Article Cite This: Cryst. Growth Des. XXXX, XXX, XXX-XXX
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Epitaxial Growth of Fat Crystals on Emulsifier Crystals with Different Fatty Acid Moieties Chinami Ishibashi, Hironori Hondoh,* and Satoru Ueno Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan S Supporting Information *
ABSTRACT: We performed in situ observations of the crystallization and melting behaviors of palm mid fraction (PMF) on sorbitan ester (SE) crystals comprising different fatty acid moieties (sorbitan tripalmitate (STP), sorbitan tristearate (STS), or sorbitan tribehenate (STB)) to reveal the epitaxial relationship between fat crystals and emulsifier crystals. The effects of SEs on the thermal behavior and polymorphism of PMF were investigated by differential scanning calorimetry and synchrotron radiation X-ray diffraction. These measurements showed that the addition of STP and STS greatly increased the crystallization temperature of PMF, which was caused by the crystallized SEs that were nucleated prior to PMF. Microscopic observations demonstrated that PMF crystals were oriented in the same direction as crystals of STP and STS because the chain lengths of the fatty acid moieties were similar to those of PMF. In contrast, when STB was used as a substrate, PMF crystals were not oriented in the same direction as STB crystals because there is a large difference between the chain lengths of STB and PMF. These results revealed that crystal growth of PMF on STP and STS crystals occurs via epitaxial growth, whereas PMF crystallization on STB crystals occurs via general heterogeneous nucleation.
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that the template films of monoacylglycerols also exhibited subcell matching and preservation of molecular orientation to n-alcohol crystals when the monoacylglycerol and n-alcohol had similar chain lengths. Emulsifiers, which have both polar and nonpolar moieties in one molecule, are commonly used as additives for fat crystallization. The addition of emulsifiers can modify the nucleation step by either promoting or retarding fat crystallization, depending on the concentration of the emulsifier, the cooling rate, and the similarity of the fatty acid compositions of the fat and emulsifier.7−12 Crystallization of fat is promoted through heterogeneous nucleation or the template effect on the surface of emulsifier crystals; such crystals act as a seeding material and as catalytic impurities for heterogeneous nucleation.11−13 Sonwai et al.11 reported that solid-state sorbitan esters accelerated early-stage coconut oil crystallization via promotion of heterogeneous nucleation, while liquid-state sorbitan esters suppressed coconut oil crystallization. Fredrick et al.12 reported that monoacylglycerols of hydrogenated palm
INTRODUCTION Fats and lipids are widely used in many industrial fields, including foods, cosmetics, and pharmaceuticals, and are crystallized to add desirable physicochemical properties to products.1,2 Solid fat crystals form a higher-order structure in products to control their hardness, texture, and stability. By control of the nucleation of fat crystals, the position, size, and morphology of crystals can be manipulated, and consequently, the physical properties of products can be controlled. Therefore, controlling the nucleation step is key to controlling the desired product characteristics. The crystallization of fat is accelerated by using additives as “templates” for the nucleation of fat crystals; this is known as the template effect.3 The template effect is more effective at promoting fat crystallization than general heterogeneous nucleation.4 Previous studies have emphasized that the template effect works well when the substrate and lipid have similar molecular shapes. Takiguchi et al.5 showed that when the number of carbons in the fatty acids of vapor-deposited thin films (template films) was equal to or two greater than that of the n-alcohol, nucleation of n-alcohol crystals was accelerated by the template films. Moreover, the molecular orientation and subcell structure of n-alcohol crystals matched those of the template films. Fujiwara et al.6 indicated © XXXX American Chemical Society
Received: July 26, 2017 Revised: October 10, 2017 Published: October 30, 2017 A
DOI: 10.1021/acs.cgd.7b01039 Cryst. Growth Des. XXXX, XXX, XXX−XXX
Crystal Growth & Design
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Figure 1. Molecular structures of the principal components of (a) PMF (1,3-dipalmitoyl-2-oleoyl-sn-glycerol) and (b) sorbitan tripalmitate.
crystals on emulsifier crystals. In this study, sorbitan esters (SEs), which have a larger hydrophilic group than monoacylglycerols, were used to evaluate the effect of the molecular structure of the emulsifier on the template effect. For this experiment, we prepared emulsifier crystals as seed crystals prior to fat crystal nucleation to distinguish between emulsifier crystals and fat crystals. The morphologies and orientations of fat crystals and emulsifier crystals were observed by a differential interference contrast (DIC) microscope and a polarized light (PL) microscope with a sensitive tint plate. In addition, the effects of emulsifiers with different fatty acid moieties on polymorphic behavior were investigated by synchrotron radiation X-ray diffraction (SR-XRD).
oil promoted the nucleation of palm oil crystals because the monoacylglycerols supplied a larger number of nucleation sites. By using synchrotron radiation microbeam X-ray diffraction, Verstringe et al.13 demonstrated that the lamellar plane of palm oil crystals, which is composed mainly of palmitic acid, oriented parallel to that of crystallized monopalmitin. Monopalmitin has a relatively smaller polar functional group, and its molecular structure is quite similar to that of the triacylglycerol (TAG). However, these studies did not distinguish clearly between the template effect and heterogeneous nucleation. When the molecular shapes of the TAG and template are similar, crystallized emulsifiers will preserve the lamellar orientation of TAG crystals and their subcell packing; the template effect would then be considered as epitaxial growth. The necessary conditions for the template effect can be summarized as follows:2,3,8 similarity in molecular shape (saturated or unsaturated fatty acids or the chain lengths between the template and fat), similar subcell packing, high thermal stability, and optimal supercooling. However, it is unknown whether the crystal growth of fat proceeds via the template effect or by general heterogeneous nucleation. Therefore, it is important to better understand the template effect on a molecular level. The purpose of the present study was to examine the effects of emulsifiers with different fatty acid moieties on the crystal growth and polymorphic behavior of fats; this should elucidate the molecular interaction between the TAG and the emulsifier molecules. Thus, we performed in situ observations of fat crystal morphologies on emulsifiers and the orientations of fat
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EXPERIMENTAL SECTION
Materials. Palm mid fraction (PMF) was obtained from Fuji Oil Co., Ltd. (Osaka, Japan). More than 50% of the fatty acid composition of PMF comprises palmitic acid, and the major TAG present in PMF is 1,3-dipalmitoyl-2-oleoyl-sn-glycerol (POP).14 Canola oil (Nisshin OilliO Group, Ltd., Tokyo, Japan) was purchased from a local supermarket. Sorbitan tripalmitate (STP), sorbitan tristearate (STS), and sorbitan tribehenate (STB) were provided by Riken Vitamin Co., Ltd. (Tokyo, Japan). The molecular structures of the principal components of PMF and STP are shown in Figure 1. The onset melting temperatures of SEs measured by differential scanning calorimetry (DSC) were 37.1 ± 0.2 °C (STP), 47.4 ± 0.1 °C (STS), and 62.6 ± 0.2 °C (STB). The lamellar and lateral hydrocarbon chain distances are designated as the long and short spacings, respectively. The long and short spacings of SEs, as observed B
DOI: 10.1021/acs.cgd.7b01039 Cryst. Growth Des. XXXX, XXX, XXX−XXX
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by SR-XRD at room temperature, were 4.82 and 0.417 nm (STP), 5.34 and 0.414 nm (STS), and 6.35 and 0.413 nm (STB), respectively, indicating that the SEs have the α form with hexagonal subcell packing. These experimental data are presented in the Supporting Information (Figures S1 and S2). Sample Preparation. PMF was mixed with 5 wt % SE and melted completely on a hot plate; this mixture was stirred manually to obtain a homogeneous solution. In this study, PMF solutions with high SE concentrations were used to detect the crystallization and melting behavior of SEs in DSC and SR-XRD measurements. Canola oil mixed with 20 wt % SE was prepared in the same way and used for microscopic observations. Differential Scanning Calorimetry. The thermal behavior of PMF and PMF−SE blends was investigated using DSC (Thermo Plus 8240, Rigaku, Japan). Samples (18 mg) were sealed in an aluminum pan, and the following temperature programs were applied: holding at 60 °C (PMF and PMF−STP blend), 70 °C (PMF−STS blend), or 100 °C (PMF−STB blend) for 10 min, followed by cooling to −10 °C at 2 °C/min, and then heating to 60 °C at 2 °C/min. Al2O3 was employed as the reference material. Three replicates were conducted for each sample. Synchrotron Radiation X-ray Diffraction. SR-XRD measurements were carried out at beamline 6A of the synchrotron radiation facility (Photon Factory) of the High Energy Accelerator Research Organization (KEK) in Tsukuba, Japan. The X-ray wavelength was 0.15 nm, and small-angle X-ray scattering (SAXS) and wide-angle Xray scattering (WAXS) patterns were measured simultaneously using PILATUS 1M and PILATUS 100K detectors (Pilatus Aircraft Ltd., Stans, Switzerland), respectively. The 2θ calibration was performed using silver behenate for SAXS and tripalmitin for WAXS. SAXS and WAXS patterns were taken every 30 s with a 20 s exposure time. A square aluminum cell with a thickness of 1 mm and a 3.5 mm × 3.5 mm window was employed as a sample holder. A liquid sample was injected into the cell, and the window was sealed with Kapton film. The samples were kept above the melting temperatures based on the DSC measurements (PMF and PMF−STP blend, 60 °C; PMF−STS blend, 70 °C; PMF−STB blend, 80 °C) for 10 min and then cooled to 5 °C at 2 °C/min and held for 10 min, followed by heating to 40 °C at 2 °C/min. The temperature of the samples was controlled using a Linkam controller (LK-600PM, Linkam Scientific Instruments, Ltd., Tadworth, U.K.). Optical Microscopy. PL microscopy and DIC microscopy were carried out to observe the orientations of fat and emulsifier crystals (BX51, Olympus, Tokyo, Japan). A sensitive tint plate was also used with PL microscopy. The temperature of the samples was controlled by a Linkam stage (model 10021, Linkam Scientific Instruments). A small amount of molten SE−canola oil blend was sandwiched between two coverslips with a 60 μm thick spacer. After the sample was melted completely at 60 °C (STP−canola blend), 70 °C (STS−canola blend), or 100 °C (STB−canola blend) for 10 min, it was cooled to 30 °C at 0.1 °C/min to grow SE crystals. Subsequently, the upper cover glass was removed, and then 3 μL of molten PMF was added to the SE crystals of the lower cover glass at 30 °C. The mixture of molten PMF and SE crystal was covered with a new coverslip. In situ observation of PMF crystallization and melting behavior was performed during the following temperature program: cooling to 5 °C at 2 °C/min, holding at 5 °C for 10 min, and then heating to 30 °C at 2 °C/min.
Figure 2. DSC thermograms of PMF and PMF mixed with 5 wt % SEs during cooling from 60 to −10 °C at 2 °C/min: (a) PMF, (b) PMF with STP, (c) PMF with STS, and (d) PMF with STB. Arrows indicate the first crystallization of PMF−SE blends.
fractions of the PMF−SE blends did not differ significantly from those of PMF. However, the crystallization temperature of the high-melting fraction of PMF with STP was around 30 °C (onset temperature; denoted by an arrow), which was clearly higher than that of PMF. The STS and STB blends exhibited different thermograms compared with PMF and demonstrated two-step crystallization. The first crystallizations started around 39 °C for STS and 55 °C for STB (denoted by arrows); the subsequent crystallizations occurred around 20 °C for STS and 17 °C for STB. The first exothermic change was presumably caused by the crystallization of SEs as described later. Figure 3 illustrates the SAXS and WAXS patterns from the SR-XRD measurements on PMF and PMF−SE blends during cooling to 5 °C at 2 °C/min and the subsequent isothermal crystallization at 5 °C for 10 min. When the molten PMF was cooled (Figure 3a), the SAXS peak at 4.73 nm (16 °C) and the WAXS peak at 0.420 nm (15 °C) appeared and were identified as the α form. The crystallization temperature of the PMF α form agreed with the crystallization temperature of the highmelting fraction of PMF obtained by DSC (15.4 ± 0.3 °C; Figure 2a). Upon further cooling, the SAXS peak at 4.73 nm gradually shifted to a lower value of 4.68 nm, indicating a closer lamellar distance at lower temperatures. An additional SAXS peak at 5.26 nm appeared at 6 °C; an additional peak was not observed in the WAXS region. This result agreed with the finding by Mykhaylyk et al.,15 who described the existence of metastable states (α2 phase) with 5.20 nm periodicity in POP, which was the transient form at the very beginning of isothermal crystallization at 253 K after quenching from the melt; the diffraction peak from α2 subcell packing was unavailable. During the isothermal conditions at 5 °C, the SAXS peak at 5.26 nm disappeared. Therefore, this was a shortlived metastable phase with an α subcell. The STP blend also exhibited an α form only during the cooling and isothermal processes (Figure 3b). However, the SAXS peak of the STP blend appeared at a higher temperature (32 °C), and the lamellar distance (5.03 nm) was a little longer compared with that of PMF. The SAXS peak of the STP blend shifted from 5.03 to 4.63 nm at around 10 °C during cooling to 5 °C. Since the lamellar distance of STP crystals was 4.82 nm at room temperature (Figure S2), the SR-XRD results indicated that STP first crystallized in the α form at 32 °C, and then
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RESULTS AND DISCUSSION Crystallization Behavior of PMF−SE Blends. DSC cooling thermograms of PMF and PMF−SE blends are shown in Figure 2. PMF exhibited two major exothermic peaks at 15.4 ± 0.3 and 8.6 ± 0.3 °C (onset temperatures) and one minor peak at −5.0 ± 0.2 °C (peak maximum temperature) during cooling to −10 °C; these peaks were designated as a high-melting fraction, low-melting fraction, and super-low-melting fraction, respectively. The crystallization temperatures of the low-melting and super-low-melting C
DOI: 10.1021/acs.cgd.7b01039 Cryst. Growth Des. XXXX, XXX, XXX−XXX
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In the PMF−STP and PMF−STS blends, the crystallization temperatures of the high-melting fraction of PMF were not determined precisely from SR-XRD measurements because the lamellar distances between STP or STS and the α form of PMF were similar (4.82 nm/5.34 nm (Figure S2) and 4.68 nm (Figure 3a)). In addition, their subcell packings were the same α form. If the structures of the emulsifier and fat are similar, crystallization of fat will be promoted with a low degree of supercooling through the crystallized emulsifier that is nucleated prior to fat. As a result, the DSC thermogram will show a broad exothermic peak. In this experiment, with the addition of STP (Figure 2b), the exothermic thermogram of the high-melting fraction gradually increased during cooling, and the peak showed a broad exothermic peak. This exothermic pattern indicated that crystallization of PMF occurred continuously after the crystallization of STP on the basis of the SR-XRD data. With the addition of STS (Figure 2c), the intensity of the exothermic thermogram of the high-melting fraction increased at 20 °C, suggesting that crystallization of PMF occurred. In contrast, the DSC cooling thermogram of the PMF−STB blend (Figure 2d) exhibited a broad exothermic peak for STB crystallization followed by a sharp crystallization peak for PMF. This occurred because the crystallization of PMF requires sufficient supercooling, which is not possible in the STP and STS blends. These results demonstrated that when the chain lengths of the fatty acid moieties of the PMF and SE were similar, the crystallization of the high-melting fraction of PMF was greatly promoted by the SE that was nucleated prior to PMF crystallization. Melting Behavior of PMF−SE Blends. Figure 4 presents the DSC heating thermograms of PMF and PMF−SE blends Figure 3. SR-XRD patterns of PMF and PMF mixed with 5 wt % SEs during cooling from 60 to 5 °C at 2 °C/min and following isothermal crystallization at 5 °C for 10 min: (a) PMF, (b) PMF with STP, (c) PMF with STS, and (d) PMF with STB. The dotted arrow indicates the crystallization of PMF in the PMF−STB blend. Unit: nm.
crystallization of PMF in the α form occurred upon further cooling. Similar behavior of the SAXS and WAXS patterns was obtained by adding STS (Figure 3c). The SAXS peak at 5.55 nm appeared at 41 °C and shifted to 4.68 nm at around 20 °C upon further cooling to 5 °C; a single peak at 0.417 nm appeared at 37 °C in the WAXS region. These results indicated that the nucleation of STS occurred before crystallization of PMF, and both STS and PMF exhibited the α form. At 6 °C, an additional SAXS peak at 5.23 nm was detected, as for PMF (Figure 3a). With the addition of STB (Figure 3d), the SAXS peak at 6.54 nm (58 °C) and the WAXS peak at 0.416 nm (52 °C) appeared, which corresponded to the α form of STB crystals. Considering the appearance of the diffraction peaks, the DSC exothermic peak (Figure 2d) observed at around 55 °C was regarded as a crystallization of STB. Upon further cooling, the SAXS peak at 4.79 nm separately appeared at 18 °C, and the intensity of the WAXS peak suddenly increased simultaneously (denoted by a dotted arrow), indicating crystallization of PMF in the α form. The SAXS peak at 5.26 nm was also observed at 8 °C, accompanying a slight shoulder in the WAXS region (0.414 nm). Therefore, the peak at 5.26 nm would have an α subcell with a lateral hydrocarbon chain distance of 0.414 nm, even though the WAXS peak was not observed for PMF (Figure 3a).
Figure 4. DSC thermograms of PMF and PMF mixed with 5 wt % SEs during heating from −10 to 60 °C at 2 °C/min: (a) PMF, (b) PMF with STP, (c) PMF with STS, and (d) PMF with STB. Dotted lines indicate the offset temperatures of the high-melting fraction of PMF in SE-PMF blends.
during heating from −10 to 60 °C at 2 °C/min. PMF (Figure 4a) showed four endothermic peaks at −3.7 ± 0.2, 11.1 ± 0.3, 19.1 ± 0.4, and 23.9 ± 0.3 °C (peak maximum temperatures). Addition of STS or STB (Figure 4c,d) did not affect the melting behavior of PMF much, and these blends exhibited heating thermograms similar to that of PMF. However, addition of STP (Figure 4b) changed the DSC heating pattern. The two middle melting peaks were combined to form one D
DOI: 10.1021/acs.cgd.7b01039 Cryst. Growth Des. XXXX, XXX, XXX−XXX
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temperature of this β′ form of the STP blend was lower than that of PMF. Hence, adding STP would reduce the melting point of the β′ form of PMF or affect the polymorphic transformation of PMF to a different β′ form. Even though the β′ crystals of PMF were melted, the SAXS peak at 4.88 nm and the WAXS peak at 0.419 nm derived from STP crystals were also observed. Thus, the broad endothermic DSC peak at around 29−34 °C indicated the melting of STP. The SR-XRD patterns resulting from adding STS and STB were similar to those of PMF (Figure 5c,d). Therefore, adding STS and STB did not influence the polymorphic behavior of PMF during the heating process. At 40 °C, the diffraction patterns derived from STS or STB crystals were observed (STS, 5.26 and 0.419 nm; STB, 6.14 and 0.417 nm). In Situ Observation of PMF Crystallization and Melting Behaviors on SEs Crystals with Different Fatty Acid Moieties. Microscopy observations were carried out to reveal differences in PMF crystal growth on SE crystals with different fatty acid moieties. Figures 6−8 present microscopy images of PMF crystallization on SE crystals during cooling from 30 to 5 °C using a DIC microscope (upper images) and a PL microscope with a sensitive tint plate (lower images). Since DIC microscopy can give high-contrast images, crystal growth of PMF at the interface of SE crystals can be clearly observed. PL microscopy with a sensitive tint plate provides information about an optical axis of birefringent crystals; the orange/blue color indicates whether there is a slow or fast direction of light in crystals.16 On the basis of the DSC results (Figure 2), crystallization of the high- and low-melting fractions of PMF should be observable by a microscope during cooling to 5 °C. At 30 °C, SE spherulites with a diameter of 20 μm were confirmed by PL microscopy with a sensitive tint plate (Figures 6a, 7a, and 8a; lower images). The spherulites should be composed of SEs only; PMF had not grown yet because the microscopic images were taken just after the addition of molten PMF to the SE crystals. Figure 6 represents a series of PMF crystallizations on STP crystals during cooling from 30 to 5 °C. Since PMF consists mainly of palmitic acid, the chain length of most of the fatty acid moieties of PMF is the same as that of STP. Thus, STP will act as a template for nucleation of PMF crystals. During cooling to 5 °C, the spherulites continuously grew from the surface of STP crystals (Figure 6b−e). This means that PMF nucleated at the surface of STP spherulites with a low energy barrier. The PL microscopy images showed that the arrangement of orange/blue color in the spherulites did not change after the crystallization of PMF. This result clearly demonstrated that the PMF crystals oriented in the same direction as the STP crystals. On the basis of the DSC and SRXRD results obtained for the STP blend, both the high- and low-melting fractions of PMF should crystallize in the α form and orient with STP spherulites. PMF crystallization on STS crystals during cooling from 30 to 5 °C is shown in Figure 7. The palmitic acid moiety in PMF is two carbon atoms shorter than the stearic acid moiety in STS. The crystal growth of PMF on STS crystals during cooling to 10 °C was the same as that on STP crystals; PMF gradually grew from the surface of STS spherulites during cooling, and the arrangement of orange/blue colors was preserved in the PMF crystals. Thus, in analogy with STP, the STS substrate made the PMF crystals orient in the same direction as the STS crystals. In addition, at 5 °C, a number of new elongated crystals occurred in the PMF−STS system (Figure 7e); these elongated crystals will be discussed later. In contrast to the results for STP and STS, the
larger peak with a peak maximum temperature of 17 ± 0.3 °C; this suggested that STP influenced the polymorphic transformation of PMF. The offset temperatures of the high-melting fraction of PMF in SE blends were decreased (denoted by the dotted lines). A wider decrease in offset temperature was observed with the shorter fatty acid moieties in the SEs. With the addition of SEs, broad endothermic peaks were observed at higher temperatures; adding STP resulted in a peak at around 29−34 °C, and adding STS and STB resulted in peaks at around 31−44 °C and 34−60 °C, respectively. These peaks are due to the melting of SE crystals. Figure 5 depicts the SAXS and WAXS patterns of PMF and PMF−SE blends obtained by SR-XRD measurements. When
Figure 5. SR-XRD patterns of PMF and PMF mixed with 5 wt % SEs during heating from 5 to 40 °C at 2 °C/min: (a) PMF, (b) PMF with STP, (c) PMF with STS, and (d) PMF with STB. The white arrow indicates a shoulder peak in the PMF−STP blend. Unit: nm.
PMF was heated from 5 to 40 °C (Figure 5a), the SAXS peak at 4.68 nm decreased and disappeared at 19 °C, whereas the alternative peak at 4.31 nm appeared and remained until 33 °C. In addition to the SAXS peak, the WAXS peak at 0.420 nm also decreased, and feeble peaks at 0.437, 0.425, and 0.393 nm appeared, suggesting the β′ form. These WAXS peaks disappeared at 32 °C. This result shows that the α form transformed into the β′ form during the heating process before melting. With the addition of STP (Figure 5b), a shoulder peak (denoted by a white arrow) was observed in the SAXS pattern during the heating process. This broad shoulder peak was also assigned as the β′ form because the WAXS region of the STP blend showed β′ subcell packing (0.437, 0.392, and 0.383 nm). However, since these peaks disappeared by 28 °C, the melting E
DOI: 10.1021/acs.cgd.7b01039 Cryst. Growth Des. XXXX, XXX, XXX−XXX
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Figure 6. Microscopy images of PMF crystallization on STP crystals during cooling from 30 to 5 °C at 2 °C/min: (a) 30 °C, (b) 20 °C, (c) 15 °C, (d) 10 °C, and (e) 5 °C. The upper images were taken using a differential interference contrast (DIC) microscope, and the lower images were taken using a polarized light (PL) microscope with a sensitive tint plate. Scale bars are 20 μm.
Figure 7. Microscopy images of PMF crystallization on STS crystals during cooling from 30 to 5 °C at 2 °C/min: (a) 30 °C, (b) 20 °C, (c) 15 °C, (d) 10 °C, and (e) 5 °C. The upper images were taken using a DIC microscope, and the lower images were taken using a PL microscope with a sensitive tint plate. Scale bars are 20 μm.
Figure 8. Microscopy images of PMF crystallization on STB crystals during cooling from 30 to 5 °C at 2 °C/min: (a) 30 °C, (b) 20 °C, (c) 15 °C, (d) 10 °C, and (e) 5 °C. The upper images were taken using a DIC microscope, and the lower images were taken using a PL microscope with a sensitive tint plate. Scale bars are 20 μm. F
DOI: 10.1021/acs.cgd.7b01039 Cryst. Growth Des. XXXX, XXX, XXX−XXX
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large hydrophilic groups, can act as templates for the nucleation of fat. The differences in crystal growth of PMF on SEs with different fatty acid moieties can be discussed in terms of the nucleation mechanisms of PMF. When the chain lengths of the fatty acid moieties of the fat and emulsifier are similar, e.g., PMF and STP or PMF and STS, the palmitic acid moiety of PMF molecules should be incorporated at the kink site of STP and STS crystals. Since PMF crystals exhibit a subcell packing similar to those of STP and STS crystals, the orientation of PMF crystals is directed toward the orientation of the substrate, such as STP and STS crystals, because of epitaxial growth. By contrast, if the chain length of the fatty acid moiety of the emulsifier is dissimilar to that of the fat, the kink sites of STB crystals cannot accept the palmitic acid moiety of PMF molecules. Hence, randomly oriented PMF crystals are nucleated at the surface of STB crystals. However, in this case also, nucleation of PMF can be promoted as a result of heterogeneous nucleation at the surface of the emulsifier crystals. When STS and STB were employed as substrates, elongated crystals were observed at 5 °C (Figures 7e and 8e). The morphology of these crystals was whiskerlike, as presented in Figure 10. We found that DIC microscopy is a powerful tool to observe the morphology of fat crystals even when PL microscopy images cannot elucidate the well-defined microstructure of crystals (Figure 10b−d; lower images). The DIC images showed that the elongated crystals grew rapidly during cooling from 6 to 5 °C (Figure 10a,b; upper images), and then the crystals disappeared within several minutes at 5 °C (Figure 10c,d; upper images). The crystallization behavior of the elongated crystals was in accordance with the appearance of the metastable phase with peaks at 5.26 and 0.414 nm (Figure 3d). Thus, these elongated crystals developed as a novel, unstable crystalline phase with α subcell packing. Mykhaylyk et al.15 pointed out the existence of metastable states (α2 phase) with 5.20 nm periodicity in POP, which was the transient form at the very beginning of isothermal crystallization at 253 K. Therefore, this transient crystal is probably another α form of POP with a whiskerlike morphology. In contrast, neither whiskerlike crystals nor the diffraction peak at 5.26 nm were observed when STP was used as an emulsifier (Figures 3b and 6). This is the case because STP has a higher degree of chain length similarity to the α form of PMF. Since STP would promote the nucleation of PMF crystals with 4.68 nm spacing better than STS and STB, STP promptly reduced the supercooling by promoting the crystal formation of PMF with a 4.68 nm spacing. Thus, the STP system did not generate whiskerlike crystals during cooling. Figure 11 presents the DIC microscopy images of the isothermal crystallization of PMF on SE crystals at 5 °C for 10 min (Figure 11a,g,m) and the heating process of PMF from 5 to 30 °C at 2 °C/min (Figure 11b−f, h−l, n−r). After isothermal crystallization of PMF at 5 °C for 10 min, needleshaped crystals were observed at the periphery of the PMF− STP crystals (Figures 6e and 11a). These needle-shaped crystals of PMF melted before the temperature reached 10 °C (Figure 11b), but some traces remained. Upon further heating to 30 °C, the size of the PMF−STP crystals gradually decreased (Figure 11b−f) until they reached the initial size of the STP spherulite; this suggested that only the α form of PMF melted (Figure 5b). In addition, the trace needle-shaped crystals surrounding the PMF−STP crystals remained until the
crystallization behavior of PMF on STB crystals was clearly different from that on STP and STS crystals during cooling. The chain lengths of the fatty acid moieties of PMF and STB are not similar: the number of carbon atoms in the fatty acid moieties in STB (behenic acid) is six greater than that in PMF. Crystallization of PMF occurred at the interface of STB spherulites around 15 °C, but the PMF crystals grew discontinuously, and a boundary was observed between the STB crystals and PMF crystals during cooling to 5 °C (Figure 8). In addition, some of the PMF crystals did not preserve the color arrangement found in the STB spherulites. This means that PMF crystals did not orient along the direction of the STB spherulites. At 5 °C, elongated crystals were also observed at the interface of PMF crystals (Figure 8e), as in the PMF−STS system. The microscopy observations clearly demonstrated the significance of matching the chain lengths in the fatty acid moieties of the SE and PMF (STP, 4.82 nm; STS, 5.34 nm; PMF α form, 4.68 nm) to produce the same orientation of PMF crystals as in the SE crystals. When the chain lengths of the fatty acid moieties in the SE and PMF were dissimilar (STB, 6.35 nm), PMF crystals were randomly oriented on the surface of STB spherulites. Combining the SR-XRD results for STP and STS blends (Figure 3b,c) shows that the polymorphs of both SEs and PMF were in the α form during cooling to 5 °C; the lateral hydrocarbon chain distances of the PMF α form and SEs were 0.420 and 0.413−0.417 nm, respectively. Although only the crystal structures of a limited number of TAGs have been determined,17 the similarity in the structural periodicity of the lamellar distance and subcell packing between SE and PMF causes the epitaxial growth (Figure 9). Therefore,
Figure 9. Schematic illustration of (a) lamellar structures and (b) subcell packing of PMF α form and SEs.
PMF would adopt the orientation and subcell packing of STP or STS crystals. For the first time, we can clearly demonstrate the epitaxial growth of a TAG on an emulsifier substrate when the chain lengths of the fatty acid moieties of the fat and emulsifier are similar. The orientation of the lamellar plane between monopalmitin and palm oil was shown by Verstringe et al.,13 but we can also confirm that the SEs oriented PMF crystals when the chain lengths in the fatty acid moieties were matched. These results indicate that emulsifiers, which have G
DOI: 10.1021/acs.cgd.7b01039 Cryst. Growth Des. XXXX, XXX, XXX−XXX
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Figure 10. Crystal growth of elongated crystals on STB crystals: (a) 6 °C; (b) 5 °C, 0 min; (c) 5 °C, 2 min; and (d) 5 °C, 4 min. The upper images were taken using a DIC microscope, and the lower images were taken using a PL microscope. Scale bars are 20 μm.
Figure 11. DIC microscopy images of the melting behavior of PMF that was crystallized around SE crystals during heating from 5 to 30 °C at 2 °C/ min: (a, g, m) 5 °C; (b, h, n) 10 °C; (c, i, o) 15 °C; (d, j, p) 20 °C; (e, k, q) 25 °C; and (f, l, r) 30 °C. Scale bars are 20 μm.
temperature reached 25 °C (Figure 11b−e). Since all of the PMF crystals melted completely at 30 °C, the crystals that remained around the PMF−STP crystals are of the β′ form of PMF (Figure 5b). Thus, the trace needle-shaped crystals were caused by a polymorphic transformation from the α form to the β′ form during the heating process. When STS crystals were used as a substrate for PMF crystallization (Figure 11g−l), the needle-shaped crystals almost disappeared after heating to 10 °C (Figure 11h). Upon heating to 20 °C (Figure 11h−j), the PMF crystals melted from the interface of the PMF crystals. The β′ form crystals derived from the α form were also observed surrounding the PMF−STS crystals during heating to 30 °C. This was similar to the PMF−STP observations, but the amount of β′ crystals in the PMF−STS system was obviously
larger than that in PMF−STP system. This implies that the polymorphic transformation of PMF was retarded in the PMF− STP system. In contrast, PMF crystallized at the surface of STB spherulites with a clear boundary between STB and PMF (Figure 11m). The PMF−STB system exhibited melting behavior and phase transformation similar to those of the PMF−STS system. (Figure 11n−r). SR-XRD measurements and DIC microscopy observations revealed that only the addition of STP retarded the polymorphic transformation of PMF from the α form to the β′ form during the heating process, although the crystal growth of PMF on STP and STS crystals did not differ during the cooling process. These differences can be explained by the higher degree of structural similarity between STP and the α H
DOI: 10.1021/acs.cgd.7b01039 Cryst. Growth Des. XXXX, XXX, XXX−XXX
Crystal Growth & Design
Article
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form of PMF in comparison with STS. Since the subcell structure of the SE crystals was always in the α form with hexagonal packing, the subcell structures of PMF and the SEs were the same during the cooling process. On the other hand, the lamellar distance of the α form of PMF (4.68 nm) was much closer to that of STP (4.82 nm) than that of STS (5.34 nm). This indicates that STP would exhibit a higher structural similarity to the α form of PMF than STS. For this reason, only STP retarded the polymorphic transformation of PMF from the α form to the β′ form.
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CONCLUSIONS We investigated the effects of SEs with different fatty acid moieties on PMF crystallization and subsequent melting behaviors to elucidate the molecular interactions between the SEs and PMF. Adding STP or STS at 5 wt % greatly increased the crystallization temperature of the high-melting fraction of PMF as a result of the crystallized STP or STS that was nucleated prior to PMF. When the chain lengths of the fatty acid moieties of the SEs were equal to or two longer than those of PMF, PMF crystals oriented along the same direction as the STP and STS crystals, suggesting the epitaxial growth of PMF on SE crystals. However, when the chain length of the fatty acid moieties of the SE was six longer than that of PMF, PMF crystals exhibited a random orientation at the interface of STB crystals, suggesting general heterogeneous nucleation of PMF. Only the addition of STP retarded a polymorphic transformation of the high-melting fraction of PMF from the α form to the β′ form; the structural similarity between STP and PMF influences the retardation of the polymorphic transformation of PMF.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.7b01039. DSC thermograms and SR-XRD patterns of SEs (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Phone: +81 82 4247935. Fax: +81 82 4247910. E-mail:
[email protected]. ORCID
Hironori Hondoh: 0000-0001-5856-1511 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors greatly appreciate Riken Vitamin Co., Ltd. (Tokyo, Japan) for providing sorbitan fatty acid esters. SR-XRD experiments were conducted under the approval of the Photon Factory Program Advisory Committee (Proposals 2014G662 and 2015G686). The authors gratefully acknowledge the help of Dr. N. Igarashi and Dr. N. Shimizu, Station Manager of beamline 6A at the Photon Factory (KEK Institute, Tsukuba, Japan).
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REFERENCES
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DOI: 10.1021/acs.cgd.7b01039 Cryst. Growth Des. XXXX, XXX, XXX−XXX
