polyethoxylated sorbitan fatty acid ester and Nanoemulsions of bioactive compounds and functional lecithin has been patented by International Flavors and food ingredients have a great potential for applications in Fragrances Inc. The flavor nanoemulsion has been used to the food industries. The emulsion-based delivery systems and prepare stable and optically clear liquid beverages including nanoemulsion edible coatings can improve the functionalities of alcoholic beverages (Lee et al., 2015). Nanoemulsions of food and also enhance their quality and shelf life. However, the natural antioxidants (extracted from fruits, vegetables or cereals) food grade nanoemulsions can find widespread application only for food preservative applications have been patented. The if its production cost is commercially feasible and meets the safety nanoemulsions of encapsulated natural antioxidants have been standards of food industry. Therefore, it is important to optimize freeze dried and applied to preserve fresh and minimally the bioactivity of the encapsulated components for scaled up processed foods. The nanoemulsion have been applied as thin, production. Further studies should focus on the biological events nanometre-size layer on the food. The nanoemulsion coating and risks associated with the use of nanoemulsion based delivery has been found to prevent gas and fluid exchange with the systems in food products and packaging applications for ensuring safety of the consumers. external environment. The edible nanocoatings extends the shelf life of fresh and minimally processed foods. It has also shown to improve the organoleptic quality of frozen foods on thawing AUTHOR CONTRIBUTIONS (Malnati et al., 2019). JA and RV were involved in design, analysis, data collection, and preparation of the manuscript. CURRENT PERSPECTIVES AND FUTURE PROSPECTS ACKNOWLEDGMENTS In the last few years, a number of studies have been carried out to ascertain the advantages of encapsulation of lipophilic and The authors acknowledge the funding agency UGC-UPE, University of Mysore. functional compounds in nanoemulsions. Nanoemulsification is Akbas, E., Soyler, B., and Oztop, M. H. (2018). Formation of capsaicin loaded REFERENCES nanoemulsions with high pressure homogenization and ultrasonication. LWT. Abdou, E. S., Galhoum, G. F., and Mohamed, E. N. (2018). Curcumin loaded 96, 266–273. doi: 10.1016/j.lwt.2018.05.043 nanoemulsions/pectin coatings for refrigerated chicken fillets. Food Hydrocoll. 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Stability of absence of any commercial or financial relationships that could be construed as a lycopene during food processing and storage. J. Med. Food. 8, 413–422. potential conflict of interest. doi: 10.1089/jmf.2005.8.413 Yang, X., Tian, H., Ho, C. T., and Huang, Q. (2011). Inhibition of citral degradation Copyright © 2019 Aswathanarayan and Vittal. This is an open-access article by oilin-water nanoemulsions combined with antioxidants. J. Agric. Food distributed under the terms of the Creative Commons Attribution License (CC BY). Chem. 59, 6113–6119. doi: 10.1021/jf2012375 The use, distribution or reproduction in other forums is permitted, provided the Yin, L. J., Chu, B. S., Kobayashi, I., and Nakajima, M. (2009). Performance original author(s) and the copyright owner(s) are credited and that the original of selected emulsifiers and their combinations in the preparation publication in this journal is cited, in accordance with accepted academic practice. of β-carotene nanodispersions. Food Hydrocoll. 23, 1617–1622. No use, distribution or reproduction is permitted which does not comply with these doi: 10.1016/j.foodhyd.2008.12.005 terms. Frontiers in Sustainable Food Systems | www.frontiersin.org 21 November 2019 | Volume 3 | Article 95 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Frontiers in Sustainable Food Systems Unpaywall

Nanoemulsions and Their Potential Applications in Food Industry

Frontiers in Sustainable Food SystemsNov 13, 2019

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REVIEW published: 13 November 2019 doi: 10.3389/fsufs.2019.00095 Nanoemulsions and Their Potential Applications in Food Industry Jamuna Bai Aswathanarayan and Ravishankar Rai Vittal* Department of Studies in Microbiology, University of Mysore, Mysore, India Nanoemulsions have small droplet size and are kinetically stable colloidal systems. They have enhanced functional properties in comparison to conventional emulsions. The composition and structure of the nanoemulsions can be controlled for the encapsulation and effective delivery of bioactive lipophilic compounds. Nanoemulsions have potential application in the food industry for the delivery of nutraceuticals, coloring and flavoring agents, and antimicrobials. The nanoemulsion formulations of active ingredients can be used for developing biodegradable coating and packaging films to enhance the quality, functional properties, nutritional value, and shelf life of foods. This review focuses on preparation of food grade nanoemulsions using high-energy methods and low-energy approaches and their characterization for physical properties, stability, and microstructure. The application of nanoemulsion formulations for sustainable food processing and improving the delivery of functional compounds, such as colorants, flavoring agents, nutraceuticals, and preservatives or antimicrobial agents in foods has Edited by: José Antonio Teixeira, been discussed. University of Minho, Portugal Keywords: nanoemulsions, encapsulation, bioactive compounds, functional foods, nutraceuticals Reviewed by: Ana C. Pinheiro, Center for Biological Engineering, INTRODUCTION School of Engineering, University of Minho, Portugal Emulsions are defined as the dispersion of two immiscible liquids, with the spherical droplets Alejandra Acevedo-Fani, forming the dispersed phase, whereas the liquid surrounding it forms the continuous phase (Tadros Riddet Institute, New Zealand et al., 2004; McClements et al., 2007; Acosta, 2009). The commonly used liquids to form emulsion *Correspondence: are water and oil. The oil droplets dispersed in an aqueous phase are known as oil-in-water (o/w) Ravishankar Rai Vittal emulsions. These emulsion systems can be used for the delivery of hydrophobic active substances. raivittal@gmail.com The water droplets dispersed in oil are called the water-in-oil (w/o) emulsions and are used for the delivery of hydrophilic compounds. Multiple emulsion systems can also be developed such as Specialty section: This article was submitted to the water-in-oil-in-water (w/o/w) and oil-in-water-in-oil (o/w/o) emulsions. The w/o/w emulsions Sustainable Food Processing, are made of large oil droplets, containing water droplets dispersed in an aqueous phase. Whereas, a section of the journal in o/w/o emulsion system, water droplets containing oil droplets are dispersed in an oil phase. Frontiers in Sustainable Food Systems Bicontinuous nanoemulsion contains microdomains of oil and water—interdispersed within the Received: 23 November 2018 system (Garti and Benichou, 2004; Weiss et al., 2006). Accepted: 07 October 2019 Emulsions are categorized as coarse emulsions, microemulsions and nanoemulsions based Published: 13 November 2019 on their droplet size and stability (Komaiko and McClements, 2016). In Table 1, the various Citation: types of emulsion systems have been mentioned. However, there is some ambiguity regarding Aswathanarayan JB and Vittal RR their description based on size. The coarse emulsions are also known as conventional (2019) Nanoemulsions and Their emulsions or macroemulsions. They have particle size of diameter >200 nm range and are Potential Applications in Food thermodynamically metastable. They break down over time due to various destabilizing factors. Industry. Conventional emulsions are optically turbid as the dimension of the droplets is similar to Front. Sustain. Food Syst. 3:95. doi: 10.3389/fsufs.2019.00095 that of the wavelength of light and hence, scatters the incident light and appears opaque. The Frontiers in Sustainable Food Systems | www.frontiersin.org 1 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications TABLE 1 | Emulsion types: physicochemical properties, stability, and preparation methods. Emulsion/coarse Microemulsion Nanoemulsion emulsion/macroemulsion Size 1–100 μM 10–100 nm <200 nm Thermodynamic Stability Metastable Stable Metastable Kinetic stability Stable Unstable Stable Optical property Turbid Transparent Transparent/slightly translucent Polydispersity High (>40%) Low (<10%) Low (<10–20%) Preparation method High and low energy methods Low energy methods High and Low energy methods Effect of temperature and pH Stable to temperature and pH Effected by changes in Stable to temperature and pH changes composition, temperature and pH changes droplets in microemulsions are <100 nm in size and are COMPOSITION OF NANOEMULSIONS thermodynamically stable. However, their stability is affected by even slight variations in environmental conditions such Nanoemulsion formulation requires the use of two immiscible as composition and temperatures. Microemulsion forms liquids and an emulsifier. One of the immiscible liquids must spontaneously as its free energy is lower than its phase-separated be oleaginous and the other aqueous in nature, and they components. They are optically transparent as the particle make up the dispersed and aqueous phase. The o/w and w/o size is lesser than the wavelength and weakly scatters light nanoemulsion consists of a core–shell structure. For example, (Anton and Vandamme, 2011). The nanoemulsions have droplet in an o/w nanoemulsion system the amphiphillic shell is dimensions similar to the microemulsions ranging from <200 made of surface-active molecules, whereas the lipophilic core and in some cases <100 nm (Saifullah et al., 2016). Similar to contains non-polar molecules. The oil phase in a nanoemulsion conventional emulsions, nanoemulsions are thermodynamically is made of triacylglycerols, diacylgycerols, monoacylglycerols, metastable as phase separation occurs over time. However, and free fatty acids. Non polar essential oils, mineral oils, nanoemulsions are conferred with kinetic stability as there lipid substitutes, waxes, weighting agents, oil-soluble vitamins, is no gravitational separation and droplet aggregation due to and various lipophilic components are also used as oil phase. the reduced attractive force between the small sized droplets The viscosity, refractive index, density, phase behavior, and (McClements and Rao, 2011). The nanoemulsions unlike the interfacial tension of the oil phase components influences the thermodynamically stable microemulsions are not affected by formation, stability, and functional properties of nanoemulsion physical and chemical variations including temperature and pH. (Tadros et al., 2004; Wooster et al., 2008; McClements and Rao, They require less amount of surfactants for their preparation. 2011). However, the long-chain triacylglycerols are preferred for The droplet size of nanoemulsion apart from determining its nanoemulsion formulation due to their low cost, availability, optical property and stability, also influences its rheological and functional, and nutritional attributes (Witthayapanyanon et al., release behavior. Hence, the nanoemulsions are more suitable 2006). The aqueous phase of a nanoemulsion is made of than microemulsions for various applications. polar solvent and a cosolvent. It determines the polarity, The present review focuses on the increased application rheology, phase behavior, interfacial tension, and ionic strength of nanoemulsions in the food industries for sustainable food of nanoemulsion. The polar solvent generally used is water, processing and packaging. The nanoemulsions have the ability whereas carbohydrates, protein, alcohol, and polyols are used to encapsulate functional compounds and active ingredients as cosolvents (Saxena et al., 2017). The aqueous phase and including antioxidants and nutraceuticals. They are also useful oil phase can breakdown due to Ostwald ripening (increase in the controlled release of flavor compounds in foods (Velikov in mean droplet size over time), flocculation, coalescence, and and Pelan, 2008; McClements and Rao, 2011; Ines et al., gravitational separation (Kabalnov, 2001; McClements and Rao, 2015). Nanoemulsion encapsulation of bioactive compounds 2011). This can be prevented by adding a stabilizer agent increase its solubility, controlled release and absorption in to nanoemulsion. The stabilizers distribute on a particle and the gastrointestinal tract, and absorption through cells (Chen can form either a monolayer, multilayer, and solid particulate et al., 2006; McClements and Rao, 2011). Nanoemulsion based nanoemulsions. Some of the stabilizers used are emulsifiers, edible nanocoatings containing flavor and coloring ingredients, weighting agents, ripening retarders, and texture modifiers. antioxidants, enzymes, antimicrobials, and antibrowning agents Emulsifiers are surface active molecules and commonly used can be used to coat foods such as meats, dairy products stabilizers in nanoemulsion preparation to protect small droplets. such as cheese, fresh produce, and fresh cuts including fruit They reduce the interfacial tension resulting in formation of and vegetables and confectionaries to improve their shelf life. small and stable nanoemulsions. The emulsifiers also prevent The nanoemulsion coatings can also prevent moisture and collision and coalescence between the droplets and increases gas exchange, minimize moisture loss and oxidation of foods the kinetic stability of the nanoemulsions (Mason et al., (Azeredo et al., 2009; Rojas-Graü et al., 2009; Salvia-Trujillo et al., 2006). The emulsifiers can be cationic, anionic, nonionic, 2015; Donsi, 2018). and zwitterionic in nature. Some examples of emulsifiers Frontiers in Sustainable Food Systems | www.frontiersin.org 2 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications are small-molecule surfactants, phospholipids, proteins, and of food grade particles including proteins, polysaccharides, and polysaccharides (McClements, 2004; Kralova and Sjöblom, 2009). flavonoids have been investigated for pickering nanoemulsion Even polymers like polyvinyl alcohol are used as emulsifiers. preparation. However, colloidal lipid particles have shown to be The surfactants can be either ionic or non-ionic in nature. the most promising pickering stabilizers in O/W emulsions as Ionic surfactant prevents droplets aggregation by electrostatic they are able to impart good physical stability. These are simple to repulsion, whereas non-ionic surfactants reduce aggregation by prepare with tunable microstructure, and can be used to prepare steric hindrances, thermal fluctuation interactions, and hydration nanoemulsions using high pressure homogenization method and (McClements, 2004; Silva et al., 2015). For o/w emulsion cross flow microfluidic device (Schroder et al., 2017, 2018). preparations, hydrophile–lipophile balance values >10 are used as a criterion for selecting emulsifiers. For example, proteins insoluble in oils are used as emulsifiers for o/w nanoemulsion PROPERTIES OF NANOEMULSIONS AND preparation. Two or more emulsifiers have also been used DESTABILIZING MECHANISM together in nanoemulsion formulation for their synergistic effect. On using both hydrophilic and lipophilic (or cosurfactant) The optical properties of nanoemulsion are important for emulsifiers, a significant decrease in surface tension has been their application in food industry. Based on their droplet size observed (Shakeel et al., 2009; Qadir et al., 2016). Similarly, nanoemulsions are optically transparent or faintly turbid. Their block copolymers that are soluble only in the dispersed phase opacity is expressed in terms of turbidity (τ) and characterized can decrease surface tension (Tadros et al., 2004). However, by transmission measurements (McClements and Rao, 2011). some surfactants have found to be irritants, which limit their The mean particle size and narrow particle-size distribution application in foods. This has resulted in the formulation of influences the opacity of nanoemulsions (Wooster et al., 2008). w/o emulsions without surfactants (Glatter and Glatter, 2013). The rheological properties of nanoemulsions modify the texture Such surfactant-free nanoemulsions can be prepared by cooling of foods (Quemeda and Berli, 2002; Walstra, 2003; Genovese the continuous phase below its melting temperature, which et al., 2007). The relative droplet size is known to influence leads to formation of kinetically stable emulsion made up of the rheological properties of nanoemulsions. For example, lyotropic liquid crystalline nanostructure (Ridel et al., 2015; beverages have low viscosity and the nanoemulsions used for Duffus et al., 2016; Chen et al., 2018). Weighting agents are their preparation should have droplets which do not increase the used in nanoemulsion preparation to impede the gravitational overall viscosity (McClements and Rao, 2011). forces and reduce sedimentation and creaming. Weighting agents The physicochemical stability of nanoemulsions is such as ester and damar gum are used in o/w emulsion as their characterized as kinetically stable systems as these breaks density matches with that of the oil phase surrounding aqueous down over time due to destabilizing physical mechanisms phase. Ripening retarders have hydrophobic action which helps (gravitational separation, flocculation, coalescence) and chemical in retarding the ripening (Schuch et al., 2014). The generally instability (Figure 1). Gravitational separation is due to different used ripening retards such as mineral oils and long chain triacyl relative densities between the dispersed and continuous phases glycerols prevent diffusion of smaller oil molecules through the and results in creaming or sedimentation. Formation of aqueous phase to form larger molecules (Sonneville-Aubrun crystalline lipids or small oil droplets causes sedimentation in et al., 2004). The ripening inhibitors have low water solubility an o/w nanoemulsions. Similarly, creaming in nanoemulsions and cause entropy of mixing for balancing the curvature effects occurs due to large particle size as the movement of droplets is (Wooster et al., 2008; Li et al., 2009). The texture modifiers used influenced by gravity. In nanoemulsions with particle diameter in the nanoemulsions interact only with the aqueous phase and <70 nm, the Brownian motion effects movement of smaller increase its viscosity by thickening it or turning it into a gel. They particles and prevents creaming (Walstra, 2003; McClements prevent the movement of oil droplets and impart creaminess and and Rao, 2011). Droplet aggregation such as flocculation or thickness to aqueous phase. Some of the commonly used texture coalescence is less in nanoemulsions due to their relatively small modifiers are biopolymers including gums, vegetable proteins, particle size (Tadros et al., 2004). In nanoemulsions, colloidal and polysaccharides (Imeson, 2011). In Table 2, the commonly interactions is related to their droplet size and occurs due to the used food grade emulsifiers and various stabilizing agents have attractive interactions (van der Waals and hydrophobic) and been mentioned. repulsive interactions (electrostatic and stearic) between two In the recent years, there is an increased interest in the use adjacent droplets (McClements, 2005; McClements and Rao, of food-grade stabilizers for the preparation of nanoemulsions 2011). Ostwald ripening occurs as the droplet size increases over (Rayner et al., 2014). Pickering stabilization has superior time due to diffusion or movement of solubilized oil molecules stability in comparison to conventional surfactant-stabilized from small droplets to large droplets through dispersed phase nanoemulsions. The pickering particles form a dense layer at (Kabalnov, 2001; McClements and Rao, 2011). The decrease the oil and water interface and by steric mechanism prevent in droplet size causes an increase in aqueous-solubility of oil droplet flocculation and coalescence (Duffus et al., 2016). The present within a spherical droplet resulting in large number of three main prerequisites for efficient pickering stabilization have solubilized oil molecules. This solubilized oil molecules move to been identified. The particle should be in 200 nm−1 μm size larger droplets leading to a gradual increase in the droplet size. range with sufficient particle charge and should have affinity The aqueous solubility of oil phase determines the stability of to emulsion continuous phase (Duffus et al., 2016). A number a nanoemulsion to Ostwald ripening (Kabalnov and Shchukin, Frontiers in Sustainable Food Systems | www.frontiersin.org 3 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications TABLE 2 | Food grade stabilizing agents used in nanoemulsions preparations for food related applications. Stabilizing agent Compound Concentration References Emulsifier (Non-ionic Lecithin (Phospholipid) and Lecithin at 1 wt.% with oil phase and Tween 80 at 2 Tan et al., 2016 surfactant) Tween 80 wt.% with aqueous phase Lecithin and Tween 80 Lecithin/Tween 80 at 0.3 molar ratio Kumar et al., 2017 PEG 40 hydrogenated castor oil; Oil:surfactant at 2:1 Nantarat et al., 2015 Sorbitan monoleate Tween 20 With aqueous phase at 0.75 wt.% Severino et al., 2015 With both phases at 1 wt.% Artiga-Artigas et al., 2017 Tween 20 and Span 80 Tween 20 and aqueous phase at 1wt.% Alexandre et al., 2016 Span 80 and oil phase at 4 wt.% Tween 80 Mixing with both phases at 3 wt.% Acevedo Fani et al., 2015 Mixing with organic phase at 7.5 wt.% Chen et al., 2017 Mixing with oil phase at 4.5 wt.% Gahruie et al., 2017 Tween 80 and Span 80 Tween 80 with aqueous phase at 1.25 wt.% and Dammak et al., 2017 Span 80 with oil phase at 3.75 wt.% Sucrose monosterate Mixing with aqueous phase at 0.5 wt.% Ruiz-Montañez et al., 2017 Emulsifier (Anionic) Sodium dodecyl sulfate with aqueous phase at 2.5 wt.% Qian and McClements, with aqueous phase at 3 wt.% Lee et al., 2013 Emulsifier (Amphiphilic) Whey protein isolate With aqueous phase at 4 wt.% Hebishy et al., 2015 Egg yolk powder Mixing with water as continuous phase at 3 wt.% Schuch et al., 2014 for W/O/W emulsion formation Mixed with aqueous phase at 3 wt.% Lee et al., 2013 Plasticizer Glycerol Mixing with nanoemulsion and biopolymer at Alexandre et al., 2016 glycerol/biopolymer ratio of 0.3 Mixing with oil phase at 2% (v/v) Alexandre et al., 2016 Mixing with biopolymer at 30 wt.% and then with Dammak et al., 2017 emulsion solutions Poly(ethylene glycol) Mixing with nanoemulsion at 0.2% (w/v) Otoni et al., 2014b Ripening retardant Sodium chloride Mixing with aqueous phase at 0.5 wt.% for W/O/W Schuch et al., 2014 inner emulsion Texture modifier Sodium alginate Addition at 1% before coarse emulsion formation by Artiga-Artigas et al., 2017 homogenizer (Ultra-Turrax) at 11,000 rpm for 2 minutes at room temperature 1992). Chemical degradation of nanoemulsions occurs due constituents, operating conditions, and preparation methods. to oxidation and hydrolysis. The large specific surface area of The emulsification process involves break up of droplets nanoemulsions makes it prone to chemical degradation. The into smaller ones, adsorption of surfactants, and collision opacity of nanoemulsions also play a role in chemical stability. of droplets. The adsorption kinetics also affects the stability The clear nanoemulsions with small droplets get easily degraded and droplet size of nanoemulsions (Silva et al., 2015). The by UV or visible light due to transparency (Dickinson, 1992; high energy methods, involve the use of mechanical devices Friberg et al., 2004; McClements, 2005). which disrupts oil phase for it to interact with water phase and form smaller oil droplets. The excessive stress generated by the mechanical device disrupts oil phase. Most of the PREPARATION OF NANOEMULSIONS food industries use high energy methods to prepare oil- in-water nanoemulsions (Gutiérrez et al., 2008). In the low The nanoemulsions have numerous droplets which increases energy methods, nanoemulsions are prepared by altering the the surface area. Therefore, large amount of energy is required temperature or composition of the oil-water system and the to create extra surface. Thus, nanoemulsion formation is not energy input is from the chemical potential of the constituents spontaneous and requires energy input. The energy required (Bouchemal et al., 2004). for the formation of nanoemulsions (1G) is estimated by the Major factors involved in nanoemulsion preparation is to expression, 1G= 1Aγ-T1S, where 1A is increase in interfacial achieve significantly low interfacial tension (<10–3 mN/m) at area, γ is surface tension and T1S is the entropy of dispersion the oil/water interface which requires the use of appropriate (Tadros et al., 2004; Schramm, 2006). surfactant. The surfactant also helps in stabilizing droplets Nanoemulsions can be prepared by either high energy produced at low interfacial tension. Fluidity at the interface or low energy methods. Its size is dependent on the Frontiers in Sustainable Food Systems | www.frontiersin.org 4 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications FIGURE 1 | Nanoemulsion formation and destabilizing mechanisms. Coalescence involves droplets combining; flocculation involves droplet aggregating but do not combine; droplets grow in size through Ostwald ripening. This destabilizing mechanism results in creaming and phase separation. is another important factor for stimulating nanoemulsion addition of emulsifier molecules and its adsorption at the formation (Bhosale et al., 2014). newly formed interfaces causes stabilization of droplets in the interaction chamber. Repeated disruption and stabilization results in production of higher number of tiny droplets. The High Energy Methods reduction in interfacial tension and maintaining low viscosity The high energy methods involve the use of a mechanical device ratio of dispersed and continuous phase decreases droplet size such as high pressure valve homogenizers, microfluidizers and (Tadros et al., 2004; Lee and McClements, 2010). An increase ultrasonicators (Figure 2). These devices are used to apply highly in concentration of emulsifier in nanoemulsions has also disruptive force for disrupting dispersed phase into tiny droplets shown to decrease droplet diameter (Windhab et al., 2005). of nanoemulsions (Maali and Hamed Mosavian, 2013). The emulsifiers can also reduce instability of re-coalescence of droplets after disruption. This is by adsorbing at the interface High Pressure Valve Homogenization Method of the droplets to form a protective layer thereby, preventing The device used in the high pressure valve homogenization the coalescence. However, the adsorption process should occur (HPVH) method, includes a positive displacement pump, at a faster rate than coalescence (Jafari et al., 2008). But the pressure valve, and chambers for homogenization and organoleptic constraints and regulations, limits the use of higher interaction. The coarse emulsion is sucked into the concentrations of emulsifier in food emulsion. Studies have homogenization chamber by the suction stroke of the pump. shown that small-molecule emulsifiers such as Tween 20 and The homogenization chamber can be a simple orifice plate, sodium dodecyl sulfate (SDS), produce smaller droplet sizes than colliding jet or radial diffuser assemblies (Stang et al., 2001; larger molecules emulsifiers such as proteins (Anton et al., 2008). Donsì et al., 2009). High pressure of upto 300 MPa generated in High pressure homogenization cannot be applied for viscous the chamber during delivery stroke causes the coarse emulsion lipids (Witthayapanyanon et al., 2006). to be pushed out through small orifice of micrometric size by the Thus, high-pressure homogenization efficiently breaks down homogenizer valve. At this stage, the factors such as turbulence, droplets and increases stability (Schultz et al., 2004). The ease shear stress, and cavitation disrupts coarse emulsions into very of applicaion, scalability, reproducibility, and high throughput finer droplets (Tesch et al., 2003; Schultz et al., 2004). Further, makes high pressure valve homogenization technique, highly the fine droplets get stabilized in the interaction chamber. suitable for producing nanoemulsions in food industries Thus, in the high pressure homogenization, emulsification (Schubert and Engel, 2004). The high-pressure homogenization occurs in two stages. Firstly, disruption of dispersed phase preparation of the nanoemulsion is more efficient and gives better results in tiny droplet formation with increased surface area quality (Liu et al., 2019). High pressure homogenization method in the homogenization chamber. In the second stage, the Frontiers in Sustainable Food Systems | www.frontiersin.org 5 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications FIGURE 2 | High energy methods such as high pressure homogenization (HPH), microfluidizer, and ultrasonication break macroemulsions into smaller droplets. was used to prepare nanoemulsions of carvacrol, mandarin, for fat globule disruption in dairy applications. However, now bergamot, and lemon. Primary emulsions of essential oils they are extensively used for food processing applications were subjected to five cycles of high pressure homogenization as nanoemulsion for delivering supplemented nutrients and at 200 MPa to obtain nanoemulsions of 133–200 nm. The as a preservation technique. It was assumed that friction nanoemulsion of carvacrol showed significant growth inhibitory heat generated in the homogenizer may cause degradation of activity against Escherichia coli O157:H7 and Salmonella temperature sensitive nutrients. Recent studies have shown that Typhimurium. Carvacrol nanoemulsion was incorporated into no thermal degradation occurs in valve due to the short residence modified chitosan to form a bioactive coating. It increased the time (3–40 ms) (Håkansson, 2019). shelf life of green beans stored at 4 C for upto 13 days (Severino et al., 2015). The o/w nanoemulsions of jackfruit pulp extract rich in carotenoids was obtained by high-pressure homogenization Microfluidization Method (400–800 bar). The nanoemulsion was stabilized by sucrose The basic principle of emulsification by microfluidization is monostearate emulsifier and exhibited a longer stability in its similar to the high pressure valve homogenizer, except for the use antioxidant activity during storage at 4 C. Nanoemulsions with of a special microchannel having dimensions in the range of 50– a high stability were produced at 800 bar. The antioxidant 300 μm to form droplets in the microfluidization method. In this activity of a jackfruit pulp extract was protected by nanoemulsion technique, the coarse emulsion is pumped with high pressure (up (Ruiz-Montañez et al., 2017). Lycopene nanodispersions with a to 270 MPa) at the inlet through a microchannel at velocities of narrow polydispersity index and good stability for application 400 m/s. The channels at the downstream gets split into two small in beverages were developed using homogenization process. branches to form Y or T junction. These branches reconnect Homogenization pressure of 500 bar reduced the particle size at far downstream to an interaction channel. In the interaction and lycopene concentration significantly, while homogenization chamber, the coarse emulsions from two steams impinges on pressure of 700–900 bar resulted in large particle sizes with each other with very high velocity. Thus, the impulsive forces −1 high dispersibility. Homogenization pressure also effected zeta generated in two streams and shear rate as high as 107 s potential and turbidity of the lycopene nanodispersion (Shariffa are sufficient for disrupting and forming fine emulsions in et al., 2017). High pressure homogenization of 50–300 MPa the interaction channel (Souto et al., 2005). The process is was used to obtain stable lentil nanoemulsions. Stable lentil repeated for more than two cycles to increase emulsification nanoemulsions was obtained with homogenizer pressure above time and pressure. Thus, the size of nanoemulsion depends 200 MPa and the best stability was achieved at 300 MPa. High- on the disruption of droplets and its recoalescence. However, pressure homogenization decreased nanoemulsion viscosity the addition of a fast-adsorbing emulsifier and increasing the under all conditions (Tabilo-Munizaga et al., 2019). High- viscosity of continuous phases can reduce the recoalescence rate pressure homogenizers were initially used in dairy industry (Jafari et al., 2007; Bae et al., 2009; Mao et al., 2010). Frontiers in Sustainable Food Systems | www.frontiersin.org 6 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications Microfluidization was used to obtain nanoemulsions of 163 nm drop size and zeta potential of –29.7 were obtained thyme, lemongrass, and sage oil dispersed in sodium alginate (Ricaurte et al., 2016). solution. The technique reduced the average droplet size of The limitations of the microfluidization techniques are nanoemulsions and resulted in ζ-potentials between −41 and increased droplet size as a result of coalescence due to longer time −70 mV. Sage essential oil nanoemulsions showed good film involved in emulsification and the use of high pressure increases property such as higher transparency, water vapor resistance, the temperature of nanoemulsions. However, in comparison and flexibility. Edible films of thyme essential oil had strong to other homogenization techniques, the efficiency of droplet antimicrobial effect on E. coli. Microfluidizers could be used to disruption by microfluidization technique is high and results in prepare nanoemulsions with active ingredients for the formation formation of fine droplets with uniform size. of edible films, with different physical and functional properties (Acevedo Fani et al., 2015). Nanoemulsion of ginger essential Ultrasonication Method oil was produced by microfluidization. Edible films reinforced In this technique, the ultrasonic agitation by sound waves with montmorillonite was activated with ginger essential oil with more than 20 kHz frequency breaks coarse droplets into nanoemulsion. The incorporation of nanoemulsions improved nanoemulsions. Sound waves applied by sonotrode produce characteristics and antioxidant activity of montmorillonite mechanical vibration and acoustic cavitation, and with the films. The incorporation of montmorillonite and nanoemulsion collapse of cavitations, strong shock waves generated break the into gelatin-based films increased the thickness and decreased coarse droplets (Behrend et al., 2000). The acoustic and shock the solubility in water, moisture content and superficial waves create high pressure and turbulence which collapse the hydrophobicity of films (Alexandre et al., 2016). Dual-channel droplets. At high frequency (mega Hz), nanoemulsions can even microfluidization have also been used for efficiently producing be prepared without using an emulsifier (Kamogawa et al., 2004; label-friendly nanoemulsions from natural emulsifiers. The Jafari et al., 2007). emulsifiers either amphiphilic biopolymers (whey protein and The device consists of an ultrasonic chamber having an gum arabic) or biosurfactants (quillaja saponinand soy lecithin) ultrasonic probe. The disruptive forces created by the ultrasonic were used to prepare corn oil-in-water nanoemulsions. The probe in combination with cavitation, turbulence, and interfacial use of dual-channel microfluidization resulted in production waves breaks the coarse emulsions flowing in the ultrasonic of nanoemulsions with the mean particle diameter decreasing chamber to fine nanoemulsions (Kentish et al., 2008). Similarly, with increasing emulsifier concentration and homogenization bench-top sonicator is used for the production of nanoemulsions pressure. Using this technique, whey protein isolate and at small scale. The piezoelectric crystal probe in the sonicator quillaja saponin were more effective at forming nanoemulsions generates intense pressure waves. An optimum level of input of fine droplets than gum arabic and soy lecithin. Low energy is required for sonification to achieve smallest droplet amount of emulsifier was required and smaller droplets were diameter. On increasing the sonication time, there is also produced (Bai et al., 2016). Dual-channel microfluidization an increase in input energy which tends to disrupt higher proved to be an efficient method for continuously producing number of droplets and decrease their size (Jafari et al., 2007). carotenoid-loaded nanoemulsions from natural emulsifiers. The other factors which influence nanoemulsion formation It was used to prepare o/w nanoemulsions of β-carotene. are concentration of emulsifier, viscosity ratio of dispersed Two types of natural emulsifier, quillaja saponins and whey and continuous phase and the amplitudes of applied waves protein isolate were used for nanoemulsions preparation by (Nakabayashi et al., 2011). It was observed that nanoemulsions this novel homogenization method. At 4 and 25 C, the prepared by high intensity ultrasonication from flax seed oil and nanoemulsions remained physically stable throughout 14 days nonionic surfactant (Tween 40) had droplet radius below 70 nm storage. At 55 C, small amount of droplet aggregation occurred (Kentish et al., 2008). Similarly, high-intensity ultrasound has in saponin nanoemulsions. Thus, microfluidization technique also been used to obtain nanoemulsions with droplet radius of can be used to encapsulate β-carotene and improve its water 20 nm. These were prepared using grade emulsifiers including dispersibility and chemical stability in foods (Luo et al., 2017). sunflower oil, Tween 80, and Span 80 (Leong et al., 2009). Microfluidization was used to produce small sized fish oil The increased ultrasonification time and decreased surfactant nanoemulsion as fish oil is rich in polyunsaturated fatty acids concentration resulted in nanoemulsions with droplet diameter (PUFAs). Coarse emulsion had a droplet size of 1.5 μm, while of 29.3 nm. The nanoemulsions were prepared with basil oil microfluidization produced smaller droplet size (≥ 200 nm). Zeta and nonionic emulsifier (Tween 80 and water) and had a high potential values increased around –30 ± 2 mV. Nanoemulsions intrinsic stability with ultrasonication time of 15 min (Ghosh with an average droplet size around 200 nm were prepared et al., 2013). with 144 MPa and two passes of microfluidization. Thus, High intensity ultrasound (150 W) has been used to prepare microfluidization could be used to develop nanoemulsions with nanoemulsions of essential oils of Zataria multiflora. The better absorption in the digestive tract (García-Márquez et al., bioactivity of the essential oil was increased by nanoemulsion. 2017). Microfluidization has also been used to encapsulate high Further, increase in the antibacterial activity could be observed nutritional value oils, such as high-oleic palm oil (Ricaurte by decreasing the nanoemulsion droplet size. The small sized et al., 2016). o/w nanoemulsions of high-oleic palm oil was nanoemulsions could be easily incorporated in the basil seed obtained by microfluidization wherein, 1–20% w/w of the gum films. Increased nanoemulsion concentration in the film oil could be easily encapsulated. Stable nanoemulsions of matrix effected the microstructure of the film and improved Frontiers in Sustainable Food Systems | www.frontiersin.org 7 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications its mechanical properties. The films also showed significant surfactant in oil is more (lipophilic) than water phase and thus antimicrobial activity against potential foodborne pathogens water-in-oil nanoemulsion is formed. Temperature at which (Gahruie et al., 2017). Nanoemulsions of resveratrol and there is a conversion of oil-in-water to water-in-oil emulsion is resveratrol-cyclodextrin inclusion complex in a phospholipid termed as phase inversion temperature (PIT) (Pathak, 2017). stabilized nanoemulsion has been prepared by ultrasonic It is also to be noted that surface tension decreases with emulsification. The resveratrol nanoemulsion and inclusion increase in temperature and fine emulsions with small sized complex nanoemulsion had an average size of 20 and 24 nm, droplets are formed. At PIT, these small droplets are not stable respectively. The nanoemulsions developed by ultrasonication as they try to coalesce and form macroemulsions. If a large had a good loading and release efficiency. The nanoemulsion amount of emulsifier is used to reduce instability, droplets do prevented degradation of resveratrol on exposure to UV not coalesce immediately, and some liquid crystal structure forms irradiation (365 nm) (Kumar et al., 2017). at PI temperature (Salager et al., 2004). Thus, although it is Studies have been carried out to compare the efficiency of possible to form emulsions near PIT, they are very unstable. To high pressure homogenization and ultrasonication to develop produce stable and fine oil-in-water nanoemulsions, a cooling the nanoemulsions of capsaicin (oleoresin capsicum). Capsaicin process is required and final nanoemulsions are required to be nanoemulsions obtained by high pressure homogenization preserved at a temperature far below PIT (Liew et al., 2010). were optically translucent with 79% efficiency and significant In addition, if emulsions formed near PIT are rapidly cooled antimicrobial activity. However, nanoemulsions prepared by or heated, kinetically stable emulsions with small droplet size ultrasonication had good physical properties and smaller droplet and narrow size distribution can be produced. PIT can be size of 65 nm (Akbas et al., 2018). determined by noting down the change in different properties of nanoemulsions such as conductivity, viscosity, and turbidity (Rao and McClements, 2010). Low Energy Methods Phase inversion temperature method was used to prepare The low-energy methods, use the energy input from chemical cinnamon oil nanoemulsions. Cinnamon oil, non-ionic potential of the components to from nanoemulsions. The surfactant, and water were heated above the phase inversion nanoemulsions form spontaneously at oil and water phase temperature of the system. It was rapidly cooled with continuous interface by gentle mixing of the components. The spontaneous stirring resulting in spontaneous formation of small oil droplets emulsification can be controlled by two methods. One of the with mean droplet diameter of 101 nm. The cooling-dilution methods is to change temperature without altering composition. method increased stability of the nanoemulsions for 31 The other method is to keep the temperature constant and vary ◦ ◦ days at 4 C or 25 C (Chuesiang et al., 2018). The effect of the compositions and interfacial properties. Thus, nanoemulsion phase inversion temperature method on biological activity of formation by low energy methods depends on physicochemical cinnamon oil nanoemulsions has been studied. The surfactant properties such as temperature, composition, and solubility concentration effected the antimicrobial activity of the cinnamon (Anton and Vandamme, 2009). The low energy methods involved oil nanoemulsions. The increase in surfactant concentration in the nanoemulsion production are phase inversion temperature from 15 to 20 wt% enhanced the antimicrobial activity of (PIT), phase inversion composition (PIC), and solvent diffusion nanoemulsion in comparison to nanoemulsions with lower method. These methods involve minimal energy generation, and surfactant concentration (10 wt%) or with bulk cinnamon oil thereby prevent the degradation of heat labile compounds. (Chuesiang et al., 2019). Phase Inversion Temperature Method The method uses phase invasion property of the molecules, Phase Inversion Composition Method wherein the emulsifiers change their hydrophilicity or In this technique, varying the composition of constituents lipophilicity as a function of temperature at fixed composition changes the hydrophilic–lipophilic behavior of emulsifier (Figure 3). Depending on the hydration of the polar heads of the (Figure 3). On adding salt to an oil-in-water nanoemulsion non-ionic surfactants, they change their spontaneous curvature with ionic emulsifier, the electric charge of surfactant changes (Anton and Vandamme, 2009). At low temperature, oil-in-water and it turns to water-in-oil emulsion system (Maestro et al., emulsion is formed. Whereas, at high temperature, water-in-oil 2008). Similarly, a water-in-oil emulsion having high salt emulsion is formed as the solubility of emulsifier in water content can be converted to oil-in-water by diluting with decreases with increase in temperature. The phase inversion water (Liew et al., 2010). This technique has low cost, does temperature is the temperature at which there is transition not require the use of organic solvents and it has high from oil-in-water to water-in-oil emulsion (McClements and thermodynamic stability (Shakeel et al., 2009). It is difficult to Rao, 2011). At a particular temperature, the curvature of use phase inversion method for highly hydrophobic compounds emulsifier layer becomes zero and solubility of emulsifier (Witthayapanyanon et al., 2006). becomes approximately equal in water and oil phase. At this Phase inversion composition method has been used to prepare stage, there is no tendency to form either oil-in-water or water- food-grade nanoemulsions enriched with vitamin E acetate in-oil nanoemulsion and constituents form a bicontinuous or with a mean particle diameter of 40 nm. This method was lamellar liquid crystalline system. At higher temperature, the more effective at producing nanoemulsions at high surfactant surfactant layer becomes concave with negative curvature due to concentration than microfluidization technique. However, the dehydration of hydrophilic nonionic surfactant. The solubility of technique was not favorable for nanoemulsions preparations with Frontiers in Sustainable Food Systems | www.frontiersin.org 8 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications FIGURE 3 | Low energy methods involves breakdown of coarse w/o macroemulsions into nanoemulsions during phase inversion. In the Phase Inversion Composition technique, dilution by water induces a phase inversion while in the Phase Inversion Temperature method, cooling or decrease in temperature induces a phase inversion. label-friendly surfactants, such as Quillaja saponin, whey protein, fine droplets. Particle size could also be reduced by increasing casein, and sucrose monoesters (Mayer et al., 2013). the temperature and stirring speed used when the oil/surfactant mixture was added to water (Saberi et al., 2013). Similarly, vitamin D nanoemulsions have been prepared by spontaneous Spontaneous Emulsification Method emulsification. The nanoemulsions with small droplet diameters In this technique, spontaneous emulsions are formed on mixing <200 nm that were stable to droplet growth at ambient water and oil together with an emulsifier by gentle stirring temperatures but unstable at high temperatures (>80 C) were at a particular temperature (Figure 4). The mixing of phases obtained. The thermal stability of the nanoemulsions was by gentle magnetic stirring causes the emulsifier to enter the increased by using a cosurfactant during the formulation (Guttoff aqueous phase leading to increase of oil-water interfacial area et al., 2015). Spontaneous emulsification has been used to prepare resulting in oil droplet formation (Anton et al., 2008). The excess oil phase is removed by evaporation under reduced pressure fish oil nanoemulsions as these are rich in omega-3 fatty acids. Transparent nanoemulsions with physical stability at 37 C and (Bouchemal et al., 2004). The drawback of this method is the oxidative stabilities at 55 C for 14 days could be prepared use of high synthetic surfactants at large scale which is not (Walker et al., 2015). Cinnamaldehyde has been encapsulated feasible economically and has regulatory and sensory issues in in self-emulsifying emulsion systems. Stable cinnamaldehyde food industry. nanoemulsions were achieved only with the addition of medium- Thus, in the low energy method of nanoemulsion formation, chain triglyceride. The encapsulation efficiency of nanoemulsions the intrinsic physicochemical properties of surfactants and the was 80% for 1 week and slow release of cinnamaldehyde could oily phase plays a major role and can be easily scaled up. In the be expected. Phase separation occurred after 12 days of storage high-energy techniques, the size distribution and composition under 37 C. Encapsulation efficiency of cinnamaldehyde in of nanoemulsion can be controlled using mechanical devices, nanoemulsions was maintained around 80% within 1 week (Tian however, there could be degradation of constituents and the production processes cannot be scaled up (Date et al., 2010). et al., 2016). The effect of high and low energy approaches on Spontaneous emulsification method has been used to obtain the physicochemical properties of nanodispersions vitamin E acetate nanoemulsions of droplet diameters <50 nm has been evaluated. Solvent displacement and high- and low polydispersity indexes. Oil phase composition and pressure valve homogenization was used to prepare lutein surfactant-to-emulsion ratio had to be optimized to produce Frontiers in Sustainable Food Systems | www.frontiersin.org 9 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications FIGURE 4 | Spontaneous emulsification is a low energy method. In this method, nanoemulsions are formed on mixing water and oil together with an emulsifier by gentle stirring at a particular temperature. nanodispersions. The particle size and particle size distribution cannot be used to produce pickering nanoemulsions. of nanodispersions prepared prepared by both methods The vapor condensation method used for pickering were not significantly different. They also had very high nanoemulsion preparation is a single step process and lutein retentions (>90%). Therefore, solvent displacement has several advantages over the conventional techniques can also be a good alternative to high-pressure valve such as use of low concentration of nanoparticles homogenization in lutein nanodispersions preparation (Kang et al., 2018). (Tan et al., 2016). Novel Nanoemulsion Preparation CHARACTERIZATIONS OF Techniques NANOEMULSIONS The conventional nanoemulsification formulation involves the break drown of larger droplets or inversion of solvents. New The physiochemical properties of nanoemulsions such as emulsification approaches are being increasingly developed to physical properties, stabilities, rheological property, and increase the range of materials formulations and operating microstructure have to be characterized for its application in conditions, and to simultaneously lower the production costs. foods as these properties are known to influence the final texture, A bottom-up approach method based on condensation taste, flavor, and stability of foods. The basic physical properties has been developed to create nanoscale emulsions. Water-in- of nanoemulsion are particle size, size distribution, charge, and oil nanoemulsions are prepared by condensing water vapor lipid crystallinity. on subcooled oil-surfactant solution. The water droplets Particle structure and size distribution of nanoemulsions is nucleate at the oil and air interface to spontaneously measured by dynamic light scattering (DLS) and small-angle X- disperse within the oil. This occurs as a result of the ray scattering (SAXS), non-invasive techniques. DLS technique spreading dynamics of oil on water which depends on the measures size distribution of small particles or droplets in oil-surfactant concentration. The nanoemulsions formed suspensions in the range of 3 nm−5 μm. DLS records the using condensation approach have a peak radii of 100 nm. intensity fluctuations that occur as a result of change of relative An oil bath is placed in a humid environment with spatial location due to Brownian motion of small particles over appropriate concentration of surfactant, and on decreasing time when light is scattered by particles (Mason et al., 2006; Fryd the temperature below the dew point, water condensation and Mason, 2012). In the DLS technique, the size distribution is induced on the oil surface resulting in nanoemulsion profiles of nanoemulsions is observed as a single and narrow formation. The process is simple, rapid, scalable, and peak as it is monodisperse (Yun Zhang, 2003). DLS measurement energy efficient with potential application in processed foods utilizes the “Mie theory” mathematical model to determine the (Guha et al., 2017). scattering pattern of the droplets contained in the emulsion. DLS Pickering nanoemulsions have been developed using data is observed as plot of particle concentration and its size the vapor condensation strategy. The use of pickering (Mcclements, 2007). DLS can also be used to evaluate the stability nanoemulsion overcomes the problems associated with of nanoemulsions in various external conditions. surfactant desorption and Ostwald ripening. The high Small-angle X-ray scattering (SAXS)/small-angle neutron energy approach prevents adsorption of the particles scattering (SANS) is used to characterize the colloidal particles on the droplets whereas, the low energy approach for its shape, size, and nanostructure (Gradzielski, 2008). The Frontiers in Sustainable Food Systems | www.frontiersin.org 10 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications SAXS pattern is a function between intensity and scattering the microstructure of nanoemulsions at a resolution of <5 nm vector and obtained at low angles (typically 0.1–10 ), whereas (Acharya and Hartley, 2012; Silva et al., 2012). Transmission SANS uses short wavelength (λ < 10Å) to measure neutron electron microscopy (TEM) which has a resolution of (0.2 nm) scattering length density of nanoemulsions (Mason et al., 2006). has been used to image materials and biological samples Particle charge or the electrical surface charge of emulsions (Luykx et al., 2008; Silva et al., 2012). It has also been used is determined by measuring the zeta potential (ζ-potential). ζ- to study the morphology and structure of the nanoemulsions potential is the electro-kinetic potential in nanoemulsions. The (Bouchemal et al., 2004; Inugala et al., 2015). However, particles with surface charge on dispersion in liquid phase attract sample preparation for TEM uses high-energy electron beam the ions of opposite charges which form a firm attachment which can cause structural damage to the materials (Luykx known as stern layer. The electrical charge on the droplet surface et al., 2008). This problem can be overcome by the use of affects the interactions between emulsion droplets and in turn cryogenic preparation (cryo-TEM) and freeze-fracture (FFTEM) the stability of nanoemulsions. The nanoemulsions having high techniques. Cryo-TEM is effective in characterizing the structure absolute value of zeta potential (negative or positive, commonly of microemulsions (Spernath et al., 2009). The FFTEM provides above 30) are electrically stabilized, and those with low zeta detailed structure of bicontinuous nanoemulsions and droplets potential coagulate (Mcclements, 2007; Mohanraj and Chen, within discontinuous nanoemulsions (Krauel et al., 2007; 2007). Acharya and Hartley, 2012). Scanning electron microscope Lipid crystallinity of nanoemulsions is measured using a (SEM) is used to characterize materials in the nanometer thermo-analytical technique, differential scanning calorimetry to micrometer scale for microstructure morphology and (DSC). The technique measures the crystalline temperature chemical composition (Goldstein et al., 2003; Ferreira et al., differences of pure oil and oil in emulsion and thus assesses 2015). SEM uses narrow electron beam to provide surface the influence of oil crystallization on the stability of the structures. Hence, SEM is used to obtain surface structure nanoemulsion (Thanasukarn et al., 2004; Rafanan, 2013). The of oil droplets of nanoemulsion (Silva et al., 2012). SEM impurities present in the oil used for emulsion causes crystal can be used to process large amounts of materials at low formation and nucleation, which in turn affects the stability magnification, and high-resolution images can be obtained of nanoemulsion. The percentage of destabilized fat in the at higher magnification (Luykx et al., 2008). The drawbacks emulsions can be calculated using the DSC instrument. It associated with SEM are high cost, use of high vacuum measures the area under the non-emulsified enthalpy peaks and high sample conductivity. Surfactants present in the during a cooling cycle, and divides it by the area under all of sample can also interfere with SEM imaging as it causes the enthalpy peaks. However, as the oil is dispersed into small coating on the particle surfaces (Luykx et al., 2008). Atomic droplets, the interfacial layer reduces the nucleation growth of oil force microscopy (AFM) microscopy is used for structural crystal (Mcclements, 2007). characterization of nanoemulsion. It detects the changes of Nuclear magnetic resonance (NMR)-based techniques have force between a shape probe and an immobilized sample and also been used to study the types, structure, and diffusion produces a high-resolution 3-D profile of the sample surface properties of components in nanoemulsions (Acharya and (Preetz et al., 2010). Hartley, 2012; Hathout and Woodman, 2012). One of the NMR techniques, the Fourier transform pulsed gradient spin echo (FT-PGSE) technique measures self-diffusion coefficients of oil, APPLICATION OF NANOEMULSIONS IN water, and surfactant molecules at a time. It is also useful in FOOD determining the connectivity in emulsions and the PGSE helps to know the phase transient between the droplet emulsion phase The technological limitations of developing functional foods and bicontinuous emulsion (Gradzielski, 2008). is the low solubility, stability, and bioavailability of the Rheology of nanoemulsions is measured using shear device bioactive compounds. Most of the bioactive food ingredients are which gives the value of apparent viscosity and the dependent susceptible to degradation during food processing and oxidative relationship between apparent viscosity and shear stress. The deterioration during storage (Xianquan et al., 2005; Shahidi and dynamic oscillation methods are used to evaluate the viscoelastic Zhong, 2010). Certain bioactives have low solubility but rapid properties of emulsions. The stable emulsions flow curves have a metabolism which reduces its bioavailability, whereas some are −1 constant value of apparent viscosity at low shear rates (∼0.01s ) volatile and sensitive to processing conditions (McClements and and a strong shear thinning follows at high shear rates (Sharma Li, 2010; Jin et al., 2016). These challenges can be overcome by et al., 2010). The rheological properties of nanoemulsions are the use of nanoemulsions to encapsulate bioactive compounds affected by the surfactants used, shape, and number density of for their use in food matrix. The encapsulation of bioactive the droplets, and interactions between the constituent droplets compounds in an oil phase or emulsifier ensures it stability, (Acharya and Hartley, 2012). Use of cosurfactants has shown bioavailability, and controlled rate of release (McClements et al., to weaken the interactions between emulsifier and the anionic 2007). The nanoemulsion based delivery system should have surfactant, resulting in reduced viscosity (Howe and Pitt, 2008). compatibility with food matrix and minimal effect on the Microstructure characterization of nanoemulsions is organoleptic properties of the food such as its flavor, appearance, carried out using microscopy imaging techniques. Electron and texture. The encapsulation of bioactive compound can microscopy (EM) techniques is used for the visualization of protect it from processing conditions and prevent its degradation Frontiers in Sustainable Food Systems | www.frontiersin.org 11 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications for long duration from temperature, light, pH, and oxidative reduced particle size and improved the polydispersity of the conditions during storage. The application of nanoemulsion nanodispersions. The β-carotene nanodispersions had high ζ- based delivery system for foods requires that the technique is potential of −27 mV at pH 7 and thus were stable against particle economically feasible for industrial scale production (Borthakur aggregation (Chu et al., 2007). O/W β-carotene nanoemulsions et al., 2016; Pathak, 2017). Table 3 provides an overview of prepared using Tween 20 as emulsifier by high pressure nanoemulsions of bioactive compounds for food applications. homogenization had mean diameters from 132 to 184 nm. Only 25% of β-carotene in the nanoemulsions degraded after 4 weeks of storage at 4 and 25 C (Yuan et al., 2008). Solvent displacement Encapsulation of Flavor and Coloring technique was used to prepare β-carotene nanodispersions of 30– Agents 206 nm using emulsifiers such as sodium caseinate, Tween 20, The flavors and coloring compounds used in food have decaglycerol monolaurate, and sucrose fatty acid ester. Though, aldehyde, ketone, and esters as functional groups which make starch casein stabilized nanodispersions had large particles size them susceptible to oxidative and photolytic degradation. but they were most stable against oxidation. This might be due Encapsulation within nanoemulsions of these ingredients can to physical barrier and the antioxidant activity of caseins (Yin prevent these deleterious effects and enhance its shelf life et al., 2009). β -carotene nanoemulsions stabilized by modified (Goindi et al., 2016). Citral, a α,β-unsaturated aldehyde with starch and spray-dried to powders after the emulsification one additional double bond is an aromatic compound and process had better storage stability. Modified starches having used as flavors in food and cosmetics. However, its degradation low film oxygen permeability had a high retention of beta- produces off-flavor compounds. Nanoemulsions of citral have carotene during storage (Liang et al., 2013). Physicochemical been prepared to increase its stability. O/W nanoemulsion of properties of β -carotene nanoemulsions have been improved citral combined with natural antioxidants such as β-carotene, by coating emulsions with starch caseinate and chitosan- tanshinone, and black tea extract had a high chemical stability epigallocatechin-3-gallate conjugates (Wei and Gao, 2016). during storage. The nanoemulsions were prepared with lecithin- Sonication-assisted method with freeze drying has been used to stabilized palm kernel lipid in pH 3 buffer 1:1 ratio of citral and prepare β-carotene nanoemulsions with high water dispersibility antioxidants. Encapsulation with antioxidants led to decreased and chemical stability (Chen et al., 2017). Carotenoids were formation of off-flavor compounds, such as α, p-dimethylstyrene extracted from Cantaloupe melon. The nanoemulsions prepared and p-methylacetophenone (Yang et al., 2011). Similarly, O/W from these carotenoids had improved water solubility and nanoemulsion system of citral and ubiquinol-10 (Q H ) had color stability. The o/w carotenoid nanoemulsions encapsulated 10 2 good chemical stability. The Q H had a protective effect on in porcine gelatin and whey protein isolate had an average 10 2 citral and prevented its chemical degradation and oxidation. particle size of 70–160 nm. Gelatin was able to increase It reduced generation of off-flavor compounds (p-cresol, α,p- water solubility of carotenoids. The yogurt containing these dimethylstyrene, p-methylacetophenone) and lipid degradation nanoemulsions as natural coloring agent was stable for 60 days products. At Q H /citral ratio of 1:1, the ubisemiquinone (Q (Medeiros et al., 2019). 10 2 10 •− )/ubiquinone (Q ) redox transition was induced, leading to pro-oxidant behavior of Q H and its ability to maintain citral’s Encapsulation of Nutraceuticals 10 2 stability (Zhao et al., 2013). Gelatin and Tween 20 have been used Resveratrol, a natural polyphenol found in grape skins, as emulsifiers to increase the stability of citral from degradation blueberries, raspberries has many functional properties such as under acidic conditions in the food industry (Tian et al., 2017). antioxidant, anticancer, and antiobesity. Nanoemulsions based β-Carotene is a precursor of vitamin A and is used as a natural delivery systems have been used to encapsulate resveratrol. colorant, and an antioxidant in the food industry. However, Encapsulation of resveratrol by spontaneous emulsification it is easily degraded by heat, light, and oxygen. Attempts are using 10% oil phase (grape seed oil and orange oil), 10% being made to increase the stability of β-carotene toward various surfactant (Tween 80) and 80% aqueous phase had 100 nm processing conditions. β-carotene nanodispersions prepared by droplet size and could carry 120 ± 10 μg/ml of resveratrol. The emulsification-evaporation technique have shown an increased encapsulated resveratrol had improved chemical stability against stability. Nanoemulsion was prepared by emulsifying organic UV-light degradation (Davidov-Pardo and McClements, 2015). solution of β-carotene in aqueous phase containing emulsifier. O/W edible nanoemulsions of vitamin D (cholecalciferol) have Stable β-carotene nanodispersions were produced with weighted been used for the fortification of dairy emulsions. Emulsifiers mean diameter ranging from 60 to 140 nm (Tan and Nakajima, polysorbate 20, soybean lecithin and their mixtures and dispersed 2005a). O/W β-carotene nanodispersions prepared using non- oil phase of soybean oil or mixtures of the oil with cocoa butter ionic emulsifier polyglycerol esters of fatty acids (PGEs) having were used to prepare nanoemulsions of mean diameters <200 nm mean diameter of 85–132 nm showed improved physicochemical with high pressure homogenizer. Vitamin D3 (0.1–0.5 μg/mL) properties and physical stability. PGE decaglycerol monolaurate were encapsulated in the oil cores of stable nanoemulsions. −1 (10 g kg ) having high glycerol polymerization could be used Whole-fat milk was fortified with vitamin D3 nanoemulsions and for highly stable β-carotene nanodispersions (Tan and Nakajima, were stable for 10 days against particle growth and gravitational 2005b). Protein-stabilized β-carotene nanodispersions prepared separation (Golfomitsou et al., 2018). O/W nanoemulsions of using emulsification-evaporation had a mean particle size of kenaf seed oil stabilized with emulsifiers sodium caseinate, beta- 17 nm. The emulsifier sodium caseinate, at a high concentration cyclodextrin, and Tween 20 had improved in vitro bioaccessibility Frontiers in Sustainable Food Systems | www.frontiersin.org 12 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications TABLE 3 | Functional compounds encapsulated into nanoemulsions for food applications. Bioactive Function Method Particle size Constituents Reference compounds Citral Flavoring agent HPH (6 cycles for 150 MPa) 109-129 nm o/w : palm kernet fat/ citral/ undecane/ lecithin Yang et al., 2011, HPH (6 cycles/ 150 MPa) 98-120 nm aqueous buffer solution; Zhao et al., 2013 HPH (3 cycles/1000 bar) 467 nm o/w: MCT/ buffer solution (citric acid/sodium Tian et al., 2017 hydroxide/sodium chloride)/ soy lecithin/ citral/ undecane o/w: medium chain triacylglycerl/ buffer solution (citric acid/sodium hydroxide/sodium chloride)/ gelatin/ Tween 20/ citral/ undecane β-Carotene Coloring agent Sequential HPH (3 cycles/ 60-140 nm o/w: Hexane/β-carotene/ Tween 20 Tan and Nakajima, 60-140 MPa), MF and 85-132 nm o/w: Hexane/β-carotene/ Polyglycerol 2005a Solvent-evaporation 17-110 nm esters of monolaurate and monooleate; Tan and Nakajima, Sequential HPH (140 MPa), 132-184 nm o/w: Hexane/β-carotene/ sodium caseinate/ 2005b MF and Solvent-evaporation 30-206 nm whey protein isolate/soy protein isolate Chu et al., 2007 MF (1-3 cycles/ 140 MPa) 114-159 nm o/w: Hexane/β-carotene/Tween 20 Yuan et al., 2008 HPH 284 nm o/w: Hexane/β-carotene/starch/ Yin et al., 2009 SD 230 nm sodium caseinate/Tween 20/decaglycerol Liang et al., 2013 HPH (10 cycles/ 150 MPa) 70-160 nm monolaurate/sucrose fatty acid ester Wei and Gao, HPH (3 cycles/ 60 MPa) o/w: β-carotene/ 2016 Ultrasonication and MF Starch/ medium chain triacylglycerl Chen et al., 2017 HPH o/w: β-carotene/ Medeiros et al., sunflower oil/ starch 2019 caseinate/chitosan-epigallocatechin-3-gallate o/w: β-carotene/ vegetable oil/ casein o/w: Caotenoid extract/ soybean oil/gelatin/ whey protein isolate Resveratrol Nutraceutical Spontaneous emulsification 100 nm o/w: resveratrol/ grape seed oil/ Orange oil/ Davidov-Pardo Tween 80 and McClements, Vitamin D Nutraceutical HPH <200 nm o/w: Vitamin D/ polysorbate 20/ soybean Golfomitsou et al., lecithin/ cocoa butter 2018 Vitamin E and Nutraceutical HPH (4 cycles/ 28000 psi) 100 nm o/w: Vitamin E/ phytosterol/ kenaf seed oil/ Cheong et al., phytosterol sodium caseinate/ beta-cyclodextrin/ Tween 20 Astaxanthin Nutraceutical Spontaneous emulsification 150–160 nm o/w: Astaxanthin/ lecithin/ caprylic-capric Alarcon-Alarcon triglyceride/ chitosan et al., 2018 Curcumin Nutraceutical HPH (20 cycles/ 103 MPa) 80 nm o/w: curcumin/ Silva et al., 2018 MCT/SDS Lecithin Nutraceutical MF (5 cycles/ 150 MPa) <400 nm o/w: curcumin/ corn oil/ sodium alginate/ Artiga-Artigas Tween 20/ lecithin et al., 2018 Oregano EO Preservative Ultrasonication (750 W) 148 nm o/w: Oregano oil/Tween 80 Bhargava et al., 2015, Ultrasonication (20KHz/400 180-250 nm o/w: Oregano oil/ clove bud oil/Tween 80/ Otoni et al., 2014b W/10 min) distilled water Orange EO Preservative Ultrasonication (20KHz/750 20-30 nm o/w: orange oil/ Tween 80/ distilled water Sugumar et al., W/10 min) 2015 Cinnamaldehyde Preservative Spontaneous emulsification 20-500 nm o/w: Cinnamaldehyde/ Tween 80/ distilled Otoni et al., 2014a water Ginger EO Preservative MF (10 cycles/1000 psi) 133 nm o/w: Ginger EO/ Tween 20/ Span 80/ Canola oil Acevedo Fani et al., 2015 Ultrasonication (20 KHz/200 57 nm o/w: Ginger EO/ Tween 80 Noori et al., 2018 W/ 5 min) Curcumin Preservative Spontaneous emulsification 40-130 nm o/w: Curcumin/ Abdou et al., 2018 Tween 80/ Glycerol/ Water Capsaicin Preservative HPH (5 cycles/140 MPa) 65 nm o/w: Capsicum oleoresin/ Tween 80/ Water Akbas et al., 2018 (Capsicum Ultrasonication oleoresin) (75% amplitude/5 min) HPH, High Pressure homogenization; MF, Microfluidization; MCT, medium chain triacylglycerl. Frontiers in Sustainable Food Systems | www.frontiersin.org 13 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications and physicochemical stability of bioactive compounds and life of chicken breast filet. The nanoemulsion based active coating antioxidants. It was observed that after 8 weeks of storage at had higher antibacterial activity than antioxidant activity. It 4 C, the nanoemulsions were stable and maintained antioxidant reduced growth of psychrophilic bacteria in refrigerated chicken activities with high percentage retention of vitamin E and filets for 12 days (Noori et al., 2018). Nanoemulsions have been phytosterols (Cheong et al., 2018). Astaxanthin, a dietary used to increase the bioavailability of lipophilic compounds. supplement is a light sensitive molecule. Nanoemulsions have High pressure homogenization and ultrasoniction were used been formulated to photostabilize the pigment for its use in foods. to prepare capsaicin (oleoresin capsicum) nanoemulsions with The chitosan-coated and carrageenan-coated nanoemulsions Tween 80 as the aqueous phase. Ultrasonication improved of astaxanthin provided it protection from UV light and physical properties as nanoemulsions with particle size below photodegradation (Alarcon-Alarcon et al., 2018). 65 nm were obtained, whereas high pressure homogenization Nanoemulsions have been used to improve the bioaccessibility resulted in preparation of nanoemulsions with good inhibitory of lipophilic bioactive compounds. Curcumin nanoemulsions activity against S. aureus and E. coli (Akbas et al., 2018). prepared with chitosan and alginate had improved antioxidant capacity during in vitro digestion and a better control over lipid digestibility by decreasing free fatty acids. Thus, these NANOEMULSION BASED FOOD nanoemulsions can be used to fortify functional foods for PACKAGING MATERIALS targeting obesity (Silva et al., 2018). Similarly, nanoemulsions of curcumin have been prepared with improved antioxidant The nanoemulsions can be incorporated into films and capacity. Lecithin-stabilized nanoemulsions of curcumin had an coatings for potential food packaging applications. The films encapsulation efficiency of 75% and were stable for 86 days in and coatings made up of biopolymer matrix constitute comparison to other surfactants such as Tween 20 stabilized the continuous phase as they provide monodispersity and curcumin nanoemulsions (Bhosale et al., 2014). stability to nanoemulsion droplets. The increase in viscosity of the continuous phase reduces the coalescence of droplets Natural Preservatives (Artiga-Artigas et al., 2017). The preparation of nanoemulsion Plant essential oils including thyme, oregano, clove and orange bioedible films includes dispersing the bioactive ingredients and their components such as thymol, carvacrol, eugenol, in a continuous phase which makes up the matrix of limonene, and cinnamon have strong antimicrobial activity the packaging films. Food grade emulsions are added and against food borne pathogens. But their application in food appropriate high or low energy techniques are used for matrix is limited due to their hydrophobicity. Nanoemulsion homogenization. The homogenized formulation is cast into formulations of essential oils can be used to overcome this films with controlled thickness and dried. Further, these problem and they can be used in foods as natural preservatives films are characterized for the structural, morphological, and in food packaging (Alexandre et al., 2016). thermal, mechanical, and barrier properties (Otoni et al., Oregano oil nanoemulsions inhibited growth of foodborne 2014a). The biopolymers used for film preparation also bacteria Listeria monocytogenes, Salmonella Typhimurium and play a role in maintaining the functional properties of E. coli O157:H7 on fresh lettuce. It was observed that oregano dispersed nanoemulsions. oil nanoemulsions disrupted bacterial membranes (Bhargava The biopolymer pectin has been used to prepare edible et al., 2015). Orange oil nanoemulsions inhibited spoilage films of cinnamaldehyde and clove essential oil nanoemulsions of apple juice by Saccharomyces cerevisiae (Sugumar et al., with antimicrobial properties. The pectin films had low water 2015). Similarly, cinnamaldehyde nanoemulsions incorporated and vapor permeability due to the decrease in ratio of in pectin edible films inhibited growth of E. coli, Salmonella hydrophilic/hydrophobic (Otoni et al., 2014a; Sasaki et al., enterica, Listeria monocytogenes, and Staphylococcus aureus 2016). Cellulose and its derivatives have been used to prepare (Otoni et al., 2014a). Nanoemulsions of clove bud and oregano edible films containing nanoemulsions of clove and oregano essential oil with 180–250 nm droplet size incorporated into essential oils (Otoni et al., 2014b). Chitosan has been used for edible methylcellulose films had improved antimicrobial activity. preparation of nanoemulsion coatings and films of essential They prevented growth of yeasts and molds and improved shelf oils with antimicrobial activity such as carvacrol, mandarin oil, life of sliced bread (Otoni et al., 2014b). Nanoemulsions of ginger bergamot oil, and lemon oil (Severino et al., 2015). Sodium essential oil incorporated into gelatin-based films have shown alginate was used to formulate films of nanoemulsion containing to improve the physical properties of the active food packaging thyme, lemongrass, and sage essential oil (Acevedo Fani et al., films (Alexandre et al., 2016). Curcumin nanoemulsions with 2015). It was also useful in preparing films containing small, mean droplet size of 40 nm pectin edible coatings increased stable and less size-dispersed nanoemulsions of corn oil (Artiga- the shelf life of chilled chicken at 4 C for 12 days. The Artigas et al., 2017). Porcine gelatin has been used in preparing nanoemulsion reduced microbial spoilage by inhibiting growth films with antioxidant activity from canola oil and ginger of psychrophiles, yeast and mold growth. Further, it showed essential oil nanoemulsions (Alexandre et al., 2016). It has reduced values of total volatile nitrogen and thiobarbituric acid, also been used to prepare films of rutin-encapsulated soybean water holding capacity and texture and higher sensory scores in oil. However, the nanoemulsified essential oil increased the comparison to the control (Abdou et al., 2018). Nanoemulsions of water vapor permeability values resulting in poor water barrier ginger essential oil with sodium caseinate coating extended shelf properties (Dammak et al., 2017). Basil seed gums have been Frontiers in Sustainable Food Systems | www.frontiersin.org 14 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications used to prepare antimicrobial films of nano-emulsified Zataria structured liquids (NSSL) have been developed by NutraLease, multiflora essential oil (Gahruie et al., 2017). a technology start-up company. Small compressed micelles, The foods have also been immersed in nanoemulsions called nanodrops are developed and these micelles serve as a formulations to test their efficacy as coating agents without carrier for fat soluble bioactives. The micelles incorporated in the aid of polymer matrix. Green beans have been coated food products pass through the digestive system effectively and with chitosan solution containing nanoemulsions of mandarin to the absorption sites without undergoing degradation. These essential oil. And further irradiated with γ-irradiation, UV- nanoemulsions have been used for developing beverages with C, and treated with ozonized water. The synergistic effect of functional compounds. NutreLease with NSSL technology has the combined treatment of nanoemulsion coating and UV-C fortified beverages with nanoemulsions of lipophilic compounds irradiation inhibited the growth of Listeria innocua in green such as β-carotene, omega-3, vitamins, phytosterols, and beans (Severino et al., 2014). Sliced breads were immersed isoflavones. It claimed that the food products had improved in methylcellulose-and clove bud or oregano essential oil bioavailability and good shelf life (NutraLease, 2011). NSSL have nanoemulsions. There was a reduction in spoilage molds been used by Shemen industries to develop Canola Active oil resulting in increase of shelf life of sliced bread (Otoni which is fortified with non-esterified phytosterols. The minute et al., 2014b). Fresh-cut Fuji apples have been immersed in micelles carry the phytosterols to the large micelles that the nanoemulsion formulation of alginate-based edible coatings of body produces from the bile acid, where they compete with lemongrass essential oil for 2 min and stored at 4 C for 14 cholesterol for entry into the micelle. The phytosterols enter days. The nanoemulsion coating had an inhibitory effect on the micelle, thereby inhibiting transportation of cholesterol from E. coli and the growth of natural flora of apple was inhibited the digestive system into the bloodstream. Similarly, Aquanova for 2 weeks (Salvia-Trujillo et al., 2015). Fresh-water rainbow has developed NovaSol beverages fortified with nanoemulsions trout was immersed for 15 min in the nanoemulsion formulation of functional compounds and natural colorants (β-carotene, containing essential oils of Zataria multiflora Boiss and packaged apocarotenal, chlorophyll, curcumin, lutein, and sweet pepper within polyethylene bags and stored at 4 C. Its shelf-life increased extract). It claimed that encapsulated compounds had enhanced and sensory attributes were preserved during the storage time stability and standardized additive concentrations (AquaNova, (Shadman et al., 2017). Similarly, rainbow trout filets immersed 2011). The product NovaSOL sustain contains nanocarrier for 3 min in nanoemulsions of rosemary, laurel, thyme, and that introduces coenzyme Q1O and alpha-lipoic acid. The sage nanoemulsions were packed in stretch films and stored at micelle (30 nm) is stable to pH and temperature variations 2 C for 24 days. The nanoemulsions were effective in inhibiting and is completely water soluble. The micelle is assumed to growth of psychrotrophs and and Enterobacteriaceae members be optimum carrier system of lipophiles and can be used for (Ozogul et al., 2017). Spoilage of cherry tomatoes by molds such efficient intestinal and dermal resorption and penetration of as Botritis cinerea has been prevented by coating it with thymol active ingredients (AquaNova, 2011). RBC Life sciences has nanoemulsions. Quinoa protein and chitosan edible coating developed NanoceuticalsTM Slim Shake Chocolate. It employs TM containing thymol nanoemulsion significantly inhibited growth NanoCluster delivery system, a nanosize powder of nutritional of fungi on cherry tomatoes after 7 days when stored at 5 C supplements. CocoaClusters developed by this method is a (Robledo et al., 2018). Similarly, anise oil loaded nanoemulsions technologically advanced form of cocoa and does not require in comparison to coarse oil and bulk oil, significantly inhibited sugar. During the process of creating NanoClusters, pure Cocoa the growth of foodborne pathogens including E. coli O157:H7 is added to the “Cluster” formation. When consumed, it reduces and L. monocytogenes (Topuz et al., 2016). the surface tension of foods and supplements to increase wetness and absorption of nutrients. Another nutritional supplement R TM Nanoemulsions in Food Industry developed by RBC Life Sciences , is called NanoCeuticals . TM A number of in vitro and in vivo food challenge studies The product NanoCeuticals , with nanoscale ingredients is have shown the advantages of using nanoemulsions in foods is claimed to have antioxidant properties. A product Nanotea as delivery systems for bioactive compounds. However, there developed by Shenzhen Become Industry & Trade Co., Ltd. are very limited examples of nanoemulsions of bioactive contains nanofine powder produced. The product is claimed to compounds incorporated into commercial food products or have antimicrobial activity and high selenium supplementation used as packaging materials. Nanoemulsions can be used for by 10 times. LivOn Labs has developed a product Lypo- TM R sustainable food processing. They can prevent the functional Spheric Vitamin C which uses smart liposomal Nanospheres ingredients from temperature, oxidation, enzymatic reactions, to encapsulate Vitamin C. The product has increased the and pH variations. Food industries such as Nestle and Unilever bioavailability of all nonliposome encapsulated forms of vitamin and a few Start-ups have used nanoemulsions in their food C (http://www.nanotechproject.org). Bottled waters with oil- products (Silva et al., 2012; Salvia-Trujillo et al., 2017). Nestle soluble flavors and enriched with electrolytes, vitamins, and have developed w/o nanoemulsions and patented polysorbates nutraceuticals with nanoemulsion technology have also been and micelle-forming emulsions for rapid and uniform thawing developed (Piorkowski and McClements, 2014). of frozen foods in the microwave (Möller et al., 2009). Unilever Granalix BioTechnologies has commercially launched TM has used nanoemulsions in ice creams for reducing fat content GranaGard a food supplement based on pomegranate oil from 16 to 1% (Martins et al., 2007; Unilever, 2011). Novel which has shown to prevent neurodegeneration diseases in nanoencapsulation method known as Nano-sized self assembled animal models. GranaGard, is a submicron pomegranate seed oil Frontiers in Sustainable Food Systems | www.frontiersin.org 15 November 2019 | Volume 3 | Article 95 Aswathanarayan and Vittal Nanoemulsions and Their Food Applications emulsion, and is an innovative formulation containing natural known to protect and increase the bioavailability of bioactive antioxidants, Punicic acid (an Omega 5 lipid), which constitutes compounds as shown by in vitro studies. But there are 80% of the nanoemuslion. The novel patented formulation was limited studies which show the actual health benefits of shown to delay disease onset and prevent neuronal death in including nanoemulsions in foods, their consumption, labeling, animal models (Binyamin et al., 2015). Therapeutics Solutions and public perception. Similarly, studies have been carried International, Inc., has produced nutraceuticals prepared from out to evaluate the use of high or low energy approach TM NanoStilbene , stable nanoemulsion of pterostilbene (75– to formulate nanoemulsions and the focus is on optimizing 90 nm) from GRAS ingredients by low energy emulsification processing parameters and ingredients used in nanoemulsion methods. The nanoemulsion of pterostilbene containing preparation. But there is not much research on reducing nanoparticles has been patented. Pterostilbene, the active the cost of production of nanoemulsions as its preparation ingredient in the Company’s patented ProJuvenol product is a and application for fortifying and packaging in foods require more potent analog of resveratrol. The nanoemulsions have a higher energy input and equipment investment. Similarly, the better solubility and stability than the parent compound (Dixon risks associated with the use of engineered nanoemulsions et al., 2017). in foods is not known. The potential toxicological effects A flavor nanoemulsion containing a hydrophobic oil droplets and biological fate of nanoparticles after digestion has not containing flavors, an aqueous phase, and a surfactant system been elucidated. including a polyethoxylated sorbitan fatty acid ester and Nanoemulsions of bioactive compounds and functional lecithin has been patented by International Flavors and food ingredients have a great potential for applications in Fragrances Inc. The flavor nanoemulsion has been used to the food industries. The emulsion-based delivery systems and prepare stable and optically clear liquid beverages including nanoemulsion edible coatings can improve the functionalities of alcoholic beverages (Lee et al., 2015). Nanoemulsions of food and also enhance their quality and shelf life. However, the natural antioxidants (extracted from fruits, vegetables or cereals) food grade nanoemulsions can find widespread application only for food preservative applications have been patented. The if its production cost is commercially feasible and meets the safety nanoemulsions of encapsulated natural antioxidants have been standards of food industry. Therefore, it is important to optimize freeze dried and applied to preserve fresh and minimally the bioactivity of the encapsulated components for scaled up processed foods. The nanoemulsion have been applied as thin, production. Further studies should focus on the biological events nanometre-size layer on the food. The nanoemulsion coating and risks associated with the use of nanoemulsion based delivery has been found to prevent gas and fluid exchange with the systems in food products and packaging applications for ensuring safety of the consumers. external environment. The edible nanocoatings extends the shelf life of fresh and minimally processed foods. It has also shown to improve the organoleptic quality of frozen foods on thawing AUTHOR CONTRIBUTIONS (Malnati et al., 2019). JA and RV were involved in design, analysis, data collection, and preparation of the manuscript. CURRENT PERSPECTIVES AND FUTURE PROSPECTS ACKNOWLEDGMENTS In the last few years, a number of studies have been carried out to ascertain the advantages of encapsulation of lipophilic and The authors acknowledge the funding agency UGC-UPE, University of Mysore. functional compounds in nanoemulsions. Nanoemulsification is Akbas, E., Soyler, B., and Oztop, M. H. (2018). Formation of capsaicin loaded REFERENCES nanoemulsions with high pressure homogenization and ultrasonication. LWT. Abdou, E. S., Galhoum, G. F., and Mohamed, E. N. (2018). Curcumin loaded 96, 266–273. doi: 10.1016/j.lwt.2018.05.043 nanoemulsions/pectin coatings for refrigerated chicken fillets. Food Hydrocoll. 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Performance original author(s) and the copyright owner(s) are credited and that the original of selected emulsifiers and their combinations in the preparation publication in this journal is cited, in accordance with accepted academic practice. of β-carotene nanodispersions. Food Hydrocoll. 23, 1617–1622. No use, distribution or reproduction is permitted which does not comply with these doi: 10.1016/j.foodhyd.2008.12.005 terms. Frontiers in Sustainable Food Systems | www.frontiersin.org 21 November 2019 | Volume 3 | Article 95

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