Abstract
Recent developments in micro and nanoencapsulation are promising tools to encounter the different limitations of essential oil formulations, enhance their functionalities, and protect them from the external environmental conditions. This review addresses the current studies and progresses related to the development of encapsulated essential oils using different systems and carrier material types. It also focuses on the formation methods used with the subsequent physicochemical characterization of the developed particles. Moreover, this review considers the factors affecting the release of essential oils with the different physicochemical release models. The choice of the appropriate formation method as well as the carrier material types and system forms were shown to highly depend on the intended purpose of the encapsulated essential oil formulation. Micro and nanoencapsulation are used to control essential oils’ release properties, enhance the various characteristics of essential oils, and allow to expand applications in different fields. This review provides the optimal conditions for micro and nanoencapsulation of essential oil formulations based on the intended end uses.
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Introduction
Researchers and consumers’ interests have increased towards the use of biosourced essential oils (EOs) with relevant and diverse functional activities. EOs are active volatile hydrophobic compounds extracted as secondary metabolites from the different parts of edible, herbal and medicinal plants. Till present, 3000 different EOs were discovered and around 300 of which were commercially used in cosmetic, perfume, food, beverage, sanitary, agronomic, medicine, and pharmaceutical industries [1]. EOs global market is increasing day by day and it was found to exceed US$ 7.51 billion in 2018 with an estimated increase greater than 9% of its annual growth rate between 2019 and 2026 [2]. Over time, several authors have reviewed the use of EOs due to their remarkable biological and fragrance properties and have reported their anti-oxidant [3,4,5,6], anti-inflammatory [7,8,9,10,11], antifungal [12], pesticidal [13,14,15], antimicrobial [16,17,18,19,20,21] and anti-cancer [7, 22,23,24] properties.
EOs broad spectrum applications have increased as they derive from natural sources, degrade easily in soil and water, are environmentally-friendly and have shown low mammalian toxicity [25,26,27]. The use of such biosourced agents ensures a sustainable development, protection and respect for the environment. Nevertheless, the direct application of EOs in their free native forms is limited due to several reasons therefore, the main challenge is to develop new strategies able to encounter the different limitations of EOs. To achieve this, micro and nanoencapsulation could be powerful approaches that enhance the different potential functionalities of EOs while decreasing the amounts being used.
The novelty of this review lies in providing the most suitable approach for the micro and nanoencapsulation of EO formulations related to their intended applications. The objectives of this review are to discuss the recent progresses in micro and nanoencapsulation to overcome EO limitations, and to focus on encapsulation: (i) forms and carrier material types, (ii) methods and physicochemical characteristics, (iii) factors affecting EO formulations release and, (iv) the physicochemical release models of EOs.
Limitations of free essential oils and their active components
The application of EOs in their free forms could be hindered due to several critical factors as their: (i) low stability and susceptibility to degradation by volatilization and/or oxidation when exposed to external deteriorating factors as oxygen, light and temperature, (ii) low water solubility and (iii) potential interactions with food components.
Oxygen presence was mainly reported to induce considerable physicochemical and compositional alterations to EOs, reducing their stability, quality and potential functionalities [28]. The peroxide values indicating the amount of oxidized compounds of eucalyptus, lavender, may chang, pine, rosemary and turpentine EOs increased mainly during the first month of storage indicating their oxidation and low stability [29]. Room storage of Thymus daenensis EO was shown to induce significant oxidation and evaporation of its major active compounds namely α-pinene, myrcene, p-cymene and γ-terpinene affecting thus its initial quality and limiting its use in cosmetic and pharmaceutical industries [30]. With an increase in storage period from 10 to 30 days, the concentration of geraniol, the main component of Rosa x damascena Mill. EO, decreased from 19.07 to 2.29%, reducing its application in flavoring and perfumery industries [31]. Also, upon air exposure, auto-oxidations of linalool and limonene were reported producing some oxidized compounds that increased skin irritation [32]. At room storage, lemon balm EO compounds having low boiling temperatures as neral, citronellal and geranial, evaporated and decreased from 18.9 to 4.0%, 25.8 to 12.6% and 27.0 to 4.6%, respectively [33]. Lemon EO samples stored in half-filled bottles containing air showed significant loss of γ-terpinene, limonene and citral [34].
Generally, light exposure induces also pronounced effects on the stability and the different potential functionalities of EOs. Storage of marjoram EO for more than 3 months in light, produced considerable changes in its chemical composition with an accumulation of oxidative compounds and a loss of its organoleptic properties making it unsuitable for use [35]. Whereas, storage in dark for 1 year did not induce any significant changes in both its chemical and organoleptic properties, suggesting that light accelerates chemical reactions and alters the stability of EOs [35]. Also, greater peroxide values were reported in rosemary and lavender EOs upon light exposure, when compared to storage in the dark at the same temperature [36]. However, in the same study, thyme EO showed pronounced stability when stored in different light and temperature conditions, which may be attributed to the high amounts of phenolic content as carvacrol and thymol that scavenge the free radicals formed during oxidation [36]. Fennel EO was extremely unstable throughout light storage with degradation of its monoterpene hydrocarbons and oxidation of trans-anethol, while lower oxidation was reported when stored in dark [37]. In another study, light and dark storages showed very similar effects on the degradation of lemon EO major compounds and the appearance of off-flavors and off-odors [34].
EOs stability was additionally found to be related to the temperature as generally, an increase in temperature accelerates chemical reactions and degradation of EOs contributing to their lower stability [28]. It was reported that after 12 weeks of storage at 38 °C, the peroxide values of rosemary EO were 8.2 times higher than the initial values, whereas at room temperature, the peroxide values were only 4.2 times greater than the initial ones, indicating the accelerated degradation of EOs at higher temperatures [36]. Also, heating nutmeg EO at 180 °C, induced significant loss of its major components (α-and ß- pinene and sabinene) with an increase in myristicin and safrole contents [38].
The direct incorporation of EOs into aqueous-based matrices and environments is limited due to their hydrophobic nature. As a result, their antimicrobial effectiveness and applications might be affected [39]. Higher concentrations will be required to achieve potential functionalities in water based matrices which may alter the organoleptic properties and exceed the acceptability thresholds [40]. The addition of solubilizers to EO compounds as carvacrol, eugenol, linalool and 2-Pentanoylfuran enhanced their solubility from 938–1399 mg/L to 1.28–5.32 mg/L in aqueous media. Subsequently, their antimicrobial activities against Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Saccharomyces cerevisiae were enhanced with lower amounts being used [40].
Another major limitation of EOs’ applications, is their potential interactions with intrinsic food constituents as proteins, lipids and other compounds that impair their applications and reduces their effectiveness [1, 41]. For example, due to the strong binding between eugenol and fat globules present in milk, higher amounts of eugenol were needed to inhibit microbial growth in milk products compared to Tryptic Soy Broth culture medium [42]. Also, the antimicrobial activity of eugenol was more effective in skim milk compared to full fat milk. 3.5 g/L eugenol were needed to completely inhibit E. coli O157:H7 bacterial cells in skimmed milk (< 0.5% fat), while 5.5 g/L eugenol were needed for a complete bacterial inhibition in full fat (4%) and reduced fat (2%) milk [42]. In zero-fat hotdogs, 10 ml/L of thyme and clove EOs induced 0.86–1.72 log reductions of Listeria monocytogenes, while in full-fat hotdogs, lower bacterial reductions were induced (0.42–1.28 logs) due to the interaction of EOs with fats present in full-fat hotdogs [43]. The anti-listerial activity of oregano and thyme EOs was also reduced when incorporated with lipid oils and potato starch at concentrations higher than 5% [44]. All these examples of the reduced activities of EOs in food matrices may be explained by the presence of fats, proteins or starches that may form a layer around bacterial cells protecting them from the action of antimicrobial agents. Additionally, EO components may migrate to the fatty compounds in food due to their hydrophobic nature, allowing bacteria to develop freely in the aqueous fraction [43, 45, 46]. As the effect of EOs is reduced in food matrix systems, higher unacceptable concentrations will be required to compensate their interaction with food components and to perform their intended functionalities [47]. Thus, EOs may develop some unpleasant unacceptable odors and flavors that alter the sensorial properties and overall acceptability of food products limiting thus their incorporation and efficiency in several applications [39, 48]. As an example, the higher concentrations of Mentha piperita EO required to achieve an antimicrobial activity induced strong odors in minced meat that decreased their overall acceptability [49]. Also, the concentrations of Salvia officinalis L. and Schinus molle L. EOs required to induce significant anti-bacterial effect in minced beef meat had notable negative effects on the flavor and odor [50]. This further required the combination of EOs with other preservation methods to reduce the concentrations being used and thus their negative sensorial impacts.
Beside these different challenges, the high concentrations of free EOs probably needed for certain applications may increase the risk of developing resistance by several microorganisms [47]. The challenge is therefore to develop new delivery systems as micro and nanoencapsulation to overcome the different obstacles of free EOs and boost their effectiveness.
Micro and nanoencapsulation of essential oils
Encapsulation is the process in which one or more active agents (coated materials, core or internal phase) are loaded in a homogenous or heterogeneous matrix (shell, wall or carrier material) at micro (1–5000 μm) or nano (< 1 μm) scale [51,52,53]. The entrapped molecules are protected from the deteriorating external conditions such as degradation, evaporation and oxidation [13, 48, 54]. Due to their reduced size and increase in surface-to-volume ratio, micro and nanoparticles allow a greater bioavailability and a facilitated diffusion of EOs into their target sites enabling efficient long-term activities [48, 55,56,57,58]. In addition, lower amounts of EOs are used in the encapsulation process which achieves lower sensorial impacts on food products, reduces the probability of developing resistance by microorganisms, minimizes their toxic effects and reduces the economic costs.
Carrier material types
In micro and nanoencapsulation of EOs, different carrier materials could be used depending on their charge, on the encapsulated material as well as on the encapsulation process and the targeted applications [59]. Carrier materials could be homogenous or heterogeneous and solids or liquids [60]. For a better encapsulation efficiency and for environmental purposes, the used carrier materials are preferable to be biocompatible, biodegradable into non-toxic products, Generally Recognized as Safe (GRAS), commercially available, non-reactive with the encapsulated active ingredients, and easily administered [53, 58]. The carrier materials used for EOs encapsulation were natural biopolymers as polysaccharides, proteins, lipids as well as synthetic ones (Fig. 1). Moreover, in some encapsulation techniques, carrier materials used were a thin layer of surfactant molecules surrounding bioactive compounds and emulsified with an aqueous solution [56, 61,62,63,64,65,66]. Surfactants could be added to form more stable EO emulsions and reduce the interfacial tension between aqueous and oil phases [66].
Polysaccharides used as carrier materials for EOs encapsulation derived mainly from plant, marine and animal sources. The polysaccharide-based carrier materials should be safe, non-toxic, biodegradable, have a good water solubility, a low viscosity at high concentrations and of low cost due to their abundancy in nature [53, 67, 68]. In comparison with other carrier types, polysaccharide-based matrices have a better thermal stability and are less sensitive to environmental changes as pH or ionic strength modifications [69].
Proteins derived from plant and animal sources are considered also good candidates for the delivery of EOs as they are widely available, easily modified, inexpensive, biodegradable, biocompatible and have good water solubility [51, 53, 67, 68]. However, most proteins are susceptible to pH, ionic strength or temperature changes, which limits their applications but could provide an advantage by enabling the controlled release of encapsulated active substances by a simple pH or heat trigger [69].
Several solid and liquid lipids were also used as carrier materials for the encapsulation of EOs. Lipid-based encapsulations showed high encapsulation efficiency, controlled release and potential applications at industrial levels [53, 58].
The other types of carrier materials used for the encapsulation of EOs were synthetic biopolymers. Most of these materials were found to be biodegradable, biocompatible, cost-efficient and non-toxic [70,71,72]. Some reviewers suggested that these polymers have shown better chemical and physical reproducibility with a higher purity than polysaccharide, protein and lipid-based carriers that presented certain limitations due to their broad range of molecular weight and variations from batch to batch [73, 74].
A combination of different types of carrier materials together has gained much attention recently in several applications. Carrier materials combination starts either by a direct interaction of materials together or by the layer-by-layer method which starts by the formation of particles made of one type of carrier material and then the adsorption of other carriers on the formed particles [75]. An example of a combination of carrier materials is whey proteins with maltodextrins that has improved thermal stability, emulsifying properties and transparency of eugenol nanocapsules [76]. Also, the combination of sodium caseinate and pectin has improved the stability and controlled release of citral from microcapsules [77]. Polylactic acid (PLA) combined with polyethylene oxide (PEO) [72], and zein combined with casein [78] ensured a controlled release of lavender, eugenol and thymol, respectively from nanocapsules. Thus, in general, the combination of different carrier materials together was proved to enhance encapsulation efficiency, loading capacity, thermal and mechanical resistance, controlled release, bio-availability and stability of the encapsulated bioactive compounds [53, 79, 80].
Carrier system forms
Several micro and nanocarrier systems have been adopted for the delivery of EOs and their bioactive compounds into specific target sites. The most common delivery system forms used are capsules, hydrogels, emulsions, liposomes, solid-lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) (Fig. 2). Each of these systems has a specific architecture and physicochemical characteristics depending on the materials used for their development and on their formation methods [53].
Capsules
Capsules are hollow vesicular systems in which bioactive compounds are entrapped within a membrane of carrier materials that forms a protective shell [58]. Proteins like whey protein, zein, casein, gelatin; polysaccharides such as maltodextrin, chitosan, pectin, alginate, cashew gum, gum Arabic; and other carrier materials as PLA, PEO, polycaprolactone (PCL), and polylactic-co-glycolic acid (PLGA) were used for the micro and nanoencapsulation of EOs in capsules [21, 42, 68, 70,71,72, 78, 80,81,82]. Capsules have shown enhanced anti-oxidant and antimicrobial activities, stability and a controlled release of the entrapped bioactive compounds [78, 83].
Hydrogels
Generally, micro and nanohydrogels are made up of oppositely charged carrier materials that are cross-linked to form a three-dimensional (3D) structure that has the ability to hold a great amount of water in different severe conditions [53]. Usually, a filled hydrogel particle contains bioactive compounds dissolved in an oil phase and entrapped within the carrier materials network. When exposed to various external stimuli, hydrogel particles ensure an effective controlled and sustained release of bioactive components while maintaining their network structure making them effective delivery systems [53, 84]. Hydrogels have also presented a high stability, high loading capacity, biocompatibility and a protection from chemical degradations for both hydrophilic and hydrophobic bioactive compounds [58, 85]. Polysaccharides like alginate, chitosan, pectin; proteins like soy and whey proteins; lipids such as soybean oil and other carrier materials as polyvinyl alcohol (PVA) were used for the encapsulation of EOs in hydrogels [60, 86, 87].
Emulsions
Emulsions are formed by the dispersion of two immiscible phases: one phase (dispersed phase) is spread as droplets into another phase (continuous phase) [53]. Emulsions could be water-in-oil (W/O) or oil-in-water (O/W) emulsions depending on the location of water and oil phases. O/W is the most suitable emulsion for the encapsulation of EOs. The incorporated bioactive compounds in the dispersed phase will be protected by the continuous phase from the different environmental conditions. Carrier materials used for the formation of EOs emulsions were polysaccharides like sodium alginate, gum Arabic; lipids like soybean oil or surfactant solutions like Surfynol, Tween, Span, Brij or lecithin [39, 61, 64, 65, 88,89,90]. Micro and nanoemulsions of EOs exhibited enhanced stability, controlled release, antioxidant and antimicrobial activities when compared to free EOs [39, 91]. Microemulsions were shown to be thermodynamically stable and appeared usually as white opaque droplets while nanoemulsions with smaller sizes, appeared as transparent to translucent droplets with a better kinetic stability favoring their use in food products [53, 66].
Liposomes
Liposomes are spherical vesicles made up of one or several phospholipid bilayers enclosing usually an internal aqueous phase [92]. Due to their amphiphilic nature, liposomes can be used to encapsulate, at the same time, both hydrophilic molecules within their internal aqueous compartment, and hydrophobic molecules as EOs within the lipid bilayer [93, 94]. Only lipid based carrier materials such as phosphatidylcholine, cholesterol and lecithin were used for the nanoencapsulation of EOs in liposomes [55, 95,96,97]. Liposomes enhanced the antimicrobial and antioxidant activities of the incorporated bioactive compounds [55, 95, 97]. In addition, they have shown targeted delivery properties [98]. However, the use of liposomes was limited due to their high production cost and as they have shown poor loading capacity and poor physicochemical stability [53, 58, 92]. Their low stability and its subsequent consequences may be improved by coating liposomes with additional carrier materials or trapping them within other carrier systems [99].
Solid lipid nanoparticles
Solid lipid nanoparticles (SLNs) are spherical nanocolloidal carriers made of completely crystallized lipid droplets in which hydrophilic or hydrophobic compounds are directly incorporated or dissolved [100]. They are prepared from oil-in-water emulsions made up of solid lipids with processing temperatures above the melting points of lipids [99]. Solid lipids used for the encapsulation of EOs in SLNs were glycerol monostearate and precirol [24, 101, 102]. SLNs encapsulating EOs were mainly stabilized by surfactant based carrier solutions as Tween 80, Span 80, Poloxamer 188 and Miranol Ultra C32 [13, 24, 54, 102]. Compared to liposomes and emulsions, SLNs presented more protection against different chemical reactions and a prolonged controlled release since the encapsulated bioactive compounds were immobilized in a solid matrix [103]. The formation of SLNs is simple and inexpensive [58]. However, they have shown under acidic conditions, poor stability, high tendency for aggregation and growth of particles during drying, possible gelation, low encapsulation load due to the crystalline structure and transitions in their fat crystalline structures during storage leading to explosions [58, 103].
Nanostructured lipid carriers
Nanostructured lipid carriers (NLCs) are a form modification of the SLNs and contain an inner phase made of a combination of lipids in both solid and liquid states where bioactive compounds are melted and/or solubilized. Five to 40% of the solid lipid phase in SNLs is replaced by liquid lipid in NLCs allowing a better solubilization of bioactive components [58, 103]. The main lipid carriers used for the formation of NLCs encapsulating EOs were cocoa butter, precirol, miglyol, virgin olive oil and sweet almond oil combined with surfactant solutions [104,105,106,107]. NLCs were developed to overcome the limitations of SNLs as they had a smaller size, presented higher loading capacity and prevented the formation of crystals and thus subsequent expulsion [103, 108]. However, before the formation of both SLNs and NLCs, lipids need to be melted at temperatures above those of the lipid melting temperature which might cause a degradation of heat-sensitive EO components [69]. Additionally, both SLNs and NLCs have a low stability under acidic conditions and their formation is not cost-effective at an industrial scale [69, 109].
As a summary for the carrier system forms used, capsules, hydrogels and emulsions were mainly used for the formation of both micro and nanoparticles with different types of carrier materials. They exhibited enhanced functional activities and controlled release when compared to EOs in their free native forms. Whereas, liposomes production was limited to lipid-based carrier materials and only nano-sized particles were produced. In SLNs and NLCs, lipids combined with surfactant solutions were mainly used as carrier materials. The latter carrier systems have shown lower stability and higher production costs at industrial scale. However, compared to emulsions and liposomes, SLNs and NLCs showed better prolonged controlled and targeted release with higher encapsulation efficiency of bioactive compounds [109]. Thus, the selection of the carrier system form depends mainly on the entrapped bioactive compounds and the target of encapsulation.
Encapsulation methods
Various techniques have been developed for the encapsulation of bioactive compounds in different carrier system forms (Table 1). The selection of the encapsulation method is highly essential and is based on the physicochemical properties of the entrapped active compounds, carrier material types, and the intended application of particles [58, 110].
The encapsulation methods could be divided into top-down as well as bottom-up approaches (Fig. 3). The top-down approaches utilize high energy mechanical external tools to reduce the size and shape of the developed particles into smaller dimensions [98]. Whereas in bottom-up approaches, low energy techniques are applied to build a molecular assembly through the association of different molecules or particles together [111, 112].
Nanoprecipitation
Nanoprecipitation, also known as solvent displacement method, relies on the addition of an organic phase containing the bioactive compound(s), organic solvent and the dissolved carrier material into a surrounding aqueous phase. Then, the carrier material immediately precipitates and the organic solvent diffuses into the aqueous phase and is then removed by evaporation (Fig. 4). PCL, PLA and PEO were used as carrier materials for the production of nanocapsules of thyme, oregano and lavender EOs by nanoprecipitation [70, 72]. This method was mainly used for the formation of nanocapsules for food and textiles applications.
Spray-drying
Spray-drying is one of the most common and adopted techniques used for the encapsulation of EOs. It is a mechanical dehydration method that converts a liquid into dried solid powder through a heating process. A primary emulsion mixture is formed and then atomized through a nozzle in a heated air chamber. The solvent will then evaporate rapidly when in contact with hot air, and thus fine solid droplets of bioactive compounds will be obtained [85, 113, 114] (Fig. 5). In fact, the drying conditions play a major role in the determination of the encapsulation efficiency and quality, thus an optimization of the process conditions is required to incorporate efficiently EOs and avoid their volatility [112, 115]. Eugenol, thymol, carvacrol, citral and Lippia siloides EOs were encapsulated in micro and nanocapsules using spray-drying [78, 80, 81]. Also, Cymbopogon citratus emulsions were prepared using this technique [90]. Spray-drying was mainly used for the formation of emulsions and capsules used for food, nutraceutical and larvicidal applications. Only polysaccharides and proteins were used as carrier materials in this method.
Ionic gelation
Ionic gelation starts by the atomization or dripping of the carrier material solution into an ionic solution under constant agitation. Then, bioactive compounds are added and dissolved in the carrier solution. Drops of this solution reaching the ionic solution are converted to spherical gel particles [116] (Fig. 6). This method is based on the interaction between oppositely charged ions to form intramolecular and inter cross-linkages. By cross-linking, particles acquire a higher mechanical strength, stability and chemical resistance which causes a controlled release of the encapsulated compounds [117]. The entrapped active components are further released by gel phase changes triggered by different stimuli as osmotic or mechanical forces, enzymes and/or pH changes [118]. The main cross-linker agents used for the encapsulation of EOs using this technique were glutaraldehyde, transglutaminase, tripolyphosphate, calcium chloride, and formaldehyde. Only chitosan based carrier materials were used for the encapsulation of EOs using ionic gelation. Carum copticum hydrogels, peppermint, green tea, carvacrol and ginger nanocapsules were produced by ionic gelation and used in food, nutraceutical, cosmetic and medical applications [83, 86, 119, 120].
Thin film hydration
Thin film hydration method was only used for the formation of liposomes. In this technique, a mixture of lipids (usually cholesterol, phosphatidylcholine or soy lecithin) is dissolved in an organic solvent which was further removed by rotary evaporation to form a dry lipid thin film [121]. The lipid film was then hydrated in an aqueous buffer solution under high agitation to form liposomes (Fig. 7). Although it is a simple technique, thin film hydration produces liposomes with low stability and wide size distribution [85]. This needs an exposure of the produced liposomes to further treatments as ultrasonication, freeze-thawing or extrusion in order to obtain homogeneous particles with reduced size. Liposomes encapsulating clove, cinnamon, carvacrol, thymol and γ-terpinene using this method were mainly used in food, cosmetic and medical applications [55, 95, 97].
Emulsification
Emulsification is another common technique used to entrap bioactive components with both hydrophobic and hydrophilic properties [58]. Two immiscible liquid phases (dispersed and continuous) are homogenized to produce emulsions. In the dispersed phase, the bioactive compounds are entrapped, while the continuous phase protects the entrapped components from the external conditions (Fig. 8). Droplet particles are either directly used in the liquid state, or freezed and spray-dried to form solid powder particles. Different types of polysaccharide, protein, lipid-based and surfactant carrier materials were used for the encapsulation of EOs using this method. Emulsification includes high energy emulsification approaches as high pressure homogenization (HPH), ultrasonication and microfluidization [58]. HPH forms a liquid coarse emulsion by high shear mixer and then forces it through a narrow gap at high speed and pressure (100–2000 bar), causing the formation of smaller droplets [79, 85]. This technique is considered the most effective method for the production of SLNs and NLCs [24]. Cymbopogon flexuosus nano-emulsions, orange hydrogels, peppermint NLCs and citral SLNs were produced by HPH [24, 54, 60, 61, 91, 105]. In ultrasonication, high frequency ultrasonic waves (> 20 kHz) are generated after the immersion of a probe in a coarse emulsion to produce intensive disruptive forces leading to the formation of small liquid droplets with uniform size [79]. Zataria multiflora SLNs, Eucaplytus, oregano, Nigella sativa L. and Thymus daenensis nanoemulsions were formed using ultrasonication [22, 62, 66, 101, 122, 123]. In microfluidization, two flows of coarse emulsions pass under high pressure through a channel and then interact in a chamber where intensive disruptive forces are produced resulting in the formation of small emulsion droplets [58]. Lemongrass, thyme and sage micro and nanoemulsions were produced by microfluidization [124, 125]. In general, the above emulsification methods are highly reproducible at large scale with no use of toxic organic solvents or high temperatures. The formed nanoemulsions present a high kinetic stability due to their extreme small size which further exhibits a better retention of EOs content on the surface of the droplets [111]. However, these high energy emulsification methods require a lot of energy consumption and sophisticated equipment. On the other hand, the low energy spontaneous emulsification is more economic, simple and does not require the use of sophisticated special equipment. However, it produces unstable emulsions when exposed to chilling, heating, drying, and requires the use of high amounts of surfactants [58]. Several EOs were encapsulated in microcapsules (citronella, thymol, carvacrol…) and in micro and nanoemulsions (eugenol, carvacrol, cinnamon bark, Pelargonium graveolens and Eucalyptus globulus…) using spontaneous emulsification [63, 65, 82, 89, 126,127,128]. Low and high energy emulsification methods were the only techniques that produced all the different forms of carrier systems for the encapsulation of EOs.
Coacervation
The main objective of coacervation, also known as phase separation, is to produce particles based on the separation of two immiscible liquid phases in a colloidal solution [129]. Coacervation could be simple if it involves the phase separation of a single polymer dissolved in an aqueous or an organic phase, or complex if the separation occurs between a mixture of oppositely charged polymers dissolved in an aqueous phase [130, 131]. To induce phase separation in simple coacervation, water miscible non-solvent or inorganic salts are added [132]. Whereas for complex coacervation, temperature or pH changes cause electrostatic attraction between oppositely charged carrier materials leading to phase separation [133]. The two separate phases produced are a coacervate phase made of concentrated insoluble carriers and another dilute or equilibrium phase which is almost free from carrier materials and contains the solvent in which the coacervate is dispersed [71]. After phase separation, the newly formed coacervate is deposited around the bioactive compounds and a hardening agent, a cross-linker, is added to consolidate particles [71] (Fig. 9). PLGA, gelatin and gum Arabic carrier materials along with octamethylcyclotetrasiloxane (OCMTS), formaldehyde, transglutaminase, glutaraldehyde or tannic acid cross-linkers were used for the formation of thyme, citronella, Zanthoxylum limonella and lavender microcapsules by coacervation [14, 15, 68, 71]. Coacervation is a simple method that does not require the use of high temperatures or solvents, however, it has a difficulty to control particles size. The formed coacervates are stable in a very narrow range of temperature and pH, and most cross-linking agents used are toxic chemicals that are forbidden in the food industry [134, 135]. As complex coacervation exhibits better functionalities and higher loading capacity than simple coacervation, it is considered a better choice for food and pharmaceutical applications [58].
Extrusion
In extrusion, a solution containing bioactive compounds and carrier material(s) passes to a gelling environment through a nozzle, syringe, pipette or atomizing disk for the solidification of particles at low temperatures [136]. The solution drops fall into a bath of a gelling agent leading to the formation of particles [137] (Fig. 10). This technique allows the encapsulation of both hydrophobic and hydrophilic heat sensitive compounds with no use of organic solvents [58, 138]. Cinnamon, thyme and clove microcapsules, in addition to thyme hydrogels were produced using this technique for pesticides and pharmaceutical applications [12, 87]. In previous studies, polysaccharides like alginate and sodium alginate, and proteins like soy proteins were the only carrier materials used in the extrusion process for the encapsulation of EOs [12, 87].
Combination of different methods
Several methods have been combined for EOs encapsulation. High energy emulsifications were the main methods combined with other techniques to provide particles with a smaller size. Microfluidization of thyme, lemongrass and sage coarse emulsions was done following HPH in order to reduce their size into nanoemulsions [125]. Cinnamon particles and citral coarse emulsions were also subject to ultrasonication after HPH to obtain smaller NLCs and nanoemulsions, respectively [88, 104]. Citral coarse emulsions formed by HPH were also spray-dried to produce microcapsules [77]. Carvacrol and thymol liposomes formed by thin film hydration were subject to ultrasonication to reduce and homogenize their sizes [96]. Thus, some combined encapsulation methods could be used to obtain targeted reduced size of particles.
Besides the various encapsulation methods proposed, none of them could be considered as an optimal absolute method for the encapsulation of bioactive compounds. The choice of the encapsulation method is related to the properties of the entrapped bioactive agents and carrier materials, the forms of carrier systems and the purpose of the encapsulation. The different advantages and limitations of each encapsulation method should be taken into account for choosing the ultimate method for the encapsulation of bioactive compounds (Table 2).
Carrier system forms could also predict the preferable encapsulation method, as some system forms are limited to specific formation methods. Emulsification was the only technique that is able to produce all types of carrier system forms. For EOs encapsulation, methods that use solvent evaporation and uncontrollable heating as thin film hydration should be avoided since EOs are heat-sensitive and volatile compounds [85]. For food applications, nanoprecipitation, thin film hydration and coacervation methods must be avoided as they use toxic organic solvents or toxic chemical cross-linkers. Additionally, the choice of the encapsulation method could be related to the intended purposes. For an enhanced stability, ionic gelation, spray-drying, nanoprecipitation, HPH, ultrasonication and thin film hydration techniques could maintain or improve the stability of the encapsulated EOs [64, 70, 72, 77, 83, 95, 107, 123]. While, extrusion and coacervation techniques did not ensure an enhanced stability of the encapsulated bioactive compounds, as the extruded particles had a large and porous structures and the coacervates were highly unstable in several conditions [58]. Regarding encapsulation efficiency and according to research studies discussed in this review, thin film hydration followed by ionic gelation methods exhibited the lowest encapsulation efficiencies of 4.16–29.2% and 4.7–45%, respectively [55, 86, 95, 97, 119, 120]. While higher encapsulation efficiency ranges of 70–96%, 55–99.6%, 84–99.84%, 57–98.2% and 72–94% were exhibited by nanoprecipitation, spray-drying, emulsification, coacervation and extrusion, respectively. The intended size of particles could also predict the choice of the encapsulation method as micro-sized particles were only formed by spray-drying and extrusion techniques. Whereas, nano-sized particles were formed by all the different encapsulation methods except of extrusion technique. Some of the studies reporting EOs encapsulation forms and methods used for different applications are presented in Table 3.
Physicochemical characteristics
After micro and nanoencapsulation of bioactive compounds, several physicochemical characteristics of the formed particles need to be evaluated as they may have an impact on the efficacy, physical stability and release mechanisms of bioactive agents [139].The most important physicochemical characteristics include size, size distribution, surface charge and morphology of the particles which depend on several external factors like pH, relative humidity and temperature, in addition to the processing operations and materials [133].
Size and size distribution
Particles size and their size distribution dictate the stability of particles over a period of time. A decrease in particles size increases the surface to volume ratio which further enhances the bioavailability and the different functionalities of the encapsulated EOs and decreases the attractive forces between droplets leading to a better stability [66, 102, 139]. The degradation rate of a carrier material is also affected by the size of particles, as an increase in particle size increases the degradation rate of particles walls [59]. In the studies reported in this review, the size of micro and nanoparticles ranged between 1.02–1880 µm and 10–811 nm, respectively.
Polydispersity index (PDI) is a measure of the particles size distribution which also predicts the stability of particles. PDI values vary between 0 and 1, with low values near zero indicating a narrow size and stable distribution of the particles. A PDI below 0.3 reveals the uniformity and homogeneity of particles [61]. Most of literature studies, reported stable particles as PDI values were below 0.3, except few studies where PDI values were between 0.358 and 0.726 indicating the non-uniformity and non-stability of the developed particles [39, 54, 102, 107, 125]. Dynamic light scattering (DLS) was the most common method used for the measurement of particle size and PDI [56, 61, 62, 64, 91, 95, 96, 102, 123, 127]. Other techniques were also used as photon correlation spectroscopy (PCS) and laser diffraction instrument [13, 77, 140].
Surface charge (zeta potential)
In an ionic solution, most particles have a layer of oppositely charged ions. When particles diffuse into a solution, this layer comes in contact with a second outer layer composed of loosely associated ions to form an electrical double layer [141]. The potential measured at the surface of this double electric layer is known as zeta potential. It is reported that zeta potential values above 30 mV (either positive or negative) indicate a good electrical stability and promote more repulsive interactions avoiding the aggregation or collision between particles [59, 83, 96, 123]. All studies indicating zeta potential values above absolute 30 mV showed a long term stability with no aggregation of particles encapsulating EOs [13, 39, 55, 81, 95, 96, 102, 123, 125, 142]. Whereas studies showing zeta potential values below absolute 30 mV presented a lower and shorter term stability of particles with a faster aggregation [61, 83, 88, 105, 120]. Zeta potential measurements were done using zeta sizer [13, 39, 77, 81, 95, 142], phase-analysis light scattering (PALS) [124, 125], and electrophoretic mobility technique [61, 70, 91, 127].
Morphology and structure
The developed particles morphology refers to their external as well as their internal structures that depend highly on the carrier materials used and on the operating conditions. Usually particles encapsulating bioactive agents are spherical but they may present some other shapes as cylindrical, ellipsoid or irregular. The shape of particles might have an impact on the stability, aggregation, optical and release characteristics of the encapsulated compounds [99]. Different microscopic techniques as phase contrast microscopy [66], atomic force microscopy (AFM) [55, 63, 66, 78, 142], scanning electron microscopy (SEM) [143,144,145], and transmission electron microscopy (TEM) [61, 62, 91, 102, 123] were used for the determination of particles topography, morphology and structure. In addition to the visualization of particles shape, the different microscopic techniques allowed an observation of the presence or absence of aggregations or fusion between particles [146].
Factors affecting essential oils release
Delivery systems release the entrapped bioactive compounds at appropriate time and site, in response to particular triggers. pH, relative humidity, temperature, particles’ size, carrier systems forms, ratio and properties of carrier materials and cross-linking agents are considered the main factors that may alter the release of the encapsulated compounds.
It is shown that pH changes alter the charge of particles encapsulating EOs [119]. In several studies, as pH changes, the degree of ionization of the different functional groups of carrier materials changes leading to charge modification. For example, at acidic pH (pH = 3), nanocapsules of peppermint, green tea, Carum copticum and carvacrol showed a significant higher release of bioactive compounds when compared to the release at higher pH values (between 7 and 11) [83, 119, 120]. The higher release of EOs in acidic medium was attributed to the ionic repulsion between the protonated amino groups (NH3+) on the neighboring chitosan chains of carrier materials, increasing the surface area of nanocapsules exposed to the external medium. Whereas at neutral and basic pH values, the deprotonation of NH3+ groups on chitosan chains caused more aggregation between particles lowering the release rate of the encapsulated active components [120]. Also, the electrostatic attraction between combined carrier systems is related to pH changes [60]. At pH values above the proteins’ isoelectric point (pI), most of amino and carboxyl groups are deprotonated resulting in net negative charges. Whereas below pI of proteins, most of these groups are protonated leading to net positive charges [60]. Thus, in systems based on a combination of proteins with other carrier materials, the attraction or repulsion between carriers is related to the pI of proteins and pH values. As an example, when proteins are positively charged (pH below pI of proteins), they will be attracted to oppositely charged polysaccharides and thus bioactive compounds will be retained inside the carrier system. Whereas, negatively charged proteins are repulsed from anionic polysaccharides leading to the release of bioactive compounds [99, 147].
Relative humidity (RH) was also found to be related to the release of bioactive compounds from carrier systems. For example, with an increase in RH from 50 to 90%, the release of thymol from PLGA microcapsules increased from 41.54 to 61.13% [128]. This increased release was mainly related to the increase in water absorption by microcapsules causing a faster degradation of carrier material and thus a greater release of the entrapped components [128].
Temperature can also alter carrier materials contraction and mobility thus affecting the release of the entrapped bioactive compounds. Based on the carrier material type and the applied temperature, different release rates have been observed. As for example, a gradual increase in environmental temperature from 40 to 80 ºC, caused a contraction of chitosan polymeric chains, a reduction of the space between particles’ pores and thus an inhibition of the release of citronella EO from microcapsules [82]. On the contrary, the release of thymol from PLGA microcapsules was greater at 25 ºC than at 4 ºC [128]. This was mainly explained by the mobility of the carrier material at higher temperatures ensuring a higher release of EOs.
The bioactive compounds release may also be related to the carrier materials and to the cross-linkers properties and ratios. An increase in chitosan carrier concentration from 0.5 to 1.5% decreased the release rate of citronella from microcapsules [82]. This was accounted for the increase in thickness of the surrounding membrane and subsequent reduction in pores sizes between chitosan molecules. Also, Zanthoxylum limonella oil release rate from microcapsules decreased when both the gelatin carrier amount and the molecular concentration of glutaraldehyde cross-linker increased [14]. An increase in PLA molecular weight from 4.5 to 10 KDa increased the release of lavender oil by 5 to 40% from nanocapsules [72]. Capsules made of PLA with higher molecular weight of 10 KDa were smaller and thus increased the release due to the higher surface area [72]. The ratio of alginate to cashew gum influenced also the release rate of Lippia siloides EO from nanocapsules. A 1:3 alginate/cashew gum ratio increased the release while a 3:1 ratio decreased the release of EO. Cashew gum was found to increase the hydrophilic properties of particles and thus increase EOs release, whereas alginate provided a more complex structure retaining EOs [81]. In addition, the bioactive compounds release was found to be related to particles size. It was reported that a decrease in microcapsules size from 225 to 11 µm, increased the release rate of citronella from approximately 22 to 50% [82]. This was mainly explained by the increase in total surface area of smaller particles allowing a faster release of bioactive components from microcapsules [82]. Controlled release of bioactive compounds was additionally found to be related to the carrier system form. SLNs and NLCs showed the most prolonged controlled release of EOs as bioactive compounds were entrapped within solid lipid carrier materials retarding their release.
Physicochemical release mechanisms
An effective encapsulation ensures a protection of the encapsulated bioactive compounds from the external conditions. After a specific trigger condition, bioactive compounds will be released at an appropriate time, concentration and speed. Several factors affect the release of encapsulated bioactive compounds including the interactions and ratio between carrier and core materials, the size as well as the viscosity of the developed particles [115]. When exposed to environmental trigger conditions, the release of encapsulated EOs from carrier systems is regulated by one or more of the following physicochemical mechanisms:
Diffusion
Diffusion is the most common release mechanism of bioactive compounds from the different carrier systems [108]. The diffusion of molecules is driven by the concentration gradient from medium of high concentration to medium of lower concentration. Generally, in diffusion-controlled release mechanisms, there is a high initial release followed by a decrease in diffusion as the distance of bioactive compounds to the surface of particles increases. Diffusion rate depends on the size of the entrapped molecules, the thickness and molecular weight of the carrier material and the medium of dispersion. Larger molecules diffuse slower than smaller ones, a thick membrane wall will also retard the release of components, and the diffusion rate is slowed in a viscous medium.
Swelling
Swelling release mechanism mainly occurs in hydrophilic based carriers like proteins and polysaccharides and particularly in hydrogels that have the ability to absorb a great amount of water [108]. Some environmental conditions may cause changes in the repulsive or attractive interactions between polymeric carrier materials leading to pores size changes and swelling of particles as a result of the absorption of fluids from the surrounding medium [99]. The increase in volume due to swelling, increases pores size and thus the entrapped active components will be released from particles by simple diffusion.
Dissolution or melting
Water-soluble carrier materials are easily dissolved in the presence of moisture or a suitable solvent, whereas lipid-based carriers release compounds upon melting by heating [79]. Once the surrounding carrier is dissolved or melted, bioactive components are released and come in contact with the external medium. The release rate depends on the thickness and nature of the carrier material [148].
Degradation
Particles made of biodegradable carriers as proteins, polysaccharides and lipids will eventually undergo enzymatic degradation under particular environmental conditions [79, 108]. As the carrier material degrades, the surrounding medium enters inside particles and leads to the release of bioactive compounds from the entrapped inner core.
Conclusions and future perspectives
A growing interest in EOs as potent biosourced and ecofriendly alternatives to the synthetic sanitizers has been evident during the last years due to their different physicochemical, biological, and functional properties. Micro and nanoencapsulation of EOs were investigated as novel promising delivery systems that could offer several benefits and overcome the different limitations of free EOs. Successful encapsulation of EOs ultimately depends on the intended purposes and applications, in addition to the selection of the carrier materials types, forms and development methods. The combination of appropriate GRAS, biodegradable and non-toxic carrier materials was found to offer advantageous formulations for the encapsulation of EOs. Several carrier system forms and encapsulation methods have highlighted the feasibility to produce micro and nano-sized stable EO particles enabling their integration and application in several fields without causing detrimental effects. SLNs and NLCs present outstanding controlled release properties but with higher production costs and lower particles stability. Whereas, the formation of capsules, hydrogels and emulsions is inexpensive and able to enhance the different functional activities of EOs but presents lower controlled release properties. It was also shown in this review to preferably avoid thin film hydration, nanoprecipitation and coacervation methods for the encapsulation of EOs as they require the use of organic solvents.
Beside the numerous advantages of encapsulation, it has raised a number of regulatory, safety and environmental issues concerning its impact on the environment and on human health. Their safety aspects are still unclear till present and require further exploration as no specific international legislation has been applied. Thus future studies should emphasize (i) the safety aspects and risk assessments of the use of micro and nanoencapsulation of EOs in several applications, (ii) the improvement of existing encapsulation methods and their production on an industrial scale, (iii) the optimization of factors affecting the release of bioactive compounds for an enhanced and more pronounced activity, and (iv) the exploration of further direct applications of EOs particles particularly in biological and food systems.
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Yammine, J., Chihib, NE., Gharsallaoui, A. et al. Advances in essential oils encapsulation: development, characterization and release mechanisms. Polym. Bull. 81, 3837–3882 (2024). https://doi.org/10.1007/s00289-023-04916-0
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DOI: https://doi.org/10.1007/s00289-023-04916-0