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LWT - Food Science and Technology 44 (2011) 1908e1914

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LWT - Food Science and Technology

journal homepage: www.elsevier .com/locate/ lwt

Nanoencapsulation of essential oils to enhance their antimicrobial activityin foods

Francesco Donsì a,*, Marianna Annunziata b, Mariarenata Sessa a, Giovanna Ferrari a,b

aDepartment of Industrial Engineering, University of Salerno, Italyb ProdAl Scarl, Competence Center on Agro-Food Productions, University of Salerno, Italy

a r t i c l e i n f o

Article history:Received 17 August 2010Received in revised form25 February 2011Accepted 2 March 2011

Keywords:Nanometric delivery systemEssential oilAntimicrobial activityHigh pressure homogenizationNanoemulsions

* Corresponding author. Tel.: þ(39) 089 96 4135; faE-mail address: fdonsi@unisa.it (F. Donsì).

0023-6438/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.lwt.2011.03.003

a b s t r a c t

This work focuses on the encapsulation of essential oils into nanometric delivery systems for incorpo-ration into fruit juices, in order to enhance their antimicrobial activity while minimizing the impact onthe quality attributes of the final product. A terpenes mixture and D-limonene were encapsulated intonanoemulsions based on food-grade ingredients, prepared by high pressure homogenization at 300 MPa.

The effect of the delivery systems on the antimicrobial activity of terpenes was investigated bydetermining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration(MBC) for three different classes of microorganisms (Lactobacillus delbrueckii, Saccharomyces cerevisiae,Escherichia coli). The increase of the antimicrobial activity resulted to depend on the formulation andmean diameter of the delivery systems as well as on the microorganisms class. Additionally, GCeMSanalysis revealed that high intensity processing for nanoemulsion production may affect the chemicalstability of several active compounds.

The application of the most efficient antimicrobial nanocapsules was tested in pear and orange juicesinoculated with L. delbrueckii. Due to the higher antimicrobial activity of the nanoencapsulatedcompounds, lower antimicrobial concentrations are required for a bactericidal action under acceleratedaging at 32 �C, with a minimal alteration of the organoleptic properties of the juice.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Nowadays, the approaches that can be adopted in food preserva-tion include: (a) aseptic handling and packaging, (b) the mechanicalremoval ofmicroorganismsbywashingorfiltration, (c) destructionofmicroorganisms by physical or chemical sanitization and finally (d)the inhibition of pathogens or saprophytes through environmentalcontrol (Davidson, Sofos, & Branen, 2005). The latter approach hasbenefited most from the recent developments in nanotechnology.

Inhibition of microbial growth through environmental control isachieved through the addition of chemical compounds (antimi-crobial preservatives) with an inhibitory or bactericidal/fungicideactivity. In the last years, natural antimicrobials have attractedconsiderable attention due to the increased consumer awarenesson the aspects of food quality and safety (Weiss, Gaysinksy,Davidson, & McClements, 2009).

Similar to most bioactive compounds, antimicrobial agents arechemically reactive species, which can cause considerable problemswhenembedded into a complex foodsystem, suchasnegative effects

x: þ(39) 089 96 4168.

All rights reserved.

on the physical stability or integrity of the food chemistry as well asthe degradation of the biological activity of bioactive compounds(McClements, 1999). This translates into the need to use concentra-tions which are high enough to inhibit microbial growth within thelimits imposed by food regulations, but at the same time minimallyalter the qualitative properties of the product (Weiss et al., 2009).

Nanoencapsulation of bioactive compounds represents a viableand efficient approach to increasing the physical stability of theactive substances, protecting them from the interactions with thefood ingredients and, because of the subcellular size, increasingtheir bioactivity.

In the case of antimicrobials, encapsulation can increase theconcentration of the bioactive compounds in food areas wheremicroorganisms are preferably located, for example water-richphases or liquidesolid interfaces (Weiss et al., 2009).

A significantly large part of current literature on the encapsu-lation of essential oils deals with micrometric size capsules, whichare used for the protection of the active compounds against envi-ronmental factors (e.g. oxygen, light, moisture, pH). For example,solid lipid microparticles have been proposed for the encapsulationof juniper oil, in order to reduce the volatility of the antimicrobialagent (Gavini et al., 2005). On the other side, biopolymers have

F. Donsì et al. / LWT - Food Science and Technology 44 (2011) 1908e1914 1909

been widely used as wall materials of microparticles in theprotection of essential oils, both in aqueous phase, such as chitosan(Pedro, Cabral-Albuquerque, Ferreira, & Sarmento, 2009), Ca-algi-nate (Wang, Gong, Huang, Yu, & Xue, 2009), as well as modifiedstarch for agrochemical applications of pest control (Glenn et al.,2010; Varona, Martin, & Cocero, 2009), or in spray-dried powders,such as milk proteins (Baranauskiene, Venskutonis, Dewettinck, &Verhe, 2006) and different polysaccharides (Adamiec & Kalemba,2006; Krishnan, Bhosale, & Singhal, 2005).

While microcapsules may guarantee excellent protection ofessential oils against degradation or evaporation, they in general donot affect antimicrobial activity. In contrast, nanometric sizedelivery systems, due to the subcellular size, may increase thepassive cellular absorption mechanisms, thus reducing masstransfer resistances and increasing antimicrobial activity.

Proof of this concept was given by the improvement of theantimicrobial activity of essential oils when encapsulated intoliposomal delivery systems (Gortzi, Lalas, Tsaknis, & Chinou, 2007;Liolios, Gortzi, Lalas, Tsaknis, & Chinou, 2009).

The encapsulation of eugenol and carvacrol into nanometricsurfactant micelles also resulted in enhanced antimicrobial activity(Gaysinsky, Davidson, Bruce, & Weiss, 2005), although the additionof micelle-encapsulated eugenol to milk resulted to be less or asinhibitory as unencapsulated eugenol (Gaysinsky, Taylor, Davidson,Bruce, & Weiss, 2007).

Among the nanometric encapsulation systems currently beingused for the delivery of bioactive compounds, nanoemulsions areparticularly suitable for food applications (McClements, 1999),owing to the possibility of formulationwith natural ingredients andthe easy industrial scalability of the production process by highpressure homogenization. Nevertheless, up to now very littlesystematic research has been conducted to evaluate their use inencapsulating antimicrobial essential oils (Weiss et al., 2009).Moreover, very often it was reported that encapsulation in nano-emulsions reduced the antimicrobial activity in comparison withunencapsulated active compounds, as shown for chitosan, anantimicrobial polysaccharide (Jumaa, Furkert, & Muller, 2002), andfor eugenol, an essential oil component (Weiss et al., 2009).

This work is based on the need for more detailed research on theformulation, design and application of nanoemulsions as antimi-crobial delivery systems. It focuses on the encapsulation in nano-emulsion-based delivery systems of two antimicrobial compounds,a terpenes mixture extracted from Melaleuca alternifolia andD-limonene, dealing with the issues of formulation and fabricationin order to retain and possibly enhance the antimicrobial activity ofthe encapsulated compounds. The most promising formulationsare tested in fruit juices, in order to evaluate the juice preservationfrom inoculated spoilage microorganisms and the possible shelf-life extension against the alteration of the quality parameters of thejuices.

2. Materials and methods

2.1. Materials

The tested antimicrobial compounds are D-Limonene (Sigma-eAldrich, Germany) and a mixture of terpenes extracted fromMelaleuca alternifolia (provided by Istituto Superiore della Sanità,Italy). In the emulsion fabrication, sunflower oil (Sagra, Italy) andpalm oil (SigmaeAldrich, Germany) were used as organic phases,while soy lecithin Solec Ip (a generous gift from Solae Italia s.r.l.,Italy), Tween 20 and glycerol monooleate (SigmaeAldrich,Germany), and CLEARGUM� CO 01 (a generous gift from Roquette,Italy) were used as emulsifying agents.

2.2. Preparation of nanoemulsions

The sunflower oil or essential oil-in-water nanoemulsions wereprepared using a High Pressure Homogenization (HPH) technique.Primary emulsions were obtained by High Shear Homogenization(HSH), using an Ultra Turrax T25 (IKA Labortechnik, Germany) at24000 rpm for 5 min. The primary emulsions were then subjectedto HPH in a Nano DeBEE Electric Bench-top Laboratory homoge-nizer (BEE International, USA) ten times at 350 MPa. When palm oilwas used as a lipid phase, the antimicrobial agents were dissolvedin the melted lipid and the temperature during processing wasalways kept about 5e10 �C above the lipid melting point. Crystal-lization of the lipid droplets was attained by rapid cooling of the hotnanoemulsions in an ice bath at the end of the HPH processing.

2.3. Droplet size measurements

Droplet size distribution was determined by photon correlationspectroscopy (PCS) at 25 �C (HPPS, Malvern Instruments, UK). Fromthe PCS data, the average droplet diameter (z-average) and thepolydispersity index (PDI) were determined. Prior to anymeasurements being taken, the samples were diluted with bidis-tilled water to a suitable concentration. Each measurement wasreplicated twice, with the means and the standard deviations beingcalculated.

2.4. Determination of MIC and MBC

Experiments were carried out on three different microbialstrains grown to the stationary phase in an aerated incubator(Haeraeus Instruments): Saccharomyces cerevisiae, Escherichia coli,Lactobacillus delbrueckii. S. cerevisiae yeast was grown in MRS broth(Oxoid, UK) at 32 �C for 48 h, E. coli in Tryptone Soya broth (Oxoid,UK) at 30 �C for 18e24 h, L. delbrueckii inMRS broth at 32 �C for 48 h.

The Minimum Inhibitory Concentration (MIC) on the threemicrobial strains was evaluated for the concentration of antimi-crobial agents in culture media ranging from 25 g/l to 0.1 g/l. Thesamples were inoculated with 100 ml of a microbial suspension(107 CFU/ml) and incubated for 24 h at 32 �C for S. cerevisiae andL. delbrueckii and at 30 �C for E. coli. The MIC value was determinedas the lowest concentration of the antimicrobial agent thatinhibited the visible growth of the test microorganism, evaluatingthe absorbance of the sample with a spectrophotometer (JascoV-650) in the range of 590e600 nm.

The Minimum Bactericidal Concentration (MBC) was deter-mined followingMIC determination. A sample of 1mlwas collectedfrom each tested sample and inoculated on sterile Plate count agar(Oxoid, UK) for E. coli and MRS agar (Oxoid, UK) for S. cerevisiae andL. delbrueckii. The plates were incubated at 37 �C for 24 h. Thehighest dilution that yielded a decrease in microbial concentrationin comparison to the control sample was considered as MBC.

Each measurement was replicated three times.

2.5. Kinetics of inactivation

The inactivation kinetics of the three microorganisms in thepresence of an encapsulated terpenes mixture were determined incomparison to a control, where terpenes were replaced bysunflower oil.

Themicroorganisms, centrifuged at 5000 rpm for 10 min at 4 �C,were resuspended in 100 ml of sterile distilled water in test tubes,with nanoemulsions being added to the desired final antimicrobialconcentrations (1.0 g/l and 2.5 g/l). The test tubes were thenincubated at 32 �C for S. cerevisiae and L. delbrueckii and at 30 �C forE. coli. After 30 min for the antimicrobial concentration of 2.5 g/l

Table 1Composition, dimension and production method of the tested nanoemulsions.

Sample Composition Process z-average[nm]

T-SL-HPH 50 g/kg terpenes,10 g/kg soy lecithin,940 g/kg water

10 HPH passesat 300 MPa 3 �C

74.4 � 2.6

T-SL-HSH 50 g/kg terpenes,10 g/kg soy lecithin,940 g/kg water

HSH at 24000rpm for 5 min

174.8 � 5.7

L-CG 50 g/kg D-limonene,100 g/kg clear gum,850 g/kg water

10 HPH passes at300 MPa 3 �C

365.7 � 7.5

L/PO-SL 50 g/kg D-limonene,50 g/kg palm oil,20 g/kg soy lecithin,880 g/kg water

10 HPH passes at300 MPa 30 �C

235.9 � 9.6

L/SO-T20/GMO 50 g/kg D-limonene,50 g/kg sunflower oil,15 g/kg Tween 20, 15 g/kgglycerol monooleate,870 g/kg water

10 HPH passes at300 MPa 3 �C

130.9 � 1.3

L-T20/GMO 50 g/kg D-limonene,7.5 g/kg Tween 20,7.5 g/kg glycerolmonooleate,935 g/kg water

10 HPH passes at300 MPa 3 �C

154.6 � 1.4

F. Donsì et al. / LWT - Food Science and Technology 44 (2011) 1908e19141910

and after 90 min and 24 h for the antimicrobial concentration of1.0 g/l, the surviving cells were evaluated by a standard plate countmethod. In brief, 1 ml of each sample was used to prepare decimaldilutions, which were plated in duplicate with Plate Count agar forE. coli and MRS agar for S. cerevisiae and L. delbrueckii. The plateswere incubated at 30 �C for 24 h for E. coli and at 32 �C for 48 h forS. cerevisiae and L. delbrueckii.

Kinetics experiments were carried out in duplicate. From thelinear regression of the common logarithm of the survival fractionvs. the exposure time to the antimicrobials, the decimal reductiontime D, defined according to Eq. (1), was calculated.

D ¼log

N0

NðtÞt � t0

(1)

2.6. GCeMS analysis

The composition of the terpenes mixture after homogenization(either HSH or HPH) was evaluated through a gas chromatography-mass spectrometry method (GCeMS). The terpenes mixture wasextracted by adding 4 ml of dichloromethane to 500 ml of nano-emulsion, followed by three vortex agitations of 10 s each. Theorganic phase was recovered with a Pasteur pipette and anhydroussodium sulphate was added to remove residual water. The extractwas micro-filtered and placed for 15 min under a nitrogen flow inorder to completely evaporate the solvent. 2 ml of the resultingterpenes were added to 2 ml of n-pentane and analyzed by Focus-GC-DSQ (Thermo Finnigan) GCeMS, equipped with a capillarycolumn Rtx-5Sil MS (30 m, ID 0.25 mm, film thickness 0.25 mm;Restek), using helium as a carrier gas (1 ml/min). The columntemperaturewas kept at 40 �C for 3min and then increased by 3 �C/min to 280 �C. The mass selective detector was used in the electronionisation mode, with the mass range between 35 and 500 beingscanned. The mass spectra were compared to both the NIST MassSpectral Libraryaswell as an in-house library for peak identification.

2.7. Fluorescence microscopy

Fluorescence microscopy observations were carried out with anEclipse TE2000S invertedmicroscope (Nikon), equippedwith a B-2Afilter (Excitation Filter Wavelengths: 450e490 nm, DichromaticMirror Cut-on Wavelength: 500 nm, Barrier Filter Wavelengths:515nmcut-on),fittedwithahigh-pressuremercuryburneras a lightsource. The images were acquired with a digital camera (DS-5MDigital Sight Camera System, Nikon), through a 20x lens (Nikon).

Nile red (SigmaeAldrich, Germany) was used as a fluorescentlipophilic stain. It excites at 485 nm, and emits at 525 nm. The NileRed was dissolved in ethanol at a concentration of 1 mg/ml;a sample of 100 ml of this solution were added to 1 ml of nano-emulsion to stain the oil droplets. 100 ml of nanoemulsion with NileRed were subsequently added to 1 ml of culture medium containing108 CFU/ml of yeast cells in a stationary phase to a final antimicro-bial concentration of 5.0 g/l. At fixed times, a drop of the sample wasmounted onto a glass slide, enclosed with a cover slit and observed.

2.8. Shelf life of fruit juices

The effect of the addition of the encapsulated antimicrobial onthe microbiological stability of two fruit juices, orange juice (Tro-picana Pure Premium PepsiCo, France) and pear juice (Yoga, Italy),was evaluated over time. The juices were inoculated with 103 CFU/ml of Lactobacillus delbrueckii. Different concentrations (from 10 g/lto 1.0 g/l) of terpenes nanoemulsions were tested under acceleratedshelf life conditions at 32 �C.

The concentration of microorganisms in each sample expressedin CFU/mlwas evaluated over time by standard plate countmethod,as previously described. Moreover, the evolution of color, pH and�Brix of the fruit juices over storage time was also evaluated byChroma Meter (CR-200b, Minolta), pH-meter (Basic 20þ, Crison)and Abbe Refractometer (Atago) respectively.

The color was measured registering the following parameters:L (brightness), a (red-green component) and b (yellow-bluecomponent). The global color difference (DE) was calculated withthe following equation:

DE ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðDLÞ2þðDaÞ2þðDbÞ2

q(2)

The values provided were the average of three replicates.

3. Results

3.1. Formulation and fabrication of stable nanoemulsions

Different formulations and fabrication methods were used toproduce stable nanoemulsions encapsulating the antimicrobialcompounds. In general, the nanoemulsions contained 50 g/kg of theactive compounds (a terpenes mixture or D-limonene), eventuallymixed in the organic phase (palm oil or sunflower oil). Soy lecithin,modified starch (CLEARGUM) or a mixture (50:50) of Tween 20 andglycerol monooleate were used as emulsifiers.

Table 1 reports all those nanoemulsions, which resulted physi-cally stable over 4 weeks with neither visible creaming norsignificant variation of the mean droplet diameter.

Only the lecithin-based nanoemulsions, whose mean dropletdiameter ranged from 75 nm (HPH processing) to 175 nm (HSHprocessing), represented a stable delivery system for the terpenesmixture, without any additional organic phase. In contrast, theproduction of stable lecithin-based nanoemulsions (240 nm)required the blending of D-limonene with palm oil (1:1).

A stable nanoemulsion (365 nm) was obtained with pureD-limonene as the organic phase only when modified starch wasused as an emulsifier.

Table 2MIC and MBC measurements of pure and encapsulated essential oils on differentmicrobial strains.

E. coli L. delbrueckii S. cerevisiae

MIC(g/l)

MBC(g/l)

MIC(g/l)

MBC(g/l)

MIC(g/l)

MBC(g/l)

Terpenes mixture(pure)

5.0 5.0 5.0 25 10 10

T-SL-HPH 1.0 5.0 10 10 1.0 5.0T-SL-HSH 5.0 5.0 5.0 10 1.0 5.0D-Limonene (pure) >25 >25 >25 >25 >25 >25L-CG 10 >25 10 >25 10 25L/PO-SL 10 >25 10 >25 10 >25L/SO-T20/GMO 5.0 >25 5.0 >25 5.0 >25L-T20/GMO 5.0 >25 25 >25 25 >25

F. Donsì et al. / LWT - Food Science and Technology 44 (2011) 1908e1914 1911

D-limonene was also encapsulated, alone or blended withsunflower oil (1:1), into stable delivery systems made of Tween 20/glycerol monooleateebased nanoemulsions, with a very fine meandroplet diameter (from 130 to 155 nm).

3.2. MIC and MBC

The MIC and MBC values of a nanoencapsulated terpenesmixture as well as nanoencapsulated D-limonene in comparison topure compounds, reported in Table 2, give a measurement of theactivity of an antimicrobial agent.

TheMIC andMBCvalues of the antimicrobial agents encapsulatedinnanoemulsions resultedalways lowerorequal topure compounds,therefore suggesting the enhancement of transport mechanismsthrough the cell membrane of the target microorganisms.

Fig. 1. Brightfield (a and c) and fluorescence micrographs (b and d) of S. cerevisiae cells expand 24 h (c and d).

The effect of the encapsulation system on the antimicrobialactivity of the terpenes mixture depended on the target microor-ganism. For S. cerevisiae, theMIC andMBC valueswere reduced from10 g/l to 1.0 g/l and 5.0 g/l respectively when the terpenes mixturewas encapsulated in both nanoemulsion T-SL-HPH and T-SL-HSH.For L. delbrueckii, the nanoencapsulation caused a reduction of onlytheMBC values for both T-SL-HPH and T-SL-HSH from 25 g/l to 10 g/l, while the MIC values remained unchanged at 5.0 g/l for nano-emulsion T-SL-HSH and increased to 10 g/l for nanoemulsion T-SL-HPH. Interestingly, encapsulation of the terpenes mixture did notsignificantly reduce the MIC and MBC values for E. coli.

The antimicrobial activity of D-limonene is significantly lowerthan that of the terpenes mixture, with the MIC and MBC valuesbeing always higher than 25 g/l. Experimental measurements wereintentionally limited to 25 g/l due to higher concentrations beingconsidered unsuitable for food applications.

In contrast with what was observed with the terpenes mixture,the encapsulation of D-limonene never reduced theMBC values, butaffected only the MIC values.

For L-CG and L/PO-SL nanoemulsions, the MIC values werereduced from >25 g/l to 10 g/l for all the microorganisms. Fornanoemulsion L/SO-T20/GMO, the MIC reached the smallest value(5.0 g/l) for all the microorganisms, which is probably due to thefine droplet diameter of the delivery system. Whereas, nano-emulsion L-T20/GMO induced a reduction of theMIC value to 5.0 g/lfor E. Coli and only to 25 g/l for the other microorganisms.

The results reported in Table 2 show that the effect of encap-sulation on the different delivery systems tested depends on theactive compounds. The encapsulation of the terpenes mixtureenhances both the bacteriostatic and bactericidal activity (MICvalues are close to MBC values) only for S. Cerevisiae and E. coli,whereas a less significant effect was observed for L. delbrueckii,

osed to nanoemulsion L-CG captured by fluorescence microscopy after 5 min (a and b)

F. Donsì et al. / LWT - Food Science and Technology 44 (2011) 1908e19141912

probably due to its thicker cell wall structure, characteristic ofgram-positive bacteria. In contrast, the encapsulation of D-limo-nene enhanced only its bacteriostatic activity (MIC values << MBCvalues) on all the tested microorganisms.

In particular, the antimicrobial activity of cyclic hydrocarbons islimited by their solubility, being available for interaction with cellsonly those molecules, which are dissolved in the aqueous phase(Sikkema, Debont, & Poolman, 1995). Therefore, essential oilcomponents, such as carvacrol, need to be dissolved in concentra-tions approaching or exceeding their maximum solubility in orderto exhibit bactericidal activity (Gill & Holley, 2006). In contrast,limonene, characterized by a solubility significantly lower thancarvacrol, exhibits only a bacteriostatic activity unless its concen-tration in the aqueous phase is increased, for example by favorablepartitioning between the aqueous and a selected lipid phase, or bysolubilization within appropriate surfactant micelles. Our resultsshowed that the use of a solid lipid emulsion (palm oil) did notaffect the limonene activity, despite the possibility that limonenemay be expelled from the emulsion droplets by solid fat crystalli-zation. In contrast, the use of Tween 20/glycerol monooleateresulted in a slight increase of the bacteriostatic activity of limo-nene, probably due to increased micelle solubilization.

The bactericidal activity of the terpenes mixture as well as thebacteriostatic activity of D-limonene were visualized by fluores-cence microscopy observations. Images of the S. cerevisiae cellsexposed to the nanoemulsion T-SL-HPH as well as the nano-emulsion L-CG, both loaded with fluorescent dye, were recordedafter 5 min and 24 h (Figs. 1 and 2).

Under a fluorescent light, the nanoemulsion droplets cannot bedistinguished when they are dispersed in an aqueous system due totheir nanometric size, with only a fluorescent halo being observed.In contrast, when the nanoemulsion droplets accumulate in the cell

Fig. 2. Brightfield (a and c) and fluorescence micrographs (b and d) of S. cerevisiae cells e(a and b) and 24 h (c and d).

membrane as well as the intracellular space, the yeast cells becamefluorescent and can be observed. Figs. 1 and 2 show that bothnanoemulsions can readily permeate the cell membrane: after5 min, the yeast cells became fluorescent, suggesting that the anti-microbial agents have reached the action sites. After 24 h, therewasa significant difference between the nanoemulsion T-SL-HPH andL-CG. The yeast cells exposed to the terpenes mixture exhibiteda shrunk cellmembrane,which suggests their death probably due tothe loss of intracellularmaterial (Fig. 2). On the other hand, the yeastcells exposed to nanoencapsulated D-limonene did not show anyapparent change in shape and size, therefore suggesting a merelybacteriostatic effect of this compound, leaving the cells alive (Fig.1).

3.3. Kinetics of inactivation

The inactivation kinetics of the three target microorganismsexposed to nanoemulsions T-SL-HPH and T-SL-HSH, encapsulatingthe terpenes mixture, were determined in water at two differentantimicrobial concentrations (1.0 g/l and 2.5 g/l).

Table 3 reports the decimal reduction time of inactivationD, defined as the time required to reduce by an order of magnitudethe number of surviving microorganisms, for E. coli, L. delbrueckiiand S. cerevisiae.

While the control systems (nanoemulsions where the terpenesmixture was substituted by sunflower oil) did not cause anymeasurable microbial inactivation over 24 h (data not reported), theterpeneseloaded nanoemulsions inactivated the three microorgan-ismswith characteristic times (D inTable 3) of theorderofminutes at2.5 g/l and of hours at 1.0 g/l. Interestingly, the nanoemulsion T-SL-HSH caused a faster inactivation than nanoemulsion T-SL-HPH,despite the larger mean droplet diameter: D of nanoemulsion T-SL-HSH is always shorter than for nanoemulsion T-SL-HPH for

xposed to nanoemulsion T-SL-HPH captured by fluorescence microscopy after 5 min

)lm/

UFC(

N

1e+0

1e+1

1e+2

1e+3

1e+4

1e+5

1e+6

1e+7

1e+8a

Table 3The decimal reduction time of inactivation of target microorganisms exposed to theterpenes mixture encapsulated in nanoemulsions T-SL-HPH and T-SL-HSH.

Decimal reduction time D [min]

E. coli L. delbrueckii S. cerevisiae

2.5 g/l T-SL-HPH 7.57 � 0.36 10.2 � 2.8 312 � 72R2 ¼ 0.977 R2 ¼ 0.655 R2 ¼ 0.587

1.0 g/l T-SL-HPH 556 � 542 714 � 12 N

R2 ¼ 0.215 R2 ¼ 0.9952.5 g/l T-SL-HSH 7.68 � 0.14 6.69 � 0.38 30.7 � 2.2

R2 ¼ 0.966 R2 ¼ 0.834 R2 ¼ 0.9241.0 g/l T-SL-HSH 909 � 23 588 � 19 N

R2 ¼ 0.992 R2 ¼ 0.975

F. Donsì et al. / LWT - Food Science and Technology 44 (2011) 1908e1914 1913

L. delbrueckii and S. cerevisiae, while no significant difference can beobserved for E. coli. The reasons for these unexpected results can befound in the degradation of the active compounds during nano-emulsion production, as discussed in the following section.

3.4. GCeMS analysis

The reported inactivation data suggest that, in contrast to whatwas expected, a straightforward correlation between the efficiencyof the antimicrobial delivery system and its mean droplet diameteris not possible. The comparison of performance of the nano-emulsions T-SL-HPH and T-SL-HSH, which share the same compo-sition, showed that the larger droplet diameter system (T-SL-HSH)induced lower MIC and MBC values for L. delbrueckii (Table 2) anda shorter D for all the microorganisms (Table 3), suggesting theoccurrence of the degradation of some active compounds duringHPH processing. Table 4 reports the GCeMS analysis of the pureterpenes mixture, which was not subjected to any fluid dynamicstresses, and the analysis of the terpenes mixture as extracted fromnanoemulsions prepared by HPH (T-SL-HPH) and HSH (T-SL-HSH),subjected, respectively, to high intensity andmild intensity stressesduring processing.

GCeMS analysis revealed that fluid dynamic stresses duringHSH and HPH processing caused the degradation of some activecompounds, such as a-fellandrene, terpinolene, p-cymene, thujene,d-terpinene, 2-carene/isoterpinolene, trans-2-caren-4-ol, carveol,

Table 4Composition of the pure terpenes mixture and the terpenes extracted from nano-emulsions T-SL-HPH and T-SL-HSH.

Component Retentionindex

Component fraction in the essential oilmixture (pure and encapsulated) (g/kg)

Terpenesmixture

NanoemulsionT-SL-HSH

NanoemulsionT-SL-HPH

a-fellandrene 14.10 1.50 0.20 0.36terpinolene 14.63 10.03 0.70 1.21p-cymene 15.06 13.94 2.01 3.82thujene 15.28 3.01 0.60 0.90d-terpinene 16.7 6.82 0.60 1.002-carene/isoterpinolene 17.99 2.81 0.20 0.40trans-2-caren-4-ol 18.33 1.40 0.20 0.30cis-a-terpineol 19.09 0.40 0.60 0.70cyclohexanol

(4-isopropil�1�methyl)-cis/cis-sabinene hydrate

20.12 1.50 3.01 3.62

carveol 20.39 0.80 0.20 0.201-terpineol 20.7 0.60 0.40 0.60cyclohexanol(4-isopropil-

1-methyl)-trans21 0.40 1.00 1.41

terpinen-4-ol 22.8 948.95 986.45 982.055-coranol 23.95 0.50 0.60 0.90thujol 27.84 3.01 2.01 2.01carvacrol 29.21 4.31 1.20 0.50

thujol, carvacrol. Interestingly, when increasing the process inten-sity from HSH to HPH, only carvacrol, which is a compound witha well-known antimicrobial activity (Ben Arfa, Combes, Preziosi-Belloy, Gontard, & Chalier, 2006), was significantly reduced.Therefore, the theoretical higher delivery efficiency of nano-emulsion T-SL-HPH, associated with its smaller mean dropletdiameter, is likely counter-balanced by the partial degradation ofsome active compounds, and in particular, carvacrol.

3.5. Addition of encapsulated antimicrobial agents to fruit juices

Nanoemulsion T-SL-HPH was added at different concentrationsto two fruit juices (orange juice and pear juice) inoculated withL. delbrueckii in order to test the microbiological stability as well asthe alteration of the chemical and physical characteristics of thejuices stored at 32 �C.

The results of the accelerated shelf life studies are reported inFigs. 3 and 4. Fig. 3 shows that for both fruit juices after 2 days, thetotal inactivation of the initial microbial load of 103 CFU/ml wasalready reached for the terpenes concentrations of 5.0 g/l and 10 g/l.At a terpenes concentration of 1.0 g/l, microorganism growth isdelayed by 5 days in orange juice and 2 days in pear juice incomparison to the control.

�Bx and pH were not significantly altered by the addition of thenanoemulsion. This was also observed during the storage period,unless significant microbial growth occurred (data not reported).

Time (days)

0 2 4 6 8 10 12 14 16 18

)lm/

UFC(

N

1e-2

1e-1

1e+0

1e+1

1e+2

1e+3

1e+4

1e+5

1e+6

1e+7

1e+8

0 2 4 6 8 10 12 14 16 181e-2

1e-1

b

Fig. 3. Inactivation curve of L. delbrueckii suspended in (a) orange juice and (b) pearjuice treated with terpenes nanoemulsion T-SL-HPH at 32 �C. Experimental data:control juice ( ), juice added with T-SL-HPH to a concentration of the terpenesmixture of 1.0 g/l ( ), 5.0 g/l ( ) and 10 g/l ( ).

0 2 4 6 8 10 12 14 16

EΔ

EΔ

0

5

10

15

20

Time (days)

0 2 4 6 8 10 12 14 160

5

10

15

20

a

b

Fig. 4. Variation over time of the global color difference of (a) orange juice and (b) pearjuice with different concentrations of terpenes nanoemulsion T-SL-HPH at 32 �C.Experimental data: control juice ( ), juice added with T-SL-HPH to a concentration ofthe terpenes mixture of 1.0 g/l ( ), 5.0 g/l ( ) and 10 g/l ( ).

F. Donsì et al. / LWT - Food Science and Technology 44 (2011) 1908e19141914

In contrast, major color deviations were observed when 10 g/lnanoencapsulated terpenes were added to the juices, while theaddition of the terpenes mixture at lower concentrations (5.0 g/land 1.0 g/l) can be considered acceptable, with it inducing muchsmaller color deviations (Fig. 4). The color remained stable over thestorage time for all those systems where no significant microbialgrowth occurred.

4. Conclusions

The encapsulation into nanoemulsion-based delivery systems oftwo essential oils, a terpenes mixture extracted from Melaleucaalternifolia and D-limonene, was investigated as a method toimprove the safety and quality of foods through the addition ofnatural preservatives.

Lecithin-based nanoemulsion resulted as being a highly efficientcarrier system for the terpenes mixture, while D-limonene wassuccessfully nanoencapsulated pure or in a blend with palm oilusing as emulsifier modified starch and soy lecithin, respectively.

The MIC and MBC values of the nanoencapsulated terpenes,tested on three different microorganisms (E. coli, L. delbrueckii andS. cerevisiae), resulted always lower than or equal to the values ofthe unencapsulated mixture. On the other hand, the nano-encapsulation of D-limonene was able to reduce only the MIC

values, without any significant variation of the MBC values incomparison to the unencapsulated D-limonene.

The terpenes nanocapsules were tested in real systems, such asorange and pear juices, inoculated with L. delbrueckii. The additionof low concentrations of the nanoencapsulated terpenes was ableto delay the microbial growth (1.0 g/l terpenes) or completelyinactivate the microorganisms (5.0 g/l terpenes) while minimallyaltering the organoleptic properties of the fruit juices.

Acknowledgements

The authors wish to thank Mariangela Falcone, Marina Fruiloand Mariarosaria Vincensi for the assistance with the chemicalanalysis. The Montana prize for Food Research (3rd edition) isacknowledged for financial support.

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