Article

pubs.acs.org/JAFC

Physicochemical Properties and Antimicrobial Efficacy of Carvacrol
Nanoemulsions Formed by Spontaneous Emulsification
Yuhua Chang,†,‡ Lynne McLandsborough,† and David Julian McClements*,†

Department of Food Science, University of Massachusetts, Amherst, Massachusetts 01003, United States

College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an, Shaanxi 710062, P. R. China

ABSTRACT: A simple cost-effective method (spontaneous emulsification) for fabricating physically stable antimicrobial
nanoemulsions from essential oils is described. These nanoemulsions (10 wt % total oil phase) were formed by titration of a
mixture of essential oil (carvacrol), carrier oil (medium chain triglyceride, MCT), and nonionic surfactant (Tween) into an
aqueous solution with continuous stirring. Oil phase composition (carvacrol-to-MCT mass ratio) had a major impact on initial
droplet diameter, with the smallest droplets (d ≈ 55 nm) being formed at 2.5 wt % carvacrol and 7.5 wt % MCT. Surfactant type
also had an appreciable impact on mean droplet diameter, with Tween 80 giving the smallest droplets (d ≈ 55 nm) from a group
of food-grade nonionic surfactants (Tween 20, 40, 60, 80, and 85). The droplet size also decreased (from >5000 to <25 nm) as
the total surfactant concentration was increased (from 5 to 20 wt %). The long-term stability and antimicrobial efficacy of
selected nanoemulsions was examined at ambient temperature. The stability of the nanoemulsions to droplet growth during
storage decreased as the carvacrol concentration in the oil phase increased. Conversely, the antimicrobial efficacy of the
nanoemulsions increased as the carvacrol concentration increased. These results have important implications for the design and
utilization of nanoemulsions as antimicrobial delivery systems in the food and other industries. They suggest that the carrier oil
concentration must be carefully controlled to obtain good physical stability and antimicrobial efficacy.
KEYWORDS: nanoemulsions, emulsions, spontaneous emulsification, encapsulation, delivery, low energy, essential oil, carvacrol,
antimicrobial

■ INTRODUCTION
Essential oils are natural compounds produced by aromatic
emulsions have similar properties to conventional emulsions
in terms of their compositions, structures, and thermodynamic
plants as secondary metabolites that have antioxidant, properties.18−20 Nevertheless, differences in droplet size mean
antiradical, and antimicrobial properties and therefore have that nanoemulsions and macroemulsions often have very
been widely used as functional ingredients in food, cosmetic, different functional properties.18−20,22 The size of the droplets
and pharmaceutical applications.1,2 The major constituents in in nanoemulsions is often much smaller than the wavelength of
commercial essential oils can be classified into three classes: light (d ≪ λ), and so they do not scatter light strongly, making
phenols, terpenes, and aldehydes.1−3 Some essential oils have them transparent or only slightly turbid. They can therefore be
been shown to exert strong antibacterial activities against food- used to incorporate lipophilic bioactive compounds into
borne pathogens,1,4,5 leading to their broad application as transparent aqueous-based products, such as clear beverages,
natural antimicrobial additives to extend the shelf life of food sauces, and syrups. The small size of the droplets in
and beverage products. The fact that essential oils are nanoemulsions also means that they typically have much better
considered to be “natural” components makes them highly stability to gravitational separation, flocculation, and coales-
desirable for use in many commercial applications due to cence than macroemulsions.18−20,22 Finally, the activity of
growing consumer demand for natural rather than synthetic encapsulated compounds may increase as the droplet size in
additives.6−8 However, the utilization of essential oils is often emulsions decreases.11,23,24
limited owing to their relatively low water solubility. A simple Nanoemulsions can be fabricated by a number of different
way to solve this problem is to encapsulate essential oils in oil- approaches, which are usually categorized as either high-energy
in-water (O/W) emulsions or nanoemulsions.9,10 These or low-energy methods.20 High-energy methods utilize
emulsion-based delivery systems have previously been used to mechanical devices that are capable of disrupting and
encapsulate various kinds of lipophilic bioactive components, intermingling the oil and aqueous phases into tiny oil droplets
including antitumor agents,5,11 anti-inflammatory agents,11 dispersed in water. In the food industry, emulsions and
vitamins,12,13 and antimicrobials.5,9,10,14−17 After encapsulation, nanoemulsions are usually produced using high-energy
the lipophilic components can be easily incorporated into methods, such as high-pressure homogenization, microfluidiza-
aqueous-based foods and beverages due to their improved tion, and sonication.25 Low-energy methods mainly rely on the
water dispersibility.
Emulsion-based delivery systems can be classified as Received: May 15, 2013
conventional macroemulsions (radius >100 nm) or nano- Revised: August 12, 2013
emulsions (radius <100 nm) depending on the dimensions of Accepted: August 21, 2013
the droplets they contain.18−21 In many respects nano- Published: September 3, 2013

© 2013 American Chemical Society 8906 dx.doi.org/10.1021/jf402147p | J. Agric. Food Chem. 2013, 61, 8906−8913

and which leads to the spontaneous formation of fine droplets due nonionic surfactant) to an aqueous phase (5 mM citrate buffer. and where they are used there is still a antimicrobial delivery systems in the food and other industries. A pH of 3. The surfactant moves from the organic phase to the water phase (red arrows).5). Adapted from McClements and Rao. and 85) were purchased from Sigma-Aldrich Co. oil−water boundary (Figure 1). For example. samples were diluted appropriately before the interfacial and bulk rheology. MCT. they have 80. 8907 dx. systems that have both high physical stability and strong ature (PIT). but other essential oils may also be used (such as thymol drinks and salad dressings. e. In some experiments.org/10. U. Louis. 40.25−28 These methods are not widely used in the implications for the design and utilization of nanoemulsions as food industry at present. such as soft study.. and then the mixture was slowly titrated into 80 g of aqueous phase at emulsions to be fabricated at room temperature using simple a rate of 2 mL/min. to prepare food industry. pH to rapid diffusion of the surfactant from the oil phase into the 3. this method involves pouring an organic brief. interested in developing antimicrobial essential oil delivery including spontaneous emulsification. phase inversion temper. particle size measurements using citrate solution (5 mM.1021/jf402147p | J. stability. nanoemulsions with stirring rather than expensive homogenization equipment.Journal of Agricultural and Food Chemistry Article Figure 1. there are also some potential acid tail group of various lengths with the two moieties being linked disadvantages of low-energy systems. they may exhibit differences in the Nanoemulsion Preparation. (St. 20 g of surfactant and 10 g of oil were mixed nanoemulsions by spontaneous emulsification has not been together and then titrated into 70 g of aqueous phase. This different surfactant-to-oil ratios (SOR) were prepared by varying the approach may therefore be quite suitable for utilization in the amount of surfactant and water in the system. nanoemulsions and the fact that relatively high surfactant-to-oil Germany). Food Chem. Unless otherwise stated. surfactant structure. The aqueous phase used to prepare low-energy spontaneous emulsification method for producing the emulsions was citrate buffer solution (5 mM. 2013. their solubility and was to investigate the major factors influencing the formation of molecular geometry. Schematic representation of spontaneous emulsification: fine oil droplets are spontaneously formed when an organic phase containing a surfactant is mixed with an aqueous phase. Medium chain triglyceride (MCT) oil the types of oils and surfactants that can be used to form stable (Miglyol 812) was purchased from Sassol Germany GmbH (Witten. pH 3.29.K. surfactant phase behavior. organic phase (containing different amounts of carvacrol. Carvacrol and nonionic surfactants (Tween 20. the formation of essential oil a system with SOR = 2. or eugenol). a number of other studies Particle Size Measurements.31 The movement of the hydrophilic temperature (∼25 °C). 61. the oil (10 g) and surfactant (10 g) were first mixed together. Double essential oil nanoemulsions suitable for utilization in foods and distilled water was used in the preparation of all solutions and emulsions. spontaneous emulsification was performed by addition of an phase (containing oil and surfactant) into an aqueous phase. lower equipment and energy costs. however.5) while magnetically stirring (500 rpm) the system at ambient aqueous phase. pH 3. reported previously.20 spontaneous formation of droplets at the boundary between oil istics. In particular.38 The purpose of the present study surface active molecules present. leading to interfacial turbulence and spontaneous oil droplet formation. Nevertheless.37. and they are simpler to Tween surfactants consist of a polyoxyethylene head group and a fatty implement.31.30 50−65% caprylic acid (C8:0) and 30−45% capric acid (C10:0) in In the current study. Each method have shown that the size of the droplets generated individual measurement was an average of 13 runs. Carvacrol was used as a model essential oil in this phase of many commercial food and beverage products. To avoid multiple depends on many factors. The manufacturer reported that the MCT used contained ratios (SOR) are typically needed to produce them. often more effective at producing very fine droplets. and phase inversion composition (PIC) antimicrobial efficacy.10. the experiments were carried out using standardized conditions: 10 wt % oil (carvacrol + surfactant from the oil phase to the aqueous phase after mixing MCT).32−36 determines the particle size from intensity−time fluctuations of a laser Previous studies using the spontaneous emulsification beam (633 nm) scattered from a sample at an angle of 173°. To our knowledge. and 80 wt % aqueous phase In these leads to the spontaneous formation of fine oil droplets at the samples. In properties. The results of this study have important methods.doi.29 Low energy approaches may have advantages MATERIALS AND METHODS over high-energy approaches for certain applications: they are Materials. we examine the potential of using the terms of its fatty acid composition.5.28. On the other hand. Malvern Instruments. 8906−8913 . MO).27. This method allows nano.25 A number of different low-energy nanoemulsions containing fine droplets.5 was used in this study to mimic the aqueous beverages. This instrument against a range of different microorganisms.25. Malvern. 10 wt % surfactant. and performance of antimicrobial nano. carried out using a method based on a spontaneous emulsification emulsions due to their different physicochemical and biological procedure described previously29 with some minor modifications. scattering effects. we were approaches have been developed to form nanoemulsions. ■ relatively poor understanding of the factors affecting their performance. including interfacial tension. including limitations on together via a sorbitol molecule. In general.5).26.). and and water phases and depend strongly on the nature of any system composition. 60. The particle size distributions and have shown that food-grade antimicrobial nanoemulsions can mean particle diameters (Z-averages) of nanoemulsions were measured using a dynamic light scattering instrument (Zetasizer be produced using high-energy methods that are effective Nano ZS. surfactant solubility character. Agric.g. Nanoemulsions formation was formation.

Initially. with the smallest droplets being formed around 25 wt % Formation. viscosity. to achieve initial cell levels photograph shows carvacrol concentrations in the lipid phase of 0. Effect of oil phase composition (wt % carvacrol in oil phase) diluted with the same amount of single strength MEB media. partitioning. Brettanomyces bruxellensis (BB). and a single yeast colony from the plate was then inoculated into 10 mL of malt extract broth (MEB) media (Becton Dickinson. Oil phase composition will influence phase composition was varied by combining different mass the formation of small oil droplets during spontaneous ratios of essential oil (carvacrol) and carrier oil (MCT) prior to emulsification due to its impact on oil phase properties (e. All experiments were carried out two or three had relatively small initial droplet diameters (d ≈ 800 nm) and times using freshly prepared samples. Otherwise.e. 20. Oil essential oil concentration. be made. around 104 CFU/mL. Note: exposure to antimicrobial treatments. Means were subjected to Duncan’s test.250. and 156 μg/mL.. MD). Five milliliters of the above media was then Figure 2. and the entire study was carried out in triplicate. Inc. Sparks. after 120 h of incubation at 25 °C.5 log reduction compared to the initial inoculation level). Emulsions and nanoemulsions were until the carvacrol concentration dropped to 156 μg/mL. respectively. and a P-value of <0.5 wt % antimicrobial efficacy of different carvacrol nanoemulsions. the initial size of the oil droplets produced in the RESULTS AND DISCUSSIONS nanoemulsions depended on the composition of the lipid Effect of Oil Phase Composition on Nanoemulsion phase. From left to right. an OD600 of 1. Determination of Antimicrobial Activity. There are a number of possible reasons for the composition on the initial size of the oil droplets formed in observed minimum in mean droplet size at an intermediate nanoemulsions produced using spontaneous emulsification.org/10.5) to conduct the following antimicrobial assay. The smallest (SC). Food Chem. IL). Systems containing 50% carvacrol in the lipid phase Statistical Analysis. creaming and oiling off were observed in these analysis was performed through subjection of data to analysis of samples after a few days of storage (data not shown).000 μg/mL (= 1 wt %) carvacrol. 10 wt % surfactant concentrations of carvacrol in each MEB media were therefore 10. 50. and Brettanomyces naardenensis (BN). the containing varying levels of carvacrol. however. Saccharomyces cerevisiae minimum value and then increased (Figure 2). A certain amount of the sterile nanoemulsions was then mixed with appropriate amounts of double strength MEB and single strength MEB media. and optimum curva- surfactant (TWEEN 80). indicating that all the droplets were able to pass through the filter pores. and were then 1/100 inoculated into MEB media that reliable measurements could not be made. 40. The droplets (d ≈ 55 nm) were obtained when the lipid phase MIC (minimum inhibitory concentration) was used to compare the contained 25 wt % carvacrol and 75 wt % MCT (i. The surviving cell numbers were monitored 15. Yeast stock cultures were kept frozen at −70 °C in 25% glycerol.. Enumeration was carried out by using a spiral plater (Spiral Biotech. which was previously adjusted to pH 3. MD). (TWEEN 80). 100 wt %. All of the nano- emulsions subject to antimicrobial assay were filtered sterilized using 0. 2.45 μm polyethersulfone membrane filters (F2500-14. to achieve 10 mL of single strength MEB media (pH 3. 60.1021/jf402147p | J. Thermo Scientific. we examined the influence of oil phase carvacrol. 8906−8913 . interfacial tension.0 (1-cm path length). Systems variance (ANOVA) using statistical software (SPSS. Oil phase composition will also influence the subsequent The initial lipid phase composition had a major influence on stability of the oil droplets to rapid growth through Ostwald the formation and stability of the essential oil nanoemulsions.g.. As a guideline. Sparks. Germany).5 wt % MCT in the system). The particle sizes distributions of the nano- emulsions did not change after the filter sterilization (data not shown). Four acid-resistant spoilage yeast strains were used As the carvacrol concentration in the lipid phase increased. 61. 5 mM citrate buffer) containing a final concentration of 10. respectively. and polarity) and surfactant ardized: 10 wt % total oil (carvacrol + MCT).0 corresponds to approximately 5 × 106 CFU/mL for cultures of yeast strains.5 by citrate buffer with a final strength of 5 mM. and so reliable particle size measurements could not each treatment. Statistical 2). The culture was then diluted to about 106 CFU/mL using fresh MEB (pH 3. 625. 30.e. Prior to stirring speed of 500 rpm at ambient temperature (∼25 °C).g.5. NY). ture). solubility. and the results are reported as were stable to visible phase separation over a few hours (Figure the mean and standard deviation of these measurements.000. As mentioned above.. 10. Norwood.5 citrate buffer solution) at a 5. 80.000. Agric. 2. emulsification. 10 wt % properties (e. Massachusetts). the target yeast strains were data for emulsions prepared using 80 or 100 wt % carvacrol in the lipid freshly subcultured and diluted to about 106 CFU/mL as stated phase were not reported because these systems were highly unstable so previously. and 80 wt % water (pH 3.500. The culture was incubated at 32 °C under mild agitation (150 rpm in a rotary shaker) for 2−3 days until the optical density (turbidity) at 600 nm (OD600) was around 1. The final prepared using 10 wt % oil (carvacrol + MCT). 312. MICs were determined as the lowest concentration of pure carvacrol (not carvacrol + 7. and this on mean particle diameter of emulsions and nanoemulsions produced procedure was continued so as to make successive 2-fold dilutions by spontaneous emulsification. It is 8908 dx. 2013. ripening and coalescence mechanisms (see below). 25. the system composition was stand.Journal of Agricultural and Food Chemistry Article Yeast Strains. These strains were obtained from the Pepsico R&D Culture Collection (Valhalla. Systems containing nanoemulsion) that inhibited growth after 5 days of incubation (at more than 60 wt % carvacrol in the lipid phase were highly least 0..doi. The unstable to droplet aggregation and underwent rapid phase antimicrobial experiments were conducted with duplicate samples of separation. The yeast strain was refreshed on malt extract agar plates (Becton Dickinson. 1. d < 100 nm) that were relatively stable to ■ visible creaming during storage over a few days. Chicago. the as target microorganisms to examine the antimicrobial effects of the mean droplet diameter initially decreased until it reached a nanoemulsions: Zygosaccharomyces bailii (ZB).05 was containing ≤40 wt % carvacrol in the lipid phase formed considered statistically significant. nanoemulsions (i. and 80 wt % aqueous phase.

We found (d = 84 nm and 55 nm. d ≈ 610 nm) or a low HLB number (TWEEN nanoemulsion formation. Tween 60. Food Chem. TWEEN 60 and 80 both had HLB It should be noted that the type of carrier oil used to stabilize numbers around 15 but gave appreciably different particle sizes the antimicrobial nanoemulsions was also important. phase composition (Figure 2). mobility of the oil−water boundary where the oil droplets are Tween 80.28 interface. From left to right.e. For example. and 20 wt % TWEEN 80. Third. This difference will spontaneous emulsification are still not clearly understood. The with smaller droplet sizes. This phenomenon would account for the fact that 15. and 25 nm at 5. 15. or optimum curvature. and affect the packing of the surfactant molecules at the oil−water further research is clearly needed in this area.Journal of Agricultural and Food Chemistry Article therefore possible that increasing the amount of carvacrol 85. and 15. the nonpolar tails in TWEEN 60 are saturated and behavior.doi. 80. emulsions. which may promote the formation of finer oil droplets were formed in the systems prepared using TWEEN droplets at the oil−water boundary. tions mean that a larger number of surfactant molecules diffuse from the organic phase into the aqueous phase when they come prepared using TWEEN 80 (d ≈ 55 nm). In general. the mean droplet diameter was relatively large (d ≈ 6. whereas the largest into contact.300 nm) were unable to form carvacrol present in the lipid phase decreased the initial size of the nanoemulsions with small droplets.9. interfacial tension. and some small droplets. The effect of surfactant concentration on particle size was investigated by preparing a series of 10 wt % oil-in-water systems with a fixed lipid phase composition (25% carvacrol + 75% MCT) and surfactant type (TWEEN 80) but different surfactant concentrations. 8906−8913 . neous emulsification. 8909 dx. 60. Effect of surfactant type on mean particle diameter and have been proposed to account for the decrease in droplet size physical stability of emulsions produced by spontaneous emulsifica. and 80.39. respectively). Agric. with smaller droplets being formed at higher surfactant concentrations (Figure 4). The reason that LCT oils were unable to form stable surfactant is known to play a major role in determining the carvacrol nanoemulsions. Tween 40. The hydrophilic−lipophilic balance (HLB) concentrations will favor different structural organizations of of the surfactants appeared to play a role in their ability to form the surfactant. Surfactants with a high HLB number (TWEEN of these arrangements are known to be more advantageous for 20.5 wt % TWEEN 80 was used. In particular. (Figure 3). HLB = rapid growth. Second.7. Oil phase = 2. and 85). 60.39. In contrast. and phase separation rapidly occurred when only 2. particle size produced and the HLB numbers of these neous emulsification that were also stable to droplet growth. For example. 10. there are other factors that need to be i. and Tween 85 as indicated. The HLB number is that stable carvacrol nanoemulsions could not be formed at any often used as a rough guide for selecting surfactants to stabilize lipid phase composition when long chain triglycerides (LCT. such as viscosity. therefore fairly linear. which accounts for the differences in physicochemical properties that impact sponta. 34. surfactants with droplets formed but also decreased their subsequent stability to intermediate HLB numbers (TWEEN 40. the spontaneously formed. Surfactants were Tween 20. HLB = 11. surfactants.0. there was not a strong correlation between the concentration small droplets could be produced by sponta. The impact of nonionic surfactant type on the formation with the aqueous phase.41 unknown. However. fact that they have fairly similar HLB numbers of ∼15. which may impact the tendency for ultrafine droplets Effect of Surfactant Type on Nanoemulsion Forma- to be spontaneously produced when the organic phase is mixed tion. Differences in the molecular characteristics (chain TWEEN 60 and 80 both have similar polar head groups and length and unsaturation) of the MCT and LCT may have led to 18-carbon-atom nonpolar tail groups. is currently formation and stability of emulsions and nanoemulsions. d ≈ 1.6.5% carvacrol + 7. 14.29 A number of physicochemical mechanisms Figure 3.40 however. 55. the physicochem.5% MCT.300 nm). whereas MCT oils were. At a particular carvacrol However. 40. phase However. whereas those in TWEEN 80 are ical mechanisms governing the size of the droplets produced by unsaturated and therefore more kinked.42 In the following experiments. where smaller droplets were reported at higher surfactant concentrations. the amount of tion. The amount of surfactant present in the systems had a major impact on the initial droplet size produced. respectively) were all able to form there was a minimum droplet size produced at a particular lipid nanoemulsions with small particle sizes (d < 100 nm). we type of nonionic surfactant had a pronounced effect on the used TWEEN 80 as a surfactant since it produced emulsions mean particle diameter of the colloidal dispersions formed with the smallest initial droplet sizes.000 nm).. and water molecules in the system. This finding is in agreement with previous studies of nanoemulsion formation using spontaneous emulsification. HLB = 16.1021/jf402147p | J. higher surfactant 85 (d ≈ 1. The smallest droplets were formed in the systems Effect of Surfactant Concentration on Particle Size. 61. 2013. Surfactant was 10 wt % surfactant present will influence the interfacial tension and in the system. Previous studies have also reported and stability of carvacrol nanoemulsions was investigated.org/10. respectively. the mean droplet size decreased and the creaming stability increased when increasing amounts of surfactant were used to form the nanoemulsions: d ≈ 168. higher surfactant concentra- photograph shows surfactant types from Tween 20 to Tween 85. the molecular geometry of a shown). oil. corn oil and canola oil) were used as carrier oils (data not considered. A that the presence of double bonds in the nonpolar chains of series of antimicrobial nanoemulsions with fixed lipid phase composition (25% carvacrol + 75% MCT) were prepared using nonionic surfactants favors the formation of nanoemulsions different surfactant types (TWEEN 20.43 First. with increasing surfactant concentration.

From left to right. then the carvacrol this reason. with the mean droplet diameter increasing rapidly over a few days (data not shown). the rate of their size buffer).e. Additionally.org/10. Figure 5. Initially. 15%.5 to 20 wt % as indicated. most commercial applications it is important that nano- emulsions remain physically stable throughout their shelf life. 107. 25%. For be stored in an undiluted form..1021/jf402147p | J. 8906−8913 . pH 3. and 80 wt % aqueous phase (pH 3. 10 wt % surfactant (TWEEN 80). where dt and d0 are (≤15 wt %). and 20 wt %. used) in a diluted form then it may be possible to incorporate Experiments were carried out on undiluted samples (10% total higher levels of essential oil into the lipid phase. Surfactant (Tween 80) concentration in emulsion varied from 2. On the other hand. these systems had different mean diameters increase was greatly reduced (Figure 5b).5) prior to storage. These systems Δd > 70% for ≥25 wt % carvacrol. the photograph shows surfactant concentrations in the system of 2. respectively. oil phase) and on samples that were diluted (×5) with buffer antimicrobial nanoemulsions are often used in a highly diluted solution (2% total oil phase). we calculated the relative increase in droplet size concentration in the lipid phase should be kept relatively low during storage: Δd = 100 × (dt − d0)/d0. If a nanoemulsion is going to 10%. it if is going to be stored (or the mean droplet diameters at time t and 0. high surfac- tant levels increase ingredient costs and lead to adverse sensory (taste) issues in commercial applications. 60. Food Chem. Oil phase = 2. Figure 4. and 80 wt % water surfactant levels.5% MCT. concentration is currently unknown and may have been due to Nanoemulsions were prepared using 10 wt % oil (carvacrol + MCT of droplet coalescence and/or Ostwald ripening at elevated varying ratios). For these reasons we used 10 wt % TWEEN 80 in the remainder of the experiments. 2013. 61.5% carvacrol + 7. 20%. and 40% carvacrol in oil phase. 55. and visible creaming over short periods (a few days). It should be noted that we also observed appreciable differences between the short-term stabilities of the carvacrol nanoemulsions formed at different surfactant concentrations. 5. and therefore 8910 dx. 15. Percentage increase in mean particle diameter of selected The physicochemical origin of droplet growth at high surfactant nanoemulsions during 30 days of storage at room temperature. respectively. Δd ≈ 13% for 20 wt % carvacrol.doi. 10. We In general.Journal of Agricultural and Food Chemistry Article The increase in droplet size in the undiluted nanoemulsions after 30 days of storage ranged from 0% to 132% depending on the initial carvacrol concentration in the lipid phase (Figure 5a).5 citrate buffer. and 99 nm for antimicrobial nanoemulsions. For undiluted (a) or diluted 5 times by citrate buffer (b). Although very fine droplets were initially formed in the nanoemulsions prepared using 15 or 20 wt % TWEEN 80. the increase in droplet size after storage was higher therefore examined the influence of storage time on the stability as the amount of carvacrol in the lipid phase increased: Δd ≈ 0 of a series of nanoemulsions that were found to be stable to for ≤15 wt % carvacrol. Agric.5 citrate buffer solution) at a stirring speed of 500 rpm at ambient temperature (∼25 °C). 30%. 75. these systems were highly unstable to droplet growth. The nanoemulsions were either Storage Stability of Carvacrol Nanoemulsions. when the same consisted of 10 wt % oil (0 to 40% carvacrol). Effect of surfactant concentration on mean particle diameter and physical stability of emulsions produced by spontaneous emulsification. form in commercial products (such as beverages). 10 wt % nanoemulsions were diluted 5 times in buffer solution (5 mM surfactant (TWEEN 80).5. This observation has due to the influence of oil phase composition on the efficiency important consequences for the practical application of of nanoemulsion formation: 133. there is little or no change in their particle size. In practice. (pH 3. This result suggests that an intermediate level of surfactant may be more suitable for preparation of physically stable antimicrobial nanoemulsions. Nevertheless. i.

the oil phase of the nanoemulsions. the carvacrol concentration in the oil phase increased. First.53 In this study. presumably by acting as an Ostwald ripening and/or even with carvacrol concentration as high as 10.35 As the total were filter sterilized to avoid the influence of any endogenous amount of MCT in a nanoemulsion increases.5) against four representative acid.33. Food Chem.000 >10. An alternative approach to enhancing the period of 5 days at room temperature.55 recent study on the effects of water-insoluble oils on the Consequently. We postulated that this reduction in efficacy was efficacy was established by measuring the minimum inhibitory caused by a decrease in the concentration of antimicrobial concentration (MIC) of carvacrol in a nutrient MEB medium molecules within the microorganisms due to partitioning into (5 mM citrate buffer.000 increasing the interfacial tension. which would alter the effectiveness of the nanoemulsions decreasing in the following coalescence stability. microfluidization. MIC was determined as long-term stability of the system would be to store the the lowest concentration of pure carvacrol (not nanoemulsion) antimicrobial as an organic phase containing essential oil and that inhibited growth after 5 days incubation. Ostwald ripening is the 20 >10.19.e.46 Indeed. The antimicrobial efficacy. MCT was beneficial not only for the spontaneous formation of carvacrol concentration in the lipid phase was ≤20 wt %. There are a number of possible reasons for this not detect any viable cells (<1 CFU/mL) for any of the four phenomenon. we determined the or MCT) in the lipid phase of antimicrobial nanoemulsions antimicrobial efficacy of a series of carvacrol nanoemulsions improved their storage stability but reduced their antimicrobial with different lipid phase compositions. For example.1021/jf402147p | J.50 Carvacrol has a finite 40 625 625 312 158 a solubility in water (∼0. 1 wt %). Initially.000 >10. Brettanomyces bruxellensis (BB). when the carvacrol droplets merge together when they collide. aqueous phases.44.e. Bretta- nomyces bruxellensis (BB). the optimum not shown).49. between droplets.45 Droplet coalescence may have been accelerated at high essential oil Table 1. Coalescence is the process whereby two or more its lipid phase (Table 1).9 In this previous study.000 >10. found that increasing the level of water-insoluble oils (corn oil sified Carvacrol Nanoemulsions.. the droplet−droplet collision frequency yeast strains after 5 days of incubation.org/10.5) against four acid -resistant yeasts as indicated: ripening.44 Second.Journal of Agricultural and Food Chemistry Article the rate of droplet growth may be relatively slow after dilution monitoring the cell numbers throughout a total incubation has been carried out. A lipophilic antimicrobial resistant yeast strains: Zygosaccharomyces bailii (ZB).10. high levels of surfactant may The results of this study are in agreement with those of a promote droplet aggregation through a depletion mechanism. 8906−8913 . will partition between oil phases.48 The addition of nonpolar triglyceride oils (such as MCT) may therefore have 10 >10. i. carrier oil (carvacrol and MCT) and then add this organic The antimicrobial efficacy of the nanoemulsions increased as phase to an aqueous product when needed. and romyces cerevisiae (SC). 2013. Dependence of the Minimal Inhibitory concentrations because of the relatively high polarity and low Concentration (MIC) of Carvacrol in Nanoemulsions with interfacial tension of this kind of oil. Finally.000 >10. The efficacy of the antimicrobial nanoemulsions curvature of the surfactant monolayer at the oil−water also depended on the strain of yeast tested.9. the nanoemulsions trations and oil−water partition coefficients.000 >10.000 1250 625 625 intervening continuous phase.47. the rate of Varying Carvacrol Concentration in the Lipid Phasea droplet coalescence in emulsions containing more polar oils MIC (μg/mL) often occurs more rapidly than those containing more nonpolar oils because of the influence of the properties of the oil carvacrol levels in lipid phase (wt %) ZB SC BB BN molecules on the optimum curvature. Ostwald ripening can be Zygosaccharomyces bailii (ZB).51 The determination of MICs was performed in a nutrient MEB and so nanoemulsions made from it are susceptible to Ostwald medium (pH 3.20. more microorganisms.000 1250 625 ones due to the mass transport of dispersed phase through the 30 10. inhibited by incorporating a highly water-insoluble oil into a relatively water-soluble oil prior to homogenization due to an entropy of mixing effect.000 >10.doi.000 >10. but at 40 wt % they could inhibit growth in all the Our results also showed that the stabilities of the yeasts with carvacrol concentration at only 625 μg/mL (Table nanoemulsions were greatly improved after dilution with 1).83 g/L at ambient temperature). we also Antimicrobial Efficacies of the Spontaneously Emul. we could water. which would there was at least a 4-log reduction in microbial numbers (data reduce the droplet aggregation rate. method.54 Fourth. dilution of the nanoemulsions may have formation. the nanoemulsions (Figure 2) but also for ensuring their long-term nanoemulsions could not inhibit growth of any of the yeasts stability. Saccharomyces cerevisiae (SC). This difference may be due to aqueous phase may increase Ostwald ripening rates because differences in the structural architecture of the yeast cells walls surfactant micelles can solubilize and transfer oil molecules or other biochemical differences between them. and nanoemulsions were added to a broth containing yeast cell therefore there will be less available to partition into the cell levels of ∼104 CFU/mL. 8911 dx. and Brettanomyces naardenensis (BN). Agric. Saccha.000 >10. pH 3. with the boundary may have been changed. high levels of surfactant in the order: BN > BB > SC > ZB.000 process whereby large droplets grow at the expense of smaller 25 >10.47 Third. 61.52 As shown previously. and antimicrobial efficacy of essential oil improved their stability by reducing Ostwald ripening and/or (thyme oil) nanoemulsions produced using a high energy coalescence rates in the system. stability..000 >10. which indicated that decreases with decreasing droplet concentration. and microorganism cell walls depending on their relative concen- Brettanomyces naardenensis (BN).000 >10. At the highest carvacrol level studied (40 wt %). lower The growth of the droplets in the nanoemulsions during amounts of carvacrol were needed to completely inhibit yeast storage may have occurred due to coalescence or Ostwald growth if the nanoemulsion contained higher carvacrol levels in ripening.000 μg/mL (= coalescence inhibitor. and then different amounts of these sterile antimicrobial agent (carvacrol) will partition into it. Growth curves were obtained by walls of the microorganisms.000 decreased the coalescence rate by decreasing the polarity and 15 >10. i.

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