Food Chemistry 214 (2017) 717–725

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Chemical profiling of the major components in natural waxes

to elucidate their role in liquid oil structuring
Chi Diem Doan a,d,⇑, Chak Ming To a, Mike De Vrieze b, Frederic Lynen b, Sabine Danthine c, Allison Brown a,
Koen Dewettinck a, Ashok R. Patel a,⇑
a
Laboratory of Food Technology and Engineering, Faculty of Bioscience Engineering, Ghent University, 653 Coupure Links, 9000 Ghent, Belgium
b
Separation Science Group, Department of Organic and Macromolecular Chemistry, Faculty of Science, Ghent University, 281 Krijgslaan, 9000 Ghent, Belgium
c
Department of Food Technology, Gembloux Agricultural University, Passage des Déportés 2, B-5030 Gembloux, Belgium
d
Department of Food Technology, College of Agriculture and Applied Science, Cantho University, Viet Nam

a r t i c l e i n f o a b s t r a c t

Article history: Elucidating the composition of waxes is of utmost importance to explain their behavior in liquid oil
Received 28 October 2015 structuring. The chemical components (hydrocarbons – HCs, free fatty acids – FFAs, free fatty alcohols
Received in revised form 19 June 2016 – FALs and wax esters – WEs) of natural waxes were analyzed using HPLC–ELSD and GC–MS followed
Accepted 20 July 2016
by evaluation of their oil structuring properties. The gel strength, including the average storage modulus
Available online 21 July 2016
and oscillation yield stress, displayed a negative correlation with FALs and a positive correlation with
HCs, FFAs and WEs. The components dictating the gel strength are HCs, FFAs and WEs in a descending
Chemical compounds studied in this article:
order of importance. The consistency of the oleogels increased with the increasing amount of FFAs and
Heptacosane (PubChem CID: 11636)
Nonacosane (PubChem CID: 12409)
HCs and the decreasing amount of WEs and FALs. The presence of more WEs results in a strong but brittle
Hentriacontane (PubChem CID: 12410) gel with a high initial flow yield stress. We believe these results might be useful in selecting the right
Palmitic acid (PubChem CID: 985) waxes to combine in certain fat-based food products.
Stearic acid (PubChem CID: 5281) Ó 2016 Elsevier Ltd. All rights reserved.
Arachidic acid (PubChem CID: 10467)
Octacosanoic acid (PubChem CID: 10470)
Myricic acid (PubChem CID: 5461027)
1-Triacontanol (PubChem CID: 68972)
1-Dotriacontanol (PubChem CID: 96117)

Keywords:
Wax
Chemical components
HPLC
GC–MS
X-ray
Gelation

1. Introduction gelling behavior at varying critical gelling concentrations of waxes,

In recent years, there has been an emerging interest in
employing waxes as effective structuring agents to entrap a high
amount of liquid oils (>90%wt) into solid-like systems, called
oleogels, which are self-standing, thermoreversible and viscoelas-
tic (Marangoni & Garti, 2011). Wax-based oleogels exhibit different
⇑ Corresponding author at: Laboratory of Food Technology and Engineering,
Faculty of Bioscience Engineering, Ghent University, 653 Coupure Links, 9000
Ghent, Belgium.
E-mail addresses: Chi.DoanDiem@UGent.be, ddchi@ctu.edu.vn (C.D. Doan),
Patel.Ashok@UGent.be (A.R. Patel).
depending on the type of wax and solvent, the wax-wax and wax-
solvent interaction, the cooling and shear rate, the temperature
and time of cooling (Blake & Marangoni, 2015b; Dassanayake,
Kodali, Ueno, & Sato, 2012). The macroscopic properties of an
oleogel are also influenced by the nature of interactions between
the microstructural components occurring at the junction zones
(permanent or transient), which exist between intersecting crystal
branches or entangled crystal fibers (Carriere & Inglett, 2000; Lee &
Inglett, 2007). The amount of such interactions can be increased or
decreased by altering the number of aggregated crystal clusters,
resulting in a change of gel properties (Carriere & Inglett, 2000).
In addition, the gelling behavior is also governed by the wax crystal

http://dx.doi.org/10.1016/j.foodchem.2016.07.123
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
718 C.D. Doan et al. / Food Chemistry 214 (2017) 717–725

morphologies (the rod-like or platelets crystals will result in the (C44) and lignoceryl lignocerate (C48) were purchased from
formation of a strong gel), which are specified by the nature and Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany. HPLC-grade
chain length of the inner chemical components present in waxes solvents used for analysis were hexane (EMD Milipore Corpora-
(Hwang, Kim, Singh, Winkler-Moser, & Liu, 2012). Therefore, a tion), methyl-tertbutylether (MTBE – Sigma-Aldrich), chloroform
better understanding of the chemical composition of waxes is of (Sigma-Aldrich) and acetic acid (Sigma-Aldrich). The standards
utmost importance to explain their behavior in liquid oil network were prepared in chloroform/hexane (1:1) solvent at concentra-
development. tions ranging between 1 and 2000 ppm, which spanned the same
Natural waxes are lipid in nature and from a chemical concentrations of wax constituents in HPLC analysis.
standpoint, they are comprised of complicated mixtures, such as:
n-alkanes or hydrocarbons (HCs), free fatty acids (FFAs), free
2.1.2. Preparation of wax samples
fatty alcohols (FALs), wax esters (WEs), ketones, and sterols
Sunflower wax (SW – melting temperature Tm = 71.7 °C) and
(Kolattukudy, 1976; Regert, 2009). However, information regard-
bees wax (BW, Tm = 49.9 °C) were provided by Koster Keunen
ing the chain length, the ratio, and percentage of components in
Holland BV Co (Bladel, The Netherlands). Oryza Sativa rice bran
each chemical class of waxes is still lacking. Furthermore, the
wax (RBW, Tm = 70.2 °C), candelilla wax (CLW, Tm = 56.8 °C),
chemical composition in each wax can vary depending on its origin
carnauba Brazilian wax (CRBW, Tm = 60.9 °C), carnauba wild wax
and extraction methods. Thus, it is important to have a complete
(CRWW, Tm = 60.7 °C) and berry wax (BEW, Tm = 16.3 °C) were
analysis related to the percentage of each chemical class
provided by Kalhwax GmbH & Co. KG (Trittau, Germany). These
(HC, FFA, FAL and WE) in each wax as well as the chain length of
waxes were dissolved in chloroform/hexane 1:1 solvent at a
different components in each wax class.
concentration of 2000 ppm for HPLC analysis.
Various techniques are available for the chemical characteriza-
tion of waxes. The different wax classes can first be separated using
thin layer chromatography (TLC) (Kanya, Rao, & Sastry, 2007) 2.1.3. Analysis of wax composition using HPLC–ELSD
before being subjected to next steps, such as Fourier Transform The analytical protocol from Hwang et al., was modified to fit on
infrared spectroscopy (Odlyha, 1995), supercritical gas chromatog- the Alliance HPLC system (Hwang et al., 2002). The three solvents
raphy (Hamilton, 1995), liquid chromatography (Asperger, used in the mobile phase were solvent A (0.2% acetic acid in
Engewald, & Fabian, 2001), gas chromatography–flame ionization hexane), solvent B (0.2% acetic acid in MTBE) and solvent C
detection (Marinach, Papillon, & Pepe, 2004), pyrolysis gas chro- (isopropanol). Two HPLC pumps were operated in gradient modes
matography (Regert, Langlois, & Colinart, 2005). TLC is a fast and by a Waters 2690 separation module combined with a gradient
widely used technique for separating complex mixtures; however, controller. A guard column (4
3.0 mm ID silica cartridge in a
the quality of achievable separation through TLC represents only Security Guard cartridge system; Phenomenex) was connected to
one side, and the unambiguous assignment of the obtained spots the main column Agilent Zorbax 5 lm Rx-Sil (4.6 mm i.d.

to defined compounds (Fuchs, Süß, Nimptsch, & Schiller, 2009). 250 mm length) and both were heated at 40 °C. The Alltech 3300
High pressure liquid chromatography (HPLC) equipped with an ELSD detector was operated at 60 °C with a nitrogen (N2) flow of
UV–vis detector and evaporative light scattering detector (ELSD) 1.7 mL/min. The temperature during HPLC–ELSD operation should
detector has also been proven to be an effective tool due to the be controlled around 40–75 °C (depending on the melting
high sensitivity regarding the achievable chromatographic resolu- temperature of the measured wax, but lower than evaporation
tion and peak tailing (Fuchs et al., 2009; Moreau, Kohout, & Singh, temperature of diluted solvents) to avoid the solidification of
2002). For substances lacking useful ultraviolet chromophore like waxes inside HPLC lines and columns, and to enhance the column
waxes, ELSD is a robust choice because it can detect non-volatile polarity (Moreau et al., 2002). By injecting 10 lL of the standard
substances that block the light path (Hwang, Cuppett, Weller, & stock at different concentrations, a calibration curve was plotted
Hanna, 2002; Megoulas & Koupparis, 2005). Therefore, the objec- to link the relation between the concentration and area for further
tive of this study is to characterize the composition of natural analysis. The elution program and washing step are respectively
waxes using HPLC–ELSD/GC–MS and, to understand the role of described in Tables 1A and S1.
different chemical constituents in oil structuring. To make a com-
prehensive case, seven natural waxes (rice bran wax, sunflower
wax, bees wax, candelilla wax, carnauba Brazilian wax, carnauba Table 1A
wild wax and berry wax) were used at their minimum gelling Gradient program on the Alliance HPLC system.
concentrations to transform liquid rice bran oil (RBO) into oleogels.
Gradient
These wax-based oleogels were comparatively evaluated by
Time (min) Solvent
exploring oscillation and flow measurements, solubility, differen-
tial scanning calorimetry, powder X-ray diffraction and polarized A (%) B (%) C (%)
light microscopy to gain a deeper insight into their crystallization 0 100 0 0
and gelation properties. 7 100 0 0
9 95 5 0
14 95 5 0
16 75 25 0
2. Materials and methods 20 75 25 0
24 40 60 0
2.1. Analytical HPLC–ELSD 28 40 60 0
30 0 40 60
35 0 50 50
2.1.1. Preparation of the standard solutions 50 40 60 0
Standard stocks including hexadecane (C16), docosane (C22), 55 75 25 0
pentacontane (C50), palmitic acid (C16), heptadecanoic acid (C17), 65 75 25 0
oleic acid (C18:1), stearic acid (C18), eicosanoic acid (C20), docosa- Solvent A: 0.2% acetic acid in hexane.
noic acid (C22), 1-triacontanol (C30), lignoceryl alcohol or Solvent B: 0.2% acetic acid in MTBE.
1-tetracosanol (C24), lignoceric acid (C24), behenyl arachidate Solvent C: isopropanol (IPA).
 C.D. Doan et al. / Food Chemistry 214 (2017) 717–725
2.2. Analytical GC–MS
2.2.1. Fraction collector
A Waters fraction collector II was attached to the HPLC for the
preparative collection of different wax classes, without connecting
to the ELSD detector. The collector was programmed to collect each
fraction at an interval of 4 min with an initial holding time of 2 min
(fraction 1 of HCs and WEs at minute 2–6, fraction 2 of FFAs at min-
ute 10–14, fraction 3 of FALs at minute 18–22). All fractions were
collected from a 50 lL injection of a 5000 ppm wax sample at a
flow rate of 1 mL/min after the separation with HPLC. Each fraction
contained 4 mL and was derived for further analysis in GC–MS.
2.2.2. Derivatization
FFAs were derived according to the method of Bicalho et al.,
using a 5%v/v acetyl chloride/methanol (AcCl/MeOH) reagent
(Bicalho, David, Rumplel, Kindt, & Sandra, 2008). One mL of reagent
was added to the sample; the reaction proceeded for 30 min at
90 °C and was terminated by adding 1 mL of bi-distilled water.
The fatty acid methyl esters (FAMEs) were extracted twice with
1 mL of hexane before the injection in the GC–MS.
AcCl reagent (0.5 mL) was added to FAL fractions and the
reaction proceeded for 30 min before AcCl was evaporated with a
nitrogen flow. The sample was re-dissolved with 1 mL of hexane
in a 1.5 mL crimp neck vial for the injection in GC–MS.
Wax esters were saponified according to a procedure of
Colombini et al., with a slight modification (Lluveras, Bonaduce,
Andreotti, & Colombini, 2009). The sample was added with 1 mL
of 10%wt KOH/EtOHaq for saponification, assisted by microwaves
at 90 °C for 2 h (power  40 W) in a microwave oven equipped
with a robotic arm for sampling (Biotage Microwave Synthesizer,
Model Initiator 8).
2.2.3. Analytical GC–MS
The major components in the derivatized samples were deter-
mined using an Agilent 7890A GC System, equipped with an
Agilent Technologies 5975C inert MSD with a triple-acid detector
and an automatic injector (Agilent technologies 7683B Series
injector). The column was a fused capillary column HP-5MS
(5%wt diphenyl/95%wt dimethyl-polysiloxane, 30 m
0.25 mm,
film thickness 0.25 lm, Agilent Technologies, Palo Alto, CA) with
the GC injector setting at 250 °C. Samples of 1 lL were injected
using a 10 lL injector under splitless mode. The temperature pro-
gram (Table 1B) was applied to all fractions. Auxiliary temperature
was set at 300 °C. The mass spectrometer was operated in the EI
positive mode (70 eV). The temperature of the MS ion source was
kept at 230 °C and the temperature of the MS quadrupole was
150 °C. The detected low and high mass were 40 and 550 m/z,
respectively.
2.3. Preparation, gelation and thermal behavior of wax-based oleogels
2.3.1. Sample preparation
Rice bran oil (RBO) (onset and peak melting temperatures,
Tm, onset = 73.95 °C, Tm = 11.14 °C) (Suriny brand, Surin Bran
Oil Company, Thailand) was purchased from a local supermarket
Table 1B
GC–MS temperature program.
Rate (°C/min) Value (°C) Hold time (min) Run time (min)
0 50 1 1
Rate 1 11 175 0 12.364
Rate 2 10 240 0 18.864
Rate 3 10 320 15 41.864
719
in Ghent (Belgium). Fatty acid composition in RBO were analyzed
by an interscience thermofocus GC (Shimadzu Co., Japan) equipped
with a TX-2330 column and a flame ionization detector, including
oleic acid (44.2%w/w), linoleic acid (30.0%w/w), palmitic acid
(19.9%w/w), stearic acid (2.1%w/w) and arachidic acid (1.1%w/w).
To prepare the oleogels, natural waxes were dispersed in RBO at
their critical gelling concentration (RBW: 5.0%wt, SW: 1.0%wt, BW:
1.5%wt, CLW: 1.0%wt, CRBW: 4.0%wt, CRWW: 2.0%wt and BEW:
1.0%wt) (Doan, Van de Walle, Dewettinck, & Patel, 2015) (Details
of determining the minimum gelling concentration is described
in Supporting information). The wax-oil mixtures were heated at
90 °C under mild agitation (200 rpm) using a magnetic stirrer
(Model EM300T, Labotech Inc., Germany) for 10–30 min until clear
solutions were obtained. The clear oily solutions were subse-
quently transferred into plastic cups before being cooled down to
5 °C at a cooling rate of 2 °C/min and stored at 5 °C overnight in
a thermal cabinet for further experiments.
2.3.2. Thermal behavior
Onset crystallization (Tc, onset), onset melting (Tm, onset), peak
melting (Tm) and peak crystallization (Tc) temperatures of neat
waxes and wax-based oleogels were determined in triplicate using
a Q1000 DSC and analyzed using the software TA Universal Analy-
sis (TA Instruments, New Castle, Delaware, USA). The cell constant
and temperature were set with indium (TA Instruments). A tem-
perature calibration was done using azobenzene (Sigma-Aldrich,
Bornem, Belgium) and undercane (Acros Organics, Geel, Belgium).
The sample (neat waxes and wax-based oleogels) was first placed
inside an aluminum pan, being sealed with an aluminum lid (TA
Instruments). The sample weight can vary from 6.0 to 8.0 mg.
The sealed aluminum pans were first located at the sample holder
and followed the thermal program: heating at 90 °C for 10 min to
erase all the crystalline history before being cooled to 5 °C at a
cooling rate of 2 °C/min, keeping isothermally at 5 °C for 20 min
and reheating to 90 °C at a heating rate of 5 °C/min.
2.3.3. Rheological behavior
All the rheological measurements were carried out using an
advanced rheometer AR2000ex (TA Instruments, New Castle,
USA). The average storage modulus (G0 LVR) in the linear viscoelastic
region (LVR) of wax-based oleogels were measured at 5 °C using a
parallel plate geometry (cross-hatched plate-plate geometry;
diameter, / = 40.0 mm; gap = 1000 lm) by logarithmically increas-
ing the oscillation stress from 0.01 to 100 Pa at a frequency of
1.0 Hz. Steady state flow measurements were performed by
logarithmically increasing the shear rate from 0.1 to 20 s1. The
obtained flow curves were fitted to the general Herschel-Bulkley
(Eq. (1)) model.
r ¼ ro þ K c_ n ð1Þ
where
r: shear stress (Pa)
ro : yield stress (Pa)
K: consistency index (Pa.sn)
c_ : shear rate (s1)
n: flow index
2.3.4. Powder X-ray diffraction spectroscopy (XRD)
Polymorphism of the wax crystals in oleogels was investigated
by XRD using a Bruker D8-Advanced Diffractometer (Bruker,
Germany) (k Cu = 1.54178 Å, 40 kV, and 30 mA), equipped with
an Anton Paar temperature control system composed of a TTK
450 low-temperature chamber connected to a waterbath (Lauda)
and heating device (TCU 110 Temperature Control Unit) (Anton
Paar, Graz, Austria). The samples were heated at 90 °C for 10 min
720 C.D. Doan et al. / Food Chemistry 214 (2017) 717–725
and subsequently cooled to 5 °C at a cooling rate of 5 °C/min. The
long and short-spacing runs were recorded at 5 °C using a Lynxeye
detector (Bruker, Germany).
2.4. Statistical analyses
The experimental data are expressed as means ± standard devi-
ation of three repetitions. The statistical tests were performed
using SPSS version 22. One-way Anova test, two-sample T-test,
Kruskall-Wallis test and Mann-Whitney U test were applied
depending on the compliance of data to a normal distribution.
Dunnet T’3 post-hoc test was used to find the significant difference
in the Anova test when no homogeneity of variances is found.
3. Results and discussion
3.1. Characterization of chemical classes in natural waxes using
HPLC–ELSD
HPLC–ELSD has proven its effectiveness as a tool for the separa-
tion of polar and non-polar lipidic classes (Moreau et al., 2002;
Nordbäck & Lundberg, 1999). The use of hexane and MTBE
combined with 0.2% acetic acid provided a better elution through
the silica column for lipidic compounds which differ in polarity
(Asikin et al., 2012).
A mixture of 10 standards (docosane, heptadecanoic acid, oleic
acid, stearic acid, eicosanoic acid, docosanoic acid, triacontanol,
tetracosanol, behenyl arachidate and lignoceryl lignocerate) was
injected into HPLC–ELSD system to determine the retention time
of each lipidic class. Because the sterol compound is acknowledged
as a minor component in wax, it can be omitted in this study
(Asikin et al., 2012). The application of the modified elution
program (Table 1A) led to distinctive retention times between
the four chemical classes in order of increasing polarity. Fatty
hydrocarbons (HCs) belong to the same chemical class as the elu-
tion solvent (0.2% acetic acid in hexane) and eluted first, followed
by WEs, FFAs and FALs (Fig. S1). For the standard mixture with high
purity, each compound could be distinguished separately in its
corresponding class, except for the two wax esters which eluted
together (Fig. S1H). For the separation of neat waxes, each peak
represented one single chemical class because all the constituents
in one class eluted together due to their similar polarity (Figs. S1A,
S1B, S1C, S1D, and S1G). Carnauba wax was an exception since
peak 4 of the FALs was clearly separated (Figs. S1E and S1F). This
may have happened because the individual alcohols in carnauba
wax significantly differ in polarity.
After the separation of four lipidic classes in HPLC–ELSD, an
integration was performed to compare the percentage of these
chemical compounds in the waxes (Table 2). RBW, SW and BEW
are considered as mono-component materials with more than
90% of the prominent constituent: WEs in RBW and SW; and FA
in BEW. SW contained 96.23% of WEs, 3.29% of FFAs, 0.32% of FALs
and 0.17% of HCs, which is different from TLC/GC–MS results
obtained by Sindhu Kanya et al., who reported that sunflower seed
Table 2
The chemical composition of the waxes analyzed through HPLC–ELSD on the Alliance system.
RBW SW BW
* a a b
HC 0.29 ± 0.29 0.17 ± 0.15 26.84 ± 1.04
WE* 93.49 ± 2.63E 96.23 ± 0.19E 58.00 ± 0.68C
FFA* 6.00 ± 2.12a,b 3.29 ± 0.16a 8.75 ± 0.75b,c
FAL* 0.22 ± 0.22A 0.32 ± 0.38A 6.42 ± 0.90B
HC: hydrocarbon, WE: wax ester, FFA: free fatty acid and FAL: free fatty alcohol.
RBW: rice bran wax, SW: sunflower wax, BW: bees wax, CLW: candelilla wax, CRBW: carnauba Brazilian wax, CRWW: carnauba wild wax, and BEW: berry wax.
*
a, b, c, d, e, A, B, C, D, E: Different letters in the same format style, within the same row, indicate significant differences at p < 0.05 according to Dunnet T’3 post-hoc test (SPSS 22).
wax from oil refineries contained 66% of WEs, 10% of FALs and 16%
of FFAs (Kanya et al., 2007). The difference can be explained by the
difference in wax origin and analysis methods. SW used by Sindhu
et al., was crude wax and purified in the laboratory, while SW in
the present study was a pure product received from Kalhwax
Company. The difference in sensitiveness between TLC and
HPLC–ELSD are also reasonable for the variation in separation
results. CRBW and CRWW, as expected, had a similar chemical
composition, with a major WE fraction and a medium amount of
FALs. BW and CLW showed a multi-component profile with respec-
tive WEs and n-alkanes being prominent. BW also contained a
considerable amount of HCs and FFAs while CLW contained
additional WEs and FFAs.
3.2. Identification of the chain length of chemical components in
natural waxes using GC–MS
3.2.1. Analysis of fatty hydrocarbons, free fatty acids and free fatty
alcohols
After the separation through the fraction collector, the hydro-
carbon fractions of BW and CLW (resulting from HPLC–ELSD) were
subjected to GC–MS without derivatization. The data in Table 3
show that the major constituents in the hydrocarbon class of BW
and CLW are odd-numbered long chains, among which C31
contributes the most to CLW composition (accounting for more
than 80%) while BW contains a considerable amount of C27, C29
and C31. These results are comparable to those previously reported
(Toro-Vazquez et al., 2007; Tulloch, 1973).
The long-chain FFAs and FALs (>20 carbons) were not volatile
enough and the presence of the free polar head group hampered
the movement through the column. By first deriving the FFAs
and FALs into their non-polar derivatives, the resultant peak shape
and resolution in GC–MS chromatogram were improved (Asikin
et al., 2012). The fractions of FFAs and FALs were derivatized by
applying the acid-catalyzed esterification and acetylation, convert-
ing them into fatty acid methyl esters (FAMEs) and alcohol
acetates, respectively. The use of GC–MS allows us to refine the
chromatograms by extracting the ions with a certain m/z value
(m/z = mass number/charge number of ions). The mass value of
74 m/z is highly characteristic in the analysis of FAMEs because
of the McLafferty rearrangement. The chain length and degree of
packing determined the order of elution for components within a
chemical class, which was clearly demonstrated by the broad range
and discriminatory capacity of the GC–MS. The smaller chain
length and lower degree of packing enabled the molecules to enter
the vapor phase, which reduced the retention time. The com-
pounds were identified by comparing the retention time of the
derivatized samples with the standard, and using the NIST library
database, which further confirmed the accuracy of the method.
Short-chain components (<14 carbons), unsaturated compo-
nents and components with an odd-alkyl chain were present in
very low amounts, indicating that the FFA portion of natural waxes
consisted largely of components with an even alkyl chain between
16 and 32 carbon atoms. The main FFA components in each wax
CLW CRBW CRWW BEW
c a a
72.92 ± 2.23 0.41 ± 0.30 0.16 ± 0.09 0.03 ± 0.01a
15.76 ± 0.35B 62.05 ± 3.03D 58.99 ± 4.57C 0.02 ± 0.02A
9.45 ± 1.14c 6.80 ± 0.76a,b 6.32 ± 3.65a,b 95.70 ± 1.11e
2.20 ± 1.02A,B 30.74 ± 2.48C 34.53 ± 3.45C 4.24 ± 1.10A,B
 C.D. Doan et al. / Food Chemistry 214 (2017) 717–725 721

Table 3
Analytical results of fatty hydrocarbons, free fatty acids and free fatty alcohols, fatty acid moieties and fatty alcohol moieties of wax esters from GC–MS.

RBW SW BW CLW CRBW CRWW BEW

Hydrocarbon
C27 // // 40.28 ± 3.43 0.15 ± 0.13 // // //
C29 // // 25.63 ± 1.90 6.26 ± 2.57 // // //
C31 // // 18.05 ± 0.85 82.48 ± 1.95 // // //
C33 // // 3.12 ± 1.19 7.68 ± 0.85 // // //
FFA
C16 20.76 ± 2.55b 33.18 ± 2.90d 30.11 ± 5.57cd 17.84 ± 1.78a 16.17 ± 1.85a 28.57 ± 6.04c 82.13 ± 2.49e
C18 5.54 ± 0.61b 20.42 ± 1.83e 6.84 ± 1.54b 1.90 ± 0.29a 17.78 ± 1.79d 5.38 ± 1.78b 11.26 ± 1.67c
C20 8.70 ± 1.58b 22.12 ± 2.05d 1.74 ± 0.64a 1.31 ± 0.29a 10.57 ± 2.14c 9.14 ± 0.65bc 0.39 ± 0.19a
C22 17.52 ± 3.12e 9.83 ± 2.26d 4.33 ± 1.39b 1.60 ± 0.17a 6.28 ± 2.40c 5.50 ± 0.35bc //
C24 28.33 ± 3.91d 4.00 ± 0.95b 27.80 ± 2.84d 0.64 ± 0.19a 15.47 ± 3.49c 13.37 ± 3.79c //
C26 3.36 ± 0.76b 2.26 ± 0.40b 6.55 ± 0.76c 0.90 ± 0.29a 6.17 ± 1.15c 5.25 ± 2.30c //
C28 3.34 ± 0.71a 2.85 ± 0.09a 7.20 ± 0.30b 3.13 ± 0.24a 10.58 ± 0.18c 9.78 ± 2.24c //
C30 3.03 ± 0.80b 0.99 ± 0.73a 6.60 ± 0.31d 31.38 ± 0.82e 4.70 ± 2.39c 4.50 ± 1.17c //
FAL
C24 25.30 ± 2.08c 25.25 ± 1.13c 12.01 ± 2.11b 1.77 ± 0.12a // // //
C26 26.15 ± 0.36d 24.11 ± 2.91c 9.46 ± 2.60b 4.53 ± 1.25a // // //
C28 16.16 ± 1.41c 17.74 ± 1.68c 12.95 ± 1.35b 11.48 ± 1.45b 6.38 ± 1.64a 5.67 ± 0.37a //
C30 15.39 ± 1.36b 10.46 ± 3.00a 29.23 ± 0.20c 42.28 ± 2.30d 13.94 ± 1.22b 13.22 ± 0.38b //
C32 9.47 ± 2.50b 5.92 ± 0.63a 20.40 ± 2.74d 14.57 ± 1.97c 65.39 ± 2.71e 68.29 ± 0.82e //
C34 4.70 ± 0.15b // 3.94 ± 0.28ab 2.83 ± 0.20a 11.17 ± 1.05c 10.95 ± 1.20c //
Wax ester fraction
FA moieties RBW SW BW CLW CRBW CRWW BEW
b a f e c d
C16 8.63 ± 1.47 4.89 ± 1.85 80.27 ± 4.50 34.16 ± 3.91 15.16 ± 5.19 19.35 ± 0.46 79.89 ± 4.55f
C18 4.97 ± 0.69a 4.84 ± 1.49a 9.54 ± 1.57b 17.44 ± 1.01d 16.69 ± 3.91d 19.00 ± 9.47e 13.25 ± 2.38c
C20 22.95 ± 1.25e 48.65 ± 6.87f 0.51 ± 0.15a 6.31 ± 3.23b 16.02 ± 0.11d 13.06 ± 2.67c //
C22 26.75 ± 2.19d 24.14 ± 1.55d 0.43 ± 0.17a 10.87 ± 2.56bc 12.19 ± 0.77c 9.47 ± 1.48b //
C24 27.83 ± 1.17e 5.87 ± 1.27b 0.47 ± 0.19a 1.20 ± 0.70a 22.38 ± 5.35d 18.65 ± 4.11c //

FAL moieties
C18 2.80 ± 0.93a 1.82 ± 0.60a 2.93 ± 0.85a 20.70 ± 7.09c 13.69 ± 3.10b 21.48 ± 1.13c 66.02 ± 2.42d
C22 6.92 ± 1.04 9.48 ± 1.43 // // 3.08 ± 0.09 2.55 ± 0.51 //
C24 29.46 ± 4.24e 34.41 ± 4.06f 25.19 ± 3.50d 2.05 ± 0.08a 3.61 ± 1.66ab 4.41 ± 0.92b 8.77 ± 2.09c
C26 23.94 ± 1.10e 30.07 ± 1.10f 14.20 ± 0.94d 8.18 ± 1.16c 1.73 ± 0.87a 1.46 ± 0.64a 3.82 ± 0.29b
C28 13.70 ± 1.31d 12.15 ± 1.98c 13.45 ± 0.51cd 18.72 ± 0.79e 3.15 ± 1.98b 1.60 ± 0.52a 3.32 ± 0.07b
C30 11.16 ± 2.92b 4.01 ± 1.59a 26.12 ± 1.70c 26.64 ± 3.05c 11.95 ± 0.74b 10.37 ± 2.25b 3.05 ± 0.08a
C32 5.31 ± 2.68b 2.20 ± 0.28a 13.57 ± 1.73d 7.99 ± 2.48c 51.74 ± 6.57f 47.89 ± 7.30e 3.53 ± 0.06ab

The symbol ‘‘//” refers to compounds of which the amount is less than the detection limit.
a, b, c, d, e: Different letters within the same row indicate significant differences at p < 0.05 – according to Dunnet T’3 post-hoc test (SPSS 22).
HC: fatty hydrocarbon, WE: wax ester, FFA: free fatty acid, FAL: free fatty alcohol, FA moieties: fatty acid moieties and FAL moieties: fatty alcohol moieties.
RBW: rice bran wax, SW: sunflower wax, BW: bees wax, CLW: candelilla wax, CRBW: carnauba Brazilian wax, CRWW: carnauba wild wax, and BEW: berry wax.
Bold values refer to the values of the major components in natural waxes.

are summarized in Table 3. Palmitic acid was present in all waxes
and contributed remarkably to the FFA compositions of BEW which
contained more than 90%wt of short-chain components (C16 and
C18). Two types of carnauba wax (CRBW and CRWW) are both
products derived from the leaves of the palm, Copernicia cerifera,
which were expected to show a similar composition. Lignoceric
acid (C24) was reported as the major FFA in carnauba wax
(Asperger et al., 2001), and there was the same proportion of multi
FFAs in both carnauba waxes used in this study. CLW is a unique
wax containing a high percentage of longer alkyl chain FFAs
(C24–C32). The length of FFAs in SW and RBW ranged from 16 to
24 carbon atoms which is congruent with Kanya et al., who
identified the predominant FFAs in crude SW to be arachidic acid
(C20) and behenic acid (C22) (Kanya et al., 2007). The chromatogram
in Fig. S2B suggests that more than 95% of the FFA fraction in BW
consisted of FFAs with an even chain length ranging from 12 to 34
carbon atoms, in which C16 and C24 were the primary constituents.
The results are consistent with those from Tulloch et al., (Tulloch,
1973).
The free FAL portion in natural waxes was largely comprised of
components with an even alkyl chain ranging between 22 and 34
carbon atoms. SW and RBW possessed the similar FAL composition,
which contained mainly C24, C26, C28 and C30 FAL. In comparison,
C24 FAL and C30 FAL were reported to be the principal alcohol
in crude SW (34.80%wt) (Kanya et al., 2007) and in RBW
(Dassanayake, Kodali, Ueno, & Sato, 2009), respectively. Triacon-
tanol (C30) was identified as the primary FAL in CLW (42.28%wt)
and BW (29.23%wt) which coincided with the findings from End-
lein and Peleikis (Endlein & Peleikis, 2011). The FAL chromatogram
of BW is clearly indicated in Fig. S2C. There was no significant
difference in FAL composition between CRBW and CRWW. The
principal components ranged from 30 to 34 carbon atoms,
which confirms the results of Asperger et al., who reported C30
(triacontanol) and C32 (dotriacontanol) to be the main FAL in
carnauba wax (Asperger, Engewald, & Fabian, 1999).
3.2.2. Fatty acid and fatty alcohol moieties from wax esters in natural
waxes
The saponification procedure severed the wax esters into fatty
acid and fatty alcohol moieties which were derived for the
characterization in GC–MS. Table 3 shows that the FA and FAL
moieties of the wax ester fraction consisted of components with
an even alkyl chain ranging between 16 and 32 carbon atoms.
The prevalent FA moieties of WEs were behenic acid (C22) and
lignoceric acid (C24) in RBW, and arachidic acid (C20) in SW, which
are comparable with the reported values (Kanya et al., 2007; Vali,
Ju, Kaimal, & Chern, 2005). In general, the chain-lengths of FA
moieties did not exceed 24 carbons. The dominant FA moieties of
722 C.D. Doan et al. / Food Chemistry 214 (2017) 717–725
WEs in BW were C16 and C18 (Fig. S2D). FA moieties of WEs in BEW
were rich in short-chain components (C16–C18) with approximately
80% of palmitic acid. FA moieties of WEs in CRBW and CRWW were
similar, with C16, C18 and C24 FA as the predominant constituents.
The FALs in the WE fraction ranged between 18 and 32 carbon
atoms (Table 3). The prevalent FAL moieties of WEs in RBW and SW
were lignoceryl (C24) and ceryl (C26) alcohol, which differs from the
report of Vali et al., who found C30 as the primary FAL of WEs in
RBW (Vali et al., 2005). However, the FAL moieties in SW are com-
parable to those reported by Kanya et al. and Carelli et al., but in
different percentages (Carelli, Frizzera, Forbito, & Crapiste, 2002;
Kanya et al., 2007). C24, C26, C28, C30 and C32 were the predominant
FALs present in WEs of BW (Fig. S2E). Triacontanyl palmitate (C46)
was established as the principal WE in BW (Tulloch, 1973), which
is confirmed in the current study by a high relative amount of C16
FA moiety (80.27%wt) and C30 FAL (26.12%wt) moiety. Triacontanol
(C30) was also the dominant FAL in WEs of CLW, followed by C18
and C28 FAL.
The results suggest that the chain length of wax esters in these
natural waxes varied from 32 to 68 carbon atoms. Most natural
WEs were comprised of a short-chain FA moiety bound to a
longer-chain FAL moiety. The hydrolysis of WEs to its building
blocks and the subsequent analysis makes it challenging to deter-
mine how the moieties are bound to each other, but it demon-
strates a rationale to explore innovative techniques for chemical
characterization of natural waxes. The suggesting predominant
WEs in waxes might be: C44–C50 in SW, C44–C52 in RBW, C40–C48
in BW, C34–C52 in CLW, C34–C56 in CRBW and CRWW, and
C34–C38 in BEW.
3.3. Macro- and microscopic analysis of oleogels prepared from rice
bran oil at minimum gelling concentration of natural waxes
The oleogels were prepared in RBO at the minimum gelling con-
centration of each wax: 1.0%wt (SW, CLW, and BEW); 1.5%wt
(BW); 5.0%wt (RBW), 2.0%wt (CRWW) and 4.0%wt (CRBW) (Doan
et al., 2015). The rheological gelling behavior of these oleogels
(the average storage modulus in LVR or G0 LVR, critical stress,
oscillation yield stress, flow yield stress and consistency index K),
Tc, onset and Tm, onset were subsequently investigated (Table 4).
Under cooling, single or mono-component lipidic classes such
as FFAs, FALs, HCs or WEs are able to form crystalline building
blocks that can immobilize a high amount of liquid oil into a
self-supporting solid-like system (oleogel) (Marangoni, 2012). As
discussed, waxes are multi-component materials with a prominent
compound and other minor constituents. Hence, the gelation
mechanism from the waxes is considered as a complicated activity
of a mixture containing multi-gelators. The wax-based oleogel is a
result from the dilution of neat wax in RBO, explaining why the
crystallization and melting peaks of the wax-based oleogels were
Table 4
Rheological and thermal properties of wax-based oleogels prepared at minimum gelling concentration.
RBW SW
Concentration of wax (%) 5.0 1.0
Viscous modulus (G0 LVR) (Pa) 54.08 ± 0.02b 433.34 ± 31.44e
Critical stress (Pa) 0.037 ± 0.005a 0.1995 ± 0.00c
Oscillation yield stress (Pa) 4.53 ± 0.41bc 11.95 ± 0.38e
Flow yield stress (Pa) 2.12 ± 0.26c 5.82 ± 0.03e
Consistency index K (Pa.sn) 2.42 ± 0.09ab 3.66 ± 0.47b
Tc, onset (°C) 56.5 ± 0.04de 54.58 ± 0.6d
Tm, onset (°C) 54.39 ± 0.17e 34.54 ± 0.42d
Tc, onset: onset crystallization temperature.
Tm, onset: onset melting temperature.
RBW: rice bran wax, SW: sunflower wax, BW: bees wax, CLW: candelilla wax, CRBW: carnauba Brazilian wax, CRWW: carnauba wild wax, and BEW: berry wax.
a, b, c, d, e, f: Different letters within the same row, indicate significant differences at p < 0.05 – according to Tukey post-hoc test (SPSS 22).
broadened and shifted to much lower temperatures as compared
to the peaks of the neat waxes (Fig. S4). As a result from the
dilution effect of various wax constituents, only the predominant
components in that wax govern the crystallization of wax-based
oleogels. A high amount of long-chain WEs causes RBW and SW
to crystallize in RBO at very high peak temperatures
(53.33 ± 0.10 °C and 49.33 ± 0.16 °C, respectively). For BW-oleogel,
the more intensive peak is the first one (Tc = 42.30 ± 0.12 °C), which
is specified by the most prominent component in BW (WEs), followed
by the second peak (Tc = 28.15 ± 0.25 °C) originating from HC fraction
(the second major chemical class in BW). For CLW-oleogel, the most
intensive crystallization peak at 39.82 ± 0.18 °C originated from HCs
(mainly 82.48% of hentriacontane C31), emerging after the smaller
peak at 43.66 ± 0.23 °C of WE portion. CRBW and CRWW possessing
similar chemical composition, were expected to display similar crys-
tallization behavior in RBO. The most prominent peaks at high tem-
peratures (Tc, CRBW = 53.89 ± 0.15 °C and Tc, CRWW = 56.09 ± 0.20 °C)
are indicative of WE fraction. For CRBW-gel, this was followed by
two minor peaks at 46.54 ± 0.21 °C (of FAs) and 36.29 ± 0.10 °C (of
FALs). However, CRWW-oleogel displayed only one minor peak at
33.75 ± 0.12 °C (of FALs). BEW-oleogel showed its late crystallization
at a low temperature (Tc, onset = 9.22 ± 0.03 °C, Tc = 6.82 ± 0.12 °C)
because of 82% of short-chain palmitic acid (C16) existing in BEW.
At the minimum gelling concentration, the oleogel prepared from
SW, BW, CLW and BEW exhibited a higher G0 LVR and critical stress
as compared to those of RBW, CRBW and CRWW. Among the strong
gels, CLW and BEW showed the highest consistency and a very low
flow yield stress at the region of low shear. In the weak gel group,
RBW displayed a higher storage modulus on linear visco-elastic
region (G0 LVR) but lower critical stress and oscillation yield stress
compared to CRWW. As expected, CRBW and CRWW, which have a
similar chemical composition, displayed similar weakness in gelation
although a higher amount of CRBW (4%wt) was required for the
gelation of RBO compared to CRWW (2%wt).
X-ray diffraction measurements were done at two ranges of
angles: wide diffraction angle (WAXD) for short-spacing runs and
small diffraction angle (SAXD) for long-spacing runs, which
provides information on the lateral and longitudinal packing of
molecules in crystalline structures, respectively (Bouzidi, Li, &
Narine, 2014) (Fig. 1). In the wide diffraction angles (2 theta > 15°),
all the wax-based oleogels (except for BEW-oleogel) exhibited
two separated intensive and narrow peaks at d-spacing
of 0.415 nm and 0.372 nm (d = n * wavelength/2sin(theta) =
1.54178/2 * sin(theta)), which are indicative of the orthorhombic
sub-cell structure (b0 morphology). The BEW-oleogel had only
one extremely small but true peak at d-value of 0.415 nm (SAXD),
which is a characteristic of a hexagonal symmetry. It is noted that
neat BEW exhibited a low melting and crystallization temperature
(16.25 ± 0.10 °C) while other waxes demonstrated higher thermal
profiles (>50 °C). According to Larsson, when the orthorhombic
BW CLW CRBW CRWW BEW
1.5 1.0 4.0 2.0 1.0
249.33 ± 47.14c 340.77 ± 36.14d 30.16 ± 0.49a 38.71 ± 1.48a 274.88 ± 30.06c
0.1585 ± 0.00b 0.3981 ± 0.00d 0.063 ± 0.00a 0.063 ± 0.00a 0.6309 ± 0.00e
2.51 ± 0.00b 8.77 ± 2.13d 0.39 ± 0.02a 0.63 ± 0.00a 6.31 ± 0.00cd
3.81 ± 0.09d 0.00 ± 0.00a 0.45 ± 0.04b 3.56 ± 0.30d 0.00 ± 0.00a
5.42 ± 0.35c 10.97 ± 0.75d 2.26 ± 0.40ab 1.37 ± 0.33a 10.96 ± 0.34d
42.4 ± 0.17c 34.78 ± 2.07b 55.95 ± 0.82d 59.73 ± 0.51e 9.22 ± 0.03a
19.6 ± 0.03b 19.94 ± 1.04b 23.45 ± 0.95c 24.56 ± 0.08c 6.54 ± 0.38a
 C.D. Doan et al. / Food Chemistry 214 (2017) 717–725
Fig. 1. X-ray diffractogram of wax-based oleogels at 5 °C.
waxes show a phase transition at temperatures close to their melt-
ing points, the orientation order of the ‘‘zig-zag” planes are lost and
the molecules can rotate around their length axis to form a hexag-
onal symmetry, which could be the case for BEW-oleogel (Larsson
& Larsson, 1994). All the wax-based oleogels showed clear peaks
(with different intensities) at the low angle region, providing
evidence for a lamellar packing. The SAXD diffractograms of the
oleogels from RBW, SW and BEW show the appearance of one
intense and strong reflection (0 0 1) peak, followed by the intensity
decrease of other higher-order peaks. This intensity reduction can
be explained by the layers of different thickness being stacked
together. In addition, Ensikat et al. stated that this intensity
reduction, or even extinction of certain (0 0 1) peaks is the unique
property of some compounds present in plant waxes, and is due
to the specific position of the oxygen atoms in the long-chain
molecules (Ensikat, Boese, Mader, Barthlott, & Koch, 2006). The
intense and narrow long-spacing peaks at the first half 2h angles
are indicative of the double-layer order structure when the com-
pounds with terminal polar groups (FAs, primary FALs) crystallize
with a head-to-head orientation (Koch & Ensikat, 2008). This is
true for the oleogels prepared from RBW, SW and BEW. The
oleogels prepared from BW, CLW, CRBW and CRWW had only
one (0 0 1) SAXD peak (Table S2) with much lower intensity, indi-
cating a significantly lower order of the layer structure, which is a
feature of mixtures of compounds with varying chain lengths
(Ensikat et al., 2006).
In 2009, Sato et al., reported that a difference in intensity
between long-spacing and short-spacing peaks (as is the case of
the oleogels from BW, CLW) is the result of a strong anisotropy
in crystal growth rates between directions vertical to the lamellar
plane and directions within the lamellar plane. The Van der Waals
interaction between the long hydrocarbon chains and the polar
ester groups (the molecular interactions within the lamellar plane)
are far stronger than the molecular interactions through the
terminal –CH3 groups. This mechanism leads to the appearance
of rod-like or platelet morphologies, which are desirable for
the formation of a strong three-dimensional gelling network
(Fig. 2) (Dassanayake et al., 2009; Hwang et al., 2012). This is con-
firmed by the strong gelation of SW, CLW, BW and BEW in RBO,
which exhibited the tiny needle-like crystals, which are actually
the edges of platelet crystals (Blake & Marangoni, 2015a). It is
important to note that the crystals shown in Fig. 2 were heated
and re-crystallized between the narrow space of the glass slide
and the cover slide. Under cooling, the edges of the platelets
723
developed in a unidirection, resulting in the needle-like form as
seen in Fig. 2. Chambers, Ritchie, and Booth (1976) reported that
WEs dominated the platelet crystals, which was confirmed by Koch
and Ensikat in 2008 (Koch & Ensikat, 2008). For RBW-oleogel,
besides the existence of some platelet-crystals, RBW-oleogel also
displayed the long dendritic crystals, which interconnected to form
a branched network with many voids resulting in the weak gelling
ability of RBW. As discussed above, RBW and SW possessed similar
chemical composition, in which the most remarkable difference is
substantially higher amount of C24-COOH and C24 moiety in RBW
than in SW (28% vs 4%, respectively). In addition, neat RBW dis-
played 2 distinct peaks on the crystallization curve as compared
to only one intensive peak of neat SW (Fig. S4A). We are convinced
that the minor crystallization peak of neat RBW at 59.05 ± 0.13 °C
originated from FA fraction (the second prominent compound in
RBW) and is related to the appearance of the big dendritic crystals.
Although this minor peak diminished on the crystallization curve
of RBW-oleogel due to the dilution effect, the related FA fraction
still existed in a relatively sufficient amount to form big denritic
crystals. Their presence would interfere with the crystallization
of the major WE peak, which is important for the formation of
platelet crystals and a strong gelling network. This explains why
RBW was not efficient in structuring RBO, this is in complete con-
trast to the results obtained by Sato et al. (Dassanayake et al.,
2009), who reported that RBW could structure olive oil and salad
oil into good gels, even at 1.0%wt. We also compared the structur-
ing properties of RBW and SW (the type used in this study) in
gelling different oils (high oleic sunflower oil, olive oil, peanut
oil, walnut oil and hazelnut oil). We found that the crystal habits
of RBW and SW were independent on the oil type. RBW always
exhibited the big dendritic crystals and formed weak oleogels
while SW always revealed the platelet crystals connecting in a
dense and strong network. It can thus be claimed that the minor
FA fraction in RBW formed the dendritic molecules which inter-
fered with the network development of platelet crystals, resulting
in a weak gel structure.
The molecular order of the minor components might influence
the wax crystal shape, and the two morphological wax types
(tubules and platelets) were able to self-assemble (Birkett &
Talbot, 2009). Clusters of self-assembled crystals might form an
aggregate in different ways leading to a stronger or weaker net-
work. The crystalline aggregates of CRBW (Fig. S3) and CRWW
(Fig. 2) oleogels revealed a similar morphology of spherulites and
dendritic morphology explaining the weaker gel formation: the
724 C.D. Doan et al. / Food Chemistry 214 (2017) 717–725
Fig. 2. Microstructure of wax-based oleogels at 5 °C.
low critical stress (0.063 Pa) and quick flowing characteristic at
very small oscillation and shear stress regions (Table 4). CRBW
and CRWW are comprised of approximately 60% of WEs, 6% of FFAs
and 30% of FALs, which were expected to positively contribute to
the oil gelation. The long-chain FALs (P24 carbons) in CRBW and
CRWW also govern the oil structuring through H-bonding; how-
ever, the activity of FALs has a threshold limit. The hydrogen bond-
ing between very long chain FALs present in CRBW and CRWW
(C30, C32, and C34) becomes stronger as the size and mass of the
FALs increases (Mudge, 2005). Such hydrogen bonding in long
chain FALs, according to Zhu and Dordick, contributes to the
overall gelator-gelator interaction which compromises the
solvent-gelator H-bonding, resulting in a poor gelation (Zhu &
Dordick, 2006).
A statistical Pearson correlation (details in Supporting informa-
tion) was executed to understand the relationship between the
gelling properties and chemical composition in waxes (Table S3).
There is a positive correlation between oscillation yield stress
and the average storage modulus (G0 LVR) which displays the
strength of the oleogels, and between the critical stress and
consistency index K which reflects the flexibility, smoothness
and consistency of the oleogels. Based on the Pearson correlation,
the gel strength including G0 LVR and oscillation yield stress has a
negative correlation with FALs and a positive correlation with
HCs, FFAs and WEs. The gel strength will increase with the decreas-
ing amount of FALs and the increasing amounts of HCs, FFAs, and
WEs. Similarly, the critical stress and consistency index K have a
positive correlation with FFAs and HCs, and a negative correlation
with WEs and FALs. The flow yield stress is positively correlated to
the amount of WEs and negatively correlated to FALs, FFAs and
HCs. More WEs will lead to a strong but brittle gel with a high
initial flow yield stress. The brittleness is determined by the
high gel strength and a narrow LVR as well as yield zone, in which
the slope of the curve gives information about the breakage
of intermolecular forces holding up the structure (Patel,
Babaahmadifooladi, Lesaffer, & Dewettinck, 2015; Uhlherr et al.,
2005). We would like to stress that the oil model used in this
research was RBO. Other liquid oils with different triacylglycerols
will differently contribute to the gelling properties of waxes, for
which the research is ongoing.
The gelator with a higher amount of FFAs and HCs can be com-
bined in food products which require consistency, smoothness and
plasticity like whipped cream, ice cream, confectionery products
(spread, fillings), and spreading margarine. However, FFAs could
be oxidized during heat processing and storage, resulting in off-
flavor. For ice cream, berry wax with a high amount of FAs (low
melting temperature) can be a good choice because of the low-
temperature working condition. Waxes with a prominent amount
of WEs (SW, CRBW, CRWW or RBW) can be utilized as a good alter-
native for shortening or baking margarine.
4. Conclusion
In this study, the crystallization and gelling activity of natural
waxes in RBO has been explained by the presence of different
chemical components in waxes. More WEs will result in a strong
and brittle gel, which is expressed in a high storage modulus and
flow yield stress of the wax-based oleogels. HCs and FFAs con-
tribute to the consistency and stability of the wax-based oleogels
(a high critical stress and high consistency index). However, the
chain length needs to be taken into account because more short-
chain FFAs (<16 carbons) result in wax crystallization at low tem-
peratures, which can limit its ability in the food application. FALs
also have an important contribution to the oil structuring. How-
ever, the strong H-bonding between very long-chain FALs (>30 car-
bons) in waxes and RBO compromises the oil-wax H-bonding and
hinders the gelation. The results of our study provide important
information relating to the elucidation of wax components in liq-
uid oil structuring, which can be exploited to select a wax with
properties desirable for food application.
Acknowledgements
This research has been funded with support from the European
Commission. This publication reflects the views only of the author,
and the Commission cannot be held responsible for any use which
may be made of the information contained therein. Authors also
thank Marie Curie Career Integration Grant (SAT-FAT-FREE) and
Vandemoortele Centre for microscope support, and colleague Iris
Tavernier and Kim Moens for scientific inputs.
 C.D. Doan et al. / Food Chemistry 214 (2017) 717–725
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2016.
07.123.
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