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Interactions at the interface between hydrophobic and hydrophilic emulsifiers: Polyglycerol polyricinoleate (PGPR) and milk proteins, studied by drop shape tensiometry
Food Hydrocolloids 29 (2012) 193e198
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Interactions at the interface between hydrophobic and hydrophilic emulsifiers: Polyglycerol polyricinoleate (PGPR) and milk proteins, studied by drop shape tensiometry _ Ibrahim Gülseren*, Milena Corredig Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 August 2011 Accepted 13 March 2012
The equilibrium interfacial tension and dilational elasticity at the soy oilewater interface were studied in the presence of a lipophilic emulsifier, polyglycerol polyricinoleate (PGPR), in the continuous oil phase, and dairy proteins, b-lactoglobulin (b-lg) or sodium caseinate, in the aqueous phase using drop shape tensiometry. The interfacial tension decreased with increasing PGPR concentration, and was <2 mN m1 at PGPR concentrations beyond 1%. The presence of proteins in the water phase, b-lg and sodium caseinate, further reduced the interfacial tension. Even at the low concentrations (0.008%) tested, PGPR dominated the interfacial elasticity, which was only slightly affected by the addition of elevated levels of protein. While in the presence of b-lg (0.1%) in isolation, the system showed a high interfacial elasticity, the addition of PGPR lowered the elasticity, suggesting that PGPR interfered with proteineprotein interactions at the interface, or caused displacement of b-lg. Interfacial elasticity at the oilewater interface showed little dependence on dilation or frequency of the sinusoidal oscillation, when the interface was dominated by PGPR. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Polyglycerol polyricinolate (PGPR) Drop shape tensiometry Dilational elasticity Sodium caseinate b-Lactoglobulin
1. Introduction Emulsifiers are compounds that lower surface tension by adsorbing onto interfaces and orienting their structure to minimize the Gibbs free energy. In other words, emulsifier molecules diffuse from the phase that they are solubilized in to the interface, overcome the adsorption barrier, adsorb at the interface and attain a thermodynamically favorable arrangement after adsorption (Serrien, Geeraerts, Ghosh, & Joos, 1992). Their ability to stabilize interfaces is due to the amphiphilic nature of their structures; the hydrophilic head group of surfactants is attracted to the aqueous phase, whereas the lipophilic tail preferentially interacts with the oil phase. In the case of proteins, the molecules rearrange on the surface of air or oil interfaces, with their hydrophobic moieties (with amino acids such as phenylalanine, leucine, and isoleucine) adsorbing at the surface and the hydrophilic portions protruding into solution (Hasenhuettl, 2008). Depending on the ability of the structures to rearrange, proteins may show different levels of unfolding at the interface (Haynes & Norde, 1995). At low protein concentrations, protein molecules have a higher probability of unfolding and rearranging extensively at the interface, reaching * Corresponding author. Tel.: þ1 (519) 824 4120x58132; fax: þ1 (519) 824 6631. _ Gülseren). E-mail address: [email protected] (I. 0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2012.03.010
equilibrium later than at high protein concentrations, where the extent of rearrangements is low (Wüstneck, Moser, & Muschiolik, 1999). Proteins can also interact extensively once adsorbed at the interface, creating films with different viscoelastic properties (Martin, Grolle, Bos, Cohen Stuart, & van Vliet, 2002). Upon aging, the elasticity of the interface can increase with increasing interactions, for example through disulphide bridging among the adsorbed protein molecules (Dickinson & Matsumura, 1991). In general, the stability of an emulsion depends on the amount of emulsifier. At relatively low concentrations, emulsifier molecules tend to cover the interface only partly, often creating bridges between different droplets, whereas at moderate concentrations, complete interfacial coverage will take place. As the emulsifier concentration is further increased, multilayer adsorption may occur. Polymeric emulsifiers, such as proteins, diffuse slowly to the interface and rearrange extensively over time, whereas small molecule surfactants diffuse faster and lower the interfacial tension very rapidly, while showing little structural rearrangements. A hydrophilic emulsifier is dissolved in an aqueous phase, and is utilized in the stabilization of lipophilic dispersions. Conversely, lipophilic emulsifiers are used in the stabilization of aqueous droplets, generally dissolved in oil phases. For lipophilic emulsifiers, the frequency of the hydrophobic moieties is significantly higher compared to the hydrophilic emulsifiers. The well-known
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hydrophilicelipophilic balance (HLB), originally defined by Griffin (1949), reflects this structural difference, and it is often employed to predict the behavior of emulsifiers. Hydrophilic emulsifiers have high HLB values (8e18), whereas hydrophobic emulsifiers are represented by low HLB (4e6). Although the HLB value defines the frequency of hydrophilic or hydrophobic moieties and the medium of dispersion well, the size of the molecule, the temperature dependence and the physical and chemical behavior, including phase transitions and critical micellar concentration are all important parameters to better predict the emulsifier functionality (Bergenståhl, 2008). Although a great deal of research has been carried out on designing stable water-in-oil-in-water emulsions and studying the influence of protein incorporation in the inner or outer aqueous phases, the investigations on the interactions between lipophilic emulsifiers and proteins are relatively scarce. According to Garti, Aserin, and Cohen (1994), the lipophilic emulsifier Span 80 adsorbed at the oilewater interface together with BSA. The investigators suggested that BSA and Span 80 molecules formed a complex at the inner droplet interface which yielded a strong film that imparted elasticity and resistance to rupture of the inner droplets. The interfacial complex formation was found to be less pronounced at the outer droplet interface. Previously, interfacial complex film formation between BSA and Span 80 was observed by Omotosho, Law, Whateley, and Florence (1986). The investigators attributed this finding to hydrogen bonding and hydrophobic interactions between the protein and surfactant molecules. In another study, Span 80 was found to be less efficient than Tween 20 in displacing proteins (b-lg and b-casein) form the oilewater interface, possibly due to the small hydrophilic head group (Cornec et al., 1998). Based on all the studies mentioned here, a somewhat cooperative interaction can be expected between lipophilic surfactants and proteins at the oilewater interface which could possibly rely on hydrophobic interactions and hydrogen bonding. Polyglycerol polyricinoleate (PGPR) is a synthetic molecule widely used as an emulsifier in the stabilization of water-in-oil (w/ o) and water-in-oil-in-water (w/o/w) emulsions. Its production is based on the esterification reaction of polymerized glycerol with condensed castor oil fatty acids (Wilson, Van Schie, & Howes, 1998). Most commercial samples are far from being monodisperse, and include a variety of hydrogen, fatty acid and polyricinoleic acid esters of polyglycerol (Dedinaite & Campbell, 2000). It has been reported that when used at concentrations >2% (Su, Flanagan, Hemar, & Singh, 2006), w/o and w/o/w emulsions attain remarkable stability. PGPR is also quite commonly used in chocolate manufacture, due to its excellent water-binding properties which inhibit the thickening of chocolate in the presence of undesired inclusions of water (Wilson et al., 1998). According to FDA definition, PGPR is generally recognized as safe (FDA, 2006). As any other food additive, the use of PGPR has to be declared on product labels, and its maximum allowed dosage is controlled about 2.6 mg kg1 of body weight per day (Wilson et al., 1998). Therefore new approaches to reduce the usage of this lipophilic surfactant are highly sought after. It has been recently shown that the PGPR concentration necessary to stabilize w/o/w emulsions can be reduced in the presence of sodium caseinate (Su et al., 2006). It was suggested that sodium caseinate and PGPR may act synergistically to stabilize the w/o interface of the w/o/w emulsion, by forming an enhanced viscoelastic interfacial layer. In addition, a different study has demonstrated that the addition of xanthan or whey protein isolate enhances the stability of w/o emulsions stabilized by lecithin, whereas highly surface active proteins gelatine and sodium caseinate cause rapid destabilization (Knoth, Scherze, & Muschiolik, 2005). It has also been shown that, at low casein concentrations, binding of lecithin to the interfacial film
enhances the packing efficiency of casein in oil-in-water emulsions (Fang & Dalgleish, 1993). The objective of the present work was to study the possible interactions between PGPR and model dairy proteins (b-lg and sodium caseinate), by determining the difference in the interfacial tension and elasticity at a w/o interface, using drop shape tensiometry. A better understanding of the synergies occurring at the interface between the proteins and PGPR will result in a better optimization of the formulations containing this synthetic emulsifier in w/o emulsions. 2. Materials and methods 2.1. Materials Highly pure b-lg used in the experiments was purified from whey protein isolate (New Zealand Dairy Products, NZMP) using preparative ion chromatography on Q Sepharose (GE Healthcare) as previously reported (Andrew, Taylor, & Owen, 1985). Sodium caseinate was used as received from NZMP (Alanate 180, Lamoyne, PA, USA). PGPR was obtained from Palsgaard (PalsgaardÒ 4150, Juelsminde, Denmark). Soy oil (SigmaeAldrich, Catalog No: S7381, St. Louis, MO, USA) phase was purified using Florisil (SigmaeAldrich, Catalog No: 46385, St. Louis, MO, USA). Using a shaking plate, the oil was mixed with Florisil at a ratio of 10:1 (for 2 h at 60 rpm). Rapidly afterwards, Florisil was removed by filtering. Prior to all tensiometry measurements, this purification step was repeated three times. To ensure the high purity of aqueous phase, HPLC grade water (Fisher Scientific, Catalog No: W5-4, Fair Lawn, NJ, USA) was used in the experiments. Appropriate amounts of proteins were dissolved in HPLC grade water using a stirrer plate (2 h). Prior to use, all the protein solutions were kept refrigerated overnight in order to facilitate complete hydration. 2.2. Drop shape tensiometry The interfacial tension (g) and modulus of dilational elasticity (ε) at the soy oilewater interface were determined using drop shape tensiometry (25 C) (Tracker, IT Concept, Longessaigne, France). After the purification of the oil phase, an aqueous droplet (approximately 10 ml) was automatically formed at the tip of a sample syringe which was immersed in a glass cuvette containing the oil phase. The shape of the droplet was automatically analyzed to record the changes in the interfacial tension over time (sampling frequency ¼ 0.5 s1), as the cuvette and syringe assembly were monitored by a CCD (charge coupled device) camera and high quality image acquisition was utilized. Dynamic interfacial tension was calculated based on the YoungeLaplace equation. All the measurements were carried out in triplicate. For the interfacial elasticity measurements, equilibration duration of 4 h was used prior to analysis. When necessary, the adsorption process was allowed to take place continuously overnight. In most cases, however, the difference in measured elastic modulus from a 4 h or an overnight equilibration was negligible. Once the equilibrium was attained, dilational elasticity was determined at a strain amplitude (DA/A ¼ 0.1, A being the droplet surface area) and a sinusoidal oscillation frequency (x ¼ 100 mHz), unless otherwise stated. This extent of dilation lies within the linear viscoelastic range (Benjamins, Cagna, & Lucassen-Reynders, 1996). In the majority of studies in the literature, similar ranges are employed for the characterization of oilewater interfaces (for example, Lucassen-Reynders, Cagna, and Lucassen (2001) and Márquez, Medrano, Panizzolo, and Wagner (2010)), the value of which being typically 0.1. We have separately shown in this study
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that, it was possible to use a dilational amplitude range of 0e0.5 (at 0e200 mHz) for the interfaces mentioned here and remain in the linear range. The range for amplitude of dilation throughout the study was 0e0.2. The method is based on the automatically controlled, sinusoidal compressioneexpansion of the aqueous droplet at a defined oscillatory frequency and amplitude of dilation. The number of data points shown was reduced for clarity. The interfacial modulus of dilational elasticity was calculated from the change in interfacial tension (dg) relative to the change in droplet surface area (dA) (Lucassen-Reynders et al., 2001):
ε ¼
dg dlnA
(1)
3. Results and discussion 3.1. Interfacial tension The interfacial tension at the soy oilewater interface in the presence of varying PGPR concentrations (0e8% by wt) in the continuous oil phase was determined using drop shape tensiometry (Fig. 1). The equilibrium interfacial tension value at the oilewater interface (0% PGPR) was found to be 30.5 0.6 mN m1. This value was coherent with the earlier findings of Gaonkar (1989) (30.7 mN m1). As expected, equilibrium interfacial tension decreased with increasing concentrations of PGPR up to about 0.8%, beyond which a plateau region was observed. The equilibrium value in the plateau was approximately 1.8 mN m1. In the present work, the plateau was reached at much lower concentrations than those recently reported, where at 4% PGPR a value of 2.91 mN m1 was obtained (Márquez et al., 2010). The source of the discrepancy may be the Florisil step performed on the oil, as well as differences in the source of PGPR. To observe the effect of the presence of b-lg or sodium caseinate in the aqueous phase and to study the interactions occurring between the protein molecules and the lipophilic emulsifier, PGPR, experiments were carried out at a constant PGPR concentration of 0.008% (Fig. 2). This PGPR concentration (0.008%) was selected as it causes only a moderate reduction in the interfacial tension (a decrease of 7.69 mN m1 compared to no surfactant (Fig. 2, solid line). The addition of proteins in the aqueous phase caused a further decrease in the interfacial tension, even at the low concentrations. In both cases, the interfacial tension decreased with increasing
Fig. 1. Interfacial tension as a function of added PGPR to oil phase at the soy oilewater interface. Solid line indicates the o/w interfacial tension in the absence of PGPR. In all cases, standard deviation was lower than 5%.
Fig. 2. Changes in interfacial tension as a function of protein concentration as proteins are added to 0.008% PGPR in soy oil e water interface. Solid line indicates the o/w interfacial tension in the presence of 0.008% PGPR without protein in the aqueous phase. Bars indicate standard deviations for at least three independent experiments.
amounts of protein added, with sodium caseinate lowering the interfacial tension more efficiently than b-lg at similar concentrations based on wt%. Since dairy proteins alone decrease the interfacial tension (Martin et al., 2002), it is not possible to claim synergistic effects under the present conditions. However, it is worth to point out that very little concentrations of proteins already caused an extensive decrease in the value of the interfacial tension. The results shown in Fig. 2 clearly suggest that the presence of the proteins and PGPR did not inhibit the adsorption of the molecules at the w/o or o/w interface. This finding is supported by the previous studies which indicated, for example, an equilibrium interfacial tension of approximately 30 mN m1 for n-tetradecane/b-lactoglobulin (104% by wt) solution interface (Yamamoto & Araki, 1997) and approximately 19 mN m1 for n-heptane/b-casein interface (Beverung, Radke, & Blanch, 1999). Furthermore, using 0.5% sodium caseinate, Benjamins et al. (1996) showed that reduction interfacial tension (i.e., interfacial pressure) determined by drop shape tensiometry at the sunflower oilewater interface was about 19.8 mN m1 after 100 min. In addition, according to Pawlik, Cox, and Norton (2010), the interfacial tension at the sunflower oilewater interface was about 26 mN m1. A similar extent of reduction (i.e., 20 mN m1) would still yield an equilibrium interfacial tension of approximately 10.7 mN m1 in the present work. At the corresponding sodium caseinate concentration, with 0.008% PGPR, the interfacial tension measured was 3.1 mN m1. This value is inarguably below what has been reported and can be achieved by the presence of only sodium caseinate at the interface. Similarly, the interfacial tension at the sunflower oilewater interface in the presence of b-lactoglobulin has been reported to be approximately 15 mN m1 (as derived from the published plots) (Wüstneck et al., 1999). The interfacial tension value measured in this work (7.3 mN m1), in the presence of PGPR, was once again considerably lower. To better investigate the interactions between these molecules, studies on the interfacial elasticity of the mixed interfaces were also carried out (see following section). The results shown in Fig. 2 are well in line with literature reports. Knoth et al. (2005) showed that sodium caseinate is more effective in lowering interfacial tension of lecithin containing medium chain triglycerides interfaces compared to whey protein isolate. This is due to the higher flexibility of sodium caseinate at the interface, in other words, the ability of the casein molecules to efficiently spread at the oil/water interface (Dickinson, 1992, Chap. 2).
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3.2. Interfacial elasticity In addition to determining the decrease in interfacial tension of the mixed interfaces, the measurement of the viscoelastic properties of the interfaces may provide better information on the interactions between PGPR and the proteins. Interfacial dilational rheology is indeed affected by the structure of the molecules and their interactions. During the measurement, after a period of equilibration, the droplet was subjected to an automatically controlled sinusoidal oscillation, at a frequency of 100 mHz and a strain amplitude of 0.1. The viscoelastic properties of the soy oilewater interfaces were studied in the presence of 0.008% PGPR over a time scale of 5000 s and the results are summarized in Table 1. In all cases, dilational measurements were carried out after 4 h of equilibration. The addition of PGPR at 0.008% did not show any significant difference in the dilational modulus, when compared to the soy oilewater interface (data not shown). PGPR does not form an elastic network at the interface, and its presence at the interface does not improve dilational elasticity. On the other hand, in the absence of PGPR, the dilational modulus for b-lg added at a concentration of 0.1%, increased over time reaching a value of 84 mN m1 after 5000 s. A value of 38 mN m1 has been reported in the case of blg added at 0.01% (Martin et al., 2002). The addition of b-lg (0.001% and 0.1%) to the aqueous phase in the presence of 0.008% PGPR only slightly increased the dilational modulus at the interface, with values that decreased over time (Table 1). Since the addition of b-lg, even at low levels, decreased the interfacial tension (Fig. 2) in the presence of PGPR, it is possible to conclude that although both molecules were adsorbed at the interface, the presence of PGPR inhibited the formation of a b-lg film at the interface. In other words, PGPR dominated the viscoelastic properties at the interface, perhaps penetrating in the water phase and preventing a full coverage of b-lg. It is important to note that the dilational modulus decreased over time, suggesting further rearrangements with time. Using a low viscosity oil, Williams and Prins (1996) showed an interfacial dilational modulus of about 20 and 65 mN m1 at a b-lg concentrations of 0.001 and 0.1%, respectively. The high value of dilational modulus for b-lg is attributed to the presence of an interfacial network of protein molecules through covalent or noncovalent interactions (Dickinson & Matsumura, 1991; Williams & Prins, 1996). In a study at the airewater interface, Martin et al. (2002) indicated a modulus value of 63 mN m1 after 1.7 h. However, Lucassen-Reynders, Benjamins, and Fainerman (2010) and Wüstneck et al. (1999) measured lower dilational moduli for b-lg, whereas Graham and Phillips (1980) noted significantly higher moduli for other globular proteins (BSA and lysozyme). The differences may be partially attributed to the differences in the operation conditions, properties of the oil phase, duration of equilibration prior to the analysis and the purity of the proteins used in the investigations. Previous studies have indicated possible interactions of lecithin emulsifiers with protein films. For example, Fang and Dalgleish
Table 1 Dilational elastic modulus as a function of drop age and protein (b-lg, Na caseinate) and PGPR concentration at the soy oilewater interface. The strain amplitude was 0.1, and sinusoidal oscillation frequency was 100 mHz. Sample
ε (t ¼ 0)
0.1% b-lg 0.1% b-lg þ 0.008% PGPR 0.001% b-lg þ 0.008% PGPR 0.1% Na caseinate 0.1% Na caseinate þ 0.008% PGPR 0.001% Na caseinate þ 0.008% PGPR
54.3 24.1 11.5 9.8 13.6 14.3
4.8 4.4 0.7 0.4 0.4 3.2
ε (t ¼ 1000 s) ε (t ¼ 5000 s) 64.6 29.2 23.7 15.9 16.4 15.9
0.1 0.3 0.1 1.1 1.0 0.2
84.5 22.8 20.9 31.8 20.4 13.5
0.7 0.9 0.2 1.9 0.2 1.6
Table 2 Dilational elastic modulus as a function of drop age and extent of dilation (0.05e0.2) at the 0.008% PGPR in soy oil e 0.1% aqueous protein (b-lg, Na caseinate) solution interface. The sinusoidal oscillation frequency was 100 mHz. Sample
ε (t ¼ 0)
b-lg e DA/A ¼ 0.2 b-lg e DA/A ¼ 0.1 b-lg e DA/A ¼ 0.05 Na caseinate e DA/A ¼ 0.2 Na caseinate e DA/A ¼ 0.1 Na caseinate e DA/A ¼ 0.05
21 24.1 24.4 14.1 13.6 12
0.6 4.4 2.7 0.6 0.4 1.6
ε (t ¼ 1000 s) 16.5 29.2 19.6 13.2 10.8 14.5
0.3 0.3 1.0 0.8 0.2 2.2
ε (t ¼ 5000 s) 19.4 22.8 13.5 17.0 15.8 17.1
0.4 0.9 1.6 1.5 0.4 2.2
(1993) have demonstrated that at low protein concentrations, lecithin may adsorb to the hydrophobic holes of the interfacial film thereby increasing the thickness of the adsorbed layer. Zhang, An, Cui, and Li (2003) have shown that the interaction between b-lg and phospholipids were primarily of hydrophobic nature. In our case, it is possible that PGPR and b-lg interact favorably at the interface mostly through hydrophobic interactions, and PGPR plays an important role in imparting the elastic properties of the film is dominant. This finding may be consistent with the fact that coalescence stability of w/o/w emulsions is controlled by PGPR concentration, and not by that of sodium caseinate added to the aqueous phase (Su et al., 2006). In the case of sodium caseinate, the dilational elasticity, even in isolation, without PGPR added, showed low values after an oscillatory period of 1000 s (Table 1) and an increase in the elasticity with time. The presence of casein molecules in the aqueous phase did not enhance the dilational elasticity, and after 5000 s, addition of 0.1% sodium caseinate induced significant changes in the dilational modulus to 32 mN m1 (Table 1). These values are in agreement with those reported in the literature for airewater interfaces with dilational moduli of 11 and 20 mN m1 at the airewater interface in the presence of b-casein after 100 s and 1.7 h, respectively (Martin et al., 2002). At a low viscosity oilewater interface, Williams and Prins (1996) measured the similar moduli as 25 and 15 mN m1 for 0.001% and 0.1% b-casein, respectively. As already shown for b-lg, also in the case of sodium caseinate, lower values of dilational modulus (especially after 5000 s) were measured for sodium caseinate in the presence of 0.008% PGPR, once again confirming the domination of the PGPR in imparting lower elasticity to the interface (Table 1). In all cases, the dilational moduli generated in the presence of blg were higher compared to that of sodium caseinate. Globular proteins such as b-lg are known to form a strong cohesive network when adsorbed at the interface, which limits the amount of both diffusional and conformational relaxation, whereas even at elevated concentration levels, the network structure formed by bcasein was suggested to be weaker with a higher frequency of molecular relaxations (Graham & Phillips, 1980; Lucassen-Reynders et al., 2010; Williams & Prins, 1996). The extent (Table 2) and frequency (Table 3) of dilation did not affect the dilational modulus. These findings suggest that the interactions between the two proteins and PGPR are not altered
Table 3 Dilational elastic modulus as a function of drop age and frequency of dilation at the 0.008% PGPR in soy oil e 0.1% aqueous protein (b-lg, Na caseinate) solution interface. The strain amplitude was 0.1. Sample
ε (t ¼ 0)
b-lg e 100 mHz b-lg e 50 mHz
24.1 21.6 13.6 14
Na caseinate 100 mHz Na caseinate 50 mHz
4.4 1.2 0.4 1.6
ε (t ¼ 1000 s) 29.2 40.9 16.4 20.5
0.3 1.8 1.0 0.8
ε (t ¼ 5000 s) 22.8 46.9 20.4 15.8
0.9 1.1 0.2 2.8
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the presence of proteins limits the ability of PGPR to pack efficiently at the interface. Therefore, the formulators of w/o and w/o/w emulsions should exercise extreme care in the partial replacement of this strongly lipophilic emulsifier. References
Fig. 3. Dilational elastic modulus as a function of drop age at soy oil e 0.1% b-lg aqueous solution interface before and after addition of PGPR to the oil phase. The strain amplitude was 0.1, and sinusoidal oscillation frequency was 100 mHz. Results were obtained after equilibration.
during dilation. In all cases, b-lg containing systems had a higher elastic modulus than sodium caseinate containing systems. The influence of PGPR addition to oil phase was also studied on a b-lg dominant interface as shown in Fig. 3. The addition of PGPR clearly affected the viscoelastic properties of the interface. After 5000 s of adsorption, PGPR (0.008%) was added to the oil phase which was characterized by a rapid reduction of the dilational modulus, after a brief period of equilibration. As the concentrated PGPR solution was diluted into the continuous oil phase, due to the relatively high viscosity, the mixing took place slowly. Possibly the turbulence in the medium could have caused the increase in the elastic modulus. The equilibrium value of the modulus (Fig. 3) was only slightly higher than that of the oilewater interface which contained 0.008% PGPR in the oil phase and 0.1% b-lg in the aqueous phase (Table 1). Therefore, the process was at least partially reversible which suggested that the sequence of addition was not influential of the final viscoelastic properties of the interface. The affinity of proteins to soy oilewater interface was shown to be lower than that to less polar oil interfaces, therefore a lesser concentration of surfactants is needed for the displacement of proteins from soy oil interfaces (Courthadon & Dickinson, 1991; Dickinson & Tanai, 1992). It has been shown that as whey protein isolate is partially displaced from the oilewater interface by monoolein or monopalmitin, dilational modulus either drastically decreases or approaches that of the lipid film (Rodríguez Patino, Navarro Garcia, Rodríguez Niño, 2001). Fig. 3 clearly shows that, even at low concentrations, PGPR dominated the oilewater interface and the value of dilational modulus. In this case also, proteins interacted with PGPR possibly through hydrophobic interactions which lowered the equilibrium interfacial tension.
4. Conclusions PGPR is a lipophilic emulsifier that is commonly used in the stabilization of water-in-oil and water-in-oil-in-water emulsions. In order to decrease the usage of PGPR, addition of proteins in the aqueous phase has been previously recommended. The present work clearly demonstrate that dairy proteins further decrease the interfacial tension in the presence of PGPR, without displacement of PGPR by protein molecules. It was concluded that the PGPR interacts with the hydrophobic moieties of the proteins, causing changes in the viscoelastic properties of the interface. Furthermore,
Andrew, A. T., Taylor, M. D., & Owen, A. J. (1985). Rapid analysis of bovine milk proteins by fast protein liquid chromatography. Journal of Chromatography A, 348, 177e185. Benjamins, J., Cagna, A., & Lucassen-Reynders, E. H. (1996). Viscoelastic properties of triacylglycerol/water interfaces covered by proteins. Colloids and Surfaces A, 114, 245e254. Bergenståhl, B. (2008). Physicochemical aspects of an emulsifier functionality. In G. L. Hasenhuettl, & R. W. Hartel (Eds.), Food emulsifiers and their applications (2nd ed.). (pp. 173e194) New York: Springer. Beverung, C. J., Radke, C. J., & Blanch, H. W. (1999). Protein adsorption at the oil/ water interface: characterization of adsorption kinetics by dynamic interfacial tension measurements. Biophysical Chemistry, 81, 59e80. Cornec, M., Wilde, P. J., Gunning, P. A., Mackie, A. R., Husband, F. A., Parker, M. L., et al. (1998). Emulsion stability as affected by competitive adsorption between an oil-soluble emulsifier and milk proteins at the interface. Journal of Food Science, 63, 39e43. Courthadon, J. L., & Dickinson, E. (1991). Competitive adsorption of lecithin and bcasein in oil in water emulsions. Journal of Food and Agricultural Chemistry, 39, 1365e1368. Dedinaite, A., & Campbell, B. (2000). Interactions between mica surfaces across triglyceride solution containing phospholipids and polyglycerol polyricinolate. Langmuir, 16, 2248e2253. Dickinson, E. (1992). An introduction to food colloids. New York: OxfordUniversity Press. Dickinson, E., & Matsumura, Y. (1991). Time-dependent polymerization of b-lactoglobulin through disulphide bonds at the oil-water interface in emulsions. International Journal of Biological Macromolecules, 13, 26e30. Dickinson, E., & Tanai, S. (1992). Protein displacement from the emulsion droplet surface by oil-soluble and water-soluble surfactants. Journal of Food and Agricultural Chemistry, 40, 179e183. Fang, Y., & Dalgleish, D. G. (1993). Casein adsorption on the surfaces of oil-in-water emulsions modified by lecithin. Colloids and Surfaces B, 1, 357e364. Food and Drug Administration (FDA). (2006). GRAS Notice No. GRN 000179. CFSAN/ Office of Food Additive Safety. Gaonkar, A. G. (1989). Interfacial tensions of vegetable oil/water systems: effect of oil purification. Journal of American Oil Chemists’ Society, 66, 1090e1092. Garti, N., Aserin, A., & Cohen, Y. (1994). Mechanistic consideration on the release of electrolytes from multiple emulsions stabilized by BSA and nonionic surfactants. Journal of Controlled Release, 29, 41e51. Graham, D. E., & Phillips, M. C. (1980). Proteins at liquid interfaces. IV. Dilational properties. Journal of Colloid and Interface Science, 76, 227e239. Griffin, W. C. (1949). Classification of surface-active agents by HLB. Journal of the Society of Cosmetic Chemists, 1, 311e326. Hasenhuettl, G. L. (2008). Overview of food emulsifiers. In G. L. Hasenhuettl, & R. W. Hartel (Eds.), Food emulsifiers and their applications (2nd ed). (pp. 1e9). New York: Springer. Haynes, C. A., & Norde, W. (1995). Structures and stabilities of adsorbed proteins. Journal of Colloid and Interface Science, 169, 313e328. Knoth, A., Scherze, I., & Muschiolik, G. (2005). Stability of water-in-oil emulsions containing phosphotidylcholine-depleted lecithin. Food Hydrocolloids, 19, 635e640. Lucassen-Reynders, E. H., Benjamins, J., & Fainerman, V. B. (2010). Dilational rheology of protein films adsorbed at fluid interfaces. Current Opinion in Colloid & Interface Science, 15, 264e270. Lucassen-Reynders, E. H., Cagna, A., & Lucassen, J. (2001). Gibbs elasticity, surface dilational modulus and diffusional relaxation in nonionic surfactant monolayers. Colloids and Surfaces A, 186, 63e72. Márquez, A. L., Medrano, A., Panizzolo, L. A., & Wagner, J. R. (2010). Effect of calcium salts and surfactant concentration on the stability of water-in-oil (w/o) emulsions prepared with polyglycerol polyricinoleate. Journal of Colloid and Interface Science, 341, 101e108. Martin, A. H., Grolle, K., Bos, M. A., Cohen Stuart, M. A., & van Vliet, T. (2002). Network forming properties of various proteins adsorbed at the air/water interface in relation to foam stability. Journal of Colloid and Interface Science, 254, 175e183. Omotosho, J. A., Law, T. K., Whateley, T. L., & Florence, A. T. (1986). The stabilization of w/o/w emulsions by interfacial interaction between albumin and nonionic surfactants. Colloids and Surfaces, 20, 133e144. Pawlik, A., Cox, P. W., & Norton, I. T. (2010). Food grade duplex emulsions designed and stabilized with different osmotic pressures. Journal of Colloid and Interface Science, 352, 59e67. Rodríguez Patino, J. M., Navarro García, J. M., & Rodríguez Niño, M. R. (2001). Protein-lipid interactions at the oil-water interface. Colloids and Surfaces B, 21, 207e216. Serrien, G., Geeraerts, G., Ghosh, L., & Joos, P. (1992). Dynamic surface properties of adsorbed protein solutions: BSA, casein and buttermilk. Colloids and Surfaces, 68, 219e233.
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Su, J., Flanagan, J., Hemar, Y., & Singh, H. (2006). Synergistic effects of polyglycerol ester of polyricinoleic acid and sodium caseinate on the stabilisation of watereoilewater emulsions. Food Hydrocolloids, 20, 261e268. Williams, A., & Prins, A. (1996). Comparison of the dilational behaviour of adsorbed milk proteins at the air-water and oil-water interfaces. Colloids and Surfaces A, 114, 267e275. Wilson, R., Van Schie, B. J., & Howes, D. (1998). Overview of the preparation, use and biological studies on polyglycerol polyricinoleate (PGPR). Food and Chemical Toxicology, 36, 711e718.
Wüstneck, R., Moser, B., & Muschiolik, G. (1999). Interfacial dilational behaviour of adsorbed b-lactoglobulin layers at the different fluid interfaces. Colloids and Surfaces B, 15, 263e273. Yamamoto, Y., & Araki, M. (1997). Effects of lecithin addition in oil or water phase on the stability of emulsions made with whey proteins. Bioscience, Biotechnology and Biochemistry, 61, 1791e1795. Zhang, Y., An, Z., Cui, G., & Li, J. (2003). Stabilized complex film formed by coadsorption of b-lactoglobulin and phospholipids at liquid/liquid interface. Colloids and Surfaces A, 223, 11e16.
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Interactions at the interface between hydrophobic and hydrophilic emulsifiers: Polyglycerol polyricinoleate (PGPR) and milk proteins, studied by drop shape tensiometry _ Ibrahim Gülseren*, Milena Corredig Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 August 2011 Accepted 13 March 2012
The equilibrium interfacial tension and dilational elasticity at the soy oilewater interface were studied in the presence of a lipophilic emulsifier, polyglycerol polyricinoleate (PGPR), in the continuous oil phase, and dairy proteins, b-lactoglobulin (b-lg) or sodium caseinate, in the aqueous phase using drop shape tensiometry. The interfacial tension decreased with increasing PGPR concentration, and was <2 mN m1 at PGPR concentrations beyond 1%. The presence of proteins in the water phase, b-lg and sodium caseinate, further reduced the interfacial tension. Even at the low concentrations (0.008%) tested, PGPR dominated the interfacial elasticity, which was only slightly affected by the addition of elevated levels of protein. While in the presence of b-lg (0.1%) in isolation, the system showed a high interfacial elasticity, the addition of PGPR lowered the elasticity, suggesting that PGPR interfered with proteineprotein interactions at the interface, or caused displacement of b-lg. Interfacial elasticity at the oilewater interface showed little dependence on dilation or frequency of the sinusoidal oscillation, when the interface was dominated by PGPR. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Polyglycerol polyricinolate (PGPR) Drop shape tensiometry Dilational elasticity Sodium caseinate b-Lactoglobulin
1. Introduction Emulsifiers are compounds that lower surface tension by adsorbing onto interfaces and orienting their structure to minimize the Gibbs free energy. In other words, emulsifier molecules diffuse from the phase that they are solubilized in to the interface, overcome the adsorption barrier, adsorb at the interface and attain a thermodynamically favorable arrangement after adsorption (Serrien, Geeraerts, Ghosh, & Joos, 1992). Their ability to stabilize interfaces is due to the amphiphilic nature of their structures; the hydrophilic head group of surfactants is attracted to the aqueous phase, whereas the lipophilic tail preferentially interacts with the oil phase. In the case of proteins, the molecules rearrange on the surface of air or oil interfaces, with their hydrophobic moieties (with amino acids such as phenylalanine, leucine, and isoleucine) adsorbing at the surface and the hydrophilic portions protruding into solution (Hasenhuettl, 2008). Depending on the ability of the structures to rearrange, proteins may show different levels of unfolding at the interface (Haynes & Norde, 1995). At low protein concentrations, protein molecules have a higher probability of unfolding and rearranging extensively at the interface, reaching * Corresponding author. Tel.: þ1 (519) 824 4120x58132; fax: þ1 (519) 824 6631. _ Gülseren). E-mail address: [email protected] (I. 0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2012.03.010
equilibrium later than at high protein concentrations, where the extent of rearrangements is low (Wüstneck, Moser, & Muschiolik, 1999). Proteins can also interact extensively once adsorbed at the interface, creating films with different viscoelastic properties (Martin, Grolle, Bos, Cohen Stuart, & van Vliet, 2002). Upon aging, the elasticity of the interface can increase with increasing interactions, for example through disulphide bridging among the adsorbed protein molecules (Dickinson & Matsumura, 1991). In general, the stability of an emulsion depends on the amount of emulsifier. At relatively low concentrations, emulsifier molecules tend to cover the interface only partly, often creating bridges between different droplets, whereas at moderate concentrations, complete interfacial coverage will take place. As the emulsifier concentration is further increased, multilayer adsorption may occur. Polymeric emulsifiers, such as proteins, diffuse slowly to the interface and rearrange extensively over time, whereas small molecule surfactants diffuse faster and lower the interfacial tension very rapidly, while showing little structural rearrangements. A hydrophilic emulsifier is dissolved in an aqueous phase, and is utilized in the stabilization of lipophilic dispersions. Conversely, lipophilic emulsifiers are used in the stabilization of aqueous droplets, generally dissolved in oil phases. For lipophilic emulsifiers, the frequency of the hydrophobic moieties is significantly higher compared to the hydrophilic emulsifiers. The well-known
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hydrophilicelipophilic balance (HLB), originally defined by Griffin (1949), reflects this structural difference, and it is often employed to predict the behavior of emulsifiers. Hydrophilic emulsifiers have high HLB values (8e18), whereas hydrophobic emulsifiers are represented by low HLB (4e6). Although the HLB value defines the frequency of hydrophilic or hydrophobic moieties and the medium of dispersion well, the size of the molecule, the temperature dependence and the physical and chemical behavior, including phase transitions and critical micellar concentration are all important parameters to better predict the emulsifier functionality (Bergenståhl, 2008). Although a great deal of research has been carried out on designing stable water-in-oil-in-water emulsions and studying the influence of protein incorporation in the inner or outer aqueous phases, the investigations on the interactions between lipophilic emulsifiers and proteins are relatively scarce. According to Garti, Aserin, and Cohen (1994), the lipophilic emulsifier Span 80 adsorbed at the oilewater interface together with BSA. The investigators suggested that BSA and Span 80 molecules formed a complex at the inner droplet interface which yielded a strong film that imparted elasticity and resistance to rupture of the inner droplets. The interfacial complex formation was found to be less pronounced at the outer droplet interface. Previously, interfacial complex film formation between BSA and Span 80 was observed by Omotosho, Law, Whateley, and Florence (1986). The investigators attributed this finding to hydrogen bonding and hydrophobic interactions between the protein and surfactant molecules. In another study, Span 80 was found to be less efficient than Tween 20 in displacing proteins (b-lg and b-casein) form the oilewater interface, possibly due to the small hydrophilic head group (Cornec et al., 1998). Based on all the studies mentioned here, a somewhat cooperative interaction can be expected between lipophilic surfactants and proteins at the oilewater interface which could possibly rely on hydrophobic interactions and hydrogen bonding. Polyglycerol polyricinoleate (PGPR) is a synthetic molecule widely used as an emulsifier in the stabilization of water-in-oil (w/ o) and water-in-oil-in-water (w/o/w) emulsions. Its production is based on the esterification reaction of polymerized glycerol with condensed castor oil fatty acids (Wilson, Van Schie, & Howes, 1998). Most commercial samples are far from being monodisperse, and include a variety of hydrogen, fatty acid and polyricinoleic acid esters of polyglycerol (Dedinaite & Campbell, 2000). It has been reported that when used at concentrations >2% (Su, Flanagan, Hemar, & Singh, 2006), w/o and w/o/w emulsions attain remarkable stability. PGPR is also quite commonly used in chocolate manufacture, due to its excellent water-binding properties which inhibit the thickening of chocolate in the presence of undesired inclusions of water (Wilson et al., 1998). According to FDA definition, PGPR is generally recognized as safe (FDA, 2006). As any other food additive, the use of PGPR has to be declared on product labels, and its maximum allowed dosage is controlled about 2.6 mg kg1 of body weight per day (Wilson et al., 1998). Therefore new approaches to reduce the usage of this lipophilic surfactant are highly sought after. It has been recently shown that the PGPR concentration necessary to stabilize w/o/w emulsions can be reduced in the presence of sodium caseinate (Su et al., 2006). It was suggested that sodium caseinate and PGPR may act synergistically to stabilize the w/o interface of the w/o/w emulsion, by forming an enhanced viscoelastic interfacial layer. In addition, a different study has demonstrated that the addition of xanthan or whey protein isolate enhances the stability of w/o emulsions stabilized by lecithin, whereas highly surface active proteins gelatine and sodium caseinate cause rapid destabilization (Knoth, Scherze, & Muschiolik, 2005). It has also been shown that, at low casein concentrations, binding of lecithin to the interfacial film
enhances the packing efficiency of casein in oil-in-water emulsions (Fang & Dalgleish, 1993). The objective of the present work was to study the possible interactions between PGPR and model dairy proteins (b-lg and sodium caseinate), by determining the difference in the interfacial tension and elasticity at a w/o interface, using drop shape tensiometry. A better understanding of the synergies occurring at the interface between the proteins and PGPR will result in a better optimization of the formulations containing this synthetic emulsifier in w/o emulsions. 2. Materials and methods 2.1. Materials Highly pure b-lg used in the experiments was purified from whey protein isolate (New Zealand Dairy Products, NZMP) using preparative ion chromatography on Q Sepharose (GE Healthcare) as previously reported (Andrew, Taylor, & Owen, 1985). Sodium caseinate was used as received from NZMP (Alanate 180, Lamoyne, PA, USA). PGPR was obtained from Palsgaard (PalsgaardÒ 4150, Juelsminde, Denmark). Soy oil (SigmaeAldrich, Catalog No: S7381, St. Louis, MO, USA) phase was purified using Florisil (SigmaeAldrich, Catalog No: 46385, St. Louis, MO, USA). Using a shaking plate, the oil was mixed with Florisil at a ratio of 10:1 (for 2 h at 60 rpm). Rapidly afterwards, Florisil was removed by filtering. Prior to all tensiometry measurements, this purification step was repeated three times. To ensure the high purity of aqueous phase, HPLC grade water (Fisher Scientific, Catalog No: W5-4, Fair Lawn, NJ, USA) was used in the experiments. Appropriate amounts of proteins were dissolved in HPLC grade water using a stirrer plate (2 h). Prior to use, all the protein solutions were kept refrigerated overnight in order to facilitate complete hydration. 2.2. Drop shape tensiometry The interfacial tension (g) and modulus of dilational elasticity (ε) at the soy oilewater interface were determined using drop shape tensiometry (25 C) (Tracker, IT Concept, Longessaigne, France). After the purification of the oil phase, an aqueous droplet (approximately 10 ml) was automatically formed at the tip of a sample syringe which was immersed in a glass cuvette containing the oil phase. The shape of the droplet was automatically analyzed to record the changes in the interfacial tension over time (sampling frequency ¼ 0.5 s1), as the cuvette and syringe assembly were monitored by a CCD (charge coupled device) camera and high quality image acquisition was utilized. Dynamic interfacial tension was calculated based on the YoungeLaplace equation. All the measurements were carried out in triplicate. For the interfacial elasticity measurements, equilibration duration of 4 h was used prior to analysis. When necessary, the adsorption process was allowed to take place continuously overnight. In most cases, however, the difference in measured elastic modulus from a 4 h or an overnight equilibration was negligible. Once the equilibrium was attained, dilational elasticity was determined at a strain amplitude (DA/A ¼ 0.1, A being the droplet surface area) and a sinusoidal oscillation frequency (x ¼ 100 mHz), unless otherwise stated. This extent of dilation lies within the linear viscoelastic range (Benjamins, Cagna, & Lucassen-Reynders, 1996). In the majority of studies in the literature, similar ranges are employed for the characterization of oilewater interfaces (for example, Lucassen-Reynders, Cagna, and Lucassen (2001) and Márquez, Medrano, Panizzolo, and Wagner (2010)), the value of which being typically 0.1. We have separately shown in this study
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that, it was possible to use a dilational amplitude range of 0e0.5 (at 0e200 mHz) for the interfaces mentioned here and remain in the linear range. The range for amplitude of dilation throughout the study was 0e0.2. The method is based on the automatically controlled, sinusoidal compressioneexpansion of the aqueous droplet at a defined oscillatory frequency and amplitude of dilation. The number of data points shown was reduced for clarity. The interfacial modulus of dilational elasticity was calculated from the change in interfacial tension (dg) relative to the change in droplet surface area (dA) (Lucassen-Reynders et al., 2001):
ε ¼
dg dlnA
(1)
3. Results and discussion 3.1. Interfacial tension The interfacial tension at the soy oilewater interface in the presence of varying PGPR concentrations (0e8% by wt) in the continuous oil phase was determined using drop shape tensiometry (Fig. 1). The equilibrium interfacial tension value at the oilewater interface (0% PGPR) was found to be 30.5 0.6 mN m1. This value was coherent with the earlier findings of Gaonkar (1989) (30.7 mN m1). As expected, equilibrium interfacial tension decreased with increasing concentrations of PGPR up to about 0.8%, beyond which a plateau region was observed. The equilibrium value in the plateau was approximately 1.8 mN m1. In the present work, the plateau was reached at much lower concentrations than those recently reported, where at 4% PGPR a value of 2.91 mN m1 was obtained (Márquez et al., 2010). The source of the discrepancy may be the Florisil step performed on the oil, as well as differences in the source of PGPR. To observe the effect of the presence of b-lg or sodium caseinate in the aqueous phase and to study the interactions occurring between the protein molecules and the lipophilic emulsifier, PGPR, experiments were carried out at a constant PGPR concentration of 0.008% (Fig. 2). This PGPR concentration (0.008%) was selected as it causes only a moderate reduction in the interfacial tension (a decrease of 7.69 mN m1 compared to no surfactant (Fig. 2, solid line). The addition of proteins in the aqueous phase caused a further decrease in the interfacial tension, even at the low concentrations. In both cases, the interfacial tension decreased with increasing
Fig. 1. Interfacial tension as a function of added PGPR to oil phase at the soy oilewater interface. Solid line indicates the o/w interfacial tension in the absence of PGPR. In all cases, standard deviation was lower than 5%.
Fig. 2. Changes in interfacial tension as a function of protein concentration as proteins are added to 0.008% PGPR in soy oil e water interface. Solid line indicates the o/w interfacial tension in the presence of 0.008% PGPR without protein in the aqueous phase. Bars indicate standard deviations for at least three independent experiments.
amounts of protein added, with sodium caseinate lowering the interfacial tension more efficiently than b-lg at similar concentrations based on wt%. Since dairy proteins alone decrease the interfacial tension (Martin et al., 2002), it is not possible to claim synergistic effects under the present conditions. However, it is worth to point out that very little concentrations of proteins already caused an extensive decrease in the value of the interfacial tension. The results shown in Fig. 2 clearly suggest that the presence of the proteins and PGPR did not inhibit the adsorption of the molecules at the w/o or o/w interface. This finding is supported by the previous studies which indicated, for example, an equilibrium interfacial tension of approximately 30 mN m1 for n-tetradecane/b-lactoglobulin (104% by wt) solution interface (Yamamoto & Araki, 1997) and approximately 19 mN m1 for n-heptane/b-casein interface (Beverung, Radke, & Blanch, 1999). Furthermore, using 0.5% sodium caseinate, Benjamins et al. (1996) showed that reduction interfacial tension (i.e., interfacial pressure) determined by drop shape tensiometry at the sunflower oilewater interface was about 19.8 mN m1 after 100 min. In addition, according to Pawlik, Cox, and Norton (2010), the interfacial tension at the sunflower oilewater interface was about 26 mN m1. A similar extent of reduction (i.e., 20 mN m1) would still yield an equilibrium interfacial tension of approximately 10.7 mN m1 in the present work. At the corresponding sodium caseinate concentration, with 0.008% PGPR, the interfacial tension measured was 3.1 mN m1. This value is inarguably below what has been reported and can be achieved by the presence of only sodium caseinate at the interface. Similarly, the interfacial tension at the sunflower oilewater interface in the presence of b-lactoglobulin has been reported to be approximately 15 mN m1 (as derived from the published plots) (Wüstneck et al., 1999). The interfacial tension value measured in this work (7.3 mN m1), in the presence of PGPR, was once again considerably lower. To better investigate the interactions between these molecules, studies on the interfacial elasticity of the mixed interfaces were also carried out (see following section). The results shown in Fig. 2 are well in line with literature reports. Knoth et al. (2005) showed that sodium caseinate is more effective in lowering interfacial tension of lecithin containing medium chain triglycerides interfaces compared to whey protein isolate. This is due to the higher flexibility of sodium caseinate at the interface, in other words, the ability of the casein molecules to efficiently spread at the oil/water interface (Dickinson, 1992, Chap. 2).
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3.2. Interfacial elasticity In addition to determining the decrease in interfacial tension of the mixed interfaces, the measurement of the viscoelastic properties of the interfaces may provide better information on the interactions between PGPR and the proteins. Interfacial dilational rheology is indeed affected by the structure of the molecules and their interactions. During the measurement, after a period of equilibration, the droplet was subjected to an automatically controlled sinusoidal oscillation, at a frequency of 100 mHz and a strain amplitude of 0.1. The viscoelastic properties of the soy oilewater interfaces were studied in the presence of 0.008% PGPR over a time scale of 5000 s and the results are summarized in Table 1. In all cases, dilational measurements were carried out after 4 h of equilibration. The addition of PGPR at 0.008% did not show any significant difference in the dilational modulus, when compared to the soy oilewater interface (data not shown). PGPR does not form an elastic network at the interface, and its presence at the interface does not improve dilational elasticity. On the other hand, in the absence of PGPR, the dilational modulus for b-lg added at a concentration of 0.1%, increased over time reaching a value of 84 mN m1 after 5000 s. A value of 38 mN m1 has been reported in the case of blg added at 0.01% (Martin et al., 2002). The addition of b-lg (0.001% and 0.1%) to the aqueous phase in the presence of 0.008% PGPR only slightly increased the dilational modulus at the interface, with values that decreased over time (Table 1). Since the addition of b-lg, even at low levels, decreased the interfacial tension (Fig. 2) in the presence of PGPR, it is possible to conclude that although both molecules were adsorbed at the interface, the presence of PGPR inhibited the formation of a b-lg film at the interface. In other words, PGPR dominated the viscoelastic properties at the interface, perhaps penetrating in the water phase and preventing a full coverage of b-lg. It is important to note that the dilational modulus decreased over time, suggesting further rearrangements with time. Using a low viscosity oil, Williams and Prins (1996) showed an interfacial dilational modulus of about 20 and 65 mN m1 at a b-lg concentrations of 0.001 and 0.1%, respectively. The high value of dilational modulus for b-lg is attributed to the presence of an interfacial network of protein molecules through covalent or noncovalent interactions (Dickinson & Matsumura, 1991; Williams & Prins, 1996). In a study at the airewater interface, Martin et al. (2002) indicated a modulus value of 63 mN m1 after 1.7 h. However, Lucassen-Reynders, Benjamins, and Fainerman (2010) and Wüstneck et al. (1999) measured lower dilational moduli for b-lg, whereas Graham and Phillips (1980) noted significantly higher moduli for other globular proteins (BSA and lysozyme). The differences may be partially attributed to the differences in the operation conditions, properties of the oil phase, duration of equilibration prior to the analysis and the purity of the proteins used in the investigations. Previous studies have indicated possible interactions of lecithin emulsifiers with protein films. For example, Fang and Dalgleish
Table 1 Dilational elastic modulus as a function of drop age and protein (b-lg, Na caseinate) and PGPR concentration at the soy oilewater interface. The strain amplitude was 0.1, and sinusoidal oscillation frequency was 100 mHz. Sample
ε (t ¼ 0)
0.1% b-lg 0.1% b-lg þ 0.008% PGPR 0.001% b-lg þ 0.008% PGPR 0.1% Na caseinate 0.1% Na caseinate þ 0.008% PGPR 0.001% Na caseinate þ 0.008% PGPR
54.3 24.1 11.5 9.8 13.6 14.3
4.8 4.4 0.7 0.4 0.4 3.2
ε (t ¼ 1000 s) ε (t ¼ 5000 s) 64.6 29.2 23.7 15.9 16.4 15.9
0.1 0.3 0.1 1.1 1.0 0.2
84.5 22.8 20.9 31.8 20.4 13.5
0.7 0.9 0.2 1.9 0.2 1.6
Table 2 Dilational elastic modulus as a function of drop age and extent of dilation (0.05e0.2) at the 0.008% PGPR in soy oil e 0.1% aqueous protein (b-lg, Na caseinate) solution interface. The sinusoidal oscillation frequency was 100 mHz. Sample
ε (t ¼ 0)
b-lg e DA/A ¼ 0.2 b-lg e DA/A ¼ 0.1 b-lg e DA/A ¼ 0.05 Na caseinate e DA/A ¼ 0.2 Na caseinate e DA/A ¼ 0.1 Na caseinate e DA/A ¼ 0.05
21 24.1 24.4 14.1 13.6 12
0.6 4.4 2.7 0.6 0.4 1.6
ε (t ¼ 1000 s) 16.5 29.2 19.6 13.2 10.8 14.5
0.3 0.3 1.0 0.8 0.2 2.2
ε (t ¼ 5000 s) 19.4 22.8 13.5 17.0 15.8 17.1
0.4 0.9 1.6 1.5 0.4 2.2
(1993) have demonstrated that at low protein concentrations, lecithin may adsorb to the hydrophobic holes of the interfacial film thereby increasing the thickness of the adsorbed layer. Zhang, An, Cui, and Li (2003) have shown that the interaction between b-lg and phospholipids were primarily of hydrophobic nature. In our case, it is possible that PGPR and b-lg interact favorably at the interface mostly through hydrophobic interactions, and PGPR plays an important role in imparting the elastic properties of the film is dominant. This finding may be consistent with the fact that coalescence stability of w/o/w emulsions is controlled by PGPR concentration, and not by that of sodium caseinate added to the aqueous phase (Su et al., 2006). In the case of sodium caseinate, the dilational elasticity, even in isolation, without PGPR added, showed low values after an oscillatory period of 1000 s (Table 1) and an increase in the elasticity with time. The presence of casein molecules in the aqueous phase did not enhance the dilational elasticity, and after 5000 s, addition of 0.1% sodium caseinate induced significant changes in the dilational modulus to 32 mN m1 (Table 1). These values are in agreement with those reported in the literature for airewater interfaces with dilational moduli of 11 and 20 mN m1 at the airewater interface in the presence of b-casein after 100 s and 1.7 h, respectively (Martin et al., 2002). At a low viscosity oilewater interface, Williams and Prins (1996) measured the similar moduli as 25 and 15 mN m1 for 0.001% and 0.1% b-casein, respectively. As already shown for b-lg, also in the case of sodium caseinate, lower values of dilational modulus (especially after 5000 s) were measured for sodium caseinate in the presence of 0.008% PGPR, once again confirming the domination of the PGPR in imparting lower elasticity to the interface (Table 1). In all cases, the dilational moduli generated in the presence of blg were higher compared to that of sodium caseinate. Globular proteins such as b-lg are known to form a strong cohesive network when adsorbed at the interface, which limits the amount of both diffusional and conformational relaxation, whereas even at elevated concentration levels, the network structure formed by bcasein was suggested to be weaker with a higher frequency of molecular relaxations (Graham & Phillips, 1980; Lucassen-Reynders et al., 2010; Williams & Prins, 1996). The extent (Table 2) and frequency (Table 3) of dilation did not affect the dilational modulus. These findings suggest that the interactions between the two proteins and PGPR are not altered
Table 3 Dilational elastic modulus as a function of drop age and frequency of dilation at the 0.008% PGPR in soy oil e 0.1% aqueous protein (b-lg, Na caseinate) solution interface. The strain amplitude was 0.1. Sample
ε (t ¼ 0)
b-lg e 100 mHz b-lg e 50 mHz
24.1 21.6 13.6 14
Na caseinate 100 mHz Na caseinate 50 mHz
4.4 1.2 0.4 1.6
ε (t ¼ 1000 s) 29.2 40.9 16.4 20.5
0.3 1.8 1.0 0.8
ε (t ¼ 5000 s) 22.8 46.9 20.4 15.8
0.9 1.1 0.2 2.8
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the presence of proteins limits the ability of PGPR to pack efficiently at the interface. Therefore, the formulators of w/o and w/o/w emulsions should exercise extreme care in the partial replacement of this strongly lipophilic emulsifier. References
Fig. 3. Dilational elastic modulus as a function of drop age at soy oil e 0.1% b-lg aqueous solution interface before and after addition of PGPR to the oil phase. The strain amplitude was 0.1, and sinusoidal oscillation frequency was 100 mHz. Results were obtained after equilibration.
during dilation. In all cases, b-lg containing systems had a higher elastic modulus than sodium caseinate containing systems. The influence of PGPR addition to oil phase was also studied on a b-lg dominant interface as shown in Fig. 3. The addition of PGPR clearly affected the viscoelastic properties of the interface. After 5000 s of adsorption, PGPR (0.008%) was added to the oil phase which was characterized by a rapid reduction of the dilational modulus, after a brief period of equilibration. As the concentrated PGPR solution was diluted into the continuous oil phase, due to the relatively high viscosity, the mixing took place slowly. Possibly the turbulence in the medium could have caused the increase in the elastic modulus. The equilibrium value of the modulus (Fig. 3) was only slightly higher than that of the oilewater interface which contained 0.008% PGPR in the oil phase and 0.1% b-lg in the aqueous phase (Table 1). Therefore, the process was at least partially reversible which suggested that the sequence of addition was not influential of the final viscoelastic properties of the interface. The affinity of proteins to soy oilewater interface was shown to be lower than that to less polar oil interfaces, therefore a lesser concentration of surfactants is needed for the displacement of proteins from soy oil interfaces (Courthadon & Dickinson, 1991; Dickinson & Tanai, 1992). It has been shown that as whey protein isolate is partially displaced from the oilewater interface by monoolein or monopalmitin, dilational modulus either drastically decreases or approaches that of the lipid film (Rodríguez Patino, Navarro Garcia, Rodríguez Niño, 2001). Fig. 3 clearly shows that, even at low concentrations, PGPR dominated the oilewater interface and the value of dilational modulus. In this case also, proteins interacted with PGPR possibly through hydrophobic interactions which lowered the equilibrium interfacial tension.
4. Conclusions PGPR is a lipophilic emulsifier that is commonly used in the stabilization of water-in-oil and water-in-oil-in-water emulsions. In order to decrease the usage of PGPR, addition of proteins in the aqueous phase has been previously recommended. The present work clearly demonstrate that dairy proteins further decrease the interfacial tension in the presence of PGPR, without displacement of PGPR by protein molecules. It was concluded that the PGPR interacts with the hydrophobic moieties of the proteins, causing changes in the viscoelastic properties of the interface. Furthermore,
Andrew, A. T., Taylor, M. D., & Owen, A. J. (1985). Rapid analysis of bovine milk proteins by fast protein liquid chromatography. Journal of Chromatography A, 348, 177e185. Benjamins, J., Cagna, A., & Lucassen-Reynders, E. H. (1996). Viscoelastic properties of triacylglycerol/water interfaces covered by proteins. Colloids and Surfaces A, 114, 245e254. Bergenståhl, B. (2008). Physicochemical aspects of an emulsifier functionality. In G. L. Hasenhuettl, & R. W. Hartel (Eds.), Food emulsifiers and their applications (2nd ed.). (pp. 173e194) New York: Springer. Beverung, C. J., Radke, C. J., & Blanch, H. W. (1999). Protein adsorption at the oil/ water interface: characterization of adsorption kinetics by dynamic interfacial tension measurements. Biophysical Chemistry, 81, 59e80. Cornec, M., Wilde, P. J., Gunning, P. A., Mackie, A. R., Husband, F. A., Parker, M. L., et al. (1998). Emulsion stability as affected by competitive adsorption between an oil-soluble emulsifier and milk proteins at the interface. Journal of Food Science, 63, 39e43. Courthadon, J. L., & Dickinson, E. (1991). Competitive adsorption of lecithin and bcasein in oil in water emulsions. Journal of Food and Agricultural Chemistry, 39, 1365e1368. Dedinaite, A., & Campbell, B. (2000). Interactions between mica surfaces across triglyceride solution containing phospholipids and polyglycerol polyricinolate. Langmuir, 16, 2248e2253. Dickinson, E. (1992). An introduction to food colloids. New York: OxfordUniversity Press. Dickinson, E., & Matsumura, Y. (1991). Time-dependent polymerization of b-lactoglobulin through disulphide bonds at the oil-water interface in emulsions. International Journal of Biological Macromolecules, 13, 26e30. Dickinson, E., & Tanai, S. (1992). Protein displacement from the emulsion droplet surface by oil-soluble and water-soluble surfactants. Journal of Food and Agricultural Chemistry, 40, 179e183. Fang, Y., & Dalgleish, D. G. (1993). Casein adsorption on the surfaces of oil-in-water emulsions modified by lecithin. Colloids and Surfaces B, 1, 357e364. Food and Drug Administration (FDA). (2006). GRAS Notice No. GRN 000179. CFSAN/ Office of Food Additive Safety. Gaonkar, A. G. (1989). Interfacial tensions of vegetable oil/water systems: effect of oil purification. Journal of American Oil Chemists’ Society, 66, 1090e1092. Garti, N., Aserin, A., & Cohen, Y. (1994). Mechanistic consideration on the release of electrolytes from multiple emulsions stabilized by BSA and nonionic surfactants. Journal of Controlled Release, 29, 41e51. Graham, D. E., & Phillips, M. C. (1980). Proteins at liquid interfaces. IV. Dilational properties. Journal of Colloid and Interface Science, 76, 227e239. Griffin, W. C. (1949). Classification of surface-active agents by HLB. Journal of the Society of Cosmetic Chemists, 1, 311e326. Hasenhuettl, G. L. (2008). Overview of food emulsifiers. In G. L. Hasenhuettl, & R. W. Hartel (Eds.), Food emulsifiers and their applications (2nd ed). (pp. 1e9). New York: Springer. Haynes, C. A., & Norde, W. (1995). Structures and stabilities of adsorbed proteins. Journal of Colloid and Interface Science, 169, 313e328. Knoth, A., Scherze, I., & Muschiolik, G. (2005). Stability of water-in-oil emulsions containing phosphotidylcholine-depleted lecithin. Food Hydrocolloids, 19, 635e640. Lucassen-Reynders, E. H., Benjamins, J., & Fainerman, V. B. (2010). Dilational rheology of protein films adsorbed at fluid interfaces. Current Opinion in Colloid & Interface Science, 15, 264e270. Lucassen-Reynders, E. H., Cagna, A., & Lucassen, J. (2001). Gibbs elasticity, surface dilational modulus and diffusional relaxation in nonionic surfactant monolayers. Colloids and Surfaces A, 186, 63e72. Márquez, A. L., Medrano, A., Panizzolo, L. A., & Wagner, J. R. (2010). Effect of calcium salts and surfactant concentration on the stability of water-in-oil (w/o) emulsions prepared with polyglycerol polyricinoleate. Journal of Colloid and Interface Science, 341, 101e108. Martin, A. H., Grolle, K., Bos, M. A., Cohen Stuart, M. A., & van Vliet, T. (2002). Network forming properties of various proteins adsorbed at the air/water interface in relation to foam stability. Journal of Colloid and Interface Science, 254, 175e183. Omotosho, J. A., Law, T. K., Whateley, T. L., & Florence, A. T. (1986). The stabilization of w/o/w emulsions by interfacial interaction between albumin and nonionic surfactants. Colloids and Surfaces, 20, 133e144. Pawlik, A., Cox, P. W., & Norton, I. T. (2010). Food grade duplex emulsions designed and stabilized with different osmotic pressures. Journal of Colloid and Interface Science, 352, 59e67. Rodríguez Patino, J. M., Navarro García, J. M., & Rodríguez Niño, M. R. (2001). Protein-lipid interactions at the oil-water interface. Colloids and Surfaces B, 21, 207e216. Serrien, G., Geeraerts, G., Ghosh, L., & Joos, P. (1992). Dynamic surface properties of adsorbed protein solutions: BSA, casein and buttermilk. Colloids and Surfaces, 68, 219e233.
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_ Gülseren, M. Corredig / Food Hydrocolloids 29 (2012) 193e198 I.
Su, J., Flanagan, J., Hemar, Y., & Singh, H. (2006). Synergistic effects of polyglycerol ester of polyricinoleic acid and sodium caseinate on the stabilisation of watereoilewater emulsions. Food Hydrocolloids, 20, 261e268. Williams, A., & Prins, A. (1996). Comparison of the dilational behaviour of adsorbed milk proteins at the air-water and oil-water interfaces. Colloids and Surfaces A, 114, 267e275. Wilson, R., Van Schie, B. J., & Howes, D. (1998). Overview of the preparation, use and biological studies on polyglycerol polyricinoleate (PGPR). Food and Chemical Toxicology, 36, 711e718.
Wüstneck, R., Moser, B., & Muschiolik, G. (1999). Interfacial dilational behaviour of adsorbed b-lactoglobulin layers at the different fluid interfaces. Colloids and Surfaces B, 15, 263e273. Yamamoto, Y., & Araki, M. (1997). Effects of lecithin addition in oil or water phase on the stability of emulsions made with whey proteins. Bioscience, Biotechnology and Biochemistry, 61, 1791e1795. Zhang, Y., An, Z., Cui, G., & Li, J. (2003). Stabilized complex film formed by coadsorption of b-lactoglobulin and phospholipids at liquid/liquid interface. Colloids and Surfaces A, 223, 11e16.