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Stability of whippable oil-in-water emulsions: Effect of monoglycerides on crystallization of palm kernel oil
Food Research International 54 (2013) 1738–1745
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Stability of whippable oil-in-water emulsions: Effect of monoglycerides on crystallization of palm kernel oil Merete B. Munk a,⁎, Alejandro G. Marangoni b, Hanne K. Ludvigsen a, Viggo Norn a, Jes C. Knudsen c, Jens Risbo c, Richard Ipsen c, Mogens L. Andersen c a b c
Palsgaard A/S, Palsgaardvej 10, DK-7130 Juelsminde, Denmark Department of Food Science, University of Guelph, 50 Stone Road East, Guelph N1G 2W1, Ontario, Canada Department of Food Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark
a r t i c l e
i n f o
Article history: Received 12 June 2013 Accepted 5 September 2013 Keywords: Palm kernel oil Emulsion stability Lipid crystallization Monoglycerides LACTEM Polymorphism
a b s t r a c t The effect of selected monoglycerides on droplet morphology and rheology of palm kernel oil emulsions was studied. Combination of lactic acid ester of monoglycerides (LACTEM) and unsaturated monoglycerides (GMU) yielded highly viscous emulsions caused by partial coalescence. LACTEM combined with saturated monoglycerides (GMS) or diacetyl tartaric acid ester of monoglycerides (DATEM) gave low-viscous emulsions. The rheological behavior was found not to be related to polymorphic transformations of fat in the emulsions. A possible relationship between structural changes of emulsions and the effect of monoglyceride crystallization behavior of PKO was examined by studying the monoglycerides' influence on crystallization of bulk PKO. Polymorphic transformation, crystallization kinetics and melting/crystallization profile of bulk PKO were analyzed. The monoglycerides had minor effect on crystallization behavior of bulk PKO compared to the major impact on emulsion stability. This indicates that emulsifiers act differently in bulk and dispersed fat. For PKO the structural changes of the emulsions were independent of the effects of monoglycerides on the crystallization of bulk fat. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Making a successful whippable fat-based emulsion requires a delicate balance between conflicting physical properties. On the one hand, to achieve voluminous and stable foam of a whipped aerated emulsion, the fat droplets must interact strongly with each other and form a stable semi-rigid network, which stabilizes foam structure. Partial coalescence plays a crucial role for foam structure formation by creating a network of fat droplets that stabilize air bubbles in foams (Barfod & Krog, 1987; Goff, 1997). On the other hand, prior to whipping, the emulsion must be pourable and thus have a perpetual low viscosity, which requires that attractive forces between droplets are weak in order to avoid flocculation, droplet coalescence and consequent creaming of larger aggregates. In addition, the emulsion should preferably have long-term stability, implying resistance to agitation and thermal fluctuations occurring during transportation and storage. Ideally, formation of a strong network of partly coalesced droplets should not happen until whipping is initiated, and should not give rise to undesirable solidification, clumping or creaming of the emulsion. To ensure stable foam with long shelf life, various types of monoglycerides are often used as emulsifying agents in whippable emulsions. The high surface activity of monoglycerides is assumed to displace proteins at the fat droplet interface. Consequently, steric repulsion is ⁎ Corresponding author. Tel.: +45 41125539. E-mail address: [email protected] (M.B. Munk). 0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.09.001
reduced and a new thinner monolayer of monoglyceride surrounds the interface, which is more susceptible for crystal penetration from one fat droplet into another. As a result, partial coalesced fat aggregates, which make up the foam network, are more likely to be formed. However, some monoglycerides seem to cause unintended properties for emulsions before the whipping. Unsaturated monoglyceride (GMU) and lactic acid ester of monoglyceride (LACTEM) have been reported to cause increased viscosity of emulsions both under shear and quiescent conditions (Allen, Murray, & Dickinson, 2008; Barfod, Krog, Larsen, & Buchheim, 1991; Davies, Dickinson, & Bee, 2000; Davies, Dickinson, & Bee, 2001). Emulsifiers will affect the adsorbed interfacial layer around emulsified droplets, which is decisive for the nature of droplet interactions among themselves and with other components in the emulsion. Additionally, emulsifiers can play a secondary role due to their influence on crystallization of the emulsified fat. Emulsifiers can also affect crystallization behavior including nucleation, crystal growth, recrystallization of polymorphic forms and melting profiles of bulk fat (Smith, Bhaggan, Talbot, & van Malssen, 2011). Crystallization of fat in the dispersed droplets can lead to changes of physical properties of emulsions, such as increased viscosity. The objective of the present work was to investigate how physical stability of whippable palm kernel oil (PKO) emulsions prior to whipping was affected by different combinations of monoglycerides. Physical stability in this context refers to a perpetual pourability of the non-whipped emulsions, which requires that the dispersed droplets remain finite and spherical. Droplet
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size determinations, confocal microscopy, and rheological measurements were used to study physical stability of emulsions. Furthermore, the effect of the monoglycerides on crystallization of bulk PKO was examined with the aim of elucidating a possible relationship between undesirable textural changes of non-whipped emulsions and the monoglycerides' effect on crystallization behavior of PKO. The latter was examined by DSC, pNMR and powder X-ray diffraction. 2. Materials & methods 2.1. Materials Emulsifiers were of commercial food grade and all were provided by Palsgaard A/S (Juelsminde, Denmark): Lactic acid ester of monoglyceride (LACTEM) made from fully hydrogenated palm oil and rape-seed oil (C16:0 and C18:0 N97% of fatty acids); unsaturated monoglycerides (GMU) made from sunflower oil (C18:1 N81%); >saturated monoglycerides (GMS) made from fully hydrogenated palm oil and rape-seed oil (C16:0 and C18:0 N 97%); and diacetyl tartaric acid ester of monoglyceride (DATEM) made from palm oil and sunflower oil (C16:0, C18:1, and C18:2 comprised 93% of fatty acids). The stabilizer mixture (Palsgaard A/S, Juelsminde, Denmark) contained microcrystalline cellulose (MCC), sodium carboxymethylcellulose (CMC) and disodium phosphate. Hydrogenated palm kernel oil (PKO) was obtained from AAK (Karlshamn, Sweden) and sodium caseinate from DMV International (Veghel, The Netherlands). Sugar was purchased from Nordic Sugar (Nakskov, Denmark) and sorbitol from Roquette (Cedex, France). 2.2. Emulsion and bulk PKO blend preparation 2.2.1. Emulsions Sodium caseinate (0.6 wt.%), stabilizer mixture (0.6 wt.%) and sugar (10 wt.%) were dispersed in water under continuous stirring and put aside for 4 h to hydrate proteins. Melted PKO (25 wt.%), LACTEM (0.55 wt.%) and GMU (0.15 wt.%), GMS (0.15 wt.%) or DATEM (0.10 wt.%) were mixed with the heated water phase (~70 °C) and the complete mix was re-heated to 82 °C. A pre-emulsion was conducted by mixing with a high-shear blender (Ultra-Turrax, IKA, NC, USA) for approximately 20 s. Homogenization was subsequently carried out on a two-stage high-pressure valve homogenizer (APV Rannie LAB-12.50, SPX, Silkeborg, Denmark) at 150/50 bar. Emulsions were rapidly cooled to 5–6 °C by a plate heat exchanger (APV U2, SPX, Silkeborg, Denmark) connected to the homogenizer. The emulsions were immediately poured into small beakers and stored at 5 °C. Control emulsions with sodium caseinate as the only emulsifier were prepared in a similar way. 2.2.2. Bulk PKO blends GMU (5.0 wt.%), GMS (5.0 wt.%) or DATEM (5.0 wt.%), either singly or in combination with LACTEM (5.0 wt.%), was added to melted PKO and the blend was stirred to ensure homogenous distribution and heated to 80 °C to remove crystal history. The blend was applied to an appropriate container (DSC-vial, NMR-tube or X-ray plate) depending on analysis method and immediately analyzed by DSC, NMR or X-ray.
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Heidelberg, Germany). Total concentration of 1 ppm BODIPY 493/503 (4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene) (Invitrogen, Carlsbad, USA) dissolved in dimethyl sulfoxide was added to emulsions to stain lipid. Excitation wavelength was 488 nm and emission bandwidth was 500–570 nm. A water immersion objective (HCX PL APO lambda blue 63.0 × 1.20 water UV) was used and image resolution was set to 1024 × 1024 pixels. A thin layer of stained emulsion was applied on a large standard cover glass and covered by another cover glass to avoid evaporation/drying. 2.3.3. Rheological measurements An ARG2 rheometer (TA-Instruments, West Sussex, England, UK) fitted with a Peltier temperature control connected to a water bath was used for all rheological measurements. Steady state flow experiments with increasing shear rate from 0.01 to 100 s−1 were performed at 5 °C and 20 °C to determine viscosity. A cone (diameter 40 mm, inclination 1.59°) and plate geometry was applied. Some emulsions exhibited a very firm texture and were not flowable. These were molded into discs of diameter 20 mm and characterized by a stress sweep with increasing oscillation stress from 0.1 to 1000 Pa at a constant shear frequency of 1 Hz. Plate (diameter 20 mm) and a plate geometry, both with roughened surfaces, to avoid slip was used. Viscosity measurements were performed at least in duplicate whereas small amplitude oscillatory shear was repeated 5 times. 2.4. Characterization of fat crystallization 2.4.1. Differential scanning calorimetry Melting and crystallization temperatures were determined for both emulsions and PKO blends by the use of DSC1 Stare System (Mettler Toledo, Schwerzenbach, Switzerland). Samples were initially held at 5 °C for 10 min, then heated to 80 °C at 2 °C/min and subsequently re-cooled to 5 °C at 2 °C/min. Empty pans were used as reference and each sample was analyzed in duplicate. 2.4.2. Crystallization kinetics Solid fat content (SFC) of PKO blends containing emulsifiers was determined as a function of time at isothermal conditions by the use of pulsed NMR spectroscopy (Bruker Minispec, Bruker, Germany). Tubes of samples were placed in water baths at 10 °C and 20 °C and SFC was measured at appropriate intervals till crystallization appeared to be completed as the crystalline fraction reached a constant level. 2–5 replicates of each sample were measured. The results were fitted to the Avrami equation by non-linear regression of Origin® 7 (OriginLab, Northampton, USA): SFC t −kt n ¼ 1−e : SFC max
ð1Þ
Where SFCt is the crystalline content (%) at time t and SFCmax the maximum crystalline content (%) achieved at the particular temperature. k is the crystallization rate constant, and n the Avrami exponent which is dependent on crystal growth mechanism with respect to nucleation and growth dimensions.
2.3. Characterization of emulsions 2.3.1. Determination of droplet size by light scattering Droplet size distribution was measured 2, 8 and 15 days after emulsion preparation (Mastersizer Microplus, Malvern Instruments, Malvern, U.K.). Measurements were conducted at room temperature on emulsions diluted approximately 1:100 with de-ionized water. At least 3 replicates were measured for each emulsion. 2.3.2. Confocal laser scanning microscopy Structure and morphology of fat globules in emulsions were examined by an inverse confocal laser scanning microscope (Leica TCS SP5,
2.4.3. Polymorphic transformation The polymorphism of crystals was examined for both emulsions and PKO blends by powder X-ray diffractometer (Rigaku, Tokyo, Japan). Radiation at λ = 1.54 Å was generated from a copper anode and operated at 40 kV and 44 mA. The scanning velocity was 0.05°/min for emulsions and 1°/min for PKO blends in the area of 1.2–35 2θ. The diffractometer was protected by a Multiflex chamber, which was equipped with Peltier temperature control connected to a water bath. Emulsions were examined at 20 °C without any preceding treatment and also after exposing the emulsion to stirring with a spatula. At least two diffraction spectra were recorded for each emulsion. Fully melted PKO blends were cooled
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to either 5 °C or 20 °C and after 10 or 15 min respectively the diffraction spectrum was recorded. The samples of PKO blends were kept and recorded the following day, day 7 and day 21.
a
2.5. Statistical analysis The results of the analyses are reported as the mean and standard deviation calculated from the replicates. The level of significance for melting and crystallization temperature is calculated by one way ANOVA and is given by the P-value. Intervals of confidences for n and k of the Avrami model is calculated from the Jacobian matrix which is an output of the non-linear fitting procedure. 3. Results 3.1. Rheological properties of emulsions Four different palm kernel oil emulsions were made using LACTEM as one of the emulsifiers. The emulsions also contained caseinate, stabilizer and sugar in order to mimic the composition of a commercial whippable emulsion. LACTEM was included because it promotes fat droplet aggregation (Westerbeek, 1989) and therefore is widely used in whipped toppings and dessert products (Si, 1991). In addition, three of the emulsions contained DATEM, unsaturated (GMU) or saturated monoglycerides (GMS), since LACTEM is often used in combination with other monoglycerides in commercial products. Furthermore, the specific combinations of LACTEM and the other monoglycerides were selected in order to create large variations in emulsion stability in terms of changes in viscosity and texture. An emulsion without added monoglycerides was prepared as a control. The differences among the emulsions were immediately discernible after preparation as observed by the products ranging from low-viscous homogeneous emulsions to thick pastes and clumping emulsions. Emulsions with LACTEM alone were found to yield semi-solid emulsions. The properties were initially qualitatively assessed by stirring the emulsions with a spatula at room temperature or at 5 °C. Stirring the LACTEM emulsion at 5 °C drastically reduced the viscosity, whereas stirring the emulsion at room temperature gave a thick paste. In addition, the process causing the instability of this emulsion appeared to be reversible, as a pourable emulsion could be obtained again by cooling the thick paste to 5 °C followed by stirring. Combining LACTEM with GMU gave very firm emulsions that appeared not to be sensitive to any combination of stirring and temperature. In fact, clotting appeared already within 10–15 min after the preparation of the emulsion and after 1-2 h at 5 °C complete solidification occurred. On the other hand, combinations of GMS or DATEM together with LACTEM yielded perpetual pourable emulsions. The control emulsion made without any added monoglyceride resulted in a low-viscous emulsion that was not affected by stirring and temperature. The observed qualitative changes in viscosity and texture were characterized by rheological measurements (Fig. 1). At 5 °C and at low shear rates, the apparent viscosity of the emulsion containing LACTEM was ~50 Pa·s (Fig. 1a). The apparent viscosity decreased with increasing shear rate, demonstrating shear thinning. However, the apparent viscosities were 5 to 10 times higher than for the control emulsion without monoglycerides. The two emulsions with either added GMS or DATEM also demonstrated shear thinning behavior and had viscosities between the control emulsion and the LACTEM-only emulsion at all shear rates. At 20 °C only the control emulsion demonstrated a true shear thinning effect comparable to measurements at 5 °C (Fig. 1b). The viscosity of the emulsion with LACTEM could not be determined at 20 °C since the texture of the emulsion was too firm. A lumpy texture in emulsions caused by LACTEM has previously been reported (Allen et al., 2008), and is probably due to partial coalescence as a consequence of protein displacement on droplet interface (Thivilliers-Arvis, Laurichesse, Schmitt, & Leal-Calderon, 2010). Emulsions with LACTEM + GMS exhibited a sudden shear thickening behavior at 20 °C at shear rates around
b
c
Fig. 1. Monoglycerides' influence on rheological behavior of emulsions. ■: Control, ▲: LACTEM + DATEM, □: LACTEM + GMS, ○: LACTEM, ▼: LACTEM + GMU. a: Viscosity at 5 °C, LACTEM + GMU was too viscous to be measured. b: Viscosity at 20 °C, emulsions with LACTEM and LACTEM + GMU were too viscous to be measured. c: The elastic modulus at 20 °C of high viscous emulsions, LACTEM and LACTEM + GMU.
0.03 s−1 (Fig. 1b). Yet, the viscosity was significantly lower compared to emulsions with LACTEM. Addition of DATEM prevented shear induced modifications until a certain degree, and at sufficient high shear rates a slight increase of viscosity occurred indicating subtle shear thickening effects. The instability of emulsions containing LACTEM + GMU was very evident since they exhibited complete solidification at all temperatures. The firm emulsions remained resistant to shear torque at 2 · 105 mN/m at 5 °C and 20 °C, and thus viscosity was not a measurable parameter.
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Fig. 2. The effect of monoglycerides on droplet size distribution in emulsions, — (solid black) control(solid black), LACTEM (long dash), The line style of "LACTEM + GMS" should be grey LACTEM + GMS (solid grey), — LACTEM + DATEM (short dash), …… LACTEM + GMU (dot).
Instead, rheological properties were characterized by small amplitude oscillatory shear. An elastic modulus, G′ ≈ 3.2 ± 0.4 · 106 Pa (Fig. 1c) was found, and similar for the firm LACTEM emulsion at 20 °C with G′ ≈ 4.5 ± 3 · 106 Pa. Although no statistical significant differences in G′ were obtained between emulsions containing LACTEM and LACTEM + GMU, it should be noted that emulsions containing LACTEM + GMU were three times firmer than emulsions made only with LACTEM (measured by back extrusion; data not shown). The higher stress and/or longer duration required to induce structural changes in emulsions with LACTEM + GMS as compared to LACTEM + GMU is in agreement with Barfod et al. (1991) who observed a high stability of emulsions containing GMS in contrast to emulsions with GMU. 3.2. Droplet size The droplet size distributions were clearly related to the viscosity of the emulsions. A monomial and fairly narrow distribution of small
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droplets was characteristic for the three low-viscosity emulsions made without added monoglycerides, a combination of LACTEM + GMS, or a combination of LACTEM + DATEM (Fig. 2). The mean diameters, d32, were respectively 0.98 ± 0.03 μm, 0.84 ± 0.03 μm and 0.65 ± 0.01 μm for the three emulsions after storage at 5 °C for 2 days. The two highly viscous and firm emulsions made either with LACTEM or LACTEM + GMU were characterized by very broad droplet distributions. The broad distributions of mainly large droplets suggest the presence of aggregates in emulsions. Prior to size distribution measurements, emulsions were diluted with water. Despite the fact that the continuous phases of all the emulsions were water, dilutions of the firm emulsions made with LACTEM and LACTEM + GMU were practically impossible despite vigorous shaking using a vortex mixer. This demonstrated extreme sensitivity towards agitation and thermal fluctuation since both types of emulsions, but particularly LACTEM + GMU, appeared as large non-dispersible flocks in water as a consequence of the preparatory steps of measurements even though the samples were held at 5 °C. Droplet size distributions were also measured after 8 and 15 days of storage at 5 °C but no significant changes occurred during this period. The differences among the emulsions in terms of microstructure were clearly visualized by confocal laser scanning microscopy (Fig. 3). The three low-viscous emulsions, without added monoglycerides, with LACTEM + GMS or LACTEM + DATEM, all exhibited a structure of small spherical fat globules (Fig. 3a, e and f), supporting the results from droplet size distributions. Conversely, the fat globules in the high viscous emulsion containing LACTEM + GMU exhibited large irregular aggregates (Fig. 3d). The emulsion with only LACTEM also contained aggregated droplets, and the sizes of the aggregates increased drastically when the emulsion was subjected to shear at room temperature (Fig. 3b and c). The physical mechanism of the latter aggregate enlargement was most likely flocculation, since reduced viscosity could be regained by cooling and shearing. On the other hand, the large coherent aggregates of the emulsions with LACTEM + GMU could not similarly be changed to give a low-viscous emulsion and thus probably resulted from partial coalescence.
Fig. 3. Micro-structure investigated by CLSM of emulsions containing various monoglycerides. Before microscopic analysis the emulsions were stored at 5 °C. The fat fraction is stained and the scale bar equals 10 μm. a: Control, b: LACTEM, low viscous, c: LACTEM, high viscous due to shear at room temperature, d: LACTEM + GMU, e: LACTEM + GMS, f: LACTEM + DATEM.
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Fig. 4. Melting and crystallization thermograms of bulk PKO. Arrows indicate defined melting and crystallization temperatures.
3.3. DSC analysis of emulsions and bulk PKO Partial coalescence of lipid droplets in emulsions requires that the lipids are partially crystallized. Since the emulsifiers potentially could modify the crystallization behavior of the palm kernel oil, the effect of the different monoglyceride combinations on the melting and crystallization behaviors of both bulk and emulsified PKO was studied by DSC. For the study of bulk PKO, the monoglycerides were added both separately in a concentration of 5 wt.% or in combination with 5 wt.% LACTEM. The concentration of monoglycerides in the bulk fat was higher than in the emulsions for two reasons. First, it was assumed that an excessive concentration would make the crystallization effect of monoglycerides more distinct if any. Second, the local concentration of monoglyceride was much higher at the droplet interface compared to the total concentration in the emulsions and therefore it was presumed that a higher monoglyceride concentration in bulk PKO would reflect the crystallization behavior more realistic. The endothermic DSC thermogram of pure PKO showed that PKO melted over a broad temperature range between 5 °C and 43 °C in accordance with the observations by Siew (2001) (Fig. 4). The onset melting temperatures could not be determined reliably due to a very gradual onset of melting. Melting temperatures were therefore defined as the peak temperature of the lowest endothermic peak as indicated in Fig. 4. In agreement with previous work, the presence of monoglycerides did not significantly affect the melting behavior of neither bulk nor emulsified PKO (P N 0.05), Table 1 (Barfod, Schrader, & Buchheim, 2000). In the cooling experiments, most of the emulsions and bulk PKO samples showed two exothermic peaks caused by crystallization; a minor one at 22–23 °C and a major one at 19–18 °C (Fig. 4 and Table 1). Apart from GMS, the monoglycerides did not have
significant effect on the crystallization temperature of bulk PKO samples (P N 0.05). Only addition of GMS increased crystallization temperature significantly of bulk PKO (P b 0.001), probably due to GMS being a high melting component (mp. N 60 °C). Formation of crystals early in the cooling experiment can have acted as seeds for nucleation of PKO. In contrast, the crystallization temperature of emulsified PKO without added monoglycerides was significantly reduced compared to bulk PKO most likely as a consequence of supercooling. The entire fat fraction in the control emulsion crystallized around 8 °C, which indicated supercooling due to the absence of nucleus monoglycerides and the presence of a thick protecting layer of sodium caseinate blocking intrusion of nucleating crystals (Palanuwech & Coupland, 2003). Similar to the bulk PKO-blends, emulsions containing LACTEM, GMU and GMS displayed a small exothermic peak around 22–28 °C, and a second exothermic peak at temperature N 8 °C (Table 1). Destabilized emulsion droplets are expected to yield a thermogram comprising the crystallization peaks of both the bulk PKO and the dispersed PKO, that is 23 °C, 19 °C and 8 °C respectively (Palanuwech & Coupland, 2003; Vanapalli, Palanuwech, & Coupland, 2002). Since none of the emulsions containing monoglycerides were crystallizing at 8 °C, droplet destabilization was apparently not the cause for the increased crystallization temperature as compared to the control emulsion. Emulsion destabilization implies that the positions of the peaks corresponding to bulk and emulsified fat are steady, and therefore the single crystallization peak at 11 °C for emulsions containing LACTEM + DATEM can neither be explained by droplet destabilization. The different crystallization behavior between emulsions with monoglycerides and the control emulsion is probably caused by monoglycerides that catalyze the nucleation event by intervening as heteronuclei and thereby increase the crystallization temperature. Davies, Dickinson, and Bee reported similar effects on crystallization of both bulk and emulsified fat with addition of emulsifiers (Davies et al., 2001).
3.4. Crystallization kinetics of bulk PKO Crystallization of bulk PKO at 5 °C occurred too fast to follow experimentally and therefore the monoglycerides' influence on crystallization kinetics of bulk PKO was examined at 10 °C and 20 °C by pulsed nuclear magnetic resonance (pNMR). By fitting the isothermal SFC as function of time to the Avrami model (Eq. (1)), estimates could be obtained for the crystallization rate constant (Avrami constant k), and the index of crystallization related to the type of nucleation and dimensionality of growth (Avrami exponent n) (Table 2). The maximum crystalline content was within 60–70% at 10 °C and 82–89% at 20 °C for all emulsions. Each monoglyceride was added in concentrations of 5 wt.% as no effect could be detected at lower concentrations. GMU and DATEM did not affect crystallization of PKO at either temperature and values of k and n similar to those of pure PKO were obtained (Fig. 5 and Table 2). Both LACTEM and GMS increased the crystallization rate of PKO. The increase in crystallization rate was also observed when
Table 1 Melting and crystallization temperatures of bulk PKO and emulsions containing various monoglycerides. Melting temperature was defined as the peak of the first endothermic peak. Most samples displayed two exothermic crystallization peaks, a minor (~22 °C) and a large one (~19 °C). Emulsifier
No monoglycerides LACTEM GMU GMU + LACTEM GMS GMS + LACTEM DATEM DATEM + LACTEM
Bulk PKO
Emulsion
Tpeak melting (°C)
Tpeak crystallization (°C)
Tpeak melting (°C)
Tpeak crystallization (°C)
23.2 23.8 24.0 23.3 24.4 23.4 22.9 23.4
22.8 23.5 22.1 22.7 25.7 26.3 23.3 23.3
24.0 ± 0.4 23.7 ± 0.6
7.8 ± 0.1 21.9 ± 0.1/17.4 ± 0.2
23.8 ± 0.5
21.8 ± 0.8/17.7 ± 0.6
23.5 ± 0.2
27.9 ± 0.4/15 ± 0.2
24.0 ± 0.6
11.4 ± 0.2
± ± ± ± ± ± ± ±
0.8 0.6 0.0 1.1 0.1 0.0 0.5 0.3
± ± ± ± ± ± ± ±
0.3/19.1 0.3/19.1 0.2/18.3 0.5/18.1 0.2/19.7 0.5/20.9 0.2/19.7 0.1/18.9
± ± ± ± ± ± ± ±
0.2 0.2 0.3 0.1 1.0 0.2 0.1 0.4
M.B. Munk et al. / Food Research International 54 (2013) 1738–1745
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Table 2 Avrami exponent n and Avrami constant k for crystallization of PKO containing various monoglycerides at 10 °C and 20 °C. PKO
10 °C
20 °C
n No monoglycerides LACTEM GMU GMS DATEM LACTEM + GMU LACTEM + GMS LACTEM + DATEM
1.8 1.5 1.9 1.6 1.9 1.6 1.3 1.5
k ± ± ± ± ± ± ± ±
0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.1
6.8 6.4 5.7 2.9 4.9 3.4 1.6 4.2
n ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗
−5
10 10−4 10−5 10−4 10−5 10−4 10−3 10−4
± ± ± ± ± ± ± ±
LACTEM was mixed with other emulsifiers, although the effect was moderated with GMU and DATEM. In contrast, combination of GMS and LACTEM resulted in the highest crystallization rate and the lowest value of n at 10 °C. For all PKO blends the Avrami exponent, n, assumed a value between 1.3 and 1.9. This indicate a similar crystal growth mechanism, which according to polarized light microscopy (PLM) (data not shown) most likely was a rod-like growth from sporadic nuclei (n = 2) (Marangoni, 2005). Thus, the tested monoglycerides seemed not to have an effect on nucleation and growth dimensions. However, the possibility that GMS induced rod-like growth from instantaneous nuclei (n = 1) (Marangoni, 2005) cannot be rejected since initial crystallization for this blend had occurred prior to the PLM examination. Image analysis (data not shown), however, suggested similar nucleation and morphology among the blends.
Fig. 5. Effect of monoglycerides on the initial crystallization kinetics of PKO at 10 °C and 20 °C. ■: No monoglycerides,○: LACTEM, □: GMS, ▲: DATEM, ▼: GMU. Insets: The complete crystallization at 10 °C and 20 °C followed over 1 h and 2 h, respectively.
3.5 3.5 2.9 1.2 3.0 3.5 4.1 2.3
∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗
−5
10 10−4 10−5 10−4 10−5 10−4 10−4 10−4
1.9 1.4 1.9 1.6 1.9 1.6 1.3 1.7
k ± ± ± ± ± ± ± ±
0.2 0.2 0.2 0.1 0.2 0.2 0.1 0.2
2.3 4.4 1.5 1.2 1.6 9.2 5.0 5.0
∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗
10−5 10−4 10−5 10−4 10−5 10−5 10−4 10−5
± ± ± ± ± ± ± ±
2.0 3.6 1.5 8.6 2.2 9.9 2.7 6.4
∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗
10−5 10−4 10−5 10−5 10−5 10−5 10−4 10−5
3.5. Polymorphic transformations of emulsions and bulk PKO The polymorphic forms were identified by reflections in powder Xray diffraction experiments corresponding to characteristic d-spacing. The α-form is characterized by d = 4.15 Å, β′ is characterized by d = 3.8 and 4.2 Å and β characterized by d = 4.6 Å (AOCS, 1995). The transformation of emulsions from pourable to thick pastes upon shear made it plausible to believe that the transformation came along with recrystallization of the fat. Therefore X-ray diffraction was carried out twice on emulsions. First emulsions were not exposed to any preceding treatments and second emulsions were stirred with a spatula until viscosity had increased. The prevalence of polymorphic forms was indifferent although the textural properties of the emulsions had changed, Fig. 6. All emulsions regardless the type of monoglycerides and pourability consisted of β′-polymorphs with traces of α. For the PKO blends the X-ray diffractograms differed a bit more, although all blends contained a mixture of α- and β′-polymorphs. The ratio of β′/α depended on added monoglyceride, cooling temperature and time after temperature quench. The diffractograms were superimposed, which complicated a straightforward quantification of the relative amount of each polymeric forms using information from the short spacing part of the diffractograms. In such case long spacing d-values in the SAXS regime have previously been used for quantification of the polymorphs (de Graef, van Puyvelde, Goderis, & Dewettinck, 2009; Fredrick, Foubert, Van De Sype, & Dewettinck, 2008; Kalnin, Schafer, Amenitsch, & Ollivon, 2004). In the present case a clear correlation was found between the reflection at 4.15 Å and a reflection at 43 Å, and the latter was thus also attributed to the αpolymorph. Likewise, a correlation was established between reflections at 3.8/4.2 Å and 35 Å, which again were attributed to the β′-polymorph.
Fig. 6. Crystal polymorphism of emulsion containing LACTEM + GMS. The upper diffractogram displays a low-viscous pourable emulsion with no pre-treatment and the lower diffractogram displays the emulsion stirred into a thick paste. The peaks corresponding to β′ (d = 4.2 Å and 3.8 Å) and α (d = 4.15 Å) are pointed out.
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Table 3 The relative ratio of β′-crystals compared to α-crystals in bulk PKO with addition of monoglycerides as a function of storage time at 5 °C and 20 °C. After day 7 the peak corresponding to α was not detectable (n.d.) for most samples and therefore results from day 21 are not included in the table. Emulsifier
No monoglycerides LACTEM GMU GMU + LACTEM GMS GMS + LACTEM DATEM DATEM + LACTEM
5 °C
20 °C
Day 0
Day 1
Day 7
Day 0
Day 1
Day 7
0.9 3.0 1.0 1.0 1.6 6.4 1.2 1.0
1.3 3.2 2.0 1.8 2.2 9.5 1.9 2.9
1.4 3.0 4.0 3.1 2.1 8.0 n.d. 4.1
1.5 3.4 2.0 1.5 2.0 2.3 3.4 1.5
1.9 4.5 6.5 2.1 2.3 4.2 n.d. 2.0
2.1 4.3 5.9 n.d. 2.8 8.8 n.d. n.d.
The relative rate of polymorphic transformation from α to β′ at temperatures of 5 °C and 20 °C was followed for a duration of 3 weeks, and the results are shown in Table 3. The time development of the polymorphic transformation from α to β′ is studied by examining the ratio: β0 Apeakd¼35 ; α Apeakd¼43
ð2Þ
which expresses the ratio between the amount the two polymorphic forms. Initially all PKO blends comprised a mix of α and β′, but during three weeks α was transformed into β′, the most stable polymorph of PKO (Rossell, 1975). The preference of β′ over β is most likely due to the wide range of fatty acid chain lengths in triglycerides of PKO (Sato, 2001; Timms, 1984). The initial fraction of α, as well as polymorphic transformation rate, was clearly dependent on crystallization temperature, storage time and addition of monoglycerides. Using a storage temperature of 5 °C caused a higher fraction of α-polymorphs as compared to 20 °C, due to low thermodynamic stability and low melting point of α (Sato & Ueno, 2011). Subsequent recrystallization into β′-polymorphs occurred, and after 7 days the reflection corresponding to α was not distinguishable for most samples. Several of the tested monoglycerides favored initial formation of β′ directly from the melt. This applied especially to LACTEM, LACTEM + GMS and DATEM, although for the latter this was only observed at 20 °C, Table 3. In addition, all monoglycerides, to varying degrees, accelerated the polymorphic transformation in agreement with previous studies (Aronhime, Sarig, & Garti, 1988; Fredrick et al., 2008; Smith et al., 2011). Overall, all tested monoglycerides promoted faster formation of the most stable polymorph. This might be related to the fact that except anionic DATEM, all applied monoglycerides were non-ionic which are suggested to induce transformation of crystals into more stable polymorphs (Bunjes, Koch, & Westesen, 2003). However, the monoglycerides appeared to be divided into two groups: GMU and DATEM (used in pure form or in combination with LACTEM) accelerated gradually the transformation from α to β′, whereas GMS and LACTEM readily caused a high level of β′ directly from the melt which remained fairly constant during the remaining experimental period (Fig. 7). 4. Discussion The stabilities of the investigated emulsions were strongly dependent on the type of monoglycerides added. Both droplet size distributions and microscopic inspection revealed formation of large fat aggregates in the two high-viscosity emulsions made with LACTEM or LACTEM + GMU. Furthermore, increased viscosity as a consequence of shear and increased temperature was linked to enlargement of the aggregates. The clusters of droplets remained non-dispersable in a water solution despite vigorous shaking, and therefore the aggregates
Fig. 7. Polymorphic transformations of α to β′-crystals during storage for 20 days at 5 °C for PKO without monoglycerides (■), with LACTEM + GMU (▼) and with LACTEM + GMS (□).
were unlikely to be held together by flocculation. Furthermore, the partial crystalline state of the dispersed fat, the irregular shapes of the aggregates and the increase in emulsion viscosity indicated that coalescence was not the responsible instability mechanism, since coalesced droplets remain spherical and do not affect viscosity (Fredrick, Walstra, & Dewettinck, 2010). Partial coalescence is therefore a likely mechanism of aggregation of fat droplets and the change of the emulsion textures. While GMU increased the instability induced by LACTEM, partial coalescence was somehow prevented by using a combination of LACTEM together with GMS or DATEM. The mechanism behind the enhanced stability remains unknown, but GMS and DATEM might increase the thickness of the adsorbed layer around the fat droplets, as a thicker layer counteracts partial coalescence (McClements, Dickinson, Ngan, Sella, et al., 1993; Palanuwech & Coupland, 2003). Susceptibility to partial coalescence was not related to polymorphic transformations of lipid crystals since the polymorphism was identical for all emulsions and remained unaltered despite large rheological changes. The impact of the monoglycerides on emulsions was only assessed at a given set of concentrations highly inspired by the compounding of commercial emulsifier mixtures. Further studies are required to establish if the reported effects will be valid for other concentrations of monoglycerides as well. A prerequisite for partial coalescence is fat crystallization in the dispersed droplets. Besides being present at the interface, crystals should be large and oriented perpendicular to the interfacial plane. The probability of a crystal piercing through the interface of another droplet is higher when the orientation is perpendicular to the interfacial plane (Rousseau, 2000). For occurrence of partial coalescence, large crystal sizes are preferable since penetration into neighboring droplets would be more efficient. Vanboekel and Walstra (1981) found that large droplet sizes increased the rate of partial coalescence due to the fact that crystals can achieve larger sizes in larger droplets. Fast cooling or efficient undercooling could therefore contribute to suppression of partial coalescence as these methods inhibit crystal growth and generate small crystals (Ghosh & Rousseau, 2010). This might explain the stability of the emulsion containing LACTEM + DATEM, which displayed a considerably higher undercooling prior to crystallization compared to the other emulsions containing monoglycerides (Table 1). Furthermore, partial coalescence increases with increasing solid fat content as a consequence of high prevalence of crystals able to protrude and pierce through adjacent droplets (Boode, Walstra, & Degrootmostert, 1993). However, the polarity of crystal faces is also of importance as protruding crystals should be wetted by oil instead of water to initiate partial coalescence (Vanboekel & Walstra, 1981). Partial coalescence also implies that fat droplets are able to approach one another, thus implying that electrostatic repulsion provided by interfacial sodium caseinate must be reduced. It is generally assumed that monoglycerides displace
M.B. Munk et al. / Food Research International 54 (2013) 1738–1745
proteins on the droplet interface and consequently the fat droplets tend to agglomerate. If protein displacement is the cause of the observed morphology and rheological changes, GMU can be expected to displace caseinate from the droplet interface more efficiently than GMS and DATEM. However, the present study provides no information about competitive displacement and further analyses are required to clarify this aspect. In contrast to the large effects on emulsion stability, the monoglycerides tested only affected the overall crystallization rate of bulk PKO to a minor extent. However, polymorphic transformations to stable PKO crystals were accelerated by addition of all monoglycerides in the bulk. Furthermore, both LACTEM and GMS increased the rate of crystallization, while GMS also initiated the nucleation stage causing crystallization to proceed at a higher temperature. In conclusion, the impact of monoglycerides on bulk PKO crystallization is far less noticeable compared to the pronounced effect observed in the dispersed state (emulsions). In particular, GMU in combination with LACTEM had a major impact on the stability of the emulsions. However, only a minor effect was observed when LACTEM + GMU were added to bulk PKO. 5. Conclusion This study shows that physical stability of emulsions is strongly dependent on the choice of emulsifiers. However, the results also point to the fact that the effects of emulsifiers in bulk fat and emulsified fat are different. Furthermore it emphasizes that emulsifiers either exert a different effect when oriented at the water–oil interface compared to orientation in bulk fat or affect interactions between droplets rather than affecting individual droplets (McClements & Dungan, 1997). In conclusion, the different processes leading to instability of emulsions, as illustrated by changes in droplet size, droplet morphology and rheological properties, cannot be explained by investigating a simplified bulk fat model system. Acknowledgment The authors gratefully acknowledge the Danish Agency for Science, Technology and Innovation for financial support. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.foodres.2013.09.001. References Allen, K. E., Murray, B.S., & Dickinson, E. (2008). Whipped cream-like textured systems based on acidified caseinate-stabilized oil-in-water emulsions. International Dairy Journal, 18, 1011–1021. AOCS (1995). X-ray diffraction analysis of fats. AOCS official method CJ2-95. Aronhime, J. S., Sarig, S., & Garti, N. (1988). Dynamic control of polymorphic transformation in triglycerides by surfactants — The button syndrome. Journal of the American Oil Chemists' Society, 65, 1144–1150. Barfod, N. M., & Krog, N. (1987). Destabilization and fat crystallization of whippable emulsions (toppings) studied by pulsed NMR. Journal of the American Oil Chemists' Society, 64, 112–119. Barfod, N. M., Krog, N., Larsen, G., & Buchheim, W. (1991). Effects of emulsifiers on protein–fat interaction in ice-cream mix during aging 1. Quantitative-analyses. Fett Wissenschaft Technologie-Fat Science Technology, 93, 24–29.
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Barfod, N. M., Schrader, K., & Buchheim, W. (2000). Water continuous fat crystal networks in ice cream mix with unsaturated monoglycerides. 2nd International symposium on food rheology and structure (pp. 1–5). Zürich, Switzerland: ETH. Boode, K., Walstra, P., & Degrootmostert, A. E. A. (1993). Partial coalescence in oil-in-water emulsions.2. Influence of the properties of the fat. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 81, 139–151. Bunjes, H., Koch, M. H. J., & Westesen, K. (2003). Influence of emulsifiers on the crystallization of solid lipid nanoparticles. Journal of Pharmaceutical Sciences, 92, 1509–1520. Davies, E., Dickinson, E., & Bee, R. (2000). Shear stability of sodium caseinate emulsions containing monoglyceride and triglyceride crystals. Food Hydrocolloids, 14, 145–153. Davies, E., Dickinson, E., & Bee, R. D. (2001). Orthokinetic destabilization of emulsions by saturated and unsaturated monoglycerides. International Dairy Journal, 11, 827–836. de Graef, V., van Puyvelde, P., Goderis, B., & Dewettinck, K. (2009). Influence of shear flow on polymorphic behavior and microstructural development during palm oil crystallization. European Journal of Lipid Science and Technology, 111, 290–302. Fredrick, E., Foubert, I., Van De Sype, J., & Dewettinck, K. (2008). Influence of monoglycerides on the crystallization behavior of palm oil. Crystal Growth & Design, 8, 1833–1839. Fredrick, E., Walstra, P., & Dewettinck, K. (2010). Factors governing partial coalescence in oil-in-water emulsions. Advances in Colloid and Interface Science, 153, 30–42. Ghosh, S., & Rousseau, D. (2010). Emulsion breakdown in foods and beverages. In L. H. Skibsted, J. Risbo, & M. L. Andersen (Eds.), Chemical deterioration and physical instability of food and beverages (pp. 260–295). Cambridge: CRC Press. Goff, H. D. (1997). Instability and partial coalescence in whippable dairy emulsions. Journal of Dairy Science, 80, 2620–2630. Kalnin, D., Schafer, O., Amenitsch, H., & Ollivon, M. (2004). Fat crystallization in emulsion: Influence of emulsifier concentration on triacylglycerol crystal growth and polymorphism. Crystal Growth & Design, 4, 1283–1293. Marangoni, A. G. (2005). Crystallization kinetics. Fat crystal networks (pp. 21–82). New York: Marcel Dekker. McClements, D. J., Dickinson, E., Ngan, S. R. K., Sella, J. E. M., et al. (1993). Effect of emulsifier type on the crystallization kinetics of oil-in-water emulsions containing a mixture of solid and liquid droplets. Journal of Colloid and Interface Science, 160, 293–297. McClements, D. J., & Dungan, S. R. (1997). Effect of colloidal interactions on the rate of interdroplet heterogeneous nucleation in oil-in-water emulsions. Journal of Colloid and Interface Science, 186, 17–28. Palanuwech, J., & Coupland, J. N. (2003). Effect of surfactant type on the stability of oil-in-water emulsions to dispersed phase crystallization. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 223, 251–262. Rossell, J. B. (1975). Differential scanning calorimetry of palm kernel oil products. Journal of the American Oil Chemists' Society, 52, 505–511. Rousseau, D. (2000). Fat crystals and emulsion stability — A review. Food Research International, 33, 3–14. Sato, K. (2001). Molecular aspects in fat polymorphism. In N. Widlak, R. Hartel, & S. Narine (Eds.), Crystallization and solidification properties of lipids (pp. 1–16). Illinois: AOCS Press. Sato, K., & Ueno, S. (2011). Crystallization, transformation and microstructures of polymorphic fats in colloidal dispersion states. Current Opinion in Colloid & Interface Science, 16, 384–390. Si, J. Q. (1991). The production of dairy analogue products using emulsifiers, stabilizers and flavours. In K. K. Rajah, & K. J. Burgess (Eds.), Milk fat production, technology and utilization (pp. 112–121). : The Society of Dairy Technology. Siew, W. L. (2001). Crystallisation and melting behaviour of palm kernel oil and related products by differential scanning calorimetry. European Journal of Lipid Science and Technology, 103, 729–734. Smith, K. W., Bhaggan, K., Talbot, G., & van Malssen, K. F. (2011). Crystallization of fats: Influence of minor components and additives. Journal of the American Oil Chemists' Society, 88, 1085–1101. Thivilliers-Arvis, F., Laurichesse, E., Schmitt, V., & Leal-Calderon, F. (2010). Shear-induced instabilities in oil-in-water emulsions comprising partially crystallized droplets. Langmuir, 26, 16782–16790. Timms, R. E. (1984). Phase-behavior of fats and their mixtures. Progress in Lipid Research, 23, 1–38. Vanapalli, S. A., Palanuwech, J., & Coupland, J. N. (2002). Influence of fat crystallization on the stability of flocculated emulsions. Journal of Agricultural and Food Chemistry, 50, 5224–5228. Vanboekel, M.A. J. S., & Walstra, P. (1981). Stability of oil-in-water emulsions with crystals in the disperse phase. Colloids and Surfaces, 3, 109–118. Westerbeek, J. M. M. (1989). Contribution of the alpha-gel phase to the stability of whippable emulsions. (Ph.D thesis). The Netherlands: Wageningen Agricultural University.
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Food Research International journal homepage: www.elsevier.com/locate/foodres
Stability of whippable oil-in-water emulsions: Effect of monoglycerides on crystallization of palm kernel oil Merete B. Munk a,⁎, Alejandro G. Marangoni b, Hanne K. Ludvigsen a, Viggo Norn a, Jes C. Knudsen c, Jens Risbo c, Richard Ipsen c, Mogens L. Andersen c a b c
Palsgaard A/S, Palsgaardvej 10, DK-7130 Juelsminde, Denmark Department of Food Science, University of Guelph, 50 Stone Road East, Guelph N1G 2W1, Ontario, Canada Department of Food Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark
a r t i c l e
i n f o
Article history: Received 12 June 2013 Accepted 5 September 2013 Keywords: Palm kernel oil Emulsion stability Lipid crystallization Monoglycerides LACTEM Polymorphism
a b s t r a c t The effect of selected monoglycerides on droplet morphology and rheology of palm kernel oil emulsions was studied. Combination of lactic acid ester of monoglycerides (LACTEM) and unsaturated monoglycerides (GMU) yielded highly viscous emulsions caused by partial coalescence. LACTEM combined with saturated monoglycerides (GMS) or diacetyl tartaric acid ester of monoglycerides (DATEM) gave low-viscous emulsions. The rheological behavior was found not to be related to polymorphic transformations of fat in the emulsions. A possible relationship between structural changes of emulsions and the effect of monoglyceride crystallization behavior of PKO was examined by studying the monoglycerides' influence on crystallization of bulk PKO. Polymorphic transformation, crystallization kinetics and melting/crystallization profile of bulk PKO were analyzed. The monoglycerides had minor effect on crystallization behavior of bulk PKO compared to the major impact on emulsion stability. This indicates that emulsifiers act differently in bulk and dispersed fat. For PKO the structural changes of the emulsions were independent of the effects of monoglycerides on the crystallization of bulk fat. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Making a successful whippable fat-based emulsion requires a delicate balance between conflicting physical properties. On the one hand, to achieve voluminous and stable foam of a whipped aerated emulsion, the fat droplets must interact strongly with each other and form a stable semi-rigid network, which stabilizes foam structure. Partial coalescence plays a crucial role for foam structure formation by creating a network of fat droplets that stabilize air bubbles in foams (Barfod & Krog, 1987; Goff, 1997). On the other hand, prior to whipping, the emulsion must be pourable and thus have a perpetual low viscosity, which requires that attractive forces between droplets are weak in order to avoid flocculation, droplet coalescence and consequent creaming of larger aggregates. In addition, the emulsion should preferably have long-term stability, implying resistance to agitation and thermal fluctuations occurring during transportation and storage. Ideally, formation of a strong network of partly coalesced droplets should not happen until whipping is initiated, and should not give rise to undesirable solidification, clumping or creaming of the emulsion. To ensure stable foam with long shelf life, various types of monoglycerides are often used as emulsifying agents in whippable emulsions. The high surface activity of monoglycerides is assumed to displace proteins at the fat droplet interface. Consequently, steric repulsion is ⁎ Corresponding author. Tel.: +45 41125539. E-mail address: [email protected] (M.B. Munk). 0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.09.001
reduced and a new thinner monolayer of monoglyceride surrounds the interface, which is more susceptible for crystal penetration from one fat droplet into another. As a result, partial coalesced fat aggregates, which make up the foam network, are more likely to be formed. However, some monoglycerides seem to cause unintended properties for emulsions before the whipping. Unsaturated monoglyceride (GMU) and lactic acid ester of monoglyceride (LACTEM) have been reported to cause increased viscosity of emulsions both under shear and quiescent conditions (Allen, Murray, & Dickinson, 2008; Barfod, Krog, Larsen, & Buchheim, 1991; Davies, Dickinson, & Bee, 2000; Davies, Dickinson, & Bee, 2001). Emulsifiers will affect the adsorbed interfacial layer around emulsified droplets, which is decisive for the nature of droplet interactions among themselves and with other components in the emulsion. Additionally, emulsifiers can play a secondary role due to their influence on crystallization of the emulsified fat. Emulsifiers can also affect crystallization behavior including nucleation, crystal growth, recrystallization of polymorphic forms and melting profiles of bulk fat (Smith, Bhaggan, Talbot, & van Malssen, 2011). Crystallization of fat in the dispersed droplets can lead to changes of physical properties of emulsions, such as increased viscosity. The objective of the present work was to investigate how physical stability of whippable palm kernel oil (PKO) emulsions prior to whipping was affected by different combinations of monoglycerides. Physical stability in this context refers to a perpetual pourability of the non-whipped emulsions, which requires that the dispersed droplets remain finite and spherical. Droplet
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size determinations, confocal microscopy, and rheological measurements were used to study physical stability of emulsions. Furthermore, the effect of the monoglycerides on crystallization of bulk PKO was examined with the aim of elucidating a possible relationship between undesirable textural changes of non-whipped emulsions and the monoglycerides' effect on crystallization behavior of PKO. The latter was examined by DSC, pNMR and powder X-ray diffraction. 2. Materials & methods 2.1. Materials Emulsifiers were of commercial food grade and all were provided by Palsgaard A/S (Juelsminde, Denmark): Lactic acid ester of monoglyceride (LACTEM) made from fully hydrogenated palm oil and rape-seed oil (C16:0 and C18:0 N97% of fatty acids); unsaturated monoglycerides (GMU) made from sunflower oil (C18:1 N81%); >saturated monoglycerides (GMS) made from fully hydrogenated palm oil and rape-seed oil (C16:0 and C18:0 N 97%); and diacetyl tartaric acid ester of monoglyceride (DATEM) made from palm oil and sunflower oil (C16:0, C18:1, and C18:2 comprised 93% of fatty acids). The stabilizer mixture (Palsgaard A/S, Juelsminde, Denmark) contained microcrystalline cellulose (MCC), sodium carboxymethylcellulose (CMC) and disodium phosphate. Hydrogenated palm kernel oil (PKO) was obtained from AAK (Karlshamn, Sweden) and sodium caseinate from DMV International (Veghel, The Netherlands). Sugar was purchased from Nordic Sugar (Nakskov, Denmark) and sorbitol from Roquette (Cedex, France). 2.2. Emulsion and bulk PKO blend preparation 2.2.1. Emulsions Sodium caseinate (0.6 wt.%), stabilizer mixture (0.6 wt.%) and sugar (10 wt.%) were dispersed in water under continuous stirring and put aside for 4 h to hydrate proteins. Melted PKO (25 wt.%), LACTEM (0.55 wt.%) and GMU (0.15 wt.%), GMS (0.15 wt.%) or DATEM (0.10 wt.%) were mixed with the heated water phase (~70 °C) and the complete mix was re-heated to 82 °C. A pre-emulsion was conducted by mixing with a high-shear blender (Ultra-Turrax, IKA, NC, USA) for approximately 20 s. Homogenization was subsequently carried out on a two-stage high-pressure valve homogenizer (APV Rannie LAB-12.50, SPX, Silkeborg, Denmark) at 150/50 bar. Emulsions were rapidly cooled to 5–6 °C by a plate heat exchanger (APV U2, SPX, Silkeborg, Denmark) connected to the homogenizer. The emulsions were immediately poured into small beakers and stored at 5 °C. Control emulsions with sodium caseinate as the only emulsifier were prepared in a similar way. 2.2.2. Bulk PKO blends GMU (5.0 wt.%), GMS (5.0 wt.%) or DATEM (5.0 wt.%), either singly or in combination with LACTEM (5.0 wt.%), was added to melted PKO and the blend was stirred to ensure homogenous distribution and heated to 80 °C to remove crystal history. The blend was applied to an appropriate container (DSC-vial, NMR-tube or X-ray plate) depending on analysis method and immediately analyzed by DSC, NMR or X-ray.
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Heidelberg, Germany). Total concentration of 1 ppm BODIPY 493/503 (4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene) (Invitrogen, Carlsbad, USA) dissolved in dimethyl sulfoxide was added to emulsions to stain lipid. Excitation wavelength was 488 nm and emission bandwidth was 500–570 nm. A water immersion objective (HCX PL APO lambda blue 63.0 × 1.20 water UV) was used and image resolution was set to 1024 × 1024 pixels. A thin layer of stained emulsion was applied on a large standard cover glass and covered by another cover glass to avoid evaporation/drying. 2.3.3. Rheological measurements An ARG2 rheometer (TA-Instruments, West Sussex, England, UK) fitted with a Peltier temperature control connected to a water bath was used for all rheological measurements. Steady state flow experiments with increasing shear rate from 0.01 to 100 s−1 were performed at 5 °C and 20 °C to determine viscosity. A cone (diameter 40 mm, inclination 1.59°) and plate geometry was applied. Some emulsions exhibited a very firm texture and were not flowable. These were molded into discs of diameter 20 mm and characterized by a stress sweep with increasing oscillation stress from 0.1 to 1000 Pa at a constant shear frequency of 1 Hz. Plate (diameter 20 mm) and a plate geometry, both with roughened surfaces, to avoid slip was used. Viscosity measurements were performed at least in duplicate whereas small amplitude oscillatory shear was repeated 5 times. 2.4. Characterization of fat crystallization 2.4.1. Differential scanning calorimetry Melting and crystallization temperatures were determined for both emulsions and PKO blends by the use of DSC1 Stare System (Mettler Toledo, Schwerzenbach, Switzerland). Samples were initially held at 5 °C for 10 min, then heated to 80 °C at 2 °C/min and subsequently re-cooled to 5 °C at 2 °C/min. Empty pans were used as reference and each sample was analyzed in duplicate. 2.4.2. Crystallization kinetics Solid fat content (SFC) of PKO blends containing emulsifiers was determined as a function of time at isothermal conditions by the use of pulsed NMR spectroscopy (Bruker Minispec, Bruker, Germany). Tubes of samples were placed in water baths at 10 °C and 20 °C and SFC was measured at appropriate intervals till crystallization appeared to be completed as the crystalline fraction reached a constant level. 2–5 replicates of each sample were measured. The results were fitted to the Avrami equation by non-linear regression of Origin® 7 (OriginLab, Northampton, USA): SFC t −kt n ¼ 1−e : SFC max
ð1Þ
Where SFCt is the crystalline content (%) at time t and SFCmax the maximum crystalline content (%) achieved at the particular temperature. k is the crystallization rate constant, and n the Avrami exponent which is dependent on crystal growth mechanism with respect to nucleation and growth dimensions.
2.3. Characterization of emulsions 2.3.1. Determination of droplet size by light scattering Droplet size distribution was measured 2, 8 and 15 days after emulsion preparation (Mastersizer Microplus, Malvern Instruments, Malvern, U.K.). Measurements were conducted at room temperature on emulsions diluted approximately 1:100 with de-ionized water. At least 3 replicates were measured for each emulsion. 2.3.2. Confocal laser scanning microscopy Structure and morphology of fat globules in emulsions were examined by an inverse confocal laser scanning microscope (Leica TCS SP5,
2.4.3. Polymorphic transformation The polymorphism of crystals was examined for both emulsions and PKO blends by powder X-ray diffractometer (Rigaku, Tokyo, Japan). Radiation at λ = 1.54 Å was generated from a copper anode and operated at 40 kV and 44 mA. The scanning velocity was 0.05°/min for emulsions and 1°/min for PKO blends in the area of 1.2–35 2θ. The diffractometer was protected by a Multiflex chamber, which was equipped with Peltier temperature control connected to a water bath. Emulsions were examined at 20 °C without any preceding treatment and also after exposing the emulsion to stirring with a spatula. At least two diffraction spectra were recorded for each emulsion. Fully melted PKO blends were cooled
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to either 5 °C or 20 °C and after 10 or 15 min respectively the diffraction spectrum was recorded. The samples of PKO blends were kept and recorded the following day, day 7 and day 21.
a
2.5. Statistical analysis The results of the analyses are reported as the mean and standard deviation calculated from the replicates. The level of significance for melting and crystallization temperature is calculated by one way ANOVA and is given by the P-value. Intervals of confidences for n and k of the Avrami model is calculated from the Jacobian matrix which is an output of the non-linear fitting procedure. 3. Results 3.1. Rheological properties of emulsions Four different palm kernel oil emulsions were made using LACTEM as one of the emulsifiers. The emulsions also contained caseinate, stabilizer and sugar in order to mimic the composition of a commercial whippable emulsion. LACTEM was included because it promotes fat droplet aggregation (Westerbeek, 1989) and therefore is widely used in whipped toppings and dessert products (Si, 1991). In addition, three of the emulsions contained DATEM, unsaturated (GMU) or saturated monoglycerides (GMS), since LACTEM is often used in combination with other monoglycerides in commercial products. Furthermore, the specific combinations of LACTEM and the other monoglycerides were selected in order to create large variations in emulsion stability in terms of changes in viscosity and texture. An emulsion without added monoglycerides was prepared as a control. The differences among the emulsions were immediately discernible after preparation as observed by the products ranging from low-viscous homogeneous emulsions to thick pastes and clumping emulsions. Emulsions with LACTEM alone were found to yield semi-solid emulsions. The properties were initially qualitatively assessed by stirring the emulsions with a spatula at room temperature or at 5 °C. Stirring the LACTEM emulsion at 5 °C drastically reduced the viscosity, whereas stirring the emulsion at room temperature gave a thick paste. In addition, the process causing the instability of this emulsion appeared to be reversible, as a pourable emulsion could be obtained again by cooling the thick paste to 5 °C followed by stirring. Combining LACTEM with GMU gave very firm emulsions that appeared not to be sensitive to any combination of stirring and temperature. In fact, clotting appeared already within 10–15 min after the preparation of the emulsion and after 1-2 h at 5 °C complete solidification occurred. On the other hand, combinations of GMS or DATEM together with LACTEM yielded perpetual pourable emulsions. The control emulsion made without any added monoglyceride resulted in a low-viscous emulsion that was not affected by stirring and temperature. The observed qualitative changes in viscosity and texture were characterized by rheological measurements (Fig. 1). At 5 °C and at low shear rates, the apparent viscosity of the emulsion containing LACTEM was ~50 Pa·s (Fig. 1a). The apparent viscosity decreased with increasing shear rate, demonstrating shear thinning. However, the apparent viscosities were 5 to 10 times higher than for the control emulsion without monoglycerides. The two emulsions with either added GMS or DATEM also demonstrated shear thinning behavior and had viscosities between the control emulsion and the LACTEM-only emulsion at all shear rates. At 20 °C only the control emulsion demonstrated a true shear thinning effect comparable to measurements at 5 °C (Fig. 1b). The viscosity of the emulsion with LACTEM could not be determined at 20 °C since the texture of the emulsion was too firm. A lumpy texture in emulsions caused by LACTEM has previously been reported (Allen et al., 2008), and is probably due to partial coalescence as a consequence of protein displacement on droplet interface (Thivilliers-Arvis, Laurichesse, Schmitt, & Leal-Calderon, 2010). Emulsions with LACTEM + GMS exhibited a sudden shear thickening behavior at 20 °C at shear rates around
b
c
Fig. 1. Monoglycerides' influence on rheological behavior of emulsions. ■: Control, ▲: LACTEM + DATEM, □: LACTEM + GMS, ○: LACTEM, ▼: LACTEM + GMU. a: Viscosity at 5 °C, LACTEM + GMU was too viscous to be measured. b: Viscosity at 20 °C, emulsions with LACTEM and LACTEM + GMU were too viscous to be measured. c: The elastic modulus at 20 °C of high viscous emulsions, LACTEM and LACTEM + GMU.
0.03 s−1 (Fig. 1b). Yet, the viscosity was significantly lower compared to emulsions with LACTEM. Addition of DATEM prevented shear induced modifications until a certain degree, and at sufficient high shear rates a slight increase of viscosity occurred indicating subtle shear thickening effects. The instability of emulsions containing LACTEM + GMU was very evident since they exhibited complete solidification at all temperatures. The firm emulsions remained resistant to shear torque at 2 · 105 mN/m at 5 °C and 20 °C, and thus viscosity was not a measurable parameter.
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Fig. 2. The effect of monoglycerides on droplet size distribution in emulsions, — (solid black) control(solid black), LACTEM (long dash), The line style of "LACTEM + GMS" should be grey LACTEM + GMS (solid grey), — LACTEM + DATEM (short dash), …… LACTEM + GMU (dot).
Instead, rheological properties were characterized by small amplitude oscillatory shear. An elastic modulus, G′ ≈ 3.2 ± 0.4 · 106 Pa (Fig. 1c) was found, and similar for the firm LACTEM emulsion at 20 °C with G′ ≈ 4.5 ± 3 · 106 Pa. Although no statistical significant differences in G′ were obtained between emulsions containing LACTEM and LACTEM + GMU, it should be noted that emulsions containing LACTEM + GMU were three times firmer than emulsions made only with LACTEM (measured by back extrusion; data not shown). The higher stress and/or longer duration required to induce structural changes in emulsions with LACTEM + GMS as compared to LACTEM + GMU is in agreement with Barfod et al. (1991) who observed a high stability of emulsions containing GMS in contrast to emulsions with GMU. 3.2. Droplet size The droplet size distributions were clearly related to the viscosity of the emulsions. A monomial and fairly narrow distribution of small
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droplets was characteristic for the three low-viscosity emulsions made without added monoglycerides, a combination of LACTEM + GMS, or a combination of LACTEM + DATEM (Fig. 2). The mean diameters, d32, were respectively 0.98 ± 0.03 μm, 0.84 ± 0.03 μm and 0.65 ± 0.01 μm for the three emulsions after storage at 5 °C for 2 days. The two highly viscous and firm emulsions made either with LACTEM or LACTEM + GMU were characterized by very broad droplet distributions. The broad distributions of mainly large droplets suggest the presence of aggregates in emulsions. Prior to size distribution measurements, emulsions were diluted with water. Despite the fact that the continuous phases of all the emulsions were water, dilutions of the firm emulsions made with LACTEM and LACTEM + GMU were practically impossible despite vigorous shaking using a vortex mixer. This demonstrated extreme sensitivity towards agitation and thermal fluctuation since both types of emulsions, but particularly LACTEM + GMU, appeared as large non-dispersible flocks in water as a consequence of the preparatory steps of measurements even though the samples were held at 5 °C. Droplet size distributions were also measured after 8 and 15 days of storage at 5 °C but no significant changes occurred during this period. The differences among the emulsions in terms of microstructure were clearly visualized by confocal laser scanning microscopy (Fig. 3). The three low-viscous emulsions, without added monoglycerides, with LACTEM + GMS or LACTEM + DATEM, all exhibited a structure of small spherical fat globules (Fig. 3a, e and f), supporting the results from droplet size distributions. Conversely, the fat globules in the high viscous emulsion containing LACTEM + GMU exhibited large irregular aggregates (Fig. 3d). The emulsion with only LACTEM also contained aggregated droplets, and the sizes of the aggregates increased drastically when the emulsion was subjected to shear at room temperature (Fig. 3b and c). The physical mechanism of the latter aggregate enlargement was most likely flocculation, since reduced viscosity could be regained by cooling and shearing. On the other hand, the large coherent aggregates of the emulsions with LACTEM + GMU could not similarly be changed to give a low-viscous emulsion and thus probably resulted from partial coalescence.
Fig. 3. Micro-structure investigated by CLSM of emulsions containing various monoglycerides. Before microscopic analysis the emulsions were stored at 5 °C. The fat fraction is stained and the scale bar equals 10 μm. a: Control, b: LACTEM, low viscous, c: LACTEM, high viscous due to shear at room temperature, d: LACTEM + GMU, e: LACTEM + GMS, f: LACTEM + DATEM.
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Fig. 4. Melting and crystallization thermograms of bulk PKO. Arrows indicate defined melting and crystallization temperatures.
3.3. DSC analysis of emulsions and bulk PKO Partial coalescence of lipid droplets in emulsions requires that the lipids are partially crystallized. Since the emulsifiers potentially could modify the crystallization behavior of the palm kernel oil, the effect of the different monoglyceride combinations on the melting and crystallization behaviors of both bulk and emulsified PKO was studied by DSC. For the study of bulk PKO, the monoglycerides were added both separately in a concentration of 5 wt.% or in combination with 5 wt.% LACTEM. The concentration of monoglycerides in the bulk fat was higher than in the emulsions for two reasons. First, it was assumed that an excessive concentration would make the crystallization effect of monoglycerides more distinct if any. Second, the local concentration of monoglyceride was much higher at the droplet interface compared to the total concentration in the emulsions and therefore it was presumed that a higher monoglyceride concentration in bulk PKO would reflect the crystallization behavior more realistic. The endothermic DSC thermogram of pure PKO showed that PKO melted over a broad temperature range between 5 °C and 43 °C in accordance with the observations by Siew (2001) (Fig. 4). The onset melting temperatures could not be determined reliably due to a very gradual onset of melting. Melting temperatures were therefore defined as the peak temperature of the lowest endothermic peak as indicated in Fig. 4. In agreement with previous work, the presence of monoglycerides did not significantly affect the melting behavior of neither bulk nor emulsified PKO (P N 0.05), Table 1 (Barfod, Schrader, & Buchheim, 2000). In the cooling experiments, most of the emulsions and bulk PKO samples showed two exothermic peaks caused by crystallization; a minor one at 22–23 °C and a major one at 19–18 °C (Fig. 4 and Table 1). Apart from GMS, the monoglycerides did not have
significant effect on the crystallization temperature of bulk PKO samples (P N 0.05). Only addition of GMS increased crystallization temperature significantly of bulk PKO (P b 0.001), probably due to GMS being a high melting component (mp. N 60 °C). Formation of crystals early in the cooling experiment can have acted as seeds for nucleation of PKO. In contrast, the crystallization temperature of emulsified PKO without added monoglycerides was significantly reduced compared to bulk PKO most likely as a consequence of supercooling. The entire fat fraction in the control emulsion crystallized around 8 °C, which indicated supercooling due to the absence of nucleus monoglycerides and the presence of a thick protecting layer of sodium caseinate blocking intrusion of nucleating crystals (Palanuwech & Coupland, 2003). Similar to the bulk PKO-blends, emulsions containing LACTEM, GMU and GMS displayed a small exothermic peak around 22–28 °C, and a second exothermic peak at temperature N 8 °C (Table 1). Destabilized emulsion droplets are expected to yield a thermogram comprising the crystallization peaks of both the bulk PKO and the dispersed PKO, that is 23 °C, 19 °C and 8 °C respectively (Palanuwech & Coupland, 2003; Vanapalli, Palanuwech, & Coupland, 2002). Since none of the emulsions containing monoglycerides were crystallizing at 8 °C, droplet destabilization was apparently not the cause for the increased crystallization temperature as compared to the control emulsion. Emulsion destabilization implies that the positions of the peaks corresponding to bulk and emulsified fat are steady, and therefore the single crystallization peak at 11 °C for emulsions containing LACTEM + DATEM can neither be explained by droplet destabilization. The different crystallization behavior between emulsions with monoglycerides and the control emulsion is probably caused by monoglycerides that catalyze the nucleation event by intervening as heteronuclei and thereby increase the crystallization temperature. Davies, Dickinson, and Bee reported similar effects on crystallization of both bulk and emulsified fat with addition of emulsifiers (Davies et al., 2001).
3.4. Crystallization kinetics of bulk PKO Crystallization of bulk PKO at 5 °C occurred too fast to follow experimentally and therefore the monoglycerides' influence on crystallization kinetics of bulk PKO was examined at 10 °C and 20 °C by pulsed nuclear magnetic resonance (pNMR). By fitting the isothermal SFC as function of time to the Avrami model (Eq. (1)), estimates could be obtained for the crystallization rate constant (Avrami constant k), and the index of crystallization related to the type of nucleation and dimensionality of growth (Avrami exponent n) (Table 2). The maximum crystalline content was within 60–70% at 10 °C and 82–89% at 20 °C for all emulsions. Each monoglyceride was added in concentrations of 5 wt.% as no effect could be detected at lower concentrations. GMU and DATEM did not affect crystallization of PKO at either temperature and values of k and n similar to those of pure PKO were obtained (Fig. 5 and Table 2). Both LACTEM and GMS increased the crystallization rate of PKO. The increase in crystallization rate was also observed when
Table 1 Melting and crystallization temperatures of bulk PKO and emulsions containing various monoglycerides. Melting temperature was defined as the peak of the first endothermic peak. Most samples displayed two exothermic crystallization peaks, a minor (~22 °C) and a large one (~19 °C). Emulsifier
No monoglycerides LACTEM GMU GMU + LACTEM GMS GMS + LACTEM DATEM DATEM + LACTEM
Bulk PKO
Emulsion
Tpeak melting (°C)
Tpeak crystallization (°C)
Tpeak melting (°C)
Tpeak crystallization (°C)
23.2 23.8 24.0 23.3 24.4 23.4 22.9 23.4
22.8 23.5 22.1 22.7 25.7 26.3 23.3 23.3
24.0 ± 0.4 23.7 ± 0.6
7.8 ± 0.1 21.9 ± 0.1/17.4 ± 0.2
23.8 ± 0.5
21.8 ± 0.8/17.7 ± 0.6
23.5 ± 0.2
27.9 ± 0.4/15 ± 0.2
24.0 ± 0.6
11.4 ± 0.2
± ± ± ± ± ± ± ±
0.8 0.6 0.0 1.1 0.1 0.0 0.5 0.3
± ± ± ± ± ± ± ±
0.3/19.1 0.3/19.1 0.2/18.3 0.5/18.1 0.2/19.7 0.5/20.9 0.2/19.7 0.1/18.9
± ± ± ± ± ± ± ±
0.2 0.2 0.3 0.1 1.0 0.2 0.1 0.4
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Table 2 Avrami exponent n and Avrami constant k for crystallization of PKO containing various monoglycerides at 10 °C and 20 °C. PKO
10 °C
20 °C
n No monoglycerides LACTEM GMU GMS DATEM LACTEM + GMU LACTEM + GMS LACTEM + DATEM
1.8 1.5 1.9 1.6 1.9 1.6 1.3 1.5
k ± ± ± ± ± ± ± ±
0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.1
6.8 6.4 5.7 2.9 4.9 3.4 1.6 4.2
n ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗
−5
10 10−4 10−5 10−4 10−5 10−4 10−3 10−4
± ± ± ± ± ± ± ±
LACTEM was mixed with other emulsifiers, although the effect was moderated with GMU and DATEM. In contrast, combination of GMS and LACTEM resulted in the highest crystallization rate and the lowest value of n at 10 °C. For all PKO blends the Avrami exponent, n, assumed a value between 1.3 and 1.9. This indicate a similar crystal growth mechanism, which according to polarized light microscopy (PLM) (data not shown) most likely was a rod-like growth from sporadic nuclei (n = 2) (Marangoni, 2005). Thus, the tested monoglycerides seemed not to have an effect on nucleation and growth dimensions. However, the possibility that GMS induced rod-like growth from instantaneous nuclei (n = 1) (Marangoni, 2005) cannot be rejected since initial crystallization for this blend had occurred prior to the PLM examination. Image analysis (data not shown), however, suggested similar nucleation and morphology among the blends.
Fig. 5. Effect of monoglycerides on the initial crystallization kinetics of PKO at 10 °C and 20 °C. ■: No monoglycerides,○: LACTEM, □: GMS, ▲: DATEM, ▼: GMU. Insets: The complete crystallization at 10 °C and 20 °C followed over 1 h and 2 h, respectively.
3.5 3.5 2.9 1.2 3.0 3.5 4.1 2.3
∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗
−5
10 10−4 10−5 10−4 10−5 10−4 10−4 10−4
1.9 1.4 1.9 1.6 1.9 1.6 1.3 1.7
k ± ± ± ± ± ± ± ±
0.2 0.2 0.2 0.1 0.2 0.2 0.1 0.2
2.3 4.4 1.5 1.2 1.6 9.2 5.0 5.0
∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗
10−5 10−4 10−5 10−4 10−5 10−5 10−4 10−5
± ± ± ± ± ± ± ±
2.0 3.6 1.5 8.6 2.2 9.9 2.7 6.4
∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗
10−5 10−4 10−5 10−5 10−5 10−5 10−4 10−5
3.5. Polymorphic transformations of emulsions and bulk PKO The polymorphic forms were identified by reflections in powder Xray diffraction experiments corresponding to characteristic d-spacing. The α-form is characterized by d = 4.15 Å, β′ is characterized by d = 3.8 and 4.2 Å and β characterized by d = 4.6 Å (AOCS, 1995). The transformation of emulsions from pourable to thick pastes upon shear made it plausible to believe that the transformation came along with recrystallization of the fat. Therefore X-ray diffraction was carried out twice on emulsions. First emulsions were not exposed to any preceding treatments and second emulsions were stirred with a spatula until viscosity had increased. The prevalence of polymorphic forms was indifferent although the textural properties of the emulsions had changed, Fig. 6. All emulsions regardless the type of monoglycerides and pourability consisted of β′-polymorphs with traces of α. For the PKO blends the X-ray diffractograms differed a bit more, although all blends contained a mixture of α- and β′-polymorphs. The ratio of β′/α depended on added monoglyceride, cooling temperature and time after temperature quench. The diffractograms were superimposed, which complicated a straightforward quantification of the relative amount of each polymeric forms using information from the short spacing part of the diffractograms. In such case long spacing d-values in the SAXS regime have previously been used for quantification of the polymorphs (de Graef, van Puyvelde, Goderis, & Dewettinck, 2009; Fredrick, Foubert, Van De Sype, & Dewettinck, 2008; Kalnin, Schafer, Amenitsch, & Ollivon, 2004). In the present case a clear correlation was found between the reflection at 4.15 Å and a reflection at 43 Å, and the latter was thus also attributed to the αpolymorph. Likewise, a correlation was established between reflections at 3.8/4.2 Å and 35 Å, which again were attributed to the β′-polymorph.
Fig. 6. Crystal polymorphism of emulsion containing LACTEM + GMS. The upper diffractogram displays a low-viscous pourable emulsion with no pre-treatment and the lower diffractogram displays the emulsion stirred into a thick paste. The peaks corresponding to β′ (d = 4.2 Å and 3.8 Å) and α (d = 4.15 Å) are pointed out.
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Table 3 The relative ratio of β′-crystals compared to α-crystals in bulk PKO with addition of monoglycerides as a function of storage time at 5 °C and 20 °C. After day 7 the peak corresponding to α was not detectable (n.d.) for most samples and therefore results from day 21 are not included in the table. Emulsifier
No monoglycerides LACTEM GMU GMU + LACTEM GMS GMS + LACTEM DATEM DATEM + LACTEM
5 °C
20 °C
Day 0
Day 1
Day 7
Day 0
Day 1
Day 7
0.9 3.0 1.0 1.0 1.6 6.4 1.2 1.0
1.3 3.2 2.0 1.8 2.2 9.5 1.9 2.9
1.4 3.0 4.0 3.1 2.1 8.0 n.d. 4.1
1.5 3.4 2.0 1.5 2.0 2.3 3.4 1.5
1.9 4.5 6.5 2.1 2.3 4.2 n.d. 2.0
2.1 4.3 5.9 n.d. 2.8 8.8 n.d. n.d.
The relative rate of polymorphic transformation from α to β′ at temperatures of 5 °C and 20 °C was followed for a duration of 3 weeks, and the results are shown in Table 3. The time development of the polymorphic transformation from α to β′ is studied by examining the ratio: β0 Apeakd¼35 ; α Apeakd¼43
ð2Þ
which expresses the ratio between the amount the two polymorphic forms. Initially all PKO blends comprised a mix of α and β′, but during three weeks α was transformed into β′, the most stable polymorph of PKO (Rossell, 1975). The preference of β′ over β is most likely due to the wide range of fatty acid chain lengths in triglycerides of PKO (Sato, 2001; Timms, 1984). The initial fraction of α, as well as polymorphic transformation rate, was clearly dependent on crystallization temperature, storage time and addition of monoglycerides. Using a storage temperature of 5 °C caused a higher fraction of α-polymorphs as compared to 20 °C, due to low thermodynamic stability and low melting point of α (Sato & Ueno, 2011). Subsequent recrystallization into β′-polymorphs occurred, and after 7 days the reflection corresponding to α was not distinguishable for most samples. Several of the tested monoglycerides favored initial formation of β′ directly from the melt. This applied especially to LACTEM, LACTEM + GMS and DATEM, although for the latter this was only observed at 20 °C, Table 3. In addition, all monoglycerides, to varying degrees, accelerated the polymorphic transformation in agreement with previous studies (Aronhime, Sarig, & Garti, 1988; Fredrick et al., 2008; Smith et al., 2011). Overall, all tested monoglycerides promoted faster formation of the most stable polymorph. This might be related to the fact that except anionic DATEM, all applied monoglycerides were non-ionic which are suggested to induce transformation of crystals into more stable polymorphs (Bunjes, Koch, & Westesen, 2003). However, the monoglycerides appeared to be divided into two groups: GMU and DATEM (used in pure form or in combination with LACTEM) accelerated gradually the transformation from α to β′, whereas GMS and LACTEM readily caused a high level of β′ directly from the melt which remained fairly constant during the remaining experimental period (Fig. 7). 4. Discussion The stabilities of the investigated emulsions were strongly dependent on the type of monoglycerides added. Both droplet size distributions and microscopic inspection revealed formation of large fat aggregates in the two high-viscosity emulsions made with LACTEM or LACTEM + GMU. Furthermore, increased viscosity as a consequence of shear and increased temperature was linked to enlargement of the aggregates. The clusters of droplets remained non-dispersable in a water solution despite vigorous shaking, and therefore the aggregates
Fig. 7. Polymorphic transformations of α to β′-crystals during storage for 20 days at 5 °C for PKO without monoglycerides (■), with LACTEM + GMU (▼) and with LACTEM + GMS (□).
were unlikely to be held together by flocculation. Furthermore, the partial crystalline state of the dispersed fat, the irregular shapes of the aggregates and the increase in emulsion viscosity indicated that coalescence was not the responsible instability mechanism, since coalesced droplets remain spherical and do not affect viscosity (Fredrick, Walstra, & Dewettinck, 2010). Partial coalescence is therefore a likely mechanism of aggregation of fat droplets and the change of the emulsion textures. While GMU increased the instability induced by LACTEM, partial coalescence was somehow prevented by using a combination of LACTEM together with GMS or DATEM. The mechanism behind the enhanced stability remains unknown, but GMS and DATEM might increase the thickness of the adsorbed layer around the fat droplets, as a thicker layer counteracts partial coalescence (McClements, Dickinson, Ngan, Sella, et al., 1993; Palanuwech & Coupland, 2003). Susceptibility to partial coalescence was not related to polymorphic transformations of lipid crystals since the polymorphism was identical for all emulsions and remained unaltered despite large rheological changes. The impact of the monoglycerides on emulsions was only assessed at a given set of concentrations highly inspired by the compounding of commercial emulsifier mixtures. Further studies are required to establish if the reported effects will be valid for other concentrations of monoglycerides as well. A prerequisite for partial coalescence is fat crystallization in the dispersed droplets. Besides being present at the interface, crystals should be large and oriented perpendicular to the interfacial plane. The probability of a crystal piercing through the interface of another droplet is higher when the orientation is perpendicular to the interfacial plane (Rousseau, 2000). For occurrence of partial coalescence, large crystal sizes are preferable since penetration into neighboring droplets would be more efficient. Vanboekel and Walstra (1981) found that large droplet sizes increased the rate of partial coalescence due to the fact that crystals can achieve larger sizes in larger droplets. Fast cooling or efficient undercooling could therefore contribute to suppression of partial coalescence as these methods inhibit crystal growth and generate small crystals (Ghosh & Rousseau, 2010). This might explain the stability of the emulsion containing LACTEM + DATEM, which displayed a considerably higher undercooling prior to crystallization compared to the other emulsions containing monoglycerides (Table 1). Furthermore, partial coalescence increases with increasing solid fat content as a consequence of high prevalence of crystals able to protrude and pierce through adjacent droplets (Boode, Walstra, & Degrootmostert, 1993). However, the polarity of crystal faces is also of importance as protruding crystals should be wetted by oil instead of water to initiate partial coalescence (Vanboekel & Walstra, 1981). Partial coalescence also implies that fat droplets are able to approach one another, thus implying that electrostatic repulsion provided by interfacial sodium caseinate must be reduced. It is generally assumed that monoglycerides displace
M.B. Munk et al. / Food Research International 54 (2013) 1738–1745
proteins on the droplet interface and consequently the fat droplets tend to agglomerate. If protein displacement is the cause of the observed morphology and rheological changes, GMU can be expected to displace caseinate from the droplet interface more efficiently than GMS and DATEM. However, the present study provides no information about competitive displacement and further analyses are required to clarify this aspect. In contrast to the large effects on emulsion stability, the monoglycerides tested only affected the overall crystallization rate of bulk PKO to a minor extent. However, polymorphic transformations to stable PKO crystals were accelerated by addition of all monoglycerides in the bulk. Furthermore, both LACTEM and GMS increased the rate of crystallization, while GMS also initiated the nucleation stage causing crystallization to proceed at a higher temperature. In conclusion, the impact of monoglycerides on bulk PKO crystallization is far less noticeable compared to the pronounced effect observed in the dispersed state (emulsions). In particular, GMU in combination with LACTEM had a major impact on the stability of the emulsions. However, only a minor effect was observed when LACTEM + GMU were added to bulk PKO. 5. Conclusion This study shows that physical stability of emulsions is strongly dependent on the choice of emulsifiers. However, the results also point to the fact that the effects of emulsifiers in bulk fat and emulsified fat are different. Furthermore it emphasizes that emulsifiers either exert a different effect when oriented at the water–oil interface compared to orientation in bulk fat or affect interactions between droplets rather than affecting individual droplets (McClements & Dungan, 1997). In conclusion, the different processes leading to instability of emulsions, as illustrated by changes in droplet size, droplet morphology and rheological properties, cannot be explained by investigating a simplified bulk fat model system. Acknowledgment The authors gratefully acknowledge the Danish Agency for Science, Technology and Innovation for financial support. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.foodres.2013.09.001. References Allen, K. E., Murray, B.S., & Dickinson, E. (2008). Whipped cream-like textured systems based on acidified caseinate-stabilized oil-in-water emulsions. International Dairy Journal, 18, 1011–1021. AOCS (1995). X-ray diffraction analysis of fats. AOCS official method CJ2-95. Aronhime, J. S., Sarig, S., & Garti, N. (1988). Dynamic control of polymorphic transformation in triglycerides by surfactants — The button syndrome. Journal of the American Oil Chemists' Society, 65, 1144–1150. Barfod, N. M., & Krog, N. (1987). Destabilization and fat crystallization of whippable emulsions (toppings) studied by pulsed NMR. Journal of the American Oil Chemists' Society, 64, 112–119. Barfod, N. M., Krog, N., Larsen, G., & Buchheim, W. (1991). Effects of emulsifiers on protein–fat interaction in ice-cream mix during aging 1. Quantitative-analyses. Fett Wissenschaft Technologie-Fat Science Technology, 93, 24–29.
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