Thyme Oil Nanoemulsions Coemulsified by Sodium Caseinate and

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Thyme Oil Nanoemulsions Co-emulsified by Sodium Caseinate and Lecithin Jia Xue, and Qixin Zhong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5034366 • Publication Date (Web): 18 Sep 2014 Downloaded from http://pubs.acs.org on September 25, 2014

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Journal of Agricultural and Food Chemistry

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Thyme Oil Nanoemulsions Co-emulsified by Sodium Caseinate and Lecithin

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Jia Xue and Qixin Zhong*

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Department of Food Science and Technology, University of Tennessee, Knoxville, TN 37996

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Please send correspondence to

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*Qixin Zhong, Professor

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Department of Food Science and Technology

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The University of Tennessee

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2510 River Drive

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Knoxville, TN 37996

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Phone: (865) 974-6196; Fax: (865) 974-7332

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E-mail: [email protected]

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1 ACS Paragon Plus Environment


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Abstract Many nanoemulsions are currently formulated with synthetic surfactants. The objective

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of the present work was to study the possibility of blending sodium caseinate (NaCas) and

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lecithin to prepare transparent thyme oil nanoemulsions. Thyme oil was emulsified using NaCas

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and soy lecithin individually or in combination at neutral pH by shear homogenization. The

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surfactant combination improved the oil content in transparent/translucent nanoemulsions, from

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1.0% to 2.5%w/v for 5% NaCas with and without 1% lecithin, respectively. Nanoemulsions

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prepared with the NaCas-lecithin blend had hydrodynamic diameters smaller than 100 nm and

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had significantly smaller and more narrowly-distributed droplets than those prepared with NaCas

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or lecithin alone. Particle dimension and protein surface load data suggested the co-adsorption of

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both surfactants on oil droplets. These characteristics of nanoemulsions minimized

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destabilization mechanisms of creaming, coalescence, and Ostwald ripening, as evidenced by no

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significant changes in appearance and particle dimension after 120-day storage at 21 °C.

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Keywords: Thyme oil, nanoemulsion, sodium caseinate, lecithin, synergistic surface activity

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Introduction Nanoemulsions are frequently studied as delivery systems of functional lipophilic

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compounds such as flavors, 1 vitamins 2 and antimicrobials 3 due to their advantages over other

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oil-containing systems. For example, compared with microemulsions, nanoemulsions can be

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formulated using a large variety of food grade ingredients such as proteins and polysaccharides

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and require relatively lower surfactant concentrations. 4 With small droplets (2 h. The samples

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were scanned using a rectangular cantilever probe (Bruker Nanoprobe, Camarillo, CA) with

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aluminum reflective coating on the backside and a quoted force constant of 2.80 N/m. The

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topography images were collected at the tapping mode.

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Quantification of Protein Surface Load

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The amount of NaCas on thyme oil droplet surface was quantified to investigate the

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effect of lecithin on surface adsorption of NaCas. To facilitate the separation of oil droplets,

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an excess amount (10% w/v) of thyme oil was directly emulsified in the aqueous phase with

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various combinations of NaCas (0, 2.5, and 5% w/v) and lecithin (0, 0.5, and 1.0% w/v).

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Particle sizes were measured right after homogenization and after storage at room

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temperature (21 °C) for 15 days. Emulsions before and after storage were centrifuged at

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12,550g for 60 min using an Eppendorf MiniSpin plus centrifuge (Hamburg, Germany).

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The bottom aqueous phase was filtrated through a 0.22 µm polyvinyl difluoride membrane

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filter. To minimize the interference of thyme oil compounds such as thymol on protein

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assay, 50 µL of the permeate was mixed with 500 µL of an acetone-hexane (1:1, v:v)


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mixture 29 to extract thyme oil and precipitate protein. After vigorous mixing and

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centrifugation at 6,700g for 10 min, the supernatant (organic phase) was discarded, and the

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bottom protein precipitate was re-dissolved in 600 µL of 100 mM NaOH and was quantified

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using the bicinchoninic acid (BCA) method (Thermo Fisher Scientific Inc., Morris Plains,

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NJ). NaCas was used as a reference protein in the BCA assay. The surface load (Γs, mg/m2)

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of protein and the volume-area (d3,2) mean diameters were calculated using eq 1 and 2,

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respectively. Γs =

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Ms d 3 , 2 6Voil

(1)

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where Ms is the mass of NaCas adsorbed on oil droplets, and Voil is the volume of thyme oil.

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The thymol oil density used in calculation was 0.932 g/mL. 30

d 3, 2

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∑n d = ∑n d i

3 i

i

2 i

i =1

(2)

i =1

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where di is the diameter of the ith group of droplets and ni is the corresponding number of

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droplets.

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Statistical Analysis

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Two independent emulsion replicates were studied throughout. Analysis of variance

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(ANOVA) test was conducted using the SPSS 16.0 statistical analysis system (SPSS Inc.,

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Chicago, IL). The least significant difference (LSD) test was used to determine the

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significant difference of mean values at a P level of 0.05.

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Results and Discussion

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Turbidity of emulsions



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Photographs of thyme oil emulsions prepared with various combinations of 5% w/v

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NaCas, 1% w/v lecithin and 10% v/v PG are shown in Figure 1. The treatments with 5% w/v

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NaCas alone and its combination with 10% v/v PG were only capable of emulsifying 1.0% w/v

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thyme oil as transparent or translucent dispersions. In comparison, after adding 1% w/v lecithin,

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emulsions with up to 2.5% w/v thyme oil were translucent when prepared with 5% w/v NaCas,

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and the clarity further improved with 10% v/v PG. The appearance generally agreed with the

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Abs600 (Figure 2). Since PG is fully miscible with water, pre-dissolving thyme oil in PG before

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emulsification likely reduces the oil/water interfacial tension, which is a main mechanism of co-

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surfactants facilitating the formation of nanoemulsions 31. Furthermore, PG had no significant

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impact on emulsion formation by NaCas but facilitated emulsion formation for the NaCas-

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lecithin blend. This is likely due to the impacts on solubility of surfactants: NaCas is water-

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soluble, while lecithin (phosphatidylcholines) is not.

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Subsequently, two concentrations (1.0 and 2.0% w/v) of thyme oil were emulsified using

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various combinations of NaCas (2.5% w/v) and lecithin (0.5 or 1% w/v) concentrations.

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Photographs are shown in Figure 3, while the Abs600 is compiled in Table 1. With 1.0% w/v

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thyme oil, emulsions prepared with combinations of NaCas and lecithin were all transparent,

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while those with 2.5% NaCas or (0.5 or 1.0% w/v) lecithin alone were turbid. As thyme oil

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concentration increased to 2.0% w/v, all samples were turbid. The Abs600 of thyme oil emulsions

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in Table 1 showed that addition of lecithin significantly decreased the turbidity of thyme oil

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emulsions prepared with NaCas (P < 0.05).

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Hydrodynamic diameters of emulsions

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Mean hydrodynamic diameters of thyme oil emulsions are compiled in Table 2. Emulsions prepared with NaCas-lecithin blend had significantly smaller particles than those


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prepared with NaCas or lecithin alone (P < 0.05). For example, compared to treatments with

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1.0% w/v thyme oil emulsified by 1.0% w/v lecithin (mean diameter = 179.7 nm) or 5.0% w/v

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NaCas alone (mean diameter = 105.5 nm), the corresponding nanoemulsion prepared with the

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NaCas-lecithin blend had much smaller droplets with a mean diameter of 66.6 nm (P < 0.05).

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These results were consistent with the visual appearance (Figure 3) and Abs600 (Table 1). Similar

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results have been reported for emulsions made with blends of NaCas and sucrose esters (with

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lipids being mono, di-, and tristearate or palmitate) 35. Small molecular weight surfactants may

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co-adsorb with NaCas or displace NaCas on droplet surfaces and therefore alter the composition

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and properties of interfaces, and the reduction of interfacial tension may facilitate the formation

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of smaller droplets. 32

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Emulsion structures studied by AFM

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Emulsions containing 1.0% w/v thyme oil prepared with 2.5% w/v NaCas, 0.5% w/v

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lecithin, or both were imaged using AFM (Figure 4). Relatively bigger particles were observed

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for the emulsions prepared with NaCas or lecithin alone (Figure 4A and 4B) when compared to

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the treatment prepared with the surfactant blend (Figure 4C). The average particle dimension

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estimated in AFM was 134.3, 147.3, and 76.9 nm for emulsions prepared with NaCas only,

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lecithin only, and both, respectively. These results were generally in agreement with those

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obtained using dynamic light scattering (Table 2).

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Zeta-potential of emulsions

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Zeta-potentials of emulsions prepared with 1.0% w/v thyme oil emulsified by NaCas

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alone, lecithin alone, and their blend were measured at pH 7 (Table 3). The emulsion

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prepared with 1% w/v lecithin only had a highly negative zeta-potential (-58.2 ± 1.2 mV),

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which is attributed to the phosphate group of lecithin. 33 In comparison, the zeta-potential of



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the emulsion prepared with 2.5% w/v NaCas only had a significantly smaller magnitude (-

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25.8 ± 6.8 mV) than that with lecithin only (P < 0.05). When 5% w/v NaCas was used in

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homogenization, the zeta-potential magnitude (-39.7±1.2 mV) was higher than that prepared

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with 2.5% NaCas and was close to the zeta-potential of NaCas solution, 34 which may

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suggest a significant amount of free NaCas in the aqueous phase. For the nanoemulsion

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prepared with 2.5% w/v NaCas and 1% w/v lecithin, the magnitude of zeta-potential (-44.6

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± 2.6mV) was between those prepared with lecithin and NaCas alone. Since free NaCas was

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not removed, the zeta-potential data in Table 3 did not provide straightforward information

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about interfacial structures. It however should be noted that the electrostatic attraction

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between negatively charged phosphate group of lecithin and positively charged amine

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groups of NaCas is still possible at neutral acidity despite both are overall negatively

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charged, as previously demonstrated for pectin and NaCas. 14

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Effect of lecithin on surface adsorption of NaCas

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The protein content on unit surface area of emulsion droplets (Γs, mg/m2) is commonly

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determined to study the surface activity of proteins and the impacts by competing or co-

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adsorbing surfactants. For 10% thyme oil emulsified by 2.5% w/v NaCas and 0-1% w/v lecithin,

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an increase in lecithin content decreased the droplet dimension but not the amount of adsorbed

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NaCas (P > 0.05), resulting in the reduced Γs (Table 4). Because a larger surface area (smaller

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droplets) requires the coverage by a greater amount of surfactants, the data suggest the co-

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adsorption of NaCas and lecithin. The Γs did not change significantly after 15-day storage at 21

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°C, suggesting there was no detachment of NaCas. With the gradual increase in lecithin content,

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the droplet dimension decreased to near those prepared with lecithin only. Therefore, lecithin is

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more surface-active than NaCas in emulsifying thyme oil. For emulsions prepared with 5% w/v


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NaCas, the droplet size (P < 0.05) and the percentage of adsorbed NaCas (P > 0.05) were smaller

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than those prepared with 2.5% NaCas, corresponding to similar Γs (P > 0.05). The Γs of

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treatments prepared with 5% w/v NaCas and 0 and 0.5% lecithin decreased (P < 0.05) after

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storage for 15 days. Because the droplet dimension of these two emulsions did not change after

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storage, the results indicated the detachment of NaCas. Conversely, the treatment prepared with

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5% NaCas and 1% lecithin showed insignificant changes in both droplet dimension and Γs (P >

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0.05).

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The competitive surface adsorption and possible displacement of NaCas by more surface-

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active lecithin are dependent on their overall concentrations and molar ratios. 22, 35, 36 In a study

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co-emulsifying 20% w/w soybean oil by 0.2-2% w/w NaCas and lecithin at lecithin: casein

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molar ratios of 0.7:1 - 49:1, 22 the gradual reduction of Γs of NaCas with increases in lecithin

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concentration was similar to the present study (Table 4), but their Γs increased with increases in

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NaCas concentration and was all below 3.5 mg/m2. The Γs in the present study was much higher

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than the reference 22 and was not significantly different at two (2.5 and 5% w/v) NaCas

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concentrations (Table 4) although the lecithin:casein molar ratios (2.8-11.4:1) in this work were

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within their range. The differences in their and the present studies can result from differences in

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the droplet dimension (300-800 nm in the reference 22), emulsification conditions, polarity of oil,

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and overall NaCas and lecithin concentrations. As for the interfacial structure, Fang and

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Dalgleigh 22 proposed that the thickness of adsorbed casein layer could increase when lecithin

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concentration increases gradually and, when there is a sufficient amount of lecithin, the oil

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droplets are directly covered mostly by lecithin that can be adsorbed by caseins protruding to the

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continuous aqueous phase. At their studied conditions, Fang and Dalgleigh 22 did not observe

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surface displacement of NaCas by lecithin, which is mostly in agreement with the present study



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(Table 4). However, in our case, Γs decreased for nanoemulsions prepared with 5% w/v NaCas

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and 0 and 0.5% w/v lecithin that also showed a decrease in droplet dimension after storage

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(Table 4). Since the thickness of NaCas layer increases with its bulk concentration, 22 the

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observations from these two emulsions can result from the detachment of caseins rather than the

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displacement by lecithin. The speculation however requires experimental verifications.

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Emulsion stability One common concern about essential oil nanoemulsions is their stability against Ostwald

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ripening because of the water solubility of essential oil components being higher than lipids

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composed of medium- or long-chain fatty acids. 31 Physically, compounds in smaller particles

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have a higher solubility, which results in the continuous dissolving and eventually disappearance

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of smaller particles, and the dissolved compounds join bigger particles that grow during storage.

249

37

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preparation to serve as inhibitors of Ostwald ripening. 31 Ripening inhibitors however lower the

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loading level and antimicrobial activity of essential oils.

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Nonpolar substances such as corn oil 38 can be blended with essential oils in emulsion

Emulsions with 1 and 2% thyme oil were studied for storage stability at 21 °C for 120

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days. Creaming was observed for emulsions prepared with NaCas alone after 2- or 3-day storage

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at room temperature and became more significant after longer storage. Creaming can be

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contributed by increases in overall particle dimension due to depletion flocculation (by

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unadsorbed NaCas) or particle growth due to coalescence and/or Ostwald ripening. 4 In contrast,

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nanoemulsions prepared with 1 and 2% thyme oil and NaCas-lecithin blend appeared transparent

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or translucent throughout storage.

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When particle size distributions of emulsions prepared with lecithin only or NaCaslecithin blend were compared before and after 4-month storage at room temperature (Figure 5),


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several trends were observed. Emulsions prepared with NaCas-lecithin blend exhibited smaller

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particles with narrower distributions than that prepared with lecithin only, indicating the better

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emulsifying activity of the blend. For these emulsions, the interfacial layer next to thyme oil is

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likely composed of both lecithin and NaCas, different from the model of sequential layers of

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lecithin and NaCas as proposed by Fang and Dalgleigh 122 that would otherwise result in bigger

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droplets for emulsions prepared with both surfactants. The growth of particle size was observed

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for all emulsions, and the growth was much smaller for emulsions prepared with the blend than

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those with lecithin alone, suggesting the better stabilizing ability of the blend. The growth of

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average hydrodynamic diameter (Table 2) however was overall not statistically significant (P >

270

0.05).

271

In addition to Ostwald ripening discussed above, coalescence can also cause the growth

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of particles during storage, particularly for small molecular surfactants such as lecithin with a

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small head group. 39 Coalescence is initiated by the aggregation of two oil droplets controlled by

274

colloidal interactions. In the present study, emulsions prepared with lecithin only had a higher

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magnitude of zeta-potential (Table 3) than those with the NaCas-lecithin blend. Since emulsions

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prepared with lecithin only and the NaCas-lecithin blend had insignificant increase in mean

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particle diameter (Table 2), it may suggest the electrostatic repulsion is effective in preventing

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particle aggregation. The protrusion of NaCas in the continuous phase of emulsions prepared

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with the surfactant blend may have provided additional steric repulsion that resulted in smaller

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net growth of droplets after storage than emulsions prepared with lecithin only (Table 2). Similar

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phenomena were reported for orange oil emulsions prepared with lysolecithin and sucrose

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monopalmitate. 29 Therefore, it is likely Ostwald ripening is the major mechanism responsible

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for particle growth of emulsions in the present study, and the narrower particle size distribution



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and thicker interface of emulsions prepared with the NaCas-lecithin blend both resulted in less

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significant particle size changes than those prepared with NaCas or lecithin only.

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The stability of emulsions prepared with a mixture of proteins and small molecular

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surfactants has been widely studied. In some cases, the interaction or displacement between

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emulsifiers resulted in the significantly decreased stability. For example, combinations of

289

caseinate and Tween® 20 at certain ratios led to severe flocculation and creaming, which

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was attributed to the depletion flocculation by unabsorbed protein. 40 Combinations of

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lecithin with other small molecular surfactants also have been shown to improve the

292

stability of emulsions. Lecithin added in emulsions stabilized by sucrose monopalmitate

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strengthened the electrostatic repulsion at acid conditions and resulted in the excellent

294

stability of emulsions against Ostwald ripening, flocculation, and coalescence. 41

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Emulsifying soybean oil by combinations of lecithin and non-ionic steric surfactants such as

296

polyethylene glycol hexadecyl ether (Brij®) increased the stability of emulsions at increased

297

ionic strengths when compared to those prepared with lecithin only. 42 This is because,

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although the surfactant blend reduced the zeta-potential magnitude of droplets when

299

compared to those stabilized by lecithin, Brij® provided steric repulsion to stabilize

300

emulsions. 42

301 302

In summary, the technological advancement has been shown in the present work for

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stable and transparent thyme oil nanoemulsions prepared using combinations of GRAS

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emulsifiers NaCas and lecithin. These transparent nanoemulsions had mean diameters smaller

305

than ~100 nm at neutral pH. The clearer and smaller droplets of nanoemulsions prepared with the

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NaCas-lecithin blend than those with individual surfactants suggested that NaCas and lecithin


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synergistically emulsified thyme oil rather than the preferential adsorption and displacement of

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NaCas by more surface-active lecithin. The interface composed of both NaCas and lecithin

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provided repulsive electrostatic and also likely steric interactions against destabilization

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mechanisms of creaming, flocculation, and coalescence and, together with narrow particle size

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distributions, minimized Ostwald ripening. These transparent nanoemulsions prepared from

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GRAS ingredients have great potential to incorporate lipophilic antimicrobials such as thyme oil

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in transparent beverages to enhance the microbiological safety.

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Acknowledgements This work was supported by the University of Tennessee and the USDA National Institute

316 317

of Food and Agriculture.

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of O/W interfacial layers. J. Colloid Interface Sci. 2006, 295, 495-503.

El Maghraby, G. M., Transdermal delivery of hydrocortisone from eucalyptus oil

Kale, N. J.; Allen Jr, L. V., Studies on microemulsions using Brij 96 as surfactant and

Rao, J.; McClements, D. J., Formation of flavor oil microemulsions, nanoemulsions and

Xue, J.; Davidson, P. M.; Zhong, Q., Thymol nanoemulsified by whey protein-

McClements, D. J.; Decker, E. A.; Choi, S. J., Impact of environmental stresses on orange

Wu, J.-E.; Lin, J.; Zhong, Q., Physical and antimicrobial characteristics of thyme oil

McClements, D. J.; Rao, J., Food-grade nanoemulsions: formulation, fabrication,

Tual, A.; Bourles, E.; Barey, P.; Houdoux, A.; Desprairies, M.; Courthaudon, J.-L., Effect


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Chuah, A. M.; Kuroiwa, T.; Kobayashi, I.; Nakajima, M., Effect of chitosan on the

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stability and properties of modified lecithin stabilized oil-in-water monodisperse emulsion

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prepared by microchannel emulsification. Food Hydrocolloids. 2009, 23, 600-610.

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nanoparticles using sodium caseinate. Food Hydrocolloids. 2014, 35, 358-366.

404

35.

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and. beta.-casein in oil-in-water emulsions. J. Agric. Food Chem. 1991, 39, 1365-1368.

406

36.

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and sodium caseinate on oil-water interfaces. J. Agric. Food Chem. 1996, 44, 59-64.

408

37.

Taylor, P., Ostwald ripening in emulsions. Adv. Colloid Interface Sci. 1998, 75, 107-163.

409

38.

Ziani, K.; Chang, Y.; McLandsborough, L.; McClements, D. J., Influence of surfactant

410

charge on antimicrobial efficacy of surfactant-stabilized thyme oil nanoemulsions. J. Agric. Food

411

Chem. 2011, 59, 6247-6255.

412

39.

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oil and oil-in-water emulsions. J. Surfactants Deterg. 2002, 5, 135-143.

414

40.

415

caseinate and Tween 20. J. Colloid Interface Sci. 1999, 212, 466-473.

416

41.

417

Formulation and properties of model beverage emulsions stabilized by sucrose monopalmitate:

418

Influence of pH and lyso-lecithin addition. Food Res Int. 2011, 44, 3006-3012.

419

42.

420

properties of mixed phospholipid–non-ionic surfactant stabilised oil-in-water emulsions.

421

Colloids Surf., A 1999, 152, 59-66.

Chen, H.; Zhong, Q., Processes improving the dispersibility of spray-dried zein

Courthaudon, J. L.; Dickinson, E.; Christie, W. W., Competitive adsorption of lecithin

Fang, Y.; Dalgleish, D. G., Competitive adsorption between dioleoylphosphatidylcholine

Pan, L.; Tomás, M.; Añón, M., Effect of sunflower lecithins on the stability of water-in-

Dickinson, E.; Ritzoulis, C.; Povey, M. J., Stability of emulsions containing both sodium

Choi, S. J.; Decker, E. A.; Henson, L.; Popplewell, L. M.; Xiao, H.; McClements, D. J.,

De Vleeschauwer, D.; Van der Meeren, P., Colloid chemical stability and interfacial

422



423

Figure captions

424 425

Figure 1. Appearance of thyme oil nanoemulsions prepared with various combinations of 5%

426

w/v NaCas, 10% v/v PG, and 1% w/v lecithin. Thyme oil concentration in each image is 0.5, 1.0,

427

1.5, 2.0, and 2.5% w/v from left to right.

428 429

Figure 2. Absorbance at 600 nm of nanoemulsions with 0.5-2.5% w/v thyme oil prepared with

430

various combinations of 5% w/v NaCas, 10% v/v PG, and 1% w/v lecithin.

431 432

Figure 3. Appearance of nanoemulsions with 1.0% w/v (top) or 2.0% w/v (bottom) thyme oil

433

emulsified by 2.5%w/v NaCas, 0.5 or 1%w/v lecithin, or both. All samples contained 10% v/v

434

PG. Labels on vial caps: S - NaCas; L - lecithin; numbers – surfactant concentrations.

435 436

Figure 4. AFM images of thyme oil (1.0% w/v) nanoemulsions prepared with 2.5% w/v NaCas

437

(A), 0.5% w/v lecithin (B), or both (C). All samples contained 10%v/v PG.

438 439

Figure 5. Particle size distributions of nanoemulsions with 1.0% w/v (A and C) or 2% w/v (B

440

and D) thyme oil emulsified by lecithin and NaCas-lecithin blend before (solid curves) and after

441

(dashed curves) storage at room temperature (21 °C) for 4 months.

442


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443

Table 1. Absorbance at 600 nm of 1 or 2% w/v thyme oil emulsified by 2.5% w/v NaCas and 0-

444

1.0% w/v lecithin.* Thyme oil

Lecithin

Absorbance

(% w/v)

(% w/v)

at 600 nm

1.0

0

1.48±0.14c

1.0

0.5

0.30±0.01d

1.0

1.0

0.27±0.01d

2.0

0

2.44±0.10a

2.0

0.5

2.21±0.16b

2.0

1.0

1.35±0.01c

445

*All samples contained 10% v/v PG. Numbers are mean ± standard deviation from duplicates. Different

446

superscript letters represent significant differences in the mean.

447



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448

Table 2. Mean hydrodynamic diameters (nm) of 1% and 2% w/v thyme oil emulsified by NaCas, lecithin or both, before and after storage at

449

room temperature for 120 days.* Thyme oil

Lecithin

(% w/v)

(% w/v)

1.0

2.0

Hydrodynamic diameter (nm) 0% NaCas

2.5% w/v NaCas

5.0% w/v NaCas

Day 0

Day 120

Day 0

Day 120

Day 0

Day 120

0

N/A

N/A

150.2±6.6c

N/A

105.5±3.5d

N/A

0.5

177.5±3.0bc

188.5±2.4abc

77.1±5.4fg

80.7±0.7efg

74.7±0.4fg

75.4±1.0fg

1.0

179.7±2.2bc

203.6±5.9a

67.5±0.8g

83.9±13.7efg

66.6±2.1g

71.8±4.5g

0

N/A

N/A

206.5±10.5a

N/A

180.7±12.6bc

N/A

0.5

170.3±2.9c

184.3±6.0abc

115.9±10.1d

113.0±0.8d

79.1±1.8efg

86.1±10.6efg

1.0

177.7±4.6bc

196.3±11.5ab

95.2±2.4def

101.6±4.9de

68.4±2.3g

72.7±1.7fg

450

*All samples contained 10% v/v PG. Numbers are mean ± standard deviation from duplicates. Different superscript letters represent significant differences

451

in the mean.

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452

Table 3. Zeta-potential (mV) of thyme oil (1.0% w/v) nanoemulsions prepared with NaCas

453

with and without lecithin at pH 7.0.* Lecithin (% w/v)

Zeta-potential (mV) 0% NaCas

2.5% w/v NaCas

5.0% w/v NaCas

0

X

-25.8±6.8c

-39.7±1.2b

1.0

-58.2±1.2a

-44.6±2.6b

-41.7±2.4b

454

*All emulsions contained 10% v/v PG. Numbers are mean ± standard deviation from duplicates.

455

Different superscript letters represent significant differences in the mean.



Page 24 of 31

456

Table 4. Volume-area mean diameter d3, 2 (nm), polydispersity index, percentage of adsorbed NaCas, and surface load of NaCas in emulsions

457

with 10% w/v thyme oil prepared with NaCas, lecithin, or both, before and after storage at 21 °C for 15 days.* NaCas

Lecithin

(% w/v)

(% w/v)

0

2.5

5.0

458

d3,2 (nm)

Polydispersity index

Adsorbed NaCas (%)

Protein surface load (Γs, mg/m2)

Day 0

Day 15

Day 0

Day 15

Day 0

Day 15

Day 0

Day 15

0.5

175.28±6.97c

182.37±7.32c

0.28±0.02ab

0.27±0.02bc

N/A

N/A

N/A

N/A

1.0

163.14±15.22c

161.38±27.09c

0.23±0.00c

0.22±0.02c

N/A

N/A

N/A

N/A

0

307.96±10.54ab

350.80±12.12a

0.31±0.02ab

0.30±0.01ab

69.82±3.36a

69.27±0.33a

8.35±0.40ab

9.43±0.04a

0.5

282.66±12.89b

350.48±8.65a

0.32±0.01a

0.30±0.01ab

68.56±2.91a

64.50±2.14ab

7.53±0.32abc

8.78±0.29a

1.0

182.42±8.52c

181.18±19.96c

0.28±0.01ab

0.29±0.01ab

67.84±1.23a

61.23±0.22abc

4.81±0.09e

4.31±0.02e

0

172.93±11.04c

170.64±11.12c

0.28±0.01ab

0.26±0.01bc

64.15±1.93abc

52.22±2.13cd

8.62±0.26a

6.92±0.28bcd

0.5

159.78±9.82c

143.11±13.98c

0.26±0.02bc

0.26±0.02bc

62.51±1.32abc

48.68±4.12d

7.75±0.16ab

5.41±0.46de

1.0

144.02±0.29c

143.53±7.30c

0.27±0.01bc

0.28±0.02ab

54.64±7.31bcd

46.67±1.43d

6.11±0.82cde

5.20±0.16e

* Numbers are mean ± standard deviation from duplicates. Different superscript letters represent significant differences in the mean.

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459 460

Figure 1.

461



Page 26 of 31

Absorbance at 600nm

2.5 NaCas NaCas+PG NaCas+Lecithin NaCas+Lecithin+PG

2.0

1.5

1.0

0.5

0.0 0.5

1.0

1.5

2.0

Thyme oil concentration(%) 462 463

Figure 2.

464


2.5

Page 27 of 31


465 466

Figure 3.



467 468

Figure 4.

469


Page 28 of 31

Page 29 of 31


Differential Intensity (%)

30

(A)

0.5% L

0.5% L+2.5% NaCas 25

0.5% L+5% NaCas 0.5% L

20

0.5% L+2.5% NaCas 0.5% L+5% NaCas

15 10 5 0 0

100

200

300

400

Diameter (nm) 470


30

(B) 0.5% L 0.5% L+2.5% NaCas 0.5% L+5% NaCas 0.5% L 0.5% L+2.5% NaCas 0.5% L+5% NaCas

25 20 15 10 5 0 0

100

200

Diameter (nm) 471 472


300

400


Page 30 of 31


30 (C) 1% L 1% L+2.5% NaCas 1% L+5% NaCas 1% L 1% L+2.5% NaCas 1% L+5% NaCas

25 20 15 10 5 0 0

100

200

300

400

Diameter (nm) 473

(D) 1% L 1% L+2.5% NaCas 1% L+5% NaCas 1% L 1% L+2.5% NaCas 1% L+5% NaCas


25 20 15 10 5 0 0

200

Diameter (nm)

474 475

100

Figure 5.

476 477


300

400

Page 31 of 31

478


Table-of–content graphic only

479 480


Thyme Oil Nanoemulsions Coemulsified by Sodium Caseinate and

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