Riham Gharib
email: gharib_riham@hotmail.com

Souha Haydar
email: shaydar@ul.edu.lb

Catherine Charcosset
email: charcosset@lagep.univ-lyon1.fr

Sophie Fourmentin

Professor, Hélène Greige-Gerges
email: greigegeorges@yahoo.com

First study on the release of a natural antimicrobial agent, estragole, from freezedried delivery systems based on cyclodextrins and liposomes

Keywords: conventional liposomes, drug-in-cyclodextrin-in-liposomes, estragole, freeze-drying, hydroxypropyl-ß-cyclodextrin

Estragole is considered as a promising candidate for food preservation due to its antioxidant and antimicrobial properties. However, its sensitivity to light and oxygen, its volatility and hydrophobicity prevent its large application. In order to increase the stability of estragole, conventional liposomes (CL) and drug-in-cyclodextrin-inliposomes (DCL) were prepared by the ethanol-injection method using Phospholipon 90H in combination with cholesterol and then freeze-dried using hydroxypropyl-ßcyclodextrin (HP-ß-CD) as a cryoprotectant. Fresh and reconstituted vesicles demonstrated nanometric vesicles size, spherical shape and negative surface charge.

Moreover, all suspensions were homogeneous. Compared to CL, DCL improved estragole encapsulation efficiency, and conserved the same loading rate during freezedrying. Freeze-dried CL and DCL retained estragole more efficiently compared to the reconstituted ones, as evidenced by release study performed by multiple headspace extraction. Finally, we demonstrated, for the first time, that HP-ß-CD did not affect the membrane fluidity of fresh and reconstituted Phospholipon 90H/cholesterol CL and DCL.

Introduction

Estragole (Est), 4-methoxyallylbenzene, is a natural phenylpropene found in many aromatic plants. It is a constituent of tarragon (60-75%), basil (2-43%), star anise (5-6%), and fennel (5-20%) essential oils [START_REF] Vincenzi | Constituents of aromatic plants: II. Estragole[END_REF]. Estragole is Generally Recognized As Safe (GRAS) and approved by the Expert Panel of the Flavor and Extract Manufacturer's Association (FEMA, USA) that concluded that the dietary intake of estragole from the consumption of spices do not pose a health risk [START_REF] Smith | Safety assessment of allylalkoxybenzene derivatives used as flavouring substances methyl eugenol and estragole[END_REF]. Estragole has a taste described as spice, cinnamon, mouth tingle, fresh, peppery and woody. It is used for the modification of spice flavors and seasonings for condiments and meats as well as in heavy fruit, root beer and anisetype flavors [START_REF] Smith | Safety assessment of allylalkoxybenzene derivatives used as flavouring substances methyl eugenol and estragole[END_REF] as well in teas to prevent flatulence and spasms. Moreover, estragole exhibits myorelaxant, anticonvulsant, anesthetic, bradycardic, vasoactive and antioxidative properties [START_REF] Smith | Safety assessment of allylalkoxybenzene derivatives used as flavouring substances methyl eugenol and estragole[END_REF][START_REF] Siqueira | Cardiovascular effects of the essential oil of Croton zehntneri leaves and its main constituents, anethole and estragole, in normotensive conscious rats[END_REF][START_REF] Soares | Effects of anethole and structural analogues on the contractility of rat isolated aorta: Involvement of voltage-dependent Ca++-channels[END_REF] Estragole proved an antifungal activity against Aspergillus niger, A. flavus, Trichoderma viride, Candida albicans, C. utilis, C. tropicalis, Cryptococcus neoformans, Trichophyton mucoides, T. tonsurans, and Blastoschizomyces capitatus [START_REF] Shin | Antifungal activity of the essential oil of Agastache rugosa Kuntze and its synergism with ketoconazole[END_REF][START_REF] Shin | Essential oil compounds from Agastache rugosa as antifungal agent against Trichophyton species[END_REF]. In addition, estragole presented fairly significant zones of inhibition against Gram-positive bacteria [START_REF] Andrade | Isolation, characterization and evaluation of antimicrobial and cytotoxic activity of estragole, obtained from the essential oil of Croton zehntneri (Euphorbiaceae), An[END_REF].

Widespread application of estragole is limited by its volatility and poor water solubility.

Moreover, external factors such as temperature, light, and accessibility to atmospheric oxygen affect the stability of estragole [START_REF] Turek | Impact of different storage conditions on the quality of selected essential oils[END_REF]. For example, the photooxidation of estragole produced 4-methoxybenzaldehyde (anisaldehyde) and 4-methoxybenzene acetaldehyde [START_REF] Bouvier-Brown | Methyl chavicol: characterization of its biogenic emission rate, abundance, and oxidation products in the atmosphere[END_REF]. Attempts were made to increase the stability of essential oil containing estragole through encapsulation of basil oil in gelatin microcapsules [START_REF] Sutaphanit | Optimisation of microencapsulation of holy basil essential oil in gelatin by response surface methodology[END_REF][START_REF] Chitprasert | Holy basil (Ocimum sanctum Linn.) essential oil delivery to swine gastrointestinal tract using gelatin microcapsules coated with aluminum carboxymethyl cellulose and beeswax[END_REF], in nano-and microemulsion [START_REF] Chitprasert | Holy basil (Ocimum sanctum Linn.) essential oil delivery to swine gastrointestinal tract using gelatin microcapsules coated with aluminum carboxymethyl cellulose and beeswax[END_REF][START_REF] Ghosh | Ultrasonic emulsification of foodgrade nanoemulsion formulation and evaluation of its bactericidal activity[END_REF] or in cyclodextrins (CD) [START_REF] Kfoury | Effect of cyclodextrins complexation on phenylpropanoids solubility and antioxydant activity[END_REF][START_REF] Kfoury | Complexation of estragole as pure compound and as main component of Basil and Tarragon essential oils with cyclodextrins[END_REF][START_REF] Kfoury | Solubility, photostability and antifungal activity of phenylpropanoids encapsulated in cyclodextrins[END_REF]. CDs increased the solubility and photostability of estragole (3-12 and 6-18 fold, respectively, depending on CD type) and also maintained its bioactivity.

Liposomes are viewed as attractive novel materials by the food and medical industries.

Interest in liposome has increased due to their reported functional properties, including their efficient encapsulation capacity, biocompatibility with food constituents and low cost of raw materials used for manufacturing [START_REF] Hsieh | Properties of liposomes prepared with various lipids[END_REF][START_REF] Laridi | Liposome encapsulated nisin Z: optimization, stability, and release during milk fermentation[END_REF][START_REF] Were | Size, stability, and entrapment efficiency of phospholipid nanocapsules containing polypeptide antimicrobials[END_REF][START_REF] Taylor | Liposomal nanocapsules in food science and agriculture[END_REF]. However, the main obstacle to the use of liposomes is oxidation of lipids during storage. The presence of water may favor bacterial growth and promote phospholipid degradation. Therefore, a way to stabilize them is to use freeze-drying process. Unfortunately, lyophilization itself can alter the liposome membrane bilayer. Therefore, it must be conducted according to a defined protocol and with added excipients that preserve the bilayer structure [START_REF] Wolfe | Freezing, drying, and/or vitrification of membrane-solute-water systems[END_REF].

The type of phospholipid and the presence of cholesterol in the bilayer affect the stability of liposome structure during lyophilization in addition to the cryoprotector [START_REF] Chen | An overview of liposome lyophilization and its future potential[END_REF].

Cholesterol is often included in liposome composition because it can modulate the physicochemical properties of the lipid bilayer by disordering the gel state of the lipid bilayer or ordering the liquid state [START_REF] Kaddah | Cholesterol modulates the liposome membrane fluidity and permeability for a hydrophilic molecule[END_REF]. Lyophilization of liposomes without addition of cryoprotectants provokes their coalescence and aggregation. It has been demonstrated that hydroxypropyl-ß-cyclodextrin (HP-ß-CD) maintains the physical and chemical stability of conventional liposomes made from Phospholipon 90H during freeze-drying.

Moreover, the presence of HP-ß-CD in the aqueous phase of DCL system showed a protective effect during freeze-drying [START_REF] Gharib | Hydroxypropyl-ßcyclodextrin as a membrane protectant during freezedrying of hydrogenated and non-hydrogenated liposomes and molecule-incyclodextrin-in-liposomes: Application to trans-anethole[END_REF].

We recently studied the encapsulation of essential oil components in conventional liposomes and in drug-in-CD-in-liposome (DCL). This system, combining the relative advantages of both carriers [START_REF] Mccormack | Drugs-in-cyclodextrins-in-liposomes: a novel concept in drug delivery[END_REF], improved the encapsulation of essential oil components such as trans-anethole [START_REF] Gharib | Preparation of drug-in-cyclodextrin-in-liposomes at a large scaleusing a membrane contactor: Application to trans-anethole[END_REF][START_REF] Gharib | Drug-in-cyclodextrin-inliposomes as a carrier system for volatile essential oil components: application to anethole[END_REF] and eugenol [START_REF] Sebaaly | Greige-gerges, Clove essential oil-in-cyclodextrin-in-liposomes in the aqueous and lyophilized states: from laboratory to large scale using a membrane contactor[END_REF] and reduced drug release compared to conventional liposomes [START_REF] Gharib | Drug-in-cyclodextrin-inliposomes as a carrier system for volatile essential oil components: application to anethole[END_REF].

In the present study, different CL and DCL formulations containing estragole were prepared by ethanol injection method using hydrogenated phospholipid in presence of cholesterol. These liposomes formulations were characterized before and after freezedrying. The size, polydispersity index, zeta potential of liposomes formulations were determined as well as the morphology using transmission electron microscopy. The loading rate of estragole was determined using HPLC. The stability of vesicles was assessed after 6 months of storage at 4ºC in powder form and the release of estragole from the inclusion complex, CL and DCL was studied by multiple headspace extraction at 60ºC. The volatility of estragole and the fluidity of the membrane may affect the estragole encapsulation. Hence, the Henry's law constant of estragole was determined at 30 and 60ºC, and the fluorescence anisotropy of 1,6-diphenylhexatriene (DPH) inserted in the lipid membranes of CL and DCL was studied for the first time before and after freeze-drying at 25 and 37ºC.

Materials and methods

Materials

Hydrogenated phospholipon 90H (90% soybean phosphatidylcholine, 4% lysophosphatidylcholine, 2% triglycerides, 2% water, 0.5% ethanol, 1% iodine) was supplied by Lipoid GmbH (Ludwigshafen, Germany). HP-ß-CD-oral grade (MS=0.85) was obtained from Roquette (Lestrem, France), thymol, absolute ethanol, cholesterol and methanol-HPLC grade were from Sigma-Aldrich (Germany) and estragole was purchased from Sigma-Aldrich (China). Water was purified on a Milli-Q system obtained from a Millipore® synergy system (Millipore, Billerica, Massachusetts, USA).

Preparation of HP-ß-CD/Est inclusion complex

7.44 mg of estragole was added to 5 mL HP-ß-CD solutions (0, 10, 25, 50, 75 and 100 mM). The mixtures were shaken at 25 °C for 24 h then filtered through a 0.45 μm membrane filter. The concentrations of estragole in the filtrates were determined by the HPLC method described below.

The complexation efficiency was calculated using the following equation: where m Est exp is the mass of estragole experimentally determined by HPLC in the inclusion complex solution and m Est i is the mass of estragole initially used to prepare the inclusion complex [START_REF] Kfoury | Investigation of monoterpenes complexation with hydroxypropyl-ß-cyclodextrin[END_REF].

Preparation of HP-ß-CD/Est inclusion complex for DCL preparations

HP-ß-CD was dissolved in ultrapure water and the required amount of estragole was added to obtain HP-ß-CD:Est molar ratio of 75:10 and 100:10; the concentration of HPß-CD were of 111 and 148 mg/mL, respectively. The mixture was stirred (120 rpm for 24 h at 25 °C), filtered through a 0.45 μm membrane, and then used in DCL preparations.

Preparation of liposomes by ethanol injection method

The liposomes were prepared by the ethanol injection method. Phospholipon 90H (10 mg/mL) and cholesterol (5 mg/mL) were dissolved in absolute ethanol. The resulting organic phase (10 mL) was then injected, using a syringe pump (Fortuna optima, GmbH-Germany), into the aqueous phase (20 mL) at 55ºC (temperature above the transition temperature of Phospholipon 90H) under magnetic stirring at 400 rpm. Spontaneous liposome formation occurred as soon as the ethanolic solution was in contact with the aqueous phase. The liposomal suspension was then left for 15 min at 25 ºC under stirring (400 rpm). Finally the ethanol was removed by rotary evaporation (BÜCHI Rotavapor R-100) under reduced pressure at 40 °C. Four batches were prepared: 1) blank-CL; 2) blank-DCL, where HP-ß-CD was dissolved in the aqueous phase at concentrations of 75 (blank-DCL 75) and 100 mM (blank-DCL 100); 3) Est-loaded liposomes (Est-CL), where estragole was added in the organic phase at a concentration of 2.5 mg/mL; 4) HP-ß-CD/Est inclusion complex-loaded liposomes (Est-DCL), where the HP-ß-CD/Est solution prepared previously (Est-DCL 75 and Est-DCL 100 correspond to DCL preparation using HP-ß-CD:Est molar ratio of 75:10 and 100:10, respectively). Each batch was prepared in triplicate and underwent characterization as described below.

Freeze-drying

Freshly prepared blank-CL, blank-DCL, Est-CL, Est DCL 75 and Est DCL 100 (5 mL) underwent ultracentrifugation at 170000 g for 1 h at 4 ºC. The supernatant was discarded and the pellet was reconstituted in 2 mL of water or HP-ß-CD solution (25 and 50 mM) for CL or in 2 mL of HP-ß-CD solution of 75 or 100 mM, for Est-DCL 75 and Est-DCL100, respectively. The liposomes suspensions and the inclusion complex HP-ß-CD/Est molar ratio of 75:10 and 100:10 were freeze-dried according to the protocol described in our previous study [START_REF] Gharib | Hydroxypropyl-ßcyclodextrin as a membrane protectant during freezedrying of hydrogenated and non-hydrogenated liposomes and molecule-incyclodextrin-in-liposomes: Application to trans-anethole[END_REF]. Finally, the vials were removed from the freezedryer, closed with rubber caps and stored at 4 ºC. The lyophilized liposomes were then reconstituted with ultra-pure water to its original volume (5 mL) prior to characterization and further analysis.

Liposome characterization

Dynamic Light Scattering Analysis

Malvern Zetasizer Nanoseries (Zetasizer Nano ZS; Malvern Instruments Ltd, France) was used to determine the mean size of liposome batches, its range of detection is between 30 nm and 10 m. All batches were diluted 10-fold with ultrapure water. The particle size distribution data were collected using the DTS (nano) software (version 5.10) provided with the instrument. The polydispersity index (PdI), which gives an indication of the width of particle size distribution, ranges from 0 (monodispersed) to 1 (very broad distribution). Data were expressed as the mean ± standard deviation. Zeta potential was calculated by Smoluchowski's equation from the electrophoretic mobility of liposomes.

All measurements were carried out at 25 °C after 3 min of equilibration and performed in triplicate.

Morphological characterization by transmission electron microscopy

Blank-DCL 75, blank-DCL 100, Est-DCL 75 and Est-DCL 100 before and after freeze-drying were imaged with transmission electron microscope (TEM) (CM 120; Philips, Eindhoven, Netherlands) operating at an accelerating voltage of 120 kV. A drop of liposomes was placed onto a carbon-coated copper grid for 3 min; the excess suspension was removed with a filter paper, leaving a thin liquid film stretched over the pores.

Negative staining using a sodium silicotungstate solution (1%) was directly made on the deposit over 30 s. The excess sodium silicotungstate solution was then removed with a filter paper and the stained samples were examined.

Determination of encapsulation efficiency and loading rate for estragole in liposomes

The EE of estragole into liposomes was determined upon HPLC analysis of free and total estragole present in the liposome suspensions. Aliquots were removed from the liposomal suspension to determine the total estragole concentration ([Est] Total ). The liposomal suspension underwent ultracentrifugation (Optima TM Ultracentrifuge, Beckman Coulter, USA) at 170000g for 1 h at 4 °C. Aliquots were removed from the supernatant to determine the concentration of free Est ([Est] F ). EE of Est was calculated as follows:

where [Est] Tot and [Est] F corresponds to the concentration of total and free estragole, determined by the HPLC method described below, respectively. The LR of estragole was calculated by the following equation:

(3)

The LR after freeze-drying was calculated using the following equation:

where m initial for Est-DCL is the initial mass of estragole used to prepare CD/Est inclusion complex. For Est-CL, m initial is the initial mass of estragole added to the organic phase during liposome preparation. m reconstituted liposomes is the mass of estragole found in the aqueous phase after freeze-drying and reconstitution of liposome.

HPLC assay of estragole

Stock standard solutions of estragole (1 mg/mL) and of the internal standard, thymol (1 mg/mL) were prepared in methanol. Aliquots were removed from the estragole stock solution and diluted in methanol to obtain final concentrations of estragole ranging from 1 to 250 µg/mL. The diluted solution of thymol (100 µg/mL) was prepared in methanol.

Two hundred µL of each filtrate (aqueous solution of HP-ß-CD/ Est inclusion complex) were added to 200 µL of thymol (100 µg/mL) and 400 µL of methanol. The samples were sonicated for 10 min at room temperature. The samples were analyzed by HPLC (Agilent Technologie 1200 series) using an analytical column C18 15 cm x 4.6 mm, 5 µm, (Agilent Technologies). The mobile phase was a mixture of methanol and water (70/30). The flow rate was fixed at 1 mL/min and the detection was set at 206 nm.

The retention times of thymol (internal standard) and estragole were 5.7 and 8.4 min, respectively. Calibration curves were constructed by plotting the estragole/thymol area ratio against the concentration of estragole in μg/mL. The linear relationships were evaluated by regression analysis with the least squares method and the correlation coefficient ranged from 0.996 to 0.999.

Fluorescence anisotropy measurements

To evaluate the effect of estragole, HP-ß-CD and freeze-drying on the membrane fluidity of vesicles composed of phospholipon 90H:cholesterol, steady state fluorescence polarization technique using a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies) was used. DPH was dissolved in tetrahydrofuran (4.30 10 -3 mol/L) then an aliquot was added to the vesicles suspension (blank-CL, blank-DCL 75, blank-DCL 100, Est-CL, Est-DCL 75 and Est-DCL 100) to obtain DPH:phospholipid molar ratio of 1:500.

The mixture was incubated overnight in the dark to allow the intercalation of the DPH molecules into the lipid bilayer. The final concentration of THF did not exceed 0.01% and showed no effect on the fluorescence anisotropy [START_REF] Whiting | Steroid hormone-induced effects on membrane fluidity and their potential roles in non-genomic mechanisms[END_REF]. The experiments were performed at 25 and 37 ºC. The probe was excited with a manual polarizer accessory (Cary Eclipse Manual Polarizer, Agilent Technologies; Malaysia) using vertically polarized light at 360 nm. The emission intensities were measured at 450 nm at both parallel and perpendicular to the polarization vector of the exciting light yielding components I VV and I VH , respectively. The Cary Eclipse Bio Software, delivered from the spectrophotometer, gives the fluorescence anisotropy (R), which is defined as where and are the fluorescence intensities of the light emitted with its polarization plane parallel ( ) and perpendiculaire ( ) to that of the exciting beam.

Release study

Multiple headspace extraction (MHE) is a dynamic gas extraction carried out stepwise [START_REF] Kolb | Static Headspace-Gas Chromatography: Theory andPractice[END_REF]. At each step, equilibrium conditions are established in the vial between the sample and its gas phase. The vials containing free estragole, Est-CL, Est-DCL 75, Est-DCL 100 or the inclusion complex (HP-ß-CD/Est molar ratio of 75:10 and 100:10) were submitted to 24 successive gas extractions at constant interval (8 min) in the same operating conditions. At each extraction, 1 ml of the vapor from the headspace of the solution was withdrawn from the vial using a gas-tight syringe and injected in the chromatographic column via a transfer line. All measurements were conducted with an Agilent G1888 headspace sampler coupled to a Perkin Elmer Autosystem XL gas chromatography equipped with a flame ionization detector and a DB624 column. Headspace operating conditions were as follows: oven temperature of 60ºC, transfer line temperature of 250ºC and nitrogen as carrier vector. GC conditions were as follows: column temperature of 160ºC and GC cycle of 8 min.

Release of estragole

After 6 months of storage of CL and DCL at 4ºC, the release of estragole from freezedried CL and DCL as well as from reconstituted powders was studied by MHE coupled to gas chromatography. Est-CL, Est-DCL 75 or Est-DCL 100 in powder form (10 mg), Est-CL, Est-DCL 75, Est-DCL 100, HP-ß-CD/Est 75:10, HP-ß-CD/Est 100:10 inclusion complex solutions and estragole (0.5 mg/mL) were placed in 22 ml sealed vials. After equilibrium, the release of estragole at time t was calculated as follows: [START_REF] Shin | Antifungal activity of the essential oil of Agastache rugosa Kuntze and its synergism with ketoconazole[END_REF] Where, A t and A 0 corresponds to the area of the chromatographic peak of estragole at time t and at the first extraction, respectively. The plot of ln (A t /A 0 ) as a function of time followed a first-order release kinetics. The release rate constant K (min -1 ) was calculated using the following equation:

Ln (A t /A 0 ) = -Kt (7)

Loading capacity determination

The amount of estragole in the inclusion complex HP-ß-CD/Est, CL and DCL was determined after 6 months of storage at 4ºC in powder form using MHE. The concentration of the analyte in the gas phase of the vial decreases at each extraction step.

Thus, the sum of the amounts of the analyte removed in the individual extractions will be equal to the total amount of analyte present in the original sample [START_REF] Kolb | Static Headspace-Gas Chromatography: Theory andPractice[END_REF].

A linear regression was constructed for the standard and the samples using the following equation: lnA t =-q(t-1) + lnA 1 [START_REF] Andrade | Isolation, characterization and evaluation of antimicrobial and cytotoxic activity of estragole, obtained from the essential oil of Croton zehntneri (Euphorbiaceae), An[END_REF] where A 1 is the area of the chromatographic peak of estragole at the first extraction; A t is the peak area of estragole at the extraction t and -q is the slope of the regression curve that illustrates ln A t as a function of (t -1).

Q is a constant parameter calculated as follows: [START_REF] Turek | Impact of different storage conditions on the quality of selected essential oils[END_REF] The sum of peak areas corresponding to the total amount of estragole present in the sample or in the standard are obtained as follows: [START_REF] Bouvier-Brown | Methyl chavicol: characterization of its biogenic emission rate, abundance, and oxidation products in the atmosphere[END_REF] The amount of estragole in the IC is calculated using the following relation: [START_REF] Sutaphanit | Optimisation of microencapsulation of holy basil essential oil in gelatin by response surface methodology[END_REF] where ∑A sample and ∑A standard are the sum of peak areas of estragole corresponding to the sample and the external standard, respectively; Std is the amount of the standard (0.005 mg).

Loading capacity (LC m Est /m powder ) of estragole is expressed as mg of encapsulated estragole per gram of the powder of the delivery system. ( 12)

Determination of Henry's law constant of estragole

The Henry's law constant (H c ) is the vapor-liquid partition coefficient expressed as followed [START_REF] Kolb | Static Headspace-Gas Chromatography: Theory andPractice[END_REF]: where C G is the concentration of estragole in the vapor phase and C L , the concentration of estragole in the aqueous phase.

The value of H C for estragole was determined experimentally by static headspace-gas chromatography (SH-GC) at two temperatures (30 and 60ºC). The GC column temperature was set at 160 ºC. The headspace settings were as follows: an oven temperature of 30 or 60 ºC and an equilibrium time of 30 min. The transfer line temperature was set at 250 ºC.

For the determination of H C in water, several headspace vials were prepared containing different amounts of water (0.5; 0.6; 1, 2, 3 and 5 mL) to which the same amount of estragole was added (10 µL from a standard solution of 10000 ppm). Using essentially the phase ratio variation method as described by Kolb and Ettre [START_REF] Kolb | Static Headspace-Gas Chromatography: Theory andPractice[END_REF], the values of H C were determined by the relationship between the reciprocal chromatographic peak areas and the vapor-liquid volumetric ratio (V G /V L , with V G and V L being the vapor volume and the liquid volume, respectively):

where A is the peak area, α is a constant incorporating several parameters and H C is the vapor-liquid partition coefficient, directly calculated from the ratio of the slope and intercept of the fit of the experimental data obtained after allowing a vapor-liquid equilibrium time of 30 min in the oven.

Statistical analysis

Statistical analysis was performed using the Student's t-test. P values equal or less than 0.05 were considered statistically significant.

Results and discussion

HP-ß-CD/estragole inclusion complex

The HP-ß-CD concentration able to solubilize the initial amount of estragole (7.44 mg) was determined; a complete solubilization of this mass leads to a solution containing

Mean size, pdI, zeta potential values of liposomes before and after freeze-drying

The fresh and reconstituted liposome suspensions were characterized and compared in terms of mean particle size, PdI and zeta potential values before and after freeze-drying, respectively and the results are listed in Table 2.

368

Before freeze-drying, the analysis of particle size showed no significant difference 369 between the various formulations, since Est-CLs and Est-DCLs had similar range of 370 particle size of (176-210 nm). Thus the presence of HP-ß-CD or estragole did not affect 371 the liposome size. These results are in accordance with the literature [START_REF] Gharib | Hydroxypropyl-ßcyclodextrin as a membrane protectant during freezedrying of hydrogenated and non-hydrogenated liposomes and molecule-incyclodextrin-in-liposomes: Application to trans-anethole[END_REF][START_REF] Gharib | Drug-in-cyclodextrin-inliposomes as a carrier system for volatile essential oil components: application to anethole[END_REF][START_REF] Maestrelli | New "drug-in cyclodextrin-in deformable liposomes" formulations to improve the therapeutic efficacy of local anaesthetics[END_REF][START_REF] Bragagni | Liposomal formulations of prilocaine: effect of complexation with hydroxypropyl-βcyclodextrin on drug anesthetic effect[END_REF]. 372

Besides, PdI values for all liposome formulations were less than 0.4, suggesting that 373 liposomes suspensions were homogenous (Table 2). The presence of HP-ß-CD or 374 estragole did not affect the zeta potential values, which are negative ranging between (-6; 375 -15 mV).

In a previous work, we evaluated the effect of hydrogenation and composition of soybean phospholipids on the characteristics and the stability of blank-CL and blank-DCL during freeze-drying [START_REF] Gharib | Hydroxypropyl-ßcyclodextrin as a membrane protectant during freezedrying of hydrogenated and non-hydrogenated liposomes and molecule-incyclodextrin-in-liposomes: Application to trans-anethole[END_REF]. The results showed that HP-ß-CD protect hydrogenated liposomes (Phospholipon 90H:cholesterol or Phospholipon 80H:cholesterol formulation) during freeze-drying. Furthermore, dispersing the pellet of CL in HP-ß-CD solution of (25 or 50 mM), and dispersing the pellet of DCL in water or in HP-ß-CD solution (at the same concentration present in the internal aqueous phase of liposome) protect the structures during freeze-drying. Here we applied the conditions to Est-CL and Est-DCL formulations for freeze-drying.

As can be seen in Table 2, after freeze-drying the size, pdI and zeta potential values of liposomes were maintained since no changes were observed for particle analysis before and after freeze drying. These results demonstrate a successful freeze-drying of estragole loaded Phospholipon 90H liposomes.

Encapsulation efficiency and loading rate values of estragole in liposomes

The encapsulation efficiency (EE) and the loading rate (LR) values of estragole loaded CL and DCL before and after freeze-drying were determined according to equations [START_REF] Smith | Safety assessment of allylalkoxybenzene derivatives used as flavouring substances methyl eugenol and estragole[END_REF][START_REF] Siqueira | Cardiovascular effects of the essential oil of Croton zehntneri leaves and its main constituents, anethole and estragole, in normotensive conscious rats[END_REF][START_REF] Soares | Effects of anethole and structural analogues on the contractility of rat isolated aorta: Involvement of voltage-dependent Ca++-channels[END_REF] and the results are listed in Table 3. respectively. In term of LR, DCL represents a better encapsulation system than CL for numerous drugs including volatile ones such as trans-anethole [START_REF] Gharib | Drug-in-cyclodextrin-inliposomes as a carrier system for volatile essential oil components: application to anethole[END_REF] and non-volatile compound like betamethasone [START_REF] Piel | Betamethasonein-cyclodextrin-in-liposome: The effect of cyclodextrins on encapsulation efficiency and release kinetics[END_REF]; celcoxib [START_REF] Jain | Multivesicular liposomes bearing celecoxibβ-cyclodextrin complex for transdermal delivery[END_REF], curcumin [START_REF] Dhule | Curcuminloaded γ-cyclodextrin liposomal nanoparticles as delivery vehicles for osteosarcoma[END_REF] and indomethacin [START_REF] Chen | Preparation, characterization and pharmacokinetics of liposomes-encapsulated cyclodextrins inclusion complexes forhydrophobic drug[END_REF].

Besides, the amount of estragole in CD/drug inclusion complex solutions increased upon increasing the HP-ß-CD:Est molar ratio from 75:10 to 100:10. Same results were observed by Maestrelli et al., [START_REF] Maestrelli | Effect of preparation technique on the properties of liposomes encapsulating ketoprofencyclodextrin complexes aimed for transdermal delivery[END_REF], where the EE values of ketoprofen increased with increasing the concentration of HP-ß-CD:ketoprofen inclusion complex when encapsulated in the aqueous phase of liposomes.

After freeze-drying, the LR of estragole in Est-DCL 75 and Est-DCL 100 were quasi maintained (Table 3).

Morphology

The TEM images showed the formation of nanometer-sized vesicles (Figure 2). The images in Figure 2 (A-D

Release studies

The release of volatile drugs from the samples (aqueous solution of a drug, delivery systems incorporating a drug in liquid or powder form) can be tracked using MHE mode.

The procedure is well described by Kolb & Ettre [START_REF] Kolb | Static Headspace-Gas Chromatography: Theory andPractice[END_REF]. It consists in carrying out a sampling sequence for the same sample. There is equilibrium between the sample and the gas phase in the vial. At every extraction the headspace determined the concentration of estragole in the gas phase. As the extractions proceed, the amount of the volatile guest present in the headspace decreases to zero, as do the areas of the corresponding chromatographic peaks. The sum of the areas obtained will therefore be equal to the amount of guest present in the sample. The MHE therefore allows the illustration of the release profile of the volatile product.

The release studies of estragole from freeze-dried HP-ß-CD/Est inclusion complex, Est-CL and Est-DCL stored at 4ºC for 6 months, were performed by the MHE method. The estragole peak areas of the external standard (0.5 ppm of estragole in water), inclusion complex HP-ß-CD:Est (75:10 and 100:10 molar ratios in water) and estragole loaded liposomes (CL and DCL in powder or reconstituted in water) are illustrated in.

The amount of estragole determined from powder samples (Est-CL, Est-DCL 75 and Est-DCL 100, appeared in the insert of figure 3) was very low suggesting either high retention of Est or weak presence of Est in all powder samples. For this reason, the powders were dissolved in 10 ml water and placed in sealed vials and analyzed by MHE.

The results showed that the estragole peak area increased for the reconstituted samples when compared to freeze-dried samples, meaning that the powder liposomes exhibit a higher retention of estragole than their aqueous dispersed forms. Compared to Est-CL, the encapsulation of estragole in lipid vesicles was improved with Est-DCL 75 and Est-DCL 100 while the HP-ß-CD inclusion complex showed the higher retention of estragole (Figure 3). Reconstituted batches were then considered for the release study and determination of the LC m Est /m powder of estragole. The incorporation of a volatile drug from a delivery system and its release could be affected by its volatility. For that, the vapor-liquid partition coefficient (H C ) of estragole was determined using HS-GC at 30 and 60ºC. The H C value of estragole was 0.03 at 30ºC and increased to 0.07 at 60ºC. Hence, the volatility of estragole increased with temperature. Thus, the HP-ß-CD/Est inclusion complex, CL or DCL improved the retention of estragole and controlled its release. These results were in agreement with Kfoury et al., [START_REF] Kfoury | Promising applications of cyclodextrins in food: Improvement ofessential oils retention, controlled release and antiradical activity[END_REF] who showed that ß-CD derivatives improved the complexation in CDs of essential oils components such as α-pinene, mycrene, limonene, p-cymene and terpinene.

Determination of loading capacity of estragole (m Est /m powder )

The amount of estragole in the inclusion complex obtained at both molar ratios (HP-ß-CD:Est 75:10 and 100:10) and in the reconstituted liposomes was quantified by SH-GC after 6 months of storage at 4ºC. The LC values expressed as m Est /m powder and calculated using equation 12, are listed in Table 5. The LC value of estragole in Est-CL was 0.01 μg of Est/mg of powder. The CL formulations contain small amount of estragole, which was not detected by HPLC. The encapsulation of estragole in DCL system was improved with LC values of 0.6 and 0.2 μg of Est/mg of powder for Est-DCL 75 and Est-DCL 100, respectively. However, the LC values of estragole in the inclusion complex HP-ß-CD/Est 75:10 and 100:10 were 10.8 and 9.3 μg of Est/mg of powder, respectively. These values were higher than those of Est-DCL 75 and Est-DCL 100. These results suggest that DCL system improve the encapsulation of estragole compared to CL. The inclusion complex HP-ß-CD/Est showed the best ability to retain high amount of estragole. Among the three systems, CD presents the higher LC followed by Est-DCL and Est-CL.

Fluorescence anisotropy

The fluorescence anisotropy measurements of DPH in liposomes give information on membrane fluidity of liposomes membrane [START_REF] Reiner | Effects of Gabaergic Phenols on Phospholipid Bilayers as Evaluated by 1H-NMR[END_REF]. The DPH anisotropy values obtained at 25 and 37ºC for blank-CL, blank-DCL 75, blank-DCL 100, Est-CL, Est-DCL 75 and Est-DCL 100 before and after freeze-drying are presented in Table 6. To the best of our knowledge, this is the first example of DPH fluorescence anisotropy measurements conducted on freeze-dried DCLs. As expected, an increase of temperature leads to an increase of membrane fluidity.

Compared to blank-CL, the presence of estragole or the inclusion complex HP-ß-CD/Est (75:10 and 100:10 molar ratio) did not affect the DPH anisotropy values at 25 and 37ºC.

The results obtained for Est-CL has been expected since a small amount of estragole was encapsulated in CL, which could not affect the membrane fluidity. Gharib, et al., [START_REF] Gharib | Interaction of Selected Phenylpropenes with Dipalmitoylphosphatidylcholine Membrane and Their Relevance to Antibacterial Activity[END_REF] demonstrated that the presence of estragole in DPPC membrane, at molar ratios Est:DPPC largely higher than those obtained in this study, reduced the DPH anisotropy values at 28, 41 and 50ºC in a concentration-dependent manner.

Besides, dispersing the pellet of CL and DCLs with HP-ß-CD solution before freeze drying did not affect the membrane fluidity, since the DPH anisotropy values remain unchanged after freeze-drying for all batches (Table 6). These results proved that HP-ß-CD could be used as membrane protectant during freeze-drying for CL and for DCL system made from saturated phospholipids.

Conclusion

The characteristics of hydrogenated-liposomes (size, pdI, zeta potential and LR of estragole) were maintained after freeze-drying using HP-ß-CD as cryoprotectant for CL and DCL systems. Moreover, HP-ß-CD did not affect the fluidity of phospholipon 90H/cholesterol. In addition, the presence of estragole within HP-ß-CD, CL or DCL reduced its volatility and allowed its controlled release. The HP-ß-CD:Est inclusion complex and Est-DCL system showed high retention of estragole and displayed a good stability. These results suggest that freeze-dried CD inclusion complexes and DCL systems could be considered as efficient carriers for essential oil components retention, preservation, storage and controlled release of estragole.

Figure 1 :

 1 Figure 1: The variation of the concentration of estragole (g/ml) with CD concentration.

  ) and (A'-D') are examples of blank-DCL 75, blank-DCL 100; Est-DCL 75 and Est-DCL 100 obtained before and after freeze drying, respectively. The TEM results are in agreement with the dynamic light scattering results since nanometric sized vesicles appeared in the images. The vesicles are oligolamellar and spherical in shape. CLs and DCLs loading estragole present similar structures to trans-anethole and eugenol loaded CL and DCL [25, 29].

Figure 2 :

 2 Figure 2: TEM images of Phospholipon 90H liposomes before freeze-drying: blank-DCL 75 (A); blank-DCL 100 (B); Est-DCL 75 (C) and Est-DCL 100 (D) and after freeze drying: blank-DCL 75 (A'); blank-DCL 100 (B'); Est-DCL 75 (C') and Est-DCL 100 (D').

Figure 3 :

 3 Figure 3: The estragole peak areas of the external standard (0.5 ppm of Est in water), inclusion complex HP-ß-CD:Est (75:10 and 100:10 molar ratios in water) and Est loaded liposomes (CL and DCL in powder or reconstituted in water). The insert represents the peak areas for CL and DCL in powder form and CL reconstituted in water.

Figure 4

 4 Figure 4 resumes the percentage of remaining estragole from the reference solution (0.5

Figure 4 :

 4 Figure 4: The percentage of remaining Est from Est external standard (0.5 ppm of estragole in water), inclusion complex HP-ß-CD:Est (75:10 and 100:10 molar ratios in water) and estragole loaded liposomes (CL and DCL reconstituted in water).

Figure 5 :

 5 Figure 5 : The plot of ln (A t /A 0 ) as a function of time for Est external standard (0.5 ppm of estragole in water), inclusion complex HP-ß-CD:Est (75:10 and 100:10 molar ratios in water) and estragole loaded liposomes (CL and DCL reconstituted in water).

Table 1 :

 1

	CE CD (%)

Complexation efficiency of estragole in HP-ß-CD as a function of CD concentration HP-ß-CD :Est molar ratio

Table 2 :

 2 Size, polydispersity index, zeta potential values for fresh and reconstituted Est-CL and 363 Est-DCL from Phospholipon 90H before and after freeze-drying. The values obtained after

	364
	365	lyophilization were compared to those before lyophilization and * indicates a value of P < 0.05
	366	statistically significant.
	367
				Before freeze-drying		After freeze-drying
			Size (nm)	pdI	Zeta (mV)	Dispersion of the pellet with	Size (nm)	pdI	Zeta (mV)
						CD 25 mM	169 ± 8	0.22 ± 0.04	-7.4 ± 1.7
	Blank-CL	179 ± 10 0.08 ± 0.01	-9.1 ± 6.3
						CD 50 mM	180 ± 17	0.15 ± 0.02* -5.0 ± 4.0*
						CD 25 mM	173 ± 0.3 0.14 ± 0.01* -6.4 ± 0.2
	Est-CL		176 ± 2	0.10 ± 0.2	-9.0 ± 0.9
						CD 50 mM	165 ± 9	0.14 ± 0.01* -2.4 ± 1.6*
	Blank-DCL 75	205 ± 3	0.23 ± 0.00	-10.7 ± 1.4	Water CD 75 mM	201 ± 2 198 ± 3	0.16 ± 0.00* -3.8 ± 0.2* 0.12 ± 0.00 -12.2 ± 3.8
	Est-DCL 75 191 ± 13 0.28 ± 0.07	-6.8 ± 6.2	CD 75 mM	187 ± 3	0.14 ± 0.07	-9.2 ± 3.7
	Blank-DCL 100	210 ± 17 0.31 ± 0.01 -15.8 ± 10.3	Water CD 100 mM	192 ± 6.9 197 ± 16	0.16 ± 0.03 0.21 ± 0.07 -10.1 ± 3.8 -8.2 ± 0.1
	Est-DCL 100	188 ± 19 0.39 ± 0.13	-12.3 ± 2.2	CD 100 mM	209 ± 16	0.14 ± 0.00* -11.7 ± 4.5

Table 3 :

 3 Encapsulation efficiency of Est for fresh CL and DCL suspensions and loading rate of Est in fresh and reconstituted CL and DCL before and after freeze-drying.Before freeze-drying, the estragole concentration in CL formulations was under limit of estragole detection by HPLC method, suggesting that Phospholipon 90H-CL were not able to encapsulate estragole by ethanol injection method. The preparation of liposomes requires heating at 55ºC, above the main transition temperature of Phospholipon 90H, which could induce the loss of volatile estragole during preparation. This is in line with H C value of estragole as it increased with temperature. The DCL carrier system improved the estragole encapsulation. Indeed, the EE values of estragole were 8.47 ± 1.21 and 11.36 ± 2.31 % for Est-DCL 75 and Est-DCL 100 respectively and the LR values of estragole were 1.58 ± 0.25 and 3.25 ± 0.68 % for Est-DCL 75 and Est-DCL 100

		Before freeze-drying	After freeze-drying
		Fresh liposomes	Reconstituted
				liposomes
		EE (%)	LR (%)	LR fd (%)
	Est-CL	ND*	ND*	ND*
	Est-DCL 75	8.47 ± 1.21	1.58 ± 0.25	1.30 ± 0.78
	Est-DCL 100 11.36 ± 2.31	3.25 ± 0.68	2.52 ± 1.15
	ND: not detected

Table 4 :

 4 Release rate constants of free estragole, HP-ß-CD/Est inclusion complex, Est-CL and

	Est-DCL formulations
	Release rate constant K
	(min -1 )
	Est 0.5 ppm	0.013
	HP-ß-CD/Est 100:10	0.008
	HP-ß-CD/Est 75:10	0.009
	Est DCL 75 reconstituted	0.010

Table 5 :

 5 Loading capacity m Est /m powder in HP-ß-CD/Est inclusion complex and in Est-CL and

	Est-DCL carrier systems after 6 months of storage at 4ºC.
	LC (μg of Est/mg of powder)
	HP-ß-CD:Est 75:10	10.8
	HP-ß-CD:Est 100:10	9.3
	Est-CL	0.01
	Est-DCL 75	0.6
	Est-DCL 100	0.2

Table 6 :

 6 DPH fluorescence anisotropy measurements for blank-CL, blank-DCL, Est-CL and Est-DCL made from Phospholipon 90H and cholesterol at 25 and 37ºC before and after freeze-drying.Values are expressed as the means of three repetitions ± SD; n.d.: not determined  P<0.05 compared to those before freeze-drying

		Before freeze-drying	After freeze-drying
		25ºC	37ºC	25ºC	37ºC
	Blank-CL	0.22 ± 0.01	0.17 ± 0.00	n.d.	n.d.
	Blank-DCL 75	0.21 ± 0.00	0.17 ± 0.00	0.21 ± 0.00	0.17 ± 0.00
	Blank-DCL 100	0.20 ± 0.00	0.18 ± 0.01	0.20 ± 0.00	0.18 ± 0.00
	Est-CL	0.22 ± 0.01	0.18 ± 0.01	n.d.	n.d.
	Est-DCL 75	0.22 ± 0.01	0.18 ± 0.01	0.22 ± 0.01	0.18 ± 0.01
	Est-DCL 100	0.21 ± 0.00	0.18 ± 0.01	0.21 ± 0.01	0.18 ± 0.01

Acknowledgements

The research was supported by the Research Funding Program at the Lebanese University and the "Agence Universitaire de la Francophonie", projet PCSI (2018-2020)

estragole (10 mM). Figure 1 shows the variation of estragole concentration in CD/Est inclusion complex solution at various HP-ß-CD concentrations. In the absence of CD, the concentration of estragole was 17.73 ± 2.82 g/ml. As we can see, a CD concentration of 100 mM was necessary to solubilize 7.44 mg of estragole. Table 1 shows the complexation efficiency of estragole at various HP-ß-CD concentrations. The CE CD values were calculated using equation 1. The CE CD value was 34% at HP-ß-CD:Est molar ratio of 10:10, then CE CD values increased with HP-ß-CD concentration, which is in agreement with Kfoury et al. [START_REF] Kfoury | Complexation of estragole as pure compound and as main component of Basil and Tarragon essential oils with cyclodextrins[END_REF]. The CE CD values of estragole at HP-ß-CD:Est molar ratio of 75:10 and 100:10 were 91 and 100%, respectively (Table 1). Consequently, the HP-ß-CD:Est molar ratios of 75:10 and 100:10 were used to prepare DCL batches. We should mention that CDs at high concentration can perturb the liposomes membrane properties and extract lipid components [START_REF] Hammoud | Cyclodextrin-membrane interaction in drug delivery and membrane structure maintenance[END_REF]. For that, we did not use a concentration of HP-ß-CD above 100 mM as our aim was to develop the DCL system loading estragole.

Hence, the limitation of CD concentration and the solubilization of estragole were mainly considered in the selection of CD:Est molar ratio.


Elissa Ephrem

Amal Najjar

Catherine Charcosset

Hélène Greige-Gerges
email: greigegeorges@yahoo.com

Use of free and encapsulated nerolidol to inhibit the survival of Lactobacillus fermentum in fresh orange juice

Keywords: Cyclodextrin, fruit juice, lactic acid bacteria, liposome, sesquiterpene CFU: colony forming unit, CL: conventional liposome, DCL drug-incyclodextrin-in-liposome, DLS: dynamic light scattering, DMSO: dimethyl sulfoxide, HP-β-CD: hydroxypropyl-β-cyclodextrin, HPLC: high performance liquid chromatography, Ner: nerolidol, MRS: de Man, Rogosa, and Sharpe, PdI: polydispersity index, SEM: scanning electron microscopy, TA: titratable acidity, TEM: transmission electron microscopy

Lactobacillus fermentum is commonly responsible for fruit juice fermentation and spoilage. The aim of this study was to investigate the potential use of nerolidol to control the spoilage of fresh orange juice by L. fermentum. Nerolidol was incorporated into hydroxypropyl-β-cyclodextrin inclusion complex, conventional liposome, and drug-in-cyclodextrin-in liposome systems. The systems were lyophilized and characterized with respect to their nerolidol content, size, and morphology. The effects of the acidity and cold storage of orange juice on the survival of L. fermentum were evaluated. Subsequently, the antibacterial activity of nerolidol in refrigerated orange juice was assessed at pH 3.3. Nerolidol showed a faster antibacterial activity at 4 000 µM (5 days) compared to 2 000 µM (8 days). Under the same conditions, the inclusion complex completely killed bacteria within 6 days of incubation at 4 000 µM, suggesting its potential 2 application in fruit juices. Nerolidol-loaded liposomes did not exhibit an antibacterial activity and altered the appearance of juice.

Introduction

Orange juice is consumed worldwide and has a number of functional attributes, being rich in bioactive compounds such as polyphenols, carotenoids, and vitamin C. Protecting orange juice from chemical and microbiological spoilage has been a major focus of the juice industry for years. Orange juice is susceptible to spoilage by a wide range of acid-tolerant microorganisms, including fungi, lactic acid bacteria, and spore-forming Alicyclobacillis [START_REF] Ephrem | Encapsulation of natural active compounds, enzymes, and probiotics for fruit juice fortification, preservation, and processing: An overview[END_REF].

Different strategies have been adopted to prevent this spoilage, including the use of food additives with or without the use of different pasteurization techniques. Various preservatives are currently added to orange juice, primarily nitrates, sorbates, and benzoates. However, because these preservatives can cause allergic responses and be converted to potential carcinogens [START_REF] Anand | Artificial preservatives and their harmful effects: looking toward nature for safer alternatives[END_REF], there is currently a need to identify alternative bioactive agents for orange juice preservation.

Nerolidol (Ner) is a sesquiterpene alcohol that is present in the essential oils of plants such as lemon grass (Cymbopogon spp.), lavender (Lavandula spp.), and tea tree (Melaleuca alternifolia). Ner has been approved for use as a flavouring agent in food products by the United States Food and Drug Administration [START_REF] Chan | Nerolidol: A sesquiterpene alcohol with multi-faceted pharmacological and biological activities[END_REF] and has been shown to exhibit antimicrobial activity against different microorganisms, including Streptococcus mutans, Staphylococcus aureus, and Salmonella enterica [START_REF] Chan | Nerolidol: A sesquiterpene alcohol with multi-faceted pharmacological and biological activities[END_REF]. However, Ner has a low aqueous solubility and photostability [START_REF] Azzi | First evaluation of drug-in-cyclodextrin-in-liposomes as an encapsulating system for nerolidol[END_REF][START_REF] Mcginty | Addendum to fragrance material review on nerolidol (isomer unspecified)[END_REF], requiring its incorporation into appropriate encapsulation systems to promote its use in food preservation.

The use of encapsulation systems (e.g., liposomes, nanoemulsions, and cyclodextrin inclusion complexes) to protect natural antimicrobials and enhance their aqueous solubility in food systems has been described in a number of previous studies [START_REF] Astray | A review on the use of cyclodextrins in foods[END_REF][START_REF] Blanco-Padilla | Food antimicrobials nanocarriers[END_REF][START_REF] Weiss | Nanostructured encapsulation systems: food antimicrobials[END_REF]. Antimicrobial-loaded encapsulation systems have also been demonstrated to be effective for fruit juice preservation in many studies [START_REF] Ghosh | Eugenol-loaded antimicrobial nanoemulsion preserves fruit juice against, microbial spoilage[END_REF][START_REF] Shah | Nanocapsular dispersion of thymol for enhanced dispersibility and increased antimicrobial effectiveness against Escherichia coli O157:H7 and Listeria monocytogenes in model food systems[END_REF][START_REF] Sugumar | Nanoemulsion of orange oil with non ionic surfactant produced emulsion using ultrasonication technique: evaluating against food spoilage yeast[END_REF][START_REF] Truong | Effect of alpha-cyclodextrin-cinnamic acid inclusion complexes on populations of Escherichia coli O157:H7 and Salmonella enterica in fruit juices[END_REF]. [START_REF] Azzi | First evaluation of drug-in-cyclodextrin-in-liposomes as an encapsulating system for nerolidol[END_REF] previously prepared hydroxypropyl-β-cyclodextrin (HP-β-CD)/Ner inclusion complex, Ner-loaded conventional liposome (CL), and Ner-in-cyclodextrin-in-liposome (DCL) systems. The prepared aqueous formulations were optimized and characterized in terms of size, encapsulation efficiency, loading rate, release rate, photostability, and storage stability and yielded preparations with high encapsulation efficiencies and loading rates. Moreover, the DCL system exhibited the lowest release rate of Ner, followed by CLs and the inclusion complexes. Significant enhancements of the photostability and storage stability (4 °C) of Ner were also observed.

Ner has been previously shown to exhibit outstanding activity against Lactobacillus fermentum in de Man, Rogosa, and Sharpe (MRS) broth (pH 6.2, 37 °C) [START_REF] Ephrem | Selection of nerolidol among a series of terpenic and phenolic compounds for its potent activity against Lactobacillus fermentum ATCC 9338[END_REF]. In this study, L. fermentum ATCC 9338, an obligate heterofermentative, gram-positive, anaerobic, thermoacidophilic lactic acid bacterium linked to fruit juice spoilage was used as a model strain.

According to [START_REF] Lawlor | Microbiological spoilage of beverages[END_REF], heterofermentative lactic acid bacteria are the major contributors to fruit juice spoilage. These bacteria can produce different products in fruit juices, including lactic acid, acetic acid, ethanol, and carbon dioxide, which are primarily responsible for the alteration of the organoleptic properties of juice [START_REF] Ashurst | Chemistry and Technology of Soft Drinks and Fruit Juices[END_REF]. [START_REF] Aneja | Microbes associated with freshly prepared juices of citrus and carrots[END_REF] reported 23.3% occurrence of lactic acid bacteria in orange juice. [START_REF] Garcia | Identification of lactic acid bacteria in fruit pulp processing byproducts and potential probiotic properties of selected Lactobacillus strains[END_REF] identified up to 24 L. fermentum strains in different fruit pulp processing byproducts, including pineapple, mango, strawberry, soursop, and Barbados cherry. L. fermentum was also isolated from unpasteurized orange juice [START_REF] Parish | Isolation and identification of lactic acid bacteria from samples of Citrus Molasses and unpasteurized orange juice[END_REF] and from pasteurized mixed fruit juice (pineapple, orange, paw-paw, and guava) stored in the presence of sodium benzoate (0.08%) [START_REF] Uzoukwu | Microbiological status of mixed fruit juice preserved with different concentrations of sodium benzoate[END_REF].

In this study, HP-β-CD/Ner inclusion complexes and Ner-loaded CLs and DCLs were prepared as previously reported by [START_REF] Azzi | First evaluation of drug-in-cyclodextrin-in-liposomes as an encapsulating system for nerolidol[END_REF]. The efficacy of free Ner, Ner solubilized in dimethyl sulfoxide (DMSO), HP-β-CD/Ner inclusion complexes, and Ner-loaded CLs and DCLs systems was investigated in orange juice against L. fermentum.

Materials and methods

Chemical and reagents

Nerolidol (98%, mixture of cis (40%) and trans (60%) isomers), thymol (>99%), absolute ethanol, methanol (HPLC-grade), and DMSO were purchased from Sigma-Aldrich (Missouri, United States). HP-β-CD was purchased from Wacker-Chemie (Lyon, France). De Man, Rogosa, and Sharpe (MRS) broth and MRS agar were purchased from Laboratorios Conda (Madrid, Spain). Lipoid E80 (80-85% egg phosphatidylcholine, 7-9.5% phosphatidylethanolamine, 2% water, and 0.2% ethanol) was purchased from Lipoid GmbH (Ludwigshafen, Germany). Citric acid was purchased from Sigma-Aldrich (Steinheim, Germany).

Bacterial strain and culture conditions

L. fermentum ATCC 9338 was purchased from the American Type Culture Collection (Virginia, USA). L. fermentum cultures were routinely maintained on MRS agar at 4 °C. Before each antimicrobial assay, fresh cultures were prepared in sterile MRS broth and incubated at 37 °C for 22 h under anaerobic conditions. A bacterial suspension was prepared by diluting the bacterial culture in MRS broth to a final concentration of 25 × 10 5 CFU(colony forming unit)/mL.

Preparation of HP-β-CD/Ner inclusion complexes, conventional liposomes and drugin-cyclodextrin-in-liposomes loaded with Ner

HP-β-CD/Ner inclusion complexes as well as liposomes (CL and DCL systems) loaded with Ner were prepared as previously described by [START_REF] Azzi | First evaluation of drug-in-cyclodextrin-in-liposomes as an encapsulating system for nerolidol[END_REF]. Ner inclusion complexes were prepared using the freeze-drying method. First, an aqueous solution (300 mL) containing HP-β-CD (25 mM) and an excess of Ner (667 mg) was magnetically stirred at 300 rpm for 24 h at room temperature. Then, the suspension was filtered (0.45 µm, cellulose acetate membrane) to remove excess Ner, after which it was frozen at -80 °C and lyophilized. Blank HP-β-CD was prepared similarly without Ner. The obtained powders were stored at 4 °C until usage.

CLs and DCLs loaded with Ner were prepared using the ethanol injection method as previously described by [START_REF] Azzi | First evaluation of drug-in-cyclodextrin-in-liposomes as an encapsulating system for nerolidol[END_REF]. Briefly, to prepare CLs, E80 phospholipids were dissolved in ethanol at 50 mg/mL (0.066 M), after which Ner was added to the organic phase at an E80 phospholipid to Ner molar ratio of 100:10. The organic phase (10 mL) was then added to 20 mL of ultrapure water at an injection flow rate of 1 mL/min and under magnetic stirring (400 rpm) at room temperature. The obtained suspension was magnetically stirred for 15 min, after which ethanol and a small volume of water were evaporated under reduced pressure at 25 °C, and a final volume of 17 mL was obtained. Blank CLs were prepared similarly without Ner.

Ner-loaded DCLs were prepared following the same method as CL except that the organic phase (10 mL) was added to 20 mL of the HP-β-CD/Ner inclusion complex aqueous solution, which was prepared as described above. The obtained DCL system had an E80:HP-β-CD:Ner molar ratio of 100:75:30. For the preparation of blank DCLs, the organic phase was added to an aqueous solution of HP-β-CD (25 mM).

The CL and DCL suspensions were centrifuged at 15 000 rpm for 30 min at 4 °C to eliminate the excess aqueous media. HP-β-CD, used as cryoprotectant, was added to the suspensions at a concentration of 25 mM [START_REF] Gharib | Hydroxypropyl-ßcyclodextrin as a membrane protectant during freeze-drying of hydrogenated and nonhydrogenated liposomes and molecule-in-cyclodextrin-in-liposomes: Application to transanethole[END_REF][START_REF] Sebaaly | Clove essential oil-in-cyclodextrin-in-liposomes in the aqueous and lyophilized states: From laboratory to large scale using a membrane contactor[END_REF], after which the samples were frozen at -80 °C and lyophilized. The obtained powders were stored at 4 °C until usage.

Characterization of delivery systems

Determination of Ner incorporation in the delivery systems by HPLC analysis

The total concentration of Ner in the encapsulation systems was determined by HPLC as previously described [START_REF] Azzi | First evaluation of drug-in-cyclodextrin-in-liposomes as an encapsulating system for nerolidol[END_REF]. The HPLC instrument used was a Hitachi LaChrom

Elite® HPLC system (L-2130 pump, L-2200 autosampler) coupled to a L-2455 diode array detector. Samples were prepared at a concentration of 1 mg/mL in water. To generate a calibration curve, standard solutions of Ner in methanol were prepared at concentrations ranging from 2.5 to 250 μg/mL, with thymol used as an internal standard at a concentration of 100 μg/mL in methanol. For HPLC analyses, 100 μL of the Ner standard solutions, liposomal suspensions, or inclusion complex solutions were added to 200 μL of methanol and 100 μL of thymol solution (100 μg/mL). The samples were then sonicated for 10 min at 4 °C and centrifuged at 15 000 rpm for 30 min at 4 °C. The supernatant (20 μL) was then analysed using a reversed-phase C18

Agilent column (150 × 4.6 mm, 5 µm), using a mobile phase of methanol/ultrapure water (75/25 v/v) at a flow rate of 1 mL/min. The absorbance was measured at 212 nm and used to calculate the mass of Ner per mg of powder.

Size, PdI, and zeta potential of liposome formulations

The mean size and polydispersity index (PdI) values of the freeze-dried CLs and DCLs suspended in water were determined by dynamic light scattering (DLS) using a Malvern Zetasizer (3600, Orsay, France). The zeta potential was determined using a Malvern Zetasizer nanoSeries. Each sample was diluted to obtain a count rate between 150 and 300 kilo counts per second. The measurements were repeated in triplicates at 25 °C.

Transmission electron microscopy analysis of liposome formulations

The morphologies of the liposomal formulations were determined by transmission electron microscopy (TEM) using a Philips CM120 microscope (Eindhoven, Netherlands) at the "Centre Technologique des Microstructures" (CTμ) at the University of Lyon (Villeurbanne, France).

The analyses were performed at an acceleration of 80 kV. One drop of each sample was placed on a copper grid coated with carbon. After 2 min, the excess sample was removed using a filter paper to obtain a thin film on the grid. Negative staining was performed using a 1% sodium silicotungstate solution. After 30 s, the excess sodium silicotungstate solution was removed with filter paper, and the sample was then left to dry at room temperature.

Scanning electron microscopy analysis

Scanning electron microscopy (SEM) analyses were performed using an FEI Quanta 250 FEG microscope at the "Centre Technologique des Microstructures" (CTμ) at the University of Lyon (Villeurbanne, France). Freeze-dried samples of HP-β-CD, HP-β-CD/Ner complexes, blank liposomes (CLs or DCLs), or Ner-loaded liposomes (CLs or DCLs) were deposited on a flat steel holder. The samples were then coated under vacuum by cathodic sputtering with copper and observed by SEM under an accelerating voltage of 10 kV.

Preparation and quality evaluation of orange juice samples

Valencia oranges (Citrus sinensis) were purchased from a Lebanese local market. Unblemished fruits were selected, washed, and squeezed using a manual hand juicer. Physicochemical parameters (pH, titratable acidity, and Brix degree) were assessed to evaluate the quality of the orange juice during the experimental procedures. The pH of orange juice was determined using a pH meter (Orion Star A111, Thermo Scientific, USA). The pH measures the total concentration of free H + ions in a solution. The titratable acidity (TA) was determined by titration with a sodium hydroxide (0.1 N) solution. The TA represents the total concentration of organic and inorganic acids present in the juice and is expressed as grams of citric acid per litre of orange juice, as citric acid is the major organic acid in orange juice. The total soluble solids (Brix degree) was determined by refractometry using a hand refractometer (713528, Carl Zeiss Jena, Germany). The Brix degree is an indicator of the sugar content in juice, where 1 Brix degree is expressed as 1 g of sucrose in 100 g of juice.

Bacterial studies

Bacterial survival and proliferation in orange juice

The ability of L. fermentum to survive or proliferate in orange juice was investigated at two different storage temperatures (4 and 25 °C). In addition, the sensitivity of L. fermentum to the pH of orange juice was investigated at pH values between 2.5 and 3.9. When needed, the pH of the orange juice samples was lowered using a citric acid solution (3 M). A 200-µL volume of a diluted bacterial suspension (25 × 10 5 CFU/mL) was added to 4.8 mL of orange juice in glass culture tubes (20 × 100 mm) under sterile conditions to yield an initial bacterial count of 10 5 CFU/mL in juice. Control tubes contained orange juice without L. fermentum. The bacterial counts were then determined at 0, 24, 48, and 96 h using the plate count method. To this end, samples were serially diluted in peptone water, and 100 µL of each dilution was spread onto MRS agar. The bacterial counts were then determined after incubating the plates at 37 °C for 22 h. Each experiment was performed in triplicate. with or without L. fermentum, served as controls for bacterial growth. In addition, control tubes containing orange juice alone were used to exclude the possibility of contamination with lactic acid bacteria. The prepared tubes were then incubated at 4 °C, and changes in the bacterial counts were followed over 10 days of incubation at 4 °C by the plate count method as described above.

Antibacterial activity of

Antibacterial activity of encapsulated Ner in refrigerated orange juice

HP-β-CD/Ner complex and Ner-loaded CLs and DCLs were tested against L. fermentum in orange juice. Each freeze-dried formulation was added to fresh orange juice (4.8 mL) under sterile conditions to yield the desired concentration of Ner (2 000 or 4 000 µM). The tubes were then inoculated with L. fermentum as previously described and incubated at 4 °C. The bacterial count was assessed over time (20 days) using the plate count method. The experiment was repeated in triplicate.

Statistical analysis

Statistical analysis was performed using Student t-test, and P values equal to or less than 0.05 were considered to be significant.

Results and discussion

3.1 Characterization of the HP-β-CD/Ner complex, CL, and DCL systems

Determination of the mass of Ner/mass of freeze-dried encapsulation system

The HPLC analysis results showed that the retained mass of Ner in the HP-β-CD/Ner complexes, CLs, and DCLs was 40, 30, and 15 µg Ner/mg, respectively.

Size, homogeneity, and morphology of freeze-dried liposomes reconstituted with water

The DLS analysis of the four liposomal formulations (blank CLs, Ner-loaded CLs, blank DCLs, Ner-loaded DCLs) showed three populations, in contrast to the two populations obtained by laser granulometry analysis of non-freeze dried suspensions [START_REF] Azzi | First evaluation of drug-in-cyclodextrin-in-liposomes as an encapsulating system for nerolidol[END_REF]. The PdI values of these samples were higher than 0.48 (Table 1), highlighting the low homogeneity of the suspensions. Moreover, the second population represents the major population and had a mean particle size of approximately 1 µm, which is higher than that previously reported by [START_REF] Azzi | First evaluation of drug-in-cyclodextrin-in-liposomes as an encapsulating system for nerolidol[END_REF]. Indeed, the lyophilization process may promote liposome aggregation and fusion. [START_REF] Gharib | Hydroxypropyl-ßcyclodextrin as a membrane protectant during freeze-drying of hydrogenated and nonhydrogenated liposomes and molecule-in-cyclodextrin-in-liposomes: Application to transanethole[END_REF] previously reported on the increment of the size and the PdI of the unsaturated lipoid S100 liposomal suspensions during the freeze drying process, unlike liposomes prepared with saturated phospholipids (phospholipon 90 H). Similarly, in this study the liposomal formulations prepared with the unsaturated lipoid E80 demonstrated an increase of the size following the freeze-drying process.

The incorporation of Ner in CL increased the size of particles, similarly to the results reported by [START_REF] Azzi | First evaluation of drug-in-cyclodextrin-in-liposomes as an encapsulating system for nerolidol[END_REF]. In contrast, Ner had no effect on the size of DCL particles. On the other hand, blank CL and DCL showed a negative zeta potential, which was not greatly affected by the presence of Ner. Also, the obtained zeta potential values were similar to those previously reported in literature for lipoid E80 liposomes [START_REF] Karn | Characterization and stability studies of a novel liposomal cyclosporin A prepared using the supercritical fluid method: comparison with the modified conventional Bangham method[END_REF]. TEM analysis supported the results obtained by DLS, as two populations were observed for blank and Ner-loaded CL and DCL systems (Fig. 1). However, particles with a mean diameter close to or higher than 2 µm were not observed by TEM for all formulations (Fig. 1). SEM analysis of the freeze-dried CL and DCL showed irregular particles structures (Fig. 2). On the other hand, untreated HP-β-CD sample appeared as amorphous spherical particles, and the HP-β-CD/Ner inclusion complexes were observed as irregular particles (Fig. 3). Similar results were obtained by [START_REF] Figueiras | Preparation and physicochemical characterization of omeprazole:methyl-beta-cyclodextrin inclusion complex in solid state[END_REF] and [START_REF] Li | Physicochemical characterization of inclusion complex of catechin and glucosyl-β-Cyclodextrin[END_REF] for untreated CD powder, images of HP-β-CD prepared in the same manner as the HP-β-CD/Ner complexes (dissolution, filtration, and lyophilization) were similar to those of the inclusion complexes. The SEM analysis results could not demonstrate the formation of the inclusion complexes, as the newly observed structures are a direct consequence of the preparation method.

Bacterial studies

Evaluation of orange juice quality parameters

Physicochemical quality parameters were assessed for all of the freshly-squeezed orange juice samples, which exhibited pH values between 3.5 and 3.9, titratable acidity (TA) values between 5 and 6 g/L of citric acid, and Brix degree values of approximately 11-12 °Brix. When orange juice was stored at 4 °C, the values of these parameters were maintained and were unaffected by the presence of L. fermentum due to the inhibition of bacterial proliferation and metabolic activity at this temperature. At 25 °C and after 96 h of incubation, the TA increased to 9.4 ± 0.45 g/L of citric acid, equivalent to that of orange juice spiked with L. fermentum, and the Brix degree was decreased by approximately 1 °Brix, whereas no significant changes were noted in the pH value (data not shown). In addition, similar variations in TA and Brix degree values were observed in the tubes without L. fermentum at 96 h due to the proliferation of naturally occurring spoilage bacteria or fungi in orange juice.

L. fermentum survival and proliferation in orange juice

L. fermentum was able to survive in freshly-squeezed orange juice (pH 3.5-3.9; TA 5-6 g/L citric acid; 11-12 °Brix) at 4 °C until the end of the experiment (96 h) (Fig. 4) and exhibited exponential growth at 25 °C, with an approximately 5.5 log increase of the bacterial count after 96 h of incubation (Fig. 4). To further investigate the tolerance of L. fermentum to acidity in orange juice, the survival and proliferation of L. fermentum was tested at 4 and 25 °C in orange juice samples in which the pH was adjusted with citric acid (3 M) to values ranging from 2.5 to 3.3. At a pH value of 3.3 (TA of approximately 12 g/L citric acid), the bacteria were able to survive at 4 °C, and only a slight proliferation was observed at 25 °C (Fig. 4). At pH values lower than or equal to 3.0 (TA > 22 g/L citric acid), L. fermentum was unable to survive in orange juice at 4 and 25 °C (data not shown). These results indicate that a low pH and/or a high organic acid content in juice may inhibit bacterial growth and even lead to cell death.

Furthermore, the effectiveness of organic acids as preservatives is enhanced at lower pH values, as they are active when present in their undissociated form. In the latter state, organic acids can easily penetrate the cytoplasmic membrane of bacteria and alter their internal pH. The pKa values of citric and malic acids, which are the primary organic acids of orange juice, are 3.13 and 3.40, respectively [START_REF] Kim | Marked synergistic bactericidal effects and mode of action of medium-chain fatty acids in combination with organic acids against Escherichia coli O157:H7[END_REF]. Thus, at pH values below 3.5, the two organic acids are primarily in their antibacterial, undissociated forms.

To avoid possible interference by other microorganisms and to perform the experiments under the storage conditions used for fresh orange juice, the antibacterial activity of Ner was assessed at 4 °C.

Antibacterial activity of Ner in refrigerated orange juice

Based on the ability of L. fermentum to survive in orange juice at a pH values higher than 3.0, the antibacterial activity of Ner was assessed in orange juice with a pH value of 3.3 (TA approximately 12 g/L citric acid) at 4 °C. Ner solubilized in DMSO was tested at concentrations between 50 and 4 000 µM. At concentrations lower than 2 000 µM (50-1 000 µM), Ner showed no antibacterial activity for up to 10 days of incubation (data not shown). At 2 000 and 4 000 µM, total bacterial death was obtained within 8 and 5 days of incubation, respectively (Fig. 5).

Surprisingly, the effective concentration of Ner and the time needed to reach total bactericidal activity in orange juice (pH 3.3, 4 °C) increased by 40-and 12-fold, respectively, compared to that observed in MRS broth (pH 6.2, 37 °C) [START_REF] Ephrem | Selection of nerolidol among a series of terpenic and phenolic compounds for its potent activity against Lactobacillus fermentum ATCC 9338[END_REF]. The less potent and delayed effect of Ner in orange juice may be due to the influence of the incubation temperature and the rich matrix of the juice. Interestingly, [START_REF] Friedman | Antibacterial activities of plant essential oils and their components against Escherichia coli O157:H7 and Salmonella enterica in apple juice[END_REF] reported that the antibacterial activity of essential oil components against Escherichia coli and Salmonella enterica in apple juice was enhanced as the incubation temperature increased from 4 to 37 °C. Some orange juice components may also be major suppressors of Ner activity. For instance, the interaction between essential oils and food components may decrease the bioactivity of essential oil components in food products [START_REF] Perricone | Bioactivity of essential oils: a review on their interaction with food components[END_REF].

Effective concentrations (2 000 and 4 000 µM) of Ner were directly added without DMSO to orange juice, and the antibacterial activities of solubilized and unsolubilized Ner in orange juice were compared. The results showed that similar antibacterial activities were obtained.

Antibacterial activity of encapsulated Ner in refrigerated orange juice

The HP-β-CD inclusion complex, CL, and DCL systems were tested for their ability to maintain or improve the antibacterial activity of Ner in orange juice (pH 3.3; TA: approximately 12 g/L citric acid; 11-12 °Brix). When Ner-loaded CLs and DCLs were added at a final Ner concentration of 2 000 µM, the antibacterial activity was considerably delayed, and no antibacterial activity was observed for up to 20 days of storage (data not shown). Compared to the CD inclusion complexes, the CL and DCL systems extended the release of Ner in water at 25 °C [START_REF] Azzi | First evaluation of drug-in-cyclodextrin-in-liposomes as an encapsulating system for nerolidol[END_REF]. This result could be due to the strong retention of the loaded agent in liposomes (CLs and DCLs) compared to the inclusion complex system. Furthermore, to be released, Ner needs to overcome the bilayered membrane of CLs and the inclusion complex in addition to the membrane in DCL. Even though this approach increases the photostability of Ner and enhances its stability during storage [START_REF] Azzi | First evaluation of drug-in-cyclodextrin-in-liposomes as an encapsulating system for nerolidol[END_REF], the delayed release abrogates the antibacterial activity of Ner in orange juice. Thus, the maintained Ner concentration in contact with L. fermentum is not effective against the bacterial cells. Moreover, the CL and DCL systems may have specific limitations in fruit juice applications at the tested concentration due to their milky appearance (Fig. 6). However, the HP-β-CD/Ner inclusion complexes did not alter the appearance of the juice (Fig. 6) and showed a total bactericidal activity in orange juice against L.

fermentum within 17 days of incubation at 2 000 µM and 4 °C (Fig. 7). Therefore, only the inclusion complex was assessed in subsequent investigations. When the HP-β-CD/Ner inclusion complexes were added at a final Ner concentration of 4 000 µM, the appearance, pH (3.3), and TA (approximately 12 g/L citric acid) of the juice were not altered. However, a slight change was observed in the Brix degree value, which increased from 11-12 to 13 °Brix. Moreover, HP-β-CD/Ner inclusion complex killed all bacteria within 6 days of incubation at 4 °C (Fig. 7). Thus, the inclusion complex showed a bactericidal activity against L. fermentum approximately that was 3-fold faster at 4 000 µM than at 2 000 µM (Fig. 7). This result may highlight the higher aqueous solubility of Ner incorporated in inclusion complexes compared to free Ner. Furthermore, the incorporation of Ner into inclusion complexes was observed to increase its aqueous solubility by more than 100-fold at 25 °C [START_REF] Azzi | First evaluation of drug-in-cyclodextrin-in-liposomes as an encapsulating system for nerolidol[END_REF].

However, HP-β-CD/Ner inclusion complexes showed delayed antibacterial activity compared to the free form (8 versus 17 days at 2 000 µM and 5 versus 6 days at 4 000 µM). This delay was even more pronounced at 2 000 µM (nine-days delay) compared to 4 000 µM (one-day delay).

This delay may be due to the release kinetics of Ner from the inclusion complexes. [START_REF] Azzi | First evaluation of drug-in-cyclodextrin-in-liposomes as an encapsulating system for nerolidol[END_REF] reported the release of approximately 45% of Ner from inclusion complexes within 8 h, followed by a slow release over 7 days at 25 °C in water. Therefore, at 4 000 µM, the available concentration of Ner for interacting with L. fermentum in juice is approximately 2 000 µM after 8 to 10 h of incubation, with complete bacterial killing observed within 6 days compared to 5 days for free Ner. [START_REF] Shah | Nanocapsular dispersion of thymol for enhanced dispersibility and increased antimicrobial effectiveness against Escherichia coli O157:H7 and Listeria monocytogenes in model food systems[END_REF] also reported a better antibacterial activity of free thymol compared to thymol nanodispersion. However, different factors limit the application of free hydrophobic molecules in food rich in water, such as fruit juice, including the appearance of undissolved particles [START_REF] Shah | Nanocapsular dispersion of thymol for enhanced dispersibility and increased antimicrobial effectiveness against Escherichia coli O157:H7 and Listeria monocytogenes in model food systems[END_REF] and the low physicochemical stability of the added agent. In this study, agglomerates of free Ner were observed in orange juice at the assayed concentrations. On the other hand, Ner has a poor photostability, which was overcome by its incorporation into HP-β-CD inclusion complexes [START_REF] Azzi | First evaluation of drug-in-cyclodextrin-in-liposomes as an encapsulating system for nerolidol[END_REF].

Conclusions

In this study, the use of HP-β-CD/Ner inclusion complexes was observed to be effective against L. fermentum in orange juice (pH 3.3; TA: approximately 12 g/L citric acid; 11-12 °Brix) at 2 21 000 and 4 000 µM, although faster bacterial killing was obtained at 4 000 µM. The addition of the inclusion complexes to the juice at 4 000 µM did not alter the appearance, pH, and TA of juice, and only a slight increase in the Brix degree value was observed. In contrast, the Nerloaded CL and DCL systems (2 000 µM) strongly altered the physical appearance of the juice and did not exhibit an antibacterial effect within 20 days. According to the results obtained in this study, we can suggest that a combination of cold storage and HP-β-CD/Ner inclusion complexes limits the bacterial spoilage of fresh orange juice. In addition, assessing the use of a combination of the three systems may be interesting, as the inclusion complexes exhibits rapid activity against the bacterium, whereas the liposomal formulations extended the presence of Ner in the juice.

  Ner solubilized in DMSO in refrigerated orange juice Ner was solubilized in DMSO and tested against L. fermentum in orange juice (pH adjusted to 3.3) at different concentrations. Twenty five microliters of DMSO-solubilized Ner was added to 4.8 mL of orange juice (pH 3.3, TA of approximately 12 g/L) in glass culture tubes (20 × 100 mm) under sterile conditions to yield a range of Ner concentrations (between 50 and 4 000 µM).Subsequently, 200 μL of a diluted L. fermentum suspension (25 × 10 5 CFU/mL) was added, and the initial bacterial count in the culture medium was approximately 10 5 CFU/mL. Each test was performed in triplicate under sterile conditions. Orange juice supplemented with DMSO (25 µL),

Fig. 1

 1 Fig. 1 TEM images of blank CL (A), Ner-loaded CL (B), blank DCL (C), and Ner-loaded DCL

Fig. 2

 2 Fig. 2 SEM images of blank CL (A), Ner-loaded CL (B), blank DCL (C), and Ner-loaded DCL

Fig. 3

 3 Fig. 3 SEM images of untreated HP-β-CD (A), HP-β-CD treated as HP-β-CD/Ner complex (B),

Fig. 4

 4 Fig. 4 L. fermentum survival and proliferation in orange juice at 4 and 25 °C, at different initial

Fig. 5

 5 Fig. 5 Antibacterial activity of Ner at 2 000 and 4 000 µM in orange juice (pH 3.3; TA:

Fig. 6

 6 Fig. 6 Appearance of orange juice stored at 4 °C in the absence (a and A) and presence of HP-β-

Fig. 7

 7 Fig. 7 Antibacterial activity of HP-β-CD/Ner complex in orange juice (pH 3.3; TA:

Table 1 : Mean size, PdI, and zeta potential values of blank CL and DCL, and Ner-loaded CL and DCL.

 1

		Population 1	Population 2	Population 3	Zeta
	Delivery				PdI
		Mean size	Mean size		Mean size	potential
	system	(%)	(%)	(%)
		(nm)	(nm)		(µm)	(mV)
					-
	Blank CL	24.6 176.3±55.1 72.9 866.3±392.4 2.6 5.0±0.6	0.51
					32.7±4.49
	Ner-loaded				-
		2.3 60.9±15.2 97.7 1114±917	-	-	0.52
	CL				33.1±3.79
	Blank DCL	7.3 130.4±33.1 76.2 925±531.5	16.5 4.2±0.9	0.50 -

Acknowledgements

The authors thank the Agence Universitaire de la Francophonie (Projet de Cooperation Scientifique Inter-Universitaire PCSI 2018-2020) and the Research Funding Program at the Lebanese University (project 2018/2020) for supporting this project.

Conflict of interest

The authors declare that there are no conflicts of interest regarding the publication of this work.