Thermal Effects on Lipids Crystallization Kinetics under High Pressure

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Thermal Effects on Lipids Crystallization Kinetics under High Pressure Musfirah Zulkurnain,† V. M. Balasubramaniam,†,‡ and Farnaz Maleky*,† †

Department of Food Science and Technology, and ‡Department of Food Agricultural and Biological Engineering, The Ohio State University, 2015 Fyffe Court, Columbus, Ohio 43210, United States ABSTRACT: The crystallization behavior of a model fat consisting of fully hydrogenated soybean oil (FHSBO) and soybean oil (SBO) is studied under isobaric cooling (100 MPa), and atmospheric conditions (0.1 MPa). To monitor the relationship between pressure and the onset of crystallization at both isobaric cooling and adiabatic compression, samples were pressurized until they achieved three different maximum temperatures (70, 80, or 90 °C) under pressure. A lower induction time of crystallization (τ > 2.1 min−1) resulting in smaller crystals was observed during adiabatic compression compared to isobaric cooling (0.4 < τ < 0.9 min−1). Moreover, different crystal size distributions were observed for the different maximum temperatures applied. Using small-angle X-ray scattering analysis, a decrease in nanoplatelet thickness was documented in samples crystallized under high pressure compared to the control samples. The impacts of high pressure processing were also evident in the samples’ polymorphic behavior, when β crystal forms were seen in all high pressure crystallized samples. The control samples and samples that partially crystallized before pressurization showed a mixture of β and β′ forms. This study suggests the practical implementation of pressure induced crystallization during adiabatic compression, which is rapid and results in a homogeneous crystallization.

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INTRODUCTION Textural properties of fats as a semisolid are determined mainly by the solid component of the system, which exist as a threedimensional colloidal fat crystal network. The understanding of structural organization present in fat materials can be related to the number of crystalline elements, their binding into the crystal unit, and the interrelationships between the individual elements or their groupings.1 The structural level of a fat crystal network involves nanoscale (0.1−100 nm), microscale (0.1− 100 μm), and macroscale (0.1−100 mm). Upon crystallization, fat crystals aggregate, grow into clusters, flocs, and finally a three-dimensional network.2−5 Heat, mass, and momentum transfer conditions during the formation of a fat crystal network have significant effects on the final microstructure and macroscopic physical properties of fats. Triglycerides (TAGs) can crystallize into different solid state structures, depending on crystallization conditions.6 Phase transition is kinetically limited by the nucleation step where supersaturation is required to overcome the kinetic energy of molecules and the interfacial free energy of the new solid phase. It is well-known that pressure, just like temperature, is one of the most essential thermodynamic variables that control overall crystallization behavior. Pressurization reduces volume of the lipid system and establishes supersaturation conditions that act as the driving force for nucleation.7 This may increase the degree of crystallization and accelerate polymorphic transformation.8 Moreover, high pressure treatment has been proposed to facilitate microscopic reordering of lipid molecules.9,10 © XXXX American Chemical Society

Studies have also shown the relationship between the method of pressurization and onset of crystallization for lipids at isothermal conditions.11,12 For instance, the qualitative observation of in situ crystal formation of oleic acid under slow step and dynamic pressurization (at a slow compression rate of 0.3 MPa/s) up to 450 MPa at 20 °C were collected by Kosciesza et al. (2010) using high pressure cell.12 It was found that stable dense solid fat slowly grows under isobaric conditions from needle-like spherulites emerging from the vessel wall, while the dynamic pressurization resulted in an instantaneous formation of poorly packed crystals. There is still more to investigate about the effects of the rapid compression step at different combinations of pressure and temperature on the formation of specific crystal structure and their polymorphic and physical properties. We previously studied some of the physical properties of a binary model fat (fully hydrogenated soybean oil (FHSBO)/ soybean oil) crystallized under rapid compression coupled with subsequent isobaric cooling and showed the direct effect of the processing on the sample’s oil loss and storage modulus.8 The effect of these processing conditions on the fat system’s properties can be further understood from the structural organization of their underlying fat crystal networks. In the present study, we intended to continue with those initial analyses and elucidate fat crystallization mechanisms induced Received: June 2, 2017 Revised: July 28, 2017 Published: August 8, 2017 A

DOI: 10.1021/acs.cgd.7b00768 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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under rapid compression coupled with subsequent isobaric cooling conditions. The onset of crystallization and crystal development (at micro-, and nanoscales) of the model fat affected by different pressure levels and maximum temperatures under pressure, Tmax were studied and correlated to their physical and functional properties.

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MATERIALS AND METHODS

Materials. Fully hydrogenated soybean oil (FHSBO) consisting of tristearin (SSS, 79.6%) as the dominant TAG was kindly provided by Bunge Canada (Toronto, Canada) and used without further purification. Soybean oil (Kroger, Cincinnati, OH) was obtained from a local market. A model fat was prepared by blending 30% (w/w) FHSBO with soybean oil. The fatty acid composition of the model fat was determined according to AOCS Ce 1−62 standard method13 to consist of 39.4% linoleic acid (C18:2n6), 29.0% stearic acid (C18:0), 15.9% oleic acid (C18:1n9), 11.1% palmitic acid (C16:0), 4.5% γlinolenic acid (C18:3n6), and 0.2% linolenic acid (C18:3n3). The fatty acid composition of the fully hydrogenated soybean oil was 91.1% stearic acid (C18:0) and 8.9% palmitic acid (C16:0). Sample Pretreatment. The model fat was melted at 90 °C for 15 min under constant stirring to erase the crystal memory before it was packaged in high-barrier pouches (0.01 m width × 0.05 m height) (Fisher Scientific, Pittsburgh, PA) and heat-sealed. For each crystallization experiment (both atmospheric and high pressure), about 8 g of sample packaged in four pouches was loaded into a sample carrier of 0.01 L polypropylene syringe (Becton Dickinson and Co., Franklin Lakes, NJ). The thermocouple was placed in the middle of the cylindrical sample carrier holder at the center where four pouches of samples were suspended in oil and the oil was in direct contact with the thermocouple. The sample carrier was insulated with two layers of tape (CVS Pharmacy Inc., Woonsocket, RI) to ensure the entire volume of the syringe content experience nearly homogeneous heat of compression during pressurization.14 Prior to each experimental run, the samples were thermally equilibrated in a water bath at 90 °C for 15 min. Sample Crystallization without High Pressure (Static Crystallization). As a control, samples were crystallized at atmospheric pressure (0.1 MPa) from 70, 80, and 90 to 30 °C for 30 min at a cooling rate of ∼4 °C/min. The experiments were conducted by immersing the samples in a temperature-controlled bath at 30 °C for 30 min, and sample’s temperature was recorded every 1 s using DasyLab software (version 7.00.04, National Instruments Corp., Austin, TX). High Pressure Crystallization. The high pressure experiments were conducted using a high-pressure kinetics tester (PT-1, Avure Technologies, Kent, WA) with a 0.054 L vertical high-pressure chamber (0.02 m internal diameter).8 The pressure chamber was immersed in a temperature-controlled bath maintained at 30 °C. Propylene glycol (Brenntag North America Inc., Reading, PA) was used as the pressure-transmitting fluid. A high-pressure intensifier (M340A, Flow International, Kent, WA) was used to achieve a pressure level up to 700 MPa at the rate of 12 MPa/s. The decompression time was at 4 s regardless of holding pressure. The pressure and temperature were monitored and recorded every 1 s using DasyLab software (Version 7.00.04, National Instruments Corp., Austin, TX). Prior to high pressure treatment, the samples were thermally equilibrated in a water bath at 90 °C for 15 min. Samples were loaded into the high pressure chamber and allowed to reach a predetermined initial temperature (Ti), in order to reach a maximum temperature under pressure (Tmax). The pressure levels were from 100 to 600 MPa (at 100 MPa interval), and the Tmax was either 70, 80, or 90 °C. Each high pressure treatment was conducted in three steps as shown in Figure 1A for Tmax = 90 °C and pressurization at 100, 200, 300, and 400 MPa. Sample’s pressure and temperature histories are shown by dotted and solid lines, respectively. Compression step increased sample’s temperature quasi-instantaneously from the predetermined Ti to Tmax of 90 °C due to the generation of heat of compression. Then, the samples were held under the targeted pressure and allowed to cool

Figure 1. Temperature and pressure histories of samples crystallized at (A) maximum temperature under pressure (Tmax) of 90 °C at (i) 100 MPa, (ii) 200 MPa, (iii) 300 MPa, and (iv) 400 MPa; and (B) Tmax of 80 °C at (1) 100 MPa, (2) 300 MPa, and (3) 600 MPa with the inset showing the onset of crystallization during adiabatic compression at 300 MPa. Ti indicates initial temperature. for 10 min by maintaining the chamber’s temperature at 30 °C, prior to depressurization to atmospheric pressure. It is important to mention that the processing time and treatment were slightly different for samples with Tmax of 70 °C at 400−600 MPa and 80 °C at 600 MPa. When subjected to pressure treatment, the sample experienced higher heat of compression values at higher pressure levels,14 and thus, a lower Ti was sufficient to achieve Tmax under pressure. For example, Figure 1B shows a representative of pressure-thermal history of the sample treated at 600 MPa and Tmax of 80 °C (also see Table 1 for corresponding Ti values). Therefore, a delay in the samples’ pressurization was applied in these samples. As shown in Table 1, some samples have Ti lower than 41.3 °C where the onset of crystallization happens at atmospheric pressure. These samples (highlighted with star symbol in Table 1) have the evidence of crystallization at atmospheric pressure prior to the compression step that introduced seed crystals in the samples before pressurization. This

Table 1. Initial temperature (Ti) of Samples Pressurized at Different Combinations of Pressure (P) and Maximum Temperature (Tmax) under Pressure initial temperature of samplea (Ti, °C) pressure (MPa) 100 200 300 400 500 600

at Tmax = 70 °C 61.2 51.4 41.6 41.1 40.5 35.8

± ± ± ± ± ±

0.3 0.2 0.3 0.2* 0.8* 0.5*

at Tmax = 80 °C 72.0 63.9 52.1 42.7 42.1 41.0

± ± ± ± ± ±

0.3 0.6 0.4 0.8 0.1 0.3*

at Tmax = 90 °C 82.0 74.5 67.3 52.2 54.1 45.7

± ± ± ± ± ±

0.4 0.3 0.2 0.7 0.4 0.2

a

Star symbol indicates sample’s initial temperature has fallen below crystallization temperature of 41.3 °C and evident crystallization event prior to pressurization.

B

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Table 2. Solid Fat Content (SFC) and Specific Enthalpy of Melting Samples As Function of Pressure and Maximum Temperature under Pressure (Tmax)a specific enthalpy of meltingb (ΔH, × 103 J/kg)

solid fat contentb (SFC, %) pressure (MPa) 0.1 100 200 300 400 500 600

Tmax = 70 °C 28.0 30.4 30.4 30.3 28.3 28.2 28.0

± ± ± ± ± ± ±

0.3B,a 0.5A,a 0.6A,a 0.5A,a 0.4B,b 0.5B,b 0.5B,b

Tmax = 80 °C 27.7 30.3 30.0 30.4 30.4 30.4 28.1

± ± ± ± ± ± ±

0.3B,a 0.4A,a 0.8A,a 0.7A,a 0.3A,a 0.4A,a 0.6B,b

Tmax = 90 °C 27.7 30.1 29.8 30.6 30.3 30.3 30.2

± ± ± ± ± ± ±

Tmax = 70 °C

0.5C,a 0.3B,a 0.3B,a 0.4A,a 0.4A,a 0.6A,a 0.3A,a

58.4 55.3 56.2 57.6 56.1 58.2 57.9

± ± ± ± ± ± ±

0.3A,a 2.2A,b 1.2A,a 2.9A,a 1.3A,a 0.1A,ab 0.6A,a

Tmax = 80 °C 58.6 59.8 56.8 56.6 56.6 58.2 54.6

± ± ± ± ± ± ±

1.2A,a 0.7A,a 0.9AB,a 2.4AB,a 2.2AB,a 1.9AB,a 3.5AB,a

Tmax = 90 °C 58.3 61.0 54.2 56.7 54.1 53.9 55.6

± ± ± ± ± ± ±

1.5AB,a 1.0A,a 2.2B,a 1.4B,a 1.7B,a 1.3B,b 0.2B,a

a Control samples were crystallized at atmospheric pressure (0.1 MPa). bMeans in the same column followed by different upper-case letters are significantly different (P < 0.05) between pressure levels for (n = 3) number of sample; means in the same row followed by different lower-case letters are significantly different (P < 0.05) between maximum temperature under pressure (Tmax).

is true for samples pressurized at 400−600 MPa with Tmax of 70 °C and at 600 MPa with Tmax of 80 °C. For the rest of this study, these samples are labeled as Group III and are used to compare the effect of high pressure crystallization of the model fat in the presence of seed crystal formed during atmospheric condition (before high pressure application). Figure 1 with all the detailed information is justified in the Results and Discussion. Solid Fat Content (SFC) Measurement. Using a pulse nuclear magnetic resonance (p-NMR) spectrometer (Bruker Minispec mq20, Bruker Corporation, MA, USA)15 samples’ SFC values were measured right after processing by placing about 0.5 g of the crystallized sample into a glass vial (Sigma-Aldrich, St. Louis, MO) stored at 20 °C. The reported data correspond to the average of five individual measurements. Thermal Properties Measurement. Thermal behavior of the crystallized samples was studied using a differential scanning calorimetry equipped with a refrigerated cooling system (Q2000, TA Instrument, New Castle, DE). About 10−15 mg of each sample was hermetically sealed in an aluminum pan, and an empty pan was used as a reference. The sample was held isothermally for 5 min at 20 °C and then heated from 20 to 80 °C at 5 °C/min. The peak melting temperature (Tm), the onset of crystallization (To) and enthalpy of melting (ΔHm) were determined using TA Universal Analysis 2000 (TA Instrument, New Castle, DE). Powder X-ray Diffraction (XRD) Analysis. The polymorphic property of the crystallized samples was determined using a powder Xray diffractometer (Rigaku Miniflex, Rigaku, Japan). The analyses were performed using Bragg-Bretano geometry with a 1.25° divergence slit, a 1.25° scatter slit, a 0.3 mm receiving slit, and copper lamp set to 40 kV and 44 mA. Approximately 1 g of each sample was placed on a glass X-ray slide, and scans were performed from 10 to 30 deg 2-theta at a scanning rate of 2°/min. The results were analyzed using PDXL software Version 2.0 (Rigaku, Japan). The identification of polymorphic forms of the samples was performed based on XRD short spacing values reported in refs 16−18. For model fat of fully hydrogenated soybean oil and soybean oil, the α form displayed a single diffraction line of d-spacing at 4.15 Å, while the β′ form is characterized by two strong diffraction lines at 3.8 and 4.2 Å. The samples β form is associated with a series of diffraction lines, one prominent at 4.6 Å and lines of lesser intensity at 3.7 and 3.8 Å. In this study, the mixture of β and β′ type crystal contents in the samples was estimated by relative intensity of the short spacing at 4.2 and 4.6 Å. A fourth order polynomial background was applied to the X-ray diffraction patterns and then fitted to a Voigt function.15 The polymorph present in the greatest proportion was qualitatively determined by a comparison of the peak heights. The model fat is a β tending fat contributed by large tristearin content from fully hydrogenated soybean oil, as reported by others.16,18,19 The polymorphic transformation of β′ → β phase was monitored during storage over 28 days. Small Angle X-ray Scattering (SAXS) Analysis. The thickness of the samples crystalline nanoplatelets or crystalline domain size (ξ)

was determined using small-angle X-ray scattering (SAXS) analysis according to Acevedo and Marangoni (2010).20 Samples were scanned from 0.5 to 3° at a scan rate of 0.05°/min using SAXS instrument (Rigaku SmartLab, Rigaku, Japan). The copper lamp was set to 40 kV and 44 mA, with 0.57 divergence slit, 0.57 scatter slit, and 0.3 mm receiving slit. From the SAXS pattern, the crystalline domain size (ξ) was calculated using the Scherrer formula:21 ξ=

Kλ FHWM cos(θ)

(1)

where K is the shape factor, in which a value of 0.9 is used for crystallites of unknown shape,22 θ is the diffraction angle of the X-rays (in rad), fwhm is the full width at half of the maximum peak height corresponding to the (001) plane (in rad), and λ is the wavelength of the X-ray, 1.54 Å for copper. PeakFit software (Seasolve, Framingham, MA, USA) was used to analyze fwhm from the measured peak. Microstructure Analysis and Particle Size Distribution. A polarized light microscope (Axio Imager.M2m, Carl Zeiss Microscopy GmbH, Germany) with high-resolution CCD camera was used to analyze microstructure of the crystallized samples. To obtain satisfactory reproducibility of slides, a definite amount of the crystallized sample was placed on each microscope slide, spread in all directions and gently secured with coverslips. Images were acquired using 20× and 50× objective lens and AxioVision software (Carl Zeiss Microscopy GmbH, Germany). The microstructure images at 20× magnification were manually thresholded, converted to grayscale, and analyzed for particle size distribution using ImageJ software (version 1.50b, National Institutes of Health, Bethesda, MD). The particle size distributions were fitted using Gaussian function in PeakFit software (Seasolve, Framingham, MA, USA) to determine mean particle equivalent diameter and standard deviation from full width at half-maximum of the Gaussian peak. The crystal clusters size was measured using Circle feature of AxioVision software on images at 50× magnification. The program assumes a circular geometry and obtains the square root of the quotient of the area to π. Statistical Analysis. One-way ANOVA was applied to analyze statistical differences between group means established at P < 0.05 using MINITAB version 16 statistical software (Minitab, Inc., State College, PA). Multiple comparisons of means were performed using Tukey’s test.

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RESULTS AND DISCUSSION The solid fat content (SFC) of all samples crystallized at different maximum temperature under pressure (Tmax) under high pressure are shown and compared in Table 2. High pressure treatments for 10 min increased the sample’s SFC by about 2.6% to 30.6% from 27.7% in the control samples. However, samples crystallized at 400−600 MPa for Tmax of 70 °C and at 600 MPa for Tmax of 80 °C (Table 2) that were C

DOI: 10.1021/acs.cgd.7b00768 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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pressurized at the presence of seed crystal, have SFC similar to those of the control samples. Hence, the effect of high pressure treatments on lipid crystallization can be categorized into two categories consistent with the nature of the samples prior to pressurization. First, samples pressurized in the state of completely melt are labeled as Group I and II that will be discussed later in this publication according to their distinct structural properties. Second, samples pressurized with the presence of some fraction of seed crystal are labeled as Group III. These two categories of samples were also distinct in their polymorphic behaviors. Figure 2A shows X-ray diffraction patterns for the wide-angle X-ray diffraction (WAXD) region for samples crystallized at Tmax of 80 °C at different pressure levels that include both samples crystallized from the melt (pressurized at 100 to 500 MPa) and samples with the presence of seed crystals (pressurized at 600 MPa). However, for clarity of presentation not all patterns were reproduced in the figure. All samples crystallized under high pressure from the melt show the presence of the β polymorphic phase from the existence of a strong diffraction peak at 4.6 Å with two peaks of lesser intensity at 3.7 and 3.8 Å. This shows that crystallization under high pressure treatment from the melt favors adoption of most thermodynamically stable polymorphic forms that can be related to an increase in a high degree of supersaturation.7,23 Moreover, the samples’ XRD pattern for 600 MPa in Figure 2A shows an additional peak at 4.2 Å, which is associated with the presence of β′ polymorph, in addition to the β polymorphic phase.16 As shown in Table 1, for Tmax of 80 °C only samples pressurized at 600 MPa had seeds prior to pressurization. A similar XRD pattern is observed for the control samples (0.1 MPa) in Figure 2A. This suggests that kinetic entrapment of TAG molecules into a less stable polymorphic phase during crystallization at atmospheric pressure prior to pressurization may retard the effect of a rapid volume reduction reaction of the high pressure treatment.24 The effects of the applied processing on the samples thermal properties are shown in Figure 2B. For the sake of reasonable clear presentation of data, only a limited number of the melting thermogram of differential scanning calorimetry (DSC) profiles is plotted in this figure for Tmax of 80 °C. The existence of β polymorphic phase in the samples crystallized under pressure from the melt (100 and 500 MPa) is confirmed from the appearance of a single exothermic peak at 62.19 °C in the melting thermogram. Moreover, the melting thermogram of sample pressurized at 600 MPa (Group 1) occurs in two steps that correspond to a shoulder at ∼50 °C suggesting the presence of β′ phase and a prominent peak at ∼62 °C corresponding to the β phase. Similar results were observed in the control samples (0.1 MPa). This is in agreement with peak melting temperature of β polymorphs reported by Ribeiro et al. (2009) for static crystallization of a similar fat mixture of 30% FHSBO/soybean oil at 63.4 °C.18 Thermal behaviors of the samples observed peak melting temperatures in the range of 61.68−62.73 °C (data not shown), with no significant trend detected. The onset temperature of the primary melting peak is in the range of 54.06−58.83 °C. However, the presence of a shoulder observed in samples with the mixture of β′ and β phases have the onset of melting temperature in the range of 42.30−44.52 °C as shown in the inset of Figure 2B. Table 2 shows specific enthalpy of melting for all the 21 samples that range from 53.9 × 103 to 61.0 × 103 J/kg. There is no significant trend detected in the position of

Figure 2. (A) X-ray diffraction patterns of samples as a function of pressure and maximum temperature under pressure (Tmax) of 80 °C. (B) Melting thermograms of samples crystallized at 0.1 MPa (atmospheric crystallization), 100, 500, and 600 MPa at maximum temperature of 80 °C. (C) Changes of X-ray diffraction patterns of high pressure crystallized samples over 4 weeks of storage for 100 and 600 MPa at maximum temperature under pressure of 80 °C.

the peak melting temperatures and specific enthalpy of melting with either increase in pressure or maximum temperature under pressure. This is because high pressure is a physical treatment that does not impart chemical modification to the fat composition.25,26 Studies have shown that physical treatment such as laminar shearing27 and rapid cooling28 did not alter the melting temperature of a similar fat system. These dissimilarities of the crystallization properties of the system under high pressure processing may be better D

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of other crystals.29 Analogous observations were reported by other researchers for similar fat systems crystallized under static conditions,18,27,30 where crystallization under mild agitation has been shown to promote the crystal aggregation. Himawan et al. (2006) associated this behavior to the network’s solid fat content. As solid fat content decreases, lower viscosity of the melt enhances molecular mobility resulting in the formation of larger crystals.31 This statement may explain our observation for the control samples in this study. The similarity in crystal morphology of large spherulitic nature agrees with their polymorphic phase, supporting the evidence that highly structured β′ seed crystals were introduced prior to pressurization. The mean cluster diameter of the samples as a function of pressure and Tmax is presented in Figure 4. The mean cluster

investigated at the microscale. Overall, the crystal morphology of high pressure treated samples can be distinguished into three distinct groups depending on different combination of pressure level and Tmax. Figure 3 shows PLM micrographs of samples

Figure 3. Microstructure at 500× magnification of samples crystallized at maximum temperature of 80 °C under (A) 100 MPa (Group I), (B) 500 MPa (Group II), (C) 600 MPa (Group III), and (D) 0.1 MPa (atmospheric crystallization).

crystallized at Tmax of 80 °C for 100, 500, and 600 MPa, illustrating representatives of Group I, II, and III, respectively, compared to the control sample. Since a similar trend was observed for other samples, for simplification only the representatives were reported. As shown in Figure 3A,B, crystallization under high pressure treatments from the melt (initial temperature > 41.3 °C) affected the crystal’s morphology differently at different pressure levels. At 100 MPa (Figure 3A), the crystal clusters were discrete with a mixture of large and small sizes, which is labeled as Group I. Interestingly, at high pressure levels above a certain pressure threshold, changes in the crystal morphology were notified on the cluster and particle size distribution. For Tmax of 80 °C, the pressure threshold was observed at 300 MPa. As shown in Figure 3B for sample at 500 MPa, the crystals visually appear smaller and embedded in well-distributed mesh of high density of small microstructural elements. The boundaries of the crystal clusters are also poorly defined. This crystal morphology is labeled as Group II. Moreover, the discrete nature of crystal clusters observed in the microstructure of Group I (Figure 3A) is less apparent in Group II’s micrographs (Figure 3B). This modification in crystal morphology has never been reported before that may be possibly created under the influence of quasi-instantaneous volume reduction that promotes homogeneous nucleation into formation of high number of small crystals. These drastic changes in the crystal morphology from Group I to Group II were more prominent at high Tmax, and the pressure threshold to induce these changes was observed at 300 and 400 MPa for Tmax of 80 and 90 °C, respectively. On the other hand, when samples’ nucleation happened at atmospheric condition prior to pressurization (Group III), a network of discrete spherulites of large structures was formed as shown in Figure 3C for sample crystallized at 600 MPa and Tmax of 80 °C. Like the control samples, atmospheric crystallization also yielded a microstructure composed of discrete spherulites of predominantly large crystal clusters, as shown in Figure 3D. These structures are possibly the aggregates of smaller elements, which are themselves aggregates

Figure 4. Mean diameter of crystal cluster as a function of pressure levels in samples crystallized at maximum temperature under pressure of 70−90 °C. Control samples were crystallized at atmospheric pressure (0.1 MPa). Different superscript letters represent significantly different diameter between pressure level at P < 0.05 for (n = 3) number of sample.

diameter in the range between 22.2 and 30.3 μm were observed at low pressure levels below the mentioned pressure thresholds that can be categorized into microstructure of Group I. Increase in pressure levels reduced these mean cluster diameters. At high pressure levels, the mean cluster diameter further reduced to the range between 12.5 and 14.9 μm that was categorized as microstructure of Group II. These mean cluster diameters were less affected by pressure levels. This is evident above the mentioned pressure thresholds of 300 and 400 MPa for Tmax of 80 and 90 °C, respectively. A closer look at the XRD pattern shows the differences between crystal properties of Group I and Group II, although both groups exhibit β polymorphic phase. For instance, as shown in Figure 2C the peak intensity of XRD patterns for sample crystallized at 100 MPa and Tmax of 80 °C (Group I) increased considerably after 4 weeks, while the sample at 500 MPa and Tmax of 80 °C did not change much. The peak intensity at 4.6 Å for the sample at 100 MPa increased by 58% from 1.59 × 104 to 2.51 × 104 a.u. that suggests the event of crystal growth in this sample during storage. The high pressure treatment significantly reduced the mean clusters diameter into three different group of sizes. For samples pressurized with the presence of seed crystals which were categorized as Group III have mean cluster diameter ranged between 33.3 and 34.9 μm (Figure 4). The mean cluster diameters are not significantly affected by pressure levels or E

DOI: 10.1021/acs.cgd.7b00768 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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and 90 °C. The mean crystal size distributions are reported in Table 3. The control samples show the largest normal distribution with mean particle size distribution in the range of 3.80−4.04 μm, and standard deviation in the range of 2.28− 2.34 μm. At Tmax of 70 °C (Figure 5A), notably two groups of particle size distributions of larger range (Group III) between 3.65 and 3.78 μm belong to samples crystallized in the presence of β′ seeds crystal and smaller ones (Group I) with a mean equivalent diameter ranged between 2.45 and 2.84 μm. The standard deviation reduced from 2.30 μm (Group III) to below 1.49 μm (Group I) that show significant reduction in particle size distribution when crystallization happens under pressure from the melt. At higher Tmax of 80 °C (Figure 5B), much smaller particle size distributions are apparent with the introduction of Group II at a combination high Tmax and high pressure levels (300, 400, and 500 MPa). Samples in Group II have a mean particle size below 2.04 μm and show no significant difference in particle size distributions between pressure levels with average standard deviation of 1.13 μm. On the other hand, at highest Tmax of 90 °C (Figure 5C), all samples crystallized from the melt show only Group I (100− 300 MPa) and II (400−600 MPa). The mean particle size of Group I reduced from 2.88 to 1.39 μm with an increase in pressure from 100 to 300 MPa, while Group II was not significantly affected by pressure levels. The particle size distributions of Group II was smaller given by standard deviation of ≤1.07 μm) compared to Group I (standard deviation ranged between 1.28 and 1.51 μm). This shows that at high pressure levels, an increase in maximum temperature significantly reduced particle size distribution of the aggregates at mesoscale. The formation of large microstructures is started from the aggregation of nanoscale primary crystals, known as nanoplatelets.2 Table 3 summarizes the domain size or the nanoplatelet thickness quantified using the Scherrer formula for all the samples. The full width at half-maximum (fwhm) of the d001 Bragg peak was determined to be at 0.004−0.006 radians at d = 44.8−45.9 Å. Using the Scherrer formula (eq 1), the crystalline domain size of the samples was calculated to be in the range of ∼27−34 nm. The d-spacing of Bragg peak (d001) from small-angle X-ray scattering pattern has been shown to correspond to the thickness of a lamella, which is the length of a unit cell in the c-axis direction. For example, the long spacing of β′ and β polymorph of C18 has been documented at 46.8 and 45.2 Å, respectively, that well corresponds to lamella thickness of the respective polymorphs.32,33 Therefore, the crystalline domain values of the samples indicate the stacking of about 6−7 lamellae per crystalline nanoplatelets. This shows an agreement with previous studies from Acevedo and Marangoni (2010)34 that showed the stacking of 7−10 platelets from fat systems containing fully hydrogenated canola oil and analogous results for milk fats by Ramel et al. (2016).35 In addition, control samples and samples in Group I have d001 spacing in the range of 46.1−47.2 Å, corresponding to thicker lamella, in this group due to the presence of less stable β′ form, compared to samples in Group I and II (all in β form) with d001 spacing in the range of 44.6−45.9 Å. However, only small differences are observed between the nanoplatelet thickness of the control samples and the samples crystallized under pressure from the melt, as shown in Table 3. Samples crystallized under high pressure treatments with β polymorph crystals have mean nanoplatelet thicknesses in the range of 25.96−27.38 nm, while samples in Group III (a

Tmax. Although these samples have similar crystal morphology with the control samples, the mean cluster diameter of the control samples are significantly larger in the range of 37.15− 42.17 μm (Figure 4). This shows that the subsequent high pressure treatment significantly reduced crystal growth compared to the atmospheric crystallization alone. The differences between the microstructure of Group III compared to control samples can be discussed further from the polymorphic transformation of β′ → β phase during storage over 4 weeks. The complete transformation of β′ → β polymorphic phase took 22 days for control samples, while samples crystallized at 600 MPa in the presence of β′ seed crystal happened in less than 14 and 7 days, for Tmax of 70 and 80 °C respectively. This is expected because of their large spherulitic crystal structure. The time needed to reach equilibrium forms of β is determined by the crystalline precursor because the solid state transformation of β′ → β involves diffusion controlled mechanism where highly structured β′ structure restricts molecular rearrangement.16 Moreover, the longer time for the polymorphic transformation to β phase in the high pressure crystallized samples at Tmax of 70 °C compared to 80 °C may reflect a larger amount β′ seeds crystal in sample at lower Tmax, which also agrees with its lower initial temperature (Table 1). Therefore, we can postulate that the subsequent high pressure treatment induces the formation of the β polymorphic phase from the remaining melt apart from the β′ fraction introduced in the sample earlier. The particle size distribution of microstructure elements was fitted into Gaussian function as shown in Figure 5 for control and samples crystallized under high pressure at Tmax of 70, 80,

Figure 5. Particle size distributions of microstructure element as a function of pressure for maximum temperature under pressure (Tmax) of (A) 70, (B) 80, and (C) 90 °C. F

DOI: 10.1021/acs.cgd.7b00768 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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Table 3. Mean Crystal Size Distributions (μ) and Nanoplatelet Thickness (ξ) of Samples As Function of Pressure and Maximum Temperature under Pressure (Tmax)a mean crystal size distributionsb (μ, μm) pressure (MPa) 0.1 100 200 300 400 500 600

Tmax = 70 °C 3.82 2.84 2.45 2.69 3.65 3.72 3.78

± ± ± ± ± ± ±

0.1A,b 0.1C,a 0.1D,a 0.1C,a 0.1B,a 0.1B,a 0.1B,a

Tmax = 80 °C 3.89 2.76 2.42 2.04 1.84 2.01 3.78

± ± ± ± ± ± ±

0.1A,ab 0.1B,a 0.1C,a 0.1D,b 0.1E,b 0.1D,b 0.1A,a

nanoplatelet thicknessb (ξ, nm)

Tmax = 90 °C 4.04 2.88 2.41 1.69 1.39 1.46 1.47

± ± ± ± ± ± ±

Tmax = 70 °C

0.1A,a 0.1B,a 0.1C,a 0.1D,c 0.1D,c 0.1D,c 0.1D,b

29.19 27.00 25.96 26.35 28.44 28.25 29.04

± ± ± ± ± ± ±

Tmax = 80 °C

0.5A,a 0.9BC,a 0.2C,b 0.2C,ab 0.5A,a 0.9AB,a 0.2A,a

29.62 27.03 27.38 26.46 26.37 26.90 28.44

± ± ± ± ± ± ±

Tmax = 90 °C

0.6A,a 0.2C,a 0.3BC,a 0.2C,b 0.7C,b 0.5C,b 0.7AB,a

29.50 27.23 27.35 27.13 26.40 26.31 26.41

± ± ± ± ± ± ±

0.3A,a 0.7AB,a 0.9AB,a 0.8AB,a 0.5B,b 0.4B,b 0.9B,b

a The mean crystal size distribution was calculated using Gaussian function, and the nanoplatelet thickness or crystalline domain size was determined by Scherrer analysis. bMeans in the same column followed by different upper-case letters are significantly different (P < 0.05) between pressure levels; means in the same row followed by different lower-case letters are significantly different (P < 0.05) between maximum temperatures under pressure (Tmax).

mixture of β′ and β phases) ranged between 28.44 and 29.04 nm. They are comparable to atmospheric crystallized samples in the range of 29.19−29.62 nm that were also a mixture of β′ and β phases. This can be explained from the orthorhombic chain packing of β′ crystal, which is less tilted than triclinic chain packing of β crystal.32,33 Therefore, differences in the polymorphic phase may contribute to the small differences at the nanoscale that may suggest that the formation of nanoplatelet is not affected by the high pressure treatments. Mechanistic Considerations. In order to understand the mechanism of lipid crystallization under high pressure, it is important to relate the structural properties of high pressure crystallized samples to their thermal histories during high pressure treatments. For each Tmax the temperature−pressure histories consisted of an adiabatic compression step followed by an isobaric cooling step and a rapid depressurization step (Figure 1A). As shown by the black arrows in Figure 1A, the onset of crystallization was detected from a sudden increase in temperature during isobaric cooling, which is associated with the release of latent heat of crystallization.36−38 The peak temperature during this phase change was taken as the crystallization temperature (Tc) and found to be at 64.5, 79.7, and 89.9 °C under isobaric conditions at 100, 200, and 300 MPa, respectively. The Tc increment with an increase in pressure is in agreement with the Clausius−Clapeyron relation.11 And one may conclude that beyond 300 MPa, the crystallization will occur at Tc > 89.9 °C. Therefore, at 400 MPa the Tc will be higher than Tmax (90 °C). This can explain why no crystallization was observed during isobaric cooling of sample pressurized at 400 MPa and Tmax 90 °C (line iv in Figure 1A). Instead, the onset of crystallization is observed during adiabatic compression step (at a lower pressure of 164.4 MPa and Tc of 68.9 °C). This phenomenon is illustrated in the inset of Figure 1A that shows a slight increase in temperature during adiabatic compression with the release of latent heat of crystallization. As summarized in Table 1, the sample at 400 MPa is pressurized at a lower Ti (52.2 °C) compared to samples at 100−300 MPa. It is important to note that the crystallization during adiabatic compression was observed at a lower pressure level (300 MPa) when Tmax was reduced to 80 °C (see Figure 1B). The pressure threshold for the onset of crystallization to happen during adiabatic compression was found at 200 MPa with reduction in Tmax to 70 °C (data are not shown). Studies have shown that an increase in pressure increases the degree of supersaturation for nucleation and consequently

increases the rate of crystallization.11,39 To investigate this concept in this study, the time required for the onset of crystallization to occur was calculated and reported in Table 4 Table 4. Inverse Induction Time of Crystallization (τ) for the Onset of Crystallization As Function of Pressures and Maximum Temperature under Pressure (Tmax) inverse induction time (τ, min−1)a pressure (MPa) 0.1 100 200 300 400 500 600

Tmax = 70 °C 0.14 0.65 2.13 2.17 0.14 0.13 0.14

± ± ± ± ± ± ±

0.01C,a 0.003B,a 0.01A,a 0.01A,a 0.002C,b 0.002C,b 0.002C,c

Tmax = 80 °C 0.13 0.35 0.91 2.14 2.15 2.13 0.15

± ± ± ± ± ± ±

0.01D,a 0.01C,b 0.01B,b 0.01A,a 0.01A,a 0.01A,a 0.002D,b

Tmax = 90 °C 0.12 0.38 0.47 0.53 2.13 2.14 2.17

± ± ± ± ± ± ±

0.02E,a 0.01D,b 0.01C,c 0.01B,b 0.01A,a 0.01A,a 0.01A,a

a

Means in the same column followed by different upper-case letters are significantly different (P < 0.05); means in the same row followed by different lower-case letters are significantly different (P < 0.05).

as the inverse induction time of crystallization. As shown in this table, the inverse induction time ranges between 0.12 and 0.14 min−1 at atmospheric pressure and increases to 0.35−0.91 min−1 for 100−200 MPa at Tmax of 80 °C when the crystallization took place during isobaric cooling (Group I). It is also shown that the rate of crystallization of Group I increases with an increase in pressure. For example, at Tmax of 90 °C, the inverse induction time increases from 0.38 min−1 at 100 MPa to 0.47 min−1 at 200 MPa, and 0.53 min−1 at 300 MPa. However, when the crystallization happens during rapid compression step (Group II) above 300 MPa, the inverse induction time drastically increases to a range between 2.13 and 2.17 min−1. In contrast, samples in Group III show low inverse induction time values (ranging between 0.13 and 0.14 min−1) comparable to control sample. This is not surprising since for Group III the onset of crystallization occurred at atmospheric pressure. The data in Table 4 can also explain the observation of different crystal morphologies of different Groups shown in Figure 3. The broad crystal size distribution of Group I (Figure 5) may suggest that the crystallization mechanism during isobaric cooling is heterogeneous in nature similar to atmospheric crystallization. This is parallel to in situ observations reported for oleic acid12 and triolein11 under isobaric conditions that exhibit heterogeneous nucleation and crystal growth similar to static crystallization. However, the G

DOI: 10.1021/acs.cgd.7b00768 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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ORCID

presence of smaller clusters in Group I (almost half in size) compared to the control samples indicates that the volume reduction under high hydrostatic pressure not only promotes formation of a denser structure of β polymorphs, but also limits the crystal growth event. This may also help to reach the maximum network density earlier compared to atmospheric conditions when the edge of adjacent clusters touches each other.11 Although Ferstl et al. (2011) reported a higher crystal growth rate with an increase in pressure,11 in this study the reduction of the cluster size alongside the increase in pressure could be reasoned by the early attainment of the maximum network density at a higher pressure level. At high pressure levels above 400 MPa, the drastic changes in the crystal morphology observed for Group II could be explained by their high rate of crystallization during the adiabatic compression step. Garside and Davey (1980) hypothesized that lipid crystallization with the domination of nucleation over crystal growth leads to the formation of a higher number of small crystals.40 Under medium pressure levels (200 and 300 MPa), crystallization was induced during adiabatic compression by reducing the Tmax. However, it is interesting to note that although the rate of crystallization was high for samples crystallized at Tmax of 70 °C and medium pressure levels the crystal size distribution was large comparable to Group I (Figure 5A). The reduction in Tmax also reduced Ti that may induce nucleation at atmospheric pressure prior to pressurization when Ti falls into the metastable region.

Farnaz Maleky: 0000-0003-4927-3627 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research was a joint contribution between The Food Safety Engineering and Lipid Analysis laboratories at The Ohio State University. M.Z. gratefully acknowledges the financial support from Ministry of Higher Education, Malaysian Government. Support in part provided by USDA National Institute for Food and Agriculture HATCH projects and the food industry is also gratefully acknowledged. References to commercial products or trade names are made with the understanding that no endorsement or discrimination by The Ohio State University is implied.

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ABBREVIATIONS TAG, triacylglyceride; FHSBO, fully hydrogenated soybean oil; SBO, soybean oil; maximum temperature SFC, solid fat content; XRD, X-ray diffraction; SAXS, small-angle X-ray scattering; fwhm, full width of half-maximum; WAXD, wide angle diffraction; PLM, polarized light microscopy

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CONCLUSION In this study, data from PLM, XRD, and SAXS all provided evidence that high pressure treatments affected the crystal morphology, crystal size distribution, polymorphic properties, and nanostructure of the fat crystal network of principally tristearin. The effects of temperature and pressure levels on the structural and physical properties depend on the rate of crystallization as where the onset of crystallization took place. While high pressure treatment favors formation of the most stable polymorphic form, rapid crystallization during dynamic compression step reduces particle size distribution compared to isobaric cooling. In particular, the crystallization during the adiabatic compression step at high pressure levels is favorable in the formation of the high density of small crystals due to a large volume reduction and high melting temperature under pressure that provides a high degree of supersaturation for nucleation. An increase in maximum temperature under pressure not only increases the pressure threshold for rapid crystallization during compression step but also increases the initial temperature to avoid nucleation prior to pressurization. Although crystallization during isobaric conditions is heterogeneous in nature, an increase in pressure reduces the crystal size due to an increase in the rate of crystallization with an increase in melting temperature at higher pressure levels. By clarifying the complexity of interaction effects between temperature and pressure during high pressure crystallization, the above findings provide direction for future research that suggests a better understanding of the phase diagram. This demonstrated that high pressure crystallization may be used to produce lipid based foods with improved texture and structural properties.

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Corresponding Author

*E-mail: [email protected]. Phone: 614 688 1491. H

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DOI: 10.1021/acs.cgd.7b00768 Cryst. Growth Des. XXXX, XXX, XXX−XXX