Competitive Displacement of Sodium Caseinate by Low-Molecular

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Competitive Displacement of Sodium Caseinate by Low-MolecularWeight Emulsifiers and the Effects on Emulsion Texture and Rheology M. B. Munk,*,†,§ F. H. Larsen,‡ F. W. J. van den Berg,‡ J. C. Knudsen,‡ and M. L. Andersen‡ †

Palsgaard A/S, Palsgaardvej 10, DK-7130 Juelsminde, Denmark Department of Food Science, University of Copenhagen, Rolighedsvej 26, DK-1958 Frederiksberg C, Denmark

‡

ABSTRACT: Low-molecular-weight (LMW) emulsifiers are used to promote controlled destabilization in many dairy-type emulsions in order to obtain stable foams in whippable products. The relation between fat globule aggregation induced by three LMW emulsifiers, lactic acid ester of monoglyceride (LACTEM), saturated monoglyceride (GMS), and unsaturated monoglyceride (GMU) and their effect on interfacial protein displacement was investigated. It was found that protein displacement by LMW emulsifiers was not necessary for fat globule aggregation in emulsions, and conversely fat globule aggregation was not necessarily accompanied by protein displacement. The three LMW emulsifiers had very different effects on emulsions. LACTEM induced shear instability of emulsions, which was accompanied by protein displacement. High stability was characteristic for emulsions with GMS where protein was displaced from the interface. Emulsions containing GMU were semisolid, but only low concentrations of protein were detected in the separated serum phase. The effects of LACTEM, GMS, and GMU may be explained by three different mechanisms involving formation of interfacial αgel, pickering stabilization and increased exposure of bound casein to the water phase. The latter may facilitate partial coalescence. Stabilizing hydrocolloids did not have any effect on the LMW emulsifiers’ ability to induce protein displacement.

1. INTRODUCTION Low-molecular-weight (LMW) emulsifiers are used in many dairy-type emulsions to promote controlled destabilization. For products such as whipped cream, controlled destabilization of fat globules is required to obtain good whippability. The fat globules form a network that surrounds air bubbles, extends throughout the aqueous phase, and thereby stabilizes foam structure. Formation of food oil-in-water (O/W) emulsions often involves an initial rapid adsorption of proteins to the surface of newly formed fat globules during the course of homogenization.1 By forming an interfacial layer, caseins are effective in preventing globules from coalescence or flocculation via steric and electrostatic stabilization mechanisms. For the food industry it is essential that the whippable cream remains liquid during transportation, storage, and handling before whipping. It is therefore important that destabilization of fat globules does not set in before the cream is whipped, since formation of a network between globules will increase the viscosity of emulsions. The effects of LMW emulsifier addition on globule aggregation is often ascribed to displacement of interfacial adsorbed proteins followed by partial coalescence of globules.2 The mechanism of competitive protein displacement from globule interfaces has been intensively studied during the past decade.3,4 During the initial stage of protein displacement, the LMW emulsifiers get access to the globule interface through many small gaps in the interfacial protein layer. Despite the © 2014 American Chemical Society

growing LMW emulsifier domains on the surface, the amount of adsorbed protein remains constant as the protein film is compressed. A high concentration of adsorbed LMW emulsifier can be present without inducing protein displacement.5 Eventually, the interfacial protein layer cannot withstand the surface pressure and collapses under release of protein molecules into the serum phase. Protein displacement in dairy-type emulsions by lactic acid esters of monoglycerides (LACTEM), unsaturated monoglycerides (GMU), and saturated monoglycerides (GMS) have been studied extensively.6−14 Generally, it has been observed that the emulsifierto-protein molar ratio is proportional to the protein desorption from the fat droplet interface. However, inconsistencies concerning the effectiveness of GMS and GMU to displace proteins have been reported. Davies et al.,9 Færgemand and Krog,15 and Fredrick et al.12 observed that GMS reduced the interfacial tension and displaced proteins more efficiently than GMU. On the other hand, according to Barfod et al.,7 protein displacement in ice cream was more efficient with GMU as compared to GMS. Several studies have shown that fat globules can aggregate at very low concentrations of emulsifiers, where barely any protein displacement is expected to occur. Furthermore, protein Received: March 27, 2014 Revised: July 2, 2014 Published: July 2, 2014 8687

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for approximately 20 s before homogenization on a two-stage highpressure valve homogenizer (APV Rannie LAB-12.50, SPX, Silkeborg, Denmark) at 150/50 bar. Emulsions were rapidly cooled to 5−6 °C by a plate heat exchanger (APV U2, SPX, Silkeborg, Denmark) connected to the homogenizer and were immediately transferred to storage at 5 °C. Emulsion Stability. Stability of emulsions in terms of gravitational separation was measured by using a light scattering optical analyzer (Turbiscan LAB, Formulaction, L’Union, France). Freshly prepared emulsions (20 mL) were transferred to transparent cylindrical glasses, and the entire height of the filled part (40 mm) of the glasses was scanned at regular time intervals during a period of 30 days. Between measurements, the glasses with emulsions were stored at 5 °C. Kinetic stability profiles of emulsions was calculated as the Turbiscan Stability Index (TSI) based on the intensities of back scattering:

displacement is not always accompanied by fat globule aggregation.12,14,16−18 This suggests that destabilization induced by addition of LMW emulsifiers is not always due to protein displacement as such but perhaps is a consequence of the simultaneous presence of both proteins and emulsifiers adsorbed to the globule surfaces. A recent study reported that caseinate was not expelled to the serum phase by addition of GMU, but instead a decreasing mobility of the surface bound emulsifiers was observed, indicating a higher concentration of GMU at the globule surfaces.18 Increased interfacial viscoelasticity and reduced globule coalescence have been associated with interactions between caseins and mixtures of saturated mono- and diglycerides at the oil−water interface in O/W emulsions.19−21 In the presence of caseinate, the polarity of lipid crystals in emulsion model systems has been found to increase according to the level of unsaturation of monoglycerides.22 This was interpreted as GMU induced adsorption of caseinate to the fat crystal surfaces. Correlations have been found between oil droplet aggregation and protein network formation, and it was proposed that the aggregation was based on interactions between adsorbed proteins at the emulsion droplet surface and nonadsorbed proteins in the aqueous phase.23,24 Sugimoto et al.24 found that aggregation of oil droplets resulting in increased emulsion viscosity was associated with an increased caseinate surface coverage, while Euston et al.23 proved that nonadsorbed whey protein took part in the oil droplet aggregation mechanism. Here we present a study of the relation between aggregation of fat globules in O/W emulsions induced by the LMW emulsifiers LACTEM, GMU, or GMS and the displacement of sodium caseinate from fat globule interfaces. The effects on the physical properties of the emulsions were evaluated by rheology, texture analysis, and liquid-state 31P NMR. The location of protein in the emulsion was evaluated by a traditional indirect method based on separating serum by involving mechanical stress in combination with studies of the polarity of the protein environment by front face fluorescence spectroscopy. Furthermore, effects of common food stabilizers such as microcrystalline cellulose and carboxymethylcellulose on the LMW emulsifiers’ ability to cause protein displacement were assessed.

TSI =

∑ i

∑h |scani − scani − 1| H

(1)

where scani is the back scattering intensity at a given height h of the glass cell, H is the total height of the glass cell, and i is the scan number. A total of 10 scans were recorded during a period of 30 days. Rheology and Texture Analysis. Steady state flow experiments with increasing shear rate from 0.01−100 s−1 were performed at 20 °C using an ARG2 rheometer (TA-Instruments, West Sussex, England, U.K.) fitted with a Peltier temperature control connected to a water bath. A cone (diameter 40 mm, inclination 1.59°) and plate geometry was applied. Reliable measurements with cone and plate geometry were not possible with emulsions containing GMU due to the formation of large macroscopic lumps which appeared to be accelerated by shear. Instead, discs of firm GMU emulsions of diameter 25 mm were molded and characterized by frequency sweep from 500−0.1 rad/s at constant strain of 0.1% using plate geometries with rough surfaces. Texture analysis was conducted using a TA.XT.Plus (Stable Micro Systems, Etten-Leur, The Netherlands), fitted with a 20 mm diameter aluminum cylinder probe, set up to record the force used to penetrate the emulsion to a depth of 25 mm at a speed of 1 mm/s. Measurements were carried out on emulsions stored in a fridge (5 °C) and emulsions tempered at room temperature (20 ± 2 °C) for 3 h. Hardness was defined as the peak compression force (N) during penetration of the emulsion. Rheological measurements and texture analysis were performed in triplicate for each emulsion at both temperatures. Light Scattering. The size distributions of dispersed fat globules were determined by light scattering (LA950-V2, Horiba Scientific, Japan). Prior to size distribution measurements, emulsions were diluted 1:100 by 5 °C water. Droplet size distribution was determined approximately 2 h after emulsion preparation and repeated on day 2 and day 30. Measurements were done at least in triplicate. 31 P NMR Spectroscopy. The 31P NMR experiments were carried out with a Bruker Avance DRX-500 (Bruker BioSpin, Rheinstetten, Germany) operating at 202.46 and 500.13 MHz for 31P and 1H, respectively, using a double tuned BBI probe equipped for 5 mm (o.d.) NMR tubes. All NMR experiments were conducted using a 30° flip-angle for the 31P pulse, and Waltz-16 1H decoupling was utilized during recording of the FID. Each experiment was carried out using a recycle delay of 2 s, 8000 scans, an acquisition time of 0.202 s, and a temperature of 10 °C. Subsequently, the FIDs were apodized by a Lorentzian line broadening of 10 Hz prior to Fourier transformation. All spectra are referenced to an external sample of 85% H3PO4 (aq) at 0.0 ppm. Four selected emulsions containing 0.3 wt % LACTEM, GMS, or GMU and one without LMW emulsifier was analyzed by 31P NMR, and the samples were prepared by mixing of 495 μL emulsion with 55 μL D2O. Protein Displacement by the Kjeldahl Method. Concentration of protein adsorbed to the fat droplet surface was determined by an indirect method separating emulsions into an aqueous and a fat phase. This was accomplished by centrifugation of 20.000 g at 5 °C for 15 min followed by extraction of subnatant, which was repeatedly

2. EXPERIMENTAL SECTION Materials. Lactic acid ester of monoglyceride (LACTEM) made from fully hydrogenated palm oil and rape-seed oil (C16:0 and C18:0 > 97% of fatty acids), unsaturated monoglyceride (GMU) made from sunflower oil (C18:1 > 81%), and saturated monoglyceride (GMS) made from fully hydrogenated palm oil and rape-seed oil (C16:0 and C18:0 > 97%) were used. All emulsifiers were provided by Palsgaard A/S (Juelsminde, Denmark). Emulsions contained 25 wt % hydrogenated palm kernel oil (HPKO) (AAK, Karlshamn, Sweden), 0.6 wt % sodium caseinate (DMV International, Veghel, The Netherlands), 10 wt % sucrose, and 1 wt % sorbitol. Unless specified, the emulsions were prepared without addition of stabilizers. Emulsions with stabilizers contained 0.6 wt % of a stabilizer mixture composed of microcrystalline cellulose (MCC), sodium carboxymethylcellulose (CMC), and sodium hydrogen phosphate (Palsgaard A/S). Emulsion Preparation. Sodium caseinate and sugar (and stabilizer) were dispersed in water under continuous stirring and put aside for 4 h in order to hydrate proteins. The emulsifier in concentrations of either 0.0, 0.2, 0.3, 0.5, or 1.0 wt % was added to molten HPKO and subsequently mixed with the heated water phase (∼70 °C), and the combined mixture was heated to 82 °C. The mixture was blended with high-shear (Ultra-Turrax, IKA, NC, U.S.A.) 8688

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Table 1. Emulsifier-to-Protein Molar Ratio, R, Average Globule Size, d32, and Turbiscan Stability Index, TSI, after 30 Days for Emulsions Containing Various Concentrations of LACTEM, GMS, and GMU LACTEM wt %

Ra

0.0 0.2 0.3 0.5 1.0

0 18 28 46 92

GMS

d32 (μm)

TSI

Ra

± ± ± ± ±

12.4 11.1 12.0 12.0 6.1

0 22 33 56 111

1.77 1.23 1.57 1.53 1.00

0.25 0.04 0.31 0.03 0.09

GMU

d32 (μm)

TSI

Ra

d32 (μm)

TSI

± ± ± ± ±

12.4 11.0 8.8 8.1 3.9

0 21 32 54 107

1.77 ± 0.25 1.12 ± 0.33 1.06 ± 0.19

12.4 4.9 4.6 1.9 1.1

1.77 1.13 0.99 0.81 0.71

0.25 0.03 0.20 0.18 0.06

a

For calculation of R, Mw of sodium caseinate is set to 23 000 g/mol, Mw(LACTEM) is 417 g/mol, Mw(GMS) is 345 g/mol, and Mw(GMU) is 357 g/mol.

rheological and texture properties.25 Some emulsions are stable fluids whereas others are very easily converted into firm textures. HPKO emulsions were made with different concentrations of the LMW emulsifiers: 0.0, 0.2, 0.3, 0.5, and 1.0 wt % (Table 1). The sodium caseinate concentration was kept constant at 0.6 wt % in all emulsions, resulting in an emulsifierto-protein molar ratio, R, ranging from 0 to 111. The long term stability of the emulsions with respect to phase separation was monitored using light scattering. Decreased intensity of backscattering in the bottom of the glass cell indicated the presence of a more diluted aqueous phase while only faint changes were observed in the parts of the curves transmitting the emulsions in the middle and top of the cells. The most significant phase separation was measured for the control emulsion without LMW emulsifiers as the backscatter intensity was decreased up to a height of 10 mm in the glass cell on day 30. A smaller extent of reduced backscatter intensity in the bottom of the cells was observed for emulsions containing LACTEM or GMS, while the backscattering curves for emulsions with high concentration of GMU did not change during the period of 30 days. The phase separation was reported as turbiscan stability index (TSI), where smaller values of TSI correspond to more stable emulsions (Table 1). For all three LMW emulsifiers, TSI decreased with increasing emulsifier concentration, implying retardation of creaming. However, LACTEM prevented creaming less efficiently than the two other emulsifiers, and the creaming rate of emulsions was only significantly delayed at a concentration of 1 wt % LACTEM. Conversely, even at low concentrations, GMU was very efficient, providing good separation stability, and at concentrations ≥0.5 wt % phase separation was almost nonexistent during a period of 30 days. GMS also retarded the creaming rate of emulsions, although the effect was more moderate than GMU. The rheological measurements showed that emulsions with LACTEM in concentrations ≥0.5 wt % gave rise to shear thickening behavior at 20 °C (Figure 1a). The sensitivity to shear increased with the concentration of LACTEM since the onset shear rate for structure formation was lower for 1.0 wt % LACTEM than for 0.5 wt % LACTEM. The sudden increase of viscosity as a function of shear is most likely reflecting aggregation of fat globules25 and was never observed for 0.2 wt % LACTEM and control emulsion without LMW emulsifiers. However, the rheological results for LACTEM containing emulsions proved difficult to reproduce as the onset shear rate for structure formation fluctuated between experiments, and the shear thickening was also in some cases observed for emulsions with 0.3 wt % LACTEM. GMS did not appear to have any effect on the viscosity of the emulsions. Even at concentrations up to 1 wt % and exposure to shear the viscosity

centrifuged and extracted with Pasteur pipet until the absence of fat. To ensure complete removal of fat, the serum phase was finally filtered through a 0.8 μm syringe filter (Sartorius stedim, Pennsylvania, U.S.A.). The serum phase was evaporated to dryness using Foss Digestor Bloc in a vial, and the nitrogen content was subsequently determined by Kjeldahl analysis. The protein concentration was calculated as the nitrogen content multiplied by 6.38. Protein analysis was performed twice on two separately prepared batches of emulsions approximately 2 h after emulsion preparation and again on day 2 and day 30. Front Face Fluorescence Spectroscopy. Fluorescence spectra were recorded using a Varian Cary Eclipse fluorescence spectrophotometer. The spectral landscapes of tryptophan were recorded from emission 300 to 500 nm with excitation wavelengths from 280 to 300 nm with intervals of 2 nm. Detector gain was set at 500 mV and width entrance and exit slits at 5 nm. The opaque emulsions were measured in a quartz cuvette in front-face fashion under an angle of 45°. The measurements were conducted at ambient temperature, but prior to measurements the emulsions were stored at 5 °C. Electrophoretic Mobility. The charges surrounding the surface of the dispersed fat droplets were measured by electrophoretic mobility using a Zetasizer (Nano-ZS, Malvern Instruments, Malvern, U.K.). Prior to measurements the emulsions were stored at 5 °C and diluted 1:50 in 5 °C water containing 10% sucrose and 1% sorbitol, followed by careful injection of the diluted sample into disposable capillary cuvettes. The measurements were carried out on day 6 after emulsion preparation. The results are the average of 6 measurements. Interfacial Tension. Interfacial tension was determined using a two-phase model system containing deionized water and sunflower oil in a ratio of 3:4. Sunflower oil was used due to the absence of solid fat. Water containing 0.9 wt % sodium caseinate was heated to 80 °C and subsequently cooled to room temperature. Oil containing 2.0 wt % LMW emulsifiers was heated in order to melt the LMW emulsifiers and subsequently cooled to room temperature. Surface tension was measured at room temperature using a Sigma 703 tensiometer (KSV instruments ltd., Helsinki, Finland) equipped with a Wilhelmy plate. The Wilhelmy plate was placed in the water/air surface, and the oil was subsequently transferred on top of the water until the Wilhelmy plate was completely covered by oil. The equilibrium time for the monolayer interface adsorption was set to 60 min. The measured interfacial tension values were corrected for the buoyancy of the Wilhelmy plate being completely immersed into oil. A minimum of four replicates were measured for each system. Statistical Analysis. The results of the analyses are reported as the mean and standard deviation calculated from the replicates. The levels of significance for globule size, zeta potential, and Kjeldahl analysis (concentration of caseinate in the serum phase segregated by emulsion centrifugation) were calculated by one way Anova and are reported as the p-values.

3. RESULTS 3.1. Stability and Rheological Properties of Emulsions. The three LMW emulsifiers LACTEM, GMU, and GMS give HPKO oil-in-water emulsions with widely varying 8689

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Destabilization being enhanced with increasing unsaturation of emulsifiers is in agreement with previous observations.26,27 Due to the lack of fluidity, emulsions with GMU could not be compared to the other emulsions with respect to viscosity. In order to compare the ability of the emulsions to form firm structures, texture analysis was conducted on all emulsions at 5 and 20 °C (Table 2). However, emulsions made with GMS Table 2. Effect of Type and Concentration of LMW Emulsifiers on Hardness of Emulsionsa LACTEM

GMS

GMU

wt %

5 °C

20 °C

5 °C

20 °C

5 °C

20 °C

0.0 0.2 0.3 0.5 1.0

L L L L L

L L L 0.04 N 0.8 N

L L L L L

L L L L L

L L 0.6 N 12 N 29 N

L L 0.7 N 10 N 23 N

a Hardness was defined as the maximum peak force to penetrate the emulsion. Penetration force was not compliable for liquid (L) emulsions.

remained liquid at both temperatures, which was also the case for emulsions with LACTEM at 5 °C. GMU gave firm emulsions at 5 and 20 °C, presumably caused by agglomerated fat globules, and the strength of the emulsions increased with increasing GMU concentration. In accordance with the rheological measurements, emulsions with 0.5% and 1.0% LACTEM had a firm texture at 20 °C without application of shear. The hardness was considerably lower compared to the emulsions containing GMU. Opposite to the emulsions with LACTEM that remained liquid at 5 °C, emulsions with GMU were characterized by higher hardness at 5 °C than at 20 °C. The average globule diameters of the emulsions were constant during the period of 30 days after the preparation of the emulsions, and the mean globule sizes were therefore calculated as the average of 12 individual measurements obtained during this period (Table 1). Increasing concentration of emulsifiers appeared to reduce the globule size, at least in the case of GMS in agreement with previous reports.11,28 The high sensitivity toward stress and temperature complicated the determination of particle size distributions by light scattering as this technique involves shear and dilution. Although effort was done to perform the measurements gently, the emulsions containing 0.5 and 1.0 wt % GMU could not be completely dispersed and were thus not measurable. 3.2. Mobility of Sodium Caseinate in Emulsions. The formation of firm emulsions has been suggested to involve formation of a protein network.18 31P NMR was therefore used to examine the LMW emulsifiers’ effect on the mobility of caseinate in emulsions. Phosphorus in the emulsions was expected only to originate from the purified sodium caseinate as phosphorylated serine residues in caseinate and inorganic calcium phosphate.29 All 31P NMR spectra were characterized by a narrow resonance (Figure 2, marked with B) located in the region 5.0− 5.3 ppm and a broader resonance or series of overlapping resonances (Figure 2, marked with A) in the range from ∼5.3 to 7.0 ppm. The narrow resonance, B, was assigned to inorganic phosphate, whereas the broad resonance, A, was assigned to phosphoserine residues.30 The latter consisted of a range of resonances, which reflected that phosphoserine was present in different types of caseins (αS1, αS2, β, and κ) as well as in

Figure 1. Effect of LMW emulsifiers on the rheological behavior of emulsions measured at 20 °C. Viscosity of emulsions containing a: LACTEM, b: GMS or GMU (0.2 wt %). c: Elastic modulus of emulsions containing GMU. Concentrations of LMW emulsifiers: ■ 0 wt % (control), ○ 0.2 wt %, ▲ 0.3 wt %, □ 0.5 wt %, ● 1.0 wt %.

of the emulsions was unaffected by GMS and was similar to that of the control emulsion made without LMW emulsifiers (Figure 1b). Similar to emulsions with LACTEM, the rheological behavior of emulsions with GMU was also dependent on the emulsifier concentration. An emulsion with 0.2 wt % GMU had a higher apparent viscosity than all the studied emulsions containing GMS or LACTEM, and the viscosity increased further as a consequence of shear (Figure 1b). At concentrations of GMU ≥ 0.3 wt % the emulsions were so solid (irrespective of shear) that the required gap between the cone and the plate was not attainable, and it was also impossible to conduct the measurement in the rheometer cup without exceeding the limiting torque at 2 × 105 mN/m. Instead, discs were molded, and the rheological properties of emulsions with 0.5 and 1.0 wt % GMU were instead characterized by the elastic modulus, G′ ≈106 Pa (Figure 1c). However, it was not possible to characterize the emulsion containing 0.3 wt % GMU by rheological measurements since it was too fluid to be molded into discs but still too solid to be studied by shear. 8690

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Figure 2. 31P NMR spectra of emulsions with 0.3 wt % LMW emulsifiers and without (control) measured at 10 °C. The group of peaks marked A are assigned to phosphoserine groups in caseinate, whereas the peak at B is assigned to inorganic phosphate.

size of fat globules. The globule size for the very firm emulsions containing 0.5 and 1.0 wt % GMU could not be determined by light scattering, and in this study protein displacement is instead reported by the concentration of caseinate in the serum phase. However, the relative differences of serum phase caseinate concentrations corresponds roughly to the relative differences of calculated protein surface load, since the variation of measured globule sizes between emulsions were fairly low (Table 1). The serum phase was separated from the remaining emulsion by centrifugation at 5 °C, since maximum protein displacement has been reported to occur at 5−10 °C.7,10,11 The serum phase from the different emulsions differed in terms of turbidity. The serum phases of the firm emulsions with GMU were transparent in agreement with a denser network which would be able to retain smaller particles. Increasing concentration of LACTEM in emulsions was found to increase the transparency of the serum phases, while emulsions with GMS gave turbid serum phases. The total concentration of caseinate in emulsions was 0.6 wt % corresponding to a concentration in the serum phase equal to 0.8 wt %. With addition of 0.2 wt % LACTEM or GMS to emulsions, the concentration of caseinate in the serum phase increased approximately 100% from 0.3 to 0.6 wt % compared to the control emulsions without LMW emulsifiers (Figure 3a,b). Further release of caseinate to the serum phase was observed with higher emulsifier concentrations up to 1.0 wt %, although the increases were not statistically significant (P >

different local conformations within each type of casein. The chemical shifts were approximately 2 ppm higher compared to those of previous 31P NMR study of caseinate in microfiltrated milk,30 which was ascribed to different environmental conditions of phosphorus in the emulsions despite comparable neutral pH values. The spectral resolution was highest in the spectrum of the control emulsion, followed by emulsions with GMS and LACTEM, whereas addition of GMU induced a rather significant line broadening. Line broadening reflects restricted mobility of caseinate in emulsions.31 Reduced mobility of caseinate as observed by 31P NMR line broadening has been suggested to indicate adsorption of caseinate to the interface of oil droplets.29,32 The mobility of phosphoserine in emulsions containing GMU was reduced, either due to adsorption of protein to the fat globule interface or entrapment in a fat globule network. On the other hand, phosphoserine in emulsions with GMS and LACTEM and without LMW emulsifiers appeared to have higher mobility, and therefore it is likely that a larger fraction of caseinate was located in the aqueous serum. 3.3. Competitive Adsorption between Sodium Caseinate and LMW Emulsifiers. Displacement of proteins from the surface of globules in emulsions is usually discussed based on the protein surface load, which is calculated as the amount of protein adsorbed per unit surface area of the fat globules. The calculation of protein surface load is based on the 8691

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GMU did not increase the concentration of caseinate in the serum phase relative to the control emulsion (Figure 3c). This is in agreement with a recent study where it was found that unsaturated monoglyceride in a concentration of 0.2 wt % did not change the protein surface load in an O/W emulsion.12 In fact, the level of protein in the serum phase decreased with increasing concentration of GMU, and this effect was gradually enhanced over time but especially within the two first days after emulsion preparation. The concentration of caseinate in the serum was significantly reduced (P < 0.05) on day 30 in emulsions containing 0.3−0.5 wt % GMU. On day 0 only 1.0 wt % GMU gave a significantly (P < 0.05) lower level of serum protein when compared to 0.2 wt % GMU. It was only possible to obtain isolated serum phase from the emulsion with 1.0 wt % GMU on day 0 despite extended centrifugation. The ease of collecting the serum phase was clearly correlated with firmness of emulsions, as only minimal serum phase could be obtained from the solid emulsions despite exposure to severe centrifugal stress. Firmness of emulsions with GMU increased gradually over time and was proportional to increasing concentrations of GMU as well. There seemed to be a correlation between firmness of emulsions with GMU and decreasing amount of caseinate in the serum phase obtained by centrifugation. The usual evaluation of protein displacement from fat globules in emulsions is based on the assumption that protein is either located nonbound in the serum phase or bound at the interfacial layer of the globules. According to this assumption, the protein surface load should then be very high for emulsions with GMU due to the low level of proteins in the separated serum phase. However, based on spin probe experiments, it has been suggested that proteins also could be retained in the emulsion due to network formation,18 which would violate the above-mentioned assumption. Furthermore, it was an issue of concern that the intense centrifugation could lead to globule collisions, causing additional aggregation and further protein release. This notion required special attention since most emulsions were highly structurally sensitive toward stress and temperature (Figure 1 and Table 2). The partitioning of protein between the fat globule interface and the aqueous serum phase was therefore also analyzed by front-face fluorescence spectroscopy as an alternative to the Kjeldahl method requiring phase separation. This method does not perturb the physical characteristics of the samples as it does not require phase separation, dilution, or any other pretreatment of the emulsions. Front-face fluorescence spectroscopy is based on obtaining emission spectra of tryptophan from which it can be deduced whether the protein tryptophan residues are in a hydrophilic or hydrophobic environment.33,34 Emission spectra in the range of 310−380 nm were fitted to Gaussian distributions, and the wavelengths of the peak positions (λmax) were registered. A plot of λmax against the concentration of caseinate in the serum obtained by centrifugation on day 0 gave a linear correlation for emulsions with LACTEM and GMS (Figure 4), showing high concentration of caseinate in aqueous serum corresponded to high λmax. This is in agreement with a study of Rampon et al.,35 who showed that λmax of tryptophan decreased when the amount of bovine serum albumin in the serum phase was reduced. It has also been established that tryptophan in hydrophilic and hydrophobic environments yields peaks in emission spectra at 333 and 319 nm, respectively.33,34 A larger proportion in hydrophilic environment would consequently

Figure 3. Effects of LMW emulsifiers and storage time on the concentration of dissolved protein in the serum phase of emulsions, determined at 5 °C. The total concentration of protein in emulsions was 0.6 wt %, corresponding to a protein concentration of 0.8 wt % in the serum phase. LMW emulsifiers: (a) LACTEM, (b) GMS, and (c) GMU. Concentrations of LMW emulsifiers: ■, 0 wt % (control, marked with dashed line); ○, 0.2 wt %; ▲, 0.3 wt %; □, 0.5 wt %; ●, 1.0 wt %. The emulsions were stored at 5 °C. The plotted results are the mean of two replicates.

0.05). The high levels of caseinate in the serum phases suggest LACTEM and GMS both efficiently displace caseinate from the fat globule surfaces. The high level of caseinate in the serum phase of LACTEM and GMS emulsions was observed immediately after emulsification (on day 0), and the concentration of caseinate in the serum phase did not change much during the following 30 days. It should be noted that the presence of caseinate was required for emulsion formation, since emulsions made with LACTEM, GMS, or GMU but without caseinate phase-separated shortly after homogenization (data not shown). 8692

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emulsions, and which is negatively charged at the pH of the emulsions (pH ∼ 7). The zeta potential increased with addition of the LMW emulsifiers, indicating that caseinates were desorbed from the fat droplet surface and probably (partly) replaced by LACTEM, GMS, or GMU. All concentrations (0.2−1.0 wt %) of LACTEM and GMS resulted in a significantly higher (P < 0.05) zeta potential as compared to the control emulsion. The zeta potential increased up to a concentration level of 0.3 wt % of LMW emulsifier, and at higher concentrations the zeta potential leveled off. This is consistent with 0.2 wt % LACTEM and GMS causing a substantial increase of caseinate in the serum phase, while at concentrations above 0.2 wt % the effect on serum protein levels was less significant. The firm emulsions containing GMU was not compatible with measurements of the zeta potential since this technique required dilution of the emulsions. Only emulsions with 0.2 wt % GMU could be studied. The zeta potential for this emulsion was lower than the zeta potential for the control emulsion, but the difference was not significant (P < 0.05). In accordance with the results from protein displacement, zeta potential measurements showed that 0.2 wt % GMU displaced proteins from the fat globule interface to a lesser extent than equivalent concentrations of LACTEM or GMS. 3.4. Interfacial Tension. Interfacial tension between aqueous caseinate suspension and sunflower oil decreased with the addition of LACTEM, GMU, or GMS (Table 3),

Figure 4. Wavelengths of peak positions for front-face fluorescence spectra of tryptophan plotted against the concentrations of protein in the aqueous serum obtained by centrifugation of emulsions. The emulsions were made with LMW emulsifiers LACTEM (red), GMS (blue), and GMU (green) in the following concentrations: ◆, 0.0 wt %; ●, 0.2 wt %; ▲, 0.3 wt %; ■, 0.5 wt %; ▼, 1.0 wt %. The encircled points were not included in the linear regression.

shift λmax to a higher value. The firm emulsions containing 0.3− 1.0 wt % GMU diverged from the linear correlation (Figure 4). According to the high values for λmax for tryptohan fluorescence in these emulsions, caseinate should be located in a highly polar environment, although only small amounts could be separated out in the centrifuged serum phase. This suggests that caseinate is not directly attached to the interfacial layer in the GMU emulsions but instead may be located in the aqueous phase where it connects and entangles the fat globules and thereby participates in forming a network together with fat globules resulting in the overall firm structure of the emulsion as suggested previously.18 The zeta potential of emulsions was determined to obtain information about changes of the molecular environment around the fat globules caused by LMW emulsifier addition (Figure 5). In the absence of LMW emulsifiers, the fat globules had zeta potential around −20.5 mV. This was assigned to caseinate, which is the only charged component in the

Table 3. Effect of LMW Emulsifiers on Interfacial Tension in a Model System Composed of Aqueous Caseinate Suspension and Sunflower Oila emulsifier control LACTEM GMS GMU a

interfacial tension (mN/m) 10.0 4.2 8.6 0.1

± ± ± ±

0.5 0.4 0.4 0.5

The control contained no LMW emulsifiers in the oil phase.

indicating the three LMW emulsifiers were (at least partly) positioned at the oil−water interface. A coexistence of emulsifiers with caseinate at the interface is assumed to exist.15,36 The LMW emulsifiers may intrude small holes in the interfacial protein network that are not accessible for the larger proteins.3 Moreover, protein layers can withstand rather high surface pressures before displacement occurs, which allows high accumulation of LMW emulsifiers on the interface.3−5 Surface pressure is the interfacial tension arising from presence of surfactants at an interface: Π = γ0 − γ, where Π is the surface pressure, γ0 is the interfacial tension of the pure oil and aqueous solvent without surfactants, and γ is the interfacial tension arising when surfactants are adsorbed between oil and water. The lowest interfacial tension was obtained with LACTEM and GMU, while GMS caused only a minor reduction. Low interfacial tension was expected to be correlated to efficient protein interfacial displacement. In the case of LACTEM and GMS, the decrease of interfacial tension may be ascribed to interfacial occupation by the LMW emulsifiers and consequently desorption of caseinate. In the case of GMU, absence of protein displacement did not seem compatible with the large reduction of interfacial tension, which is in agreement with the observation by Fredrick et al.12 However, either GMU may pack closely at the interface without interrupting the protein layer, or the protein molecules were displaced from the

Figure 5. Zeta potential as a function of LMW emulsifier concentration for emulsions containing LACTEM (■), GMS (○), and GMU (△). 8693

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LMW emulsifiers. Presence of stabilizers did not change the fact that LACTEM and GMS efficiently expelled caseinate from the interface to the serum phase. Regardless of stabilizers, GMU still gave a reduced amount of caseinate in the serum phase. The largest variation with respect to the presence of stabilizers was seen for control emulsions without LMW emulsifiers. The concentration of caseinate in serum was lower with addition of stabilizer, which could be explained by reduction of globule size and consequently larger interfacial area. However, reduced globule size was a common phenomenon for all the emulsions with stabilizers. Therefore, it seems more reasonable that reduced molecular mobility due to increased viscosity of the continuous phase caused caseinate to remain at the interfacial layer when no other surface-active components were present.

interface but remained among the fat globules in the fat globule network, therefore not being detected in the centrifuged serum phase. The latter would be in agreement with the conclusions from the front face fluorescence results (Figure 4). 3.5. Stabilizers’ Effect on Competitive Displacement. Stabilizers are usually added to food emulsions such as ice cream and whippable emulsions in order to increase viscosity and thereby prevent creaming and collapse of air bubbles. In this study stabilizers were excluded because the high viscosity was not compatible with many of the performed analysis, e.g., electrophoretic mobility, front face fluorescence spectroscopy, and 31P NMR. Stabilizers are, however, common ingredients in cream products, and it is therefore highly relevant to study whether they affect the competitive displacement between proteins and LMW emulsifiers. Emulsions were prepared with 0.6 wt % stabilizer mixture and containing 0.5 wt % LACTEM, 0.5 wt % GMS, or 0.5 wt % GMU as well as one control emulsion also with the stabilizer mixture but without LMW emulsifiers. The stabilizers increased the stability toward creaming as expected (Table 4). In fact, the control emulsion

4. DISCUSSION Instability of emulsions in terms of aggregation of fat globules has been suggested to result from either fat crystallization or displacement of proteins from the fat globule interfaces. However, a recent study showed that the effect of LMW emulsifiers on fat crystallization of bulk HPKO was not related to the LMW emulsifiers’ ability to initiate fat globule aggregation in HPKO emulsions.25 The present study has demonstrated that the viscosity increase and solidification of HPKO-based emulsions is not due to protein displacement by competitive surfactants. Protein displacement as such did not induce fat droplet aggregation, which was clearly illustrated by emulsions containing GMS. Caseinate was efficiently expelled to the serum phase by GMS; however, the emulsions remained fluid and stable toward shear despite concentrations of GMS up to 1 wt %. Efficient displacement of protein from the droplet interface and absence of shear-induced destabilization of emulsions by GMS was also observed by Pelan et al.14 and Davies et al.9 However, according to the phase diagram of GMS it is crystalline in the temperature range of the experiment.37 As the protein becomes displaced from the surface, nanocrystals of monoglyceride absorbed at the surface could stabilize the emulsion by a Pickering mechanism. Emulsions with LACTEM were sensitive to shear resulting in thickening of the emulsion. LACTEM also led to high levels of caseinate in the serum phase. The characteristics of the fat globule network causing the thickening differed from the network formed with GMU, as the strength was considerably lower, and the structure formation was dependent on temperature and shear. The mechanisms responsible for formation of a network of aggregated fat globules were therefore assumed to be different for the LACTEM and GMU emulsions. In the temperature interval of 10 to 20 °C the interfacial elastic modulus of the interfacial layer composed of LACTEM is approximately 10 times higher compared to the interfacial elastic modulus characterizing the interfacial layer of GMU.27 This large difference is probably related to the phase behavior of the two emulsifiers, since GMU forms a cubic structure and LACTEM forms an α-polymorphic crystals in the studied temperature interval.38,39 It has been suggested that below the melting temperature of LACTEM (T < 42 °C), formation of an α-crystalline gel at the oil−water interface takes place which link globules together and thereby promotes flocculation.39 This mechanism implies desorption of protein from fat globules, which complies with the results of zeta potential measurements, protein displacement, and interfacial tension in this study.

Table 4. Turbiscan Stability Index, TSI, and Average Globule Size, d32 (μm), for Emulsions Containing 0.6 wt % Stabilizers and 0.5 wt % LMW Emulsifiersa

a

emulsions

TSI

control LACTEM GMS GMU

0.7 1.0 0.8 1.7

d32 0.82 0.76 0.46 1.25

± ± ± ±

0.03 0.18 0.16 0.45

The control contained no LMW emulsifiers.

without LMW emulsifiers became the most resistant to creaming with addition of stabilizers, whereas it was the most unstable in the absence of stabilizers. Moreover, stabilizers affected the droplet size which were approximately 50% smaller compared to the corresponding emulsion without stabilizer (Tables 1 and 4). The concentration of caseinate in the serum phase obtained by centrifugation was determined and compared to corresponding emulsions without stabilizer mixture (Figure 6). It seemed that stabilizer mixture did not affect the competitive displacement between proteins and

Figure 6. Effect of stabilizers on the concentration of protein in the centrifuged serum phase of emulsions determined at 5 °C. The open symbols represent emulsions made with 0.6 wt % stabilizers and the closed symbols represent emulsions without stabilizers. (◆, ◇) No LMW emulsifiers added (control), (■, □) 0.5 wt % LACTEM, (●, ○) 0.5 wt % GMS, (▲, △) 0.5 wt % GMU. 8694

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globule interface to the aqueous serum phase. LACTEM induced shear instability of emulsions, which was accompanied by protein displacement. High stability was characteristic for emulsions with GMS, but concurrently protein was displaced from the interface. Emulsions containing GMU were semisolid but did not have high levels of protein in the separated serum. This implies that instability is not equivalent to protein displacement and vice versa, but instability can occur as a consequence of modified interfacial layer. Pickering stabilization, formation of interfacial α-gel, and increased exposure of bound casein to the aqueous phase were suggested to be the mechanisms leading to different stability and protein displacement behavior of GMS, LACTEM ,and GMU, respectively. Furthermore, stabilizing hydrocolloids did not have any effect on protein displacement by LMW emulsifiers.

The effect of GMU on the emulsions was opposite to GMS. Solidification of emulsions was induced by GMU, but the thickening was not accompanied by protein displacement from the globule interfaces as only very low concentrations of soluble caseinate were found in the separated serum phase obtained by centrifugation. Similar results have been found by Pelan et al.14 who showed that destabilization of fat droplets caused by GMU did not involve proportional protein displacement. However, other studies have found correlations between instability of emulsions induced by GMU and increasing protein displacement.8,40 The low casein concentration in the separated serum phase, combined with the results from measurements of interfacial tension and zeta potential, suggests GMU positions itself at the globule interface. However, caseinate will still be anchored at the interface, but each caseinate molecule will cover a smaller interfacial area and will become more stretched out into the serum phase. The 31P NMR experiments showed caseinate had a low mobility in the GMU emulsions indicating that it was still bound to the globule surfaces, whereas the frontface fluorescence experiments indicated that most of the protein was located in the aqueous environment. This suggests a larger part of the caseinate structure is exposed to water while still being bound to the surface of the globules in the GMU emulsions. The higher exposure into the aqueous phase also implies a smaller part of the caseinate is in direct contact with the globule interface. Alternatively, caseinate could also be trapped in an extremely dense fat network or be entrapped in the aqueous cubic phases of GMU.41,42 However, the latter would not explain the increased viscosity of emulsions, which reflect fat globule aggregation, and the change of mobility of components at the globule surfaces.18,25 Partial coalescence of fat globules may still explain the formation of a dense fat network in the GMU emulsion; however, it remains unknown how GMU modifies the emulsion system in order to increase its ability to undergo fat aggregation. Caseinate was suggested to facilitate closer approach between two globules that must take place in order to achieve partial coalescence by entangling the fat globules. GMU might interact directly with caseinate and consequently attach a part of the molecule to the fat globule interface or induce conformational changes of caseins that enhance participation in a fat globule network.19−21 Another possible effect of GMU was that it adsorbs onto HPKO crystals situated at the interface and consequently enhances crystal penetration through the interface as proposed by Davies et al.8 The present study also demonstrated that discussion of protein displacement from globule interfaces based on changes in protein concentrations in serum separated from emulsions by centrifugation should be done with caution. Front-face fluorescence experiments demonstrated that a major amount of protein in the GMU emulsions was present in the aqueous phase, although this was not reflected in the protein concentration of the separated serum. However, the combination of the two techniques appears to be needed in order to provide information about the formation of protein networks in the aqueous phase which strongly bind proteins, and which furthermore can have pronounced effects on the texture and rheological properties of emulsions.

■

AUTHOR INFORMATION

Present Address §

Department of Food Science, University of Copenhagen, Rolighedsvej 26, 1958 Frederiksberg C, Denmark. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The Danish Agency for Science, Technology and Innovation is gratefully acknowledged for financial support to M.B.M to carry out this research.

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REFERENCES

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