Research Article

The influence of the type of oil phase on the self-assemblyprocess of g-oryzanol þ b-sitosterol tubules in organogelsystems

Hassan Sawalha1,2, Giel Margry1, Ruud den Adel3, Paul Venema1, Arjen Bot3, Eckhard Floter3,4 and

Erik van der Linden1

1 Laboratory of Physics and Physical Chemistry of Foods, Department of Agrotechnology and Food

Sciences, Wageningen University, Wageningen, The Netherlands2 Chemical Engineering Department, An-Najah National University, Nablus, Palestine3 Unilever Research and Development Vlaardingen, Vlaardingen, The Netherlands4 Food Process Engineering, Department of Food Technology and Food Chemistry, Technical University

Berlin, Berlin, Germany

Mixtures of g-oryzanol and b-sitosterol were used to structure different oils (decane, limonene,

sunflower oil, castor oil, and eugenol). The g-oryzanol and b-sitosterol mixtures self-assemble into

double-walled hollow tubules (�10 nm in diameter) in the oil phase, which aggregate to form a

network resulting in firm organogels. The self-assembly of the sterol molecules into tubules was

studied using light scattering and rheology. By using different oils, the influence of the polarity of the

oil on the self-assembly was studied. The effects of temperature and structurant concentration on the

tubuler formation process were determined and the thermodynamic theory of self-assembly was

applied to calculate the change in Gibbs free energy (DG0), enthalpy (DH0), and entropy (DS0)

resulting from the aggregation of the structurants was determined. The self-assembly was found to be

enthalpy-driven as characterized by a negative DH0 and DS0. A decreasing polarity of the oil promotes

the self-assembly leading to formation of tubules at higher temperatures and lower structurant

concentrations.

Keywords: Critical aggregation concentration / Enthalpy / Entropy / Light scattering / Organogels

Received: November 20, 2011 / Revised: October 15, 2012 / Accepted: October 30, 2012

DOI: 10.1002/ejlt.201100395

1 Introduction

Structuring of edible oils with crystalline triacylglycerols, i.e.

fats, for production of foods (e.g. margarine and table

spreads) can contribute to the risk for cardiovascular diseases

due to the high saturated fatty acids content in these fats [1–

3]. In the last decade, much effort has been invested to

identify alternative structurants [4–6]. Mixtures of plant

sterols (g-oryzanol and b-sitosterol) were proposed as a pro-

mising candidate to replace these crystalline triacylglycerols

[7, 8]. Both compounds can be obtained from edible sources:

b-sitosterol is a minor component in several vegetable oils

and g-oryzanol can be found in rice bran oil [7]. In addition,

(esterified) plant sterols are used as cholesterol lowering

agents as they interfere with absorption of cholesterol in

the intestine [9].

Several publications reported on the use of mixtures of

g-oryzanol and b-sitosterol for structuring of organogels and

emulsions [7, 8, 10–15]. The g-oryzanol and b-sitosterol in

sunflower oil were found to self-assemble into hollow tubules

with an average diameter of �10 nm which aggregate to

form a network resulting in a transparent and firm organogel

[7, 8, 16]. In a water-in-oil emulsion, the formation of the

tubular microstructure was disturbed because the water mol-

ecules bind to the sitosterol to form sitosterol monohydrates,

which hinder the self-assembly of the sterol molecules

into tubules [10, 12, 13]. Recently, the thermodynamic

Correspondence: Hassan Sawalha, Chemical Engineering Department,

An-Najah National University, P.O. Box 7, Nablus, Palestine

E-mail: hassan.sawalha@gmail.com

Fax: þ970 9 2345 982

Abbreviation: CAC, critical aggregation concentration

Eur. J. Lipid Sci. Technol. 2013, 115, 295–300 295

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parameters of the self-assembly of sterols into tubules in

sunflower oil were determined: binding energy, enthalpy,

and entropy change. The self-assembly into tubules was

found to be enthalpy-driven [14].

The objective of the present study was to extend this work

and to investigate the effect of the polarity of the oil phase on

the thermodynamics of sterol self-assembly. To do so, orga-

nogels were prepared in several oils with different polarities.

Light scattering and rheology were used to study the start of

the self-assembly process as a function of sterol concentration

and temperature. From these measurements, the changes in

Gibbs free energy, enthalpy, and entropy associated with the

self-assembly were determined.

2 Materials and methods

2.1 Materials

In the current study, g-oryzanol powder was obtained from

Tsuno Rice Fine Chemicals, Wakayama, Japan and tall oil

sterol granules (78.5% b-sitosterol, 10.3% b-sitostanol, 8.7%

campesterol, and 2.5% of other minor sterols) was provided

by Unilever, the Netherlands. The following oils were used

for the preparation of the organogels: decane (purity >99%,

Sigma–Aldrich, The Netherlands), limonene (purity >97%,

Sigma–Aldrich), sunflower oil (Reddy, NV Vandemoortele,

Breda, the Netherlands), castor oil (Sigma), and eugenol

(purity>99%, Aldrich, see Table 1 for the dielectric constant

and chemical structures of the oils). All materials were used as

received.

2.2 Methods

2.2.1 Preparation of the organogels

Mixtures of oryzanol and sitosterol at a fixed ratio of (60

oryzanol:40 sitosterol w/w) were dissolved in the oil phase at

�1008C using a magnetic stirrer. The structurant solution

was cooled down according to the cooling profile specified

below to follow the aggregation using the light scattering or

rheology.

2.2.2 Light scattering

The light scattering, Malvern (Nano) Zetasizer 3 (Malvern

Instruments, UK), was used to study the aggregation process

of oryzanol and sitosterol into tubules in different oils. Hot

structurant solution was poured in the preheated (808C)

cuvette. Measurements were performed by cooling the

solution in the cuvette from 80 to 08C stepwise with a cooling

rate of 28C/step. Before each measurement, the solution was

kept at a fixed temperature for �8 min after each cooling

step to equilibrate.

2.2.3 Rheology

Anton PaarModular Compact Rheometer (MCR300) with a

coaxial cylinder measuring geometry (Cuvette cell, CC 17)

was used to perform the rheological measurements. The

Cuvette cell was preheated to 808C before a hot structurants

solution was poured into the cell and kept at 808C for 20 min

to equilibrate. The tubuler formation process was followed by

cooling the solution from 80 to 08C using a cooling rate of

0.28C/min. The measurements were performed in the linear

regime with a strain of 0.001 and frequency of 1 Hz.

2.2.4 Scanning electron microscope (SEM)

Field emission scanning electron microscope (Magellan 400,

FEI, Eindhoven, the Netherlands) was used to visualize the

tubular morphology of the organogels. The method reported

by Sawalha et al. [13] was followed in preparing the samples

for SEM tests.

3 Results and discussion

3.1 Aggregation as measured by light scattering andrheology

The aggregation of oryzanol and sitosterol in different oils

was studied using light scattering and rheology. Figure 1

shows an example of the scattered light intensity as a function

of temperature during cooling of solutions with different

Table 1. Dielectric constant and chemical structure of various oils

Oil Chemical structure Dielectric constant [13]

Decane CH3(CH2)8CH3 2.0

Limonene 2.4

Sunflower oil

3.1

Castor oil 4.5

Eugenol 4-(H2C¼CHCH2)C6H3-2-(OCH3)OH 10.4

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concentrations of structurants in castor oil. The figure shows

that upon cooling of the solutions, the scattered light intensity

remains constant and sharply increases at a specific tempera-

ture. The temperature at which the intensity suddenly

increased is dependent on the structurant concentration. A

sharp increase in the scattered light intensity is indicative to

the formation of aggregates. The so-called critical aggrega-

tion concentration (CAC) of structurants is the concentration

below which aggregates do not exist, while above the CAC

aggregates do exist. In our case, the aggregates are linear

assemblies that assume a tubular shape [14]. One can

measure the CAC as a function of temperature. For each

structurant concentration, there exists a temperature at

which a sudden increase in light scattering occurs. The

specific structurant concentration equals the CAC at that

temperature.

The resulting CAC’s as obtained from light scattering

data are summarized in Table 2. The table shows that the

dependency of the CAC on temperature also is a function of

the type of oil used. Decane shows high aggregation tem-

peratures as compared to the other oils, whereas terpenes

(i.e. limonene and eugenol) show relatively low aggregation

temperatures (for the same concentrations).

Rheological measurements were also performed to study

the aggregation process. The storage moduli (G0) of the

structurant solutions in various oils were measured during

cooling and the results (for castor oil as an example) are

shown in Fig. 2. During cooling, at a given concentration

structurant, a sharp increase in the G0 was observed at a

certain temperature. This sudden increase is a consequence

of the formation of a network of aggregates. Since only a very

low amount of linear aggregates already leads to network

formation, this point practically marks the aggregate for-

mation. Taking the reasoning above for sudden increase in

the intensity of scattered light, the sudden increase of G0

also indicates the CAC. The CAC data of the various oils

obtained from the G0 data are given in Table 2. The Table

shows a good agreement between the CAC data as obtained

from light scattering and rheology.

3.2 Appearance of the organogels

From visual observation of the organogels, we noticed that

the gels prepared with high polarity oils, i.e. sunflower oils

and eugenol were generally transparent, whereas gels pre-

pared with decane and limonene turn opaque after short time

of preparation. Furthermore, the transparency of the gels

generally decreases with increasing the concentration of

the gelators. These differences might be ascribed to the

differences in the chemical structure of the oils, which affects

the solubility and mutual interaction of the sterols.

Figure 1. The scattered light intensity of 16, 18, 20, 22, and

24% w/w (60 oryzanol: 40 sitosterol) solutions in castor oil as a

function of temperature during cooling.

Table 2. Aggregation temperature at different structurants

concentration during cooling of structurants solutions in different oils

measured by light scattering and rheology

Oil

Structurants

concentration

% w/w

Taggregation (K)

light scatteringa)Taggregation (K)

rheologya)

Decane 8 327 326

10 331 329

12 334 330

14 340 333

16 339 335

Limonene 14 279 275

16 279 276

18 284 276

20 285 283

22 284 285

Sunflower 8 289 287

10 299 298

12 307 306

14 314b) 314

16 318b) 321

Castor oil 16 287 285

18 295 296

20 301 303

22 307 310

24 313 313

Eugenol 28 278 276

30 281 282

32 287 288

34 295 291

a) Some of the samples were measured in duplicate and in this case

the average temperature was taken.b) The data points were taken from the work of Sawalha et al. [14].

Eur. J. Lipid Sci. Technol. 2013, 115, 295–300 Self-assembly process of g-oryzanolþb-sitosterol tubules 297

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3.3 Morphology of organogels

The morphology of the tubular microstructure in organogel

was visualized with SEM. Figure 3 shows SEM images of

organogel sample prepared with 32%w/w sitosterol–oryzanol

mixture in sunflower oil. Bundles of tubules with an average

size of �60 nm/bundle could be clearly seen in Fig. 3a. The

bundles were visualized at a higher magnification as shown in

Fig. 3b. Individual linear tubules with an average diameter of

7–15 nm (including the tungsten coating of 3 nm) and aver-

age length ranging from 5 to 10 mmwere observed. The SEM

results confirm that mixtures of sitosterol and oryzanol

indeed self-assemble to form tubules in the oil phase, which

is in agreement with small-angle X-ray scattering and small-

angle neutron scattering results published recently [13, 16].

The small-angle X-ray scattering and small-angle neutron

scattering interference patterns for organogels prepared with

the same type of oils used in the present study were similar

which indicates similar tubular structures in all oils.

3.4 Thermodynamics of tubule formation

In the present section, the data of the CAC and aggregation

temperature, presented in Table 2, will be used to determine

the change in Gibbs free energy DG0, enthalpy DH0, and

entropy DS0 that is associated with tubule formation.

The sterol monomers are assumed to self-assemble into

one-dimensional tubular aggregates like the steps in a spiral

staircase [14]. The change in Gibbs free energy was

therefore calculated using the following equation from the

one-dimensional self-assembly theory [17, 18]:

xN ¼ N x1exp�DG0

RT

� �� �N

(1)

here xN is the molar fraction tubular aggregates containing N

sterols as building blocks, x1 is the sterol monomeric molar

fraction of the building blocks that are not incorporated into

the tubules. [16, 17]. Since xN cannot become larger than one

(it follows that x1 cannot become larger than exp(DG0/RT).

This identifies the molar fraction of monomeric building

blocks (sterols) at the CAC as x1, CAC � exp(DG0/RT). All

additional building blocks (sterols) added in excess of the

CAC will be incorporated into tubules.

Figure 2. Storage modules (G0) of 16, 18, 20, 22, and 24% w/w

(60 oryzanol: 40 sitosterol) solutions in castor oil as a function of

temperature during cooling.

Figure 3. SEM micrograph of an organogel prepared with 32% (60:40 mixture of oryzanol/sitosterol) in sunflower oil.

298 H. Sawalha et al. Eur. J. Lipid Sci. Technol. 2013, 115, 295–300

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To calculate the changes in Gibbs free energy DG0,

enthalpy DH0 upon aggregation involved in the tubular for-

mation process, the following equations were used:

DG0 ¼ RTlnx1;CAC(2)

DH0 ¼ �RT2 @ lnx1;CAC

@T

� �(3)

For the calculation of the entropy change DS0, the following

equation was used:

DG0 ¼ DH0 � TDS0 (4)

where DS0 is the change in molar entropy upon aggregation.

The values of lnx1,CAC were plotted against the corre-

sponding aggregation temperature for the various oils as

shown in Fig. 4. The structurant molar fraction was calcu-

lated based on the average molecular weight of (60:40 ory-

zaol:sitosterol) and the temperature is taken as the average of

the Taggregation measured by light scattering and rheology

shown in Table 2. The data was fitted using Eq. (3) and

the change in enthalpy and entropy was determined (see

Table 3). Figure 4 represents the (mole fraction sterols vs.

temperature) phase diagram of the tubule formation in differ-

ent oils. Here, the phase transition lines of the aggregation

process are plotted. The enthalpy and entropy changes of

the aggregation process in all oils were negative (i.e. DH0<0

and DS0<0, see Table 3) in line with the expectation that

the tubule formation is an enthalpy driven process. The

results also show that the changes in enthalpy DH0 and

entropy DS0 decrease with decreasing oil polarity (see

Fig. 5). For example, with decane (the lowest polarity among

the oils used, see Table 1 for dielectric constant) the DH0 and

DS0 were at least five times lower compared to eugenol (the

most polar oil used). This is in line with the change in Gibbs

free energy DG0 which also decreases with decreasing the

polarity of the oil indicating that the tubules (and resulting

gels) are more thermodynamically stable in oils with lower

polarity.

4 Conclusions

The effect of oil phase polarity on tubule formation by

g-oryzanol/b-sitosterol mixtures was investigated. The

g-oryzanol and b-sitosterol monomers exhibited self-assem-

bly and a CAC depending on the type of oil. The self-assem-

bly was found to be enthalpy driven where both DH0 and DS0

are negative. The values of DG0, DH0, and DS0 related to the

self-assembly were dependent on the polarity of the oils; the

lower the polarity, the lowerDG0,DH0, andDS0. The stability

against temperature changes of the tubules (and resulting

gels) increases with decreasing the polarity of the oil.

The main conclusion of this study is that polarity and

chemical structure of the oil phase play an important role in

the aggregate formation by sterols molecules in organogel

systems.

The authors have declared no conflict of interest.

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-3

-2

-1

00.001 0.002 0.003 0.004

ln(x

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