Food Hydrocolloids 25 (2011) 868e878

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Ability of whey protein isolate and/or sh gelatin to inhibit physical separation and lipid oxidation in sh oil-in-water beverage emulsion
Ali R. Taherian*, Michel Britten, Hassan Sabik, Patrick Fustier
Agriculture and Agri-Food Canada, Food Research and Development Center, 3600 Casavant West, St-Hyacinthe, Quebec J2S 8E3, Canada

a r t i c l e i n f o
Article history: Received 31 May 2010 Accepted 11 August 2010 Keywords: Whey protein isolate Fish gelatin Lipid oxidation Physical separation Rheology Beverage emulsions

a b s t r a c t
The effect of pH on the capability of whey protein isolate (WPI) and sh gelatin (FG), alone and in conjugation, to form and stabilize sh oil-in-water emulsions was examined. Using layer-by-layer interfacial deposition technique for WPIeFG conjugate, a total of 1% protein was used to prepare 10% sh oil emulsions. The droplets size distributions and electrical charge, surface protein concentration, ow and dynamic rheological properties and physiochemical stability of emulsions were characterize at two different pH of 3.4 and 6.8 which were selected based on the ranges of citrus and milk beverages pHs, respectively. Emulsions prepared with WPIeFG conjugate had superior physiochemical stability compare to the emulsions prepared with individual proteins. Higher rate of coalescence was associated with reduction in net charge and consequent decrease of the repulsion between coated oil droplets due to the proximity of pH to the isoelectric point of proteins. The noteworthy shear thinning viscosity, as an indication of occulation onset, was associated with whey protein stabilized sh oil emulsion prepared at pH of 3.4 and gelatin stabilized sh oil emulsion made at pH of 6.8. At pH 3.4, it appeared that lower surface charge and higher surface area of WPI stabilized emulsions promoted lipid oxidation and production of hexanal. Crown Copyright 2010 Published by Elsevier Ltd. All rights reserved.

1. Introduction Among the functional ingredients u-3 and u-6 fatty acids in sh oil which contain eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) has been claimed for their health benets. These benets include reduced susceptibility to mental illness, protection against heart disease, and improved brain and eye function in infants (Krutulyte et al., 2008; Ritter-Gooder, Lewis, Barber-Heidal, & Waltz-Hill, 2008; Sir, Kpolna, Kpolna, & Lugasi, 2008). As a result, food products containing these polyunsaturated fatty acids which positively affecting human health can be classied as so called functional food (Kolanowski, Swiderski, & Berger, 1999). However, the u-3 and u-6 fatty acids are subject to rapid and/or extensive oxidation and other chemical changes by exposure to air, light or heat during processing (Jacobsen, Bruni Let, Nielsen, & Meyer, 2008; Medina, Cascante, Torres, & Pazos, 2008). The outcomes are production of aldehydes, ketones, alcohols and hydrocarbons (Coupland & McClements, 1996) that render unacceptable colours, odours and avours in polyunsaturated fatty acid

* Corresponding author. Tel.: 1 450 768 3329; fax: 1 450 773 8461. E-mail address: ali.taherian@agr.gc.ca (A.R. Taherian).

(PUFA) containing foods and nutraceutical products. In addition, products of lipid oxidation, such as hexanal, propanal, acrolein and malonaldehyde, among others, possess adverse health effects due to their cytotoxic and genotoxic effects (Giroux, St-Amant, Fustier, Chapuzet, & Britt, 2008; Huber, Vasantha Rupasinghe, & Shahidi, 2009). Therefore, successful incorporation of u-3 fatty acids into processed foods would most likely be in the form of lipid dispersions which are referred to as oil-in-water emulsions (Dalgleish, 2006). Small spherical oil droplets, in an oil-in-water emulsion, could be stabilized in the aqueous phase by surface-active hydrocolloids such as proteins, arabic gum and modied starch (Sun & Gunasekaran, 2009; Taherian, Fustier, Britten, & Ramaswamy, 2008). The surface-active hydrocolloid is adsorbed at the interface between oil and the aqueous phase to lower surface tension, increase force of repulsion and prevent oil droplets from aggregation. Proteins extracted from a variety of natural sources can be used as emulsiers in foods because of their ability to facilitate the formation, improve the stability, and produce desirable physicochemical properties in oil-in-water emulsions (Surh, Decker, & McClements, 2006; Surh, Ward, & McClements, 2006). Owing to its hydrophobic and hydrophilic regions, whey protein isolate has been widely used as an emulsier for its ability to adsorb rapidly at the oilewater interface and provide protection for oil

0268-005X/$ e see front matter Crown Copyright 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2010.08.007

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droplets through a combination of electrostatic and steric interactions (Matsumiya, Takahashi, Inoue, & Matsumura, 2010; Sun & Gunasekaran, 2009). Such adsorbed layers around the surface of oil droplets are responsible for stabilizing the vast majority of food emulsions against occulation and coalescence. The unfolding of protein molecules at the oilewater interface leads to changes in secondary and tertiary structure, and to the exposure of residues which would normally be buried within the native globular structure (Dickinson & Matsumura, 1991). Gelatin, a derivative of animal collagen, is a relatively high molecular weight protein which is prepared by sweltering animal tissues in the presence of either acid (Type A, pI w 7e9) or alkaline (Type B pI w5). The relatively high isoelectric point (pI  7.0) of Type A gelatin allows the creation of oil-in-water emulsions with positively charged droplets. As a result, Type A gelatin may be suitable for preparing oil-in-water food emulsions with high oxidative stability since it could repel iron ions from oil droplet surfaces over most of the pH range typically found in foods (Surh, Decker, et al., 2006; Surh, Ward, et al., 2006). Gelatin as an emulsier has been subject of several studies (Cheng, Lim, Chow, Chong, & Chang, 2008; Lobo, 2002; Ries, Ye, Haisman, & Singh, 2010; Surh, Gu, Decker, & McClements, 2005). The aims of this work were rst to nd the evidence for preferential adsorbtion of the WPI over FG using deposition technique and characterize the physiochemical properties of the omega-3 sh oil emulsions as an inuence of pH and understand the factor that determine the efciencies of WPI and FG, alone and conjugated, for providing the steric and electrostatic stabilization against coalescence and occulation.

2.2. Preparation of stock solutions Buffer solutions were prepared based on the method by Colowich and Kaplan (1995). The pHs of buffer solutions were adjusted at 3.4 (juice beverage) and 6.8 (milk beverage) using citric acid (0.1 M) and dibasic sodium phosphate (0.2 M) solutions mixed in appropriate ratios. 2.3. Preparation of emulsions Prior to preparation of emulsions, sh oil was thawed in a refrigerator at 4  C for 12 h. Pure protein emulsions were then prepared by slow addition of 3 g WPI or FG to 267 g buffer solution in a pre-homogenize vessel and successive blending at high speed for 2 min using a commercial blender (Waring, ON, Canada). Protein solutions were then placed in a screw cap bottle and kept overnight at 4  C (WPI) or room temperature (sh FG) for complete hydration. Fish gelatin was stored at room temperature to prevent low temperature gelation. Following day, pre-weighed 30 g sh oil was slowly added into a 500 ml beaker containing hydrated WPI or FG solution while blending at low speed. A coarse emulsion (300 mL total volume) was then made by blending sh oil and protein solution for 3 min at high speed. Oil droplets size was further reduced with the aid of high pressure homogenizer (Emulisiex-C5, Avestin, ON, Canada) at 4000 psi for 3 passes. Final protein and fat content in the emulsions were respectively 1 and 3%. The prepared emulsions were transferred into screw cap glasses bottle and tested right after preparation. The rest of emulsion was loaded with 0.02% sodium azide and stored at 4  C before conducting the second series of test. For the preparation of WPIeFG conjugate emulsions, the method of Aoki et al. (2005) was adopted with slight modications. WPI (1.5 g) and FG (1.5 g) were separately hydrated in 133.5 g buffer solutions. Fish oil (30 g) was rst added into the hydrated whey protein, while agitating, and blended for 3 min at high speed. The coarse emulsion was homogenized at 4000 psi for 3 passes to prepare primary emulsion. The primary emulsion was then diluted in hydrated FG following by blending for 3 min at high speed and high pressure homogenization at 4000 psi for 3 passes. Prepared emulsions were tested right after preparation and within 3 weeks for assessment of size growth kinetics. 2.4. Electrical charge and oil droplet size The electrical charge (z-potential) and mean diameter of emulsion droplets were determined using a commercial instrument capable of electrophoresis and dynamic light scattering measurements (Zetasizer Nano-ZS, Malvern Instruments, Worcs., UK). Prior to conducting the measurements, emulsions were diluted 1: 250 (using double distilled water) in order to prevent multiple scattering effects in size measurement. Viscosity of diluted emulsion was measured at constant shear rate of 0.1 s1 and 25  C for 1 min to consider viscosity effect in z-potential assessment. Each individual z-potential data point was calculated from the average of at least 6 readings made on the duplicate samples. 2.5. Assessment of emulsion protein load: effect of protein concentration Emulsions were rst prepared at 4 level of protein concentrations (0.2, 0.6, 1, and 1.5 wt%) using the identical preparation methods provided earlier. The concentration of adsorbed and free protein at the interface in the emulsions was then determined by centrifugation of emulsions at 25,200 g during 60 min at 5  C using Beckman model J2.21 and rotor model JA-20.1 (Beckman Centrifuge, USA) to

2. Material and methods 2.1. Materials Fish oil (OmegaPure, Houston, TX) containing 32e37% omega-3 fatty acid was kindly donated by NEX-XUS (Montreal, PQ). Based on the claim by OmegaPure the sh oil contains 35.2% omega-3 fatty acids and fatty acid prole was as follow:

Fatty acid Myristic, C14:0 Palmitic, C16:0 Palmitoleic, C16:1 Stearic, C18:0 Oleic, C18:1 Linoleic, C18:2 (n-6) Alpha Linoleic, C18:3 (n3) Stearidonic, C18:4 (n-3) Arachidonic, C20:4 (n-3) Eicosapentaenoic, C20:5 (n-3) Docosapentaenoic, C22:5 (n-3) Docosahexaenoic, C22:6 (n-3) Other

Area% of total fatty acid 8.2 19.1 11.7 3.0 13.2 2.2 1.6 3.5 1.7 13.8 2.2 11.8 7.0

Right after receiving the sh oil, 36 30 g sh oil was weighed in 36 screw cap bottles and store at 18  C. Fish gelatin (275 FG 30) and whey protein isolate (Hilmar 9400) were respectively provided by Rousselot Inc (Wisconsin, WI) and Hilmar Ingredients (Hilmar, CA). Food grade citric acid and disodium phosphate dehydrate (donated by Canada Colors and Chemicals Limited, Brampton, ON) were used to adjust the acidity and 0.02% sodium azide to reduce the risk of contamination in prepared emulsions.

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separate the oil droplets from the serum phase. With the aid of a syringe, serum phase was collected and the quantity of protein was assessed using kjeldahl method (Kjeltec auto 1030 analyser, Kjeldahl, Tecator). Using a Zetasizer (Nano-ZS, Malvern Instruments, Worcs., UK) the mean particle size was determined as the surface-weighted P P ni d2 mean diameter, d32 ni d3 i= i , where ni is the number of particles with diameter di. The surface protein concentration (mg/m2) was then calculated from the mean diameter (d 32) of the oil droplets and the difference in the amount of protein used to prepare emulsion and those measured in the subnatants and sediment after centrifugation.

over 15 day storage (complete information is available in Mengual, Meunier, Cayr, Puech, & Snabre, 1999). In addition, 60 ml of each emulsion sample was placed in a 100 ml Wainthropp tube to observe the separation in parallel with instrumental assessment. 2.7. Flow and dynamic rheological measurements Measurement of rheological parameters was based on the methods by Taherian et al. (2007a, 2007b) using an AR1000 Rheometer (TA Instrument, New Castle, DE, USA) equipped with a 2 degree cone of 60 mm diameter. Emulsions were subjected to ow test, measuring shear viscosity (hg) as a function of shear rate ranging from 0.1/s to 100/s at 22  C. Flow behaviour index (n) and consistency coefcient (m) were calculated using the power law model. Prior to dynamic rheology assessment a stress ramp at 0.01e100 Pa and 1 Hz frequency was conducted to nd out linear region. Dynamic rheological properties tests were then conducted at constant temperature of 22  C, 0.5 Pa stress and a range of frequency from 1 to 25 rad/s to assess storage modulus (G0 ), loss modulus (G00 ) and delta degree (G00 /G0 ). The duplicate samples along with 3 measurements for each emulsion were considered. 2.8. Optical microscopy Using a glass test tube, emulsions were gently agitated to ensure homogeneity prior to analysis. A drop of emulsion was then placed on a microscope slide and covered with a cover slip. Images of freshly prepared emulsions were taken using a Nikon Eclipse E600 microscope coupled with a Nikon digital DXM 1200 camera (Nikon Corporation, Japan). The Nikon ACT-1 version 2.12 software was used to process the images. Pictures were taken from three different elds on each slide and representative micrographs were presented. 2.9. Assessment of oxidative stability A volatile secondary oxidation compounds (hexanal, purchased from SigmaeAldrich, Oakville, ON, Canada) was selected as an indicator for sh oil oxidation and was extracted from emulsions by solid-phase microextraction (SPME). Emulsions were stored in screw cap bottles at 25  C and exposed to 150 W UV light using a 40 cm 50 cm 60 cm open box (Macbeth, Judge II, New Windsor, NY). At the day rst and after 3 and 6 months, 1 g of sample was transferred into a 10-mL screw-top headspace clear vial; the vial was sealed with a magnetic screw cap containing a polytetrauoroethylene (PTFE)/silicone septum (Varian, Mississauga, ON, Canada). The SPME ber (85 mm Carboxen/PDMS, Supelco, Oakville, ON, Canada) was inserted into the headspace of the vial for 44 min at 40  C. The SPME operations were automated using an MPS2 multipurpose sampler (Gerstel Inc., Baltimore, MD). Volatile compounds were desorbed by inserting the ber into the injection port 1078/1079 of a Varian CP-3800 gas chromatograph (Palo Alto, CA) in splitless mode (Glass insert SPME, 0.8 ID; Varian, Mississauga, ON, Canada) for 3 min at 300  C. The GCeMS system used in this study consisted of an ion trap mass spectrometer equipped with an electronic impact (EI) ionization source controlled with Saturn 2000 mass spectrometry detector (Varian Inc., Palo Alto, CA). A Varian CP-Sil 8CB-MS capillary column (5% phenyl/95% dimethylpolysiloxane; 30 0.25 mm; 25 mm lm thickness) was used with Helium as carrier gas at a ow rate of 1.0 mL/min. The column oven was set initially at 35  C for 3 min, heated to 80  C at a rate of 6  C/min, increased to 280  C at a rate of 20  C/min and then held at 280  C for 2 min. The total time of analysis was

2.6. Measurement of surface protein composition The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) technique was used to identify the proteins subunits present in dried cream, based on their molecular weights, under denaturation condition. Oil droplets in emulsions were rst isolated to measure the adsorbed or incorporated proteins. Cream layers of emulsions were separated by centrifugation at 25200 g for 40 min at 5  C using a Beckman centrifuge (Beckman model J2.21, and rotor model JA20.1, Beckman Instruments, Fullerton, CA). The separated cream was transferred into another sample tube, dispersed well in the DI water and centrifuged at 87,000 g for 30 min at 4  C. The washing procedure was repeated again and the resultant cream layer was freeze-dried prior to quantication. Approximately 1 mg of each protein was accurately weighed, and an exact volume of sample buffer (Bio-Rad No. 161-0791) was added to obtain a concentration of exactly 1 mg protein per milliliter. For the washed cream samples the mixture was stored overnight at room temperature to allow the separation into a cream layer at the top and an aqueous serum at the bottom. The serum was then recovered and loaded with 50 ml of 2-mercaptoethanol (Bio-Rad No. 161-0792). The solution was mixed well and immersed in boiling water for 5 min. The resulting samples were microcentrifuged to remove any insoluble matter. 2.6.1. Assessment of protein A Bio-Rad Criterion Cell was used with Bio-Rad 4e12% BiseTris Gel as the medium, and dilution was performed using XT MOPS buffer (Bio-Rad No. 161-0788) at a constant voltage of 200 V in accordance with the manufacturers recommendations. A volume of 20 ml of the sample was used, and the molecular weights were estimated with a low-range standard. The proteins were visualized by staining with Coomassie Blue R-250 (Bio-Rad No. 161-0436). 2.6.2. Optical characterization of emulsion stability Emulsion stability was quantied based on the method provided by Taherian, Fustier, Ramaswamy (2007a, 2007b). Emulsions were subjected to stability test by pouring 6 ml of each emulsion into a at-bottom cylindrical glass tube (100 mm height, 16 mm internal diameter) and subjecting to an optical scanning instrument (Quick Scan, Coulter Crop., Miami, FL). The transmission of monochromatic light (l 850 nm) from the emulsions was measured as a function of their height. Separation rate was quantied by conducting a total of 10 scans (each scan was repeated 5 times throughout 10 min) within 15 days for each tube. This quantication was based on the migration rate of the oil droplets from the bottom to the top of the sample which induces a progressive fall in concentration at the sample bottom (clarication) and therefore increases the transmission. The resulting positive peaks were then transferred to Microsoft Excel and separation rate was calculated as slope of transmission mean values

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22.5 min. The mass spectrometer was operated in the mass range from 30 to 200 at a scan rate of 1.00 s/scan. Calibration curves were done using standards of hexanal and propanal in a media of whey and gelatin at pHs 3.4 and 6.8. Hexanal retention time was around 6.4 min. The quantication was realized by total ion current (TIC) mode. 2.10. Statistical analysis All experiments were repeated at least 6 times and the data were analyzed using T-test and differences were considered to be signicant at p  0.0005. Statistical analysis was done using Microsoft Excel and experiments were performed in duplicate. 3. Results and discussion 3.1. Effect of z-potential on emulsion stability The droplet charge quantication is normally done by applying an electrical voltage to the particle and measuring the speed of induced movement (Malhotra & Coupland, 2004). Fig. 1 indicates that the intensity of zeta potential of oil droplets, for all studied emulsions, was notably inuenced by pH. As the pH increased from 3.4 to 6.8 the net electrical charge of adsorbed protein on the surface of sh oil droplets changed from positive to negative. The electrical charge at low pH is below the isoelectric points of both WPI (pI z 4.6e5.6) and FG (pI z 7e9) indicating that H and OH ions are potential determining ions for z-potential dependency on pH (McClements, 2005). At pH of 6.8 the net negative charge intensity of FG-coated droplets is 3.5 0.1 mV which is greatly lower than that of whey protein isolate (56 2.1 mV). The low negative charge intensity of FG-coated droplets is related to the balancing of positive charges by the negative charges due to proximity of its isoelectric point to the pH of the system as indicated by Kittiphattanabawon, Benjakul, Visessanguan, Kishimura, and Shahidi (2010). Conversely, the net positive charge intensity of FG stabilized droplets at pH 3.4 is 42.7 1.1 mV and that of WPI is 18.0 0.4 mV. The surface charge of the droplets in both WPI and FG-coated droplets is governed by the ionization degree of amino groups (eNH2) and carboxyl groups (eCOOH) of protein molecules depending on the pH of the surrounding aqueous phase (Surh, Decker, et al., 2006; Surh, Ward, et al., 2006). At the pH closed to the isoelectric point, z-potential became zero, indicating that the number of cationic charged groups was equal to the number of anionic charged groups and therefore the net surface charge of the

droplets is neutral. A further increase in pH causes the droplets to gain a net anionic charge, which increases as the number of anionic charged group increases and cationic charged group decreases (Kulmyrzaev & Schubert, 2004). The negative charge of the gelatin covered emulsion droplets, therefore, might be due to the negatively charged amino acid surrounding the oil droplet (Aewsiri et al., 2009). The relatively higher negative z-potential of whey protein isolate coated droplets at pH 6.8 as well as higher positive zpotential of sh gelatin coated droplets at pH 3.4 may account for greater intensity of the electrostatic repulsion force and hence superior stability of emulsion. Study by Gu, Decker, and McClements (2007) indicated that the z-potential of the droplets covered with b-lactoglobulinecarrageenanegelatin as tertiary emulsions at pH 6 was 38 1 mV. The negative zeta potential was related to the adsorbtion of cationic gelatin molecules to the surfaces of the anionic b-lactoglobulinecarrageenan coated droplets. The zpotential of the droplets coated with WPIeFG conjugates were 44 1.0 mV and 64.4 1.7 mV for pH 3.4 and pH 6.8 respectively, which may account for adsorbtion of the cationic gelatin molecules to the surfaces of anionic whey protein coated droplets. 3.2. Protein concentration at the interface The purpose of conducting this test was to determine directly the amount of protein adsorbed at the oilewater interface in emulsions by analyzing the cream phase. Fig. 2 illustrates the surface concentrations of protein in emulsions as a function of WPI, FG, and WPIeFG concentrations in deionized (DI) water determined by Kjeldahl method. Increasing the concentrations of WPI and FG in emulsions made with either individual or conjugated proteins augmented the interfacial protein load. The results are in agreement with Ye (2008) and Raikos (2010) which indicated that proteins will adsorb to the oil interfaces in proportion to their concentrations in the aqueous phase. At all concentrations, however, FG appeared to be, somewhat, less readily adsorbed and the total surface protein concentrations of emulsions made with WPI were slightly higher, compare to those made with FG. This may also be related to higher proportion of hydrophobic residues, short peptides, and the open molecular structure in whey protein isolate compared to sh gelatin. The surface sh gelatin and whey protein isolate concentrations increased from 4.16 mg/m2 up to 7.23 mg/m2 and from 4.30 mg/m2 to 7.75 mg/m2, respectively, as the total protein concentration increased from 0.2% to 1.5%. Beyond 1%, the surface whey protein isolate and sh gelatin slightly increased. Conjugating WPI and FG increased interfacial protein concentration from 4.92 mg/m2 up to 8.84 mg/m2 and, similarly, slightly increased as concentration

Fig. 1. Zeta potential of whey protein isolate (WPI), sh gelatin (FG) and conjugates as an inuence of pH.

Fig. 2. Interfacial protein load of emulsions made with whey protein isolate (WPI), sh gelatin (FG) and whey protein isolate sh gelatine (WPI FG) in DI water.

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augmented from 1% to 1.5%. The outcomes suggested that whey proteins adsorbed in preference to sh gelatin, especially when protein concentrations were below 1% for preparing the emulsions. This was also in agreement with Koupantsis and Kiosseoglou (2009) and Ye (2008) as the adsorption behaviours of whey protein in the emulsions formed with whey protein and polysaccharide or whey protein and sodium caseinate were similar. Hunt and Dalgleish (1994) also suggested that a protein concentration of 1% is adequate to provide monolayer coverage of the interfacial area for 20 wt% soy oil during homogenization. Increasing the protein concentration, however, increases the unadsorbed protein concentration leading the enhancement of depletion occulation and physical separation. The different amounts of individual proteins adsorbed at the surface may also be attributed mainly to different states of protein molecular structure at the surface. Greater adsorbtion of whey proteins at low concentration may be due to less spreading of the globular whey protein molecules on the surface; in particular, b-1actoglobulin may have been adsorbed at the surface as a dimer structure as stated by Ye (2008). Fig. 3, presenting SDS-PAGE patterns of adsorbed proteins in emulsions prepared with WPI or/and FG in deionized (DI) water (pH w 6) and buffers at pH of 3.4 and 6.8 with a total protein concentration of 1 wt%. The low molecular weight bands related to whey protein (b-1actoglobulin w 18 kDa and a-lactalbumin w 14.4 kDa) appeared as 4 bands in the base of the gels whereas high molecular weights proteins, which occupy the large fractions of sh gelatin, are observed on the top of the gels. The patterns of adsorbed proteins for emulsions prepared with conjugate of WPIeFG in DI water and at pHs of 3.4 and 6.8 look more clear and similar. Similar gel quality was observed by Taylor et al. (2006) after addition of whey protein isolate into the gelatin sample. The patterns for emulsion prepared with either protein and in DI water and pH of 6.8 are also resemblance. The concentrations of b-1actoglobulin were 62.88%, 73.92% and 86.62% for WPI added emulsions at pHs of 3.4, 6.8 and DI water respectively. Conjugating WPI and FG slightly increased the surface concentration of b-1actoglobulin up to 68.58% and 76.07% at pH 3.4

and 6.8, correspondingly, even though the concentration of WPI was half of that used for emulsions made with WPI alone (0.5% vs 1%). The concentration of a-lactalbumin remains unchanged (w11%) for emulsions containing WPI and prepared in buffer solutions. The whey protein subunits concentrations for the same emulsion prepared with DI water were slightly lower. The 3 intense bands appeared on the top of the gels for emulsion prepared with either FG or WPIeFG are associated with high molecular weight proteins of 166 kD, 125 kD and 115 kD present in FG. The concentrations of proteins at 166 D and 125 kD were found to be considerably greater for emulsions made with FG at pH 3.4 compare to those prepared at pH 6.8 (20.67 vs 14.48 and 41.63% vs 22.85% respectively). Accordingly, concentrations of high molecular weight proteins (166 kD and 125 kD) were greater in WPI-FG added emulsions at pH 3.4 (7.58% and 3.77% vs 5.54% and 1.81% correspondingly), whereas 115 kD protein concentrations were comparable for emulsions made with WPIeFG at either tested pH (w14%). Overall concentrations of these proteins were comparable for emulsions prepared with DI water or buffer at pH of 6.8. This suggest that pH and its intimacy to the isoelectric point of protein play a major role in the amount of adsorbed protein to the surface of oil droplets. In addition, more exibility of amphiphilic whey protein isolate compare to sh gelatin (Jiang, Li, Chai, & Leng, 2010) could cause faster adsorbtion during homogenization and makes this protein the dominant species among the stabilizing layers. These above results show evidence for preferential adsorption of the b-1actoglobulin and a-lactalbumin where it appear in the base of SDS-gel for emulsion prepared with either whey protein isolate or conjugate of whey protein isolate and sh gelatin. 3.3. Effect of particle size distribution on emulsion stability Britten and Giroux (1991) stated that the coalescence becomes a slow destabilizing mechanism when preparing the o/w emulsions with low concentrations of proteins as the only emulsifying agent and under quiescent conditions. On this basis, emulsions were aged under quiescent conditions at room temperature. The mean average droplet size (Z-average size) was considered to compare emulsions stability and compute the rate of coalescence (Dc). Changes in Z-average were monitored during 3 weeks considering duplicate measurements and a total of 3 tests for each measurement (Table 1). Studies by Sherman (1983), Ye, Hemar, and Singh (2004), Paraskevopoulou, Boskou, and Kiosseoglou (2005) and Taherian et al. (2008) pointed out that the rate of coalescence of emulsion droplets (Dc) mainly follows the rst-order kinetics (Eq. (2))

Nt N0 expDc t

(1)

where N0 and Nt are the numbers of droplets per unit volume of emulsion initially and time t, respectively, and Dc is the rate of droplets coalescence. Using mean average droplet size measured after preparation and 3 consecutive weeks the rate of coalescence (Dc) can be determined by plotting 3(ln (Dt/D0)) vs. time (t) using Eq. (2):

ln Dt ln D0

Dc t 3

(2)

Fig. 3. SDS-PAGE patterns of the aqueous serum of whey protein isolate (WPI), sh geletin (FG) and mixture of why protein isolate sh gelatine (WPI FG) after separation and centrifugation, in DI water and pHs of 3.4 and 6.8, ranging in size from 175.66 kDa at the top to 14.40 kDa at the bottom.

where D0 and Dt are the average droplet sizes initially and at time t, respectively. As are shown in Table 1, emulsion stabilized by whey protein isolate at pH 3.4 indicated the highest rate of particle growth and coalescence. Deposition of gelatin on whey protein coated droplets appreciably (r 0.0005) reduced the rate of coalescence by 6.4 times at the identical pH. The reduction in the rate of coalescence

A.R. Taherian et al. / Food Hydrocolloids 25 (2011) 868e878 Table 1 Comparison of droplet size growth for sh oil emulsions. Emulsions pH Average particle size (nm) Day-1 WPI WPI FG FG WPI FG WPI FG 3.4 6.8 3.4 6.8 3.4 6.8 408 5.5 338.9 5.3 612.5 13.5 1110.3 15.8 593.7 10.6 474.3 9.2 Day-7 1138 11.5 440.87 14.3 808.4 13.5 2462.2 19.7 677.2 17.6 518 12.6 Day-14 3299 24.7 597.6 9.4 1224 24.5 3098.6 25.4 822.3 22.1 568.5 8.5 Day-21 4999 35.4 698.6 11.1 1364 18.8 4098.6 22.7 856.1 23.2 655.7 13.1 Dc

873

0.064 0.021 0.022 0.031 0.01 0.008

for whey protein coated droplets at pH 6.8 and after deposing gelatin was also predominant (r 0.0005). The fact that the emulsion droplets were coated by whey protein with appreciable electrical charge suggests that electrostatic repulsion may play an important role in stabilizing them against droplet aggregation (McClements, 2005). Nevertheless, statistical analysis indicated that viscosity of sh gelatin is signicantly higher (r 0.0005) than that of whey protein isolate covering oil droplets. This suggests that steric interactions have also played an essential role in retarding the droplets coalescence. Study by Hernndez-Balada, Taylor, Phillips, Marmer, and Brown (2009) also indicated that addition of a small amount of gelatin to whey protein isolate resulted in a dramatic rise of viscosity, higher gel strengths, and the appearance of high molecular weight bands due to inter-protein crosslinking in SDSPAGE gel patterns compare to either gelatin or whey protein treated separately. They suggested that the reducing environment partially unfolds the whey proteins, increases access to glutamine and lysine side chains, and the gelatin chains crosslink the whey proteins to form a network. Comparing the rate of coalescences for emulsions prepared with FG or WPI and sh oil at identical pH of 3.4 and volume ratio (4 z 0.1) shows that sh gelatin coated droplets are less susceptible to coalesce, while at elevated pH of 6.8 the rate of coalescence is inferior for whey protein coated droplets. This phenomenon is directly related to the both changes in the conformation of the protein molecule and the net charge of the adsorbed protein layers at the interface as inuence of the pH. As Pearce and Kinsella (1987) stated, the ability of the protein to be adsorbed at the surface of oil droplets depends upon its capacity to unfold and spread over the interface to stabilize the new area created. As a result, the conformational changes of the protein molecule are extremely important, because they are related to properties such as surface hydrophobicity, protein exibility, solubility, degree of disulphide bonds, degree of hydrogen interactions and other stabilizing forces. Furthermore, Fachin and Viotto (2005) studied the effects of pH and heat treatment on emulsifying properties of whey protein concentrate and concluded that emulsifying properties were considerably improved when the heat treatment was done after ultraltration at pH 7 compared to pH 6. They related this improvement to the greater denaturation of whey proteins at this pH. Likewise, study by Yamauchi, Shimizu, & Kamiya (1980) specied that the stability of whey protein emulsions enhanced when pH is increased from 5 to 7 and noted that this is, most likely, due to

an increase in electrostatic repulsion of the charge protein. Therefore at pH 3.4, closed to the isoelectric point of whey protein and pH 6.8 near to the isoelectric point of sh gelatin, the net charge is reduced and consequently the repulsion between the oil droplets coated by these proteins is decreased resulting in higher rate of coalescence. 3.4. Effect of rheological properties on stability of emulsion 3.4.1. Flow rheology Flow behaviours of emulsions were characterized by measuring the shear-rate dependent viscosity over 21 days time period. The ow curves were tted to the power law and the determination coefcients (R2) were more than 0.98 (not shown), indicating a high level of linear relation between measuring points. Table 2 illustrates representative data for the dependency of ow properties on applied shear rate for the studied emulsions as inuences by pH and aging time. In all cases, the emulsions showed shear thinning behaviour and the viscosity decreased along with increasing the shear rate. WPI stabilized emulsions at pH 3.4, among the other emulsions, demonstrated the highest apparent viscosity and lowest ow behaviour index after 21 days aging at room temperature. For this emulsion the apparent viscosity decreased from 9828.04 mPa s at 0.1 s1 to 22.42 mPa s at 100 s1 at day-21 with the ow behaviour index of 0.12. WPI stabilized emulsion under acidic condition also showed the highest rate of coalescence suggesting time dependent induction of weak cold-set gelation and reduction of electrostatic repulsion due to intimacy of the solutions pH to the isoelectric point (pI) of protein. Cavallieri and Lopes da Cunha (2008) reported that gelation process of whey protein involves two distinct stages; the rst stage is associated with an initial setting up of the gel network up to the gel point, and the second stage is linked to a structural development through bond strengthening and/or local rearrangements, which is time and pH dependant. Our results also indicated that loss in viscosity for protein solutions with pH close to isoelectric point is time dependent but structural development is nonhomogenous causing creation of two dissimilar phases, one rich in occulated and coalesced droplets trapped in a weak gel network and the other rich in soluble solid with water like viscosity. The higher amount of gel, which is directly related to the higher degree of coalescence, caused more increase in viscosity at shear rate of 0.1 s1 following by sharp decrease as shear rate develop to 100 s1.

Table 2 Changes in consistency coefcient (m) and ow behaviour index (n) of emulsions as function of pH and storage time. Emulsion WPI WPI FG FG WPI FG WPI FG pH 3.4 6.8 3.4 6.8 3.4 6.8 m (mPa) day-1 13.0 0.3 3.6 0.2 38.0 1.2 28.0 0.8 20.8 0.1 24.1 0.6 m (mPa) day-7 62.0 1.1 4.50 0.2 48.0 1.5 65.0 1.3 23.5 0.2 26.9 0.2 m (mPa) day-14 634.0 10.0 5.1 0.5 62.0 1.9 102.0 2.3 27.8 0.4 29.3 0.4 m (mPa) day-21 1290.0 11.5 5.4 0.2 72.0 2.3 128.0 4.2 31.3 0.2 33.9 0.1 n day-1 0.72 0.01 0.97 0.02 0.75 0.01 0.81 0.01 0.99 0.02 0.97 0.00 n day-7 0.49 0.01 0.95 0.01 0.70 0.01 0.70 0.01 0.97 0.01 0.95 0.01 n day-14 0.24 0.01 0.93 0.01 0.66 0.01 0.56 0.00 0.96 0.01 0.94 0.01 n day-21 0.12 0.00 0.90 0.01 0.60 0.01 0.46 0.00 0.92 0.02 0.93 0.01

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Moreover, previously reported data have shown that both increase in viscosity and decrease in ow behaviour index of emulsion as an inuence of aging is directly related to the onset of occulation, close proximity of the droplets and enhancement of the coalescence (Dickinson & Stainsby, 1988; Taherian et al., 2007a, 2007b; Vingerhoeds et al., 2009). When the ocs are small, increase in viscosity is minimal and occulation is reversible upon application of slight shear. In the case of strong occulation and coalescence of droplets the aggregation is irreversible and seems to be driven upon electrostatics interactions as was pointed out by Sillettia, Vingerhoedsa, Nordeb, and van Aken (2007). Contrary to WPI stabilized emulsions, rheological parameters for FG stabilized emulsion were more affected at pH 6.8 and increase in consistency coefcient of FG, as an inuence of aging, was higher. The value of n decreased from 0.81 at day-1 to 0.46 at day-21 indicating unset of occulation and coalescence at this pH. According to Bae et al. (2009), solubility of proteins is the lowest near their isoelectric point since intramolecular electrostatic interactions would be maximal at this point, causing the dipolar molecule to adopt the most compact conformation. The proximity of FG isoelectric point to the pH 6.8 and subsequent increase in electrostatic interactions could, therefore, explain such occurrence.

Diluting WPI-coated droplets into hydrated FG, at both pHs of 3.4 and 6.8, minimize the differences between apparent viscosities at day-1 and day-21 and showed, practically, Newtonian ow behaviour for all tested emulsions. This indicates that occulation and coalescence, despite pH, were minimal for emulsions made with conjugated WPI and FG. 3.4.2. Dynamic rheology Dynamic rheological properties were assessed through frequency development of delta degree (G00 /G0 ) over 21 days time period. Emulsions were rst pre-sheared at 100 s1 during 1 min to ensure homogeneity of the sample before subjection to frequency sweep (Fig. 4). The accuracy of storage modulus (G0 ) and loss modulus (G") was assured by investigating the linear region, where the dynamic parameters (G0 , G" and phase shift angle d) are independent of the magnitude of applied stress, and selection of proper measuring parameters via conduction of a stress sweep test. As illustrated in Fig. 4, the effect of aging was prominent for emulsion made with WPI in acidic pH. The delta degree, at the highest level of applied frequency, increased from 35.79 degree up to 54.27 degree indicating loss of elasticity. Comparing all gures, it can be noted that the emulsions stabilized with conjugated proteins show the lowest diversity between delta degrees at all levels of applied frequency.

100 WPI pH 6.8 Delta de gree (G"/G')

De l ta d e gree (G"/G')

100 WPI pH 3.4 80 60 40 20 0 Day 1 Day 7 Day 14 Day 21 0 5 10 15 Frequency (rad/s)

100

80 60 40 20 0 0 5 10 15 Frequency (rad/s)
100

Day 1 Day 7 Day 14 Day 21 20 25 30

20

25

30

FG pH 6.8
D elta d egre e (G"/G') D e l ta de g re e (G"/G') 80 60 40 20 0 0 5 10 15 Frequency (rad/s)
100

FG pH 3.4 80 60 40 20 0 20 25 30 0 5 10 15 Frequency (rad/s) 20 25 30 Day1 Day 7 Day 14 Day 21

Day1 Day 7 Day 14 Day 21

100

WPI-FG pH 6.8
80

WPI-FG pH 3.4
Delta degree (G"/G') 80 60

Delta degr ee (G "/G')

60

Day1
40

Day1
40 20 0

Day 7 Day 14 Day 21

Day 7 Day 14 Day 21

20

0 0 5 10 15 Frequency (rad/s) 20 25 30

10

15 Frequency (rad/s)

20

25

30

Fig. 4. Frequency development of delta degree (G00 /G0 ) for emulsions prepared with whey protein isolate (WPI), sh gelatin (FG) and conjugates of FG WPI as inuences of pH and storage time.

A.R. Taherian et al. / Food Hydrocolloids 25 (2011) 868e878

875

14 WPI+FG pH=3.4 (a) 12 WPI+FG pH=6.8 (b) WPI pH=3.4 (c) 10 Transmission (%) 8 6 4 WPI pH=6.8 (d) FG pH=3.4 (e) FG pH=6.8 (f)

2 0 0 2 4 6 8 10 Time (day) 12 14 16 18 20

Fig. 5. Creaming velocity proles for emulsions prepared with whey protein isolate (WPI), sh gelatin (FG) and conjugates of FG WPI as inuences of pH and aging.

Furthermore, the corresponding frequency developments of delta degree for emulsions stabilized with FG at pH 3.4 and WPI at pH 6.8 diverge in lower extend compare to FG stabilized emulsion at pH 6.8 and WPI stabilized emulsion at pH 3.4. The results are in agreement with the outcomes of rheological ow tests indicating gain of viscosity and loss of elastic components for emulsions made at the pH close to the isoelectric points of either protein. In general, emulsions prepared with conjugated WPI-FG designated higher viscosity, elasticity, and physical stability compare to those prepared with WPI or FG alone. This suggests diluting WPIcoated droplets in hydrated FG enhance the interfacial lm viscosity and elasticity due to segregative interactions between WPI and FG (Fitzsimons, Mulvihill, & Morris, 2008) on the surface of double coated emulsions droplets resulting in superior stability. 3.4.3. Effect of pH and aging on physiochemical stability of emulsions Emulsions droplets are in continual motion and frequently collide with one another leading to occulation or coalescence. Proteins lower surface tension at the interface that is formed during the emulsication process and form a macromolecular layer surrounding the dispersed droplets which structurally stabilizes the emulsions and reduce the rate of coalescence (Juliane Floury, Desrumaux, & Lardires, 2000). Fig. 5 illustrates the rate of aggregations combined with visual observation in 100 ml Wainthropp tubes for studied emulsions during 18 days with 2 days time interval. Each point in the graph represents the average of 3 measurements of transmitted light from 6 cm emulsion height which was placed in a at-bottom cylindrical glass tube. The slopes indicate the rate of droplets migration rising to the upper part of the tube and collide between each other. Palazoloa, Sorgentinib, and Wagner (2005) and McClements (2005) quoted that the total collision frequency between emulsion droplets is the contribution of the Brownian movement and the shifting of the droplets under gravitational force. Once cream phase is formed, the coalescence process is mainly governed by uctuations prole and interfacial lm movement due to a long contact between oil droplets. Coalescence likelihood increases when uctuations become large enough to form holes that can pass from one droplet to another. At this situation the magnitude of the shape uctuations is governed by the interfacial tension, lm rheology and mechanical applied forces.

The steeper slope, therefore, signies lower resistance of droplets interfacial lm against occulation and coalescence during the migration process. The shallower slopes, on the other hand, represent more stable emulsions suggesting that the thickness of the continuous phase around the droplets (interstitial continuous phase) was enough to avoid contact between droplet lms, and interfacial uctuations did not lead to the exclusion of interstitial water. As the highest rate of aggregation is associated to whey protein stabilized emulsion at pH 3.4, the results are in excellent agreement with previously mentioned coalescence rate presented in Table 1. The results obtained during stability studies conrmed rheological observations, and once again the emulsion stabilized by whey protein and sh gelatin conjugate at elevated pH demonstrated the lowest aggregation rate and, hence, the greater stability. The microstructures of studied emulsions obtained after preparation are compared in Fig. 6. As can be observed, the micrographs display evidence of aggregated droplets of WPI-coated droplets at pH 3.4 and FG-coated droplets at pH 6.8 compare to whey protein and sh gelatin stabilized emulsions at pH 6.8 and 3.4 respectively. This conrms the effect of closeness of pH to the isoelectric point of each protein. The micrographs for emulsion prepared with conjugation of both proteins show similarity between particle size distribution and conrm the segregation effect of whey protein-sh gelatin interfacial lm. Fig. 7 shows the hexanal concentration for tested emulsions after 3 and 6 months along with pictures of corresponding emulsions taken after 6 month. Hexanal is a characteristic secondary oxidation product of linoleic acid (Ries et al., 2010) and was measured to nd out extend of sh oil oxidation in proteins coated droplets. For emulsion made with whey protein at pH 3.4 the hexanal production was above all the other emulsions with the concentration of 2.54 ppm after 3 month which almost doubled up to 4.21 after 6 month aging under ultraviolet light. Deposition of sh gelatin on whey protein coated droplets at identical pH reduced the hexanal production down to 0.19 and 0.70 after 3 and 6 months respectively. Conversely, formation of hexanal for sh gelatin coated droplets was higher at pH 6.8 with concentrations of 1.74 ppm and 2.25 ppm compare to the one at pH 3.4 at 0.52 ppm and 1.41 ppm after 3 and 6 months correspondingly. Whey proteinsh gelatin coated droplets at pH 6.8 was the lowest at 0.06 ppm after 3 month and 0.18 ppm after 6 month aging at ultraviolet and room temperature conditions.

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Fig. 6. Microstructures of emulsions containing whey protein isolate (WPI), sh gelatin (FG) and conjugates of FG WPI as inuence of pH (pictures taken at the day-1, cut and resize to t the page).

Previous studies showed that both whey protein and sh gelatin are able to reduce lipid oxidation either by steric or by electrostatic stabilization. In the case of steric stabilization the thickness of the interface play the most important role. In electrostatic stabilization, extend of lipid oxidation is controlled by magnitude and the sign of

droplet charge (McClements & Decker, 2000). Protein emulsied droplets may have either a positive or negative surface charge depends on the pH of continuous phase. With a negative surface charge, emulsion droplets will attract the positively charged ions (Na, Ca2, Fe2, Fe3) and bring them into closer proximity to the

Fig. 7. Physiochemical stability of emulsions containing whey protein isolate (WPI), sh gelatin (FG) and conjugates of FG WPI after 3 and 6 month exposure to the UV light at room temperature.

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877

u-3 sh oil, thereby enhancing lipid oxidation. Conversely, droplet

carries positive charge may repel co-ions and retard lipid oxidation. Nevertheless, the solubility of mineral ions generally increases at decreasing pH which could potentially promote lipid oxidation (Jacobsen et al., 2008; Jongjareonraka, Benjakula, Visessanguanb, & Tanak, 2008). Study by Hu, McClements, and Decker (2003) indicated that particle size, inuencing surface area, droplet charge, causing either attraction or repulsion of transition metals, thickness of emulsier layer at the interfacial region of emulsion droplet, impacting interactions between lipids and aqueous phase prooxidant, and chemical components of proteins that enabling to scavenge free radicals or chelate prooxidant metals are the major factors affecting lipid oxidation rate in oil-in-water emulsions. Jacobsen et al. (2008) suggested that the antioxidative mechanism of protein in the interfacial region, such as binding trace metal ions from the lipid phase and freeradical scavenging activity, may involve a dynamic exchange process with protein molecules from the continuous phase. Our study showed that, the hexanal production for emulsion stabilized by whey protein was signicantly higher (r < 0.0005) than that stabilized by sh gelatin. The higher oxidative stability of sh gelatin stabilized emulsions at acidic pH could, therefore, be due to the higher surface charge (42.7 1.1 mv vs 18.05 0.42 mv) and lower surface area (612.5 13.5 nm vs 408 5.5 nm) of emulsions. Overall, the greatest stability was observed for WPI FG at pH 6.8, which corresponds to the highest negative charge of this conjugate (zeta potential w60 mV). Also there was a good correlation between the hexanal concentration and emulsion creaming velocity. In addition, physiochemical stability of whey protein-sh gelatin stabilized emulsions were signicantly greater (r < 0.0005) than either whey protein or sh gelatin stabilized emulsions and, hence, the stabilization effects could be consider as both steric and electrostatic. 4. Conclusions This study showed that deposition of sh gelatin over whey protein coated sh oil droplets has a major impact on the physiochemical stability of emulsion. This was attributed to both steric effect of whey protein-sh gelatin conjugate and electrostatic repulsion between the sh oil droplets which prevents them from coming into close proximity. Addition of whey protein could improve the stability of emulsions by increasing the electrostatic repulsion at pH 6.8 and is useful for delivering omega-3 sh oil into the milk beverages. On the other hand, adding sh gelatin to cover oil droplets perk up the stability of emulsions through steric stabilization at pH 3.4 and could be valuable for delivering omega-3 sh oil into the fruits beverages. The conjugate WPIeFG can be used as an effective emulsier for formulating food emulsions under acidic conditions and the results from this study may have practical applications for the design of industrial dispersions to deliver functional ingredients into the beverages. References
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