Article pubs.acs.org/Langmuir
Synchrotron Radiation Macrobeam and Microbeam X-ray Diffraction Studies of Interfacial Crystallization of Fats in Water-in-Oil Emulsions Paul Wassell,*,†,‡ Airi Okamura,§ Niall W.G. Young,†,‡ Graham Bonwick,‡ Christopher Smith,∥ Kiyotaka Sato,§ and Satoru Ueno§ †
Nutrition and Health, Danisco/DuPont, Brabrand, Denmark Department of Biological Sciences, University of Chester, U.K. § Laboratory of Food Biophysics, Hiroshima University, Japan ∥ Department of Food and Tourism Management, Manchester Metropolitan University, U.K. ‡
S Supporting Information *
ABSTRACT: Using macrobeam and microbeam techniques, we performed synchrotron radiation X-ray diffraction (SR-XRD) analyses of fat crystallization in water-in-oil (W/O) emulsion, in combination with DSC and polarized optical microscopic observation. Particular focus was on the crystallization of the fats around water droplets in the W/O emulsion systems using food emulsifiers of polyglycerol polyricinoleate (PGPR) alone (PGPR emulsion), and PGPR and monobehenoylglycerol (MB) (PGPR +MB emulsion). We obtained the following results: (1) macrobeam SR-XRD confirmed that adding MB promoted fat crystallization during cooling, (2) microbeam SR-XRD indicated that the lamellar planes of fat crystals near the water and oil interfaces are arranged almost parallel to the interface planes in both PGPR emulsion and PGPR+MB emulsion, and (3) adding MB resulted in the formation of tiny fat crystals because it promoted crystallization, which occurred both in the bulk oil phase and at the W/O interfaces. The present study is the first to apply microbeam SR-XRD to observe the microscopic features of fat crystallization in W/O emulsion, following fat crystallization in the oil droplets in the oil-in-water (O/W) emulsion (Arima, S.; Ueno, S.; Ogawa, A.; Sato, K. Langmuir 2009, 25, 9777− 9784).
1. INTRODUCTION Fat crystallization in dispersed systems is important because of inherent structural and functional implications with regard to size, orientation, network, and polymorphic behavior.1−5 Specifically in water-in-oil (W/O) emulsion, the formation of fat crystal networks prevents water droplet coalescence with crystallization-promoting emulsifiers at the water−oil interface and in the continuous oil phase.6,7 Previous studies indicate that the composition, polymorphism, solid fat content, and microstructure of triacylglycerols (TAGs) all play crucial roles in the structure of lipid-based products.1,8 Fat crystallization at the interface is critical in developing further understanding of the fundamentals of heterogeneous nucleation through hydrophobic and hydrophilic interactions.9 Focusing exclusively on the fat phase can address the complete issue of fat crystallization at the interface. Understanding interfacial stability requires balanced emulsifier technology.2,10 Surfactants having long-chain saturated fatty acid moieties have been found to be the most effective, due to their melting characteristics at the water−oil interface, as observed in O/W emulsion droplets.11,12 This enables them to act as templates to promote heterogeneous interfacial crystallization. © 2012 American Chemical Society
Practically, some emulsifiers can template with other surfactants, resulting in increased strength of the interfacial membrane between the water and oil phases so that the surfaces of the water droplets are either partially or wholly covered, forming monolayers for heterogeneous crystallization. This aspect has been analyzed in oil-in-water (O/W) emulsions,12 but has not been investigated in W/O emulsions (Figure 1). To accommodate recent consumer demands to replace trans fats with suitable alternatives and to reduce saturated fat content, it is necessary to analyze the crystallization properties of fats in relation to food emulsifiers, which are employed for emulsification and crystallization modification. In the production of low-fat spreads, it is common practice to use two emulsifiers (e.g., polyglycerol polyricinoleic acid (PGPR) and monoacylglycerol (MAG)).13−16 It is assumed that MAG may act as the templating agent, whereas PGPR assures emulsion stability. It has already been established that studying the Received: November 15, 2011 Revised: January 31, 2012 Published: February 16, 2012 5539
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types of synchrotron radiation X-ray diffraction (SR-XRD) using macrobeam and microbeam, DSC, and polarized optical microscopy. In particular, we employed small-angle SR-XRD using X-ray microbeam (SR-μ-SAXD), which enabled us to observe the microscopic features of fat crystals near the W/O interface. Microbeam X-ray diffraction analysis can provide microscopic information about crystallized materials on the order of micrometer to submicrometer dimensions.25−27 For lipid crystals, studies have been conducted on spherulites,28 O/W emulsion,11,12 granular crystals in fat spread,29 and binary mixture phases of lipids.30 The present work is the first to focus on the interfacial crystallization of fats in W/O emulsion, demonstrating the interactions between fats and food emulsifiers in the continuous oil phase and at the water and oil interface.
Figure 1. Interfacial crystallization at the water and oil interface in emulsion.
2. MATERIALS AND METHODS
behavior of saturated and unsaturated emulsifiers together with their polymorphism and time−temperature effects can provide information on their interactions in systems with and without a water phase.2,17−20 Similarly, studies on the kinetic stabilization of emulsions with low concentrations of continuous phase crystals indicate that polymorphic form and morphology can be manipulated in a multidisciplinary approach featuring dilution, tempering regimes, and the presence of PGPR under different crystallization conditions, here taken to mean the difference between static and dynamic crystallization.6,10,21 It was further suggested that systems such as PGPR and saturated MAGs are unlikely to influence the crystal lattice structure unless PGPR is coupled with agitation, which restricts or even retards polymorphic transformation. Recently, the effects of PGPR and MAG on the network and interfacial crystallization have been studied for 80% fat phase W/O emulsions.22,23 Results indicate that pickering crystals formed by a glycerol monostearate (GMS) may stabilize the dispersed aqueous phase via different mechanisms, compared to PGPR. At decreasing temperature, MAG forms surface-active crystals that are thought to be transported to the interface before crystallization in the surface zone with the transport of monomers of MAG from the bulk of the oil phase. This crystal can therefore be expected to form faster than nucleation and crystal growth in the oil. The effect of temperature on interfacial tension is more pronounced with long-chain MAGs than with short-chain MAGs.6,9,14 PGPR is highly surface active and has a large complex structure ((molecular weight (MW) range >4000 g/mol)), and MAG has a smaller molecular structure (MW 580 g/mol) than PGPR.22,24 Interactions of the two emulsifiers have an interesting effect on interfacial and bulk crystallization behavior. To our knowledge, the influence of PGPR and MAG containing behenic acid moiety (monobehenate (MB)) has not been analyzed to determine the effects of interfacial heterogeneous crystallization of low fat (35% oil phase) W/O emulsions. To clarify the effects of emulsifier additives on low-fat W/O emulsion (water/oil = 65%/35%, hereafter called 35% W/O emulsion), we tested two real emulsions, using two sets of emulsifiers. One emulsion used PGPR, a polyglycerol ester of polycondensed fatty acids from castor oil (PGPR 90-K); the other emulsion used PGPR and MB, which is a distilled saturated MAG based on fully hydrogenated high-erucic rapeseed oil (GRINDSTED CRYSTALLIZER 110-K) containing a large amount of behenic acid moiety. We applied two
Preparations of 35% W/O emulsions were assembled using ingredients described in Tables 1 and 2. Emulsifier additives were provided by Danisco/DuPont (Grindsted, Denmark).
Table 1. Sample Recipes for 35% W/O Emulsions
water (tap) salt water phase total fat blend: solid fat (interesterified: palm stearin/lauric kernel)/liquid oil (RBD rapeseed oil) = 1/3 other fat ingredients GRINDSTED PGPR 90-Ka GRINDSTED CRYSTALLIZER 110-Kb fat phase total a
GRINDSTED glycerol ester b GRINDSTED behenoyl (MB) (HEAR).
PGPR alone
PGPR + MB
64.0% 1.0% 65.0% 34.6%
64.0% 1.0% 65.0% 34.45%
0.4%
0.4% 0.15% 35.0%
35.0%
PGPR 90-K: polyglycerol polyricinoleate, a polyof polycondensed fatty acids from castor oil. CRYSTALLIZER 110-K: distilled saturated monobased on fully hydrogenated high-erucic rapeseed oil
2.1. Preparation of W/O Emulsions. The 35% W/O emulsion compositions were processed on a Gerstenberg-Schröder pilot plant:
Table 2. Properties of Fats Used for W/O Emulsions SFC %a 10 °C 20 °C 25 °C 30 °C 35 °C 40 °C slip melte °C iodine valuef saturated %g monounsaturated %g polyunsaturated %g trans %g
interesterified palm stearin/ lauricb
liquid rapeseed oilc,d
72−80 49−55 23−28 3−4 37−42 74 21 5 max 2
110−121 7 62 30 max 1
a
SFC = solid fat content (%), IUPAC 2.150a. b= Cargill GmbH., Hamburg, Germany. c= AarhusKarlshamn (AAK), Denmark. d= Cloudpoint (°C) −16, ASTM D97 SS-EN (23015)m. e= AOCS Cc 3−25. f= IUPAC 2.205. g= IUPAC 2.304. 5540
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3-tube pilot perfector 3 × 57 (serial no. 11803). Design pressure 80− 120 bar. Refrigerant: ammonia (NH3) from SPX Flow Technology Copenhagen A/S, Vibeholmsvej 22, Brøndby, Denmark. Specifications are as follows: Scraped surface heat exchanger (SSHE): diameter 57 mm length/430 mm, annular space 6.5 mm, volume 0.42 L, cooling surface area per tube 0.0567 m2, RPM (each tube) 200−1000, cooling (max) −25 °C, high-pressure three-piston pump (model P-3-15/35Labo; capacity 20−210 L/h, maximum pressure 160 bar), centrifugal pump CS 25 (serial no. 48258) with a 115 mm internal propeller, 0.55 KW Bi Polar motor 0.75 hp motor (2900 rpm), and AISI316 stainlesssteel heated pump housing (model T82). The 35% W/O emulsion was assembled as follows. In the water phase, the water was heated to 80 °C, and all dry ingredients were slowly added to the water while stirring intensively for 4 min. The water was then cooled to 40 °C. The oil/fat/emulsifier, beta carotene (2% solution), was heated to 80 °C, stirred, and then cooled to 40 °C. Table 3 lists the laboratory pilot-scale process conditions used for the 35% W/O emulsions.
Table 3. Details of Laboratory Pilot-Scale Process Conditions Used for 35% W/O Emulsions Gerstenberg and Agger A/S processing parameters for 3-tube lab scale perfector
35% W/O
oil phase temperature (°C) water phase temperature (°C) emulsion temperature (°C) centrifugal pump % capacity high-pressure pump (kg/h) cooling (NH3) tube 1 (°C) cooling (NH3) tube 2 (°C) SSHE (rpm) tube 1 SSHE (rpm) tube 2
40 40 40 20 to 30 25 −15 −15 1000 1000
Figure 2. Data analyses of SR-μ-SAXD patterns. (a) 2D SR-μ-SAXD pattern. (b) Lamellar direction in a fat crystal. (c) χ extension pattern. peaks from the crystals whose lamellar planes are aligned along the same direction at χ = 200° (arrow in Figure 2c). The degree of orientation of the lamellar planes of TAG crystals can be evaluated by calculating the half width of χ value (Δχ). A smaller Δχ yields a higher degree of orientation of the lamellar planes, as evidenced by the fat crystals in the O/W emulsion.12 The μ-SAXD measurement was performed at BL-4A of the Photon Factory, the synchrotron radiation facility of the High-Energy Accelerator Research Organization (KEK) in Tsukuba, Japan. The full details of the μ-SAXD method have been reported previously (Ueno et al. 2008). Briefly, the X-ray microbeam wavelength was 0.11 nm, and the beam area was 5 × 5 μm2. The emulsion sample was sealed in a 50 μm-thick cell made of mica covered with polyethylene terephthalate (PET) film, and set on a temperature-controlled stage. The sample was thermally treated using a Linkam furnace (Linkam, U.K.) as follows. First, the sample was kept at 60 °C for 5 min. The temperature was then reduced to 5 °C at a rate of 2 °C/min and kept at 5 °C during the μ-SAXD measurement. In principle, small-angle and wide-angle diffraction patterns can be simultaneously observed to obtain the lamellar distance and subcell structure, as we reported previously. However, it was necessary to decrease the distance between the sample and the 2D detector to 30 cm so that both the small- and wide-angle diffraction patterns could be imaged on a 2D detector with a 6 inch × 6 inch area. With the microbeam position fixed, the measured sample was moved by an x−y−z stepping motor for observation by an optical microscope (magnification ×200). The sample was moved automatically within a 2D plane in 5 μm steps. 2.4. DSC Measurements. DSC analyses were performed using a differential scanning calorimeter model XRD-DSCII (Rigaku, Tokyo, Japan). First, 10 mg of the sample was weighed in an aluminum pan. Before analysis, the sample was heated to 60 °C at a rate of 10 °C/min and held under isothermal conditions for 5 min to erase its previous thermal history. The sample was then cooled to 0 °C at a rate of 2 °C/ min and heated again to 60 °C to obtain the cooling and heating thermograms. All experiments were performed under a nitrogen flow of 50 mL/min, and an empty aluminum pan was used as a reference in all runs. Duplicated experiments were conducted for each emulsion, and the same results were obtained. 2.5. Polarized Optical Microscopic Observation. Water droplet distribution and fat crystal morphology were observed using a CX31-P POM (Olympus Co., Tokyo, Japan) with a DP 12 digital camera (Olympus). The POM was set under the crossed Nicols condition. Samples were set in a Linkam (TU-600PM, Cambridge,
2.2. Macrobeam X-ray Diffraction (XRD) Measurements. In order to observe the macroscopic crystallization behavior of fats in the emulsion, synchrotron radiation (SR)-XRD experiments with a normal macrobeam (beam width 0.5 × 0.5 mm2) were carried out at two different beamlines (BL-15A) of the SR source Photon Factory (PF) in the National Laboratory for High-Energy Physics (Tsukuba, Japan). For both beamlines, a double-focusing camera was operated at a wavelength of 0.15 nm. Two different detectors were used: a CCD camera for small-angle data and a PSPC for wide-angle data. SAXD and WAXD measurements were performed simultaneously. The SAXD pattern was used to determine the chain length structure of the TAG, and the WAXD pattern enabled us to identify the polymorphic forms. The temperature program applied to the sample was controlled by a Mettler DSC-FP84 (Mettler Instrument Corp., Greifensee, Switzerland) with FP99 software. A 2 mm-thick sample was placed in a stainless-steel sample cell with Kapton film windows. 2.3. Microbeam Small-Angle X-ray Diffraction Measurements. The basic principle of SR-μ-SAXD has been reported elsewhere.27 Briefly, we can construct 2D images of a micrometerdimension in real space by scanning the X-ray microbeam on a thin section of the sample in two dimensions with steps on the order of the beam size, and by collecting each two-dimensional (2D) XRD pattern with a 2D X-ray sensitive area detector (Figure 2). The polymorphic structure can be assessed by measuring the long spacing value, which is calculated by the diffraction angle (2θ) extension (Figure 2a). In addition, the lamellar plane direction (Figure 2b) of the fat crystal can be assessed by measuring the azimuthal angle (χ) extension pattern at a fixed 2θ position (Figure 2c). When all the fat crystals are highly oriented, two sharp 2D diffraction peaks (arc peaks) should appear because of the preferred orientation of crystals. In this case, the average direction of the lamellar planes of the fat crystals is directed normal to the direction connecting the two arc peaks. For example, in Figure 2c, sharp arc peaks appear at χ = 110° and χ = 290°, which are superimposed by a 180° rotation. We refer to this set of two peaks as twin peaks. These twin peaks correspond to the symmetric diffraction 5541
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U.K.) furnace set to a temperature of 5 °C and placed on the sample stage of the POM.
promoted the crystallization of solid fat in the 35% W/O emulsion. The enthalpy values of crystallization were calculated as 1.32 J/g for the PGPR emulsion and 3.51 J/g for the PGPR + MB emulsion. As the enthalpy value of the PGPR + MB emulsion is larger than that of the PGPR emulsion, we may conclude that the extent of crystallization was increased by the addition of MB. 3.2. Macrobeam SR-XRD Measurements. The melting and crystallization of the PGPR emulsion were examined by macrobeam SR-XRD (Figure 4). At 5 °C, the SAXD and WAXD patterns exhibited a long spacing peak of 4.07 nm (Figure 4a), a strong peak of 0.462 nm, and a weak peak of 0.386 nm (Figure 4b). The WAXD peaks correspond to β polymorph, which represents T∥ subcell structure. The reason for the presence of β form in the emulsion sample is that the sample was stored over a long period and the most stable form of the fat crystals in the emulsion was formed. When the emulsion was heated, both SAXD and WAXD patterns disappeared at 40 °C, due to the melting of the β form. During cooling, crystallization was detected with the occurrence of the SAXD peak of 4.65 nm at 14 °C, corresponding to the exothermic peak of crystallization observed in the DSC cooling pattern (Figure 3a). The long spacing of 4.65 nm means that the polymorphic form of these crystals is α form, as the chain axis in the TAG crystals is normal to the lamellar interface.1 In addition, the α form tends to crystallize faster than the other more stable forms during the ambient rate of cooling. Despite the strong SAXD peak, no strong peaks were detectable in the WAXD area during cooling (Figure 4b). This may be due to the weak diffraction of the hexagonal subcell packing of α form in the W/O emulsion. Figure 5 depicts the melting and crystallization of the PGPR + MB emulsion examined by macrobeam SR-XRD. Similar to the PGPR emulsion, the SAXD patterns confirmed the presence of the β form at 5 °C, as evidenced by a long spacing peak of 4.07 nm (Figure 5a), and the WAXD patterns of strong
3. RESULTS AND DISCUSSION 3.1. DSC Thermograms. Figure 3 depicts the DSC cooling thermopeaks of two emulsions using PGPR alone and PGPR +
Figure 3. DSC cooling thermopeaks taken for (a) PGPR emulsion and (b) PGPR + MB emulsion.
MB. Although not shown here, the DSC heating thermograms did not indicate any differences between the two emulsion samples, whereas the cooling thermopeaks were remarkably different. The effects of the emulsifiers are revealed in the kinetic processes of fat crystallization in the W/O emulsion. Crystallization occurred at 16.1 °C in the PGPR emulsion, whereas the crystallization temperature increased to 17.9 °C in the PGPR + MB emulsion. Also, the exothermic peak was split from single (PGPR emulsion) to double (18 and 10 °C in the PGPR + MB emulsion). This splitting may have been caused by the promotion of the crystallization of a high-melting fraction of solid fat by the addition of MB. This result indicates that MB
Figure 4. Macrobeam SR-XRD patterns of PGPR emulsion taken during heating and cooling. Unit: nm. Experiment noise is denoted by arrows. 5542
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Figure 5. Macrobeam SR-XRD patterns of PGPR + MB emulsion taken during heating and cooling. Unit: nm. Experiment noise is denoted by arrows.
peaks of 0.462 nm and weak peaks of 0.386 nm (Figure 5b). No strong peak was detectable in the WAXD area when the strong SAXD peak of 4.65 nm appeared. However, a weak WAXD peak of 0.383 nm appeared when the SAXD peak of 4.15 nm appeared below 10 °C. We assume that this form is a lowtemperature sub-α form.31 It was evident that the macrobeam XD patterns taken during cooling from 55 °C exhibited two-stage crystallization, as the first peak (4.65 nm) appeared at 16 °C and the second peak (4.15 nm) appeared at 10 °C. This two-stage crystallization observed in the SR-XRD experiment also corresponds to split DSC exothermic peaks taken during cooling (Figure 3b). Although the temperatures of the occurrence of the two crystals differ by 2 °C, the increase in crystallization temperature from 14 °C (PGPR emulsion) to 18 °C (PGPR + MB emulsion) is caused by promotion of the crystallization of the high-melting fraction of solid fat with the addition of MB. 3.3. μ-SAXD Measurements-1: Azimuthal Angle (χ) Extension Patterns. PGPR emulsion. Figure 6 depicts 2D μ-SAXD patterns taken at 131 positions near a water droplet in the PGPR emulsion. By observing the emulsion sample during the μ-SAXD experiment in which an optical microscope was attached, we could roughly determine the position of the water droplet. In addition, the precise position of the interface line between the water and oil phases was drawn by carefully detecting the μ-SAXD peaks at all the positions examined. Thus, the ellipsoid-shaped area of the water phase is denoted by the dotted line in Figure 6. We focused on the detailed structures of the μ-SAXD patterns taken in the oil phase very close to the W/O interface. For this purpose, we precisely analyzed the μ-SAXD patterns at positions 1−14 around the water droplet (Figure 6). In addition, the μ-SAXD pattern at position A, far from the W/O interface in the oil phase, was analyzed. To compare the directions of the W/O interface at different positions with the directions of the lamellar planes of the crystals, which are determined by analysis of the azimuthal angle extension of the
Figure 6. μ-SAXD patterns at different positions near a water droplet in PGPR emulsion. Water−oil interface is denoted by the dotted line.
2D μ-SAXD patterns, the starting direction of the azimuthal angle (χ = 0°) is denoted by the arrow in the inserted box in Figure 6. The d-spacing values of fat crystals at the positions of 14 and A in Figure 6 were determined by observing diffraction angle 2θ- (q-)extension patterns as shown later. The details of the μ-SAXD patterns taken at positions 14 and A are depicted as three-dimensional (3D) images and azimuthal extension patterns in Figure 7. Positions 14 and A were chosen because they reveal the most typical μ-SAXD patterns obtained from the fat crystals near the W/O interface and the bulk oil phase. In every case, the weakest X-ray scattering intensity did not reach zero, indicating that a certain noise level of X-ray scattering could not be avoided. However, clear differences in 5543
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Figure 7. (a) 3D μ-SRXD patterns and (b) azimuthal angle (χ) extension patterns of PGPR emulsion taken at positions 14 and A in Figure 6.
the diffraction intensity distribution patterns were detectable between the two positions. At position 14, two sharp arc peaks appeared in the 3D images. Correspondingly, two peaks appeared at χ ∼ 168° and χ ∼ 348°, which are superimposed by a 180° rotation, although the peak at χ ∼ 348° is split into two, due to scattering noise. The direction of the lamellar planes of the fat crystals at position 14 is along the center position of the twin peaks at χ = 78° (which is equivalent to 258°, not shown), denoted by the arrow in Figure 7. It is evident that this χ value is almost the same as that of the direction (χ = 75°) parallel to the W/O interface plane near position 14 in Figure 6. The sharp peak indicates that the lamellar planes of the fat crystals in this area are highly ordered. The value of Δχ of position 14 was calculated as ∼28° according to the peak at χ ∼ 168°. By contrast, the pattern at position A has no sharp peak, indicating that the lamellar planes of the fat crystals in the microbeam area at this position are directed randomly. The same results were obtained at the other positions far from the W/O interface. Figure 8 illustrates the χ extension patterns and the directions of the lamellar planes of the crystals, which were determined by analyzing the 2D μ-SAXD patterns taken at the 14 positions around the WO interface noted in Figure 6. Clear twin peaks are confirmed at positions 1−4, 8−12, and 14, among which all positions except position 4 revealed that the values of the χ directions indicating the lamellar directions of the crystals are more or less parallel to the directions of the W/ O interfaces (noted in parentheses at every position). Although not fully understood, no clear twin peak is observed at positions 5, 6, and 13, and four peaks are observed at position 7. At position 4, the lamellar direction makes an angle of 70°. Despite these exceptions, we discovered that the lamellar directions of the fat crystals near the W/O interface tend to be parallel to the W/O interface plane. Thus, we can conclude that the fat crystals near the W/O interface in the W/O emulsion using PGPR alone as the emulsifying reagent reveal that the lamellar planes of the fat crystals are almost parallel to the interface plane. Considering that such a tendency was not
Figure 8. χ extension patterns at positions 1−14 in Figure 6. The χ value at the W/O interface near every position is indicated in parentheses after the position number.
observed at the positions in the bulk oil phase, we may assume that the PGPR membrane at the W/O interface may induce the interfacial heterogeneous crystallization illustrated in Figure 1. For reconfirmation, complementary data of the PGPR emulsion are presented in the Supporting Information (Figure S1). PGPR + MB Emulsion. We performed the same microbeam X-ray diffraction experiments for the PGPR + MB emulsion as for the PGPR emulsion. The two emulsions had basically common results in terms of lamellar directions of the fat crystals at the positions near the W/O interface, which were confirmed by analysis of the χ extension patterns at various positions surrounding the water droplets. However, the intensity of the diffracted X-ray beams was less in the PGPR + MB emulsion than in the PGPR emulsion, probably because the addition of MB caused the formation of tiny fat crystals (see POM micrographs below). This effect made analysis of the 5544
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results of μ-SAXD data from the PGPR + MB emulsion rather difficult, particularly for the Δχ values. Figure 9 plots 2D μ-SAXD patterns taken at 89 positions near a water droplet in the PGPR + MB emulsion. Similar to
The d-spacing values of fat crystals at the positions of 3 and B in Figure 9 were determined by observing diffraction angle 2θ(q-)extension patterns as shown later. Figure 10 presents the details of three-dimensional (3D) images of the μ-SAXD patterns and the χ extension patterns taken at positions 3 and B in Figure 9. These two selected positions reveal the most typical μ-SAXD patterns obtained from the fat crystals near the W/O interface and the bulk oil phase. In both cases, the weakest X-ray scattering intensity did not reach zero because of the noise level of X-ray scattering. However, two sharp peaks appeared in the 3D images at position 3 (Figure 10a), and two corresponding peaks appeared at χ ∼ 70° and χ ∼ 250° in the χ extension pattern (Figure 10b). From the twin peaks, we can determine the lamellar planes of the crystals at χ ∼ 160° (arrow in Figure 10b), which is parallel to the W/O interface plane near position 3 in Figure 9. The values of Δχ of position 3 are 15° according to the peak at χ ∼ 70° and 20° according to the peak at χ ∼ 250°. Both these values are smaller than Δχ = 25° taken at position 14 near the W/O interface in the PGPR emulsion in Figure 8. The sharper peaks indicate that the lamellar planes of the fat crystals at position 3 in the PGPR + MB emulsion are more highly ordered than those at position 14 in the PGPR emulsion. However, such a comparison of Δχ values could not be made for the other positions of the PGPR + MB emulsion because of their rather broad twin peaks. Therefore, we cannot draw any conclusion about the preferred orientation of the lamellar planes of the fat crystals near the W/O interface in the PGPR + MB emulsion, in comparison with those in the PGPR emulsion. The μ-SAXD pattern at position B in Figure 9 did not exhibit any sharp twin peaks, indicating that the lamellar planes of the fat crystals in the microbeam area at this position are directed randomly. The same results were obtained at the other positions far from the W/O interface in Figure 9. Figure 11 depicts the χ extension patterns and the directions of the lamellar planes of the crystals at the 14 positions in Figure 9, which were determined according to those in Figure 8. Clear twin peaks are confirmed at positions 1, 2, 6−8, and 14. It is evident that that the values of the χ directions indicating
Figure 9. μ-SAXD patterns at different positions near a water droplet in PGPR + MB emulsion. Water−oil interface is denoted by the dotted line.
Figure 6, we could determine the position of the water droplet by carefully detecting the μ-SAXD peaks at all positions examined. The nearly circular water phase is denoted by a dotted line in Figure 9. The μ-SAXD patterns at positions 1−14 near the WO interface around the water droplet were precisely analyzed. In addition, the μ-SAXD pattern at position B far from the W/O interface in the oil phase was analyzed. The starting direction of the azimuthal angle (χ = 0°) is also denoted by the arrow in the inserted box in Figure 9.
Figure 10. (a) 3D μ-SRXD patterns and (b) χ extension patterns of PGPR + MB emulsion taken at positions 3 and B in Figure 9. 5545
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presented in Figures 7 and 8. Regardless of whether the positions are close to, or far from the W/O interface, sharp peaks having long spacings of 4.06−4.08 nm were observed. These values correspond to β form within experimental error (±0.02 nm), as noted in the macrobeam SR-XRD measurements (Figures 4 and 5). 3.5. Polarized Optical Microscopic Observation. We observed the crystal morphology and dimensions of the fat crystals in the PGPR emulsion and PGPR + MB emulsion with a crossed Nicols polarized optical microscope (Figure 13). Clear differences were detectable between the crystal morphologies of the two emulsions. Figure 13a depicts circular water droplets with diameters 30− 60 μm, and fat crystals in the oil phases between the water droplets in the PGPR emulsion. Bright images in the oil phase correspond to fat crystals, and large aggregates with dimensions of several micrometers are visible (typical ones are denoted by arrows). Figure 13b depicts circular water droplets with diameters 30−60 μm in the PGPR + MB emulsion, similar to the PGPR emulsion. However, the crystal morphology is different in that no large crystal aggregate is observed in the oil phase in the PGPR + MB emulsion. Instead, thin layers of bright images are attached to the water phases (arrows). This morphological change can be explained by the promotion of fat crystallization with the addition of MB, which was also observed by macrobeam SR-XRD (Figure 5). The addition of MB increased the crystallization temperature, as confirmed by the cooling experiments of SR-XRD and by the reduction in crystal size due to increased rates of crystal nucleation. To summarize, the present study revealed the following results. First, the additive of MB promoted fat crystallization as observed by DSC and macrobeam SR-XRD analysis. Second, microbeam SR-XRD indicated that the lamellar planes of fat crystals near the water and oil interfaces are arranged almost parallel to the interface planes in both the PGPR emulsion and the PGPR + MB emulsion, and no appreciable differences between them were confirmed. Third, POM observation indicated that adding MB eliminated the formation of large crystal aggregates, resulting in the formation of tiny fat crystals. The μ-SAXD experiments about the interfacial crystallization which occurred with PGPR alone as well as with PGPR + MB are similar to those for fats in the oil droplets in O/W emulsion caused by the addition of high-melting sucrose emulsifiers examined with μ-SAXD.12 Although PGPR contains large amounts of liquidus fatty acid moieties at the temperatures examined in the present μ-SAXD experiments, it can be assumed that certain molecular interactions between PGPR and fat molecules may be operating at the W/O interface to cause interfacial crystallization to some extent, and the addition of MB may strengthen this interfacial crystallization. Such interactions may include not only hydrophobic chain−chain interactions between the fatty acid moieties of the emulsifiers and fat molecules, but also hydrophilic interactions between the polar groups of the emulsifiers and glycerol groups of fat molecules, as recently discussed by Ghosh and Rousseau.7 Regarding the effects of adding MB on intensifying the interfacial crystallization illustrated in Figure 1, which we expected to observe at the beginning of the present study, no significant evidence was observed, except for the promotion of fat crystallization in the continuous oil phase and close to the water phase perimeter. One reason may be low adsorption of MB at the W/O interface in cooperation with PGPR membranes, whose adsorbability exceeds that of MB.
Figure 11. χ extension patterns at positions 1−14 in Figure 9. The χ value at the W/O interface near every position is indicated in parentheses after the position number.
the lamellar directions of the crystals are almost parallel to the directions of the W/O interfaces. At the other positions, no clear twin peaks were obtained because of broadening of the peaks. However, we note two facts: (1) the six positions at the W/O interface confirmed good agreement between the χ values of the lamellar planes of the fat crystals and the W/O interface direction, and (2) such a tendency was not observed at the positions in the bulk oil phase. Thus, we assume that the PGPR and MB membrane at the W/O interface may induce the interfacial heterogeneous crystallization illustrated in Figure 1. For reconfirmation, complementary data of the PGPR + MB emulsion are presented in the Supporting Information (Figure S2). 3.4. μ-SAXD Measurements-2: Diffraction Angle Extension Patterns. The polymorphic structures of the fat crystals examined by μ-SAXD measurement can be determined by observing the diffraction peaks in the diffraction angle 2θ (q) extension patterns. Figure 12 depicts the diffraction angle extension patterns taken at the positions in the PGPR emulsion and the PGPR + MB emulsion whose χ extension patterns are
Figure 12. 2θ extension patterns of μ-SAXD in (a) PGPR emulsion (two positions in Figure 6) and (b) PGPR + MB emulsion (two positions in Figure 9). Unit: nm. 5546
dx.doi.org/10.1021/la204501t | Langmuir 2012, 28, 5539−5547
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Figure 13. Polarized optical micrographs of (a) PGPR emulsion and (b) PGPR + MB emulsion. Scale bar: 25 μm. and Dairy Analogs. Office of Premarket Approval (HFS-200); Center for Food Safety and Applied Nutrition, Food and Drug Administration: Washington, DC, 2005. (14) Garti, N.; Binyamin, H.; Aserin, A. J. Am. Oil Chem. Soc. 1998, 75, 1825−1831. (15) Hancock, H. I. Macromolecular Surfactants; Academic Press: London, 1984; pp 287−321. Hodge, S. M.; Rousseau, D. J. Am. Oil Chem. Soc. 2005, 82, 159. (16) Wilson, R.; Van Schie, B. J.; Howes, D. Food Chem. Toxicol. 1998, 36, 711. (17) Basso, R. C.; Ribeiro, A. P. B.; Masuchi, M. H.; Gioielli, L. A.; Gonçalves, L. G.; Santos, A. O.; Cardoso, L. P.; Grimaldi, R. Food Chem. 2010, 122, 1185. (18) Brubach, J. B.; Jannin, V.; Mahler, B.; Bourgaux, C.; Lessieur, P.; Roya, P.; Ollivon, M. Int. J. Pharm. 2007, 336, 248. (19) Vereecken, J.; Meeussen, W.; Foubert, I.; Lesaffer, A.; Wouters, J.; Dewettinck, K. Food Res. Int. 2009, 42, 1415. (20) Vereecken, J.; Meeussen, W.; Lesaffer, A.; Dewettinck, K. Food Res. Int. 2010, 43, 872. (21) Young, N. W. G.; Wassell, P.; Wiklund, J.; Stading, M. Int. J. Food Sci. Technol. 2008, 43, 2083. (22) Ghosh, S.; Rousseau, D. J. Colloid Interface Sci. 2009, 339, 91. (23) Ghosh, S.; Tran, T.; Rousseau, D. Langmuir 2011, 27, 6589. (24) Dedinaite, A.; Campbell, B. Langmuir 2000, 8, 2248−2253. (25) Riekel, C.; Burghammer, M.; Davies, R.; Gebhardt, R.; Popov, D. Fundaments of Soft Condensed Matter Scattering and Diffraction with Microfocus Techniques. In Applications of Synchrotron Light to Scattering and Diffraction in Materials; Ezquerra, T. A., GarciaGutierrez, M., Nogales, A., Gomez, M., Eds.; Springer: Heidelberg, 2009; Vol. 776, pp 91−104. (26) Riekel, C. Rep. Prog. Phys. 2000, 63, 233. (27) Riekel, C.; Burghammer, M.; Müller, M. J. Appl. Crystallogr. 2000, 33, 421. (28) Ueno, S.; Nishida, T.; Sato, K. Cryst. Growth Des. 2008, 8, 751. (29) Tanaka, L.; Tanaka, K.; Yamoto, S.; Ueno, S.; Sato, K. Food Biophys. 2009, 4, 331. (30) Bayes-Garcia, L.; Calvet, T.; Cueva-Diarte, M. A.; Ueno, S.; Sato, K. Cryst. Eng. Commun. 2011, 13, 6694. (31) Yano, J.; Sato, K.; Kaneko, F.; Small, D. M.; Kodali, D. M. J. Lipid Res. 1999, 40, 140.
Complementary studies using, for example, interfacial rheology techniques must be applied to further clarify these properties.
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ASSOCIATED CONTENT
S Supporting Information *
Additional figures and details about the processing software. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS The experiments were performed with the approval of the Photon Factory Program Advisory Committee (Proposals. 2010G114 and 2010G115). The authors gratefully acknowledge the help of Professor A. Iida, Station Manager of Beamline 4A at the Photon Factory (KEK Institute, Tsukuba, Japan). The authors also appreciate the experimental support provided by Dr. Y. Shinohara and Professor Y. Amemiya (University of Tokyo).
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REFERENCES
(1) Sato, K.; Ueno, S. Curr. Opin. Colloid Interface Sci. 2011, 16, 384. (2) Wassell, P.; Bonwick, G.; Smith, C. J.; Almiron-Roig, E.; Young, N. W. G. Int. J. Food Sci. Technol. 2010, 45, 642. (3) Rogers, M. A. Food Res. Int. 2009, 42, 747. (4) Pernetti, M.; van Malssen, K. F.; Flöter, E.; Bot, A. Curr. Opin. Colloid Interface Sci. 2007, 12, 221. (5) Wassell, P.; Young, N. W. G. Int. J. Food Sci. Technol. 2007, 42, 503. (6) Rousseau, D. Food Res. Int. 2000, 33, 3. (7) Ghosh, S.; Rousseau, D. Curr. Opin. Colloid Interface Sci. 2011, 16, 421. (8) Narine, S. S.; Marangoni, A. G. Food Res. Int. 1999, 32, 227. (9) Krog, N.; Larsson, K. Fat Sci. Technol. 1992, 94, 55. (10) Wassell, P.; Wiklund, J.; Stading, M.; Bonwick, G.; Smith, C.; Almiron-Roig, E.; Young, N. W. G. Int. J. Food Sci. Technol. 2010, 45, 877. (11) Shinohara, Y.; Takamizawa, T.; Ueno, S.; Sato, K.; Kobayashi, I.; Nakajima, M.; Amemiya, Y. Cryst. Growth Des. 2008, 8, 3123. (12) Arima, S.; Ueno, S.; Ogawa, A.; Sato, K. Langmuir 2009, 25, 9777. (13) Byers, B. GRAS NotificationPGPR Polyglycerol polyricinoleic acid for Use in Margarines, Low Fat Margarines, Spreads, Creamers 5547
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