Revealing the Influence of Tempering on Polymorphism and Crystal

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Revealing the Influence of Tempering on Polymorphism and Crystal Arrangement in Semicrystalline Oil-in-Water Emulsions Kim Moens,* Nathalie De Clercq, Stefanie Verstringe, and Koen Dewettinck Laboratory of Food Technology & Engineering, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium ABSTRACT: The influence of tempering (i.e., successive heating and cooling) on crystallization properties of semicrystalline milk fat-in-water emulsions was studied by X-ray diffraction (XRD), differential scanning calorimetry (DSC), and cryoscanning electron microscopy. Melting profiles obtained by DSC indicate a fractionation process of milk fat in every individual fat droplet. The long spacings, as observed by XRD, were useful in the designation of polymorphic changes to a shift in fatty acid composition of the crystals. An increased layer thickness of the 3L-crystals at 20 and 30 °C tempering points to a preferential incorporation of long-chain unsaturated fatty acids over short-chain fatty acids. Additionally, at 30 °C, an increased layer thickness of the 2L-crystals assumes the incorporation of longer-chain saturated fatty acids. The highly mixed crystals, an inherent property of milk fat, seem to be fractionated in purer crystals grouping triacylglycerols with a similar fatty acid composition. Moreover, a different crystal organization was obtained for both tempering temperatures, where tempering at 20 °C appears to result in a coarser structure with well-defined lamellar regions near the interface, while tempering at 30 °C seems to lead to a finer, more homogeneous crystal structure. The observed effects on the microstructural level will have implications on storage stability and applicability.

1. INTRODUCTION Oil-in-water emulsions are commonly used in different industrial products such as foods, cosmetics, pharmaceutics, etc. In this study we will focus on milk fat-in-water emulsions which have the specific property of containing a semicrystalline dispersed fat phase. This creates the ability to transform these emulsions into complex products like whipped cream, ice cream, and butter by means of partial coalescence. Obviously, the fat crystal network in the droplets is of paramount importance to the process of partial coalescence and is characterized by the ratio solid to liquid fat, arrangement of the fat crystals and crystal size, polymorphism, and morphology.1 The occurrence of partial coalescence should be balanced as it is wanted in the production of many complex products, while it should be minimalized during storage in order to prevent destabilization of the emulsion. Milk fat is a particularly complex fat. It contains a wide range of fatty acids differing in chain length and degree of saturation, and thus many different triacylglycerols (TAG)2 favoring the formation of mixed crystals3 which may exist in different polymorphic forms.4−9 The group of Ollivon has extensively investigated polymorphism of milk fat in emulsions with different droplet sizes,10−13 at different cooling rates,11,12,14,15 and under isothermal conditions.10,16 They analyzed the polymorphic transitions occurring in a milk fat emulsion during quench cooling and subsequent isothermal storage at 4 °C.16 It was seen that the α-crystals that were formed during cooling partly underwent a transition to β′ during the isothermal period. Finally, after 6 days at 4 °C, α-crystals, two types of β′-crystals, and traces of β-crystals were observed. Those polymorphs were © XXXX American Chemical Society

related to a predominant 2L-structure and with a 3L-structure that was present to a lesser extent. It was suggested that the 2L longitudinal organization could be attributed to the β′-crystals and the 3L longitudinal organization to the α-crystals. This crystallization mechanism is confirmed by Fredrick et al.17 who concluded that the 2L-structure indeed can be linked to β′crystals and the 3L-structure to the α-crystals. The longitudinal stacking of the crystals is highly related to the fatty acid structure.18,19 The high-melting fraction of milk fat contains mainly TAGs with three long-chain saturated fatty acids.5 Because of the similarity in chain length and saturation, they crystallize preferentially in a 2L-structure.18,19 TAGs containing two long-chain saturated fatty acids and one shortchain fatty acid or one long-chain unsaturated fatty acid belong to the middle-melting fraction and TAGs containing one longchain saturated fatty acid and two short-chain fatty acids or two long-chain unsaturated fatty acids belong to the low-melting fraction.5 The dissimilarity in chain length and degree of unsaturation favors the formation of 3L-structures. Different strategies are possible to influence the fat crystal network of the dispersed phase, including cooling rate,11,20,21 solid fat content,22 oil−water interface23,24 and droplet size.10−13,21,25 Moreover, tempering is shown to be an interesting tool for modifying the fat crystal network as well. Tempering is the application of a specific time−temperature profile in which the temperature is first increased partly melting Received: May 13, 2015 Revised: August 11, 2015

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DOI: 10.1021/acs.cgd.5b00665 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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light microscopy but only after tempering repeatedly. Different tempering cycles were probably needed to increase the crystal size to be visualized using light microscopy. Increasing the temperature disintegrates the continuous fat crystal network in the droplets, releasing fat crystals. Those crystals seem to move to the energetically more favorable interface where they grow during subsequent cooling leading to the observation of more fat droplets of the L- and M-type.22 This research will investigate the hypothesis that partial melting of fat crystals during tempering may enable the TAGs to be reorganized in groups with similar fatty acids leading to the formation of purer crystals. It is likely that this process also influences the shape and orientation of the crystals in the droplets. The effect of two tempering temperatures (Tmax), namely, 20 and 30 °C, on the melting behavior, polymorphism, and crystal arrangement of the fat crystal network in milk fat-inwater emulsions was investigated.

the fat and then decreased again inducing heterogeneous crystallization of the liquid fat.22,26−34 To date, studies on tempering of emulsions were mainly focusing on partial coalescence and whipping properties but little on the intrinsic changes of the fat crystal network. The pioneering research of Boode-Boissevain22 on tempering of emulsions showed that tempering increases the susceptibility to partial coalescence if the emulsion contains minimally 25% fat and is semicrystalline at the tempering temperature. For milk fat-in-oil emulsions, the latter condition is fulfilled when considering tempering temperatures below 40 °C. Drelon, Gravier, Daheron, Boisserie, Omari, and Leal-Calderon26 studied the effects of applying a tempering step after whipping of natural cream and found from the melting profile that a better separation between the high- and the low-melting fraction of milk fat was obtained. In addition, they concluded from the differential scanning calorimetry (DSC) results that no significant polymorphic changes occurred during tempering. Nevertheless, X-ray diffraction measurements remain necessary to draw conclusions about polymorphism. The latter technique was used by Mutoh, Nakagawa, Noda, Shiinoki, and Matsumura28 to study the influence of tempering on fat polymorphism in vegetable fat-in-water emulsions. For some of the vegetable fats, they observed a polymorphic transition to the more stable β-crystals. Tempering may induce changes in polymorphic forms through melt-mediated or solid-state transformations. This is known for chocolate where the goal of tempering is to obtain the preferred βV-crystals.35 To the best of our knowledge, no literature is available on the effect of tempering on milk fat polymorphism in emulsions. However, the effect of the cooling rate was studied in the past. In the case of bulk milk fat, it was shown that during slow cooling TAGs can immediately form β′-crystals.8,36 However, for emulsified milk fat, crystallization primarily occurs in the α-form which will then recrystallize into β′-crystals14 and finally partly transform in β-crystals.16 Purer crystals are formed during slow cooling because TAGs with a similar fatty acid composition have sufficient time to arrange them together in crystals.8 Besides polymorphism, the position of the fat crystals is also likely to be affected. In most emulsions the contact angle of the crystals implies that the interfacial free energy is minimized when crystals are situated at the interface.37 Consequently, if crystals are capable of moving because they are not part of a continuous network, they will preferably be located at the interface. Fat droplets are classified according to the position of the fat crystals: N-type (needle-shaped crystals in the whole fat droplet), L-type (crystals are situated parallel to the crystal boundary), and M-type (combination of needle-shaped crystals and crystals parallel to the crystal boundary).38 Processing conditions may influence the position of the fat crystals. Lopez, Bourgaux, Lesieur, Bernadou, Keller, and Ollivon11,39 investigated the morphology of emulsion droplets subjected to different cooling rates. It was shown that fast cooling resulted in a large amount of small crystals in N-type of droplets. The slower the cooling rate the more layered crystals parallel with the interface appear (M-type and L-type) because, in contrast to higher cooling rates, crystals have time to optimize their position. Similar observations were done by Truong, Morgan, Bansal, Palmer, and Bhandari21 for nanoemulsions. Tempering also allows fat crystals to be positioned at the interface if they are released of the crystal network. Boode-Boissevain22 could observe a repositioning of the fat crystals by using polarized

2. MATERIALS AND METHODS 2.1. Materials. Emulsions were prepared by recombining anhydrous milk fat (FrieslandCampina butter, Noordwijk, The Netherlands), sweet cream butter milk powder (Westbury Dairies, Westbury, United Kingdom), carrageenan (Satiagel ME4, Cargill Deutschland GmbH, Krefeld, Germany), and potable water. Sodium azide (Acros organics, Geel, Belgium) was added to prevent microbial spoilage. 2.2. Production of Milk Fat-in-Water Emulsions. Sweet cream butter milk powder (7.6%) was dissolved in potable water and kept overnight for full hydration. Carrageenan (0.01%) and sodium azide (0.01%) were added to this aqueous phase and preheated to 50 °C. The anhydrous milk fat (35%) was melted and preheated to 50 °C. The pre-emulsion was prepared by thoroughly mixing the aqueous phase with the fat phase using an Ultra-Turrax (IKA-Werke, Staufen, Germany) (10 000 rpm) for 10 min at 50 °C. This pre-emulsion was then homogenized using a two-step laboratory scale homogenizer (APV cooling systems, Alberslund, Denmark). The pressure applied in the first step was 30 bar followed by a second step at a pressure of 10 bar to break up possible homogenization clusters. Finally, the emulsion was rapidly cooled (−10 °C/min) to 5 °C and stored in a thermostatic cabinet at 5 °C for at least 7 days to complete fat crystallization before being used for tempering. The size of the emulsion droplets was measured after 7 days with laser light diffraction according to the method described in Fredrick, Moens, Heyman, Fischer, Van der Meeren, and Dewettinck.24 The Sauter diameter (D3,2) of the emulsion droplets was 2.99 ± 0.16 μm. 2.3. Time-Resolved Synchrotron X-ray Diffraction. Polymorphic behavior of milk fat in the dispersed state during tempering was investigated by synchrotron X-ray diffraction (XRD). Small-angle Xray scattering (SAXS)/wide-angle X-ray diffraction (WAXS) measurements were performed on the Dutch-Belgian (DUBBLE) beamline BM26B at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France). The experiments were conducted at a fixed wavelength λ of 1.24 Å. A linear 300 K photon counting Pilatus detector was used for WAXD, whereas a large aread high sensitive photon counting 2D Pilatus detector was used to collect the SAXS images. The samples were enclosed in glass capillaries, and the temperature was controlled by a Linkam hot stage. The emulsion was stored at 5 °C before analysis. Capillaries were precooled at 5 °C to avoid heating during sample preparation. Those capillaries were filled with the emulsion and immediately inserted in the precooled sample holder. After an equilibration period of 10 min at 5 °C, the sample was heated at 10 °C/min to Tmax (20 or 30 °C) and kept isothermal for 30 min. Subsequently, the sample was cooled at −10 °C/min to 5 °C and kept isothermal for 30 min. Scattering patterns were taken every 30 s during the whole experiment. Known reflections of standard silverbehenate,40 and alpha aluminum samples were used to calibrate the SAXS and WAXD scattering angles, 2θ. The SAXS patterns are presented as a function of s (1/Å), with s = 1/d = 2 sin θ/λ and d the B

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Figure 1. SAXS patterns of emulsion tempered at 30 °C: (A) heating at 10 °C/min from 5 to 30 °C, (B) isothermal at 30 °C for 30 min, (C) cooling at 10 °C/min from 30 to 5 °C, and (D) isothermal at 5 °C for 30 min. Polymorphic forms presented between [ ] are assumed to be present in very small amounts. sample was transferred again to the precooled (5 °C) measuring cell of the DSC, and the melting profile was analyzed by heating the sample at 5 °C/min to 55 °C. The total melting heat (J/g), the melting heat of the individual melting peaks (J/g), and the peak maximum of the individual melting peaks (°C) were determined by integration using a horizontal linear baseline. Measurements were performed in triplicate. 2.5. Nuclear Magnetic Resonance. A Maran Ultra 23 MHz pulsed-field gradient nuclear magnetic resonance (NMR) instrument (Oxford Instruments, Tubney Woods, Abingdon, UK) was used to measure the solid fat content of the emulsion before and after tempering. The initial solid fat content was determined after storage of the emulsion for at least 7 days. The solid fat content after tempering was measured after an isothermal period of 30 min at 5 °C. An indirect SFC measurement was performed to eliminate the contribution of the aqueous phase to the FID signal. A detailed description of this method is given in Fredrick, Van de Walle, Walstra, Zijtveld, Fischer, Van der Meeren and Dewettinck.17 2.6. Cryoscanning Electron Microscopy. The arrangement of the crystals in the fat droplets was visualized using scanning electron microscopy with cryogenic sample preparation. The emulsion was mounted on an aluminum stub and quickly frozen in slushed nitrogen (−210 °C). The time between sample preparation and freezing was kept as short as possible to avoid crystal melting. Then, the sample was fractured at −150 °C, sputter-coated with platinum, and finally

distance. The WAXD patterns are presented as a function of d (Å), with d = λ/2 sin θ. All scattering patterns were corrected for the detector response, normalized to the intensity of the primary beam, and corrected by the sample absorption before performing the background subtraction. The XRD patterns were further analyzed using the PeakFit software (SeaSolve Software inc., Framingham, USA). 2.4. Differential Scanning Calorimetry. A Q1000 DSC with a refrigerated cooling system (TA Instruments, New Castle, USA) was used to study crystallization and melting during and after tempering of the emulsion to 20 and 30 °C. The DSC was calibrated with indium (TA Instruments, New Castle, USA), azobenzene (Sigma-Aldrich, Bornem, Belgium), and undecane (Acros organics, Geel, Belgium) prior to analysis. Nitrogen was used to purge the system. Aluminum pans were filled with 5−15 mg of the emulsion and hermetically sealed. An empty pan was used as a reference. DSC was used to temper the emulsion by using the following t,T-program: (i) equilibration at 5 °C for 10 min, (ii) heating at 10 °C/min to Tmax (20 or 30 °C), (iii) isothermal at Tmax for 30 min, (iv) cooling at −10 °C/min to 5 °C, (v) isothermal at 5 °C for 30 min. The melting heat (J/g) and crystallization heat (J/g) were determined by an integration using a horizontal linear baseline. After completion of this t,T-program, the sample was removed manually and immediately transferred to a thermostatic cabinet at 5 °C. After 7 days of storage at 5 °C, the C

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Figure 2. WAXD patterns of emulsion tempered at 30 °C: (A) heating at 10 °C/min from 5 to 30 °C, (B) isothermal at 30 °C for 30 min, (C) cooling at 10 °C/min from 30 to 5 °C, and (D) isothermal at 5 °C for 30 min. Polymorphic forms presented between [ ] are assumed to be present in very small amounts.

the presence of two types of β′-crystals (indicated as β′1 and β′2).6 Furthermore, β-crystals are represented by the small peak around 4.6 Å.6 The α-, β′-, and β-peaks are superimposed to a broad peak corresponding to the liquid TAGs. Lopez, Bourgaux, Lesieur, and Ollivon16 demonstrated the existence of similar crystal polymorphs in milk fat-in-water emulsions stored at 4 °C for a longer period. On the other hand, Bugeat, Briard-Bion, Perez, Pradel, Martin, Lesieur, Bourgaux, Ollivon, and Lopez10 reported the presence of an extra β polymorphic form and no α polymorphic form in emulsified milk fat. Those differences could be attributed to the smaller droplet size in the latter study. 3.1. Tempering to 30 °C. Step 1 (Figure 1A and 2A): Upon heating, several changes in the short and long spacings occur. The SAXS pattern indicates that both 3L-crystals and 2L-crystals start to melt at a temperature of 15 °C. At 30 °C mainly 2L-structures exist in addition to traces of 3L-structures. This is partially in agreement with Bugeat, Briard-Bion, Perez, Pradel, Martin, Lesieur, Bourgaux, Ollivon, and Lopez,10 Lopez, Bourgaux, Lesieur, and Ollivon16 who also observed simultaneous melting of the 2L and 3L until 20−25 °C, but they noted a complete melting of the 3L polymorphic form, although in

observed with a Jeol JSM-7100F scanning electron microscope (Jeol (Europe) B.V., Zaventem, Belgium).

3. RESULTS AND DISCUSSION The evolution of long and short spacings and the melting and crystallization events during tempering of the milk fat-in-water emulsion were characterized. Besides, the microstructural organization of the fat crystals in the droplets after tempering was visualized with cryo-SEM. Tempering was executed at two tempering temperatures (Tmax), 20 and 30 °C, and is divided in four steps: (i) heating at 10 °C/min from 5 °C to Tmax; (ii) isothermal at Tmax for 30 min; (iii) cooling at 10 °C/min from Tmax to 5 °C; (iv) isothermal at 5 °C for 30 min. The polymorphic forms initially present at 5 °C are shown in Figures 1A and 2A. Two peaks are distinguished in the SAXS pattern: a small peak at d = 54.9 Å corresponding to 3Lstructure (typically d between 55 and 75 Å7,17,41,42) and a larger peak at d = 39.9 Å corresponding to a 2L-structure (typically d between 40 and 50 Å7,17,41,42). The WAXD pattern shows a broad peak at 4.1−4.3 Å covering both the α-polymorphic form (typically d = 4.15 Å6) and the β′-polymorphic form (typically d = 4.2 Å6). Around 3.8 Å a double peak is observed, suggesting D

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Figure 3. SAXS (A) and WAXD (B) patterns of emulsion obtained at the beginning and the end of each step of the tempering procedure (30 °C). From the bottom to the top of the graph, the first pattern is initially obtained at 5 °C, the second is obtained immediately after heating to 30 °C, the third is obtained after 30 min isothermal at 30 °C, the fourth is obtained immediately after cooling to 5 °C, and the fifth is obtained after 30 min isothermal at 5 °C.

crystals and the isothermal growth at 30 °C of those β′12Lcrystals. Furthermore, the double chain layer thickness increases to d = 41.6 Å, suggesting the incorporation of TAGs with long-chain fatty acids that were melted during step 1. Mainly mixed crystals are formed in milk fat because of the wide variety of fatty acids, especially at the high cooling rates applied during processing.3 The WAXD pattern shows that the broad peak at 4.1−4.3 Å evolves into three overlapping but well-separated peaks representing the α-, β′1-, and β′2-crystals, pointing out an evolution from highly mixed crystals to purer crystals during tempering at 30 °C. Crystal melting in step 1 releases TAGs which can be incorporated into the remaining crystals at 30 °C due to similarity of the fatty acids resulting in purer crystals. Step 3 (Figure 1C and 2C): During cooling, a slight increase of the 2L-peak is noted upon 20 °C, while the WAXD pattern shows an increase in β′2-crystals. Growth of those β′22L-crystals is observed as a broadening of the β′1-peak at 3.8 Å and an increase in intensity of the β′2-peak at 4.35 Å. Subsequent cooling to below 20 °C induces growth of the β′12L-crystals established by the parallel increase in intensity of the 2L-peak and the β′1-peak. Step 4 (Figures 1D and 2D): Crystallization is continued during the isothermal period at 5 °C. After approximately 1 min, a 3L-structure at s = 0.014 Å−1 (d = 72 Å) suddenly arises in combination with an explicit increase of the α-peak. It follows that new nuclei are formed in an α3L polymorphic form. Thereafter, those peaks gradually decrease in intensity simultaneously with the increase of the original 3L-structure at s = 0.016 Å (d = 64.4 Å) and its second order peak (3L002) at s = 0.031 Å−1 (d = 32.7 Å). This shift in 3L-peak from 72 to 64.4 Å was also observed by Lopez, Bourgaux, Lesieur, and Ollivon,16 who attributed this to a change in tilt of the TAG chains in the lamellar structure which is observed during an α to β′ transition. It is likely that this transition occurs because the 2L-peak and the β′1- and β′2-peaks increase during the remaining isothermal time. In addition, a slight increase of the β-crystals is noted during the isothermal period at 5 °C, suggesting a β′ to β transition. Figure 3 depicts the SAXS and WAXD pattern at the beginning and end of each step of the tempering process,

this study traces of this 3L polymorphic form seem to persist. The WAXD pattern shows that initially the top of the broad peak at 4.1−4.3 Å is shifted to a higher d-value, indicating the melting of α-crystals, and then the intensity of the β′2-peak (around d = 3.8 Å) and the β′1-peak decreased. At 30 °C predominantly β′1-crystals are present. Presumably, traces of α-, β′2-, and β-crystals remain present. The lower melting point of β′2-crystals compared to β′1-crystals could be explained by the difference in fatty acid composition of both fractions. Although it is generally accepted that β-crystals have a higher melting point than β′1-crystals, the opposite is noted in this study as well as in other studies.10,16 According to Walstra,3 compound β′-crystals can be more stable than pure β-crystals. Besides the decrease in intensity, the position of the 2L- and 3L-peaks was shifted. The 2L-peak remained at the same position until 25 °C and then shifted, simultaneously with the melting of β′1crystals, to a higher d-value (d = 40.5 Å). This increase in layer thickness was previously observed by Bugeat, Briard-Bion, Perez, Pradel, Martin, Lesieur, Bourgaux, Ollivon, and Lopez,10 Lopez, Bourgaux, Lesieur, and Ollivon16 during heating of milk fat emulsions that were stored at 4 °C for a longer period. This shift was assigned to the selective melting of TAGs containing short-chain fatty acids which were incorporated in the highmelting crystals (β′1). Consequently, the liquid fraction could be enriched with TAGs containing short-chain fatty acids. Especially the high cooling rate applied in this research favors the formation of mixed crystals which could lead to the incorporation of low-melting TAG in high-melting crystals. An even more apparent shift is noticed for the 3L-structure. The majority of 3L-crystals melt, but the remaining peak shifted from d = 54.9 Å to around d = 64.4 Å. This shift could be attributed to the selective melting of TAGs containing two short-chain fatty acids which are typical for the low-melting fraction,5 whereas TAGs with two unsaturated fatty acids could represent the traces of 3L-crystals present at 30 °C. This shift could also be explained by the release of TAG with unsaturated fatty acids from β′1 upon melting which are forced in a 3Lstructure. Step 2 (Figure 1B and 2B): A gradual, simultaneous increase in intensity of the 2L-peak and the β′1-peak is noted, demonstrating the double chain length structure of the β′1E

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Figure 4. DSC curves recorded (A) during tempering of the emulsion to 30 °C and subsequent cooling to 5 °C and (B) during melting of the emulsion tempered to 30 °C after 7 days of storage at 5 °C.

Figure 5. SAXS patterns of emulsion tempered at 20 °C: (A) heating at 10 °C/min from 5 to 20 °C, (B) isothermal at 20 °C for 30 min, (C) cooling at 10 °C/min from 20 to 5 °C and (D) isothermal at 5 °C for 30 min. Polymorphic forms presented between [ ] are assumed to be present in very small amounts.

summarizing the main effects of tempering at 30 °C discussed in the paragraphs above. A more distinct separation between the α, β′1-, and β′2 -peak (Figure 3B) and between the 2L- and 3L-peak is observed (Figure 3A). Most probably this is explained by an increase in organization of the crystals and an

increase in crystal purity. Milk fat crystallization in emulsions is known for its lack in organization and the formation of mixed crystals,3 but during tempering the TAGs seem to reorganize themselves into better structured and purer crystals grouping TAGs with a similar fatty acid structure. This hypothesis is F

DOI: 10.1021/acs.cgd.5b00665 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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Figure 6. WAXD patterns of emulsion tempered at 20 °C: (A) heating at 10 °C/min from 5 to 20 °C, (B) isothermal at 20 °C for 30 min, (C) cooling at 10 °C/min from 20 to 5 °C and (D) isothermal at 5 °C for 30 min. Polymorphic forms presented between [ ] are assumed to be present in very small amounts.

2.7% to 46.2 ± 2.3% after tempering at 30 °C. This is explained by several postcrystallization mechanisms occurring during further cooled storage like solid-state polymorphic transitions and even additional crystallization of TAGs which did not crystallize immediately.17 Crystallization can be divided in two steps: non-isothermal crystallization during cooling (main crystallization peak) and isothermal crystallization (second crystallization peak). In combination with the XRD data, it is deduced that during cooling growth of the β′2L-crystals takes place, whereas during the isothermal period nucleation of α3Lcrystals occurs. The melting profile obtained 7 days after tempering was compared to the melting profile of the untempered emulsion (Figure 4B). The broad melting peak of the untempered emulsion consists of one melting peak at 23.2 ± 0.1 °C and a shoulder at higher temperatures. The tempered emulsion starts to melt at a lower temperature, and the peak maximum of the first peak is shifted to 21.4 ± 0.2 °C. On the other hand, the tempered emulsion shows a second well-distinguishable melting peak at 34.3 ± 0.2 °C with a melting onset at 31.3 ± 0.3 °C, which is just above Tmax. The XRD results show that this highmelting fraction corresponds to the β′1-crystals. Furthermore, it

supported by narrowing of the 2L-peak during tempering which may be attributed to an increased order.16 Finally, tempering influenced the layer thickness. Last-mentioned is a function of the chain length of the fatty acids and the tilt of the chains depending on the polymorphic form.41 When comparing the long spacings obtained before (Figure 3A, bottom pattern) and after (Figure 3A, upper pattern) tempering at 30 °C, it is noted that the layer thickness of the 2L- and 3L-structures increased assuming a redistribution of TAG between the crystals10 leading to an enrichment of the solid phase with long-chain fatty acids and an enrichment of the liquid phase with shortchain fatty acids. The melting and crystallization events recorded by DSC during tempering to 30 °C are shown in Figure 4A. The melting peak represents melting of two fractions, which is confirmed by the XRD results, indicating the successive melting of α3L- and β′2L-crystals. The crystallization heat is lower than the melting heat (data not shown) from which it is concluded that not all the TAGs recrystallize during cooling and the subsequent isothermal period of 30 min. The same conclusion could be based on the solid fat content measured by pNMR. The solid fat content of the emulsion decreased from 53.4 ± G

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Figure 7. SAXS (A) and WAXD (B) patterns of emulsion obtained at the beginning and the end of each step of the tempering procedure (20 °C). From the bottom to the top of the graph, the first pattern is initially obtained at 5 °C, the second is obtained immediately after heating to 20 °C, the third is obtained after 30 min isothermal at 20 °C, the fourth is obtained immediately after cooling to 5 °C, and the fifth is obtained after 30 min isothermal at 5 °C.

Figure 8. DSC curves recorded (A) during tempering of the emulsion to 20 °C and subsequent cooling to 5 °C and (B) during melting of the emulsion tempered to 20 °C after 7 days of storage at 5 °C.

Step 2 (Figure 5B and 6B): Isothermal crystallization at 20 °C of the β′12L-crystals is noted, suggesting the incorporation of TAGs released during melting of the highly mixed α3Lcrystals. Step 3 (Figure 5C and 6C): During cooling, the growth of β′2L-crystals is noted by the increase of the 2L-peak and the β′peaks. It is difficult to determine from the WAXD pattern which type of β′-crystals grows. Step 4 (Figure 5D and 6D): Together with the increase of the α-peak, the 3L-peak increases and at the same time moves to a higher d-value (d = 60.4 Å). It is plausible that during tempering the TAGs which are melted from the α3L-crystals have enough time to rearrange themselves, resulting in the favorable incorporation of the long-chain fatty acids over the short-chain fatty acids during recooling. Besides, the β′2Lcrystals progressively grow during this isothermal period. The β-crystals seem to be remained during tempering to 20 °C. Figure 7 shows the SAXS and WAXD pattern at the end and the beginning of each step of the tempering process emphasizing the main effects of tempering the emulsion to 20 °C discussed in the paragraphs above. The increase in layer thickness of the 3L-structure is clearly noticeable and might be related to the preferential inclusion of long-chain fatty acids over the short-chain fatty acids. Although the layer thickness of

is assumed that the separation of the high-melting peak is mainly established by the movement of TAGs containing longchain saturated fatty acids from the highly mixed low- and middle-melting crystals to the high-melting crystals. This results in a higher purity of both fractions observed by a clear peak separation in the DSC recordings, a lower melting onset and melting point of the low-melting fraction, and a higher final melting point of the high-melting fraction. 3.2. Tempering to 20 °C. As mentioned above, the emulsion at 5 °C is composed of a 2L-structure and a 3Lstructure and four polymorphic forms: α, β′1, β′2, and β. Step 1 (Figure 5A and 6A): Upon heating to 20 °C, four interesting simultaneous events are noted: (i) the intensity of the 3L-peak decreases, (ii) the intensity of the 2L-peak increases, (iii) the broad peak in the WAXD pattern at 4.1− 4.3 Å, representing both the α- and β′-crystals, shows a shift to a higher d-value, and finally (iv) a clear increase in β′1 is noticed around 3.8 Å. The simultaneous occurrence of those events implies first that the α-crystals have a 3L-structure and β′1 a 2Lstructure. Second, it implies that a melt-mediated polymorphic transition from α3L-crystals to β′12L-crystals occurs. This is confirmed by the results in Section 3.1 (Figure 1A and Figure 2A) but when exceeding 20 °C those β′12L-crystals partly melt. H

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Figure 9. Cryo-SEM images of the emulsion before tempering (A), after tempering to 20 °C (B and C), and after tempering to 30 °C (D and E). Arrows indicate the lamellar regions near the interface observed after tempering to 20 °C. Magnifications: (A) X8000; B (X10000); (C) X12000; (D) X8000; (E) X6500.

the 2L-structure remains unchanged, the increase in intensity of the 2L-peak and the β′-peaks during the whole process points at a continuous growth of those crystals. The melting and crystallization events recorded by DSC during tempering of the emulsion to 20 °C are shown in Figure 8A. A somewhat higher melting heat compared to crystallization heat is observed suggesting postcrystallization also after tempering to 20 °C. The latter was confirmed by the solid fat content measured by pNMR. The solid fat content of the emulsion decreased from 53.4 ± 2.7% to 46.4 ± 1.3% after tempering at 20 °C. The melting profile obtained 7 days after

tempering was compared to the melting profile of untempered emulsion (Figure 8B). The initial decrease in heat flow is stronger compared to untempered emulsion. The melting profile contains one well-separated peak with a melting point around 18 °C and one broad peak at higher melting temperatures. The XRD results show that heating to 20 °C only melted the α3L-crystals, whereas β′2L grew progressively. From this it is concluded that the melting peak at 18 °C attributes to the melting of α3L, while the broad melting peak at higher temperatures represents the β′2L-crystals. The more I

DOI: 10.1021/acs.cgd.5b00665 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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distribution of fat crystals is observed before tempering and after tempering to 30 °C. However, well-defined lamellar regions near the interface are detected in fat droplets subjected to tempering at 20 °C suggesting L- and M-type of fat droplets. The latter observation is comparable to the suggested morphology of fat droplets obtained after slow cooling.11,21 These results contribute to the hypothesis that the remaining fat crystals at Tmax move to the energetically more favorable interface where they grow during subsequent cooling, resulting in L- and M-type fat droplets.22,28,30,31 However, it should be noted that Boode-Boissevain22 observed this movement after tempering to 35 °C and then repeatedly to 25 °C, while in this study this movement was only observed for tempering to 20 °C and not for tempering to 30 °C. It could also be seen from the images that the crystal network observed for 20 °C tempered emulsion is more course compared to the 30 °C tempered emulsion, adding to the expected difference in crystal size.

clear separation could be due to the increased purity of the α3L-crystals after tempering. 3.3. Comparison of 20 and 30 °C Tempering. XRD and DSC measurements have shown that the polymorphic evolutions occurring during tempering are different for both temperatures. Increasing the temperature to 20 °C melts only the α3L-crystals. In the case of 30 °C tempering, exceeding 20 °C melts first the β′22L-crystals and then partly the β′12Lcrystals. Isothermal crystallization of the remaining β′-crystals at Tmax occurs at both temperatures. The long spacings provide valuable information regarding the type of fatty acids included in the crystals. The layer thickness of the 2L-crystals progressively increases during the whole tempering process at 30 °C which is attributed to the inclusion of TAGs with long-chain fatty acids while this is not occurring at 20 °C. It follows that those long-chain fatty acids must originate from the β′22L-crystals as these only melt when the temperature exceeds 20 °C. The 3L layer thickness increases for both Tmax putting forward that after melting of the α3Lcrystals those TAGs reorganize themselves during the isothermal period, resulting in a preferential inclusion of TAGs with long-chain unsaturated fatty acids over TAGs with short-chain fatty acids. Consequently, the purity of the α3Lcrystals increases. This is confirmed by the melting profiles as for both temperatures the melting onset of the first melting peak (representing the α3L-crystals) is clearly decreased. On the other hand, the WAXD patterns after tempering at 30 °C point out a more apparent separation of the β′-peaks as well, what may be attributed to an increased purity of those crystals which was not observed for 20 °C. The melting profile of emulsion tempered at 30 °C shows indeed a well-separated melting peak of the high-melting fraction corresponding to the β′12L-crystals. The differences in polymorphism and layer thickness of the longitudinal stackings are likely to result in differences in microstructural arrangements of the fat crystals. It is concluded for 20 °C that they are mainly the crystals persisting at 20 °C that grow during cooling and subsequent isothermal storage, indicating that the crystal size may be increased. Conversely, tempering at 30 °C results not only in crystal growth of the β′crystals but also in nucleation. Therefore, the crystal size is probably less increased as compared to 20 °C tempering. To verify the latter assumption, the domain size of the 2Lstructures was calculated. TAGs are grouped according to their longitudinal stacking in lamella which are characterized by the layer thickness (d) providing information about the incorporated TAGs. Those lamella are on their turn combined into domains whose size is related to the full width at halfmaximum.43 Tempering at 30 °C increased the domain size from 0.044 ± 0.001 μm to 0.058 ± 0.001 μm, which corresponds to an increase of the amount of lamella per domain from 11.0 ± 0.1 to 14.2 ± 0.3. Tempering at 20 °C increased the domain size from 0.044 ± 0.001 μm to 0.063 ± 0.001 μm and an increase of the amount of lamella per domain from 10.8 ± 0.2 to 15.4 ± 0.3. These results confirm the increase in crystal size during tempering which was also put forward by Mutoh, Nakagawa, Noda, Shiinoki, and Matsumura28 for vegetable creams and point out a greater increase for tempering at 20 °C compared to 30 °C. Cryo-SEM imaging was used to verify the morphology of the fat droplets. Figure 9 shows the cross-section of a fat droplet before tempering (A), after tempering to 20 °C (B and C), and 30 °C (D and E), unraveling the internal fat crystal network. A random

4. CONCLUSIONS The focus of this research was to study the influence of tempering on the fat crystals in fat droplets of a semicrystalline oil-in-water emulsion. Interestingly, tempering induces a fractionation process in every individual fat droplet, hence influencing its crystallization and melting properties. The effect on fat polymorphism, crystal size, and crystal orientation is different for the examined temperatures. The emulsion tempered at 20 °C showed mainly an increase in α3L-crystal purity, which could be explained by the preferential inclusion of TAGs containing long-chain unsaturated fatty acids over the ones containing short-chain fatty acids. The cryo-SEM image shows a coarser crystal structure with lamellar regions near the interface. Besides the increased α3L-crystal purity, the emulsion tempered at 30 °C showed an increased β′2L-crystal purity. Apparently, melting of the β′22L-crystals releases TAGs which may fit better in β′12L, but were incorporated in the β′22Lcrystals at the high cooling rate applied during production. The shift of those TAGs from one β′-fraction to the other increased the crystal purity of both fractions. The cryo-SEM image shows a finer, less organized crystal structure. The observed effects of tempering on the crystallization properties of semicrystalline oil-in-water emulsions may have major implications on its applicability. First, the physicochemical stability of the product prior to use is an important characteristic which may be influenced by its thermal history. Second, the use of such emulsions for whipping applications is determined by its susceptibility to partial coalescence. The latter is greatly influenced by the crystal structure in the fat droplets and therefore by its thermal history (publication in progress).

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +32 9 264 6198. Fax: +32 9 264 6218. E-mail: Kim. [email protected]. Web: http://www.fte.Ugent.be. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the ESRF (Grenoble, France) for the use of the synchrotron facilities. The Dutch-Belgian Beamline (DUBBLE) research group at the ESRF and the Dutch organization for scientific research (N.W.O.) are acknowledged J

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for their help and continuous support of the DUBBLE project (ESRF, Grenoble, France). Hercules foundation is acknowledged for its financial support in the acquisition of the scanning electron microscope JEOL JSM-7100F equipped with the cryotransfer system Quorum PP3000T and Oxford Instruments Aztec EDS (Grant Number AUGE-09-029). Benny Lewille is greatly acknowledged for his assistance with the experiments.

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DOI: 10.1021/acs.cgd.5b00665 Cryst. Growth Des. XXXX, XXX, XXX−XXX