Water Emulsions Affecting the Deposition, Clearance

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Properties of Oil/Water Emulsions Affecting the Deposition, Clearance and After-Feel Sensory Perception of Oral Coatings Sara Camacho, Elyn Hollander, Fred van de Velde, and Markus Stieger J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505653t • Publication Date (Web): 14 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Journal of Agricultural and Food Chemistry

Properties of Oil/Water Emulsions Affecting the Deposition, Clearance and After-Feel Sensory Perception of Oral Coatings

Sara Camacho a,b, Elyn den Hollander b, Fred van de Velde a,c, Markus Stieger a,b,* a

TI Food and Nutrition, P.O. Box 557, 6700 AN Wageningen, The Netherlands

b

Wageningen University, Agrotechnology and Food Sciences Group, P.O. Box 8129, 6700 EV Wageningen, The Netherlands

C

NIZO food research BV, P.O.Box 20, 6710 BA Ede, The Netherlands

*

Corresponding author at: Wageningen University, Agrotechnology and Food Sciences Group, Division of Human Nutrition, P.O. Box 8129, 6700 EV Wageningen, The Netherlands. Tel.: +31 317 481694. E-mail address: [email protected]

ACS Paragon Plus Environment


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Abstract

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The aims of this study were to investigate the influence of (i) protein type, (ii) protein content

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and (iii) viscosity of o/w emulsions on the deposition and clearance of oral oil coatings and

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after-feel perception. Oil fraction (moil/cm2tongue) and after-feel perception differed

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considerably between emulsions which do not flocculate under in mouth conditions (Na-

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caseinate) and emulsions which flocculate under in mouth conditions (lysozyme). The

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irreversible flocculation of lysozyme stabilized emulsions caused slower oil clearance from

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the tongue surface compared to emulsions stabilized with Na-caseinate. Protein content had a

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negative relation with oil fraction for lysozyme stabilized emulsions and no relation for Na-

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caseinate stabilized emulsions, immediately after expectoration. Viscosity differences did not

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affect oil fraction, although the presence of thickener decreased deposition of oil on tongue.

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We conclude that after-feel perception of o/w emulsions is complex and depends on the

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deposited oil fraction, the behavior of proteins in mouth and thickeners.

14 15 16 17

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Keywords: oral coating, in vivo fluorescence measurements, emulsion, Na-caseinate,

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lysozyme, xanthan, after-feel perception


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Introduction

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Oral coatings are defined as residues of foods and beverages that are retained in the oral

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cavity after swallowing. Oral coatings are known to influence taste, texture and after-feel

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perception of foods.1-5 The formation and clearance of oral coatings on the tongue surface is

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hypothesized to depend on several factors such as the composition of the food (type and

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amount of fat, protein and polysaccharide), the physical-chemical properties of the food

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(rheological properties, pH) and the morphology of the tongue which can vary between

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individuals.1-9

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Several studies have reported relations between oral coatings and after-feel perception.1,5,8-10

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After-feel perception of oil/water (o/w) emulsions was found to follow a semi-logarithmic

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relationship to the oil fraction deposited on the tongue surface (moil/cm2tongue) after

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expectoration of the emulsion.5 Perceived fattiness of custards was found to increase with

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increasing amount of oral coating.1 Likewise, perception of creamy and coating after-feel and

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fatty and slippery mouth-feel increased with increasing oil content of o/w emulsions.10 It was

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hypothesized that the retention of emulsion droplets onto the oral mucous layer gives rise to

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lubrication in the oral cavity leading to an enhancement of perception of fat related attributes.

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When gum arabic was added as a thickener to low oil content o/w emulsions, the perception

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of coating after-feel, fatty and slippery mouth-feel increased.10 This suggests that thickeners

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contribute to sensory perception of fat related attributes in o/w emulsions. It was hypothesized

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that a thickener can form a layer similar to oil on oral surfaces or imitate an oil layer due to

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increased viscosity in mouth. 8 Conversely, addition of guar gum to o/w emulsions resulted in

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a slightly reduced oil retention on the tongue surface.8 No differences were reported in oil

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retention on the tongue between dressings differing in type of thickeners (starch, xanthan and

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a mixture of the two).11



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In addition to viscosity, the behavior of o/w emulsions in mouth may also affect coating

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deposition and after-feel perception. The behavior of o/w emulsions in mouth depends on the

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protein used to stabilize the o/w emulsions.12,13 Flocculation of o/w emulsions can occur when

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emulsions are mixed with saliva.12,13 Whether the flocculation is reversible or not depends on

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the surface charge of the emulsion droplet, pH, salts and composition of salivary biopolymers.

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Saliva is charged negatively at neutral pH.13 Emulsion droplets stabilized with highly negative

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charged proteins do not flocculate with salivary biopolymers, while weakly negative charged

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to neutral surface charged emulsion droplets experience reversible flocculation.13 Positively

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surface charged emulsion droplets flocculate irreversibly with negative biopolymers in saliva

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due to bridging interactions.13 Previous work focused on the in mouth behavior of lysozyme, a

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protein with a net positive charge at neutral pH. It was found that flocculation due to

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electrostatic interactions occurred when o/w emulsions stabilized with lysozyme were mixed

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with saliva.13

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In addition to protein charge, also protein content can play a role on the coating deposition

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and after-feel perception. The effect of protein-poor (or unstable) against protein-rich (or

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stable) o/w emulsions on the adhesion and spreading of fat on the tongue was previously

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reported.6 It was found that o/w emulsions stabilized with little protein had a stronger

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adhesion on the tongue (i.e., more stable against rinsing with saliva) compared to o/w

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emulsions stabilized with high concentration of protein. It was hypothesized that this was due

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to difference in stability of the interfacial layer of the emulsion against rupture, and thus

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creating coalescence of the oil droplets.6

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Although the mentioned studies have indicated relations between o/w emulsion properties,

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such as viscosity, emulsifier type and content, and oral coatings, the mechanisms on how

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different macronutrients influence the deposition and clearance of oral oil coatings and how

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they relate to after-feel perception are still not understood. The aims of this study are to


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investigate the influence of (i) protein type, (ii) protein content and (iii) viscosity of o/w

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emulsions on the clearance of oral oil coatings and after-feel sensory perception. The

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deposition and clearance of oral oil coatings from the tongue surface was determined using in

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vivo fluorescence measurements. The after-feel perception was determined using progressive

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profiling. To study the influence of protein emulsifier type on oil coatings, o/w emulsions

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stabilized with proteins differing in flocculation behavior when mixed with saliva were

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prepared. Lysozyme which forms irreversible agglomerates upon mixing with saliva and Na-

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caseinate which is hypothesized to form no agglomerates upon mixing with saliva were

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used.13 To study the influence of protein content on clearance and after-feel sensory

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perception of oral oil coatings, three concentrations of protein were used (all in excess to

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stabilize the water/oil interface). We hypothesize that excess protein has a blending effect

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with saliva resulting in faster clearance of oil deposits from the tongue surface. To study the

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influence of viscosity of o/w emulsions on the clearance and after-feel sensory perception of

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oral oil coatings, three o/w emulsions stabilized with Na-caseinate were thickened with

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varying concentrations of xanthan gum. Emulsions stabilized with Na-caseinate were chosen

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to study the influence of viscosity, independently from other factors, to rule out possible

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effects of emulsifier-saliva biopolymers. Xanthan gum was chosen as it is commonly used as

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a thickener in various food liquid foods.



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Materials and Methods

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Materials

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Sunflower oil (Perfekt, purchased from local retailer), Na-caseinate (Excellion sodium

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caseinate S, DMV International, The Netherlands, typical protein content: 91%), lysozyme

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hydrochloride (The Protein Company, Belgium, protein content: > 99%), sucrose (AH basic,

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purchased from local retailer), xanthan gum (Keltrol Advanced Performance, CP Kelco,

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Denmark), curcumin (7% w/w curcumin dissolved in propylene glycol and polysorbate; L-

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WS; Sensient, The Netherlands), NaOH (Merck, Germany) and tap water were used. All

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ingredients were food grade. Pig’s tongues were obtained from the VION Food Group (The

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Netherlands).

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Preparation and Characterization of o/w emulsions

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Table 1 shows the composition of all the o/w emulsions. All o/w emulsions were prepared in a

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food-grade environment. The oil phase of the o/w emulsions consisted of sunflower oil (10%

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(w/w)). The aqueous phase of the o/w emulsions consisted of protein solution (concentrations

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see table 1) with 8% (w/w) sucrose. The aqueous phase was prepared by first dissolving

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sucrose in tap water, and later dissolving either Na-caseinate or lysozyme, at room

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temperature. The aqueous phase with dissolved lysozyme was brought to a pH of 6.9 by

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addition of 1M NaOH. Protein concentration in the aqueous phase varied (0.2%, 3.0% and

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5.8% (w/w)). The 0.2% (w/w) protein concentration was chosen, as the lowest concentration,

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as it allows to prepare stable emulsions with low concentrations of “free-protein” in the

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aqueous phase, i.e., protein that is not adsorbed into the oil droplets surface. The 3.0% (w/w)

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protein concentration was chosen, as the middle concentration, as it is a protein concentration

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common in dairy drinks. The 5.8% (w/w) protein concentration was chosen, as the highest

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concentration, as it creates emulsions with high concentration of “free-protein” in the aqueous


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phase and has an equal difference of protein concentration between the emulsion with 3.0%,

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as the emulsion with 0.2%.

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The aqueous phase was then pre-homogenized with the oil phase (sunflower oil) using an

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Ultra Turrax (IKA® RW 20 Digital) at 4000 min-1 for 3 minutes, and homogenized with a

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two-stage homogenizer (Niro-Soavi S.p.A. NS1001L2K) at pressures between 250 and 350

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bar. All o/w emulsions had a neutral pH. The o/w emulsions thickened with xanthan gum

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were prepared from an o/w stock emulsion and mixed at a ratio of 1:1 with pre-prepared

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aqueous xanthan gum solutions. Xanthan gum solutions were prepared by heating water to

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70°C and adding the xanthan gum under agitation. The xanthan gum thickened o/w emulsions

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contained 10% (w/w) oil, 3% (w/w) Na-caseinate and 8% (w/w) sucrose in the aqueous phase

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and varying concentrations of xanthan gum (see table 1).

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Emulsions were stored after preparation in a refrigerator at 4°C and used for further studies no

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later than three days after preparation. Curcumin was added to the emulsions as a hydrophobic

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fluorescent dye prior to the in vivo measurements. One µL of 7% curcumin per gram of o/w

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emulsions was added under magnetic stirring immediately before testing.

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Particle size distribution was determined by light scattering (Malvern Instruments, Goffin

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Meyvis) using a refractive index for sunflower oil of 1.469. Particle size of the different o/w

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emulsions was measured for every batch made for the fluorescence measurements. Sample

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was added to the light scattering measurement device until the obscuration reached 10%. d3,2

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was obtained as a measure of oil droplet size and averaged over measurements of 3-4 batches.

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For every emulsion, the number of oil droplets was estimated by calculating:

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ܰ‫ ݏݐ݈݁݌݋ݎܦ ݈ܱ݅ ݂݋ ݎܾ݁݉ݑ‬ሺܰሻ =

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number of oil droplets in each emulsion, the surface area of the emulsion can be estimated by

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calculating: ܵ‫ × ܰ = ݊݋݅ݏ݈ݑ݉ܧ ݂݋ ܽ݁ݎܣ ݂݁ܿܽݎݑ‬4 × ߨ × ‫ ݎ‬ଶ. Based on previous studies we can

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estimate the amount of protein needed to cover the surface area of the emulsions.14 We

௏௢௟௨௠௘ ௢௙ ை௜௟ ర ×గ×௥ య య

where ‫ ݎ‬is radius of the oil droplet.


By knowing the


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estimate that for the o/w emulsions with lower protein concentrations (0.2% w/w) about 1

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mg/m2 (Na-Caseinate) to 1.5 mg/m2 (lysozyme) of protein is adsorbed onto the surface of the

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oil droplets. For the higher protein concentrations used in our study (> 2.25% (w/w)), we

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estimate that about 3.2 mg/m2 of protein (Na-Caseinate and lysozyme) is adsorbed onto the

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surface of the oil droplets.14 This corresponds to a minimum protein concentration absorbed

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on the oil droplet interface of 0.05% (w/w) for low and 0.15% (w/w) for high protein

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concentrations. Therefore, all protein concentrations used in our study to emulsify the o/w

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emulsions are assumed to be higher than the minimum protein concentration required to cover

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the oil droplet surface (see Table 1). All o/w emulsions used contained an excess of protein as

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emulsifier.

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Flow curves of all o/w emulsions were determined in duplicate at 20°C at shear rates ranging

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from 0.01 to 1000 s-1 using a rheometer (Anton Paar GmbH, Physica MCR 301) with a double

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gap geometry. All samples displayed shear thinning behavior. The viscosities at a shear rate

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of 63s-1 (closest value in the range of the reported in-mouth shear rate (50-100 s-1) 15,16) were

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extracted from the flow curves and are reported in table 1.

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Light microscopy (Carl Zeiss, Axioskop 2 Plus) was used to characterize the structure of the

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o/w emulsions before and after mixing with human saliva at a magnification of 40x. Saliva

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was collected according to reported procedure.13 Unstimulated saliva was collected for 30

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minutes in the same morning as the experiments from one healthy subject. First the subject

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rinsed her mouth with water and then collected the saliva with closed lips and expectorated

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into ice chilled beakers.13 Mixtures between o/w emulsions and saliva were prepared at room

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temperature at a ratio of 1:1 as in previous reports.17,18

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In vivo fluorescence measurements to determine oil fraction deposited on tongue surface

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Fluorescence Measurements and Calibration Curves

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The method used in the present study has been previously described in detail.5 Fluorescence

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measurements were made with a single point measurement (Fluorolog Instruments SA Inc,

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Jobin Yvon Spex) at an excitation wavelength of 440 nm, an emission wavelength of 495 nm

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with a slit width of 0.95 mm and a measurement time of 0.1s. A fluorescence remote read

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fiber optic probe was used to measure on tongue surfaces (in vivo for determination of oil

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fraction, and ex vivo for calibration curves). A plastic ring (diameter 16.9 mm, height 5 mm)

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was attached to the end of the probe to ensure that the distance between the probe and the

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tongue surface remained constant. The probe was put perpendicularly to the tongue surface

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and with slight pressure to avoid deformation of the papillae and the coating. For all

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measurements the background (fluorescence intensity of the tongue surface without o/w

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emulsion) was measured first. The background measured immediately before the

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measurement of the o/w emulsion was subtracted afterwards from the o/w emulsion

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measurement.

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To convert the fluorescence intensity measured on the subjects tongue surface to the oil

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fraction deposited on the tongue surface, i.e. mass of oil per area of tongue (mg/cm2),

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calibration lines were made following the procedure described previously.5 Briefly,

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calibrations were made with pieces of the middle part of the pig’s anterior tongue at 37.5oC.

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O/w emulsions at room temperature were spread on the surface of the pig’s tongue (2x2 cm)

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and fluorescence intensity was measured. For each measurement a fresh piece of tongue was

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used.5 As the protein type and content affects the fluorescence intensity of curcumin,

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calibrations (fluorescent intensities vs. oil fraction on tongue surface) were made for each of

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the nine o/w emulsions. Calibration lines consisted of six data points, for targeted oil fractions

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of 0.10 mg/cm2 (Vemulsion=4µL), 0.20 mg/cm2 (Vemulsion=8µL), 0.50 mg/cm2 (Vemulsion=20µL), ACS Paragon Plus Environment


mg/cm2

(Vemulsion=25µL),

0.88

mg/cm2

(Vemulsion=35µL),

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0.63

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(Vemulsion=40µL). All calibration measurements were performed in triplicate and are shown in

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the appendix.

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Presentation of o/w emulsions

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O/w emulsions were presented to the subjects at room temperature in three blocks. Each block

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consisted of two sessions of about one hour during which each o/w emulsion was presented in

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triplicate. The o/w emulsions were presented in randomized order over subjects per block.

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During the first block the o/w emulsions stabilized with 0.2, 3.0 and 5.8% (w/w) Na-caseinate

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were assessed. During the second block the o/w emulsions stabilized with 0.2, 3.0 and 5.8%

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(w/w) lysozyme were assessed. Finally, during the third block the o/w emulsions stabilized

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with 3% Na-caseinate and thickened with 0.05, 0.2 and 0.5% (w/w) xanthan gum were

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assessed.

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Determination of oil fraction deposited on tongue surface

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Twenty untrained subjects (7 men, 13 women, mean age 23.4 ± 3.2 years) assessed the nine

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o/w emulsions in triplicate. Only subjects without tongue piercings and braces participated in

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the study. Participants gave informed written consent and received a financial compensation

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for their participation.

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Subjects were asked to ingest 20 mL of the o/w emulsions and let it flow freely in the mouth

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for 30 s. Then, subjects expectorated the o/w emulsions and the fluorescence intensity was

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measured on the front and back part of the anterior tongue at 0, 15, 30, 45, 60, 90, 120 and

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180 s after expectoration. Before presentation of the next o/w emulsion, subjects cleaned their

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tongue with a tongue scraper and crackers were provided together with warm water (40°C)

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and room temperature water to rinse the oral cavity. A break of approximately 5 min was

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made between measurements.


and

1.0

mg/cm2

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Determination of after-feel perception of oral coatings

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During the fluorescence measurements, subjects performed progressive sensory profiling of

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after-feel attributes fatty film, sweetness and creaminess at 0, 30, 60, 90, 120 and 180 s after

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expectorating the o/w emulsions. The definition of attributes and evaluation protocol were

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explained to the subjects (Table 2). The three attributes for the progressive profiling were

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chosen as they have been shown to relate to the after-feel perception of o/w emulsions after

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expectoration.5,9,10 A continuous 100 mm Visual Analogue Scale was used anchored with

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“little” and “very” at a distance of 5 mm from the ends.

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Statistical data analysis

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SPSS® Statistics version 19 was used for the statistical analysis. Descriptive statistics were

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used to obtain the mean and standard error (SE). Outliers (z>2) were removed from the data.

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Fluorescence intensity data was normalized with a square root transformation. The effect of

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position of probe (within subject factor; front and back), o/w emulsion (within subject factor;

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0.2, 3.0, 5.8% lysozyme; 0.2, 3.0, 5.8% Na-caseinate; 0.05, 0.2, 0.5% xanthan), time (within

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subject factor; 0, 15, 30, 45, 60, 90, 120, 180s after expectoration) and interactions on oil

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fraction deposited on the tongue were tested by repeated-measures ANOVA. For the sensory

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data, a repeated-measures ANOVA was used to investigate the effect of o/w emulsion (within

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subject factor; 0.2, 3.0, 5.8% lysozyme; 0.2, 3.0, 5.8% Na-caseinate; 0.05, 0.2, 0.5% xanthan)

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and time (within subject factor: 0, 30, 60, 90, 120, 180s after expectoration) and interactions

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on “fatty film”, “sweetness” and “creaminess” intensity. A significance level of p

Water Emulsions Affecting the Deposition, Clearance

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