Effects of Glycerol and Tween 60 on the Crystallization Behavior

Effects of Glycerol and Tween 60 on the Crystallization Behavior, Mechanical Properties, and Microstructure of a Plastic Fat Jerrold W. Litwinenko, Anand Pal Singh, and Alejandro G. Marangoni*

CRYSTAL GROWTH & DESIGN 2004 VOL. 4, NO. 1 161-168

Department of Food Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada Received July 19, 2003;

Revised Manuscript Received August 20, 2003

W This paper contains enhanced objects available on the Internet at http://pubs.acs.org/crystal. ABSTRACT: The addition of 0.10% glycerol or Tween 60 increased the yield force of a 70:30 (w/w) mixture of triolein/HMF (high-melting fraction of milkfat) at 5 °C. Further increases in glycerol or Tween 60 caused a decrease in yield force. Solid fat content (SFC) versus time profiles at 30 °C (glycerol) and 28 °C (Tween 60) allowed for the determination of Avrami rate constants of crystallization (k) and Avrami exponents (n). Higher Avrami constants and lower Avrami indices (higher rate of crystal growth), smaller crystallites, and a shorter induction time of nucleation were observed in samples containing 0.10% Tween 60 relative to the control and 0.50% Tween 60. A 0.10% glycerol addition, on the other hand, yielded opposite results. The increase in hardness induced by small additions of Tween 60 could be attributed to decreases in crystallite size at constant SFC and fractal dimension. The increase in hardness induced by small additions of glycerol could only be attributed to increases in the crystalmelt interfacial tension, at constant SFC and fractal dimension, despite increases in crystallite size. Introduction Polyoxyethylene sorbitan monostearate (Tween 60) and glycerol are commonly used in the food industry as additives. Glycerol is widely used as a humectant, plasticizer, and starch and sugar crystallization modifier, while Tween 60 is usually used as an emulsifier. The effects of nonionic surfactants on emulsion properties have been studied extensively, but little is known about their effects on the crystallization behavior and physical properties of bulk fat systems. Emulsifiers have been shown to alter the kinetics of polymorphic transformations in triacylglycerols (TAGs) such as tristearin,1 as well as cocoa butter2 and sunflower oil.3 Nonionic surfactants are also known to delay the formation of bloom in chocolate.4 Emulsifiers can also affect TAG crystallization kineticssthey can increase or decrease the rates of nucleation and growth. Surfactant particles can serve as crystallization seeds, or form mixed crystals with TAGs, thus delaying or enhancing TAG nucleation. However, surfactants can also coat the faces of growing crystals, thus slowing crystal growth.5 Changes in the kinetics of crystallization have been shown to have profound effects on both microscopic and macroscopic properties of various fat systems.6-8 Since the effects of crystal seeding and coating can also occur simultaneously, crystallization kinetics can be affected in a complex fashion, usually resulting in trend reversal as a function of surfactant concentration.9 The aim of this study is to investigate the effects of glycerol and Tween 60 on crystallization kinetics, mi* To whom correspondence should be addressed: Alejandro G. Marangoni, Department of Food Science, University of Guelph, Guelph, ON N1G2W1, Canada. Ph: (519) 824-4120 x 54340. Fax: (519) 8246631. E-mail: [email protected]. Website: http://www.uoguelph.ca/∼amarango.

crostructure, melting properties, polymorphism, and mechanical properties of a plastic fat system. Experimental Section Sample and Chemicals. A model bulk fat system composed of triolein and the high melting fraction of milkfat (HMF) was prepared at a ratio of 70:30 (w/w). HMF was prepared from anhydrous milkfat from Gaylea Foods (Guelph, ON, Canada) by solvent fractionation10 and was melted and blended with practical grade triolein from Sigma (St. Louis, MO). Two different batches of HMF were prepared from anhydrous milkfat and a 2 °C difference in melting point was observed. Polyoxyethylene sorbitan monostearate (Sigma, St. Louis, MO), commonly referred to as Tween 60, was added to the blend at levels in the range of 0.1 to 3.0% (w/w) and the effects observed. Glycerol (Sigma, St. Louis, MO) was added in the range from 0.03 to 3.0% (w/w) to the 70:30 triolein/HMF mixture. High-purity nitrogen and helium used for calorimetry experiments were from BOC Gases (Guelph, ON, Canada). Lipid Composition. To verify the composition of the model system components, gas-liquid chromatography (GLC) was performed using a Shimadzu GC-8A (Tokyo, Japan) and a flame-ionization detector operated at 360 °C as previously described.11 The HMF triacylglycerides were determined based on carbon number (minus glycerol) and fatty acid (FA) composition was determined following derivatization of the samples to fatty acid methyl esters.12 The injector port temperature was 230 °C and runs were performed by ramping from 60 to 210 °C at 5 °C/min. Rheology. Rheological measurements at small deformations were made using a CarriMed CSL2 500 and AR 2000 rheometers (TA Instruments, Mississauga, ON, Canada) with a 2-cm flat plate attachment. The sample platform temperature was controlled, allowing for sample temperature to be maintained during analysis. Melted samples were poured into cylindrical molds of uniform diameter (20 mm) and thickness (3.2 mm), and were crystallized and stored at 5.0 °C for 24 h. Sample compression was set to 10% to ensure good contact between the parallel plate attachment and the fat sample, as previously described.13 The linear viscoelastic region (LVR) for each sample was first determined by performing oscillatory stress sweeps from 0.65 to 400 Pa, at a constant frequency of

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1 Hz. Storage moduli (G′) were determined within the LVR of the samples. Large deformation mechanical measurements (compression test between parallel plates) were made using a materials tester (SMS Materials Test, Texture Analyzer model MT-LQ, Surrey, UK) with a 50 N load cell. Molten fat samples, with crystal history erased, were poured into molds. Upon crystallization at 5 °C for 24 h, cylindrical samples (10 mm diameter × 5.7 mm height) were obtained. Samples were compressed uniaxially at a crosshead speed of 5 mm/s until failure occurred. Sample temperature was maintained during compression by circulating cooled water through the base-plate. The peak breaking force (N) was recorded. Solid Fat Content (SFC). SFC was measured by pulsed nuclear magnetic resonance (pNMR) with a Bruker PC/20 series and MQ 20 NMR analyzer (Bruker, Milton, Canada). Melting profiles of the fat systems with various levels of glycerol and Tween 60 were obtained by first holding the samples at 80 °C for 30 min to erase any crystal history. The melted fats were thoroughly mixed, placed in NMR tubes (four replicates), and crystallized at temperatures ranging from 0 to 42 °C for 24 h. Differential Scanning Calorimetry (DSC). To examine the effects of glycerol and Tween 60 on the crystallization and melting behavior of the fat, DSC analysis was performed with a DuPont 2910 and Q 1000 DSC (TA Instruments, Mississauga, ON, Canada). Nitrogen and helium purge gases were used to prevent condensation in the cell and an empty pan was used as reference. Fat samples (7-9 mg), with crystal history erased were loaded in aluminum pans, hermetically sealed, cooled from 80 to 5 °C at a rate of 10 °C/min and stored for 24 h at 5 °C prior to analysis. Melting was then performed on these samples by heating the pans from 5 to 60 °C at a constant temperature ramp of 5 °C/min. The melting temperatures (Tm) were determined from the peak maxima. Comparisons of peak melting temperature and peak shape were made to assess the possibility of effects of glycerol and Tween 60 on polymorphism and/or fractionation. Powder X-ray Diffraction Spectroscopy (XRD). Powder X-ray diffraction spectroscopy was performed using an EnrafNonius KappaCCD diffractometer (Nonius, Delft, The Netherlands) with an FR590 X-ray generator. Selected samples that showed large differences in both crystallization kinetics and rheology (0.10 and 0.50% Tween 60) were drawn into 1.0-mm glass capillary tubes (Charles Supper Co., Natick, Maine, USA). Samples were crystallized at 5 °C and held for 24 h prior to analysis. To determine initial polymorphic form, another set of samples were melted and held to erase crystal memory, quenched at 5 °C and examined after just 7 min of storage. The d spacings were calculated by comparing the spacings of the rings in these images to those of a standard (CaSO4‚2H2O). Polymorphic forms were determined from the d spacings. Crystallization Kinetics. The effects of glycerol and Tween 60 on crystallization kinetics were first investigated at 30 and 28 °C, respectively, by plotting SFC by pNMR against time. Four replicates of each sample were melted in NMR tubes at 80 °C for 30 min to erase any crystal history, and then held at 60 °C for 30 min. Crystallization curves were obtained by placing the tubes in a water bath at 30 and 28 °C and taking SFC readings at the appropriate time intervals. The crystallization curves were then fitted to the Avrami equation by least squares nonlinear regression.14 Avrami’s theory describes changes in the volume of the crystal mass as a function of time during a phase change. The Avrami equation is used to quantify fat crystallization kinetics as:

SFC(t) ) SFC∞[1 - exp(-ktn)]

(1)

where SFC(t) and SFC∞ are the SFC (%) at time t and the maximum SFC after crystallization was completed, respectively. Fitting the SFC data to this model allows for the determination of the rate constant of crystallization (k) at a particular temperature, and information on the mechanism of nucleation and crystal growth via the exponent (n).15,16

Induction Time of Crystallization (τ). Induction times were determined using a cloud point analyzer (Fats and Oils analyzer PSA-70V-HT, Phase Technology, Richmond, BC, Canada). A total of 150 µL of each sample was preheated to 80 °C for 30 min, and then pipetted into the sample chamber, which was preheated to 70 °C. The sample was given time to equilibrate at 70 °C before being cooled at a rate of 10 °C/min to crystallization temperatures of 30.0, 32.0, 33.0, 35.0, 36.0, and 37.0 °C for glycerol and 28.0, 30.0, 31.0, 32.0, and 33.0 °C for Tween 60. On the basis of scattered light intensity measurements, the apparatus provides information on the changes in crystal mass as a function of time. Induction times (τ) of crystallization were determined by extrapolating from the linearly increasing portion of the curve to the time axis. Every crystallization was carried out on freshly prepared sample to improve reproducibility. From induction times, apparent free energies of nucleation were assessed using the Fisher-Turnbull equation as described in previous studies.16 Polarized Light Microscopy (PLM) and Image Analysis. A small droplet (about 10 µL) of melted fat (80 °C for 30 min) was placed on a preheated (80 °C) glass slide, using a preheated capillary tube. A preheated glass cover slip was carefully placed over the sample to produce a film of uniform thickness. The slides were then stored for 10 min at 60 °C. The samples were then quenched to 5 °C and stored for 24 h prior to imaging. Sample temperature during imaging was maintained by the use of a thermostatically controlled microscope platform (Linkam, UK). When viewed by polarized light microscopy (PLM) (Olympus BH, Tokyo, Japan) the birefringent solid microstructural elements of the network could be directly observed and digital images were acquired via a black and white Sony XC75 CCD camera and LG-3 capture board (Scion Corporation, Frederick, USA). Gray scale images were then inverted, thresholded, and analyzed using a particle counting algorithm using NIH Image (National Institute of Health, Bethesda, USA) to determine a particle-counting fractal dimension, Df. The algorithm works as follows: the number of distinct particles (N) is first counted within the entire image of known length (L). At 5% increments, the image is cropped to smaller lengths and the number of particles again counted. This cropping and counting procedure is repeated until the length of the image is 35% of the original size. The slope of the linear regression of the log-log plot of the number of particles (N) within each region of length (L) corresponds to Df. It is important to note that two counts are performedsone that includes particles touching the edges of each region of interest and one that excludes those at the edges. The average of the Df values obtained by counts excluding and including particles at the edges best represents the spatial distribution of mass. The noninverted, thresholded gray scale images were also analyzed using a commercially available algorithm (TruSoft International Inc., St. Petersburg, USA, http://www.trusoftinternational.com) to determine the box-counting fractal dimension (Db).17 In this method, a grid is laid over the image and the number of boxes that are occupied by mass are counted. The size of the boxes that make up the grid is then iteratively reduced and the occupied boxes counted. The number of occupied boxes (N) versus the length of the boxes that make up the grid (L) is plotted on log-log axes, and the slope corresponds to -Db. Mean particle sizes were determined using image analysis toolkit plug-ins (Reindeer Graphics Inc., North Carolina, USA) for Adobe Photoshop 6.0 (Adobe Systems Inc., San Jose, USA). Calibration was performed by using the IP‚Measure: Calibration filter on an image of a calibration micrometer. Mean particle sizes were obtained by using the IP‚Features: Distribution filter with output on the basis of feature area. Kinetic microscopy experiments were carried out on the samples that showed large differences in crystallization kinetics and rheological properties (control, 0.1%, and 0.5% Tween 60). Crystal memory was erased by heating to 80 °C for 30 min, and samples were subsequently cooled from 60 to 28 °C at a rate of 10 °C/min. Images were acquired every 15 s and compiled into animations, allowing for qualitative and quan-

Effects of Glycerol and Tween 60 on Plastic Fat

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Table 1. Fatty Acid and Triacylglycerol Composition of the High-Melting Fraction of Milkfat Determined by Gas-Liquid Chromatography fatty acid

composition (wt %)

triacylglycerol (carbon number)

composition (wt %)

8:0 10:0 10:1 12:0 14:0 14:1 15:0 15:1 16:0 16:1 17:0 18:0 18:1 18:2 18:3 20:0

0.34 1.58 0.08 3.36 15.33 0.94 1.70 0.41 43.17 1.71 1.04 19.88 8.52 0.76 0.34 0.55

42 44 46 48 50 52

4.96 10.80 19.00 26.22 24.57 12.41

titative comparisons of crystallization kinetics and the final microstructures that resulted. The timed images acquired during the crystallization processes were inverted, thresholded, and the percentage of black pixels (representing birefringent crystal mass) computed. The normalized percentage of black pixels was then plotted against time and the kinetics of crystallization examined. The final microstructures that resulted were also subjected to the same static analyses: the fractal dimensions were determined by the methods as previously outlined; and the number of particles and mean particle size were determined by image analysis as previously described. All the isothermal crystallization studies in the case of the glycerol system were carried out at 30 °C while for the Tween 60 system, they were carried out at 28 °C. This 2 °C difference for the isothermal studies was chosen due to a 2 °C difference in the melting point for the two batches of HMF used in the two systems even though the lipid composition was similar.

Figure 1. Changes in breaking force (N) of HMF/triolein as a function of (A) glycerol and (B) Tween 60 concentration at 5 °C after 24 h of crystallization.

Results and Discussion Sample Composition. TAG and FA compositions of the HMF used in this study are shown in Table 1. Mechanical Properties. The addition of glycerol and Tween 60 significantly affected the mechanical properties of the fat (Figure 1). The addition of 0.030.20% glycerol resulted in an increase in breaking force relative to the control, while from 0.25 to 0.75% glycerol, a gradual decrease in the breaking force was observed. Above 0.75% glycerol, the breaking force increased again. Similarly, the addition of 0.10-0.25% Tween 60 resulted in an increase in breaking force, whereas at 0.50%, there was a minor softening effect relative to the control. Further increases in the levels of glycerol and Tween 60 beyond 0.50% resulted in an increase in the breaking force. On the other hand, glycerol and Tween 60 addition did not significantly affect the storage modulus (G′) of the material. The G′ at all concentrations remained constant at 8.3 ( 2.6 MPa (glycerol, n ) 44) and 7.6 ( 1.1 MPa (Tween 60, n ) 35). Solid Fat Content (SFC). For plastic fats, the solid fat content (SFC) has a large impact on mechanical properties.18 SFC is highly dependent on processing and crystallization conditions. Faster cooling rates, and/or crystallization at higher degrees of supercooling often result in the formation of fat crystal networks with a higher solids’ content.6-8,19 Generally, a higher SFC translates into a harder fat.18-20

Figure 2. Solid fat content versus temperature melting profiles for HMF/triolein containing various levels of glycerol and Tween 60.

At all crystallization temperatures studied, the addition of glycerol or Tween 60 did not have a significant effect on the resulting SFC (Figure 2). This would indicate that the observed differences in hardness were not due to variations in the amount of solids present in the network. Instead, these results suggested that differences in mechanical properties might have been induced by changes in polymorphism or microstructure. Differential Scanning Calorimetry (DSC). DSC was used to investigate the possible influence of polymorphism on the observed changes in mechanical properties. Changes in the shape of the melting profile could be suggestive of changes in polymorphism. All samples had very similar thermogram shapes, and all displayed the same peak melting temperature of 42.3 °C (glycerol) and 41.9 °C (Tween 60). This suggested that differences in hardness were not due to changes in polymorphic form. To confirm this, powder X-ray diffraction was performed on HMF/triolein containing Tween 60. Powder X-ray Diffraction (XRD). Powder XRD was performed on selected samples of HMF/triolein

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Figure 4. Changes in the induction time of nucleation (τ) as a function of glycerol/Tween 60 concentration.

Figure 3. Changes in turbidity as a function of time as measured in a cloud point analyzer at for systems containing different concentration of (A) glycerol and (B) Tween 60.

containing Tween 60, both immediately after crystallization at 5 °C to determine the initial polymorph formed, as well as after 24 h of storage at 5 °C. Samples showing the largest changes in hardness were chosen (0.10 and 0.50% w/w Tween 60). Both samples were found to nucleate in the metastable R form as characterized by a single wide-angle reflection (“short spacing”) at 4.15 Å. This confirmed that differences in hardness were not due to differences in the initial polymorphic form. Samples stored at 5 °C for 24 h prior to analysis were found to be in the more stable β polymorphic form, as indicated by a single wide-angle reflection at 4.6 Å. Thus, differences in breaking force were not due to changes in fat crystal polymorphism either. Instead, we hypothesize that differences might be due to changes in microstructure. Crystallization Kinetics. Turbidimetry, pulsed nuclear magnetic resonance (pNMR), and polarized light microscopy (PLM) were used to examine crystallization kinetics at 28 °C (Tween 60) and 30 °C (glycerol). Differences in crystallization kinetics were observed by turbidimetry (Figure 3). The curves suggested that, depending on the concentration, addition of glycerol or Tween 60 enhanced or delayed the crystallization process. At the 0.10% level, the crystallization process was significantly delayed in the glycerol system and accelerated in the Tween 60 system relative to all other concentrations. These opposite trends were surprising since mechanical properties were affected in a similar fashion (Figure 1). Induction times of crystallization were obtained by extrapolating the linearly increasing portion of the curves obtained by turbidimetry to the time axis (Figure 4). Changes in the crystallization kinetics patterns with respect to the concentration of glycerol/Tween 60 fol-

Figure 5. Fisher-Turnbull plot at various levels of (A) glycerol and (B) Tween 60.

lowed opposite trends. The longest induction time was observed for 0.1% glycerol in HMF, while the shortest induction time was observed for 0.1% Tween 60 in HMF (Figure 4). When fats are crystallized at high degrees of supercooling, or at high cooling rates, crystal aggregates tend to be smaller and more numerous, resulting in a more rigid network.2,19 In this study, however, both the supersaturation and the rate of cooling were equal for all samples, and thus differences in crystallization kinetics must be due to glycerol or Tween 60 addition. Induction times were determined for the control, and samples containing 0.10, 0.25, and 0.50% glycerol or Tween 60 at the additional temperatures of 30, 31, 32, 33, 35, 36, and 37 °C. It was hoped that from these measurements, apparent activation free energies of nucleation and crystal-melt interfacial tensions could be obtained. Unfortunately, Fisher-Turnbull plots (Figure 5) did not yield a distinct linear region in either system, other than HMF/triolein+0.25% glycerol, making computation of apparent free energies of nucleation and crystal-melt surface free energies impossible. Another well-established technique to monitor crystallization is pulsed NMR. This technique yields growth


Figure 6. Solid fat content versus time profiles during isothermal crystallization of HMF/triolein containing various levels of (A) glycerol and (B) Tween 60.

curves of SFC as a function of time for different concentrations of glycerol or Tween 60. Crystallization kinetics could then be quantified using the Avrami model. By fitting SFC-time data to the Avrami model, parameters that describe the kinetics of crystallization and the nucleation and growth modes can be obtained. Plots of SFC as a function of time for isothermal crystallizations at 30 °C (glycerol) and 28 °C (Tween 60) show small differences in the kinetics of solids formation with respect to the different additive concentrations (Figure 6); however, all samples achieved the same final SFC, in agreement with previous findings (Figure 2).


Fitting these SFC-time curves to the Avrami model yielded estimates of the Avrami exponents (n) (Figure 7A,B) and Avrami constants (k) (Figure 7C,D). The Avrami rate constants trends (k) were opposite for the glycerol and Tween 60 systems, in agreement with the turbidimetry studies. Generally, high values of k and low values of n are associated with an increased rate of crystallization and a more instantaneous nucleation process with a shorter induction time. This, in turn, would yield smaller and more numerous crystals, a greater number of crystal-crystal interactions, and thus a harder fat.19 The opposite would also be true. These relationships were obeyed in these systems, except that for the case of glycerol. Even though low values of k were obtained and larger and less numerous crystals were observed, a harder fat, albeit at 5 °C, was obtained. Static Microscopy and Image Analysis. Following crystallization and storage at 5 °C for 24 h, samples containing 0.10% glycerol contained the largest crystals (Table 2, Figure 8C) relative to the control (Table 2, Figure 8A), while for 0.10% Tween 60 (Table 3, Figure 9B), smaller crystals relative to the control were observed (Table 3, Figure 9A). An increase in glycerol content beyond 0.10% lead to decreases in crystal size (Table 2, Figure 8D). An addition of 0.50% Tween 60 (Table 3, Figure 9C), on the other hand, leads to an increase in crystal size relative to the 0.10% addition. The particle-counting fractal dimension (Df) increased from ∼1.97 at 0 and 0.03% glycerol to 2.10 at 0.1 and 0.25% glycerol, while the box-counting dimension (Db) remained fairly constant for all glycerol concentrations (Table 2). On the other hand, Df and Db were similar for all Tween 60 concentrations at 5 °C (Table 3). Micrographs obtained after 15 min at 30 °C (glycerol, Figure 8E-H) and 28 °C (Tween 60, Figure 8D-F) yielded more dramatic differences in the number of crystals, and mean crystallite diameters (Tables 2 and 3). The addition of 0.1% glycerol resulted in larger and

Figure 7. Changes in Avrami parameters as a function of glycerol (A, C) and Tween 60 (B, D) concentration.

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Figure 8. Polarized light micrographs of the HMF/triolein systems containing various levels of glycerol after 24 h at 5 °C (A-D) and 15 min at 30 °C (E-G). (A, E) control, (B, F) 0.03%, (C, G) 0.10%, and (D, H) 0.25%. Table 2. Effect of Glycerol Addition on Microstructural Parameters Including Particle Size and Fractal Dimensions Determined by Image Analysis crystallization temp (°C)

glycerol (%)

mean particle size (µm2)

no. of particles (N)

Df

Db

5a

0.00 0.03 0.10 0.25 0.00 0.03 0.10 0.25

40 30 86 69 163 302 326 150

4845 4630 3300 3646 1386 685 679 1555

1.97 1.98 2.10 2.10 1.99 1.92 1.93 1.96

1.87 1.87 1.88 1.87 1.75 1.76 1.76 1.75

30b

a

Following storage for 24 h. b Following storage for 15 min.

less numerous crystals (Figure 8G) relative to the control (Figure 8E). At 0.25% glycerol (Figure 8H), smaller and more numerous crystals were observed as compared to the 0.10% level (Figure 8G). These results for the 0.1% glycerol sample were consistent with the trends observed in Avrami parameters (lower k and higher n) and longer induction times of nucleation. The Df for HMF/triolein was higher than for HMF/triolein containing 0.10 or 0.25% glycerol (Table 2). The Db remained fairly constant for the different glycerol levels (Table 2). On the other hand, the addition of Tween 60 at the 0.10% level resulted in smaller and more numer-

ous crystals (Figure 9E) relative to the control (Figure 9D). In the case of 0.50% Tween 60, larger and less numerous particles were detected (Figure 9F). These results corresponded to the trends observed in the Avrami parameters (higher k and lower n) and shorter induction times of nucleation. Image analysis of micrographs at 28 °C (Tween 60) and 30 °C (glycerol) confirmed the qualitative results described above (Figure 10). Kinetic Microscopy and Image Analysis. The 0.10 and 0.50% Tween 60 samples displayed large differences in crystallization kinetics and microstructure at 28 °C. To better visualize differences in crystallization kinetics, real-time polarized light microscopy of the isothermal crystallization process at 28 °C was carried out. Time-lapse image capture followed by time-scaled reanimation of the still images allowed for visualization of the crystallization processes at 0.10 and 0.50% Tween levels, and can be viewed on the Internet (See web enhanced object, Figure 9). Dramatic differences in kinetics were observed, similar to those shown by turbidimetry. The 0.10% sample displayed enhanced and more rapid nucleation and crystal growth relative to the 0.50% sample, resulting in the formation of smaller, more numerous crystals. The apparent final percentage of crystal mass, or percentage of black pixels of the thresholded images were nearly equal, agreeing with our SFC results by pNMR. To depict the kinetics of crystallization of these samples graphically, the still images that made up the dynamic processes were thresholded and the percentage of black pixels computed, normalized, and plotted against time (Figure 11). This analysis yielded crystallization kinetics curves showing differences in both induction times and rates of crystal growth similar to those shown by turbidimetry. The crystallization conditions for turbidimetry and microscopy are similar (sample size, heat transfer), and thus it is encouraging that these results agreed well. The addition of glycerol or Tween 60 at levels ranging from 0.03 to 3.0% affected the macroscopic mechanical properties of the bulk fat system at 5 °C, and mainly differences in crystallite size accompanied the changes in mechanical properties. The box-counting and particlecounting fractal dimension did not change statistically (P > 0.05) at 5 °C upon glycerol or Tween 60 addition. At lower degrees of supercooling (30 or 28 °C), differences in crystallization kinetics and microstructure were more readily observed, and agreed qualitatively with results obtained at 5 °C. Changes in mechanical properties were due to altered kinetics of crystallization, resulting in differences in the number and size of crystallites. Impurities can modify crystallization kinetics by acting as a template for heterogeneous nucleation, or by cocrystallizing with the TAGs, thus enhancing nucleation and growth. Impurities may also adsorb onto growing crystal faces, interfering with mass transfer to the crystal surface, thus decreasing the rate of growth9. This may also result in changes in crystal-crystal interactions. In many cases, as was observed in this experiment, the overall effects appear to be due to a combination of all these effects. These findings may be explained using a model



Figure 9. Polarized light micrographs of the HMF/triolein systems containing various levels of Tween 60 after 24 h at 5 °C (A-C) and 15 min at 30 °C (D-F). (A, D) control, (B, E) 0.10%, and (C, F) 0.50%. W A movie in MOV format is available showing the addition of Tween 60 at 0.1% (w/w) levels (left panel) leading to an increase in the rate of nucleation and growth of the high-melting fraction of milk fat in triolein at 28 °C, relative to 0.5% (w/w) levels (right panel), and the control (not shown). This movie clearly shows the enhancement in crystal nucleation and growth rates induced by the surfactant. Table 3. Effect of Tween 60 Addition on Microstructural Parameters Including Particle Size and Fractal Dimensions Determined by Image Analysis crystallization temp (°C)

Tween 60 (%)

mean particle size (µm2)

no. of particles (N)

Df

Db

5a

0.00 0.10 0.50 0.00 0.10 0.50

34 27 37 59 32 58

4267 4579 3648 2355 2790 2102

2.00 1.99 2.00 1.91 1.91 2.00

1.90 1.90 1.90 1.67 1.63 1.70

28b

a

Following storage for 24 h. b Following storage for 15 min.

developed by our group that describes the relationship between mechanical properties and microstructure in fats,21-23

σo ) λΦ1/d-D

(2)

where σo is the yield stress, Φ is the volume fraction of

solids (Φ ) SFC/100), d is the Euclidean embedding dimension, D is the mass fractal dimension of the network, and λ is a complex term inversely proportional to the size of the primary crystallites (a) and directly proportional to the crystal-melt interfacial tension (δ),

λ∼

6δ a

(3)

In this work, we have shown that increases the yield force of HMF/triolein upon Tween 60 addition are not due to changes in the amount of solid fat or decreases in the mass fractal dimension of the fat crystal network, but rather due to decreases in primary crystallite size, despite possible decreases in crystal-melt interfacial tension (see below). Subsequent decreases in the breaking force were accompanied by increases in primary crystallite size. On the other hand, glycerol addition was accompanied by increases in crystallite size. This should have lead to a decrease in the breaking force of the fat, as predicted by eq 3. The opposite was true though.

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to measure crystal-melt interfacial tension and highlights the importance of this parameter in the design and manufacture of polycrystalline materials. Acknowledgment. The financial assistance of the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Ontario Ministry of Agriculture and Food (OMAF) are gratefully acknowledged. Note Added after ASAP Posting An earlier version of this paper posted on the ASAP website on October 2, 2003 had an incorrect caption to Figure 5. The caption has been corrected in this new version posted November 13, 2003. References

Figure 10. Changes in mean particle area of birefringent features in polarized light micrographs as a function of glycerol (A) and Tween 60 (B) concentration.

Figure 11. Increase in the normalized percentage of black pixels in thresholded polarized light images of HMF/triolein as a function of time, at various levels of Tween 60.

Thus, increases in breaking force must be primarily due to increases in the crystal-melt interfacial tension at constant solid fat content and fractal dimension (eq 3). The interfacial tension effects must be large enough to overcome the effects of increasing crystallite size. An increase in crystal-melt interfacial tension would also lead to a lower free energy of nucleation and thus a lower nucleation rate. This would lead to less numerous and larger crystallites. Addition of Tween 60, on the other hand, would lead to a decrease in the crystal-melt interfacial tension, thus leading to an increase in the nucleation rate. This, in turn, would have resulted in smaller and more numerous crystallites. These two predictions are consistent with our results. This research points to the need to develop reliable methods

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CG034136V

Effects of Glycerol and Tween 60 on the Crystallization Behavior

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