Essential oil encapsulations: uses, procedures, and trends

Abstract

Recently there has been an increased interest towards the biological activities of essential oils (EOs). However, EOs are unstable and susceptible to degradation when exposed to environmental stresses like oxygen, temperature, and light. Therefore, attempts have been made to preserve them through encapsulation in various colloidal systems such as microcapsules, nanospheres, nanoemulsions, liposomes, and molecular inclusion complexes. This review focuses on various techniques used for the encapsulation of EOs, potential applications in food, and their behaviours/trends after encapsulation. The encapsulation efficiency, particle size, and physical stability of EOs encapsulated in colloidal systems is dependent on the kind of technique and the type and concentration/ratio of emulsifier/wall material used. Moreover, the benefits associated after encapsulation, namely bioavailability, controlled release, and protection of EOs against environmental stresses are discussed. The applications of encapsulated EOs are also summarized in this review. Encapsulated EOs are promising agents that can be used to increase the anti-microbial, antifungal, antiviral, and pesticidal activities of EOs in real food systems, to study their action mechanism, and to provide nonlethal therapeutic agents to treat several diseases.

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1. Introduction

EOs are a diverse group of natural aromatic compounds isolated mostly from non-woody plant materials by hydro-distillation, solvent–solvent extraction, and liquid CO₂ extraction.¹ They contain terpenoids, especially monoterpenes (C10), sesquiterpenes (C15), and diterpenes (C20), along with a variety of aliphatic hydrocarbons (low molecular weight), acids, alcohols, aldehydes, and esters.^2–4 They are characterized by main constituents present in higher concentrations rather than components in trace quantities. For example, EOs of clove contains 85% eugenol, and 10–12% eugenol acetate, and these determine its biological activity.⁵ Due to these versatile compounds EOs possess bactericidal, fungicidal, antioxidant, virucidal, and anticarcinogenic properties. EOs have already been utilized to control bacterial and fungal contaminations.^6–8 Fu and others (2007)⁶ evaluated the bactericidal and fungicidal potential of clove and rosemary EOs alone and in combination. They found significant inhibition of S. epidermidis, E. coli, and C. albicans at MIC values of 0.062–0.500% (v/v), 0.125–1.00% (v/v) for clove and rosemary EOs, respectively. Similarly, other researchers have also used EOs as antibacterial and antifungal agents.^7–13 Gortzi and others (2008)¹⁴ used Myrtus communis extract to inhibit the oxidation of sunflower oil. The results showed considerable reduction in oxidation at 160 ppm, and the antioxidant activity of extracts further improved when encapsulated in liposome. EOs possess volatile constituents which are sensitive to oxygen, light, humidity, and heat. To increase their stability and functional performance EOs can be encapsulated.

Encapsulation has been widely used for protection, target delivery, and enhanced biological functions of bioactive compounds.^15–17 Wang and others (2009)¹⁷ prepared carvacrol loaded microcapsules, designed to target the intestine for enhanced antimicrobial activity and increased bioavailability. They found <20% oil released in stomach and rest was completely released in intestine. Similarly, variety of researchers reported sustained release characteristics of EOs after encapsulation in different matrices.^{15,16,18–20} Encapsulation not only provides controlled release, but also increased the bioavailability of bioactive compounds/drugs. The same trend was observed, when different EOs constituents (peppermint oil, eugenol, carvacrol and thymol) were nanoencapsulated, which resulted in enhanced antimicrobial activity compared to bulk oil.^21,22 To achieve these benefits, EOs have been encapsulated by using various chemical, physicochemical and mechanical procedures. Among these liposomes,¹⁴ molecular inclusion,^23,24 coacervation and complex coacervation,^25,26 spray drying,²⁷ emulsification,^21,28 ionic gelation²⁹ and emulsion extrusion¹⁷ have been used by many researchers to encapsulate EOs. Encapsulation consists of two important things namely core (bioactive) & wall material/emulsifier (that protects bioactive). The latter is of great importance because the stability, release behavior of core (bioactive) depends on its physiochemical nature, and further on type, and parameters of encapsulation technique. Different types of protein,^26,30 polysaccharide^21,31 and synthetic^22,32 emulsifiers/wall materials have been used to encapsulate the EOs/constituents. This article will focus on various procedures employed for EOs encapsulation, benefits after entrapment, uses in food, and their trends.

2. Essential oils and their properties

EOs are complex natural, volatile aromatic compounds, characterized by two or three major components at fairly high concentrations (20–70%) compared to other components present in trace amounts. Biological activities of EOs are mainly due to main components that are present in high concentration. For example, Artemisia alba EO posses camphor 24%, while Mentha piperita contain menthol and menthone 59% and 19%, respectively. Because of these components, EOs have been largely employed for their well-known antibacterial, antifungal, antioxidant, and anticarcinogenic applications. Walsh and others (2003)⁸ reported antimicrobial properties of natural bactericidal compounds eugenol, thymol, triclocarban (TCC), and didecyldimethylammonium chloride (DDDMAC) against E. coli, S. aureus and P. aeruginosa. Eugenol, thymol, and alkyl dimethyl amine oxides (ADMAO) were effective against E. coli, S. aureus, while TCC showed activity only against S. aureus. The observed minimum inhibitory concentration values (MIC) of eugenol, and thymol against E. coli, S. aureus and P. aeruginosa were 0.05, 0.1 and >0.1% (v/v), respectively. Strong inhibitory action of eugenol, carvacrol, thymol, diacetyl, and cinnamic acid against E. sakazakii has also been confirmed.³³ Fu and others (2007)³⁴ suggested inhibition of S. epidermidis, S. aureus, B. subtilis, E. coli, P. vulgaris, P. aeruginosa, C. albicans and A. niger by clove and rosemary EOs alone and in combination. The MIC values for clove & rosemary EOs were in the range of 0.062% to 0.5% (v/v) and 0.125–1% (v/v). Bactericidal action of EOs have also been confirmed by many studies.^9–11,13

EOs possess promising antifungal activity, and have potential to replace synthetic preservatives as revealed by many researchers.^35–41 Bansod and Rai (2008),⁴² suggested fungicidal action of Cymbopogon martini, Eucalyptus globules, and Cinnamomun zylenicum EOs against A. niger, and A. fumigatus. The MIC values were 0.06, 0.12, and 0.12% (v/v), respectively. Amiri and others (2008),⁴³ reported eugenol (2 mg ml⁻¹) induced mycelial growth inhibition of P. vagabunda, P. expansum, Bortrytiscinerea, and M. fructigena. The average growth inhibition varied between 88.6–90% at 4 °C and 72.5–84.4% at 20 °C, respectively. EOs also possess antioxidant activity as evidenced by many studies.^44–50 Viuda-martos and others (2010)⁵¹ used EOs for in vitro evaluation. Among five spice EOs oregano, thyme, rosemary, sage, and clove, clove EO showed strong antioxidant potential as it inhibited (98.74%) DPPH radical. Antioxidant potential of cumin (Cuminum cyminum L.) stem, leaves, and flowers EOs have also been reported. They found that cumin flower acetone extract was more effective antioxidant interms of DPPH radical scavenger, lipid peroxidation inhibitor, and reducing agent with IC₅₀ value 4, 32 and 8 μg ml⁻¹, respectively.⁵²

EOs have been also utilized as potential source of anticarcinogenic agents.^53–60 The EOs from lemon grass (Cymbopogon flexuosus), sage (Salvia officinalis), katafa (Cedrelopsis grevei), and bugleweed (Lycopus lucidus) have been reported to be cytotoxic to human cancer cell lines.^61–63 Sylvestre and others (2006),⁶⁴ found significant tumor growth reduction of human lung carcinoma cell line A-549 and human colon adenocarcinoma cell line DLD-1, when treated with Croton flavens leaf EO. The GI50 values were 27 ± 4 μg ml⁻¹ for A-549 and 28 ± 3 μg ml⁻¹ for DLD-1, respectively. Similarly, Ashour (2008)⁶⁵ reported anticarcinogenic potential of Eucalyptus sideroxylon and Eucalyptus torquata leaf, stem, and flower EOs against human hepatocellular carcinoma cell line (HEPG2), and human breast adenocarcinoma cell line (MCF7). Inspite of all these characteristics, EOs have certain limitations such as low water solubility, high volatility, and strong odor that limit their applications in food and pharmaceutical industry. To overcome such barriers, EOs can be encapsulated to retain their stability, flavor retention, and functional properties.

3. Encapsulation of essential oils (EOs): techniques/strategies

Encapsulation of EOs has been carried out by variety of chemical,¹⁵ physicochemical²⁴ and mechanical procedures.^21,22,28,30 Commonly used procedures to encapsulate EOs are summarized in Fig. 1.

Fig. 1 Showing different processes and materials utilized for encapsulation of essential oils, parameters (outside the boxes) that affect the encapsulated product and its characteristics.

3.1 Chemical procedures

Among chemical approaches liposomes have been widely used for the encapsulation of EOs. Liposomes are normally prepared by mixing lipids in organic solvents and subsequent drying either by rotary evaporator, spray drying or by lyophilization. Phospholipids have typically been used for the preparation of liposomes. For thymol and carvacrol egg L-α phosphatidylcholine & cholesterol were mixed in methanol, solvent removed at 35 °C under nitrogen stream & the lipid film obtained was hydrated to prepare multilamellar vesicles (MLV) from unilamellar vesicle. A total of 1.07 mg carvacrol was added in liposomes and the encapsulated percentage was 4.16% (0.045 mg). The liposomes incorporated carvacrol & thymol showed enhanced antimicrobial activity and its long term retention favored its stability in liposomes as evident from stability study.⁷¹ In another study Myrtus communis extract was incorporated in liposomes prepared by L-α phosphatidylcholine & cholesterol mixed in chloroform–methanol solution to determine the antioxidant activity. Whereas, phosphatidylglycerol was replaced with L-α phosphatidylcholine for antimicrobial activity determination. The liposomes prepared were spherical in shape and the size was in the range of 270–300 nm.¹⁴ Similarly, Sinico and others (2005)³² prepared MLV of A. arborescens EO (2.5 mg ml⁻¹) using soya phosphatidylcholine in chloroform. They reported less incorporation of EO in Brij 30 based vesicles, while soya phospholipid vesicles entrap 60–74% of A. arborescens EO with size in the range of 70–150 nm. The liposomes incorporated A. arborescens oil significantly reduced herpes simplex virus-1 (HSP-1) at 100 μg ml⁻¹ while free/unencapsulated oil at similar dose showed poor activity. This result also confirmed the stability of oil in liposomes with increased bioactivity. In addition to chemical procedures various researchers used physicochemical procedures to encapsulate EOs.^25,26

3.2 Physicochemical procedures

Coacervation is a physico-chemical process that involves phase separation of one or more hydrocolloids from solution and subsequent deposition of newly formed coacervate phase around the active ingredient suspended in the same reaction media. Rosmarinus and thymus EOs were dispersed in 10% gelatin solution prepared at 40 °C, mixture was emulsified using high shear mixer with subsequent addition of sodium sulphate (20% wt/wt) to obtain coacervate phase at 5 °C under continuous stirring for 1 hour. Further, glutaraldehyde (1 mmol per g gelatin) was added at pH 8 under stirring at 750 rpm at 5 °C for 3 hours and finally microparticles were filtered and freeze dried. The microcapsules prepared were spherical with an average diameter of 60 μm and retained 75% of oil in microcapsules. However, significant increase in morality of Indian meal moth (P. interpunctella) was recorded as concentration of microcapsules increased in the diet.²⁶ Similarly, citronella oil was also encapsulated by simple coacervation as described earlier except formaldehyde solution (37% v/v) that was used to rigidize citronella oil entrapped gelatin microcapsules. The microcapsules showed slow release of citronella oil (CO) and after 10 hours 70% was released. The results of this study showed sustained release and protection of oil from environmental factors.³¹ In another study, lavender oil microcapsules were prepared using complex coacervation of collagen hydrolysate (CH), chitosan (C) crosslinked with glutaraldehyde. Briefly, both CH (5 g) and C (2.5 g) were dissolved separately in distilled water (100 ml), heated until transparency achieved. Both CH–C solution (1/0.0, 0.75/0.25, 0.5/0.5 & 0.25/0.75) were mixed under stirring (800 rpm) at 30 °C. After that temperature increased to 42 °C, lavender oil (2–10 ml) was added drop wise and coacervate formed by the addition of sodium sulphate (10% wt/v) at pH 7. Further microcapsules were cross-linked using glutaraldehyde (0.1–0.3 mmol g⁻¹) under stirring at 42 °C for 6–8 hours, temperature reduced to 30 °C and microcapsules washed with 0.1% Tween 80 solution to remove surface oil & finally freeze dried. The microcapsules prepared were spherical and encapsulation efficiency was 36.84–73.73%.⁶⁷ Complex coacervation was also used to prepare camphor oil loaded microcapsules of gelatin blended with gum arabic and further fabricated with polystyrene. The microcapsules showed encapsulation efficiency between 80–100% and particle sizes were 85.7, 167.2 & 294.7 μm respectively.²⁵

Ayala-Zavala and others (2008)²³ prepared cinnamon and garlic oil loaded microcapsules using molecular inclusion/β-cyclodextrin inclusion complex method. Briefly, β-cyclodextrin (50 g) was dissolved in ethanol–water mixture (1 : 2) at 55 °C, after that each EO dissolved in ethanol (10% w/v) was slowly added to warm β-cyclodextrin solution in variable weight ratios (0 : 100, 4 : 96, 8 : 92, 12 : 88 & 16 : 84). Finally, resultant mixture was stirred without heating for 4 h and precipitated oil–β-cyclodextrin microcapsules were filtered and dried in convection oven (50 °C). The microcapsules of both cinnamon & garlic oil showed encapsulation efficiency of 94.82, 93.76%, respectively and were effective against A. alternata fungus. Choi and others (2009)²⁴ prepared eugenol loaded microcapsules using β-cyclodextrin & 2-hydroxyl propyl (2HP) β-cyclodextrin by molecular inclusion method. They reported higher encapsulation efficiency of eugenol–β-cyclodextrin (90.9%) than eugenol–2HP-β-cyclodextrin (89.1%) and confirmed that hydrophilic chain of 2-hydroxyl propyl group in 2HP-β-cyclodextrin was not efficient for the inclusion of lipophilic compounds like eugenol. Another research group encapsulated flax seed oil in β-cyclodextrin inclusion complexes in variable ratios (5 : 95–20 : 80 wt/wt). They obtained maximum load (95.8 mg of oil per g β-cyclodextrin) of flaxseed oil at 20 : 80 ratio and further no change in the composition of oil was observed after inclusion in β-cyclodextrin as evidenced from gas chromatography results. These results showed protection of eugenol and favored its encapsulation in inclusion complexes as suitable approach.⁸¹ PO,¹⁹ lemon oil⁸² in β-cyclodextrin inclusion complexes have also been encapsulated as described earlier. Hill and others (2013)⁸³ prepared β-cyclodextrin inclusion complexes of eugenol, trans-cinnamaldehyde, clove extract, cinnamon bark extract and eugenol–trans-cinnamaldehyde via freeze drying. The particle sizes were in the range of 0.86–2.00 μm, showed spherical morphology with entrapment efficiency of 41.7–84.7%. They confirmed inhibitory action of microcapsules against S. enterica, Typhimurium & L. innocua but unencapsulated/free oil and extracts showed poor inhibition at same concentrations. These results showed better retention and protection of lipophilic compounds in inclusion complexes.

3.3 Mechanical procedures

Among these spray drying is the low cost, commercial process which is mostly used for the encapsulation of EOs. In spray drying, core material is dispersed in polymer solution, and sprayed into a hot air chamber. Arana-Sanchez and others⁷⁴ spray dried OEO emulsion prepared using β-cyclodextrin, fed at room temperature with an inlet air temperature of 105 °C, and pump flow rate was 1.1 ml min⁻¹. Microcapsules showed spherical to ovoid shape with size in the range of 0.71–20 μm, 1.42–28.14 μm, and 1.07–38 μm, respectively. The formulation with bigger size showed increased encapsulation efficiency (81.03%) compared to smaller sized capsules (0.71–20 μm) with 53.90%. Thymol loaded emulsion prepared with whey protein–maltodextrin conjugate was also spray dried with an inlet air temperature of 150 °C, compressed air pressure of 600 kpa, air flow rate of 35 m³ h⁻¹, and feed rate was 6.67 ml min⁻¹. The encapsulation efficiency was 73.8–82.8% at 10% volume fraction of oil phase, but reduced to 67.6% when oil phase fraction increased to 30%. They also reported loss of thymol due to inlet temperature that was equivalent to its vapor pressure (8.0 kpa), and concomitant loss of capsule shape i.e., ruptured wall that happened during spray drying as evident from AFM images.^75,76 Another research group prepared powdered eugenol nano-dispersions by spray drying at 6.67 ml min⁻¹ feed rate and outlet temperature was 80–90 °C. They reported stable and transparent nano-dispersions having small particle size.^75,76 Basil oil emulsion emulsified with gum arabic was spray dried using emulsion feeding rate of 0.7 l h⁻¹, drying temperature of 180 °C, and vapor pressure of 0.4 MPa.⁷⁷ The droplets mean diameter was 1.17–2.87 μm, and oil retention varied from 56.43–88.28%. Baranauskiene and others³⁰ spray dried peppermint oil (15.25% wt/wt) dispersed in wall material solutions of n-octenyl succinic anhydride modified starches. The dispersions were dried in Buchi mini spray drier at inlet air temperature of 200 ± 10 °C, outlet temperature of 120 ± 10 °C, and compressor pressure of 400 mm/H₂O. They also reported increased retention of oil (39.2–97.4%) was associated with small size droplets compared to bigger ones. On the other hand, more oil evaporated/lost during atomization as modified starches take longer time for film formation around droplets during spray drying. Clove extract (2.5 g) emulsion prepared with maltodextrin (12 g) and gum arabic (6 g) was spray dried at inlet & outlet temperature of 150 °C & 86 °C, respectively. The powder particles showed (1–15 μm) shriveled morphology with yield percentage of 62%, when air flow, emulsion feed rate, and atomization pressure were kept at 40 mm, 6.67 ml min⁻¹, −45 mbar, respectively. Rosemary EO emulsified with whey protein isolate, and inulin was fed into spray dryer at 0.9 l h⁻¹ with an inlet temperature of 170 °C to prepare encapsulated powders. The microencapsulation efficiency was 28.97–38.34%, and the retention of oil was more when whey protein, and inulin concentration was 3 : 1 and 1 : 1. However, no increase in oil retention was observed with higher inulin concentration. They reported increased efficiency of EO during spray drying that was dependent on wall material type and its emulsifying capability.⁷⁸ In another study rosemary EO dispersed in gum arabic solution (1 : 4 oil–wall) was spray dried to prepare encapsulated powder.⁷⁹ Spray drying was done at an inlet temperature of 171 °C, feed rate of 0.92 l h⁻¹, and atomizing air pressure was kept at 40 l h⁻¹. They reported efficient drying at higher inlet temperature, and increased wall material concentration, which resulted in more powder recovery. They observed loss of oil content from particles due to volatilization during drying by atomization because of delay in the formation of semi permeable membrane when carrier concentration was low. However, maximum oil retention was 36.95% when wall material (gum arabic) concentration was 19.3%. Similarly, Beirao-da-costa and others (2013)¹⁸ spray dried OEO emulsion, the capsules prepared were in the range of 3–4.5 μm and some capsules represent ruptured wall due to high inlet temperature. Najafi and others (2011)⁸⁰ compared spray and freeze drying method for the encapsulation of cardamom oil using HI CAP 100 and skim milk powder as wall materials. The spray drying retained more volatiles (91–94%) than freeze drying (84–86%) and microcapsules prepared were of high quality compared to freeze dried microcapsules. However, in case of skim milk powder, microcapsules particle size (13.97–19.91 μm) was smaller than HI CAP 100 (15.41–21.87 μm) but, former released volatile contents much faster during drying than later one. Therefore, spray drying was recommended as suitable method for the encapsulation of EOs.

Similarly, various researchers used high energy emulsification approach for the generation of EOs loaded nanoemulsions has been carried out by many researchers. For example, Liang and others (2012)²¹ for the preparation of PO loaded nanoemulsion, blended PO with medium chain triglyceride (MCT), and mixed with purity gum 2000 solution 12% (w/w) to prepare coarse emulsion using Ultra Turax (a high speed blender). Finally, coarse emulsions passed through high pressure homogenizer at 150Mpa, and 5 processing cycles, showed particle size <200 nm and were effective against S. aureus & L. monocytogenes till 24 hours at 0.25% (v/v) concentration. They confirmed similar composition of PO components before and after encapsulation. The long term inhibition of bacterial growth by PO nanoemulsions and their better protection promoted high energy emulsification a suitable method for encapsulation of EOs. Similarly, another research group prepared D-limonene, cinnamaldehyde and carvacrol loaded nanoemulsions (130–293 nm) using soy lecithin (3%), pea proteins (3%), sugar ester (1%) and glycerol monooleate with Tween 20 (0.5 : 0.5%) as emulsifying agents. The formulations with glycerol monooleate with Tween 20 showed prolonged bactericidal action against S. cerevisiae, E. coli & L. delbrueckii because of more availability of antimicrobial compound by this emulsifier compared to other emulsifiers. D-Limonene & terpenes mixture from Melaleuca alternifolia based nanoemulsions (74–365 nm) were also prepared using Tween 20 & glycerol monooleate, soy lecithin and cleargum individually as emulsifiers. Soy lecithin based emulsions formulations of D-limonene & terpene mixture were efficient and delayed growth of L. delbrueckii for 5 days in orange & 2 days in pear juice compared to control when added at concentration of 1 g l⁻¹.²⁸ Terjung and others (2012)²² also reported preparation of eugenol and carvacrol loaded nanoemulsions using high pressure homogenization technique. They observed antimicrobial activity of eugenol and carvacrol loaded variable particle sized emulsions (80, 200, 1000 and 3000 nm) against E. coli & L. innocua. They reported complete killing of bacterial cells when treated with bigger particle size (3000 nm) due to more concentration of antimicrobial compound and vice versa happens with smaller particle size (80 nm). Rosemary EO loaded emulsions prepared by high pressure homogenization was stable for 50 days at ambient conditions.⁸⁴ Chang and others (2012)⁸⁵ prepared thyme oil (10% v/v) loaded nanoemulsions with average particle size of (160, 170 nm) using high pressure homogenization. They reported decreased antimicrobial efficacy because of increased concentration of ripening inhibitors type (corn oil or medium chain triglyceride MCT oil) in lipid phase and when 70% ripening inhibitor was added in lipid phase the minimum inhibitory concentration against Zygosaccharomyces bailii containing corn & MCT oil were 750 and 3000 μg ml⁻¹, respectively. These results showed physical stability of thyme oil in nanoemulsions droplets but decreased antimicrobial activity was due to partition coefficient. Vegetable,⁸⁶ soybean,⁸⁷ corn or octadecane,⁸⁸ castor,⁸⁹ citronella, basal & vetiver oil⁹⁰ based nanoemulsions have also been prepared by using high pressure homogenization technique.

3.4 Other encapsulation methods

Eugenol and carvacrol grafted chitosan nanoparticles were prepared via Schiff base reaction.²⁹ Briefly, chitosan nanoparticles (CH-NPs) were prepared using ionic gelation method. For this 0.5% w/v chitosan in 1% v/v acetic acid at pH 4.1, TTP solution (2.5 mg ml⁻¹) was added drop wise in chitosan solution under stirring at 10 000 rpm, separated by centrifugation and were freeze dried. Further, eugenol and carvacrol aldehydes were grafted on CH-NPs via Schiff base reaction. A 30 ml methanol, 100 mg CH-NPs with excess of eugenol & carvacrol aldehydes and reaction mixture was refluxed for 48 hours. Finally, eugenol and carvacrol grafted nanoparticles were separated by centrifugation and vacuum dried at 42 °C for 12 hours. Among eugenol (235 nm) & carvacrol (260 nm) grafted CH-NPs, eugenol grafted nanoparticles showed antioxidant activity at lower EC50 (2.6 mg ml⁻¹) value than carvacrol grafted nanoparticles (>4.0 mg ml⁻¹). On the other hand, eugenol grafted CH-NPs showed higher antimicrobial efficacy than carvacrol grafted CH-NPs. In another research, OEO was encapsulated in chitosan nanoparticles by two step process i.e., oil in water o/w emulsion and ionic gelation of chitosan with sodium tripolyphosphate (TPP). In this chitosan (1% w/v) in acetic acid (1% v/v) solution was stirred and Tween 80 was added to get homogenous mixture under stirring at 42 °C for 2 hours. After that OEO (0.04, 0.08, 0.16 & 0.32 g) was dissolved in CH₂Cl₂ (4 ml) and dropped in chitosan solution during homogenization to prepare oil in water emulsion. TTP solution was then added drop wise into agitated emulsions till 40 minutes. Finally, nanoparticles were centrifuged (9000g), suspension was sonicated in ice bath (0.7 s working & 0.3 s rest) and freeze dried at −35 °C for 72 hours. The prepared nanoparticles were spherical (40–80 nm) and the encapsulation efficiency was 21–47%.²⁰ In the orifice method citronella oil loaded microcapsules were prepared as follows, 0.5 wt% yellow dye was added in citronella oil, citronella oil (2 ml) was added in chitosan solution (0.2, 0.5, 1 & 1.5%) under stirring. After that 0.1–1.5 wt% NaOH was added slowly and the prepared microcapsules were centrifuged to remove excessive material and kept in 5 wt% natural coconut oil for 10 days. Finally microcapsules were vacuum oven dried at 30 °C for overnight. They concluded that encapsulation efficiency affected by chitosan concentration and when these were 0.5, 1 & 1.5% the encapsulation efficiencies were 98.2, 95.8 and 94.7%, respectively.¹⁵ Carvacrol loaded microcapsules were also prepared by using Ca-alginate hydrogel using emulsion extrusion method. For this alginate (20 g l⁻¹) in deionized water, carvacrol & Tween 80 were added to a final concentration of 200 and 0.5 g l⁻¹ respectively. The mixture was emulsified using blender, emulsion was extruded into collecting water bath using 20 g l⁻¹ CaCl₂ in encapsulator with 500 μm nozzle. The microcapsules kept for 30 minutes in CaCl₂ for hardening, washed and air dried for 30 h at 22 °C. The microcapsules showed strong antimicrobial activity against E. coli in vitro and during in vitro digestion only 20% carvacrol released in gastric fluid while remaining in intestinal fluid after 6 hours of incubation.¹⁷

Co-crystallization is another method used for the encapsulation of orange peel oil. In this process crystal structure of sucrose is modified from a perfect crystal to conglomerate. The resultant structure has porous configuration which can accept the addition of second ingredient. For orange peel oil sucrose syrup of 70° Brix was concentrated to 95° Brix under hot magnetic stirring, orange peel oil was added at variable ratios (100, 150, 170, 200 and 250 g oil per kg sucrose) using shear mixer till crystallization achieved. When crystallization started, heating was stopped and heat of crystallization eliminated water to make granular product. The encapsulation efficiency of sucrose syrup was >90% and oxidized flavor were observed in formulations that have antioxidants prior to co-crystallization. This method is rarely used for the encapsulation of food ingredients due to lack of versatility and health concerns related to sucrose.⁹⁰ In the emulsion evaporation method.⁹¹ Poly(DL-lactide-co-glycolide) PLGA (50 mg) in dichloromethane (2 ml) with trans-cinnamaldehyde or eugenol (16% w/w) was prepared. After that aqueous phase containing poly vinyl alcohol (PVA: 0.3% w/v) mixed with organic phase and oil in water (O/W) emulsion was prepared using homogenization. Further, emulsions were sonicated at 2 °C for 10 minutes at 70 W energy output and organic phase evaporated using rotary evaporator. Finally, nanoparticles loaded with eugenol and trans-cinnamaldehyde were purified through ultrafiltration and freeze dried. The nanoparticles were in the size range of 200 nm with encapsulation efficiency of both compounds ranging from 92–98% and were effective against Salmonella and Listeria spp. These results favored encapsulation of EO using this approach because of its ability to protect and increased the shelf stability of encapsulated compound. Zein nanospheres loaded with thyme, cassia & oregano EO were prepared using phase separation method. For this each oil (250 mg) with zein (1 g) dissolved in ethanol (85%), solution was rapidly mixed with 0.01% silicone fluid till single phase formed and the opaque solution containing oil was lyophilized overnight. The nanospheres prepared were of irregular shape and the encapsulation efficiencies of oils were in the range of 65–75%. This technique is rapid, enabled protection of EOs and controlled its release in stomach, small intestine & large intestine.⁹² Emulsification external gelation method was used for the preparation of turmeric oil loaded nanocapsules.⁹³ Briefly, turmeric oil solution in ethanol (20 mg ml⁻¹) was mixed with alginate solution (0.6 mg ml⁻¹) containing Tween 20 (1%/v) to prepare o/w emulsions. For gelification of oil droplets emulsions were combined with 0.67 mg ml⁻¹ CaCl₂ solution under stirring and another polymer chitosan (0–0.6 mg ml⁻¹ acetic acid) was subsequently added, oil loaded alginate-chitosan nanocapsules were equilibrated overnight and solvent was removed using rotary evaporator. The nanocapsules prepared were in the size range of 162–667 nm and were stable for 120 days under room temperature and 4 °C. Yeast cell has been successfully used for the encapsulation of limonene. To carry out this, yeast cells cytoplasmic material was removed by plasmolyser, cells were washed with water, centrifuged and spray dried. Finally, yeast cells (80 g) were infused with limonene solution (9.1% w/w). The oil was successfully encapsulated as evident from transmission electron microscope (TEM) images and proved to be a suitable approach for the encapsulation of water soluble bioactives.⁶⁸

3.5 Emulsifiers/wall materials used for essential oils

A variety of carbohydrate, proteins, and gums based emulsifiers or wall materials have been used for the encapsulation of EOs (Table 1). Among carbohydrates, β-cyclodextrin was used to prepare OEO loaded emulsions that were spray dried to obtain stable microcapsules having size in the range of 1.07–38 μm.⁷⁴ Similarly a variety of researchers used β-cyclodextrin to prepare inclusion complex around hydrophobic EOs that protect them against oxidation, heat damage and increased their antibacterial efficacy for a longer time period.^24,81–83 Beirao-da-costa and others (2013)¹⁸ used inulin as wall material for the encapsulation of OEO, microcapsules prepared were spherical (3–4.5 μm) and enabled sustained release of oil till 200 minutes. N-Octenyl succinic anhydride modified (HI CAP 100, Purity Gum 2000, Capsul & N-lock) and hydrolyzed starches (EnCapsul 855) used for the encapsulation of PO. Modified starches showed increased retention, better stability of oil in spray dried powder³⁰ and nanoemulsions²¹ (<200 nm). Eugenol and Carvacrol were encapsulated by chitosan and tripoly phosphate (TPP) ionic cross linking and the nanoparticles produced (217, 235 & 260 nm) showed better retention of compounds incorporated. Further, nanoparticles were stable for extended time period before and after incorporation of EO components.²⁹ Another research group encapsulated orange peel oil using sucrose syrup, the granular co-crystallizate showed 90% encapsulation efficiency and showed better protection against oxidation.⁹⁰ In case of proteins, gelatin was used to encapsulate citronella, thyme and rosemary oil, microcapsules (60 μm) prepared were spherical and exhibited controlled release of oil (10 hours).^26,31 Parris and others (2005)⁹² encapsulated thyme, oregano and cassia oil using zein, nanoparticles prepared had an average diameter of 100 nm and showed oil yield from 65–75%. They reported less release of oil from zein nanospheres during in vitro digestion in stomach, slow release in small intestine and rapid release in large intestine. Soy lecithin (3%) and pea proteins (3%) have been used as emulsifiers for the encapsulation of limonene, trans-cinnamaldehyde and Melaleuca alternifolia terpenes. The nanoemulsions prepared were stable in terms of particle size and had particle size in the range of 184–239 nm.²⁸ Corn oil nanoemulsions have also been prepared using sodium caseinate and β-lactoglobulin and were physically stable having particle diameter 150 nm.⁸⁹ Among gums acacia, gum arabic has been used for the encapsulation of ginger, basil and rosemary EO.^77,94 Gum arabic (1 : 4 w/w oil to wall ratio) emulsified emulsions of both oils exhibited low viscosity and particles prepared after spray drying showed retention of basil and rosemary oil (56.43–90.6% : 7.15–47.57%), respectively. They also reported more loss of EO from larger droplets compared to smaller ones during spray drying due to longer time of film formation around droplets. Najafi and others (2011)⁸⁰ compared protein (skim milk powder) and carbohydrate (HI CAP 100) based wall material for the encapsulation of cardamom oil. They reported narrow droplets size (13.97–19.91 μm) prepared from HI CAP 100 compared to skim milk powder (15.41–21.87 μm) and subsequently more loss of volatiles occurred in skim milk encapsulated powders (Table 2).

Table 1 Summary of different encapsulated EOs, encapsulation techniques, emulsifiers used and characteristics of EOs after encapsulation

Essential oil	Reference	Technique used	Emulsifier type/wall material	Characteristics	Encapsulation efficiency (%)	Level/quantity	Particle size
Ginger oil	Kadam et al. 2011	Spray drying	Acacia gum	—	—	20 gram	55.9–32.1 μm
Oregano oil	Arana-sanchez et al. 2010	Spray drying	β-Cyclodextrin	Spherical capsules with no dent	75–85	14.3% wt/wt	1.07–38 μm
Oregano oil	Beirao-da-costa et al. 2013	Spray drying	Inulin	Spherical capsules	—	—	3–4.5 μm
Rosemary oil	Fernandes et al. 2013	Spray drying	Gum arabic	Spherical with irregular surface but no dent	47.57	1 : 4 oil : wall ratio	13.6 μm
Rosemary oil	Fernandes et al. 2014	Spray drying	Whey & inulin	Amorphous	37.7 ± 0.2	25%	11.5–11.0 μm
Clove extract	Chatterjee & Bhattacharjee 2013	Spray drying	Maltodextrin & gum arabic (12 : 6 g)	Shriveled	65	2.5 g	1–15 μm
Peppermint oil	Baranauskiene et al. 2007	Spray drying	HI cap, N-lock & capsule	Spherical	88.8–99.7	15.25% wt/wt	—
Basil oil	Garcia et al. 2008	Spray drying	Gum arabic	—	90.61	10–25% wt/wt	1.28 μm
Eugenol oil	Shah et al. 2012	Spray drying/emulsion evaporation	Maltodextrin–WPI maillard conjugates	—	7.3%	10% v/v	127 nm
Thymol oil	Shah et al. 2012	Spray drying/emulsion evaporation	Maltodextrin–WPI maillard conjugates	Spherical shell	51.4	10% v/v	1–5 μm
Cardamom oil	Najafi et al. 2011	Spray drying	Skim milk powder and HI CAP 100	Spherical	75–86	5% wt/wt	13.97–21.87 μm
Origanum dictamnus oil	Liolis et al. 2009	Liposome	—	—	4.16	—	—
A. arborescens oil	Sinico et al. 2005	Liposome	Brij 30	—	60–74	12.5 mg ml^−1	70–150 nm
Myrtus extract	Gortzi et al. 2008	Liposome	—	Spherical	—	—	285 nm
Cinnamon & garlic oil	Ayala-Zavala et al. 2008	Molecular inclusion	β-Cyclodextrin	—	93.76, 94.82	0–9.54 g	—
Eugenol	Choi et al. 2009	Molecular inclusion	β-Cyclodextrin, hydroxy propyl (HP)–β-cyclodextrin, poly caprolactone & pluronic 80	Hexagonal & spherical	90.9, 89.1 & 100	1 : 1 ratio	319.7 ± 5.1, 312 ± 5.3, 321.2 ± 1.5
Flax seed oil	Elkader and Aggor 2013	Molecular inclusion	β-Cyclodextrin	—	94.77	5/95–20/80 ratio	—
Lemon oil	Padukka et al. 2000	Molecular inclusion	β-Cyclodextrin	—	—	12%	—
Eugenol, trans-cinnamaldehyde, cinnamon bark and clove bud extract	Hill et al. 2013	Molecular inclusion	β-Cyclodextrin	Spherical, smooth & agglomerated	41.7–84.7	1 : 1 ratio	2.006, 0.860, 1.229, 1.39 μm
Peppermint oil	Ciobanu et al. 2013	Molecular inclusion	α, β, γ & HP–β-cyclodextrin	—	—	1 : 1 ratio	—
Lavender oil	Ocak B 2012	Coacervation	Collagen hydrolysate, chitosan & glutaraldehyde	Spherical	60.09	2–10%	50 μm
Rosmarinus and thymus vulgaris oil	Passino et al. 2004	Coacervation	Gelatin	—	98	2 : 1 ratio	60 μm
Citronella oil	Solomon et al. 2012	Coacervation	Gelatin	—	60	—	—
Camphor oil	Chang et al. 2003	Complex coacervation	Gelatin & gum arabic	—	99.6	20–50 ml	85.7, 167.2 & 294 μm
Lemon grass oil	Leimann et al. 2009	Coacervation	SDS
Peppermint oil	Rong et al. 2012	Emulsification	Purity gum 2000	Spherical	—	12%	<200 nm
D. limonene & trans-cinnamaldehyde	Donsi et al. 2012	Emulsification	Lecithin, pea proteins, sucrester and combination of glycerol monooleate with Tween 20	—	—	2%	<250 nm
D. limonene & melaleuca alternifolia terpenes	Donsi et al. 2011	Emulsification	Soy lecithin, cleargum & Tween 20 and glycerol monooleate	Spherical	—	50 g kg^−1	74.4–365.7 nm
Eugenol & carvacrol	Terjung et al. 2012	Emulsification	Tween 80	—	—	5–50 wt%	<100 nm
Corn oil	Qian & McClements 2011	Emulsification	SDS, Tween 20, sodium caseinate & β-lactoglobulin	—	—	5 wt%	60 & 150 nm
Rosemary oil	Rodriguez-rozo et al. 2012	Emulsification	Span 20, Tween 20 &80	—	—	50 ml	2–5 μm
Thymol & carvacrol	Guarda et al. 2008	Emulsification	Gum arabic & Tween 20	—	—	1–10%	4.5 μm
Soybean oil	Hamouda & Baker 2000	Emulsification	Triton X-100	—	—	64%	400–800 nm
Thyme oil	Chang et al. 2006	Emulsification	Tween 80	—	—	10% wt/wt	<200 nm
Citronella, basil & vetiver oil	Nuchuchua et al. 2009	Emulsification	Montanov 82	—	—	20% wt/wt	150–220 nm
Eugenol & carvacrol	Gaysinsky et al. 2005	Emulsification	Surfynol	—	—	0.85% eugenol, 0.55% carvacrol	5.30–16.45 nm
Eugenol & carvacrol	Chen et al. 2009	Ionic gelation	Chitosan & TPP	—	—	70%	217, 260 & 235 nm
Oregano oil	Hosseini et al. 2013	Nanoemulsion & ionic gelation	Tween 80, chitosan & TPP	Spherical	21–47	0.04–0.32 g	40–80 nm
Citronella oil	Hsieh et al. 2006	Orifice method	Chitosan	Spherical with no dent	94.7–98.2	2 ml	11, 131 & 225 μm
Carvacrol	Wang et al. 2009	Emulsion extrusion	Sodium alginate	Spherical	80	20% v/v	0.7–1.2 mm
Neem oil	Jerobin et al. 2012	Nanoemulsion coated beads	Tween 20 & sodium alginate	Spherical with dent	66.5–81.8	1–8 ml	1.28–1.49 mm
trans-Cinnamaldehyde & eugenol	Gomes et al. 2011	Emulsion evaporation	PVA	Spherical	92.56 & 98.27	16% wt/wt	173–79 nm
Rosemary & thyme oil	Moretti et al. 2002	Phase separation	Gelatin & Tween 80	Spherical and agglomerated	99.2 & 98.6	0.25–1% wt/wt	58.5 & 56.7 μm
Oregano, thyme & cassia oil	Parris et al. 2005	Phase separation	Zein	Irregular	65–75	10–20%	20–120 nm
Orange peel oil	Beristain et al. 1996	Cocrystallization	Sucrose syrup	Granular	94.6	100–250 oil per kg sugar	—
Turmeric oil	Lertsutthiwong et al. 2009	Emulsification gelification and solvent removal	Tween 80, chitosan & alginate	—	68.5	20 mg ml^−1	162–667 nm
Limonene	Dardelle et al. 2007	Yeast as delivery vehicle	—	—	—	9.1% wt/wt	—

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Table 2 Reported applications of encapsulated essential oils

Essential oil	In vitro/vivo	Application	Reference
Peppermint oil	In vitro	L. monocytogenes & S. aureus	Liang and others 2012
Eugenol	In vitro	E. coli & L. innocua	Terjung et al. 2012
Terpenes mixture from M. alternifolia	In vivo (orange & pear juice)	L. delbrueckii	Donsi et al. 2011
Carvacrol, D-limonene & trans-cinnamaldehyde	In vitro	E. coli, S. cerevisiae & L. delbrueckii	Donsi et al. 2012
Oregano oil	In vitro	E. coli, S. aureus & P. aeruginosa and antioxidant activity	Arana-sanchez and others 2010
Eugenol	In vivo (bovine milk)	E. coli	Shah et al. 2012
Eugenol & trans-cinnamaldehyde	In vitro	Salmonella & Listeria spp S. aureus, epidermidis	Gomes et al. 2011
Thymol & carvacrol	In vitro	Mutans, viridians, E. coli, E. cloacae, K. pneumoniae, P. aeruginosa, C. albicans, glabrata, tropicalis and L. monocytogene	Liolis et al. 2009
Eugenol & carvacrol	In vitro	E. coli & S. aureus	Chen et al. 2009
Thymol & carvacrol	In vitro	E. coli, S. aureus, L. monocytogene, S. cerevisiae & A. niger Zygosaccharomyces bailii	Guarda et al.
Thyme oil	In vitro		Chang et al. 2006
Myrtus communis extract	In vitro	S. aureus, epidermidis, mutans, viridians, E. coli, E. cloacae, K. pneumoniae, P. aeruginosa, C. albicans, glabrata, tropicalis and L. monocytogene	Gortzi et al. 2008
Citronella, basil & vetiver	In vitro	Aedes aegypti	Nuchuchua et al. 2009
Carvacrol	In vitro	E. coli	Wang et al. 2009
Cinnamon & garlic oil	In vitro	A. alternata (fungus)	Ayala-Zavala et al. 2008
Clove extract	In vivo (soybean oil)	Antioxidant	Chatterjee and Bhattacharjee 2013
Lemongrass oil	In vitro	S. aureus & E. coli	Leimann et al.
Thyme & rosemary	In vitro	L. dispar (forest pest)	Moretti et al. 2002
Thymus & rosemary	In vitro	P. interpunctella	Passino et al. 2004

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Inspite of individual use of protein/carbohydrate based emulsifiers or wall materials, mixtures of carbohydrate : protein, carbohydrate : gums and protein : gums have also been used to encapsulate EOs for better retention and protection. Fernandes and others (2014)⁷⁹ used whey protein isolate (WPI)–inulin blends for the encapsulation of rosemary EO. They reported better protection of oil particles in powder against oxidation due to low moisture content as WPI concentration in blend increased. WPI : inulin blends (1 : 1 & 3 : 1) were the effective carriers for the entrapment of rosemary oil and had particle size in the range of 11.5–11.9 μm. Similarly, WPI : maltodextrin maillard conjugates (1%/v) were used for the encapsulation of thymol oil,⁷⁵ microcapsules prepared were spherical having size in the range of 1–5 μm and oil retention increased as concentration of maillard conjugates increased from 1–11.1% w/v. Chatterjee and Bhattacharjee (2013)²⁷ used maltodextrin : gum arabic (12 : 6 g) mixture for the encapsulation of eugenol rich clove extract, microcapsules (1–15 μm) showed shrivelled morphology and maximum retention of eugenol was 65%. They reported increased percentage of carbohydrate (4.8) in relation to gum (2.4) that was effective in increasing the retention of oil.

In addition to above mentioned food grade emulsifiers/wall materials, variety of synthetic emulsifiers have also been used for the encapsulation of EOs. Sinico and others (2005)³² used Brij 30 (5.75 mg ml⁻¹) as surfactant for the synthesis of A. arborescens oil loaded liposomes. They reported lower retention of oil (66.09%) in niosomal bilayers prepared with Brij 30 compared to phospholipid based liposome (74.15%). However, liposomes and niosomes were stable for one year when stored at 4–5 °C. The agglomeration of lemon grass oil microcapsules was overcome by the addition of 0.4 wt% sodium dodecyl sulphate (SDS) due to repulsion mechanism as it is ionic surfactant.⁵⁵ Donsi and others (2011)²⁸ prepared D-limonene & terpenes mixture from Melaleuca alternifolia based nanoemulsions using natural (soy lecithin, cleargum) and synthetic emulsifiers (glycerol monooleate with Tween 20). Nanoemulsions of terpenes, D-limonene prepared with soy lecithin had particle size (74 & 240 nm) while Tween 20/glycerol monooleate based emulsions droplet diameter was (130–155 nm). Moreover, all emulsions formulations were physically stable for a period of 4 weeks, showed no visible creaming and consistent particle diameter. Corn oil nanoemulsions prepared with synthetic emulsifiers (Tween 20 & SDS) showed smaller droplet size 60 nm while in case of sodium caseinate & β-lactoglobulin the particle size raised to 150 nm. However, later nanoemulsions were physically more stable than former ones.⁸⁸ Similarly Span 60,⁸⁴ Tween 80^84,22 & Tween 20, Montanov 82,⁸⁹ Triton X-100 (ref. 87) have been used to encapsulate EOs.

3.6 Encapsulation and its benefits

3.6.1 Encapsulation of essential oils (EOs) for controlled release. Encapsulation represents a viable and efficient approach to increase the physical stability of EOs, protection from evaporation, and because of narrow size range enables controlled release & enhanced bioactivity. Chang and Dobashi (2003)⁶⁶ prepared eucalyptus oil loaded alginate, and calcium chloride complex capsules of 1–2.5 mm, using interfacial insolubilization reaction. They showed controlled, slow release of eucalyptus oil after encapsulation as analyzed by using incubation and finger crash force technique. Briefly, capsules were placed in between the fingers and crashed; the crashing force was standardized by taking 10 volunteers (Chinese students) index finger readings. The force was measured, and determined as 5.4 × 10⁶ dyne. In another study, camphor oil was encapsulated to attain its sustained release, using complex coacervation method. Microcapsules of variable sizes (294.7 ± 14.2, 167.2 ± 11.2, 85.7 ± 8.7 μm) at different homogenization speed 500, 1000 & 2000 (rpm) were prepared. They observed that microcapsules prepared at 500 rpm & 0.75 oil/wall ratio showed 99.6% (wt/wt) encapsulation efficiency. Moreover, they reported that camphor oil sustained release properties were directly dependent on cross linking agent called polystyrene.²⁵ Hosseini and others (2013)²⁰ reported controlled release pattern of oregano essential oil (OEO) loaded nanoparticles, prepared by using two step method that involves oil in water emulsion, and then ionic gelation of chitosan and tripolyphosphate (TPP). They observed rapid release of oil from smaller nanoparticles, and 82% of OEO released during 3 hours even though at low concentration of OEO (0.1% w/w chitosan), but its release was slowed down at higher chitosan concentration (0.8% w/w), and reduced from 82% to 12% during 3 hours. Similarly, contrasting results were obtained when citronella oil was microencapsulated using chitosan, NaOH, and coconut oil as cosurfactant. They observed slow, and sustained release in microcapsules (225 ± 24 μm) that have larger size, and higher concentration of chitosan than smaller (131 ± 20, 11 ± 3 μm) ones.¹⁵ Moreover, slow release of neem oil (azadirachtin) loaded nanoemulsions based beads coated with gum arabic, and polyethylene glycol (PEG), having particle size in the range of 1.28 ± 0.006–1.49 ± 0.004 mm have also been reported. They showed increased release of azadirachtin (44.2, 66.8, 79.4 & 100%) from nanoemulsions after 6, 12, 18 and 24 hours. However, significant decrease in azadirachtin release from nanoemulsions (31.6, 40.45, 51.6 & 70.6% and 41.6, 54.2, 66.8, 80.6%, respectively) was attained after being coated with gum arabic, and PEG. They concluded that gum arabic coating on nanoemulsions was better for controlled and slow release of azadirachtin than PEG.⁶⁷ Beirao-Da-Costa and others (2013)¹⁸ also reported controlled release of OEO when encapsulated using inulin. The microcapsules prepared (3–4.5 μm) showed increased release of oil during first 75 min but, later it became slow till 200 min and after that lag phase appeared. Release of lavender oil was also controlled by encapsulating it in collagen hydrolysate, chitosan, and glutaraldehyde as cross linker. They observed slow release of lavender oil from microcapsules by increasing concentration of chitosan, and cross linker. Protection of aroma compounds, and controlled release, and their increased bioavailability have also been confirmed by other researchers.^19,68,69

3.6.2 Encapsulation of essential oils (EOs) for increased bioavailability. In addition to controlled release characteristics, various researchers have also reported increased bioavailability of EOs after encapsulation in variable matrices. For example, Liang and others (2012)²¹ encapsulated peppermint oil (PO) in starch based nanoemulsions to increase its stability, and bioavailability. The nanoemulsions with particle size of 200 nm showed enhanced bactericidal activity against L. monocytogenes, and S. aureus compared to bulk PO. This was attributed to greater solubility, and more availability due to subcellular size of particles. Similarly, increased bactericidal action due to more absorption of D. limonene, carvacrol, eugenol, and cinnamaldehyde by L. delbrueckii, S. cerevisiae, and E. coli cells from nanoemulsion has also been reported.^22,28,70 Increased absorption of EOs after encapsulation lowered the amount of oil required to kill microorganisms. Meanwhile, increased bactericidal action of OEO attained at 25.0 × 10⁻⁸ g ml⁻¹, when incorporated in liposome and this quantity was equivalent to 6.0 × 10⁻³ g ml⁻¹ unencapsulated OEO. These results showed enhanced batericidal activity of OEO in liposomes against human pathogenic bacteria (S. aureus, S. epidermidid, S. mutans, S. viridans, P. aeruginosa, E. coli, E. cloacae, K. pneumonia), fungi (C. albicans, C. tropicalis, C. glabrata) and food borne pathogen (L. monocytogenes) because of increased uptake/bioavailability by living cells.⁷¹ Increased absorption of EOs in nanoemulsions by bacterial cells S. aureus, B. cereus, E. coli and P. mirabilis has also been reported by other researchers.^72,73

In addition to increased absorption of EOs by bacterial cells, A. arborescens EO loaded unilamellar (78 ± 11, 104 ± 19, 123 ± 21 nm) and multilamellar liposomes (232 ± 25, 252 ± 29 304 ± 21 nm) also showed increased antiviral potential against herpes simplex 1 (HSP) virus due to increased absorption after encapsulation. They confirmed poor antiviral activity of unencapsulated, and unilamellar liposomes incorporated A. arborescens EO at 100 μg ml⁻¹. However, antiviral activity significantly increased when it was incorporated in multilamellar liposomes and EC50 value reduced to 18.5 μg ml⁻¹.³²

3.6.3 Encapsulation of essential oils (EOs) for increased stability. Encapsulation not only provides controlled release, and improved bioaccessibility of EOs but, also increased their stability^21,22,28,70 as they are susceptible to conversion and degradation after exposure to environmental stresses⁹⁷ as shown in Fig. 2. Various researchers reported increased bioactivities (antimicrobial & antiproliferative) of EOs in encapsulation matrix compared to free oil even at same or lower concentration, that suggests their resistance against conversion (oxidation, isomerization, polymerization, thermal rearrangements etc.) and degradation.^17,22,70 For example, Liang and others²¹ prepared peppermint oil (PO) loaded nanoemulsions, and observed decrease (5%) in main constituent of PO (menthol) after quantification using GC-MS compared to PO. Inspite, of decrease in main constituent of PO, the nanoemulsions showed enhanced and long term bactericidal growth inhibition against L. monocytogenes and S. aureus compared to free PO even at same MIC value. These results suggest the better stability of EOs after encapsulation. Similarly, other researchers^28,98 also reported greater bactericidal activities of EOs in nanoemulsions based encapsulation system, even after minimal losses of their constituents during processing. On the other hand, carvacrol loaded calcium alginate microcapsules showed better stability, when passed through gastrointestinal digestion model. Further, retention of carvacrol antimicrobial activity against E. coli after intestinal digestion of microcapsules suggests encapsulation being a suitable approach to prevent EOs from degradation and conversion.¹⁷ Similarly, Ocimum basicilicum and Origanum vulgare EOs showed better stability against degradative action of oxygen and temperature, when encapsulated in β-cyclodextrin inclusion complex and microparticles.^99–101 Moreover, the better retention, and increased protection of EOs in colloidal matrix depends on the type, and parameters of technique/procedure used that ultimately affect the bioavailability, and controlled release of active compound.

Fig. 2 Possible conversion reactions in essential oils (Redrawn from Turek & Stintzing, 2013).

4. Encapsulated essential oils applications

Encapsulated EOs have been used in vitro and in vivo applications for food by many researchers. Liang and others (2012)²¹ used PO loaded nanoemulsions (0.25% v/v) to inhibit the growth of food borne pathogens L. monocytogenes and S. aureus. They observed long term inhibition of bacterial growth when treated with PO nanoemulsions even though MIC values of both bulk oil and PO nanoemulsions were same. Similarly, eugenol & carvacrol loaded nanoemulsions (800 ppm) were also used to inhibit the growth of E. coli & L. innocua and strong inhibition occurred with emulsions having droplet size 3000 nm. Improved bactericidal action (E. coli, L. delbrueckii & S. cerevisiae) of carvacrol, cinnamaldehyde and D-limonene nanoemulsions has also been reported.²⁸ In vitro application of OEO loaded microcapsules against E. coli (0.20–0.05), S. aureus (0.10–0.05) and P. aeruginosa (0.20–0.10) decreased minimum bactericidal concentration of OEO two to fourfold compare to pure oil. They also reported four-eightfold increase in antiradical activity after encapsulation.⁷⁴ Similarly, increased antibacterial potential was also observed when eugenol and carvacrol grafted nanoparticles were used against E. coli & S. aureus.²⁹ Growth of food borne pathogens (Salmonella & Listeria spp) was also reduced to a greater extent when exposed to eugenol and trans-cinnamaldehyde loaded nanoparticles (10–20 mg ml⁻¹).⁹¹ OEO loaded liposomes caused growth inhibition of gram positive (S. aureus, epidermidis, mutans & viridians), gram negative (E. coli, E. cloacae, K. pneumoniae & P. aeruginosa), three human fungal pathogens (C. albicans, glabrata & tropicalis) and L. monocytogene at concentration 25.0 × 10⁻⁸ g ml⁻¹ that was equivalent to unencapsulated 6 × 10⁻³ g ml⁻¹.⁷¹ The results of this study showed encapsulated EOs are better to use in food and other applications to overcome the challenge of sensory attributes variation that happened due to the use of higher EO concentrations. In addition to above mention in vitro applications encapsulated EOs have also been used in vivo as natural preservative to increase the shelf life. Donsi and others (2011)²⁸ used Melaleuca alternifolia loaded nanoemulsions in orange and pear juices to extend the shelf life of juices. The nanoemulsions with variable terpenes concentrations of 5 g l⁻¹, 10 g l⁻¹ completely inhibited the initial microbial (L. delbrueckii) load 10³ CFU ml⁻¹ whereas, 1 g l⁻¹ only delayed bacterial growth till 5 days in orange juice and 2 days in pear juice compared to control. Similarly, nano-dispersed eugenol was also used to increase the shelf life of milk (4% fat, 2% fat & skimmed milk <0.5% fat). Nanodispersed eugenol completely inhibited bacterial growth of E. coli at 3.5 g l⁻¹, while 4.5 g l⁻¹ was not effective to inhibit the bacterial growth in full fat milk. However, 5.5 g l⁻¹ completely inhibited bacterial growth of E. coli in all milk types. In case of L. monocytogenes nano-dispersed eugenol at 5.5 g l⁻¹ concentration was not effective as against E. coli but, showed better efficacy than free eugenol and reduced bacterial growth in full fat milk to 2.6 log CFU ml⁻¹ and 5.5 log CFU ml⁻¹ in case of free eugenol.⁷⁶

In order to control herpes simplex virus1 (HSP1), Artemisia arborescens EO loaded liposomes showed excellent alternative to drugs. Liposomes inhibited HSP1 growth at EC50 dose of 5.95 μg ml⁻¹ that was equivalent to 100 μg ml⁻¹ of free EO.³² Nuchuchua and others (2009)⁸⁹ reported prolonged action of citronella, basil and vetiver oil nanoemulsions against Aedes aegypti both in vivo and in vitro. Nanoemulsions applied to 3 × 10 cm² area of human skin, well spreaded due to small droplet size (50–220 nm) and increased the protection time to 4.7 hours (basil : vetiver : citronella 5 : 5 : 10 w/w%). In another study, carvacrol loaded microcapsules (calcium alginate) used to control the enteric diseases in pig. In vitro release study of microencapsulated carvacrol showed limited release in stomach, slow release in small intestine and more release in large intestine. However, in vitro antibacterial test showed similar minimum bactericidal concentration (200 μl l⁻¹) of both free and encapsulated carvacrol against E. coli with K88 pili.¹⁷ Moretti and others (2002)⁹⁶ used thymus and rosemary oil loaded nanoemulsions against Limantria dispar, a cork oak forest pest. They reported 100% mortality rate after emulsions treatment till 7 hours.

Improved bioactivity of liposomes encapsulated Myrtus communis extract was reported by¹⁴ when incorporated in sunflower oil at concentration of (160 ppm), the encapsulated extract showed 25% higher oxidation protection factor (1.5 after incubation of 29.5 h) compared to free extract (1.2 after incubation of 23.5 h). However, liposomes encapsulation concentration was not equivalent to free extract. They also confirmed antioxidant potential of liposomes encapsulated Myrtus communis in terms of onset of thermo oxidation process using differential scanning calorimetry (DSC) and the temperature ranges for control, free extract & liposome encapsulated extract were 218, 237 and 272 °C, respectively. In case of soybean oil the microencapsulated eugenol rich clove extract powder showed similar antioxidant protection values (0.085 ± 0.006) as observed in BHT and unencapsulated clove extract. On the other hand both BHT and unencapsulated clove extract showed pro-oxidant activity due to excessive antioxidants in soybean oil that degrade linoleic acid and ultimately forms free radicals by the decomposition of hydrogen peroxide. However, encapsulated clove extract incorporated soybean oil showed no pro-oxidant activity and therefore, recommended to use as natural antioxidant in food rather synthetic ones.²⁷

5. Encapsulated essential oils behaviours/trends

The composition of PO in purity gum 2000 based nanoemulsions before and after encapsulation using high pressure homogenization was quantified to interpret the loss of oil constituents during processing. They reported quite similar composition of pure and encapsulated PO.²¹ Sweet orange oil microencapsulated by complex coacervation (soybean protein isolate : gum arabic mixture) method also showed complete retention of flavour compounds in microcapsules as in pure oil (D-limonene 89.5% in pure & 90.97%) in microcapsules.⁹⁵ Similarly, Arana-Sanchez and others (2010)⁷⁴ also reported no degradation of OEO components when emulsions were spray dried and analysed using GC-MS. The percentage composition of OEO constituents pure & extracted from microcapsules were quite similar (p-cymene 34.68–34.66%, thymol 9.42–19.52% & carvacrol 7.34–7.36%, respectively). Moretti and others (2002)⁹⁶ also confirmed similar profile of thymus and rosemary EO constituents before and after encapsulation. However, terpenes mixture of Melaleuca alternifolia was quantified using GC-MS after processing through high shear and high pressure homogenizer.⁷⁰ They observed degradation of active compounds (α-fellandrene 1.50–0.36, terpinolene 10.03–1.21, carvacrol 4.31–0.50 g kg⁻¹). In another study, changes in the composition of PO constituents were observed when liquid emulsified and spray dried products were analysed using GC & GC-MS. β-Pinene was decreased 2–3 times compared to pure EO and percentage of oxygenated terpenol menthol increased in processed products from 47.5–50.1%.³⁰ Gaysinsky and others (2005),⁹⁷ reported temperature stability of micellar encapsulated eugenol & carvacrol. They observed with increased eugenol concentration in micelle, the temperature stability decreased i.e., at 0.1% eugenol the micelles were stable at 90 °C but at 0.9% eugenol the micelles were stable at 60 °C. Eugenol encapsulated in β-cyclodextrin, 2HP-β-cyclodextrin & PCL using molecular inclusion and emulsion diffusion method showed irradiation induced stability as evident by TGA after 60 days of storage under light and without light in desiccator at 25 °C. Both β-cyclodextrin–eugenol, 2HP-β-cyclodextrin–eugenol complexes after O₂ injection during TGA analysis showed significant weight gain (7.9 & 15.2%) at 20–150 °C and it was attributed to free oxidation reaction occurred from free eugenol with oxygen that injected as purged gas.²⁴ In the emulsion ionic gelation of OEO (carvacrol), improved thermal stability was confirmed after encapsulation at elevated temperature of 340.6 °C.²⁰ The result from this study favours the use of encapsulated EOs in various food applications even at elevated temperature during processing.

6. Conclusions and future perspectives

Encapsulation is therefore an efficient approach to protect the EOs from light, air and humidity, because these interactions lead to oxidation or volatilization and reduced biological activities. Moreover, encapsulation increases the solubility of oil, provides controlled release and makes it more bioavailable. Spray drying and emulsification are the most versatile and commercially available techniques that had been used widely for EOs encapsulation. The encapsulated EOs showed enhanced antimicrobial, antifungal, antioxidant, antiviral and pesticidal activities. The use of encapsulated EOs in food, cosmetic and pharmaceutics could be an economic benefit and also fulfill the consumer concern regarding safety. The use of encapsulated EOs in cosmetics and pharmaceutics is lacking. Further, research is required to underpin recent analytical approaches in order to gain deeper understanding of oxidation, isomerization and thermal rearrangements processes and strategies to avoid them. Moreover, identification of products generated from these processes appears to be a valuable future objective. Further, encapsulated EOs can be used to increase the bioactivities of EOs in real food systems, to study their action mechanism on cell membranes, and to provide non-lethal therapeutic agents to treat several diseases.

Acknowledgements

This work was financially supported by National 863 Program 2011BAD23B02, 2013AA102207, NSFC 31171686, 30901000, 111 project-B07029 and PCSIRT0627.

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