EMULSION OF SYSTEMS CONTAINING EGG YOLK , POLYSACCHARIDES AND VEGETABLE OIL

This work characterizes the emulsifying properties of systems containing egg yolk (0.1; 1.0 and 2.5 % w/v) and polysaccharides (xanthan gum, carrageen, pectin and carboxymethylcellulose) and three different vegetable oils (sunflower, canola, and palm oils). Emulsifying activity and emulsion stability were measured of each combination and it was found the effect of the oil on emulsion stability correlated to the amount of monounsaturated fatty acid. Additionally, increased egg yolk concentration increased emulsifying activity by reducing coalescence of oil droplets. Lastly, 2.5% egg yolk and 0.2% polysaccharide generated emulsions with high emulsifying activity, excellent stability, and droplet size of 4.32 μm.


INTRODUCTION
An emulsion is a macroscopic dispersion of two liquids where one is a continuous part dispersed throughout small drops of the other (MORR, 1981).Proteins with an appreciable number of hydrophobic groups are effective emulsifying agents because they promote aggregation of oil droplets.However, proteins containing both hydrophobic and hydrophilic regions are superior emulsifiers because they significantly reduce load surface tension.This allows oil droplet breakage and formation of thin films around the surfaces of emulsified droplets (WALSTRA, 1983;TCHOLAKOVA;DENKOV;DANNER, 2004;WILDE et al, 2004).
Interactions between anionic polysaccharide and protein form a conjugate which relies on hydrophilic properties of polysaccharides to thicken and stabilize aqueous medium adjacent to the emulsion interface.Therefore, it helps to stabilize drops formed by protein and prevent coalescence during the emulsification formation and subsequent storage (SHEPHERD et al., 1995;McCLEMENTS, 2004).Most hydrocolloids can act as stabilizers of oil-in-water emulsions, but only a few can act as emulsifiers.Functionality of emulsifiers demands a substantial surface activity at the oil-water interface, and the ability to facilitate the formation and stabilization of fine droplets during and after emulsification (WILLIAMS; PHILLIPS, 2004).
Emulsion stability generally occurs according to the oil volume fraction, type of oil, temperature, pH, type and quantity of emulsifier(s), ionic strength, and the presence of other ingredients such as sugar and carbohydrates (MORR, 1981;McCLEMENTS, 2005).This objective of this work was to evaluate emulsifying properties of egg yolk and four anionic polysaccharides (xanthan gum, pectin, carboximethylcellulose, and carrageen) and three vegetable oils (sunflower, canola, and palm).

Determination of emulsifying properties
To determine emulsifying activity and emulsion stability, conductivity data measurements over time were divided in two stages respectively.The first stage was measured for all but the final 2 min of homogenization, and was used to determine emulsifying activity.The second stage began immediately after homogenization and was used to determine emulsion stability (ES) through the oil volume fraction of the creamed phase ( ).Kato et al. (1985) defined the emulsifying activity (EA) of a protein as the difference between conductivity of the protein solution prior to homogenization (C 0 ) and the minimum conductivity achieved during homogenization (C 2 ) (Equation 1).Anton and Gandemer (1997) proposed a mathematic model to determine  through its relationship with conductivity (Equation 2): Where V is the volume of the aqueous solution used in the emulsion formulation (20 mL), v is the volume of used oil (7 mL), CF is the conductivity of the emulsion and C 0 is the initial conductivity of the aqueous solution.All samples were made in duplicate and measurements were repeated twice.Statistical analyses were performed with SAS ® software.
After evaluating the stability of the emulsion, microscopy was performed on a 1 mL aliquot to observe drop size and the distribution of the emulsion.Images were generated on an LV 150 (Nikon, USA) equipped with a 20× objective and assembled camera (DS-Fi1, Nikon, USA) and software (NSI-Elements D 3.0,USA).

Evaluation of emulsifying activity
Figures 1, 2, and 3 summarize EA values as a function of EY concentration and that they gradually increase with increasing EY concentrations.Greater concentrations of protein molecules form more dense interfaces, which are absorbed more quickly around oil drops (formed during homogenization) and further reduce coalescence (SURH; DECKER; McCLEMENTS, 2006;DICKINSON, 2009).This is thought to occur due to the presence of amphipathic phospholipids in EY decreasing interfacial tension among formed oil drops (ALUKO; KEERATIURAI; MINE, 1998).Regarding polysaccharide concentration, greater concentrations of polysaccharides in the system reduce EA for the three studied protein concentrations (experiments 3, 6, and 9 in Figures 1 and 2; and experiments 6 and 9 of Figure 2).This observation may be due to increasing system viscosity leading to reduced adsorption of proteins on the surfaces of oil drops formed by homogenization (McCLEMENTS, 2006;WANG;WHANG;OZKAN, 2010).EINHORN-STOLL et al. (2005) observed that conjugates of whey protein isolate (WPI) with xanthan gum or pectin had lower emulsifying activity; however, the polysaccharide was capable of forming a strong 3dimensional network around the droplets which prevented coalescence.

Concentrations of EY and PO
When the effect of oil type on EA is investigated, palm oil resulted in smaller EA values (around 1.4 mS/cm in the experiments 4 and 7) than sunflower or canola oils.The mechanism of this phenomenon is poorly understood, but Farr (2000) suggested that monounsaturated fatty acid, specifically oleic acid, further facilitates interactions between hydrophobic protein groups and oil droplets.This hypothesis is supported by the oleic acid content in canola oil [55-58% (MORAES; BELL, 1993)] being greater than sunflower oil [35-45% (BITTENCOURT; SADER; MORAIS, 1998)] and palm oil [15-25% (BORA et al., 2003)].
Tukey tests (p<0.05) were used for means comparisons, where one can observe that some EA polymers showed the most significant (p<0.05)what for certain studied oils.The CA was more efficient emulsifier at lower concentrations (0.1 and 0.2%) for sunflower oil and 0.1% for canola oil.CMC was a superior emulsifier at 0.1% concentration for palm oil and 0.4% for sunflower oil.Finally, the PEC was superior at 0.4% concentration for sunflower oil and GX of 4.0% for canola oil.

Emulsion stability
Figure 4 shows photomicrographs of emulsions with their corresponding droplet size after homogenization.It can be clearly seen that different concentrations of OP and EY directly influenced the size of oil droplets and subsequently-studied ES.
Tables 1 to 3 present fractional volumes in the cream phase.While evaluating the effects of PO and EY concentrations for each one of the studied vegetable oils, lower values of  indicate better emulsion stability.Canola oil was the one gave the lowest values of  because of its high fraction of monounsaturated fatty acids.
Figure 4 (A and C) shows that low concentrations of polysaccharide (CMC) and 1.0% EY were not sufficiently to confer EA (Figure 2), leading to instability in the formed emulsion (Table 2) with a considerably larger droplet diameter.This is primarily because low protein concentrations are inefficient and form layers around the oil droplets.As the number of hydrophobic connections around droplet is insufficient, coalescence gradually increases until the emulsion collapses and phase separation occurs (McCLEMTS, 2006;JONES;McCLEMENTS, 2010).However, Figure 4B shows that 0.2% XG and 2.5% EY, which have high EA, led to a considerable reduction in droplet diameter and subsequently higher stability (RANGSANSARID; FUKADA, 2007; ANTON; GANDEMER, 1997;LIZARRAGA;PAN;SANTIAGO, 2008).Reduced coalescence caused by the interaction of a thin protein layer around the droplets with the three-dimensional polysaccharide network bound to water (TIPPETTS; MARTINI, 2012).
Polynomial model for the oil volume fraction of the creamed phase  , as a function of mass of egg yolk and polysaccharides, was fitted to the experimental data.The general quadratic model (Equation 3) was first analyzed and the no significant parameters were eliminated on the basis of the t-test with p>0.05.Table 4 shows the predictive models based on equation model (3) for each used oil.

Concentrations of EY and PO
where  is the oil volume fraction of the creamed phase, w 1 is the mass of egg yolk lipoprotein, w 2 is the mass of polysaccharides and  1 ,  2 ,  3 ,  4 ,  5 , , and  7 were obtained by nonlinear regression.The mathematical polynomial model was well adjusted to the experimental data, as observed in table 4.This is an indicative of a good fit to the experimental data.
St a t i s t i ca l a n a l yses r evea l e d t h a t ea ch polysacch ari de genera ted emul si ficat ions wi th different characteristics using the different oils.This be h a vi or i s p r i m a r i l y d ue t o t h e i n h e r e n t characteristics of two studied polysaccharides: (i) the non-polar chemical groups attached to different hydrophilic polysaccharide backbone, or (ii) the presence of a protein component coval ently or physically bound to the polysaccharide (DICKINSON, 2009).

CONCLUSIONS
This study examined EY concentration and polysaccharide species in formation of emulsions.We observed that the combination of low EY concentrations with low polysaccharide concentrations resulted in unstable emulsions with large droplet size.However, higher EY and polysaccharide concentrations yielded stable emulsions.It is clear that oil, specifically monounsaturated fatty acids, are extremely important in emulsion formation and facilitate the emulsification process.The polynomial model used to predict the volume fraction of the cream oil phase of emulsions had a good fit to the experimental data and holds promise for further studies to assess the functional properties of this new emulsion.

Table 1 -
Φ values for systems using palm oil in different concentrations of egg yolk and polysaccharides.

Table 3 -
Φ values for systems using canola oil in different concentrations of egg yolk and polysaccharides.

Table 4 -
Models to calculate oil volume fraction of the emulsions creamed phase Φ, (p>0.05).

Table 2 -
Φ values for systems using sunflower oil in different concentrations of egg yolk and polysaccharides.