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J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 1 2 5 – 1 1 3 4
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Evaluation of a functional food preparation based on chitosanas a meal replacement diet
Nidal A. Qinnaa, Faisal T. Akayleha, Mayyas M. Al Remawib, Basma S. Kamonaa,Hashem Tahac, Adnan A. Badwanc,*
aFaculty of Pharmacy and Medical Sciences, Petra University, P.O. Box 961343, Amman, JordanbCollege of Pharmacy, Taif University, P.O. Box 888, Taif 21944, Saudi ArabiacDelass Natural Products Co., a subsidiary of the Jordanian Pharmaceutical Manufacturing Co., Plc., P.O. Box 94, Naor 11710, Jordan
A R T I C L E I N F O A B S T R A C T
Article history:
Received 9 October 2012
Received in revised form
7 March 2013
Accepted 11 March 2013
Available online 6 April 2013
Keywords:
Chitosan
Diet
Emulsion
Food
Meal replacement
Obesity
1756-4646/$ - see front matter � 2013 Elsevihttp://dx.doi.org/10.1016/j.jff.2013.03.009
* Corresponding author. Tel.: +962 6 5727 207E-mail address: crl@jpm.com.jo (A.A. Bad
The ability of chitosan to entrap large amounts of water when dispersed in an oily phase
was utilized to formulate a novel meal replacement functional food. Furthermore, the pro-
posed preparation can be fortified with nutrients. The purpose of this formulation was to
produce an edible low calorie pseudo-fatty rich meal that can enhance the feeling of satiety
when ingested. Different concentrations of chitosan and pectin were tested to find out a
stable preparation with acceptable physical characteristics. It was found that a preparation
containing 1% chitosan and 6% pectin is suitable to be consumed as a meal replacement
diet. The safety of such preparation was assessed by repeated dose administration to rats.
A set of other in vivo experiments was performed to assess the ability of this preparation to
enhance satiety. The ingestion of chitosan preparation resulted in reduced body weight,
food and water intake, and reduced faecal excretion in the emulsion administered rats
(p < 0.05). Furthermore, serum lipids of tested rats were not essentially changed. Accord-
ingly, the investigated chitosan emulsion could be introduced as a low calorie, relatively
stable and a safe functional food preparation for enhancing satiety when ingested as a
meal replacement diet.
� 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Obesity is recognized as a worldwide heath problem that is on
the rise in both adults and children (Ogden et al., 2006;
Stewart, Reilly, & Hughes, 2009). Such problem has been asso-
ciated with a range of physical and psychological symptoms,
therefore many obesity treatment guidelines have been con-
sidered (Clapham, Arch, & Tadayyon, 2001; Ogden & Flanagan,
2008). Lifestyle interventions to achieve weight loss comprise
dietary control and increased physical activity. This is accom-
panied with modification of food intake habits to reinforce
weight reduction behaviours (Cannon & Kumar, 2009). Dietary
control generally focuses on inducing a state of negative
er Ltd. All rights reserved
; fax: +962 6 5727 641.wan).
energy balance by cutting down the caloric intake from diet.
Nevertheless, overcoming hunger feeling in controlling diet
intake is considered a challenge. Hunger is often referred to
as a set of intestinal and stomach contractions described as
emptiness. However, considerable cross talk between the
gut and the brain has been reported as a so-called gut–brain
messages that can serve to stimulate or inhibit feeding
(Schellekens, Dinan, & Cryan, 2009; Swartz, Duca, & Covasa,
2009). Moreover, the brain centrally controls the state of
hunger and satiety through numerous molecular targets
(Clapham et al., 2001).
Unfortunately, most of the developed centrally acting
drugs for treating obesity possess a high ratio of ‘‘Risk over
.
1126 J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 1 2 5 – 1 1 3 4
Benefit’’ that could lead to many unwanted effects, and there-
fore, alternative strategies other than therapy must be sought.
It has been recognized that meal replacement strategy is a
choice. Such functional food preparations are considered more
attractive than drugs and offer a safe alternative method for
controlling hunger and appetite during body weight reduction
(Clifton, 2008). Meal-replacement is a form of low-calorie diet
in which one or two full calorie meals daily are replaced with
a low-calorie snack that usually has reduced energy content
and consists most commonly of protein with a small amount
of fat and carbohydrate. These diets offer both a dietary change
and a behaviour modification strategy, and are popular among
people trying to lose weight (Heymsfield, 2010).
Repeatedly, marine resources have been recognized as rich
sources of structurally diverse biologically active compounds
such as bioactive peptides with great application potential in
marine functional foods (Harnedy & FitzGerald, 2012; Panges-
tuti & Kim, 2011). Chitosan and chitin, as functional biopoly-
mers from marine crustaceans, are well established safe food
and drug additives in many countries (Baldrick, 2009). Chito-
san is an N-deacetylated form of chitin (Sajomsang, 2010).
The free amine groups (–NH2) of chitosan are protonated as
(�NHþ3 ) in the acidic stomach. The appearance of these posi-
tive charges causes electrostatic attraction with negatively
charged fatty acids. Thus, dietary fat would be excreted in
the faeces rather than being absorbed. This made chitosan
as ingredient in many dietary weight-loss supplements
(Kanauchi, Deuchi, Imasato, Shizukuishi, & Kobayashi, 1995).
However, the ability of chitosan in reducing body weight
using chitosan fatty acid complexation is still debatable as
many contradictory results obtained from both human and
animal studies have been published (Yun, 2010). Nevertheless,
the ability of chitosan to form a hydrogel (Athamneh et al.,
2012) was the attractive property which initiated the current
investigation. Generally, hydrogel polymers are highly swol-
len and capable of forming networks that can absorb and ad-
sorb large amounts of water and drastically increase in
volume (Eftaiha et al., 2010). Consequently, the non-harmful
chitosan was previously used to entrap relatively high
amounts of water that was contained in a delivery system
composed of oleic acid as continuous phase (Athamneh
et al., 2012). Such principal can be used to introduce a low cal-
orie, water insoluble, anorexic and oral indigestible system
for enhancing satiety.
Therefore, the scope of the present study was to formulate
a stable emulsion which can be ingested as a meal replace-
ment functional food. This can fulfill the requirement to over-
come some of challenges associated with psychological and
cognitive therapeutic interventions for managing obesity
through enhancing satiety. The prepared emulsion was eval-
uated in terms of physical stability, safety and its efficacy in
weight reduction ability in rats.
2. Materials and methods
2.1. Materials
Oleic acid (cis-9-octadecenoic acid), hydrochloric acid (HCl)
and formaldehyde were purchased from Merck KGaA, Darms-
tadt, Germany. Sodium hydroxide (NaOH) and acetic anhy-
dride (C4H6O3) were purchased from Acros Organics, Fair
Lawn, NJ, USA. Olive oil was purchased from a local market
in Amman, Jordan. Citrus pectin was purchased from Cesalpi-
nia SpA, Bergamo, Italy. Sorbitol 70% (Sorbidex C) and glycer-
ine were purchased from Cargill Srl, Vigonza, Italy. Triton X-
100 was purchased from Sigma–Aldrich Corp., St. Louis, MO,
USA. All chemicals were used as received without any further
purification. Water was double distilled and deionized (mea-
sured conductance was less than 2 lS/cm).
2.2. Preparation and characterization of chitosan
Low-molecular-weight chitosan having a molecular weight of
30 kDa was prepared according to Elsayed, Remawi, Qinna,
Farouk, and Badwan (2009) by HCl acid depolymerization of
a high-molecular-weight chitosan (250 kDa, 93% DDA) having
an average particle size of 170 lm obtained from Hongjiu Gin-
seng Ltd., Dalian, China. Briefly, 10 g of the high-molecular-
weight chitosan was dissolved in 1 L of 2 M HCl. This solution
was heated on a magnetic stirrer hot plate (at 100 �C and
750 rpm) for 1 h under reflux. After cooling, the precipitate
was separated and washed several times with approximately
3 L of ethanol (96%, v/v) by centrifugation at 4000 rpm (3436g)
for 5 min (accuSpin� 3 centrifuge, Fisher Scientific, Schwerte,
Germany) until neutral pH (6.9–7) was reached and then
freeze dried for 24 h (Hetopower dry PL 9000 freeze drier,
Thermo Fisher Scientific, Inc., Waltham, MA, USA). The
molecular weight of the prepared chitosan was calculated
using a previously reported viscosity average molecular
weight (Mv) determination method for chitosan (Elsayed
et al., 2009).
In order to prepare an 80% DDA chitosan grade, the ob-
tained chitosan was dissolved in distilled water (1%, w/v)
and the pH was adjusted to 6.5 by dropwise addition of 6 M
NaOH. Chitosan solution was reacted with acetic anhydride
in a 1:1 M ratio (km), using magnetic stirring (500 rpm) at
room temperature for 10 min (Kubota, Tatsumoto, Sano, &
Toya, 2000). The reacted solution was then dialysed against
4 L of distilled water under gentle stirring at room tempera-
ture for 24 h using dialysis tubes having a molecular-weight
cut off of �30 kDa (Medicell International Ltd., London, UK).
Finally, the dialysed chitosan solution was poured in petri
dishes and dried overnight in an oven at 40 ± 3 �C and trans-
ferred to an amber-airtight glass bottle which was stored at
room temperature until used. The DDA of the prepared chito-
san was confirmed with a British Pharmacopoeia UV-spectro-
scopic method (B.P., 2007).
2.3. Preparation of the emulsified chitosan
The components of the chitosan emulsion are listed in Table 1.
Different amounts of chitosan HCl (30 kDa, 80% DDA) were
dissolved in 15 mL distilled water and their pH values were
adjusted to 6.5 by dropwise addition of 2 M NaOH solution.
Clear transparent solutions were obtained. The prepared
aqueous solutions were added dropwise with vigorous mixing
to an oily mixture of 2.5 g oleic acid and 5 g olive oil using a
Silverson homogenizer (model L4RT, Silverson Machines
Ltd., Chesham, UK) at 4000 rpm. Each formed dispersed
Table 1 – Composition of formulations.
Components (%) F01 F02 F03 F04 F05 F06
Chitosan 1 1 1 2 4 6
Oleic acid 2.5 2.5 2.5 2.5 2.5 2.5
Olive oil 5 5 5 5 5 5
Citrus pectin 3 6 9 6 6 6
Glycerine 15 15 15 15 15 15
Sorbitol 70% 17.5 17.5 17.5 17.5 17.5 17.5
Distilled water 56 53 50 52 60 48
Total weight (g) 100 100 100 100 100 100
J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 1 2 5 – 1 1 3 4 1127
system was left at room temperature until required. On the
other hand, different amounts of citrus pectin (3, 6 or 9 g)
along with 15 g of glycerine and 17.5 g of sorbitol were dis-
solved in the remaining amount of distilled water (quantity
sufficient to 100 g final emulsion weight) and gradually added
to the prepared dispersed system and homogenized until a
creamy viscous off-white paste was obtained.
2.4. In vitro evaluation of the prepared chitosan emulsions
2.4.1. Measurement of rheological properties of the formedemulsionsViscosities were determined at 25 �C with an MRC 301 cone and
plate viscometer, Anton Paar GmbH, Graz, Austria. The cone
used had an angle of 0.994� and a diameter of 50.005 mm.
The measurements were performed for different formulations
listed in Table 1 in triplicates over a shear rate range of approx-
imately 2–250 s�1 with a gap width of 0.05 mm.
2.4.2. Physical stabilitySamples of the emulsion formulations were kept in a refriger-
ator (4 �C), at room temperature and in an oven at 40 �C for 10
and 20 days, respectively. They were periodically tested for
physical changes like phase separation, development of objec-
tionable colour, odor, consistency and general appearance.
2.4.3. Stress stabilityStability of the formulated emulsions to centrifugation was
determined in test tubes filled with the prepared chitosan emul-
sions and centrifuged at 4000 rpm (3436g) for 20 min using accu-
Spin� 3 centrifuge, Fisher Scientific, Schwerte, Germany. The
volume of the separated phase was aspirated and recorded.
2.5. Evaluation of chitosan emulsion as a mealreplacement diet in rats
2.5.1. Animal handlingAdult male Sprague Dawley (SD) rats with an avarage weight
of 220 ± 20 g were purchased from Yarmouk University (Irbid,
Jordan) and accommodated at Petra University (Amman, Jor-
dan) animal house unit under standard temperature, humid-
ity and photoperiod light cycles. All rats were acclimatized for
10 days before experimentation date. All experiments were
carried out in accordance with the guidelines of the Federa-
tion of European Laboratory Animal Sciences Association
(FELASA). The study protocol was revised and approved by
the Ethical Committee of the Higher Research Council at the
Faculty of Pharmacy, Petra University (Amman, Jordan).
2.5.2. Safety evaluation of the prepared chitosan basedpreparationA repeated dose subacute safety study was conducted on rats
utilizing one oral chitosan preparation dose level of 3 mL/
250 g rat body weight. Adult male S.D rats were randomized
into two groups (n = 8). The first group was assigned as a con-
trol group receiving distilled water (3 mL/250 g) while the sec-
ond group was assigned as the test group fed orally with the
prepared dispersion system using a stainless steel oral gavage
needle. The body weights of all rats were recorded daily just
after feeding. The study lasted for 30 days were the rats were
checked twice daily for mortality, morbidity and any reported
behavioural changes.
At the end of the study period, all rats were sacrificed and
blood was collected from each rat by cardiac puncture. Whole
blood and serum samples were sent directly to a referral animal
biochemical laboratory (First Medical Labs, Amman, Jordan) and
subjected to complete blood count (CBC) analysis and serum
clinical assays listed in Table 2. Furthermore, gross anatomical
examination was performed for all animals. Liver and heart
biopsies (n = 2/group) fixed in formaldehyde (10%, v/v) were sec-
tioned, mounted on slides, and stained with hematoxylin and
eosin for histopathological examination under light microscope.
2.5.3. In vivo testing of the orally delivered chitosanbased preparation2.5.3.1. Body weight, food, water and faeces evaluationsprotocol. Ten adult male SD rats were randomized into two
groups placed in clean cages and served 100 g of food placed
in special dishes to reduce spillage and offered 250 mL of
clean tap water. Food intake, water consumption, weight of
faeces and body weights were recorded daily for each group
between 6:00 PM and 7:00 AM. At this time any spilled food
were collected, air-dried if necessary, and weighed. The col-
lected faeces were left at room temperature and weighed after
24 h of collection. The study was performed for 12 days di-
vided into two equivalent time periods namely; the Control
period and the Test period. At the Control period, both groups
were administered 3 mL/250 g body weight distilled water for
6 days. Later, one of the groups was switched to receive the
oral chitosan emulsion (3 mL/250 g) while the other group
(control group) continued to receive distilled water till the
end of the Test period.
Table 2 – Biochemical and haematological findings of rats treated with chitosan emulsion (F02) compared to control groupfollowing 30 days of administration. Results are expressed as mean ± SEM (n = 8). No statistically significant differenceswere seen in all tested parameters (p > 0.05). GPT, glutamate pyruvate transaminase; GOT, glutamic-oxaloacetictransaminase; GGT, gamma-glutamyl transpeptidase; ALP, alkaline phosphatase; RBC, red blood cells; HB, haemoglobin;WBC, white blood cells; MCV, mean corpuscular volume.
Parameter (Unit) Control group Chitosan emulsion group
Liver function tests GPT (IU/L) 41.6 ± 4 32.2 ± 2
GOT (IU/L) 202 ± 17.2 193.6 ± 19.8
GGT (IU/L) 0.9 ± 0.3 0.9 ± 0.2
ALP (IU/L) 164.1 ± 38 109.2 ± 19.9
Kidney function tests Creatinine (mg/dL) 0.5 ± 0.1 0.6 ± 0
Urea (mg/dL) 38.8 ± 2.9 42.5 ± 2.4
Glucose (mg/dL) 123.6 ± 15 95.8 ± 8.1
Serum lipids Cholesterol (mg/dL) 67.8 ± 5.4 60.8 ± 2.4
Triacylglycerols (mg/dL) 75.8 ± 6.5 111.1 ± 16.2
Serum proteins Total protein (g/dL) 8.5 ± 0.1 8.6 ± 0.1
Albumin (g/dL) 4.1 ± 0.1 4.2 ± 0.1
Blood analysis RBC (· 1012/L) 6.8 ± 0.4 7.2 ± 0.3
HB (g/dL) 13.6 ± 0.7 14.1 ± 0.6
WBC (· 106/L) 4.2 ± 1 3.6 ± 0.6
Neutrophils (%) 7.7 ± 1.2 7.5 ± 0.5
Lymphocytes (%) 72.5 ± 2.9 77.5 ± 1.1
Monocytes (%) 18.2 ± 1.6 14.8 ± 0.7
Platelets (· 109/L) 822.5 ± 200.3 945.2 ± 118.3
MCV (fL) 57.6 ± 0.7 56.3 ± 0.9
1128 J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 1 2 5 – 1 1 3 4
2.5.3.2. Biochemical assay of serum lipids. Rats were random-
ized into two groups (n = 6) where the first group received the
oral chitosan emulsion (3 mL/250 g body weight) once daily at
6:00 PM while a second control group was administered dis-
tilled water in the same manner. After seven days of treat-
ment, fasted rats (16 h) were sacrificed under ether
anaesthesia and blood was collected directly from the heart
by cardiac puncture after exploration of the thorax. Serum
samples were prepared and stored at �20 �C for lipid profile
analysis. Total cholesterol, triacylglycerols and low-density
lipoprotein (LDL) were determined by enzymatic methods
using Analyticon� Biotechnologies AG kits, Lichtenfels, Ger-
many, while high-density lipoprotein (HDL) was measured
by using Labkit�, Chemelex S.A., Barcelona, Spain. The absor-
bance was measured at 546 nm wavelength using RA-50
Chemistry Analyzer, Technicon-Bayer Healthcare, New York,
NY, USA.
2.5.3.3. Extraction and analysis of liver total cholesterol andtriacylglycerol. Liver lipids were extracted according to meth-
ods described by Haug and Hostmark (1987). At the end of the
experiment described in the previous section, all liver organs
were removed, weighed and kept at �20 �C until processed.
The liver (0.5 g sample) was homogenized with 5 mL of iso-
propanol and centrifuged for 15 min at 2500 rpm (601g) using
a Hettich EBA 20 centrifuge, Tuttlingen, Germany. The super-
natant was collected and subjected to lipid analysis. Total
cholesterol and triacylglycerols were measured as described
above.
2.5.3.4. Extraction and analysis of faecal lipids. Folch, Lees,
and Sloane Stanley (1957) method was used to extract lipids
from faeces where, at the last day of treatment, the faeces
were collected from the cages after 16 h of dose administra-
tions and dried in an oven at 45 �C for 24 h. The dry faeces
of each group were crushed into powder using a mortar and
pestle. Five samples of 0.5 g of dry faeces powder were ex-
tracted in glass tubes with 5 mL of chloroform: methanol
solution (2:1, v/v). The tubes were vigorously shacked for
30 s then sonicated for 5 min at room temperature. Later,
the tubes were centrifuged for 5 min at 5000 rpm (2404g)
and the supernatants were collected in clean tubes and dried
in an oven at 45 �C for 24 h. In order to analyze the faecal cho-
lesterol and triacylglycerol content, the dry tubes were recon-
stituted in 10% Triton X-100 dissolved in normal saline and
analyzed as described earlier.
2.6. Statistical analysis
The data are expressed as mean values ± standard error of
means (SEM). Student’s t-test was used for statistical evalua-
tion (SPSS 17, Chicago, IL, USA). p < 0.05 was considered
significant.
3. Results
3.1. Characterization and stability of the chitosan basedpreparation
Six runs of formulations were carried out to produce chitosan
preparations with varying percentages of pectin (F01, F02 and
F03) and chitosan (F04, F05 and F06). Based on the physical sta-
bility observations, F01 preparation showed extensive phase
separation that was detected both visually and after centrifu-
gation under all tested storage conditions. On the other hand,
F02 and F03 preparations were the most physically stable when
A
B
Fig. 1 – Photographs from the physical stability studies
showing (A) phase separation of F01 post centrifugation
compared to F02, (B) changes in colour and texture of
chitosan emulsions when different concentrations of
chitosan were used and after 20 days of storage at room
temperature. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web
version of this article.)
J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 1 2 5 – 1 1 3 4 1129
stored at room temperature and under refrigeration with no
evidence of phase separation under centrifugation (Fig. 1A).
No development of objectionable odor and colour changes or
microbial growth post 20 days of storage was observed for
these preparations. However, when stored at 40 �C for 20 days,
F02 and F03 preparations showed signs of physical instability
where phase separation was detected visually in the stored
samples even without centrifugation.
As for F04, F05 and F06 preparations, it was revealed that
increasing the amount of chitosan resulted in an intense
change in the colour and the texture of the preparations at
all tested storage conditions as illustrated in Fig. 1B without
any detected phase separation.
The increased amounts of chitosan and pectin in the pre-
pared systems clearly showed shear-thickening behaviour
where the apparent sheer stress increased with increasing
shear rate (Fig. 2A and B). Fig. 2C and D confirmed the shear
dependency of the tested preparations with higher viscosities
being observed with those containing higher percentages of
chitosan and pectin. As expected, the increase in the disper-
sion medium’s viscosity resulted in higher physical stability
of the prepared emulsions over different storage conditions.
It was concluded from the above physical characterization
studies that F02 was the preparation appropriately selected
for further testing.
3.2. Safety study results
No mortality was observed following the administration of
the oral chitosan based preparation. In addition, monitoring
the treated animal groups for 30 days did not show any auto-
nomic or behavioural changes. There were no significant
changes in blood biochemistry values such as glucose, glu-
tamic-oxaloacetic transaminase (GOT), glutamate pyruvate
transaminase (GPT), gamma-glutamyl transpeptidase (GGT),
alkaline phosphatase (ALP), urea, creatinine, cholesterol, tria-
cylglycerols, total protein, and albumin (p > 0.05) (Table 2).
However, elevated triacylglycerols levels were observed in
the oral chitosan emulsion treated group compared to the
control group (p = 0.08). In addition, repeated administration
of the preparation did not reveal any significant haematolog-
ical changes in the tested whole blood samples (p > 0.05). Con-
cerning the measured rat’s body weights (Fig. 3A), normal
increase in weights was noticed in the control group. Never-
theless, results indicated that the administration of this prep-
aration during the study period prevented the normal
increase in body weights seen in the control group (p < 0.05).
3.3. Body weight, food, water and faeces evaluations
Measured body weights of rats during the Control period
stayed in the normal range of initial weights (Fig. 3B). How-
ever, decline in body weights started immediately following
switching to the oral chitosan based preparation on the sixth
day of experimentation. Although the decline in body weights
seemed to possess clinical significance, non significant value
was calculated statistically when day 6 was compared with
day 12 (p = 0.077). On the other hand, food and water con-
sumption along with faeces weights were decreased signifi-
cantly (p < 0.05) during the test period (Fig. 4).
3.4. Assay of serum lipids
Serum cholesterol and LDL levels increased when rats in-
gested the oral chitosan preparation compared to the control
group (Fig. 5A). Nevertheless, this increase was not statisti-
cally significant (p = 0.09). Non significant changes in serum
triacylglycerols and HDL levels were also recorded in this
study (p > 0.05).
3.5. Assay of liver total cholesterol and triacylglycerols
Liver cholesterol level was significantly elevated in rats admin-
istered the oral chitosan preparation compared to the control
group where the levels reached 30.8 ± 1.9 and 25 ± 1.1 mg/dL,
respectively (Fig. 5B). Conversely, triacylglycerols level was
not affected by the oral preparation ingestion (p > 0.05).
3.6. Assay of faecal lipids
As illustrated in Fig. 5C, both levels of faecal cholesterol and
triacylglycerols determined for rats administered the oral
0
200
400
600
800
1000
1200
0 50 100 150 200 250Shear Rate (1/s)
Shea
r Stre
ss (P
a)
F04 F05 F06B
0
50
100
150
200
250
0 50 100 150 200 250
Shear Rate (1/s)
Visc
osity
(Pa.
s)
F01 F02 F03C
0
50
100
150
200
250
0 50 100 150 200 250
Shear Rate (1/s)
Visc
osity
(Pa.
s)
F04 F05 F06D
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400
600
800
1000
1200
0 50 100 150 200 250
Shear Rate (1/s)
Shea
r Stre
ss (P
a)
F01 F02 F03A
Fig. 2 – Viscosity and shear stress plots of the formulation. Each data point represents mean ± SEM of three determinations.
1130 J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 1 2 5 – 1 1 3 4
chitosan preparation were increased significantly compared
to control levels (p < 0.01).
4. Discussion
In general, long term low calorie intake would enhance fat
stores to provide the energy needed by the body to function
properly thus, fat will be lost and body weight will drop off.
Among many proposed strategies for weight reduction,
researchers and dieticians succeeded in demonstrating the
potentiality of using meal replacement diets to treat obesity
(Ashley, St Jeor, Perumean-Chaney, Schrage, & Bovee, 2001;
Heymsfield, van Mierlo, van der Knaap, Heo, & Frier, 2003).
Functional food is repeatedly defined as any food or food
component that provides health benefits beyond basic nutri-
tion (Ngo, Lee, Kim, & Kim, 2009). Therefore, many efforts
are undertaken to recognize meal replacement diets as func-
tional foods. In obesity, for example, a potential role for func-
tional diets could be achieved by introducing foods that help
in the management of controlling hunger, contribute to more
inefficient use of ingested energy, and foods that affect the
glucose-insulin levels in the body (Hill & Peters, 2002). In fact,
meal replacement diets are currently governed by a European
Commission Directive (96/8/EC) and accordingly named ‘meal
replacement for weight control’. Later, and through Directive
(2007/29/EC), it is allowed to make claims referring to a reduc-
tion in the sense of hunger or an increase in the sense of sati-
ety if the claims comply with EU legislation.
The current research investigates a low calorie system that
can be used as a meal replacement functional food or, more
broadly, as a carrier of other functional foods where water
or fat soluble agents and bioactive compounds such as miner-
als, vitamins, amino acids or proteins can be added as fortify-
ing substances along with other enhancing additives such as
taste modifiers, flavoring and coloring agents.
The rational behind such strategy is to form an edible
paste with minimum amount of olive oil fortified with oleic
acid. This gives a texture of pseudo fatty rich mass allowing
the stomach to feel a real fatty meal having a butter-like con-
sistency, taste and texture. It has been reported that such
presence of lipids in the gut can stimulate strong feedback
signals that could affect satiety in a process termed ‘ileal
brake’ (Maljaars, Peters, Mela, & Masclee, 2008; Olsson, Sund-
berg, Viberg, & Haenni, 2010). Furthermore, short-term die-
tary studies have shown that human satiety is affected by
the volume of food eaten rather than its weight or energy con-
tent (Clifton, 2008; Rolls et al., 1998).
Chitosan is a polymer which is not absorbed or metabo-
lized in the body; this makes its addition of a great value.
Chitosan is considered the main ingredient in preparing our
proposed meal replacement functional food due to its ability
to entrap high amounts of water when dispersed in the oily
phase. Chitosan had been also introduced to the market as
a dietary supplement for body weight reduction due to its re-
ported ability to bind to dietary fats (Sumiyoshi & Kimura,
2006). However, results obtained from clinical trials indicated
that the effect of chitosan on body weight is minimal and un-
likely to be of clinical significance (Jull, Ni Mhurchu, Bennett,
Dunshea-Mooij, & Rodgers, 2008; Mhurchu, Dunshea-Mooij,
Bennett, & Rodgers, 2005). As for its calorie content, chitosan
is hardly digestible and therefore, it is free of calories (Zeng,
Qin, Wang, Chi, & Li, 2008). This might be attributed to the
lack of chitosan’s degrading enzymes, such as chitosanase,
in the human intestine which renders the ingested chitosan
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110
115
120
0 3 6 9 12 15 18 21 24 27 30Days on test (24 h)
Initi
al B
ody
Wei
ght (
%)
Oral Chitosan EmulsionControl
80
85
90
95
100
105
110
0 1 2 3 4 5 6 7 8 9 10 11 12
Days on test (24 h)
Initi
al B
ody
Wei
ght (
%)
Control period Test periodB
A
Fig. 3 – Percentage of change in body weights of (A) rats
administered daily doses of oral chitosan emulsion (F02)
compared to control group receiving same amount of
distilled water during the repeated dose subacute toxicity
study (p < 0.05). Each data point represents mean ± SEM
(n = 8) and (B) a group of rats administered initially distilled
water (Control period) then switched to receive oral chitosan
emulsion preparation (Test period). Each data point
represents mean ± SEM (n = 5).
80
90
100
110
120
130
140
Control Period Test Period
Wat
er C
onsu
mpt
ion
(mL)
A
0
10
20
30
40
50
60
Control Period Test PeriodFo
od C
onsu
mpt
ion
(g)
B
0
5
10
15
20
25
Control Period Test Period
Feac
es W
eigh
t (g)
C
Fig. 4 – Water consumption volume (A), food consumption
weight (B) and faeces weight (C) during the control period
and following the oral administration of the formulated oral
emulsion. Both periods lasted for six days. Each bar
represents mean ± SEM (n = 5). *p < 0.05.
J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 1 2 5 – 1 1 3 4 1131
undigestable and therefore, may behave as a dietary fibre in
food and nutraceuticals industries (Shahidi, Arachchi, & Jeon,
1999).
The oily phase in the current system was composed of ol-
ive oil and oleic acid. In fact, triacylglycerol esters of oleic acid
compose the majority of olive oil. Other oil sources of mono-
unsaturated fatty acids are canola, peanut, sunflower, corn,
soybean and safflower oils (Nicklas, Hampl, Taylor, Thomp-
son, & Heird, 2004). Olive oil was chosen for its claimed
healthy effects, especially improving cardiovascular risk fac-
tors, that can in particular be attributed not only to the high
relationship between unsaturated and saturated fatty acids
in olive oil but also to the antioxidant property of its phenolic
compounds (Tripoli et al., 2005). Free oleic acid was proven to
possess a better surface activity interacted with chitosan
solution (Assaf et al., 2011) and thus, its presence in small
quantity (2.5%, w/w) stabilized the formed emulsion systems
in our preliminary formulations. Although oils other than ol-
ive oil, particularly canola oil, can be used, the calculated cal-
orie intake derived from the oily phase, mainly due to olive
oil, was estimated to be around 44 kcal/100 g of chitosan
preparation.
Citrus pectin was used herein as a thickening agent.
Increasing pectin concentration resulted in increased viscos-
ities in the present preparations (Fig. 2). In addition, due to its
large water-binding capacity, pectin was previously reported
to enhance satiety by prolongation of the gastric emptying
half-time and therefore, the use of pectin was suggested in
treatment of disorders related to overeating (Di Lorenzo, Wil-
liams, Hajnal, & Valenzuela, 1988). Pectin possess low calorie
amount (estimated 19 kcal/100 g chitosan emulsion) and re-
ported to be safe as a food additive (Sorensen, Pedersen, &
Willats, 2009).
0
20
40
60
80
100
120
Cholesterol Triacylglycerol
Seru
m le
vel (
mg/
dL) Control
Oral Chitosan Emulsion
0
20
40
60
80
100
120
140
Cholesterol Triacylglycerol HDL LDL
Seru
m le
vel (
mg/
dL) Control
Oral Chitosan Emulsion
0
20
40
60
80
100
120
140
Cholesterol Triacylglycerol
Seru
m le
vel (
mg/
dL) Control
Oral Chitosan Emulsion
*
A
B
C
Fig. 5 – Measurements of lipid profiles in serum (A), livers (B)
and faeces (C) of rats following six days of repeated oral
administration of chitosan emulsion (F02). Each bar
represents mean ± SEM (n = 5). *p < 0.05, **p < 0.01.
1132 J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 1 2 5 – 1 1 3 4
Glycerine and sorbitol were incorporated in the formula-
tions as sugar replacers. As sugar alcohols, both are metabo-
lized slowly by the body and therefore could be used in low
calorie diets and diabetic foods (Livesey, 2003; Wang & van
Eys, 1981). Furthermore, sorbitol was found to exert some
laxative effects by accelerating colonic transit time regardless
of food intake (Skoog, Bharucha, Camilleri, Burton, & Zins-
meister, 2006); which would be considered a beneficial effect
added to the proposed meal replacement functional food
when used in restricted food regimes that might be accompa-
nied with constipation.
In the current study, the stability results highlighted the
importance of optimizing the used concentrations of both
pectin and chitosan in the prepared emulsions. While low
concentrations of chitosan showed extensive phase separa-
tions in the tested preparations preliminarily, increasing the
concentration of chitosan in the investigated emulsions re-
sulted in noticeable changes in its colour following storage.
Therefore, it was decided to incorporate pectin to improve
the reduced physical stability that may arise from using the
low amount of chitosan (1%). However, it was found that
increasing the amount of the added pectin highly affected
the consistency of the preparation. Consequently, F02 for-
mula having 1% chitosan and 6% pectin was chosen as the
optimal preparation.
Concerning the safety evaluation, the results indicated
that repeated dose administration of the optimal chitosan
oral emulsion preparation (F02) at 3 mL/250 g dose level did
not change significantly any of the tested clinical parameters
compared to the control group (Table 2). None of the adult
male rats exhibited any marked change behavioural signs
and symptoms when checked daily for 30 days. However, re-
duced food intake was noticed following the emulsion admin-
istration. Such reduction in food intake reflected the reduced
normal increase of body weights of the emulsion treated ani-
mals when compared to the water treated rats. In general,
excessive fat intake induces fat accumulation in the heart
and liver and results in atherosclerosis and fatty liver in these
organs, respectively. In the current investigation, histopathol-
ogical examinations confirmed that hearts and livers were
found to be normal with no detected fat deposits when rats
were fed with chitosan emulsion for 30 days.
In another a set of in vivo experiments, reductions in body
weight were again detected when rats were switched to re-
ceive oral chitosan emulsion for six days (Fig. 3B). The reduc-
tion in body weights was also accompanied with a significant
reduction in food and water intake along with reduced
amount in faeces defecation (Fig. 4). Consequently, both
safety and efficacy studies highlighted the ability of the pro-
posed meal replacement diet to reduce body weights of tested
animals. This can be by reduced food and water intake that
might be attributed to the large volume of the administered
preparation or by increased satiety due to the presence of this
pseudo-fatty rich meal. However, further investigations on
such mechanisms are still warranted.
Moreover, it was found that the serum lipid profile of rats
fed with the present preparation was not affected signifi-
cantly, which indicate the low ability of the emulsion to be ab-
sorbed. This reduced absorption was also confirmed by the
significant increase in faeces cholesterol and triacylglycerol
content post emulsion administration (Fig. 5). Due to its po-
tent fat-binding capacity, chitosan was found previously
capable of lowering plasma and liver triacylglycerol and total
cholesterol and was shown to increase faecal neutral steroid
and bile acid excretion in rats (Zhang, Liu, Li, & Xia, 2008).
In terms of industrial viability, producing emulsions is
considered common in food manufacturing practice. As such,
the investment and operational expenses associated with
manufacturing such a preparation are not expected to repre-
sent a significant burden on the manufacturer’s part. More-
over, in terms of the materials used, the proposed emulsion
is comprised of components that are widely used in the food
industry and water constitutes more than 50% of the
preparation.
All these factors suggest that the proposed functional food
preparation is cost-effective and can be produced on com-
monly used machinery in food industry.
J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 1 2 5 – 1 1 3 4 1133
5. Conclusion
The present work is proposing a meal replacement functional
food which can also be used as a base to add other low calorie
additives due to its biphasic composition. This food prepara-
tion has a function beyond its nutritional value in terms of
having a fatty texture similar to butter which can influence
the individual’s psychology by feeling to ingest fats where
the stomach by sensing rich fatty meal would stimulate feed-
back mechanism to affect satiety. Such edible preparation has
no negative impact on body fluid profile and is not harmful to
ingest, consequently, can be used in body weight control.
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