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J Am Oil Chem Soc DOI 10.

1007/s11746-011-1965-y

ORIGINAL PAPER

The Role of Mixing Temperature on Microstructure and Rheological Properties of Butter Blends
Patrizia Buldo Lars Wiking

Received: 20 June 2011 / Revised: 8 October 2011 / Accepted: 20 October 2011 AOCS 2011

Abstract The present study demonstrated that the rheological properties of butter blends can be modied by the applied mixing temperature. Blends were prepared by mixing 10 or 25% of rapeseed oil (RO) with butter, at three different temperatures (13, 18 and 23 C). Afterwards the blends were stored at 5 C until analyzed. Microstructure, rheological properties, melting behavior and solid fat content (SFC) of the blends were examined. The viscoelastic properties of the blends were measured by rheological oscillation analysis. Mixing at 23 C always resulted in the softest products, hence the lowest rmness, independently of the content of rapeseed oil. By increasing the percentage of RO and the mixing temperature, a decrease in melting point (Mp) and in SFC was observed in the blends. The microstructure of the blends was analyzed with confocal laser scanning microscopy (CLSM), which explains the effect on the rheological behavior. The microstructure analysis showed that a high content of RO and high processing temperatures produce a less dense crystal network and a change in protein/water distribution. Furthermore, this study shows that the addition of RO to butter and the high mixing temperature solubilize some of the milk fat triacylglycerides (TAG), which are not able to re-crystallize fully. A high mixing temperature is shown to inhibit the ability to rebuild the rigidity of the crystal network in butter blends. Keywords Butter blend Mixing temperature Crystallization Rheology Microstructure Solid fat content Rapeseed oil
P. Buldo L. Wiking (&) Department of Food Science, Aarhus University, Blichers Alle 20, P.O. box 50, 8830 Tjele, Denmark e-mail: lars.wiking@agrsci.dk

Introduction Blending milk fat with vegetable oils is mainly used to change the physical properties of milk fat-based products. The blended products, or cold-spreadable products, have improved spreadability at refrigerator temperature. Furthermore, butter blends are used to enhance the nutritional properties of butter products, such as a desirable fatty acid (FA) prole, and vice versa they increase the buttery avor of vegetable fats. Rapeseed oil (RO) is a good vegetable oil for use in cold-spreadable products. It has a high content of oleic acid, and it is avorless and odorless. The manufacturing process of butter blends involves mixing of butter and vegetable oil just after churning. The microstructure of butter blends consists of a dispersion of fat crystals in a continuous liquid oil phase [1]. The fat crystal network outside the globules is, to some extent, interrupted by intact milk fat globules, their membranes preventing the enclosed fats from being incorporated into the liquid/solid fat dispersion [2, 3]. A part of the crystalline fat is inside the globules, and this part is presumed not to contribute to the rmness of butter. Additionally, water and air droplets are dispersed in the liquid phase. In pure fat systems, several models have been formulated to describe the fat crystal network structure. The rst theory dates back to Van den Tempel [4], and it was reformulated in 1979 [5]. The last model proposed that the structure of the fat crystal network resembles that of a occulated colloid, thus it is constituted of aggregates instead of straight chains of fat particles held together by attractive Van der Waals forces. Due to the impossibility of microstructure quantication, Vreeker et al. [6] suggested that fat crystal networks had a fractal nature and introduced the fractal dimension to quantify the relationship between the mass of a cluster and its size. Afterwards, Marangoni0 s

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group [7, 8] further developed that concept. They stated that viscoelastic lipid systems have a fractal attribute, thus a fractal dimension was used to describe and quantify the microstructure of the fat crystal network and further correlate to rheological properties. Recently, by using empirical models, Liang et al. [9] have demonstrated that the solid fat content has a relatively larger effect than the microstructure on the rheological properties of model lipid systems. The crystallization behavior of anhydrous milk fat is strongly affected by the processing conditions. Some of the processing conditions that have been explored are crystallization and storage temperature [1012], cooling rate [13, 14] and addition of emulsiers [15, 16]. Butter is a more complex fat-based system as it is also composed of partly damaged fat globules with crystallized fat inside, and the literature is scarce in regard to the effect of processing treatments on microstructure and texture of butter. The aim of the present study was to explain the effect of mixing temperature during butter blend manufacturing on rheological properties, melting behavior, and microstructure of butter blends. Furthermore, interactions between rapeseed oil concentration and mixing temperatures were examined. We also intended to predict the relationship between either melting point (Mp) or level of solid fat content (SFC) and the processing conditions (mixing temperature and RO concentration).

the same temperature as for the blends but where no shear was applied, were used as control samples. The nal products were stored at 5 C for a minimum of two weeks. Melting Prole of Butter Blends The melting prole of butter blends was analyzed by a Q 2000 Differential Scanning Calorimeter (DSC, TA Instrument, UK). Between 30 and 39 mg sample material was loaded in a hermetic aluminium pan. An empty, sealed pan was used as reference. Two different isothermal crystallization temperatures (?5, ?0) were studied. The crystallization temperature was held isothermally for 30 min, afterwards, the temperature was increased to 60 C at a rate of 5 C/min. The melting point of the low and the high melting fraction was measured from the minimum peak of the thermographs of butter blends and references. DSC analyses were performed at least in triplicate. Solid Fat Content Determination of Butter Blends SFC of butter blends was measured by a low eld pulsed Nuclear Magnetic Resonance Spectrometer (NMR, Resonance Maran Ultra, UK). The samples were equilibrated at 0 C in a water bath for 45 min and at 5 C in a refrigerator before performing the analysis. The sample determinations were run in quadruplicate. Rheological Oscillation Analyses of Butter Blends

Materials and Methods Materials Unsalted butter was supplied by Arla Foods (Viby J, Denmark). Rapeseed oil (RO) was obtained from DLG Food Oil (Dronninglund, Denmark). Nile red was from Sigma Aldrich (St. Louis, MO) and FITC from Merck (Darmstadt, Germany). Preparation of Butter Blends Model systems of butter blends were prepared by mixing 10 and 25% of RO with butter at three different temperatures, 13, 18, and 23 C, respectively. In addition, pure butter was incubated and mixed at the same temperatures. In total, nine treatments were performed. In both butter blends, the water content was adjusted to 16%. Butter, RO, and water were incubated at the three different temperatures and then mixed together in a table top blender (Hobart in Offenburg, Germany) equipped with a mixer balloon whisk. Blending was performed for 1 min at mediumhigh speed. Butter samples, mixed at similar temperatures and durations as for the blends, and butter samples incubated at Oscillatory experiments were performed on a rheometer (AR-G2, TA Instrument, US based) using 40-mm parallel plate geometry. Temperature control (25 C 0.1 C) was achieved by a Peltier element located at the base of the measurement geometry. The linear viscoelastic region (LVR) of the system was rst determined by performing an oscillatory stress sweep test from 0.1 to 1,000 lNm with a constant frequency of 1 Hz. A frequency sweep test was performed with a frequency from 0.1 to 100 Hz, and torque amplitude was chosen based on the LVR previously identied (50 lNm). The frequency sweep prole was obtained by the measuring elastic modulus (G0 ), viscous modulus (G00 ) and complex modulus (|G*|). For each treatment, seven to nine repetitions were performed. Rheograms were obtained by plotting the logarithm of the moduli (Pa) as a function of the logarithm of the angular frequency (rad/s). Microstructure Analysis of Butter Blends Imaging was performed using an inverted Leica confocal scanning laser microscope (CLSM; Leica TCS SP2, Germany) at room temperature. Nile red and FITC were used as the uorescence stain agents for fats and proteins,

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respectively. Both dyes were dissolved in acetone to a nal concentration of 0.01%. The dye solutions were added to a pre-cooled (4 C) glass slide and the acetone allowed to evaporate before the sample was added. Sample slides were stored at 4 C, for approximately 30 min, before microstructure analysis. Argon 488 nm and HeNe 543 nm laser lines were used for excitation to induce uorescence emission. 409 (N.A. 1.00) and 1009 (N.A. 1.30) oil immersion objective lenses were used to acquire the images. The images were recorded at a speed of 400 Hz and 8-bit resolution. Statistical Analysis Data were statistically analyzed by two-way analysis of variance (SAS Institute, Cary, NC), to determine the relationship between the mixing temperature and the percentage of RO on the level of SFC and the Mp. Principal component analysis (PCA) and a partial least square (PLS) analysis were performed by SIMCA P? version 12 (Umetrics, Umea, Sweden) to predict the level of SFC and the Mp of butter blends based on the mixing temperature and the percentage of RO added to butter. A full cross validation was used. All variables were centered and autoscaled prior to analysis.

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Rheological Properties of Butter Blends RO was blended into partly crystallized butter at 13, 18, or 23 C. Figure 1 shows the complex modulus |G*| of the butter blends after storage for a minimum of two weeks at 5 C. Blends mixed at 23 C obtained the signicantly lowest |G*| scores, i.e. less rm, compared with the blends mixed at lower temperatures. No general effect on |G*| was observed between mixing temperatures at 13 and 18 C, although |G*| of butter blends with 10% RO was lower for the 13 C mixing than the 18 C (Fig. 1b). Butter samples in which no RO was added, and shear was applied, obtained a signicantly larger, complex modulus (Fig. 1a). As expected, increasing the RO content in the butter blends signicantly decreased the complex modulus |G*| (Fig. 1c). Moreover, butter samples in which no shear was applied, obtained a signicantly lower |G*| than the same samples in which shear was applied (Fig. 1a). The effect of mixing temperature also inuenced the rheological behavior of butter and butter blends. Increasing the mixing temperature decreased |G*|. The elastic modulus (G0 ) was greater than the viscous modulus (G00 ) in all blends (data not shown). This indicates that the elastic behavior prevails over viscous behavior, which is normal
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Fig. 1 Complex modulus |G*|, of reference butter samples (a), butter blends with 10% of rapeseed oil (b) and butter blends with 25% of rapeseed oil (c) as a function of angular frequency during a torque sweep at a frequency of 50 lNm. The reference samples were mixed (empty symbols) and non mixed (full symbols) at 13 (open circles), 18 (inverted triangles), and 23 C (open squares), respectively. Commercial butter at 5 C was also analyzed (open diamonds). The error bars represent the standard error

for plastic fats [17]. By increasing the content of RO, the difference between (G0 ) and (G00 ) decreases, which demonstrates how the structure of the system becomes less

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elastic and more viscous by increasing the amount of RO. It has previously been demonstrated that the rheologically cohesive structure of the crystal network could still be preserved by addition of 20% of canola oil added to anhydrous milk fat at 5 C, meaning that anhydrous milk fat can accommodate a large proportion of oil without losing integrity [17]. We found that addition of a lower percentage of RO to butter signicantly decreased G0 , probably due to a structurally weaker network in consequence of the higher mixing temperature used. The complex modulus increased with increasing frequency (Fig. 1). The low frequency dependence of |G*| describes the presence of a network with low possibility of rupture of junction zones within the frequency range used [12]. The increase of G0 and decrease of G00 , with increasing frequency, indicate the change from a mainly viscous to a mainly elastic behavior. The rheological behavior of butter and butter blends was also inuenced by the shear applied during processing. Aggregation of fat crystals is facilitated by shear since, it increases the collision frequency between fat crystals [18]. If a fat crystal network has already been formed, shear force can act on the solid bridges formed between crystal fats, leading to crystal network damage [18, 19]. The resulting network fragments are attracted to each other by Van der Waals forces, leading to internal rearrangement. The shear force applied during the mixing process of our butter blends may have facilitated and increased the collision frequency between fat crystals, creating a stronger network. On the other hand, some of the bridges between crystals and crystal aggregates of partially crystallized butter could also have been broken by applied shear. In the latter case, either a weaker network with less interaction between crystals and crystals aggregates may be formed, or a new crystal network formed by re-crystallization and/or re-aggregation of the fragments may occur. The data obtained (Fig. 1a) showed that the |G*| obtained after applying shear to butter had a larger value than the butter without shear. Accordingly, it can be assumed that a new and stronger crystal network is formed after applying shear to the system. During the storage period at 5 C, which follows the mixing process, part of the melted milk fat is crystallized, consequently sintering between the crystal fragments occurs [18]. Since the solid bridges formed by sintering are much stronger than Van der Waals bonds, the structure will in time be rmer and more stable [18]. Non sheared butter had |G*| values similar to that of blends with 25% RO. This indicates that, besides the mixing temperature used, the presence of shear leads to a rmer structure. Furthermore, within the same percentage of oil added to butter, the mixing temperature had a clear effect on the rheological properties of blends mixed at 23 C. This indicates that the high mixing temperature disrupts large

parts of the crystal network in the continuous lipid phase, and therefore the shear-induced rebuilding is retarded. Melting Behavior of Butter Blends In order to understand the co-crystallization between milk fat and RO, the solid fat content and the melting point of the butter blends were analyzed after isothermal crystallization at 0 and 5 C, respectively. The chosen temperatures lie around the storage temperatures of the products and in an interval where milk fat is still crystallizing. For both crystallization temperatures, the DSC analysis showed that all butter blends are characterized by two melting peaks (Figs. 2, 3). The presence of two melting peaks corresponds to the medium and high melting fractions of triglycerides [2022]. Addition of RO resulted in a shift towards lower melting temperatures in both melting peaks (Fig. 2). This has also recently been observed in anhydrous

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Fig. 2 Thermographs of reference butter samples (continuous line), butter blends containing 10% (long-dashed lines) or 25% (shortdashed lines) of rapeseed oil mixed at 13 C and further crystallized at 0 C (a) or at 5 C (b). The heating rate was 5 C/min

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Fig. 3 Thermographs of reference butter blends containing 25% of rapeseed oil mixed at 13 C (continuous line), 18 C (long-dashed lines), or 23 C (short-dashed lines) and further crystallized at 0 C (a) or at 5 C (b). The heating rate was 5 C/min

milk fat/RO blends by Aguedo et al. [23]. Interestingly, an increase in mixing temperature also lowered the Mp for both melting fractions (Fig. 3). The Mps for all the butter blends are listed in Table 1. Both mixing temperature and percentage of oil added to butter contribute, although to a different extent, to the melting points of the blends that are isothermally crystallized at 0 and 5 C. The contribution from the amount of RO is slightly stronger than the one from the mixing temperature. Moreover, interaction between the variables was detected only after isothermal crystallization at 5 C (Table 1). Increasing both the concentration of RO and the mixing temperature signicantly decrease the SFC of butter blends at both isothermal crystallization temperatures (Table 1). Furthermore, interaction between the RO concentration and the mixing temperature signicantly affected SFC. As expected, the isothermal crystallization temperatures were shown to inuence the level of SFC signicantly but only slightly the Mp of the butter blends. The same effects of

RO and mixing temperatures were observed between the two isothermal crystallization temperatures. PLS analysis was used to obtain a better overview of the variable interactions and predict their effects. The root mean square error of estimation (RMSEE) was used as a measure of the ability of the model to furnish accurate predictions. The counterplots reveal that the effect of mixing temperature on Mp of the lower melting fraction is larger in blends crystallized at 0 C than those crystallized at 5 C, where more RO is required to decrease the melting point (Fig. 4ab). Interaction between mixing temperature and RO content was only observed at the crystallization temperature of 5 C. The SFC was slightly inuenced by mixing temperature when the blends had been crystallized at 0 C (Fig. 4c), while at a crystallization temperature of 5 C (Fig. 4d), a stronger effect of the mixing temperature on SFC was observed. At a higher RO concentration, interaction between factors affected SFC. Despite the fact that the conclusions from the PLS and ANOVA analyses are similar, the PLS gives more information about where the specic interaction takes place i.e. the level of RO concentration. Consequently, it can be used to predict combinations where an optimized effect of either mixing temperature or oil concentration can be utilized. The decrease in SFC and Mp in butter blends by addition of RO is presumably caused by solubilization of some of the solid milk fat TAG into the oil phase [17, 24]. Moreover, a higher mixing temperature was also responsible for the decrease in SFC and Mp in butter blends. The partial melting of milk fat during mixing mainly affects the medium melting fraction, and a higher mixing temperature, of course, results in more melting. The more melt, the more efcient the RO can be distributed in the blend, and the solubility effect becomes larger. The SFC in cream is lower than in anhydrous milk fat with similar temperature treatment and storage [25]. No increase in SFC after mixing the control butter was observed (Table 1), arguing in favor of the ratio between continuous and globular fat not being affected by mixing temperature. Comparing the level of SFC with the rheological properties of the different butter blends isothermally crystallized at 5 C, it was noticed that they were related to each other. In previous studies, it has been reported that SFC has a profound effect on the viscoelastic properties of butter blends, thus they are dependent on the temperature [12, 17, 26]. Vithanage et al. [11] found that, by increasing the temperature from 10 to 20 C, the level of SFC decreases by 50% in butter and 25% in spreadable blends and spreads. In the latter study, the levels of SFC as well as the rheological properties decrease with increasing temperature. In this study, we assumed that part of the melted crystal fractions is unable to fully re-crystallize to the original form at a subsequent temperature increase, such as post

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J Am Oil Chem Soc Table 1 Melting points (Mp), of the rst (Mp1) and second (Mp2) endothermic, and solid fat content (SFC) of butter and butter blends mixed with two concentration of rapeseed oil (RO) at different mixing temperatures (Mixing T) Mixing T (C) % RO Mp1 (C) 0 C 0 10 Mp1 (C) 5 C 25 0 10 25 Mp2 (C) 0 C 0 10 25 Mp2 (C) 5 C 0 10 25 SFC (%) 0 C 0 10 25 SFC (%) 5 C 0 10 25 13 16.83 0.07 16.50 0.06 15.57 0.10 17.03 0.03 16.13 0.05 15.92 0.15 33.45 0.17 33.45 0.12 32.27 0.13 33.07 0.12 32.12 0.01 32.08 0.16 45.68 0.31 42.40 0.82 33.53 1.01 42.83 0.19 37.19 1.28 31.23 1.03 18 16.74 0.08 16.08 0.15 15.32 0.16 16.55 0.02 16.27 0.05 15.61 0.15 33.14 0.16 33.07 0.24 31.95 0.18 32.47 0.01 32.63 0.09 31.44 0.18 46.34 0.30 39.64 0.54 33.65 0.61 42.20 0.09 35.74 0.45 31.09 1.18 23 Mixing T 16.51 0.21 15.78 0.06 15.04 0.13 16.77 0.18 16.17 0.11 15.19 0.07 33.51 0.25 33.16 0.02 32.59 0.17 33.42 0.10 32.80 0.04 32.31 0.34 46.60 0.69 40.19 0.42 28.43 0.21 40.33 0.55 34.68 0.55 25.93 0.15 *** *** *** *** *** *** *** *** ** * *** ns * *** ** ** *** ns Effect %RO MixingT X %RO

The melting points were analysed after isothermal crystallization at either 0 or 5 C, and SFC was also measured at these two temperatures * P \ 0.05, ** P \ 0.01, *** P \ 0.001

processing temperature or storage temperature. The solid fat dissolved in the liquid oil cannot participate in the formation of the solid fat crystal network. Consequently, it cannot contribute to the elastic properties of the blends. Thus, a high mixing temperature may inhibit the availability to rebuild the rigidity of a crystal network in butter and butter blends. Effect on Microstructure and Link to Rheological Properties of Butter Blends Besides the effect of the Mp and the level of SFC of butter blends, it is known that the microstructure of the fat crystal network has an effect on the physical properties of the products [27]. To have a complete overview of the role of mixing temperature on the rheological properties of butter blends, their microstructure was studied. Micrographs of butter blends stored at 5 C are shown in Fig. 5. The fat crystal network appears as black shadows on the background of the pictures. This was tested by melting the sample which lead to more of the red area at the expense of the black shadows. Butter blends mixed at 23 C resulted in a less dense fat crystal network than blends mixed at lower temperatures, i.e. 13 and 18 C. A less dense crystal network was also observed in blends with 25% of RO when

compared with the ones with a lower percentage of RO, i.e. 10% (Fig. 5ghi vs. def). No signicant changes occurred between mixing temperature of 13 and 18 C on the fat crystal network, although in butter blends with 10% of RO, the crystal network appears to be denser in the blend mixed at 18 C than in those mixed at 13 C (Fig. 5e, d). As expected, the most dense crystal network was observed in blends where no RO was added (Fig. 5abc). Besides the fat crystal network of the blends, a change in protein/water distribution was observed by increased mixing temperatures and RO addition. The size and shape of water droplets do not inuence the consistency of butter [2830], and therefore the altered water distribution does not explain the observed effects on the rheological properties. By increasing the mixing temperature, a less dense fat crystal network was observed and thereby a more liquid fat area was seen in the system. Liquid fat in the system hinders the interactions between crystals, consequently weaker links are formed [31]. When crystals and crystal clusters are not connected to neighboring clusters, or if they form weaker links between each other, they are not included in the crystal network, and they do not contribute to the mechanical properties of the fat-based product [3133]. In other words, a thinner network and weak links between

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Fig. 4 Contour plots for the prediction of melting point in butter blends isothermally crystallized at 0 C (a) or at 5 C (b); and of level of solid fat content in butter blends isothermally crystallized at 0 C (c) or at 5 C (d). The value in each box represents the predicted value

at the specic combination of mixing temperature and amount of rapeseed oil added to the butter. The RMSEE values were 0.22 (a), 0.24 (b), 1.02 (c), 1.08 (d)

crystals lead to a lower |G*|. In this study, the rheological properties of the butter blends investigated were explained by the micrographs obtained by CLSM. In butter blends mixed at temperatures lower than 23 C and/or with a percentage of RO lower than 25%, the crystal clusters are strongly connected to each other by forming a fat crystal network. Consequently, |G*| of these blends was higher than in butter blends mixed at 23 C and/or with 25% of RO. In the latter blends, only a weak and disrupted crystal network was observed. These results showed that an increase in mixing temperature leads to microstructure changes, which result in a less rm structure of butter blends. The high mixing temperature leads to a high degree melting of the low and medium melting fraction of milk fat, and the following re-crystallization at 5 C rebuilds a thinner crystal network in the continuous fat phase as more milk fat is dissolved in the oil, which is supported by the lesser SFC observed at higher mixing temperatures.

The result from this study demonstrated that microstructure and rheological properties of butter and butter blends can be modied by adjusting mixing temperature. By increasing the percentage of RO and/or the mixing temperature, |G*| of butter blends, and thus their rmness, decreases. The addition of RO to butter and the mixing temperature used during butter blend preparation solubilize some of the milk fat TAG. Part of the melted crystal fractions is unable to fully re-crystallize to the original form at a subsequent temperature increase, thus the solid fat that is dissolved in the liquid oil cannot participate in the formation of the solid fat crystal network. Consequently, it cannot contribute to the elastic properties of the blends. This was demonstrated by a decrease in SFC and in Mp in all butter blends. Both, a high concentration of RO and a high mixing temperature, plus the interaction between them, presumably change the composition of crystallized milk fat, and thus the viscoelastic behavior of butter blends. Moreover, it was demonstrated that the

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Fig. 5 CLSM images of: reference butter samples (abc), butter blends contain 10% (def) and 25% (ghi) of rapeseed oil at the three different mixing temperatures (13, 18, and 23 C). The black

shadow on the image background represents the crystal network of the samples. The red area is the liquid fat of the system, and the green spots are protein/water phases. The bar scale indicates 30 lm 3. Precht D, Peters KH (1981) The consistency of butter.1. Electronmicroscopic studies on the inuence of different cream ripening temperatures on the frequency of denite fat globule types in cream. Milchwissenschaft-Milk Sci Int 36:616620 4. Van den Tempel M (1961) Mechanical properties of plasticdisperse systems at very small deformations. J Colloid Sci 16:284296 5. Van den Tempel M (1979) Rheology of concentrated suspensions. J Colloid Interface Sci 71:1820 6. Vreeker R, Hoekstra LL, den Boer DC, Agterof WGM (1992) The fractal nature of fat crystal networks. Colloids Surf 65:185189 7. Narine SS, Marangoni AG (1999) Fractal nature of fat crystal networks. Phys Rev E 59:19081920 8. Marangoni AG, Rousseau D (1996) Is plastic fat rheology governed by the fractal nature of the fat crystal network? J Am Oil Chem Soc 73:991994 9. Liang BM, Shi YP, Hartel RW (2008) Correlation of rheological and microstructural properties in a model lipid system. J Am Oil Chem Soc 85:397404

co-effect of mixing temperature or oil concentration can be predicted by PLS analysis.


Acknowledgments The authors wish to thank ARLA FOODS, the Ministry of Food, Agriculture and Fisheries, the Danish Dairy Research Foundation and FOOD Denmark for nancing the Ph.D. project. Mette Marie Lkke is acknowledged for the PLS analysis.

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