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Jana 2016
Jana 2016
Physical characterization of crystalline networks formed by binary blends of
waxes in soybean oil
PII: S0963-9969(16)30317-9
DOI: doi: 10.1016/j.foodres.2016.08.003
Reference: FRIN 6357
Please cite this article as: Jana, S. & Martini, S., Physical characterization of crystalline
networks formed by binary blends of waxes in soybean oil, Food Research International
(2016), doi: 10.1016/j.foodres.2016.08.003
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Sarbojeet Jana, Silvana Martini*
Department of Nutrition, Dietetics, and Food Sciences, Utah State University, 8700 Old
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Main Hill, Logan, Utah, 84322-8700
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* Corresponding Author, Tel: 435-797-8136. Fax: 435-797-2179. E-mail:
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silvana.martini@usu.edu. Website: http://www.MartiniResearch.com.
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Abstract
The objective of this study is to analyze the physical properties of 2.5% (wt. basis)
binary wax in soybean oil (SBO) system. Differential scanning calorimetry, pulsed
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nuclear magnetic resonance, rheology, and polarized light microscopy were used to
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measure melting profiles, solid fat content, viscoelastic parameters, and crystal
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morphology, respectively. Binary blends were prepared using beeswax (BW), rice bran
wax (RBW), and sunflower wax (SFW) in 0, 20, 50, 80 and 100% proportions. Melting
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behavior of binary waxes was significantly affected by the type and proportion of wax
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used. Melting Ton and Tp for RBW/SFW and RBW/BW blends were significantly higher
than those observed for SFW/BW. Enthalpy values suggest that different molecules
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present in the wax affect intermolecular interactions in the binary blends by either
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there is certainly a softening effect when different proportions of RBW/BW and SFW/BW
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parameters (G', G") results show that RBW has the highest G' value (3.1x10 4 ± 1x103
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Pa) followed by SFW (2.7x104 ± 0.2 x104 Pa) and BW having the lowest (90.7 ± 74.4
Pa). Higher G’ values in all proportions of RBW/SFW binary system in SBO indicate
significantly more solid-like behavior than any other combinations. However, blending
of two different waxes does not necessary result in a linear increase in elastic properties
and in some cases no changes in elasticity is observed as the amount of the high
Keywords: Physical properties, Binary-Wax, Beeswax, Rice bran wax, Sunflower wax,
Soybean oil.
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Abbreviations: Beeswax (BW), rice bran wax (RBW), sunflower wax (SFW), soybean
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1. Introduction
Wax in oil systems have been popular for their potential use as trans-fat and saturated
fat replacers. High melting waxes crystallized in low melting oils form a crystalline
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network that entraps oil forming semi solid materials. Several studies have been
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performed on natural waxes such as beeswax (BW), sunflower wax (SFW), candelilla
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wax (CW), and rice bran wax (RBW) and different edible oils (Hwang et al., 2012, Toro-
Vazquez et al., 2007, Dassanayake et al., 2009, Jana et al., 2014, and Martini et al.
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2015). In addition, the use of wax/oil systems in a number of food products has been
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explored and is summarized in Table 1. These studies showed that crystallization
behavior and physical properties of the different natural waxes used for food application
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strongly depends on the type of wax (Dassanayake et al., 2009, Martini et al., 2015, and
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Hwang et al., 2013) and oil (Dassanayake et al., 2009, Jana and Martini, 2014 and Lupi
et al., 2013) used. Martini et al. (2015) and others (Lupi et al., 2013, Hwang et al., 2012,
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and Martini et al., 2008) confirmed that wax concentration also affects the physical
properties of the material. The crystallization behavior and the functional properties of
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the wax materials differ significantly due to their different chemical composition. For
example, paraffin wax (PW) is formed mainly of high molecular weight n-alkanes; RBW
consists mainly of long chain aliphatic esters, while SFW and BW are a mixture of n-
alkanes, esters, free fatty acids, and aliphatic alcohols. Waxes are high melting point
materials (Tm = 50−80 °C) and have low solubility in vegetable oils and therefore
crystallize rapidly when placed at room temperature. Toro-Vazquez et al. (2007) studied
thermal and physical characteristics of candelilla wax (2% wt. basis) in safflower oil
which forms a gel upon crystallization. They concluded that structural organization of
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organogel is dependent on the cooling rate, thermodynamic driving force, and gel
setting temperature. They also mentioned that chemical composition of the oil and wax
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Dassanayake et al. (2009) studied rice bran wax and carnauba wax in olive and salad
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oil (canola: soy bean oil = 50:50). This research group has shown that different types of
waxes are responsible for different hardness and viscosity and those parameters are
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explained by crystallization behavior and thermal kinetics. These researchers confirm
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that analysis of different physical properties is needed to better understand the
range of physical properties when crystallized in an oil our hypothesis is that we can
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broaden the physical properties of these wax/oil systems by using wax mixtures. BW,
RBW, and SFW are popular natural waxes with significantly different chemical
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compositions and their use has been explored for different food applications (Table 1).
binary wax/oil systems. BW, RBW, and SFW are used as waxes and soybean oil (SBO)
is used as the oil source in the wax/oil system. A concentration of 2.5% (% wt. basis) of
binary wax (0, 20, 50, 80, and 100%) in SBO is used based on preliminary data in our
laboratory. Thermal properties such as melting temperatures (onset [Ton] and peak [Tp])
and enthalpies, viscoelastic properties such as storage modulus (G’), loss modulus
(G’’), solid fat content (SFC), and crystal morphology were measured.
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Beeswax, sunflower wax, and rice bran wax were supplied by Koster Keunen LLC
(Watertown, CT, USA). Pure Wesson soybean oil was purchased from local
supermarket. Chemical compositions and melting temperatures (Tm) of the waxes have
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been previously reported (Hwang et al. 2012, and Jana & Martini 2016). In short, BW
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(Tm = 60.5 ± 3.0 °C) is composed of wax esters, hydroxyl esters, hydrocarbon, free fatty
acids, and di-esters. RBW (Tm = 80.8 ± 0.8 °C) is composed only by saturated wax
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esters. Lastly, SFW (Tm = 75.5 ± 0.0 °C) is composed of wax esters, hydrocarbons, free
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fatty acids, and fatty alcohols. Binary wax systems were prepared by mixing BW, RBW,
and SFW waxes in different proportions (0, 20, 50, 80 and 100% w/w) to cover the
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entire range of concentration in the binary blends. The binary systems (RBW/BW,
the vial and closed with an appropriate lid. Vials were then placed in a sonication water
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bath for 5 – 10 min and placed on a vortex mixer for 1-2 min to allow for complete
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dissolution of the waxes in the hexane. The vial lids were then loosened and placed
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under airflow thin-wall fume hood for a week to evaporate the hexane. Samples of 2.5%
(wt. basis) of the binary waxes in soybean oil (SBO) were used in this study. A 2.5%
concentration of binary wax was used to obtain a semisolid material with intermediate
physical properties as reported elsewhere (Martini et al., 2015). Binary waxes were
mixed with the oil and heated to 100 °C in an oven for 30 min to allow for complete
melting and dissolution of the wax blends in the oil. Samples were thoroughly stirred
with a hot stirring bar while at 100 °C, left at 100 °C for another 15 min, and then placed
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The melting profile was measured at 25 °C using DSC (TA Instruments DSC model Q20
1963 with RCS cooling system, New Castle, DE, USA). The DSC baseline and
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temperature were calibrated with a pure indium standard. Approximately 10 mg of the
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sample were placed in Tzero aluminum pans, covered and sealed with Tzero aluminum
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lids. The samples were heated from 25 °C to 100 °C at 5 °C/min to analyze the melting
profile of the samples. TA Universal Analysis software was used to analyze the melting
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onset (Ton), melting peak temperatures (Tp), and melting enthalpy (H).
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2.3. Theoretical Enthalpy Calculation
Theoretical enthalpies were calculated using enthalpy values of each single wax
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H Aexp P
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H Ath
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Where HAth is the theoretical enthalpy in the binary mixture from component A, HAexp
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percentage of component A in the blend. For a wax blend composed of A and B waxes,
H T th H Ath H B th
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determine the solid fat content (SFC) of the samples. Samples were placed in NMR
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Triplicate runs were performed for each set of samples, and three tubes were measured
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in each run.
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2.5. Rheology Measurements
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viscoelastic properties of the material. Oscillatory tests were performed at 25 °C after 24
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h of storage by a strain sweep step to obtain storage (G’) and loss (G”) moduli. A 40-
mm-diameter parallel-plate geometry (1000 microns gap and 1 Hz frequency) was used
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for the samples. A plastic spoon was used to transfer the samples to the rheometer
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plates. For each type of wax blends and soybean oil combination, 3 experimental
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replicates were performed and 3 rheology measurements were taken from each
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replicate.
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evaluated. The crystals were observed using a polarized light microscope (Olympus
BX41, Olympus Optical Co., Tokyo, Japan). A small amount of sample containing
crystals was placed on a glass microscope slide and covered gently with a glass cover
slip. Digital images of the polarized specimens were captured using Lumenera’s Infinity
2 (Lumenera Corp., Nepean, Canada). The crystal pictures were taken at 20X
magnification.
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Crystal morphology was quantified by calculating fractal dimension. Benoit 1.3 software
(TruSoft Int’l Inc., St. Petersburg, FL, USA) was used to determine that fractal
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dimension using the box-counting method. The microscopic images were transformed
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to a greyscale mode and thresholded using Adobe Photoshop CS2 (Adobe Systems
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Inc., San Jose, CA, USA) as described by Campos (Campos, 2005). The box-counting
fractal dimension, Db, value was calculated as the negative slope of the linear
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regression curve of the log–log plot of the number of occupied boxes Nb vs. the side
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length lb [Marangoni et al., 2002, Tang et al., 2006a and 2006b]. D b values were
calculated in duplicate and the mean value and standard deviations are reported.
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Data are reported as mean values and standard deviations of replicated experiments.
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Two-way ANOVA was used to test significant differences between treatments using a
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level of significance of 0.05. Statistical analyses were performed using GraphPad Prism
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(Prism 6.01; GraphPad Software Inc., La Jolla, Calif., U.S.A.). Significant differences
were tested using two-way ANOVA and Tukey’s post hoc test (α = 0.05).
Figure 1 shows the DSC melting profiles of all the binary waxes (2.5% wt. basis) in SBO
tested in this study. A single melting peak is observed for all binary waxes tested
independently from the type or proportion of wax used. Figure 1A shows the melting
behavior of SFW/BW binary blend in SBO. It is observed that the melting peak is shifted
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to higher temperatures as the amount of SFW increases in the wax mixture from 0% to
100%.
Figure 1B shows the melting profile of RBW/BW blends in SBO. Similar to the SFW/BW
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blends an increasing trend in melting peak temperatures is observed from 0% to 100%
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blends with a sharper melting peak observed for the 100% RBW/BW melting curve. In
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addition sharper melting peaks are observed as the amount of RBW increases in the
blend which can be attributed to the homogeneous chemical composition of the sample.
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In this case, 100% RBW/BW is composed of only wax esters but 100% SFW/BW is
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composed of wax esters, hydrocarbons, free fatty acids, and fatty alcohols. The
presence of these various molecular entities in the SFW/BW blends results in a broader
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melting profile compared to the RBW/BW ones. SFW/BW or RBW/BW (0%) or 100%
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BW also show a wide melting peak (Figure 1A and Figure 1B) since BW is a mixture of
different molecular entities such as wax ester, hydrocarbons, free fatty acids, di-esters,
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and hydroxyl esters. Figure 1C shows the melting profile of RBW/SFW blends in SBO.
The increasing trend in melting peaks is not quickly noticeable as it was observed in
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Figure 1A and 1B. In this case, the melting temperatures of both the waxes (RBW and
SFW) are close with melting points of 79.8 ± 0.1 °C and 75.8 ± 0.2 °C for RBW and
SFW, respectively. Another difference in the RBW/SFW melting curves is the sharp
peak formed in all the concentrations except for the 0% RBW/SFW (or 100% SFW)
blend in SBO. Because RBW and SFW have wax esters as the major component
(above 50% of the total composition), it is very likely that melting curves in RBW/SFW in
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In previous research that evaluated phase behavior of binary waxes in the absence of
oil (Jana & Martini, 2016) two melting peaks were observed in 100% BW and only one
peak in 100% SFW and 100% RBW (Table 2). As previously discussed in this study
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only one peak is observed in BW when crystallized in SBO. In addition, even if only a
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single melting peak is observed in SFW and RBW crystallized in the absence or
presence of SBO (Jana & Martini, 2016) the melting peaks observed in the presence of
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SBO appear at lower temperatures. It is likely that the shift towards lower melting
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temperatures observed when waxes are crystallized in the presence of oil is due to the
dissolution of different molecular entities in the oil. Similar results regarding the
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decrease in Ton and Tp values with low concentrations of waxes in oil was previously
Figure 2 shows the comparative data analysis of the melting profile peaks observed in
Figure 1. Figure 2A shows the melting peak temperatures of all the binary waxes in
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SBO. The decreasing trend in peak melting temperature from 100% to 0% of all the
decreasing pattern in SFW/BW blends in SBO. This behavior can be explained by the
enrichment in the lower melting point wax (BW) in the system. RBW/BW blend in SBO
showed a slow decreasing pattern from 100% to 20% and a sharp decline at 0% from
20%. This behavior suggests that the crystallization and therefore the melting behavior
of these blends is mainly driven by RBW. RBW/SFW in SBO system follows a similar
uniform decreasing trend as SFW/BW; however since RBW and SFW have similar
melting points the degree of change is slightly smaller than the one observed for
SFW/BW blends. These results suggest that even though the crystallization in these
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samples is driven by RBW, SFW also plays an important role and some degree of
interaction between the molecular entities present in these samples might occur. Figure
2B shows the onset temperatures of all the binary waxes in SBO. In general, onset
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melting temperatures follow similar pattern in all the combinations as the one observed
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for the peak melting temperatures. Figure 2C shows a comparative analysis of the
enthalpies in all the binary waxes in SBO. The sample composed of 80% SFW/BW
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blend in SBO shows the highest enthalpy (3.5 ± 0.2 J/g) which decreased significantly
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(p < 0.05) as the content of SFW decreased in the sample and the 0% SFW/BW (100%
BW) shows the lowest enthalpy (1.1 ± 0.1 J/g). It is interesting to note that there is no
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particular pattern observed in enthalpy values for this wax combination. That is, there is
enthalpy values for the 80% SFW/BW blend. This result suggests that the presence of
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80% SFW promotes the crystallization in the system resulting in a synergistic effect.
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The enthalpy values in RBW/BW blend in SBO show a rapid decreasing pattern from
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100% to 20% and then stays constant at 0%. This decrease (p < 0.05) in enthalpy
concentrations equal or greater than 50% since when RBW is present at low
between the 0 and 20% RBW/BW samples. The enthalpy pattern in RBW/SFW blends
these 2 waxes as evidenced by a gradual increase in enthalpy as the wax with higher
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molecular entities present in each blend. Martini et al. (2015) suggested that enthalpy
changes observed in wax/oil systems is a function of wax type due to different chemical
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compositions of the waxes. The presence of a high amount of wax esters in SFW and
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RBW could be a reason of the higher enthalpy values observed in blends containing
SFW and RBW compared to enthalpy values obtained for blends with BW which have
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lower amounts of wax ester and are richer in n-alkanes, free fatty acids, and fatty
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alcohols. As the amount of SFW decreases in RBW/SFW mixtures the enthalpy values
also decrease (p < 0.05). For example, enthalpy values for the 20 and 80% RBW/SFW
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(80 and 20% of SFW) decreased from 3.8 ± 0.2 to 2.9 ± 0.5 J/g. In addition, binary wax
blends containing BW resulted in lower enthalpy values due to the lower enthalpy
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associated with this type of wax. It is not clear why enthalpy of 80% SFW/BW is higher.
It is likely that in this case, the presence of SFW induces the crystallization of the
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system resulting in a higher overall enthalpy value. Overall, these results suggest that
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wax esters in the wax blends are responsible for driving super-saturation of crystals in
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binary wax/SBO system. However, the presence of other molecular entities present in
the waxes also affects the crystallization behavior since, especially in the blends that
contain BW, the decrease in enthalpy is not linearly correlated to the amount of BW
added. Other studies have also suggested that esters play an important role in driving
the crystallization of waxes and that also the presence of minor components might
affect their crystallization. For example, Dassanayake et al. (2009) reported that RBW
forms stronger oleogels compared to carnauba and candelilla due to the higher content
of esters. In addition, Hwang et al. (2012) showed that ester-containing waxes showed
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better gelling properties and resulted in stronger oleogels. Lastly, Hwang et al. (2014)
with the amount of total polar compounds. From these studies it is evident that ester
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concentration and the presence of minor components affect the crystallization behavior
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of waxes and therefore their physical properties, however further research is needed in
this area to pinpoint what are the exact molecular interactions that occur during wax
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crystallization that ultimately affect their physical properties.
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When two components of different chemical composition, and therefore different melting
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enthalpies, are mixed, one would expect that enthalpy values will change linearly based
on the ratio of both components in the blend. This is true if the components are fully
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miscible and form solid solutions. In order to establish if molecules present in the
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compare theoretical and experimental enthalpy values (Figure 3). Figure 3A shows
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regression line for experimental enthalpy values in SFW/BW samples (Figure 3A) does
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not follow the theoretical one. In addition, a low R2 value was obtained (R2 = 0.64) for
the experimental enthalpy regression line. This lack of linearity and the deviation from
the theoretical values suggest that SFW and BW are not totally miscible and that only a
partial co-crystallization occurs when these two waxes are mixed. SFW is composed of
approximately 70% wax esters and other minor components such as free fatty acids,
fatty alcohols and also hydrocarbons (Hwang et al., 2012; Jana & Martini, 2016). These
minor components might play an important role in the crystallization behavior of the
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evidenced by the broad melting peak observed in Figure 1A and by the differences
RBW/BW and RBW/SFW enthalpy comparison lines and in both cases the linear
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regression lines formed by the experimental data points are parallel to the theoretical
data points line. R2 values are high in both correlations (0.92 and 0.90 in RBW/BW and
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RBW/SFW, respectively). Although R2 value is 0.90 in the case of RBW/SFW blend the
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narrow confidence interval explains how well the experimental data fits the theoretical
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ones. This suggests that RBW and SFW are fully miscible and behave as solid
solutions. These results corroborate our previous results obtained in binary waxes
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crystallized without oil (Jana & Martini, 2016). On the contrary, R2 value of 0.92 for
RBW/BW shows a wide confidence interval and the theoretical values fall outside this
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always fall below the theoretical ones suggesting that molecular entities present in RBW
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temperature obtained for the binary wax blends in SBO. The SFC content (%) almost
stays constant in all the different concentrations for all combination of waxes in SBO at
10 °C. Figure 4A shows the SFC vs. temperature graph of SFW/BW in SBO system.
The 0% SFW/BW blend in SBO line decreases sharply from 10 °C to 20 °C and then
keeps decreasing until 45 °C (% SFC = 0). SFC values as a function of temperature for
100% SFW/BW blend in SBO show a wider curve, this means that the wax blend
remains solid at temperatures as high as 60 °C. The wider curves become narrower
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gradually from 100% of the concentration to 0% of the SFW/BW blend. This is expected
because SFW in SBO has higher melting temperature and BW in SBO has lower
melting temperature. There is a trend observed in SFC content at room temperature (25
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°C) that as the amount of SFW is increased, the SFC content increases accordingly. As
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SFC content is directly dependent on the amount of crystals formed in the system, the
SFC values at room temperature gives an insight to formulate real food products. A
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similar trend is observed at 35 °C (close to body temperature) indicating wax blends are
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not fully melted at body temperature and could provide a waxy mouthfeel to the product.
Figure 4B shows SFC vs temperature profile for RBW/BW blends in SBO systems. The
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SFC curve for 100% BW in SBO shows a sharp decline as discussed before but 100%
RBW shows a wider curve slightly different than the one observed for SFW with SFC
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greater than zero for 60 and 65 °C. The difference in SFC content in 100% BW and
RBW is due to the higher melting point of RBW. The SFC content for 0 and 20%
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RBW/BW blends at 50 °C is zero but 50, 80 and 100% blends show higher than 0.5%
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SFC content at that temperature. Similar to the SFW/BW blends, all RBW/BW had SFC
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> 0 at 35 °C. Figure 4C shows SFC vs temperature profile for RBW/SFW blends in SBO
wider curve is observed in all the RBW/SFW blends compared to the SFW/BW and
RBW/BW blends. All the concentration curves stretched flat until 45 °C and then there is
a small drop at 50 °C. This indicates that the amount of crystals formed in all the
value (>1.0) for 0 and 20% RBW/SFW between 55 °C and 60 °C. These samples
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approximately 1 %( Figure 4C). As previously discussed SFC values at 35 °C for all the
blends were not significantly different; however, a significantly lower melting enthalpy (p
< 0.05) was observed for the 0, 20, and 50% RBW/SFW blends (data not shown).
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These results suggest that even though a significant amount of wax is still present at
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body temperature less energy is needed to melt the 0-50% blends and therefore they
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Figure 5 shows iso-solid diagrams obtained from the SFC data reported in Figure 4.
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These iso-solid diagrams help understand phase behavior of binary mixtures of fats and
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to evaluate possible softening effects. As expected, higher SFC iso-solid lines (i.e.
1.5%) always occur at lower temperature than iso-solid lines with lower SFC in all the
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binary wax compositions. Interestingly, iso-solid lines for the RBW/SFW blends occur at
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higher temperature (starting at 49 °C for 1.5%) compared to the other wax blends
tested. This suggests that enthalpies of RBW/SFW blends should be higher than other
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blends and this pattern is seen in Figure 2C. Iso-solid diagrams in Figure 5 show a
softening effect when SFW and RBW are added to BW. This softening effect is more
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pronounced at 1.5% SFC and occurs at approximately 50% SFW and 80% RBW
addition suggesting a eutectic behavior. No softening effect was observed for the
RBW/SFW blends suggesting that these waxes form solid solutions. These results are
in accordance with our previous research on phase transitions of bulk waxes (Jana &
Martini, 2016) and suggest that the heterogeneous chemical composition in BW limits
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Viscoelastic properties are affected by the amount of crystalline material and by crystal
morphology. Figure 6 shows the viscoelastic parameters in all the binary wax in SBO
systems at 25 °C. Storage modulus (G’) values indicate solid-like behavior of wax/oil
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system whereas loss modulus (G’’) indicates liquid-like behavior. Figure 6A shows G’
values of the binary wax in SBO systems at all binary wax proportions. G’ value for
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100% SFW blend in SBO is significantly higher (p < 0.0001) than 100% BW blend (or
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0% SFW/BW in SBO). SFW/BW at 80% blend shows the maximum G’ values in the
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SFW/BW system. This behavior is similar to the one observed for the enthalpy data
(Figure 2) where 80% SFW/BW composition shows maximum enthalpy. This indicates
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that 80% composition of SFW/BW in SBO would form the most elastic crystalline
network. As previously discussed it is likely that wax esters present in the 80%
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SFW/BW mixture are driving the properties of the crystalline network formed. The
crystalline network formed might be richer in wax esters that have a higher enthalpy and
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that result in a more elastic material while maintaining a constant SFC. SFW/BW of 50%
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and 20% mixture does not follow a significant change in G’ values. All the SFW/BW
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SFW/BW). In the RBW/BW system, 100% RBW shows significantly higher G’ values
than 100% BW. Interestingly, G’ values in all the other RBW/BW blends (20, 50, and
80%) are not significantly different (p > 0.05). But these values are significantly lower
than 100% RBW/BW (p < 0.05). This means that the 100% RBW possess the most
solid-like material than all the other concentrations. These results suggest that RBW is
driving the crystallization behavior of RBW/BW blends. 100% RBW and 100% SFW
does not show a significant difference in G’ values (p > 0.05) but 20%, 50% and 80% of
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RBW/SFW in SBO system shows high G’ values than any other concentration mixture
(p < 0.05) suggesting a synergistic effect when these two waxes are mixed. It is
observed that G’ values decrease as the amount of RBW increases from 20% to 100%
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RBW/SFW. Overall, RBW/SFW binary system in SBO shows significantly more solid-
like behavior than any other combinations. Figure 6B shows G’’ values of the binary wax
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in SBO systems at all concentrations. It is observed that data presented in Figure 6B
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follows a similar trend to the one presented in Figure 6A with lower G’’ values observed
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compared to G’ values. This signifies that all the systems analyzed show more solid-like
behavior than viscous-like behavior. Rheology data shows that blending of two different
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waxes does not necessary result in changes in elastic properties. In fact, in many cases
the addition of high melting wax to a low melting one does not result in a change in G’
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values.
Figure 7 shows the crystal morphology of 2.5% binary waxes in SBO for all the binary
longer than the ones observed in the 20% SFW/BW blends. There is no noticeable
difference in the shape of the crystals obtained for the 20% and 80% SFW/BW blends.
But crystals of 50% of the SFW/BW blend shows a difference in size and arrangement.
Previous research on bulk SFW/BW (without oil) showed that crystal morphology
noticeable different in crystal size, shape, and density compared to the same blends
crystallized in SBO and shown in Figure 7 (Jana & Martini, 2016). RBW/BW system
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shows smaller crystals in 50, 80, and 100% RBW/BW blends and crystals observed for
the 0% and 20% RBW/BW blends are larger with a less dense crystalline network than
the rest of the samples. It is observed that crystal size becomes bigger from 0% to 20%
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but there is a rapid decrease in crystal size from 20% to 50% of the RBW/BW
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concentration. Crystal structures in this binary blend do not show any similarity when
compared to the crystals obtained when the bulk blends were crystallized without SBO
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(Jana & Martini, 2016). 20% of RBW/SFW crystals show similarity in sizes with 0% and
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80% of the crystals show same sizes as 100% of RBW/SFW. But crystals in 50% of
RBW/SFW are different than all the other crystals where agglomerated structures can
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be observed (note the large white spots in Figure 7). Overall, individual waxes when
present in the binary wax blends do not drive any major crystal morphology changes.
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The ‘white-spots’ observed for the 50% RBW/SFW blend are the indication of densely
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Figure 8 shows fractal dimension analysis of the crystal morphology pictures discussed
in terms of crystal shape, size, area fraction and the interaction among these three
factors. Box counting method is mostly used for lipid fractal dimension and has been
previously reported in several studies (Marangoni et al., 2002, Tang et al., 2006a and
2006b). From the multiple comparison analysis done for Db values from fractal
among the binary waxes and their different concentrations except for the 50%
RBW/SFW. 100% RBW shows no significant difference (p > 0.05) in Db values with 50%
RBW/SFW. The only significantly higher Db value observed in 50% RBW/SFW system
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can be confirmed by the ‘white-spots’ in Figure 7. ‘White-spots’ are the indication how
area fraction and size and shape give strongest crystal network. Fractal dimension
analysis in Figure 7 shows lower values for all the wax blends when compared with the
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previous research where waxes were crystallized from the melt without SBO (Jana &
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Martini, 2016). This difference in Db values is due to the microstructural factors such as
crystal size, shape, area fraction and any interaction among these. It likely that wax
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solubility/supersaturation in the SBO discussed in this study plays an important role in
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changing crystal morphology compared to waxes crystallized from the melt. Similar Db
values between 1 and 2 were reported for highly crystalline materials by other authors.
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Awad and Marangoni (2005) have reported Db values between 1 and 2 for palm oil and
cocoa butter while Blake and Marangoni (2015) have reported similar Db values for
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oleogels formed with RBW, SFW, and candelilla wax. Finally, Tang and Marangoni
(2006) have used computer simulations to explain Db values in this same range.
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4. Conclusion
The present study exemplifies how wax chemical composition is responsible for the
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enthalpy values, solid fat content, and viscoelastic behavior help interpret how different
molecular entities in the waxes might affect their crystallization behavior. Our results
show that wax esters play a significant role in the crystallization behavior of the binary
wax blends. Waxes with similar chemical composition behave as solid solutions while
mixtures of waxes with different chemical composition deviate from the ideal behavior.
This study shows that blending waxes does not necessarily result in linear changes in
physical properties and that inhibition and synergism might occur in the different blends.
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that can result in particular physical properties to be used in food applications. However,
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performed in the future to ensure consumer acceptance. In addition, future studies on
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the effect that minor components present in natural waxes such as fatty acids, alcohols
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Acknowledgements
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Financial support for this research was provided by the Utah Agricultural Experiment
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Station Utah State University. This paper was approved as journal paper number 8884.
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Yilmaz, E.,& Öğütcü, M. (2014). Comparative analysis of olive oil organogels containing
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Figure Captions:
Figure 1: DSC melting profile of 2.5% (wt. basis) binary wax in soybean oil (SBO).
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Binary wax proportions of 0, 20, 50, 80 and 100% are tested. The first melting curve
from the top indicates 0% of the first component in the wax mixture and the bottom
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curve indicates 100% of the first component in the wax mixture. The 3 rd line from the top
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indicates the 50% of the binary wax. Melting profile of SFW/BW, RBW/BW, and
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Figure 2: Peak melting temperature (Tp), onset melting temperature (Ton) and melting
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and 100%. Columns with same letters indicate that values are not significantly different
(α = 0.05).
square symbol) enthalpies for binary wax blends in SBO. The dotted line indicates the
linear regression line for experimental enthalpy points. Theoretical and experimental
enthalpies of SFW/BW, RBW/BW and RBW/SFW blends in SBO are shown in A, B, and
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C respectively. Confidence interval (95%) for the experimental data is shown in light
Figure 4: Solid Fat Content (SFC) as a function of crystallization temperature for binary
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wax mixtures in SBO are plotted from temperature-controlled pulse-NMR data. SFC of
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SFW/BW, RBW/BW, and RBW/SFW blends in SBO are shown in A, B, and C
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respectively.
Figure 5: Iso-solid lines for the binary mixtures in SBO are drawn using interpolation
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method from SFC data in Figure 4. Iso-solid lines represent same solid fat content at
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0.5, 1, and 1.5%. Iso-solid diagrams of SFW/BW, RBW/BW and RBW/SFW in SBO are
Figure 6: Viscoelastic parameters such as storage modulus (G’) and loss modulus (G’’)
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scale. Columns with same letters indicate that there is no significance difference among
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others (α = 0.05).
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Figure 7: Crystal Morphology of 2.5% binary wax in SBO oil at 20X magnification at 25
°C after 24h of sample incubation in a water-bath. All the binary wax in SBO system
Figure 8: Fractal dimension (Db) of the crystals is analyzed using Box-plot counting
technique at different concentrations at 25 °C. Columns with same letters indicate that
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10%) + Olive oil
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Beeswax (3%, 7% and 10%)
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+ Olive oil
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Botega et al. (2013) Rice bran wax (10%) + Solid Fat in Ice cream
Sunflower oil
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Jang et al. (2015) Candelilla wax (3 – 6%) + Shortening in baked goods
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Canola oil
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Patel et al. (2014) Shellac (2%) + Sunflower oil Spreads, chocolate paste and
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cakes
Emulsion in 20% water
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Table 2: Melting temperatures (Tp, °C) of binary mixtures used in this study, as reported
by Jana and Martini 2016. Two melting values indicate two melting peaks observed.
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SFW/BW 53.0±0.5 52.0±0.6 52.1±0.2 49.0±0.0 75.8±0.2
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61.2±0.7 62.2±0.5 60.6±4.0 73.2±1.0
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RBW/BW 53.0±0.5 52.1±0.2 55.5±0.1 53.65±3.4 79.8±0.1
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61.2±0.7 60.78±1.0 73.8±1.7 78.1±0.9
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RBW/SFW 75.8±0.2 75.1±1.3 77.0±2.0 77.8±2.1 79.8±0.1
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Figure 1
2 .5 % S F W /B W in S B O 2 .5 % R B W /B W in S B O
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4 4
A B
H e a t F lo w ( W /g )
H e a t F lo w ( W /g )
2 2
0% 0%
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0 0
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50%
50%
-2 -2
100%
100%
-4 -4
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30 45 60 75 90 30 45 60 75 90
T e m p e r a tu re ( ° C ) T e m p e r a tu re ( ° C )
2 .5 % R B W /S F W in S B O
C
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H e a t F lo w ( W /g )
2
0%
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50%
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-2
100%
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30 45 60 75 90
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T e m p e r a tu re ( ° C )
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Figure 2
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T p o f M e ltin g T o n o f M e ltin g
80 80
A B
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0%
a a a a a a
b b 20%
c c c a a
a a
T p M e ltin g ( ° C )
T o n M e ltin g ( ° C )
60 d 60 a a
a a ,b
50%
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e b b
f f
b
80% b ,c
40 c c c
100% 40
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20 20
0 0
S F W /B W R B W /B W R B W /S F W
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E n th a lp y o f M e ltin g o f 2 .5 % B in a ry w a x e s in S B O
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C a a
a ,b
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E n th a lp y ( J / g )
b ,c b ,c b ,c
3 c c
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S F W /B W R B W /B W R B W /S F W
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Figure 3
S F W /B W
R B W /B W
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THEO 5
2
R = 0 .6 4
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2
EXP R = 0 .9 2
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E n th a lp y ( J /g )
E n th a lp y ( J /g )
3 3
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2 2
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1 1
A B
0 0
0 20 40 60 80 100 0 20 40 60 80 100
C o n c (% ) C o n c (% )
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R B W /S F W
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R = 0 .9 0
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E n th a lp y ( J /g )
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0 20 40 60 80 100
C o n c (% )
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Figure 4
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S F W /B W R B W /B W
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2 .5 2 .5
0%
A 20%
B
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2 .0 2 .0
50%
80%
% SFC
1 .5
% SFC
1 .5
100%
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1 .0 1 .0
0 .5 0 .5
0 .0 0 .0
10 20 30 40 50 60 70
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10 20 30 40 50 60 70
T e m p e r a tu re ( ° C ) T e m p e r a tu re ( ° C )
R B W /S F W
2 .5
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2 .0
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10 20 30 40 50 60 70
T e m p e r a tu re ( ° C )
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Figure 5
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S F W /B W R B W /B W
0 .5 %
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60 60
1 .0 %
T e m p e r a tu r e ( ° C )
T e m p e r a tu r e ( ° C )
1 .5 %
45 45
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30 30
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15 15
A B
0 0
0 20 40 60 80 100 0 20 40 60 80 100
B le n d ( % ) B le n d ( % )
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R B W /S F W
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T e m p e r a tu r e ( ° C )
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B le n d ( % )
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Figure 6
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G ' v a lu e s o f 2 .5 % b in a r y w a x in S B O G " v a lu e s o f 2 .5 % b in a r y w a x in S B O
6 5
10 10
A 0% B a b
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a a ,b c
b
10 5 c 20% 4 d
d d d
d 10 e
e
e e
e e e e f
e 50% f
4 f
10 f
f
G '' ( P a )
10 3
G ' (P a )
80%
10 3
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100%
f f 10 2
2
10
f f
10 1
10 1
10 0 10 0
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S F W /B W R B W /B W R B W /S F W S F W /B W R B W /B W R B W /S F W
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Figure 7
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Figure 8
B o x - c o u n tin g F r a c ta l D im e n s io n A n a ly s is
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2 .0
0% C onc.
F r a c ta l D im e n s io n ( D b )
20% C onc.
1 .5 a
50% C onc.
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a ,b a ,b
b
b b b b b
b b
b b b b 80% C onc.
1 .0
100% C onc.
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0 .5
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0 .0
R B W /B W S F W /B W R B W /S F W
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Graphical Abstract: Melting enthalpy (A), elastic modulus (B), and iso-solid diagrams
(C) of binary blends of waxes in soybean oil. H: melting enthalpy; G’: Elastic modulus,
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SBO: Soybean oil, SFW: Sunflower wax, BW: Beeswax, and RBW: Ricebran wax. Bars
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in panels A and B with the same letter indicate that values are not significantly different
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( = 0.05). Percentages shown in panel C correspond to solid fat content values.
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C
S F W /B W
2 .5 % B in a r y w a x e s in S B O 0 .5 % 1 .0 % 1 .5 %
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T e m p e r a tu r e ( ° C )
5
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A a
0% 45
a
a ,b
4 20%
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b 30
b ,c b ,c b ,c 50%
H ( J /g )
15
3 c c 80%
100% 0
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d 0 20 40 60 80 100
2 d
d
B le n d ( % )
e e
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e
1
R B W /B W
0 60
T e m p e r a tu r e ( ° C )
S F W /B W R B W /B W R B W /S F W
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45
30
2 .5 % b in a r y w a x in S B O 15
6 0 .5 % 1 .0 % 1 .5 %
10 0
B 0% 0 20 40 60 80 100
a a ,b
b
10 5 c 20% B le n d ( % )
d d d
d
e e e e
e 50%
10 4 R B W /S F W
G ' (P a )
80%
3
10
100% 60
T e m p e r a tu r e ( ° C )
f f
10 2 45
1
10 30
10 0 15
S F W /B W R B W /B W R B W /S F W 0 .5 % 1 .0 % 1 .5 %
0
0 20 40 60 80 100
B le n d ( % )
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Highlights
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properties of wax/oil systems.
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compared to the theoretical values expected.
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Blending of two different waxes does not necessary result in a change in elastic
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properties.
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