Journal of Functional Foods 39 (2017) 250–256

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Journal of Functional Foods

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Rice bran wax oleogel: A potential margarine replacement and its MARK
digestibility effect in rats fed a high-fat diet
⁎
Wanwanut Limpimwonga, Thanutchaporn Kumrungseeb, , Norihisa Katob, Noriyuki Yanakab,
Masubon Thongngama
a
Department of Food Science and Technology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand
b
Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: Rice bran wax (RBW)–rice bran oil (RBO) oleogel is an alternative structured fat used to replace saturated and
Oleogel trans fats. Our study investigated the effects of the RBW crystal network structure of oleogel on lipid digestibility
Rice bran wax compared with margarine (MG) and beef tallow. After high-fat diet feeding for 4 weeks, the RBW–RBO oleogel
Crystal network group showed a decrease in adipose tissue accumulation, triacylglycerol (TAG) levels in serum and liver and
Lipid digestibility
total cholesterol (TC) in liver, an increase in excreted TAG, TC and bile acid contents in faeces, and reduced
Hypolipidaemic activity
Hypocholesterolaemic activity
alanine aminotransferase and total bilirubin levels compared with the control and MG groups. Oleogel decreased
TAG levels by around 30% in serum and liver and increased excreted TAG levels by 30% in faeces compared with
rats fed the individual components (RBW and RBO). These results suggest that the gel network is a key con-
tributor to the decrease in lipid digestibility.

1. Introduction
It is well documented that a high intake of trans and saturated fatty
acids (TFAs and SFAs) can lead to diseases such as cardiovascular dis-
ease (CVD), obesity, type 2 diabetes mellitus and hypertension
(Estadella et al., 2013). In both Western and developing countries, diets
rich in fat are associated with such diseases, especially obesity and
CVD, which is the number one cause of death globally (WHO, 2017).
Therefore, according to the American Heart Association, reducing
consumption of TFAs and SFAs, but increasing consumption of un-
saturated fatty acids (USFAs) from vegetable oils is recommended
(Lichtenstein et al., 2006). Moreover, the requirement to label the TFA
content in food products effected in 2006 by the US Food and Drug
Administration increased public awareness and forced food manu-
facturers to eliminate TFAs and reduce SFAs from their products (Doell,
Folmer, Lee, Honigfort, & Carberry, 2012). However, replacing TFAs
and SFAs with oils rich in USFAs can affect product quality, since
shortening, margarine (MG), butter, lard and beef tallow generally
provide a colloidal structure of fat crystals and other desirable func-
tionalities in food products (Rogers, 2009).
Oleogelation is a novel and effective technology to structure liquid
oil into semisolid fat (Marangoni & Garti, 2011). In recent years, this
technology has been applied to the food industry in an attempt to
produce oleogels as novel replacements of conventional shortenings
rich in TFAs and SFAs (Rogers, 2009). Oleogels can be made by the
addition of oleogelators such as 12-hydroxystearic acid, ethylcellulose
and rice bran wax (RBW) at low concentrations (< 10%) to oils such as
canola oil and rice bran oil (RBO) (Dassanayake, Kodali, Ueno, & Sato,
2012; Martins, Cerqueira, Fasolin, Cunha, & Vicente, 2016; Tavernier
et al., 2017). The gelator–oil mixture is heated to above the melting
temperature of the used gelator to completely dissolve it and the mix-
ture is left to cool down. During the cooling process, the gelator forms a
thermoreversible three-dimensional gel network that traps liquid oil,
thereby changing the mixture from a liquid to a semisolid state, called
an oleogel (Dassanayake et al., 2012). In addition, depending on the
different types of gelators used, oleogel structures can be divided into
three groups: crystalline particle network, self-assembled fibrillar net-
work and polymer network (Marangoni & Garti, 2011). RBW can act as
a gelator forming a crystalline particle network oleogel (β′-form)
(Dassanayake et al., 2012). RBW crystals formed in oleogels have a
long, needle-like or platelet-like shape (the most desirable gelation
shape) and can form a dense crystal network by van der Waals inter-
actions as gelator–gelator interact and enable the entrapment of a large
volume of liquid oil within its structure (Dassanayake et al., 2012).
Although van der Waals interactions are weaker than hydrogen bonds,
Lupi et al. (2016) reported that the large number of van der Waals
interactions among alkyl chains of long-chain fatty alcohols could
produce a stronger gel than monoacylglycerol, which mainly comprises

⁎
Corresponding author at: Thanutchaporn Kumrungsee, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Japan.
E-mail address: kumrung@hiroshima-u.ac.jp (T. Kumrungsee).

http://dx.doi.org/10.1016/j.jff.2017.10.035
Received 31 July 2017; Received in revised form 17 October 2017; Accepted 17 October 2017
Available online 02 November 2017
1756-4646/ © 2017 Elsevier Ltd. All rights reserved.
W. Limpimwong et al. Journal of Functional Foods 39 (2017) 250–256

hydrogen bonding. In addition, longer wax ester chain increases the 2. Materials and methods
thermal stability of oleogel (Hwang, Kim, Singh, Winkler-Moser, & Liu,
2012). Owing to the edible property and general application in food 2.1. Materials
products, this research focused on oleogel prepared using RBW and
RBO, in which RBW is an efficient oleogelator (Blake, Co, & Marangoni, RBW (melting point: 77–82 °C, acid value: ≤13, saponification
2014; Dassanayake et al., 2012). In addition, RBW is a potent agri- value: 75–120, iodine value: ≤20, ester content: 92–97%, FFA: 0–2%,
cultural by-product (Vali, Ju, Kaimal, & Chern, 2005), while RBO is free fatty alcohol: 0%, hydrocarbon: 0% and resins/others: 3–8%) was
reported to be rich in USFAs and phytochemical compounds, including purchased from Koster Keunen Inc. (Connecticut, USA). RBO containing
γ-oryzanol, tocols and phytosterols (Cicero & Derosa, 2005; Perez- γ-oryzanol (> 2500 ppm), phytosterols (> 15,000 ppm) and vitamin E
Ternero, Alvarez de Sotomayor, & Dolores Herrera, 2017). Conse- (tocopherols and tocotrienols) was received from Thai Edible Oil Co.,
quently, the use of RBW and RBO for oleogel production could have Ltd. (Samutprakarn, Thailand). Cholesterol-free MG containing 81.7%
health and economic benefits. fat, produced from refined palm oil and refined coconut oil (55% and
Oleogels consist of lipid-based materials (Marangoni & Garti, 2011), 27%, respectively), was purchased from Three Top Chemical & Foods
so it is of interest to assess their lipid digestibility. Principally, most Corp., Ltd. (Samutsakorn, Thailand). Beef tallow was purchased from
triacylglycerol (TAG) (95%) is absorbed within the gastrointestinal Nacalai Tesque Inc. (Kyoto, Japan).
tract and the rest (5%) enters the colon and is excreted in the faeces
(Jackson & McLaughlin, 2009). Lipid digestion takes place in the sto- 2.2. RBW–RBO oleogel preparation
mach and small intestine. Gastric lipase is responsible for approxi-
mately 10–30% of lipolysis, hydrolysing TAG to free fatty acids (FFAs) The oleogel containing 5.0% (w/w) RBW was prepared by dissol-
and diacylglycerols, and the remaining lipids are consequently digested ving RBW in RBO at 90–100 °C in a water bath for 20 min to completely
by pancreatic lipase, resulting in FFAs and monoacylglycerols dissolve the RBW. The mixture was cooled to 25 °C using a circulating
(Wilde & Chu, 2011). Factors affecting lipolysis include composition, water bath to solidify the mixture. After the RBW–RBO oleogel was
physical state and droplet surface area of lipid droplets as well as the formed during the cooling stage, it was kept at 25 °C overnight before
structure of the matrix surrounding the lipid phase (McClements, mixing with other ingredients (Table 1) to make the diet (Dassanayake
Decker, & Park, 2009). Therefore, the physicochemical properties of et al., 2012). We used 5.0% (w/w) RBW as at this concentration there
oleogels, including structure, composition (type and concentration of was no distinct wax feeling in the model cake used and it retained
gelators) and mechanical strength have a great effect on the rate of lipid desirable cake properties (unpublished data).
hydrolysis (O'Sullivan, Barbut, & Marangoni, 2016). To date, the
number of studies focusing on digestion of oleogels is limited. In vitro 2.3. Animals and experimental design
studies of oleogel digestibility suggest that the structure and strength of
oleogel networks, such as ethylcellulose oleogel, has suppressive effects A total of 32 male Sprague–Dawley rats (3 weeks old; 40–50 g body
on the rate of enzymatic lipid digestion (O'Sullivan, Davidovich-Pinhas, weight) were purchased from the Hiroshima Laboratory Animal Centre
Wright, Barbut, & Marangoni, 2017). In human study, a single oral in- (Hiroshima, Japan) and maintained according to the “Guide for the
gestion of a test meal containing monoacylglycerol oleogel showed that Care and Use of Laboratory Animals” established by Hiroshima
oleogel lowered lipid digestibility, and levels of TAG and FFA of oleogel University and approved by the Ethics Committee of the university
groups were lower compared with the control group, possibly due to (Ethical Approval No. C15-12). The rats were housed individually in
modification of the physical structure of the oleogels (from liquid oil to stainless steel cages in a windowless and air-conditioned room
semisolid fat) (Marangoni et al., 2007). Although there are some in vitro (23–24 °C) under a 12 h/12 h light/dark cycle. After acclimatisation
and human studies of oleogel digestibility as mentioned above, there with a non-purified commercial rodent diet (MF, Oriental Yeast, Tokyo)
are no studies of RBW–RBO oleogels as well as in vivo studies over a for 7 days, the rats were fasted overnight, weighed and randomly di-
long-term feeding period. vided into four homogeneous groups of eight rats each, and fed high-fat
Wax esters, including RBW, are long-chain fatty alcohols esterified diets for 4 weeks consisting of different fat sources: beef tallow (con-
with long-chain fatty acids (Vali et al., 2005), which are known to be trol), MG, 5.0% (w/w) RBW–RBO oleogel and individual components
resistant to lipase hydrolysis and poorly digested by many mammals, (5.0% RBW and 95.0% RBO, w/w). The four high-fat diets were
including humans (Hargrove, Greenspan, & Hartle, 2004; Ling,
Nichols, & But, 2009). As the presence of a structuring or crystal net- Table 1
work in the lipid phase could slow the release of oil to the oil–water The composition of experimental high-fat diets containing 30% fat content.
interface where digestive enzymes are present, delaying the exposure of
Constituent Experimental diets (%, w/w)
the oil to the enzymes (O'Sullivan, Barbut, & Marangoni, 2016), the
RBW crystal network in the lipid phase of oleogel may influence lipid Control MG Oleogel RBW + RBO
digestion profiles. We hypothesised that the RBW crystal network in
oleogel may have the ability to limit the accessibility of lipase to oil Beef tallow 30.0 – – –
trapped within the network, leading to a lower lipid digestibility MG – 36.6c – –
Oleogel – – 30.0 –
compared with MG and beef tallow (control), which were structured by RBO – – – 28.5
the TFA and SFA networks. RBW – – – 1.5
Therefore, the objectives of this study were: (1) to investigate the Caseina 20.0 20.0 20.0 20.0
effect of different dietary fat sources, including RBW–RBO oleogel, MG L-cystine 0.3 0.3 0.3 0.3
Vitamin mixtureb 1.0 1.0 1.0 1.0
(a commercial fat for bakery products structured by a network of TFAs
Mineral mixtureb 3.5 3.5 3.5 3.5
and SFAs), and beef tallow (a fat source used in a high-fat diet-induced Cellulose 5.0 5.0 5.0 5.0
obesity rat model as a control group), on the digestion, absorption and Sucrose 20.0 20.0 20.0 20.0
excretion of lipid; and (2) to determine whether the RBW crystal net- Corn starch 20.2 20.2 20.2 20.2
work structure of the oleogel could affect the lipid digestibility com-
MG, margarine; RBO, rice bran oil; RBW, rice bran wax.
pared with the individual components (RBW and RBO) using an in vivo a
Casein: net protein content, 87% (w/w).
rat model. A high-fat diet-induced obesity rat model was used to re- b
American Institute for Nutrition-93.
present the dietary habits of food rich in fat and to assess the anti- c
MG containing 81.7% fat content was added with more content than others in order
obesity effects of the oleogel. to get the total 30% fat content in the diet.

251
W. Limpimwong et al.
prepared by mixing the dried ingredients (Table 1), then adding the fats
(beef tallow, MG, RBW–RBO oleogel and RBO) without melting and
manually mixing until the diets were homogenous. Diets were stored at
4 °C to maintain the oleogel in a semisolid state. The amount of each
high-fat diet formula was increased at weekly intervals to 15 (week 1),
17 (week 2), 19 (week 3) and 20 g/day (week 4). Food intake was
monitored daily, and the animals’ body weight was recorded two times
per week throughout the feeding period. Faeces were collected during
the last two days. At the end of the feeding period, all rats were sa-
crificed by decapitation under isoflurane anaesthesia (between 01:00
p.m. and 03:00 p.m.) after removal of food (08:00 a.m.). Trunk blood
from each rat was collected in a glass tube immediately after decap-
itation. Serum was obtained by centrifugation at 2000g for 20 min and
stored at −80 °C. Liver and adipose tissue were removed immediately,
weighed and stored at −80 °C until analysed. The percentage of tissue
weights was calculated as [tissue weight (g)/final body weight (g)] ×
100.
2.4. Blood parameter analysis
The blood parameters measured in this study were chosen mainly to
assess lipid profile, glucose levels, liver function and tissue damage.
TAG, total cholesterol (TC), FFA, high-density lipoprotein cholesterol
(HDLC) and low-density lipoprotein cholesterol (LDLC) were lipid
parameters, while aspartate aminotransferase (AST), alanine amino-
transferase (ALT), gamma-glutamyltransferase (γ-GT) and total bilir-
ubin were liver function parameters, and lactate dehydrogenase (LDH)
was a tissue damage marker. TAG and TC concentrations were en-
zymatically quantified using commercial enzymatic kits (Wako Pure
Chemicals, Osaka, Japan). HDLC, LDLC, FFA, glucose, AST, ALT, γ-GT,
LDH and total bilirubin levels were determined using a Beckman
Coulter AU480 analyser (Beckman Coulter, Krefeld, Germany), which is
an automated chemistry instrument for turbidimetric, spectro-
photometric and ion-selective electrode measurements. Briefly, 200 μL
serum was used to measure these parameters according to the manu-
facturer’s protocol.
2.5. Hepatic lipid analysis
Lipid was extracted from liver using a modified Folch method.
Briefly, fresh liver (500 mg) was minced and homogenised in 2.0 mL of
0.14 M KCl solution for 2 min. Then, 0.5 mL of homogenised solution
was added into 9.5 mL of chloroform/methanol (2:1, v/v) and shaken at
4 °C overnight prior to filtering. The filtrate (3.5 mL) was mixed with
0.75 mL of 0.02% CaCl2 solution and centrifuged at 2000g for 10 min.
The mixture was separated into two phases; the upper phase was re-
moved and the lower phase was collected and mixed with 0.75 mL of
chloroform:methanol:0.02% CaCl2 (3:48:47, v/v) then centrifuged at
2000g for 10 min. The lower phase was then collected, diluted with
1.0 mL of methanol and 2.0 mL of chloroform:methanol (2:1, v/v), and
evaporated at 60 °C (Folch et al., 1957). The samples were dissolved in
0.09 mL of tert-butanol and 0.06 mL of triton X-100:methanol (1:1, v/v)
before quantifying the hepatic TAG, TC and free cholesterol (FC) con-
centrations using commercial enzymatic kits (Wako Pure Chemicals,
Osaka, Japan) (Danno, Jincho, Budiyanto, Furukawa, & Kimura, 1992).
2.6. Faecal lipid analysis
Wet faeces were freeze-dried overnight and ground. Dried faeces
(0.250 g each) were used for lipid extraction according to the method
used in the hepatic lipid extraction described above. Faecal TAG and TC
concentrations were measured using commercial enzymatic kits (Wako
Pure Chemicals, Osaka, Japan).
Faecal bile acids (faecal acidic steroids) were extracted from the
dried faeces with hot ethanol (70 °C) for 2 h with shaking then cen-
trifuged at 2000g for 10 min. The supernatant was collected for faecal
252
Journal of Functional Foods 39 (2017) 250–256
bile acid measurement using commercial enzymatic kits (Wako Pure
Chemicals, Osaka, Japan).
2.7. Statistical analysis
All data were analysed using the Statistical Package for the Social
Sciences programme version 17.0. To measure the effect of fat sources,
analysis of variance was performed with the significance at p < .05,
and the significant differences among mean values was determined by
Tukey honest significant difference test. To determine the effects of the
RBW structure on lipid digestibility, an independent-samples t-test was
performed with the significance at p < .05 and p < .01. Pearson’s test
was carried out to determine correlations between the analysed para-
meters.
3. Results and discussion
3.1. Effects of fat sources on lipid digestibility
For 4 weeks, rats were fed one of three high-fat diets consisting of
different fat sources: beef tallow (control), MG and RBW–RBO oleogel.
The control group was fed beef tallow (30%), a fat usually used in a
high-fat diet-induced obesity model. This was used to observe the anti-
obesity effects of a commercial semisolid fat for bakery production
(MG) and a novel alternative semisolid fat (RBW–RBO oleogel). The
percentage of SFA, monounsaturated (MUFA), polyunsaturated fatty
acids (PUFA) and TFAs of fat sources is shown in Table 2.
3.1.1. Growth, food intake, dried faeces and tissue weights of rats fed high-
fat diets
As shown in Table 3, the final body weight, body weight gain and
food intake did not differ among the three groups. Liver tissue weights
of the three groups were also not significantly different. The epididymal
adipose tissue weight of the oleogel group was significantly lower than
that of the control group (p < .05), but was not different from the MG
group. The perirenal and total adipose tissue weight of the oleogel
group were significantly lower than those of the control and MG groups
(p < .05). Interestingly, the dried faeces weight of the oleogel group
was significantly higher than that of the control and MG groups
(p < .05). Due to similar weight gain resulting from all three high-fat
diets, this suggested that there were no anti-obesity effects observed.
The dried faeces weight from rats in the oleogel group was higher than
in the other groups. This might be due to the crystal network from RBW
being able to reduce lipid digestion and absorption, leading to the ex-
cretion of lipid in faeces. A previous study showed RBW and wax esters
were able to resist lipase hydrolysis (Hargrove et al., 2004; Ling et al.,
2009); therefore, it is plausible that the increase in dried faeces weight
might have resulted from unabsorbed or undigested fat or wax ester.
However, for future studies, the feeding period should be extended
(> 4 weeks) to conclusively examine anti-obesity effects, as the present
Table 2
The percentage of saturated, monounsaturated, polyunsaturated and trans fatty acid of fat
sources.
Beef tallowa MGb RBOc
SFAs 59.4 69.9 23.6
MUFAs 37.2 23.1 43.5
PUFAs 3.4 7.0 32.9
TFAs 3.9 2.9 0.6
MG, margarine; RBO, rice bran oil; SFAs, saturated fatty acids; MUFAs, monounsaturated
fatty acids; PUFAs, polyunsaturated fatty acids; TFAs, trans fatty acids.
a
Source: Yılmaz and Dağlıoğlu (2003).
b
Sample was analysed by Institute of Food Research and Product Development at
Kasetsart University.
c
Source: Thai Edible oil CO., Ltd.
W. Limpimwong et al. Journal of Functional Foods 39 (2017) 250–256

Table 3 respectively). These results suggested that the oleogel had the potency
Growth, food intake and tissue weights of rats fed high-fat diets. to lower the lipid digestibility of the diet. Our results are in agreement
with previous clinical studies that showed that monoacylglycerol gel-
Control MG Oleogel
based emulsion resulted in lower post-prandial TAG and FFA levels in
Initial body wt (g) 109 ± 2 107 ± 3 106 ± 2 blood compared with an oil–water mixture (Marangoni et al., 2007),
Final body wt (g) 358 ± 4 356 ± 7 356 ± 6 and the post-prandial responses of serum TAG and FFA levels in a 12-
Body weight gain (g/4 wk) 249 ± 5 249 ± 5 250 ± 5
hydroxystearic acid oleogel group were significant lower than those in
Food intake (g/4 wk) 591 ± 1 591 ± 4 590 ± 1
Liver wt (g) 15.0 ± 0.4 15.9 ± 0.6 15.0 ± 0.5 butter and MG groups (Hughes, Rush, & Marangoni, 2011). Taken to-
Liver wt (%) 4.20 ± 0.12 4.46 ± 0.11 4.21 ± 0.10 gether, the results shown in Table 3 and Fig. 1 imply that the oleogel
Epididymal adipose tissue (a, g) 3.21 ± 0.20b 3.00 ± 0.27ab 2.39 ± 0.12a could lower lipid digestibility and subsequently decrease fat accumu-
Epididymal adipose tissue (a, 0.90 ± 0.06b 0.84 ± 0.06ab 0.67 ± 0.03a lation by decreasing adipose tissue and increasing lipid excretion in
%)
faeces. The hypolipidaemic effects of the oleogel could be attributed to
Perirenal adipose tissue (b, g) 4.30 ± 0.18b 4.14 ± 0.40b 3.18 ± 0.16a
Perirenal adipose tissue (b, %) 1.20 ± 0.04b 1.16 ± 0.09b 0.89 ± 0.03a three possible factors including fatty acid composition of the fat
Total adipose tissue (a + b, g) 7.51 ± 0.18b 7.15 ± 0.63b 5.58 ± 0.22a sources, phytochemical compounds in RBO and the structure of the
Total adipose tissue (a + b, %) 2.10 ± 0.03b 1.99 ± 0.14b 1.56 ± 0.04a oleogel.
Dried faeces (g/2 d) 3.29 ± 0.16a 3.33 ± 0.11a 4.57 ± 0.15b
Previous studies showed that the oleogel network could limit the
All values are expressed as means ± SE (n = 6–8 rats/group).
release of liquid oil within the network and restrict access of lipase to
Means with different superscript are significantly different by Tukey honest significant the entrapped oil (Duffy et al., 2009; O'Sullivan, Davidovich-Pinhas,
difference test (p < .05). MG, margarine. Wright, Barbut, & Marangoni, 2017). In addition, as mentioned earlier,
the crystal network of RBW could also be resistant to lipase (Hargrove
study showed possible anti-obesity effects, such as low adipose tissue et al., 2004; Ling et al., 2009); therefore, it could not be digested and
weight and high faeces excretion in the oleogel group. absorbed, unlike the beef tallow (control) and MG that could be easily
hydrolysed by lipase. Thus, this could explain the lower lipid digestion
of the oleogel than the control and MG. In addition, it has been reported
3.1.2. FFA and TAG profiles
that USFAs and phytochemical compounds such as γ-oryzanol in oils
The oleogel group showed a decrease in serum FFA levels compared
play important roles in reducing serum and liver TAG and lipid ab-
with the control group (p < .10), but showed no such trend compared
sorption (Cicero & Derosa, 2005). As RBO is rich in MUFAs, PUFAs
with the MG group (Fig. 1). TAG levels in serum and liver of the oleogel
(Table 2) and oryzanol (Cicero & Derosa, 2005), it could be assumed
group were significantly lower than those of the control and MG groups
that the hypolipidaemic effects of the oleogel might also be partly at-
(serum: reduced by 57% and 56%, respectively, p < .05; liver: reduced
tributable to its fatty acid composition and phytochemical compounds.
by 54% and 44%, respectively, p < .05). In contrast, TAG levels in the
faeces of the oleogel group were significantly higher those that of the
control and MG groups (increased by 127% and 115%, p < .05,

Fig. 1. The levels of FFA in serum and TAG in serum, liver and faeces of rats fed high-fat diets with beef tallow (control), MG and RBW–RBO oleogel. Error bars indicate the SE of the
mean for n = 6–8 rats/group, and letters indicate significant difference between values by Tukey honest significant difference test (p < .05). MG, margarine; FFA, free fatty acid; TAG,
triacylglycerol.

253
W. Limpimwong et al. Journal of Functional Foods 39 (2017) 250–256

Table 4 Table 5
Effect of dietary fats on serum, liver and faeces parameters. Biochemical serum parameters related to liver and tissue damages.

Control MG Oleogel Control MG Oleogel

Serum AST (U/L) 127 ± 8 136 ± 10 118 ± 5

TC (mmol/L) 1.57 ± 0.06 1.44 ± 0.10 1.46 ± 0.07 ALT (U/L) 46.0 ± 1.8b 43.3 ± 2.8b 35.0 ± 0.6a
HDLC (mmol/L) 1.11 ± 0.03 1.17 ± 0.07 1.28 ± 0.06 γ-GT (U/L) 0.35 ± 0.08 0.20 ± 0.14 0.16 ± 0.08
LDLC (mmol/L) 0.27 ± 0.03 0.26 ± 0.02 0.33 ± 0.02 LDH (U/L) 501 ± 53 561 ± 56 442 ± 25
Glucose (mmol/L) 11.0 ± 0.1 10.9 ± 0.2 10.8 ± 0.2 Total bilirubin (µmol/L) 1.37 ± 0.09b 1.20 ± 0.05b 0.97 ± 0.04a

Liver
All values are expressed as means ± SE (n = 6–8 rats/group).
TC (µmol/g liver) 9.61 ± 0.80b 5.86 ± 0.31a 5.69 ± 0.30a
FC (µmol/g liver) 4.62 ± 0.27 4.53 ± 0.28 3.68 ± 0.29 Means with different superscript are significantly different by Tukey honest significant
difference test (p < .05). MG, margarine; AST, aspartate aminotransferase; ALT, alanine
Faeces aminotransferase; γ-GT, gamma-glutamyltransferase; LDH, lactate dehydrogenase.
TC (µmol/g dried faeces) 23 ± 2a 21 ± 1a 104 ± 6b
Bile acids (µmol/g dried faeces) 4.11 ± 0.30ab 3.37 ± 0.29a 5.05 ± 0.31b
acids excreted in the faeces. The increase in excreted TAG, TC and bile
All values are expressed as means ± SE (n = 6–8 rats/group). acid could also confirm the hypothesis of the digestion resistance of the
Means with different superscript are significantly different by Tukey honest significant oleogel compared with the control (beef tallow) and MG.
difference test (p < .05). MG, margarine; TC, total cholesterol; HDLC, High-density li-
poprotein cholesterol; LDLC, low-density lipoprotein cholesterol; FC, free cholesterol.
3.1.4. Biochemical indicators of liver and tissue function
As shown in Table 5, serum levels of AST, γ-GT and LDH were not
3.1.3. Serum, liver and faecal cholesterol profiles, serum glucose and faecal
significantly different among the three groups. The serum levels of ALT
bile acids
and total bilirubin in the oleogel group were significantly lower than in
As shown in Table 4, serum levels of HDLC, LDLC and glucose did
the control and MG groups (ALT: reduced by 24% and 19%, respec-
not differ among the three groups. Levels of serum TC were not sig-
tively, p < .05; and total bilirubin: reduced by 29% and 19%, respec-
nificantly different among the three groups, but the levels of liver TC
tively, p < .05). These results suggested that the oleogel might have
were significantly lower in the oleogel and MG groups than in the
beneficial effects on liver function by reducing ALT and total bilirubin
control group (reduced by 41% and 39%, respectively, p < .05). In-
levels in serum. As shown in Table 2, beef tallow and MG contained
terestingly, the faecal TC level of the oleogel group was strikingly
higher levels of TFAs and SFAs than RBO. It could be assumed that TFAs
higher than that of the control and MG groups (increased by 352% and
and SFAs in beef tallow and MG playing an important role to increase
395%, respectively, p < .05). The TC profiles were consistent with the
liver damage parameters, ALT and total bilirubin, compared with RBO
TAG profiles (Fig. 1), in which the oleogel reduced the accumulation of
that has significantly lower levels of TFAs and SFAs. This assumption is
TC and TAG, but increased the excretion of both in faeces. Cholesterol is
supported by other studies demonstrating that dietary TFAs and SFAs
a product of lipid digestion (Bauer, Jakob, & Mosenthin, 2005); there-
increased ALT and/or AST, indicating liver damage (Dhibi et al., 2011;
fore, low lipid digestion of the oleogel might have consequently con-
Machado et al., 2010; Obara et al., 2010). Furthermore, these studies
tributed to low levels of TC. In addition, the hypocholesterolaemic ef-
also suggested that high levels of dietary TFAs and SFAs led to hy-
fect of the oleogel might be partly attributed to phytosterols, γ-oryzanol
percholesterolaemia and hypertriacylglycerolaemia, which was corre-
and tocotrienols in RBO, which have been reported to inhibit dietary
lated with increased levels of liver damage. These results are in
and biliary cholesterol absorption and lower cholesterol synthesis in the
agreement with our results demonstrating that beef tallow and MG,
liver (Bhaskaragoud et al., 2016; Theriault, Chao, Wang,
with high amounts of TFAs and SFAs, led to an increase in TAG, TC and
Gapor, & Adeli, 1999). The TC-lowering effects of MG could be attrib-
ALT levels compared with RBO that contains a low amount of SFA and
uted to the bioactive compounds in plant oils (refined palm oil and
no TFA, but high amounts of USFAs, which are reported to play an
refined coconut oil) used in its production. Refined palm and coconut
important role in the protection of liver damage (Sullivan, 2010). In
oils contain tocotrienols and phenolics (May & Nesaretnam, 2014;
addition, antioxidative phytochemical compounds such as γ-oryzanol
Prasanth Kumar & Gopala Krishna, 2015), which were found to de-
and tocols (tocopherols and tocotrienols) in RBO may exert antioxidant
crease cholesterol synthesis in the liver (Kumarappan, Rao, & Mandal,
and anti-inflammatory effects in liver (Rana, Vadhera, & Soni, 2004;
2007; Theriault et al., 1999). The oleogel group showed a decreasing
Theriault et al., 1999).
trend of FC in the liver (p < .10) compared with the control group.
In the present study, the oleogel did not affect other indices of liver
Since the accumulation of FC in liver is related to liver damage
functions and tissue damage, such as serum AST, γ-GT and LDH. The
(Arguello, Balboa, Arrese, & Zanlungo, 2015), this FC lowering effect of
reason for this apparent lack of effect is unknown. It was reported that
the oleogel may suggest liver protective effects.
dietary TFAs, PUFAs and SFAs had no effects on ALT and AST levels in
Bile acid in the faeces of the oleogel group was higher than that of
mice fed a high-fat diet (Machado et al., 2010), while phytochemical
the MG group. This might be due to the fact that the phytosterols and γ-
compounds in RBO exhibited a decrease of AST levels with anti-lipid
oryzanol in RBO could interfere with bile acid reabsorption in the en-
peroxidative effects, but did not alter ALT levels in rats fed a high-
terohepatic circulation, leading to an enhancement of faecal bile acid
cholesterol diet (Ha et al., 2005). Recent accumulating evidence in-
excretion (Bhaskaragoud et al., 2016). Consequently, to maintain bile
dicates that the ratio of serum AST to ALT is affected by types of liver
acid levels in the enterohepatic circulation, cholesterol from the liver
disease as well as the time-course and aggressiveness of the disease
may have been used to synthesise bile acids (Maldonado-Valderrama,
(Botros & Sikaris, 2013). Therefore, it is of interest to examine the ef-
Wilde, Macierzanka, & Mackie, 2011). This may explain the negative
fects of oleogel and other fat sources on other animal disease models
correlation between the levels of bile acid in faeces and TC levels in
with extended feeding periods and disease aggressiveness as well as on
liver.
other anti-lipid peroxidative parameters.
Taken together, RBO in the oleogel might play an important role in
improving cholesterol pattern profiles by lowering the accumulation of
TC and enhancing excretion of TC and bile acids in faeces. These results 3.2. Effect of the RBW crystal network structure of oleogel on lipid
may help to confirm the speculation that the increased weight of dried digestibility
faeces in the oleogel group compared with the control and MG groups
might be partly due to undigested and unabsorbed TAG, TC and bile In this study, the structure of fat was fabricated using RBW and RBO
to change its state from liquid oil to semisolid fat. RBW was used as an

254
W. Limpimwong et al. Journal of Functional Foods 39 (2017) 250–256

Table 6 fine crumb structure. These results implied that the RBW crystal net-
Effect of the RBW crystal network structure of oleogel on adipose tissue weight and work in the oleogel still existed and was not completely destroyed by
serum, liver and faeces parameters.
mechanical force during the cake batter preparation, showing the
Individual components Oleogel RBW–RBO oleogel was still in a semisolid state and functioning as fat in
(RBW and RBO) the cake, similar to MG. According to other food application studies,
when oleogel was used as an MG substitution in bakery products, the
Adipose tissue
oleogel crystal network was still present in the product even after
Epididymal adipose 0.75 ± 0.05 0.67 ± 0.03
tissue (a, %) vigorous mixing. This implied that the RBW crystal network remained
Perirenal adipose tissue 1.03 ± 0.08 0.89 ± 0.03 intact after mixing (Hwang, Singh, & Lee, 2016). From our results, there
(b, %) are two possible mechanisms that could prevent or retard lipid diges-
Total adipose tissue 1.77 ± 0.12 1.56 ± 0.04 tion of oleogel. Firstly, the crystal network could limit accessibility of
(a + b, %)
lipase to RBO. Secondly, the crystal formation of the oleogel may play a
Serum role. As mentioned earlier, oleogel consists of needle-shaped crystals
FFA (µmol/L) 453 ± 44 363 ± 26
that form via van der Waals interaction between long hydrocarbon
TAG (mmol/L) 2.43 ± 0.18 1.59 ± 0.16** (−35%)
TC (mmol/L) 1.53 ± 0.09 1.46 ± 0.07 chains and polar ester groups (Dassanayake et al., 2012). These might
impact on the delay of lipolysis and/or reduce the lipid digestibility by
Liver
TAG (µmol/g liver) 37.1 ± 4.1 26.1 ± 2.5* (−30%) lipase hydrolysis.
TC (µmol/g liver) 5.85 ± 0.34 5.69 ± 0.30 Reduced lipid digestibility is an effective strategy to manage weight
Faeces
and reduce the risk of obesity via decreased calorie intake. Moreover,
TAG (µmol/g dried 3.02 ± 0.08 3.93 ± 0.38* (+30%) incomplete lipolysis allows the undigested fats to reach the ileum and
faeces) stimulate the ileal brake mechanism, which is able to modulate hunger,
TC (µmol/g dried 110 ± 6 104 ± 6 satiety and appetite, affect calorie intake and reduce the risk of obesity
faeces)
(Wilde, 2009). Therefore, it may be beneficial to decrease lipid di-
Bile acids (µmol/g dried 4.03 ± 0.40 5.05 ± 0.31
faeces) gestibility to avoid a deleterious impact on health.
Faecal bile acid levels in the oleogel group (Table 6) were slightly
All values are expressed as means ± SE (n = 8 rats/group). higher (25%) than the individual components group (p < .10). A sig-
Comparisons between groups were performed using independent-samples t-test. RBW, nificant positive correlation between the level of bile acids and excreted
rice bran wax; RBO, rice bran oil; FFA, free fatty acid; TAG, triacylglycerol; TC, total TAG level in faeces (r = 0.57, p < .05) was observed. Therefore,
cholesterol.
higher bile acid levels in the faeces of the oleogel-fed rats could be
* p < .05.
** p < .01. attributed to the crystal network of the oleogel that may reduce lipid
digestibility. Principally, bile acids play an important role in lipid di-
oleogelator to form a crystal network and hold RBO inside its network. gestion and absorption by emulsification of lipid droplets during in-
However, the RBW oleogel may have been unstable, since its crystal testinal lipid digestion, removal of lipolysis products from the surface of
network can be destroyed by mechanical force, leading to oil leakage lipid droplets by the formation of mixed micelles and transportation of
from the network. The oleogel group clearly showed lower lipid di- lipolysis products to be absorbed at the jejunal epithelium. The majority
gestibility compared with the control and MG groups. It was therefore of bile acid (about 95%) is reabsorbed at the distal ileum and circulated
investigated whether this effect could be attributed to the RBW crystal back to the liver (enterohepatic circulation), and the rest (5%) enters
network or the individual components (RBW and RBO). the colon and is excreted from the body (Maldonado-Valderrama et al.,
As shown in Table 6, the oleogel-fed rats showed strongly different 2011; Wilde & Chu, 2011). The increased bile acid levels of the oleogel
effects on TAG profiles in serum, liver and faeces compared with the group suggest it is possible that bile acids, which are emulsified on the
rats fed the individual components. In the oleogel group, serum and surface of undigested TAG, could pass through the small intestine and
liver TAG levels were reduced by about 30%, correlating with the in- be excreted with TAG from the body.
crease in excreted TAG level in faeces (about 30%) compared with the
group fed the individual components. Moreover, it showed that the 4. Conclusions
serum FFA levels of oleogel-fed rats was slightly lower (20%) than the
rats fed the individual components (p < .10). However, there were no RBW–RBO oleogel is a novel alternative structured fat that could
statistically significantly differences (p > .05) for TC in serum, liver effectively be utilised to replace SFAs/TFAs in food products. Our
and faeces, bile acids in faeces, and both types of adipose tissue weight findings contribute to the understanding of the effects of different
and total adipose tissue weight. These results also suggested that the dietary fat sources: 5.0% RBW–RBO oleogel, MG (a commercial semi-
oleogel could affect the rate of lipid digestion. solid fat for bakery products) and beef tallow (a fat used in the high-fat
Oleogelator–oleogelator and oleogelator–liquid oil interactions are diet-induced obesity rat model; control). The oleogel did not show anti-
required for the oil gelation to form a crystal network (Co & Marangoni, obesity effects, but showed hypolipidaemic, hypocholesterolaemic and
2012). Subsequently, liquid oil within the network can be divided into liver protective effects, in which the RBW crystal network structure and
two types: free oil and bound oil. Free oil is the oil trapped within the the health benefits of RBO are key contributors. Consequently,
crystal network, while bound oil is the oil interacting with the gelators RBW–RBO oleogel could be utilised as an alternative semisolid fat for
through intermolecular interactions (Co & Marangoni, 2012). When substitution of MG in bakery products with healthy and lower calorie
mechanical force was applied during diet mixing, the free oil could leak diets, and oleogels have recently been successfully demonstrated to
out from the oleogel structure and be easily digested by lipase. The replace commercial fats in bakery and food products such as cookies
different lipid profiles of the oleogel and individual component (RBW and ice cream (Hwang et al., 2016; Zulim Botega, Marangoni,
and RBO) groups (Table 6) suggested that the free oil was still partially Smith, & Goff, 2013). In a further study, in vivo experiments using RBW
trapped within the RBW crystal network, although RBW–RBO oleogel oleogel-incorporated food products will be necessary to investigate
was mixed with the diet and passed through the stomach and small their impact on lipid digestion profiles.
intestine. From our study (unpublished data), 5.0% RBW–RBO oleogel
could be used for MG substitution in cake as it acted as semisolid fat in Acknowledgements
the cake batter and exhibited similar cake textural properties, with a
This work was financially supported by the Capacity Building of

255
W. Limpimwong et al.
Kasetsart University Students on Internationalization Program,
Department of Food Science and Technology, Faculty of Agro-Industry,
Kasetsart University, Thailand and Funds for the Development of
Human Resources in Science and Technology” under MEXT, Japan
through the “Home for Innovative Researchers and Academic
Knowledge Users (HIRAKU)” consortium, Hiroshima University, Japan.
Conflict of interest
The authors declare that there are no conflicts of interest.
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