Accepted Manuscript

Phytosterols-induced viscoelasticity of oleogels prepared by using

monoglycerides

Mohd Dona Bin Sintang, Sabine Danthine, Allison Brown, Davy

Van de Walle, Ashok R. Patel, Iris Tavernier, Tom Rimaux, Koen
Dewettinck

PII: S0963-9969(17)30419-2
DOI: doi: 10.1016/j.foodres.2017.07.079
Reference: FRIN 6876
To appear in: Food Research International
Received date: 21 March 2017
Revised date: 27 July 2017
Accepted date: 31 July 2017

Please cite this article as: Mohd Dona Bin Sintang, Sabine Danthine, Allison Brown,
Davy Van de Walle, Ashok R. Patel, Iris Tavernier, Tom Rimaux, Koen Dewettinck ,
Phytosterols-induced viscoelasticity of oleogels prepared by using monoglycerides, Food
Research International (2017), doi: 10.1016/j.foodres.2017.07.079

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 ACCEPTED MANUSCRIPT

Phytosterols-induced viscoelasticity of oleogels prepared by using monoglycerides

Mohd Dona Bin Sintanga,b, Sabine Danthinec, Allison Brownd, Davy Van de Walled, Ashok R
Patela, Iris Taverniera, Tom Rimauxe, Koen Dewettincka,d
a
Vandemoortele Center Lipid Science and Technology, Laboratory of Food Technology and
Engineering, Faculty of Bioscience Engineering, Ghent University
b
Department of Food Technology and Bioprocess, Faculty of Food Science and Nutrition,

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Universiti Malaysia Sabah, Malaysia
c
Department of Food Science and Formulation, Universite de Liege, Passage des Deportes,

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Gembloux, Belgium
d

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Laboratory of Food Technology and Engineering, Faculty of Bioscience Engineering, Ghent
University, Ghent, Belgium
e
Vandemoortele R&D, Izegem, Belgium

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Corresponding author: MohdDonaBin.Sintang@UGent.be and Koen.Dewettinck@UGent.be
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Abstract
Monoglycerides (MGs) and phytosterols (PS) are known to form firm oleogels with liquid oil.
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However, the oleogels are prone to undergo polymorphic transition over time that lead to
crystals’ aggregation thus, compromises physical properties. Thus, we combined MGs with PS to
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control the crystallization and modify the morphology of the combination oleogels, as both
components are reported to interact together. The oleogels were prepared at different ratio
combinations and characterized in their rheological, thermal, morphology, and diffraction
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properties. The results showed that the 8:2 MGP:PS exhibited higher storage modulus (G') than
the MGP mono-component. The combination oleogels exhibited effects on the crystallization
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and polymorphic transition. Consequently, the effects led to change in the morphology of the
combination oleogels which was visualized using optical and electron microscope. The resultant
effect on the morphology is associated with crystal defect. Due to observable crystals of MGP
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and PS, it is speculated that the combination oleogels formed a mixed crystal system. This was
confirmed with diffraction analysis in which the corresponding peaks from MGP and PS were
observed in the combination oleogels. However, the 8:2 oleogel exhibited additional peak at
35.41Å. Ultimately, the 8:2 was the optimum combination observed in our study. Interestingly,
this combination is inspired by nature as sterols (phytosterols) are natural component of lipid
membrane whilst MGP has properties similar to phospholipids. Hence, the results of our study
not only beneficial for oil structuring, but also for the fields of biophysical and pharmaceutical.
Keywords: chain ordering; Monoglycerides; Oleogels; Phytosterols;
Viscoelasticity-enhancement
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1. Introduction
An oleogel is a self-assembled structure formed by the entanglement of one or more structuring
units such as crystals, fibrillar networks, or suspended polymer strands. The use of oleogels
provides an alternative to saturated and trans-fat usage in conventional lipid-based food products.
Different approaches and techniques have been discovered recently which spans from mono
component to mixed component oleogels (Blach et al., 2016; Davidovich-Pinhas, Barbut, &
Marangoni, 2016; Gravelle, Davidovich-Pinhas, Zetzl, Barbut, & Marangoni, 2016; Lopez-

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Martinez, Charo-Alonso, Marangoni, & Toro-Vazquez, 2015; Patel, Babaahmadi, Lesaffer, &
Dewettinck, 2015; Patel et al., 2014; Patel, Schatteman, De Vos, Lesaffer, & Dewettinck, 2013;

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Patel, Schatteman, Lesaffer, & Dewettinck, 2013). Approaches to structure oils with crystalline
non-fat particles have gained considerable interest among food scientists as these systems closely

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resemble conventional fat structured systems (A. Bot & Agterof, 2006; Doan, Van de Walle,
Dewettinck, & Patel, 2015; Lopez-Martinez et al., 2015; Patel et al., 2015). The quest to find

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new edible structurants has become more challenging due to the regulatory approval of chemical
compounds that typically create good oleogels but with restricted edible applications.
Monoglycerides (MGs) are lipid molecules consisting of single fatty acids esterified to the
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glycerol backbone. Different MGs vary in their type and length of carbon chain of fatty acids.
MGs when dispersed in oil, form an elastic gel upon cooling to the Krafft temperature (Chen &
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Terentjev, 2009; Chen, Van Damme, & Terentjev, 2009; Verstringe, Moens, De Clercq, &
Dewettinck, 2015). MGs can create four different phases when dissolved in a hydrophobic
matrix (oil): isotropic, inverse lamellar, sub-α crystalline, and β-crystalline phases (Chen et al.,
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2009). Upon cooling, MGs first form an inverse lamellar phase in which the glycerol heads are
densely packed in a hexagonal manner in planes in the middle of bilayers. Below the
crystallization point, lamellar phases transform into the sub-α crystalline phase with aliphatic
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chains packed in an orthorhombic configuration. The gel-like properties of those two phases are
similar with no significant difference in the rheological response (Chen et al., 2009). However,
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both phases are only metastable and tend to transform into a triclinic packing of MGs resulting in
a β-crystalline phase (Chen & Terentjev, 2009; N. K. O. Ojijo et al., 2004).
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Monoglyceride oleogels in the β-crystalline phase exhibit large crystal aggregation, which
compromises the oil binding capacity of the gel (Chen & Terentjev, 2009). Namely, the β-
crystalline phase formation causes a separation of the D- and L-isomer of monoglycerides (Chen
& Terentjev, 2009). Several studies have therefore focused on controlling the crystallization and
polymorphic behavior of monoglycerides in oil to mitigate this undesired aggregation by for
example applying shear nanostructuring (Da Pieve, Calligaris, Co, Nicoli, & Marangoni, 2010)
or by combining monoglycerides with ethylcellulose (Lopez-Martinez et al., 2015).
Ethylcellulose can bind with monoglycerides through hydrogen bonding and as such modify its
crystallization behavior. The addition of ethylcellulose also improves the rheological properties
of oleogels (Lopez-Martinez et al., 2015).
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Phytosterols (PS) are naturally occurring cell wall stabilizing components in plants that are
commercially used as cholesterol-lowering agents. β-sitosterol is the most common PS and exists
in 3 different crystalline forms: anhydrate, hemihydrate, and monohydrate, depending on the
water content (Christiansen, Rantanen, von Bonsdorff, Karjalainen, & Yliruusi, 2002; von
Bonsdorff-Nikander et al., 2005). Similar to cholesterol (COH), PS is a sterol based molecular
structure consisting of a steroid skeleton with a hydroxyl group attached at the carbon number 3
(C-3) of the A-ring and aliphatic side chain at carbon number 17 (C-17) of the D-ring (Moreau,
Whitaker, & Hicks, 2002). In oil structuring, β-sitosterol and γ-oryzanol have been shown to

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form unique structures in canola oil that are capable of trapping oil via capillary action between
the self-assembled γ-oryzanol and β-sitosterol co-crystals (Bot & Agterof, 2006; Bot, den Adel,

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& Roijers, 2008; Co & Marangoni, 2012).

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The ability of γ-oryzanol and PS to co-crystallize and form hollow tubules with 10.9 nm
diameter and 1.5 nm wall thickness, explains why the combination is capable of immobilizing
liquid oil (Bot et al., 2012). These tubules may co-assemble and create a continuous three-

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dimensional network over macroscopic length scale with non-polar solvent immobilized
internally (Bot & Flöter, 2011). Bot and Agterof examined a series of sterols and found that
dihydrocholesterol, cholesterol, β-sitosterol, and stigmasterol produced firm oleogels. They
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concluded that the presence of the hydroxyl group was critical for the formation of the gel. The
ring structure with no double bonds accelerated the gel formation, whereas a double bond
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resulted in no gel formation (Bot & Agterof, 2006; Bot, den Adel, Roijers, & Regkos, 2009). PS
have also been combined with other compounds. For instance, Han et.al. studied the structuring
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properties of a sitosterol and lecithin mixtures. Their findings suggest that lecithin induced a
change in the assembly of β-sitosterol in high linoleic sunflower oil (HLSO) and altered the
physical properties of oleogels (Han et al., 2014).
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Comprehensive understanding of the effect of cholesterol on the phospholipid membrane acts as

a basis to hypothesize that a combination of MGs and PS may produce mixed-component
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oleogels with higher elastic/storage modulus (Bin Sintang, Rimaux, Van de Walle, Dewettinck,
& Patel, 2017; Kouzounis, Lazaridou, & Katsanidis, 2017; Lopez-Martinez et al., 2015; Yang,
Chen, & Yang, 2017). Since, cholesterol improves the physical properties of phospholipid
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membrane, by condensing (chain ordering) alkyl tails of phospholipids (Cao, Tokutake, &
Regen, 2003). The formation of mixed-component oleogel is based on the ability of MGs and PS
to form hydrogen bonding, forming complexes with different thermal behavior (Gater et al.,
2013). In this article, we prepared the oleogels by reducing the ratio of MGs and replaced with
PS, whilst the total concentration was remained constant. The present paper reports some novel
findings on the effects of oil structuring properties, crystallization process, morphology, and
diffraction properties of combination oleogels. In addition, we addressed the possible interaction
which provides explanation to the improvement in oil structuring properties of MGs, PS and
their combinations reported earlier (Bin Sintang et al., 2017). The combination of PS and MGs
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offers an interesting food-grade oil structuring alternative to saturated fats and encourage further
exploration of potential application of PS.
2. Materials and methods
2.1. Materials
Refined sunflower oil and monoglycerides from hydrogenated palm oil (MGP) (C16:0; 59.1%,
C18:0; 38.5%) were supplied by Vandermoortele Lipids N.V., Belgium. CardioAid™ (non-
esterified phytosterols including 50.4% β-sitosterol, 25.0% campesterol, and 15.3% stigmasterol)
was provided by ADM (USA).

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2.2. Oleogel preparation

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Oleogels were prepared by using MGP, PS, and combinations thereof in ratios of 10:0, 8:2, 7:3,
6:4, and 0:10 (wt %) in sunflower oil. The total structurant(s) concentration is 10 wt%. The

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mixture was first heated to 100°C under continuous stirring for approximately 20-30 minutes
using a magnetic stirrer (Model EM3300T, Labotech Inc., Germany) followed by cooling at
room temperature for 20 minutes. The samples were finally stored at 5°C for one-week. An

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oleogel of MGP mono-component at 6 wt% (6:0) was prepared for thermal study.
2.3. Rheological measurement
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The linear viscoelastic region (LVR) of the prepared oleogels were measured by small amplitude
dynamic measurement using the rheometer AR 2000ex (TA Instruments, New Castle, DE) with
the Advantage application software. The experiment was performed using 40 mm cross-hatched
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parallel-plate geometry and a gap size of 1000 μm. Frequency sweep tests (1 to 100 Hz) were
conducted at constant stress value in the linear response region (Oscillatory stress = 4 pa). The
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measurement was conducted in triplicate at 5°C after one week stabilization time.
2.4. Differential scanning calorimetry (DSC)
The crystallization kinetics in the different mixed oleogels were analyzed using Q1000 DSC (TA
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Instruments, New Castle, DE). The DSC was calibrated with indium (TA Instruments, New
Castle, DE), azobenzene (Sigma-Aldrich, Bornem, Belgium) and undecane (Acros organics,
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Geel, Belgium) before analysis. The oleogels of MGP, PS, and their combinations prepared at a
total structurant of 10%wt were sealed in hermetic pans, and an empty pan was used as a
reference. The samples were heated to 150°C and held for 2 minutes to eliminate crystal history,
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followed by a cooling step to 5°C (at 10°C/min). The samples were then kept isothermal for 1
hour and 5 hours before heating to 150°C (at 5°C/min). The same pans were kept for one week at
5°C before heating to 150°C (at 5°C/min). For better illustration of the effect, 6wt% (6:0
MGP:PS) of MGP mono-component was studied using the same procedures. All the analysis was
done in triplicate. The comparison between the oleogels was based on peak temperature
(crystallization and melting).
2.5. Optical microscope
Crystal morphology was observed under normal and polarized light using a Leica DM2500
microscope (Leica Microsystems, Belgium) equipped with a color camera Leica MC170 HD.
Small amount of the oleogels was placed on a glass slide, then covered with a cover slid. The
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slide was then mounted under the microscope and visualized using 10X lens. Temperature
control stage; Linkam T95 System Controller (Linkam Scientific Instrument Ltd., Surrey, UK)
was used to regulate the temperature to 10°C.
2.6. Cryo-scanning electron microscopy (CRYO-SEM)
The microstructure of the oleogels at 5°C was visualized by using scanning electron
microscopy. After one week in the fridge, the oleogels were suspended in excess ethanol
(at 5°C) to remove liquid oil from the structure (12-hours). The oil was removed using the
adapted method by Maleky et.al, and Acevedo and Marangoni (Acevedo & Marangoni,

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2010; Maleky, Acevedo, & Marangoni, 2012). The extracted oleogels were then filtered

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to remove excess ethanol and a small piece of the filtered oleogel sample was then
mounted on an aluminium stub. The samples were then quickly frozen in slushed nitrogen

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(-210°C), fractured, sputter-coated with platinum and observed with a Jeol JSM-7100 F
scanning electron microscope (Jeol Europe) B.V., Zaventem, Belgium). The accelerating
voltage was 3 KV.

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2.7. Powder X-ray diffraction (P-XRD)
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The molecular organization of the oleogels was determined by XRD spectroscopy using a
D8 Advance Diffractometer (Bruker, Germany) equipped with an X-ray generator
Kristalloflex K780 (Bruker, Germany) (l Cu λ=1.54178Å, 40kV, 30mA), and a
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temperature control unit (TCU110 system, Anton Paar, Austria) connected to a water
bath. This system allows adjustments of the temperature of the thermostated
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diffractometer chamber TTK 540 (Anton Paar, Graz, Austria). Both small angle X-ray
diffraction (SAXD) and wide angle X-ray diffraction (WAXD) were recorded in this
analysis. The Diffrac Plus V4 software was used for data integration.
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2.8. Statistical analysis

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The peak melting temperatures are expressed as means ± standard deviation of three repetitions
and were analyzed using one factor analysis of variance (ANOVA). The equality of variances
were verified using Levene’s test prior to usage of Tukey’s test to compare the mean values at p
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< 0.05 significance level. The peak melting temperatures were compare between the oleogels at
the same isothermal period (column) and on the oleogel with different isothermal period (row).
The latter was to elucidate the evolution in the melting profile of the oleogels as function of time.
3. Results and discussion
3.1. Oleogel properties
The highest G’ was seen for the 8:2 ratio compared to the other ratios after one week at
5°C (Figure 1). The PS mono-component oleogel showed phase separation after one week
and so they were excluded from frequency sweep tests. The overall increase in the moduli
values for 8:2 MGP:PS over mono-component MGP oleogels suggests that the presence
of PS influenced the association colloidal structure (bilayers) formed by MGP as seen
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previously in COH and MGs mixtures (Crilly & Earnshaw, 1983; Larsson, Gabrielsson,
& Lundberg, 1978; Michalak, Muzzio, Milianta, Giacomini, & Lee, 2013) (Figure 2). In a
previous study, the 8:2 MGP:PS was also shown to be the most efficient combination in
terms of network strength (Bin Sintang et al., 2017). Recently, studies have reported
similar improvement in the rheological properties of oleogels prepared using mixtures of
MGs and PS (Kouzounis et al., 2017; Yang et al., 2017). It has been reported that plant
sterols are known to act as regulators of the physical properties of the phospholipid
membrane (Cao et al., 2003). Due to the structural similarities of phospholipid

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membranes and MGs bilayers, we hypothesize that the MGP and PS complex together in
the bilayer structure via hydrogen bonding (Gater et al., 2013). The arrangement of

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monolayer changes as the PS molecules align themselves between the MGP molecules.
The PS then condenses the alkyl tail of MGs which results in an oleogel with better

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viscoelasticity. Similarly, cholesterol is known to be able to alter the phase behaviour of
the MGs (Larsson et al., 1978) which upholds our hypothesis, Figure 2.

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3.2. Differential scanning calorimetry (DSC)
To investigate the effect of PS on the crystallization of MG, we used DSC to obtained the
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peak crystallization and melting temperatures of the oleogels. Additionally, the effect on
crystallization and melting behavior is a good indication to evaluate the complexation of
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MGs and PS (condensing effect) (Kamal & Raghunathan, 2012) thus, verify our
hypothesize (Figure 2). Upon cooling, the MGP mono-component was first to crystallize
followed by the 8:2, 7:3, and 6:4 MGP:PS oleogels (Figure 3). This result implies that the
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formation of inverse lamellar (α-crystal) occurs faster in mono-component oleogel, and

that the incorporation of PS in the MGs system causes a decrease in the association rate of
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MGs into ordered structures. It has been reported that the MGs in hydrophobic solution
self-assemble first into inverse lamellae (α-crystal) followed by a transition to sub-α
crystals (Chen & Terentjev, 2009; Chen et al., 2009; Verstringe et al., 2015). Hence, for a
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more thorough understanding, the 6:0 MGP:PS oleogel was also analysed under similar
conditions.
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Interestingly, the 6:0 ratio showed a faster crystallization temperature compared to the 6:4
MGP:PS ratio (Figure 3). This clearly points out the putative interaction between the PS
and MGP, especially in the lamellar phase. At that stage, the PS may interact with the
MGP molecules and solubilize together in the lamellar structures. On the other hand, the
sub-α formation temperatures (13°C) were only slightly affected by the PS as indicated by
a change in the peak shape. Bhattacharya and Haldar showed that the transition
temperature changed in the phospholipid membranes containing COH (Bhattacharya &
Haldar, 2000), which primarily due to the condensing effect (Kamal & Raghunathan,
2012). As discussed in literature, the chain ordering and hydrogen bonding by COH in
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phospholipid membrane result in depression of transition temperatures. The results found

in our systems are thus in agreement with the findings reported in the studies.

During melting of different mixtures, a significant effect on the melting transition was
observed in PS-incorporated oleogels (Figure 4 and Table 1). Studies have shown that
DSC is useful to elucidate the polymorphic transition using stop-and-return and
isothermal crystallization approaches (Foubert, Fredrick, Vereecken, Sichien, &
Dewettinck, 2008; Verstringe, Danthine, Blecker, Depypere, & Dewettinck, 2013). Figure
4 shows the melting profiles of oleogels after one-hour, five-hour, and one-week of

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isothermal holding times at 5°C, with the values summarized in Table 1. The 10:0 MGP

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mono component gel melted at a higher temperature followed by 6:0, 8:2, 7:3, and 6:4,
after one-hour. After five-hour, the 8:2 MGP:PS melted at a temperature significantly

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higher than the melting temperature after one-hour period. Similarly, the Oleogels of 7:3
and 6:4 exhibited significant change in the melting temperatures for different isothermal
periods. Meanwhile, the 6:0 MGP mono-component oleogel showed insignificant change

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in the melting temperatures for different periods (Table 1). The results can be explained
by two different possibilities: i) the MGP continued to crystallize during the isothermal
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period, and/or ii) a polymorphic transition of MGP into more stable polymorph, which
was slow in the combination oleogels. The latter can be elucidated by comparing the
melting evolution between the MGP mono-component (10:0) to the combination oleogels.
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In Figure 4a, there is a small peak appeared prior to the main melting peak of 10:0
MGP:PS. However, this pattern was only observed in the combination oleogels (7:3 and
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6:4 MGP:PS) after five-hour and one-week isothermal periods (Figure 4b and 4c). By
comparing the different patterns in the melting profile (Foubert et al., 2008), it is clear
that the combination oleogels undergo slow polymorphic transition than MGP mono-
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component.

A small peak observed in the previous melting properties of 10:0 MGP:PS oleogel
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disappeared after one-hours, leaving only a sharp main melting peak. Although the
temperature was slightly lower than the previous result, which was mainly due to a
change in peak shape. After one-week at 5°C, the melting profile of the oleogels were
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again analyzed and the results are displayed in Figure 4.c and Table 1. The 10:0 and 6:0
MGP:PS showed no change between the melting temperature for five-hour and one-week.
For the 8:2, 7:3 and 6:4 oleogels, the melting temperature increased significantly, with
appearance of an additional small peak before the main peak of 7:3 and 6:4 oleogels.

A visible difference between the 6:0 and 6:4 MGP:PS in their melting profiles (Figure 4
and Table 1). The melting temperature of the 6:0 was significantly higher than 6:4
MGP:PS, which indicates the transition is faster in oleogel without PS. In Table 1, there is
no significant difference in the melting temperatures of 10:0 and 6:0 mono-component
between five-hour and one-week isothermal periods. Additionally, the PS not only affect
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the crystallization but also the transition to a stable state. After one-week the melting
temperatures of the combination oleogels were lower than the mono-component olegels,
which significantly lower in 6:4 oleogel. This difference indicates that there is a possible
formation of eutectic mixtures between the MGP and PS.

Our thermal investigation results are in agreement with the results found in the literature
(Kamal & Raghunathan, 2012). Kamal and Raghunathan studied the phospholipid-
phytosterol membranes system and showed that the presence of sterols lowers the main
transition temperature (Kamal & Raghunathan, 2012), indicated with the depression in

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crystallization temperature.

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3.3. Microstructure visualization

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The morphology of the crystals was visualized using optical and polarized light
microscopy. It is important to visualize the morphology and crystal networks, which
reflect the spatial distribution of crystals in the oleogels. It has been reported that spatial

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distribution of crystals influences the rheological properties of oleogels (Marangoni &
Rousseau, 1996; Tang & Marangoni, 2006) thus, providing the explanation for the change
in rheological properties (Figure 1). Additionally, the visualization provides the overview
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on the observed effect of the crystallization on the crystal morphology of the oleogels. It
is generally agreed the presence of impurities will affect the crystal morphology due to
crystal defect. The crystal defect occurs when foreign molecules (impurities) attach on the
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surface of growing crystals, affecting crystal habit (Smith, Bhaggan, Talbot, & van
Malssen, 2011; von Bonsdorff-Nikander et al., 2005). In our combination, both MGP and
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PS are able to crystallize thus, act as impurities that can affect crystal habit (Christiansen
et al., 2002; Engel & Schubert, 2005; Zychowski et al., 2016).
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The optical images were taken directly after one week at 5°C from the oleogels without
annealing, Figure 5. Figure 5a shows that the MGP mono-component formed clustered-
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like crystals and in agreement with the literature (Chen & Terentjev, 2009; N. K. O. Ojijo
et al., 2004). Whereas, PS crystallized into three different morphologies consisting of the
elongated plate (needle-like), plate, and spherulite crystals (Figure 6). According to Blake
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and Marangoni (2015), these morphologies can be a different orientation of crystalline

platelets (Blake & Marangoni, 2015). The morphology of the crystals showed slight
changes as the portion of MGP was increasingly replaced by PS. In addition to MGP
crystals, PS were also crystallized as separate entities, forming two mixed crystalline
units in the oleogels prepared at 8:2, 7:3, and 6:4 MGP:PS (Figure 5b to 5d). Thus, the
MGP and PS crystals were interconnected to one another forming a three-dimensional
network that structured the sunflower oil. The shape and size of the PS crystals changed
with respect to the concentration and ratio of PS used. PS at 5wt% exhibited similar
morphology as the 10wt% (Figure S2, Supplementary document). Thus, the change in
morphology of PS in the combination oleogels was due to the presence of MGP, affecting
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the crystal growth (von Bonsdorff-Nikander, Karjalainen, Rantanen, Christiansen, &

Yliruusi, 2003). The presence of mixed crystals from MGP and PS explains the
improvement in the rheological properties of 8:2 MGP:PS (Figure 1), which also agrees
with the literature (Kouzounis et al., 2017; Yang et al., 2017). At 8:2 MGP:PS oleogel,
needle-like crystals appeared together with the MGP crystals. As the ratio of PS increased
(7:3 and 6:4 MGP:PS oleogels), the spherulite crystals emerged alongside the MGP
crystals, Figure 5c and 5d. The difference in the crystal morphology agrees with the
crystal defect theory. Studies have shown that the presence of emulsifiers affect the

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crystal habit of PS, which agrees with our results (Engel & Schubert, 2005; von
Bonsdorff-Nikander et al., 2005).

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With the use of electron microscopy, an in-depth visualization of the internal structure

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was performed. Figure 7 displays the SEM images of the oleogel formed by MGP, PS,
and their combinations. It is observed that the MGP structure is composed of layers which
appear to form a lamellar structure (arrow). These layers’ stack together to create hollow

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spaces that can entrap liquid sunflower oil (Verstringe et al., 2015). These inverse
lamellar structures coordinate themselves into a configuration that forms a three-
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dimensional network. Meanwhile, PS exhibited large plate crystals having width
dimension of ~10µm (Figure 6a), which agree with the polarized images (Figure 6b).
However, it is difficult to visualize the morphology of individual PS in the combination
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oleogels, as both MGP and PS exhibit plate-like crystals with different dimensions
(Figure 6, 7, and S5). Additional electron microscope images are given in the
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supplementary document that demonstrate the morphology on the surface of oleogels

(Figure S3 and S4).
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As previously discussed, the MGP crystallized during cooling whereas the PS only began
to crystallize at 5°C, Figure 3 and S1. This temperature-time lag disturbs the
crystallization process as well as the post-crystallization, especially with respect to the
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crystal habit, as a result of crystal defect. Therefore, there is strong relationship between
the observed effect on crystallization (Figure 3) on the morphology (crystal habits)
(Figure 5 and 6b), in which this combined effect resulting in higher G' of 8:2 oleogel
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(Figure 1). Accordingly, we speculate that the combination oleogels forms mixed crystals
oleogels, which also supported with the melting profile after one-week (Figure 4c).

3.4. Molecular organization

The molecular organization of crystals was analyzed by using X-ray diffraction (XRD)
studies. It has been speculated previously that the combination oleogels form mixed-
xrystal systems. Thus, the XRD patterns at 5°C are valuable to support the results from
the previous analyses. Table 2 and Figures 8 and 9 summarize and display the diffraction
values and patterns for the oleogels prepared by using MGP, PS, and combinations.
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In the MGP mono-component oleogel, a single diffraction peak in the small angle region
indicates interplanar distance of the MGP bilayers is approximately 49.11 Å (Figure 8). In
the wide-angle region, the main peak at 4.55 Å along with other peaks are evidence of β-
polymorphic form, Figure 9. The peaks indicate that in-plane ordering of MGP aliphatic
chains is in triclinic configuration. Based on the XRD patterns, mono-component PS
crystals also contain a pseudo bilayer structure of repeating units, Figure 8-9 and S6. The
order is induced by the stacking of the individual sterane cores which leaves some
conformational freedom for the alkyl chain (Bot & Flöter, 2011). The bilayer thickness

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was measured as 36.88 Å in small angle along with a peak at 18.43 Å, which correspond
to the length of PS molecules (Rossi, Sacanna, & Velikov, 2011). Meanwhile, multiple

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peaks were recorded in the wide-angle region for PS mono-component gel. As discussed
earlier, PS can exist in anhydrate, hemihydrate and monohydrate forms.

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The combination oleogels also formed a bilayer structure (Figure 8), in which the
corresponding peaks of MGP and PS mono-components appeared. Yet, the 8:2 showed

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the formation of a new peak at 35.41 Å, whilst the 7:3 and 6:4 MGP:PS oleogels
displayed only a shoulder-like peak. This new peak could be associated with the
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formation of complexes between MGP and PS, as illustrated in Figure 2. However, it
should also be considered here that the formation of new peak in the mixed-component
system of 8:2 could be due to the transition in the crystalline form(s) of PS (anhydrous,
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monohydrate, and hemihydrate) (Rossi et al., 2011). This can be explained by considering
the effect of MGP which acts as the emulsifier that influences the crystallization of PS
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thus (Engel & Schubert, 2005; von Bonsdorff-Nikander et al., 2005), changing the crystal
habit of the latter and supported with the microscopy images (Figure 5, 6 and S2). The
observed change in the SAXD and WAXD pattern might help to explain the change in the
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melting temperature of PS, which known to melt at approximately 140°C (Vaikousi,

Lazaridou, Biliaderis, & Zawistowski, 2007). Yet, detailed crystallography investigation
is required.
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In the wide-angle region, the main peak at 4.55 Å along with other peaks are evidence of
β-polymorph and observed in all oleogels (Da Pieve, Calligaris, Panozzo, Arrighetti, &
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Nicoli, 2011). The combination oleogels exhibited additional visible peaks in the wide-
angle region, which resemble the corresponding peaks of PS (Figure 9). This indicates
that the PS also crystallized separately as different crystals, even at a low level (8:2
oleogel). This is supported with the reported solubility of PS in liquid edible oil of 2-3%
w/w at 25°C (Vaikousi et al., 2007), which explains a diffraction patterns in the
combination oleogels. However, there was slight change in the diffraction patterns of the
combination oleogels, as marked with arrows in Figure 9 and S6. The fact that the 8:2
oleogel displayed new peak at 35.41Å, which also agrees with the observed change in the
WAXD, especially to the peaks corresponding to PS. Yet, detailed diffraction analysis on
selected combination is currently in progress. Similarly, change in the diffraction pattern
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was also observed when PS are crystallized in fat-blends (Acevedo & Franchetti,
2016)and with supercritical liquid (Moreno-Calvo et al., 2014).

Previous studies have shown that the MGs concentration does not affect the inter-plane
spacing and subcell packing of the crystals (Da Pieve et al., 2011). The results shown here
indicate that the slight change in the bilayer of 8:2 could be due to the solubilisation of PS
molecules in the bilayer matrix of MGP. Earlier investigations reported that the
incorporation of cholesterol induces a reduction or increase in the bilayer thickness based
on the concentration of the cholesterol and the type of phospholipids (Bhattacharya &

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Haldar, 2000), and this agrees with our results. The variation in thickness of the bilayers

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is believed to be due to the increase in the tilt angle of the lipid molecules, and this was
observed in our samples (mixed component oleogels), supporting the previous results.

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This observation indicates that in this system, the sterol molecules modify the flexibility
of the alkyl chain of MGP (Bhattacharya & Haldar, 2000).

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The use of MGs to structure edible liquid oil has been studied extensively (Chen &
Terentjev, 2009; Chen et al., 2009; Da Pieve et al., 2010; Da Pieve et al., 2011; Lopez-
Martinez et al., 2015; Lopez-Martinez et al., 2014; Ojijo, Neeman, Eger, & Shimoni,
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2004). However, the combination of MGs and PS is relatively new. PS alone could not
structure edible oil, as the crystals are prone to contract and fail to form a network (Co &
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Marangoni, 2012). MGs do form firm gels but their aggregation during aging, which is
related to the formation of a β-crystalline phase, compromises the oil binding properties
(rheological properties) (Chen & Terentjev, 2009; N. K. O. Ojijo et al., 2004). Hence,
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controlling the crystallization behavior may control the aggregation (Chen & Terentjev,
2009; N. K. O. Ojijo et al., 2004). Several studies have therefore been performed to
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control the crystallization behavior of MGs either through combination with

ethylcellulose (Lopez-Martinez et al., 2015) or shear nanocrystallization (Da Pieve et al.,
2010).
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It is interesting to note that the combination of MGP with PS has an influence on the
crystallization, transition, rheology and morphology of the oleogels, and discussed in the
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previous sections. The PSs influence the crystallization and the transition of MGP to a
more stable polymorph. These effects are manifested in the non-isothermal and the
isothermal crystallization discussed in the thermal behavior. It has been reported that the
gelation of MGs molecules in oil involves intermolecular hydrogen bonding (Chen &
Terentjev, 2009; Lupi et al., 2016). Thus, it is likely that the effect of the PSs on the
crystallization of MGP is attributed to the ability of both molecules to interact via
hydrogen bonding (Gater et al., 2013). The modified molecular assembly affected the
microstructural development of the combination oleogels, which most likely to form a
mixed crystal system. This conclusion is based on the diffraction results obtained from
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both SAXD and WAXD, in which the corresponding diffraction peaks of MGP and PSs
appeared in the combination oleogels.

As discussed previously, the PSs reduced the clustering of crystals and as such the
physical properties of the 8:2 MGP:PS improved. It has been discussed and explained that
crystal defect may cause change in the crystal morphology. Thus, the crystal defect is the
best explanation for how the intermolecular hydrogen bonding occurs between DM and
PSs and how the condensing effect of the alkyl tail influences the crystal morphology and
diffraction patterns of the combination oleogels (Smith et al., 2011; von Bonsdorff-

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Nikander et al., 2003). In other words, a better spatial distribution of crystalline network

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in the presence of PS, improves the viscoelastic properties (Kouzounis et al., 2017;
Lopez-Martinez et al., 2015; Marangoni & Rousseau, 1996; Tang & Marangoni, 2006).

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Meanwhile, the effect of MGP on the crystallization of PS can be observed in the change
in morphology of crystal and diffraction patterns of corresponding peaks of PS in the
combination oleogels. Yet, detailed crystallography study is required to clearly define the

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resultant crystal structures. Currently, there are several studies reported on the formation
of new PS crystals, but without detailed explanation (Acevedo & Franchetti, 2016;
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Moreno-Calvo et al., 2014).

In the mixed crystal system of stearyl alcohol (SO) and stearic acid (SA), interaction
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between SO and SA affected the crystallization and the morphology and thereby the
hardness of the combination oleogel was higher (Blach et al., 2016). Additionally, Lopez-
Martinez and co-workers studied the combination of monoglyceride and ethylcellulose
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and found the latter to delay transition from sub-α to β-polymorph. As a consequence, the
interaction between monoglycerides and ethylcellulose improved modulus of oleogel
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(Lopez-Martinez et al., 2015). The combination of MGP and PS offers an interesting

solution to the existing problems of crystal aggregation in MGP as well as high-
temperature melting of PS. Interestingly, this combination is inspired by nature as sterols
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(phytosterols) are natural component of lipid membrane whilst DM has properties similar
to phospholipids.
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4. Conclusions

In this study, 8:2 ratio of MGP:PS emerged as the strongest oleogel with higher storage
modulus compared to the MGP mono component oleogel. As seen with phospholipid
membranes where the chain-ordering and hydrogen bonding by sterol regulate the
permeability and rigidity of membranes, similarly, at an optimum ratio, the PS
synergistically improve the viscoelasticity of MGs oleogels based on the chain ordering
and packing alteration effect on MGs.

The changes in crystallization temperature and diffraction pattern strongly support the
complexation and condensing effect of PS on the alkyl tail of MGP. With respect to the
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polymorphic form, all the oleogels showed characteristics of β-polymorph but with better
stability. Based on the results, the interaction between MGP and PS resulted in the change
of crystallization which ultimately led to modification in the morphology of the oleogels.
The change in morphology is associated with crystal defect caused by PS on the growing
crystals of MGP. This is supported by the difference in the crystallization, in which only
MGP crystallized during cooling as suggested by the crystallization pattern/peak
(correspond only to MGP). The change in crystal habit and morphology led to
improvement in the G' of the 8:2 MGP:PS oleogel. With increasing PS levels, the Gʹ

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values were lower than that of 8:2 gel as well as 10:0 MGP:PS oleogel, supported with
the appearance of spherulite-crystals in the 7:3 and 6:4 MGP:PS oleogels. Hence, a

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correct balance is important in producing an oleogel with improved viscoelasticity that is
based on mixed crystals approach. Additionally, it is hypothesized that the combination

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oleogels formed a mixed crystal system which clearly observed in the microscopy and
diffraction results. However, the SAXD of 8:2 MGP:PS oleogel exhibited additional peak

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at 35.41Å. Therefore, it is interesting to conduct an in-depth analysis on the molecular
structure of the complexes together with the phase behavior of the MGP and PS
combinations, which are currently under investigation in our group and will be reported
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soon.

Acknowledgements
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Author wants to thanks Ministry of Higher Education Malaysia and Universiti Malaysia
Sabah for PhD scholarship. Vandemoortele is recognized for its financial help in the
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acquisition of the Leica polarized light microscope and resources. Hercules foundation is
recognized for its financial support in the acquisition of the scanning electron microscope
JEOL JSM-7100F(grant number AUGE-09-029).
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6554. doi: 10.1021/acs.jafc.6b01722

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Figure 1. The frequency sweeps of the oleogels prepared by using MGP, PS, and their
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combinations. The measurement was conducted at 5°C after one week storage inside a
fridge at 5°C to account for any polymorphic transitions of crystals over time.
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Figure 2. The diagram above illustrates the possibility of inter-chain reaction between
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MGs and PS in combination oleogels at molecular level, forming a mixed crystal system.
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Figure 3. DSC thermogram of oleogels cooled from high temperature to 5°C. The
oleogels were cooled with a cooling rate of 10°C/min. At 5°C, the oleogels were allowed
to crystallize for different periods.
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Figure 4. DSC thermogram of oleogels heated from 5°C to high temperature after
isothermally crystallized for a) 1 hour, b) 5 hours, and c) 1-week.
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Table 1. The table summarizes the mean for main peak melting temperatures of oleogels
isothermally crystallized for different time periods at 5°C. (N=3)
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MGP:PS 1 Hour 5 Hours 1 week

10:0 64.64 ±0.29d,B 63.32 ±0.7c,A 63.43 ±0.15b,A

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8:2 52.62 ±0.3b,A 57.83 ±1.29b,B 63.02 ±0.19b,C

7:3 49.97 ±1.5b,A 58.86 ±1.33b,B 62.26±1.46b,C
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6:4 45 ±2.94a,A 51.13 ±1.52a,B 59.31±1.68a,C

6:0 61.47 ±0.37c,A 61.95 ±0.71c,A 61.33±0.31b,A
0:10 66.90 ±2.76d,A 67.86 ±1.62d,A 66.54±1.5c,A
Small letters = Statistical analysis between the oleogels for the same isothermal period (Column)
Capital letters = Statistical analysis between the isothermal periods for the same oleogel (Row)
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Figure 5. The optical light microscopy images of oleogels prepared by using MGP, PS,
and their combinations. From top left, a) 10:0, b) 8:2, c) 7:3, and d) 6:4 MGP:PS.
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Figure 6. The (a) electron and (b) polarized light microscopy of PS disperse in sunflower
oil at 0:10 (MGP:PS). The images were taken after one-week storage at 5°C.
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Figure 7. The electron microscopy images of oleogels prepared by using MGP, PS, and
their combinations. From top left, a) 10:0, b) 8:2, c) 7:3, and d) 6:4 MGP:PS
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(arrow=lamellar layer; star=trace of liquid oil; circle=plate-like crystals). The oleogels

were fractured.
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Figure 8. The SAXD diffraction patterns of MGP, PS, and combination oleogels prepared
at 10:0, 8:2, 7:3, 6:4, and 0:10 MGP:PS.
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Figure 9. The WAXD diffraction patterns of MGP, PS, and combination oleogels
prepared at 10:0, 8:2, 7:3, 6:4, and 0:10 MGP:PS.
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Table 2. The table summarizes the diffraction values measured in the oleogels after one-
week storage. The analysis was conducted at 5°C.
MGP:PS SAXD (Å) WAXD (Å)

10:0 49.11 4.55, 4.44, 4.33, 4.11, 3.95, 3.8, 3.7

8:2 48.62, 36.94, 5.86, 5.68, 4.55, 4.44, 4.33, 4.11, 3.95, 3.8, 3.7
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7:3 48.13, 36.85 5.9, 5.76, 5.1, 4.8, 4.71, 4.64, 4.55, 4.33, 4.11, 3.8, 3.74

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6:4 48.16, 36.91 7.46, 7.24, 5.9, 5.76, 5.1, 4.8, 4.71, 4.64, 4.55, 4.33, 4.11,
3.8, 3.74

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0:10 36.88 7.46, 7.24, 5.9, 5.88, 5.76, 5.54, 5.1, 4.8, 4.71, 4.65, 4.55,
4.05

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Graphical abstract

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Highlights
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 Condensing effect of monoglycerides’ bilayer by phytosterols leading to enhanced

viscoelasticity
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 Monoglycerides and phytosterols complexation through hydrogen bonding

 Monoglycerides influence crystallization of phytosterols
 Phytosterols influence molecular arrangement of monoglycerides
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