Physical characteristics of submicron emulsions upon partial displacement of whey protein by a small molecular weight surfactant and pectin addition

    Physical characteristics of submicron emulsions upon partial displacement of whey protein by a small molecular weight surfactant and pectin addition O. Kaltsa, P. Paximada, I. Mandala, E. Scholten PII: DOI: Reference:

S0963-9969(14)00650-4 doi: 10.1016/j.foodres.2014.10.005 FRIN 5517

To appear in:

Food Research International

Received date: Accepted date:

11 July 2014 5 October 2014

Please cite this article as: Kaltsa, O., Paximada, P., Mandala, I. & Scholten, E., Physical characteristics of submicron emulsions upon partial displacement of whey protein by a small molecular weight surfactant and pectin addition, Food Research International (2014), doi: 10.1016/j.foodres.2014.10.005

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ACCEPTED MANUSCRIPT Physical characteristics of submicron emulsions upon partial displacement of whey protein by a small molecular weight surfactant

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and pectin addition

Food Science & Nutrition, Agricultural University of Athens, Iera Odos 75, 11855,

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O.Kaltsaa, P. Paximadaa, I. Mandalaa ,E. Scholtenb1

Athens, Greece , [email protected]

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Physics and Physical Chemistry of Foods, Wageningen University, Bornse

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Weilanden 9, 6708 WH, Wageningen, the Netherlands

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Corresponding author. Tel.: 0317-482288 E-mail address: [email protected] (Elke Scholten)

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Abstract

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O/W emulsions (6% wt olive oil) were prepared at pH 3.3 using different WPI:Tween

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20 weight ratios (1:0, 3:1, 1:1, 1:3, 0:1) at 1% wt total concentration. The emulsion

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droplet size was found to decrease with an increase in Tween 20. A minimum droplet size of d3,2 300 nm was found for Tween systems alone, similar to that found (360

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nm) for a 1:1 WPI:Tween 20 combination (p<0.05). This specific composition showed a value for the interfacial tension close to that of Tween 20 alone. However, the

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emulsions presented low stability regardless of the WPI:Tween 20 ratio. To increase their stability, pectin was added, in various concentrations (0.2, 0.4 and 0.6% wt),

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using the Layer by Layer technique. In the presence of pectin, the ζ-potential of the

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oil droplets became negative; indicating that negatively charged pectin was

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absorbed onto the positively-charged droplet surface forming a secondary layer. The additional layer resulted in a wide range of emulsion stability. For all pectin

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concentrations, the 1:1 ratio of WPI:Tween 20 showed the highest stability. In most emulsions, extensive aggregation of oil droplets was observed, and their viscosity increased. Insufficient amounts of pectin to form the secondary layers led to bridging flocculation phenomena of oppositely charged pectin and proteins, leading to aggregation of the oil droplets. The higher the concentration of pectin, the greater the stability of the emulsion due to higher viscosity. All in all, the addition of a second layer consisting of pectin can be used to increase the stability of an emulsion containing emulsion droplets in the sub-micron range. Keywords WPI; Tween 20; submicron emulsion; pectin; stability 2

ACCEPTED MANUSCRIPT 1. Introduction Generally, most emulsions are thermodynamically unstable systems from a

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physicochemical point of view, rapidly or slowly separating into two immiscible

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phases over a period of time (McClements, 2005; Herrera, 2012). To increase the

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stability, several strategies can be used, such as decreasing the droplet size to slow down the creaming process (gravitational separation), or increasing interfacial

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coverage (thickness) to prevent coalescence.

Besides increasing the stability, the droplet size of the emulsion also plays a major

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role in many of the emulsion properties, such as encapsulation efficiency, rheology and color (McClements, 2005; Mason et al, 2006). Hence, it is not surprising that

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there is still a need to produce emulsions with droplets in the submicron range. To

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create small droplets, low molecular weight emulsifiers (LMWE) are often used. They

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have the ability to decrease the interfacial tension to a large extent, which facilitates the break-up of the emulsion droplets during the homogenization process. An

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example of such a low molecular weight surfactant is Tween 20 (Polyoxyethylene 20 sorbitan monolaurate), which is non-ionic, water-dispersible and extensively added in food emulsions. Alternative emulsifiers are globular proteins, which are also surface active but larger in size than conventional surfactants. Especially, whey protein isolate (WPI) is one of the most common emulsifiers used in the food industry. It is a complex mixture of different individual proteins, with the most common being β-lactoglobulin (~55%) and α-lactalbumin (~24%) (Dickinson, 1997). Normally, β-lactoglobulin dominates the functional characteristics of whey proteins because of its relatively high concentration. Due to their large size (compared to LMWE) protein-stabilized interfaces provide more steric stabilization, which makes 3

ACCEPTED MANUSCRIPT these emulsions less prone to aggregation. Furthermore, another major difference between a protein film and a surfactant film is their mechanical strength.

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Surfactants generally do not interact as strongly as proteins at an interface.

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A biopolymer film normally has good viscoelasticity which provides good

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protection to the droplet, while a surfactant film hardly has any (Wilde et al., 2004). Finally, the stability of the emulsions is increased for higher charge density of the

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proteins (electrostatic repulsion) compared to non-ionic LMWE. Although WPI can produce stable emulsions under various conditions, their droplet sizes are usually

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much larger than in the case of LMWE under the same emulsifying conditions (Karaiskakis et al., 2013; McClements et al, 2012), and production of emulsions with

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droplets in the submicron range is harder to accomplish. By mixing these two

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emulsifiers in different ratios, Chen and Dickinson (1993) already showed that it is

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possible to make stable emulsions with smaller droplet sizes. In food emulsions containing olive oil as the dispersed phase to form O/W

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emulsions, production of submicron emulsion droplets is even more difficult, as olive oil is much more viscous than other vegetable oils. Hence, it requires more energy to produce droplets of sub-micron size and it is difficult to make stable emulsions. Moreover, olive oil is often less processed than most seed oils as it contains more surface-active impurities that may influence its emulsification ability. However, with an appropriate mixture of proteins and LMWE, the droplet size and interfacial thickness can be altered, and may lead to a simultaneous decrease in droplet size and increase in steric stabilization, which has already been reported in literature (Friberg & Larsson, 1997; Lindman, 2001).

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ACCEPTED MANUSCRIPT Another strategy to increase the stability of emulsions (with droplets in the submicron range) is the addition of a secondary layer to increase the steric stabilization.

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For protein-stabilized emulsions this can be done by a layer-by-layer (LbL)

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electrostatic deposition technique, which has recently been used to create

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multilayer interfaces around oil droplets, consisting of charged emulsifiers and oppositely charged polyelectrolytes, under certain pH conditions (Li et al., 2010). It

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has been reported that emulsions containing oil droplets surrounded by multilayer interfaces have better stability to environmental stresses, such as pH, ionic strength

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and temperature than conventional o/w emulsions with single-layer interfaces (Guzey & McClements, 2006; McClements, 2006; Zinoviadou et al., 2012a;

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Zinoviadou et al., 2012b). For the addition of a secondary layer, anionic

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polysaccharides, such as pectin, are often used. Pectin is a well-known food additive

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which is mainly used for its gelling and stabilizing abilities. It has been shown to adsorb to protein-coated droplets at pH values below the pI of the proteins by using

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the LbL technique (Li et al., 2010; Neirynck et al., 2004). Even though the layer-bylayer technique has been used to increase the stability of protein-stabilized emulsions, the effect of a protein-LMWE mixture on emulsions interface composition on LbL technique is quite unknown. However, the combination of these strategies might be used to decrease droplet size and increase emulsion stability simultaneously. Moreover, even though the interaction between whey proteins and other LMW emulsifiers has been widely studied (Dickinson and Wasket, 1989, Dickinson & Euston 1990, Dickinson et al. 1990, Goddard and Ananthapadmanabhan, 1993, Chen and Dickinson, 1995, Dickinson and Hong, 1995; Chen and Dickinson, 1998; Chen et al., 2000; Roth, Murray, and 5

ACCEPTED MANUSCRIPT Dickinson,2000; Nakamura et al., 2004; Tran Le et al., 2011; Nikiforidis and Kioseoglou, 2011; Munk et al., 2014; Zhao et al., 2014), there are limited data

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considering the stability of emulsions in the presence of a gum stabilizer which

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usually co-exists in a salad dressing formulation (Riscardo et al., 2003), milk-based

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beverages and alcoholic beverage emulsions (Tarko and Tuszyoski, 2007; Espinosa and Scanlon, 2013). Furthermore, one of the most interesting areas in this field of

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emulsification has been the interaction between proteins and emulsifiers. Although, both proteins and emulsifiers can stabilize emulsions alone, their individual

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mechanisms of stabilization are incompatible, often resulting in unstable systems in their simultaneous presence at the interface in a process often known as

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competitive destabilization (Wilde et al., 2004; Haling, 1981). Thus, multiple

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compound interactions’ investigation would be interesting. In our case, Tween can

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reduce the droplet size, whereas WPI and pectin can control the stabilityof the emulsion; small droplets of increased stability can be achieved.

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Therefore, the objective of this study was to investigate the effect of different compositions of WPI and Tween 20 on the average droplet size of olive oil-containing emulsions, and to investigate the stability of these emulsions upon addition of a secondary pectin layer.

2. Materials and methods

2.1 Materials Whey protein isolate (BiPro) was obtained from Davisco (Davisco Foods International, USA), pI~5.2. Tween 20 was purchased from Sigma (St. Louis, USA)

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ACCEPTED MANUSCRIPT (critical micelle concentration 60 mg/l, molecular weight 1.225 daltons) and pectin from Fluka (Buchs, Switzerland) with a degree of esterification of 70-75% and

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molecular weight (0.3-1)x105 DA. The olive oil was bought from the local

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supermarket and used as such, while for the interfacial measurements it was active

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carbon-treated (Carbograph Extract, Grace, Deerfield, IL, USA) to ensure complete purification. Citric acid monohydrate was purchased from Merck (Darmstadt,

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Germany). All the other chemicals used were of analytical grade. All aqueous solutions were prepared with ultrapure water (Millipore Corporation, Billerica, MA,

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2.2 Solution preparation

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USA).

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A 6 mM, pH 3.3 citrate stock buffer solution was prepared by dissolving citric acid

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monohydrate in ultrapure water and then adjusting the pH using 1M NaOH. WPI stock solutions (1.1% wt) were prepared by dissolving the appropriate amount of

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powder into buffer solutions under continuous stirring and stored in the refrigerator at 4oC overnight to ensure complete hydration. Tween 20 stock solutions (1.1% wt) were also prepared with the same procedure. Appropriate amounts of the emulsifiers’ stock solutions were mixed to prepare different WPI:Tween 20 weight ratios (1:0, 3:1, 1:1, 1:3, 0:1). Pectin stock solutions (0.5, 1 and 1.5 % wt) were prepared by dissolving the into the previous mentioned buffer solution (pH 3.3) under continuous stirring at 70oC for 1 hour and then left to cool down at room temperature. Thiomersal (0.01% wt) was added in the protein solutions as an antimicrobial agent (Huang, Kakuda & Cui, 2001). 2.3 Emulsion preparation 7

ACCEPTED MANUSCRIPT Primary oil-in-water emulsions in a ratio of 10:90 were prepared. Specifically, 10 g of extra virgin oil was added to 90 g of the aqueous solution emulsifier solution 1.1 %

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wt (WPI:Tween 20 mixtures of 1:0, 3:1, 1:1, 1:3, 0:1) at pH 3.3 and subsequently

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mixed in a high-shear blender (16800 rpm, 2min, Ultra Turrax T25, IKA, Germany),

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followed by one pass and 8 min of recirculation at 200 bar through a lab-scale highpressure homogenizer (LabhoScope Homogenizer, Delta Instruments, The

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Netherlands). The resulting emulsions were further diluted with the appropriate amount of buffer (pH 3.3) or pectin solutions under continuous stirring to achieve a

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final concentration of 6% wt. oil, 0.6% wt emulsifier(s) and 0, 0.2, 0.4 or 0.6% wt pectin in the secondary emulsions. The pH of the emulsions was adjusted at 3.3 with

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1M NaOH or 1M HCl, where necessary. This pH was used as it is the most typical for

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dressings. All emulsions were prepared in duplicate following the same procedure. In

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some cases three or more samples were independently prepared to confirm the results found. Acidic conditions were used for simulating food systems such as

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dressings.

2.4 Droplet size analysis The droplet size distribution of the different emulsions was measured using dynamic light scattering (Coulter Laser LS 230, Coulter Scientific Instruments, Miami, USA). Water circulating in the system was kept at 25oC using a waterbath. The dispersant fluid was deionized water. The size distribution was expressed as the surfaceweighted mean diameter or Sauter diameter (d3,2). At least three measurements were performed on freshly prepared primary emulsions, and two runs were performed for every sample to gain an averaged value. 8

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2.5 Measurement of the dynamic surface properties

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The interfacial tension of oil-solvent as a function of time was measured using a drop

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tensiometer (ADT, Itcocept, Longessaigne, France), where a purified olive oil droplet

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(7μL) was allowed to form on a tip of a needle in a solution containing buffer and WPI, Tween 20 or WPI and Tween 20 in a 1:1 ratio (Benjamins et al., 1996;

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Zinoviadou et al., 2012). The oil phase waspurified in order to determine the net effect of the emulsifier(s). The results are presented as interfacial pressure, Π=γο-γ,

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where γο is the interfacial tension of the solvent (in this case 33.1 mN/m for purified olive oil) and γ is the measured interfacial tension. The results were reported as the

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average of at least three measurements.

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2.6 ζ-potential measurements

ζ-potential measurements were carried out in a Dynamic Laser Light Scattering

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instrument (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK) at 25 oC. As the ζ-potential is related to the electrophoretic mobility of the particles, the analyzer calculates the ζ-potential from the measured velocity using the Smoluchowski equation. The samples were previously diluted (1:100) with the same buffer solution to avoid multiple scattering effects. The measurements are reported as the mean of at least two differently prepared injections, with five readings per injection.

2.7 Emulsion stability The gravitational stability of emulsions upon storage at 4oC was followed by measuring the backscattering (BS) intensity along the height of an optically 9

ACCEPTED MANUSCRIPT transparent tube using a Turbiscan MA 2000 apparatus (FormulAction, Toulouse, France). Emulsion samples (approximately 6ml) were brought into test tubes, sealed

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with a plastic cap and stored in the fridge (4oC). Measurements were performed with

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samples. Τhe creaming Index (CI) was calculated as:

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intervals of 14 days. The results are reported as the average of at least three

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Where, Hs is the height of the serum layer/ and He is the total height of the emulsion.

2.8 Light microscopy

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Samples of freshly prepared secondary emulsions were placed on a microscope slide,

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covered with a cover slip and observed under 40x magnification using an optical microscope (Zeiss, Axioscope, Germany). Several photos were taken from random

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sample positions.

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2.9 Bulk rheology

Rheological measurements of the emulsions were performed on a Physica MCR 501 rheometer (Anton Paar, Graz, Austria) using a double-gap geometry (DG-26.7) with a controlled shear rate in the range of 0.1-1000 s-1. The viscosity measurements are reported as the average of at least three recordings per sample at 20oC, while the error margin was not higher than 2%. All rheological measurements were completed before any visual destabilization effect in the emulsions took place. 3. Results and discussion 3.1 Effect of WPI:Tween 20 composition on oil droplet size

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ACCEPTED MANUSCRIPT The average volume-surface apparent diameter, d3,2, of the emulsions prepared with the different WPI:Tween 20 ratios is presented in Table 1. The results show that

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mainly two different droplet diameters are observed: around 600 nm for emulsions

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containing more WPI and around 300nm for the ones containing more Tween 20. For

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emulsions prepared with a 3:1 ratio (WPI:Tween 20), the value of the droplet diameter was practically equal to that of emulsions prepared with only WPI (1:0)

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(p<0.05), but the PDI (polydisperisty index

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two-fold increase. This suggests that the composition of the interface has been subjected to significant alteration. Two possible explanations could be suggested for

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this finding: competitive adsorption but insufficient quantity of Tween 20 to produce

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fine emulsions or partial displacement of whey protein, both leading to broader droplet distributions. It is known that at low concentrations (c< 0.01 wt%, molar

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ratio R< 0.5), low molecular weight surfactants are not always able to displace proteins from the interface and interfere with interactions within the protein layer,

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whereas at higher concentration (c>0.3, R>10) complete displacement of the protein from the droplet occurs (Demetriades & McClements, 1998, Dickinson, Euston, & Woskett, 1990). As the Tween 20 concentration was increased, the droplet size decreased up to ~300 nm. This was expected as Tween 20 is a low molecular weight surfactant, which is known to reduce the emulsion droplet size in the submicron range. This is a result of the ability of the surfactant to lower the interfacial tension to a large extent, indicating that less energy is needed to break up the droplets to reduce the droplet size (McClements, 2005). The results show that the addition of limited amounts of WPI does not lead to much larger droplet sizes. When mixed in a

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ACCEPTED MANUSCRIPT mass ratio of 1:1 or 1:3 WPI:Tween 20, the droplet size does not change significantly (values of 391 and 328 nm respectively p>0,05), as a result of the presence of WPI.

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Figure 1 shows the droplet size distribution. The compositions with an excess of WPI

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(1:0 and 3:1) have a more polydisperse droplet size distribution and even bimodal in

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just one case (3:1), as suggested by the high PDI shown in Table 1, indicating droplet coalescence. A bimodal distribution could be explained in terms of emulsifier

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shortage or process deficiency. The homogenization process, such as a short time scale or the type of homogenizer employed can influence the droplet size

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distribution of the produced emulsions (Euston, Finnigan & Hirst, 2001). The emulsions were homogenized for 8 min, and we assume that this sufficient for all

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emulsions to gain homogeneity; e.g. differences in droplet size and distribution are

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therefore expected to be a result of the composition only. The 1:1 ratio showed the

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narrowest droplet size distribution of all compositions, indicating a good uniformity of the droplet compositions with an excess of Tween 20 (1:3 and 0:1) as well as the

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1:1 tend to be monomodal, with similar polydispersities. It is believed, as stated by Mackie et al. (1999), that when there is a mixture of emulsifiers (protein and surfactants) at the interface, there are separate protein and surfactant domains. When a surfactant is added afterwards a progressive displacement of the protein occurs (Nikiforidis and Kioseoglou, 2011), as the surface pressure is increased. The surfactant can then apply a surface pressure against the surrounding protein effectively squeezing the protein from the interface and this phenomenon is concentration dependent. At specific concentrations both systems co-exist, and protein might be squeezed, but still remains at the interface, both creating a compact lamella around the droplets. 12

ACCEPTED MANUSCRIPT 3.2 Effects of surfactants on the interfacial tension The time evolution of the interfacial pressure of olive oil/water interfaces with only

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WPI, only Tween 20 and a ratio of 1:1 WPI:Tween 20 is presented in Fig. 2.

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As far as the Tween 20 system alone is concerned, the interfacial pressure (Π)

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increased rapidly, reaching a value of 28 mN/m in 100s. This was followed by a slow increase in Π over time to a final value of 30 mN/m after 2700s, showing

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reorganization of the molecules at the interface. Such a phenomenon has previously been reported for the adsorption of low molecular weight surfactants on oil/water

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interfaces (Bos & van Vliet, 2001). Bos et.al. (2001) showed that Tween 20 molecules adsorb relatively fast at freshly formed interfaces, and have the ability to reduce the

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interfacial tension to a much greater extent than high molecular weight emulsifiers,

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such as proteins. This is confirmed in our study, as the interfacial pressure of protein

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solutions alone is much lower than for the Tween 20 solution. As can be seen in Fig. 2, the pure WPI solution was found to increase the interfacial pressure to a value of

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26 mN/m within the time frame of the experiment. The results show that the adsorption kinetics for WPI only is much lower, indicating a slower diffusion to the interface due to their considerable larger size in comparison with Tween 20. When WPI and Tween 20 were jointly present, the results show that the mixture is dominated by the Tween 20, as the results resemble those of the Tween 20 most, although the amount of Tween is haft of that of the pure system; the interfacial pressure increases to a value of 29 mN/m, similar to that of the Tween 20 only. As Fig. 2 shows, the initial change in the surface pressure is higher for the mixture than for Tween 20 alone (time =0), but the diffusion rate to the interface is slowed down later on, consistent with an increase in size due to competition of WPI and Tween 20 13

ACCEPTED MANUSCRIPT to be absorbed at the interface. This high increase in interfacial pressure can explain that for this specific ratio small droplet sizes were found, similar to the droplets with

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Tween 20 only. Tween 20 and WPI, seems to be present onto the surface

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simultaneously, and Tween presence might accelerate the deposition of the WPI on

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the oil particles. Their simultuaneous presence seems to positively influence the

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interfacial properties of the interfaces such as size and narrow size distribution.

3.3 Effect of pectin addition on droplet charge

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In order to increase the stability of the emulsion droplets, negatively-charged pectin in various concentrations was added to the emulsion droplets, covered with

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different WPI:Tween 20 compositions. Αs the pH of the emulsions was adjusted to

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3.3, which is lower than the isoelectric point of WPI, WPI was positively charged and

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interacted with the negatively charged pectin. The difference in the charges of WPI and pectin and their complexation cause changes in the charge density of the emulsion droplets. In Fig. 3, the ζ-potential of these emulsions for the different

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WPI:Tween 20 compositions is depicted. As expected, in the absence of pectin, the overall charge of the oil droplets of emulsions containing only WPI was positive (+35.9 mV), as the WPI bears a positive charge at this pH. On the other hand, the overall charge of the oil droplets containing only Tween 20 (0:1) was found to be neutral within the experimental error, which was expected since Tween 20 is a non-ionic surfactant (McClements, 2005). As far as the intermediate ratios are concerned, the 3:1 ratio (WPI:Tween 20) shows a similar ζ-potential (+35mV) to that of the droplets with WPI alone and therefore it can be concluded that this interface is dominated by the presence of WPI, and only 14

ACCEPTED MANUSCRIPT limited amount of Tween 20 is present. For this composition, the Tween 20 does not have the ability to compete with the adsorption of the proteins. When the Tween 20

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concentration was increased, the ζ-potential value was lowered, indicating co-

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adsorption of Tween 20 with proteins, decreasing the total amount of protein and

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the accompanying charge density. At a ratio of 1:1, the ζ-potential decreased to a value of 25mV, and to 10 mV for a ratio of 1:3. These results indicate that the more

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Tween 20 is present, the less protein is adsorbed at the interface, and therefore the ζ-potential value decreases to values close to 0 mV. Data found confirmed previous

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suggestions about droplet size and co-existence of the two ingredients. The positive charge of the interface can be used to adsorb a secondary layer of

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negatively-charged pectin. By pectin addition, the ζ-potential turned from positive to

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negative for all its concentrations and in all samples as depicted in Figure 3. In the

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case of sufficient amount of adsorbed proteins, pectin molecules were absorbed onto the droplet surface and formed a second layer. At higher pectin concentrations

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(0.4 and 0.6%wt), the overall charge of the oil droplets slightly increased compared to the charge for low concentrations of pectin. The lower the amount of the proteins at the interface (from 1:0, 3:1, to 1:1), the lower the ζ-potential change by pectin increase, meaning that droplet surfaces saturation by the adsorbed pectin, occurred at lower pectin concentrations, since protein amount was also lower. Previous studies with WPI-stabilized emulsions have shown that the droplet surfaces can be saturated at 0.25 wt% pectin concentration (Moreau et al., 2003). For emulsions with higher Τween 20 coverage, the ζ-potential exhibited small negative values. This indicates that the secondary pectin layer did not form to large extent, which was expected because of the absence of positively-charged proteins on the surface of the 15

ACCEPTED MANUSCRIPT droplets. This small negative value can be attributed to the presence of pectin in the continuous phase, which is probably measured. Similar findings on the capability of

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pectin to absorb onto positively charged droplets with WPI have been previously

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reported by others (Cho, Decker & McClements, 2009; Dickinson, 2001; Li et al.,

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2012).

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3.4 Effect of WPI: Tween 20 composition and pectin on storage stability 3.4.1 Phase separation

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To evaluate the stability of the different emulsions upon storage, the creaming index for emulsions of various pectin concentrations and WPI:Tween 20 ratios was

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recorded for a 14 days period, and is presented in Fig. 4.

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Without added pectin, the emulsions had a significantly low creaming index (CI),

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indicating lower degree of phase separation. The addition of pectin increases instability and the different ratios of WPI:Tween 20 showed no large differences in

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creaming index (12-18%). The instability is likely to be because of the presence of pectin that can induce aggregation between the oil droplets due to bridging interactions, in particular at low concentrations. The addition of a higher pectin amount (0.6%wt) led to a decrease in CI of the emulsions containing higher amounts of WPI (Fig. 4). This could be attributed to the full coverage of droplets with pectin leading to stable emulsions against depletion flocculation (Neirynck et al., 2007). Specifically, pectin was in the appropriate concentration to saturate the droplet and therefore caused steric and electrostatic stabilization. Moreover, the viscosity of the continuous phase is increased. On the other hand, when higher amount of pectin (0.4 and 0.6%wt.) was added to 16

ACCEPTED MANUSCRIPT emulsions with an excess of Tween 20, larger values of creaming index were observed (Fig. 4). The larger degree of phase separation occurred due to the fact

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that pectin cannot adsorb on the oil droplet and hence, it remains in the continuous

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phase, inducing depletion interactions and thus extensive aggregation. Similar

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phenomena were observed by Protonotariou et al. (2013) in WPC –xanthan containing emulsions, where the neutral xanthan acts as the depletion agent.

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Figure 5 shows similar results, but given as the percentage of phase separation within the 14 day period for the different pectin concentrations added. Without

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addition of pectin (Fig. 5a), all emulsions showed less extended phase separation. When a small amount of pectin (0.2% wt.) was added to the emulsions, all of them,

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except for the 1:1 ratio, phase-separated relatively fast (Fig. 5b). For higher amount

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of pectin (Fig. 5c and 5d), the emulsions with excess of Tween 20 were more

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unstable than the emulsions with excess of WPI, probably due to lack of repulsive interactions between the droplets and the depletion flocculation process.

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From the results in Figure 4 and 5, we can conclude that the 1:1 ratio seems to form the most stable emulsions stabilized by both protein and pectin. Its creaming index was lower than the other WPI and Tween compositions regardless of the concentration of pectin. Furthermore, the 1:1 ratio seems to form more stable emulsions when it contains pectin than in its absence. This behavior can be attributed to the increase in viscosity of the continuous phase due to the addition of pectin in the emulsions, comparing to those without pectin.

3.4.2 Structural organization

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ACCEPTED MANUSCRIPT In order to understand the differences in the stability of the emulsions, the microstructure of the emulsions was investigated. In Figure 6, micrographs of

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emulsions containing various concentrations of pectin (0-0.6%wt.) are

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demonstrated.

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It is clearly seen that in the absence of pectin no flocculation can be observed, and phase separation is solely an effect of droplet creaming due to lower viscosity of the

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continuous phase, leading to increased creaming velocities as explained by Stokes law. Upon the addition of pectin, in both an excess of WPI and an excess of Tween

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20, aggregation phenomena took place. For the emulsions with a higher concentration of WPI, the presence of negatively charged pectin caused bridging

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interactions with the positively-charged oil droplets, while for the emulsions with

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excess of Tween 20 (1:3, 0:1) pectin remained in the continuous phase and caused

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aggregation due to depletion interactions. In the 1:1 ratio, no aggregation was observed, indicating that no extensive bridging or depletion interactions occur.

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These results are in agreement with the results from the storage stability. The more extensive the aggregation, the faster the creaming occurred. To conclude, without addition of pectin, the stability of the emulsion is independent on the composition of the interfacial layer (WPI: Tween 20 ratio), and showed similar values in all cases. The addition of pectin altered the stability of the emulsions, but only for a composition of 1:1 WPI:Tween 20, this was found to be beneficial when considering emulsions containing excessive Tween 20. 3.5 Effect of WPI: Tween 20 composition and pectin on rheology Structural changes due to aggregation of emulsion droplets have a large influence on the viscosity of the emulsions. The viscosity of the emulsions prepared with different 18

ACCEPTED MANUSCRIPT WPI: Tween 20 compositions and stabilized with 0-0.6% wt. pectin is presented in Fig. 7.

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In the absence of pectin, most emulsions exhibited a nearly Newtonian-like behavior

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as their viscosity was practically independent from the shear rate (Fig. 7a). Only

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when the emulsion was prepared with WPI only, a non-Newtonian behavior was observed. This Newtonian-like behavior is typical for non-aggregated emulsion

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droplets as has been previously reported (Panaras et al., 2011; Xu et al., 2012; Zinoviadou et al., 2012).

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These results are in agreement with the microscopic results (Fig. 6). The viscosity of the emulsions prepared with different pectin concentrations increased compared to

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emulsions containing no pectin due to the dominant effect of aggregation between

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the emulsion droplets at low shear rate. In addition, the greater the concentration of

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pectin in the emulsions, the higher their viscosity was due to its thickening effects and shear thinning behavior was observed (Fig. 7 b, c &d).

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In the presence of pectin and at high WPI concentrations (indicated by  and), high viscosity values were obtained, as observed in Figure 7 b, c & d. This is in accordance with the microscopic results, where extensive aggregation was observed. Due to the extensive aggregation, a certain amount of the continuous phase is entrapped within the aggregated structure, and can lead to an increase in the effective volume fraction of these entities, thereby increasing the viscosity. Progressive deformation and disruption of the aggregates do occur with applying a shear field, resulting in a shear thinning behavior (Xu et al., 2012; Zinoviadou et al., 2012). These findings are in agreement with the microstructure of the emulsions as observed by the microscope, where many aggregates are observed. 19

ACCEPTED MANUSCRIPT As expected from the lack of aggregation in the emulsions with a 1:1 WPI:Tween 20 composition (indicated by ), the flow behaviour did not change to a large extent

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with the addition of pectin. For all concentrations of pectin, the flow behavior did

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not depend on the shear rate, and shows a Newtonian-like behavior. However,

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viscosity values increased significantly by pectin concentration increase .

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4. Conclusions

Sub-micron emulsions containing extra virgin olive were produced using different

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compositions of the oil-water interface. WPI and Tween 20 were used as emulsifiers in different ratios to change the composition of the interface and thereby control the

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droplet size. The results showed that a specific ratio of WPI:Tween 20 of 1:1 can

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form emulsions not only with sub-micron droplet sizes but also stable against

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aggregation when pectin is added. A secondary polysaccharide layer in the form of negatively charged pectin was added in various concentrations (0-0.6% wt.) with the

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Layer by Layer technique (LbL). Extensive aggregation led to unstable emulsions in most of the cases, due to either bridging or depletion interactions. However, the emulsions prepared with a 1:1 ratio of WPI:Tween 20 showed an increase in their stability, and no aggregation due to bridging or depletion was observed. These results show that emulsions properties can be altered by forming complex droplet interfaces by simultaneous addition of proteins, low molecular surfactants and polysaccharides. This may have advantages in the design of emulsions fulfilling the demand for low droplet size and increased emulsion stability.

Acknowledgements 20

ACCEPTED MANUSCRIPT This work is part of the “Nonastru” project (11ΣΥΝ-2-718), implemented within the National Strategic Reference Framework (NSRF) 2007-2013 and co-financed by

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National (Greek Ministry - General Secretariat of Research and Technology) and

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Community Funds (E.U.-European Social Fund).

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ACCEPTED MANUSCRIPT Caption of figures Fig. 1. Droplet size distribution of primary emulsions prepared with various

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WPI:Tween 20 ratios: (…) 1:0, (― ‐) 3:1, (‐‐‐) 1:1, (―) 1:3 and (‐∙‐) 0:1.

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Fig. 2. Interfacial pressure as a function of time for aqueous solutions containing different WPI: Tween 20 ratios (1:0, 1:1, 0:1) at the olive oil/water interface. Fig. 3. Effect of the different concentration of pectin on the ζ-potential of o/w

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emulsions prepared with various WPI: Tween 20 combinations. Fig. 4. Creaming index as a function of pectin concentration for o/w emulsions (0.6%

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wt extra virgin olive oil) after 14 days of storage at 4oC.

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Fig. 5. Phase separation as a function of time for o/w emulsions stabilized by

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different WPI: Tween 20 ratios: () 1:0, () 3:1, () 1:1, () 1:3 and () 0:1 and containing 0% (a), 0.2% (b), 0.4% (c) and 0.6% pectin (d).

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Fig. 6. Micrographs of o/w emulsions prepared with different WPI:Tween 20 ratios (from left to right 1:0, 3:1, 1:1, 1:3 and 0:1) in the absence of pectin (a) and

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containing 0.2% (b), 0.4% (c) and 0.6%wt. pectin. Fig. 7. Viscosity as a function of shear rate for o/w emulsions stabilized by different WPI: Tween 20 ratios: () 1:0, () 3:1, () 1:1, () 1:3 and () 0.1 and containing 0% (a), 0.2% (b), 0.4% (c) and 0.6% pectin (d). Error margins where within the limit of 2%.

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ACCEPTED MANUSCRIPT Caption of tables Table 1. Droplet mean diameters (d3,2) and PDI values of primary emulsions prepared

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with various WPI: Tween 20 combinations.

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ACCEPTED MANUSCRIPT Fig. 1

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20

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5 0 0.03

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0.3 Droplet Size (μm)

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Cumulative volume (%)

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28 26 24 22 0

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Interfacial Pressure (mN/m)

Fig. 2

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WPI 1:1 Tween

10000

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time (s)

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ζ - potential (mV)

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Fig. 3

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Pectin 0% Pectin 0.2% Pectin 0.4% Pectin 0.6%

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0 -10 1:0

3:1

1:1

1:3

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Fig. 4

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60

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40

0 3:1

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1:3

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1:0

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Pectin 0% Pectin 0.2% Pectin 0.4% Pectin 0.6%

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Creaming Index (%)

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Fig. 5

a

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20 0 0

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Storage time (days)

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c

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Storage time (days)

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Phase Separation (%)

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d

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Phase Separation (%)

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4

6

8

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Storage time (days)

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3:1

1:1

1:3

(a)

0:1

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1:0

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Fig. 6

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(b)

(c)

(d)

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ACCEPTED MANUSCRIPT

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0,01

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Viscosity (Pa s)

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a

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Fig. 7

0,001 1

10

100

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Shear rate (s-1)

b

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Viscosity (Pa s)

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Shear rate (s-1)

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Table 1

d3,2 (nm)

PDI (-)

1:0

611 ± 107

2.32 ± 0.99

3:1

594 ± 79

5.07 ± 1.40

1:1

359 ± 19

0.82 ± 0.36

1:3

330 ± 31

1.12 ± 0.31

0:1

302 ± 15

0.96 ± 0.30

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WPI :Tween 20

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ACCEPTED MANUSCRIPT Highlights

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o/w emulsions of various oil-water interface composition Combination of low molecular surfactants and proteins Reduce in droplet size at sub-micron level Layer by layer technique using charged polysaccharides Control of stability by multiple compound interactions

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    

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