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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

0

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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.

R E F E R E N C E S

Ashley, J. M., St Jeor, S. T., Perumean-Chaney, S., Schrage, J., &Bovee, V. (2001). Meal replacements in weight intervention.Obesity Research, 9(Suppl. 4), 312S–320S.

Assaf, S. M., Al-Jbour, N. D., Eftaiha, A. F., Elsayed, A. M., Al-Remawi, M. M., Qinna, N. A., Chowdhry, B., Leharne, S., &Badwan, A. A. (2011). Factors involved in formulation of oilydelivery system for proteins based on PEG-8 caprylic/capricglycerides and polyglyceryl-6 dioleate in a mixture of oleicacid with chitosan. Journal of Dispersion Science and Technology,32, 623–633.

Athamneh, N. A., Tashtoush, B. M., Qandil, A. M., Al-Tanni, B. M.,Obaidat, A. A., Al-Jbour, N. D., Qinna, N. A., Al-Sou’od, K., Al-Remawi, M. M., & Badwan, A. A. (2012). A new controlled-release liquid delivery system based on diclofenac potassiumand low molecular weight chitosan complex solubilized inpolysorbates. Drug Development and Industrial Pharmacy, http://dx.doi.org/10.3109/03639045.2012.707205. [Epub ahead of print].

Baldrick, P. (2009). The safety of chitosan as a pharmaceuticalexcipient. Regulatory Toxicology and Pharmacology, 56, 290–299.

Cannon, C. P., & Kumar, A. (2009). Treatment of overweight andobesity: Lifestyle, pharmacologic, and surgical options. ClinicalCornerstone, 9, 55–68 [discussion 69–71].

Clapham, J. C., Arch, J. R., & Tadayyon, M. (2001). Anti-obesitydrugs: A critical review of current therapies and futureopportunities. Pharmacology & Therapeutics, 89, 81–121.

Clifton, P. M. (2008). Dietary treatment for obesity. Nature ClinicalPractice Gastroenterology & Hepatology, 5, 672–681.

Di Lorenzo, C., Williams, C. M., Hajnal, F., & Valenzuela, J. E. (1988).Pectin delays gastric emptying and increases satiety in obesesubjects. Gastroenterology, 95, 1211–1215.

Eftaiha, A. F., Qinna, N., Rashid, I. S., Al Remawi, M. M., Al Shami,M. R., Arafat, T. A., & Badwan, A. A. (2010). Bioadhesivecontrolled metronidazole release matrix based on chitosanand xanthan gum. Marine Drugs, 8, 1716–1730.

Elsayed, A., Remawi, M. A., Qinna, N., Farouk, A., & Badwan, A.(2009). Formulation and characterization of an oily-basedsystem for oral delivery of insulin. European Journal ofPharmaceutics and Biopharmaceutics, 73, 269–279.

Folch, J., Lees, M., & Sloane Stanley, G. H. (1957). A simple methodfor the isolation and purification of total lipides from animaltissues. Journal of Biological Chemistry, 226, 497–509.

Harnedy, P. A., & FitzGerald, R. J. (2012). Bioactive peptides frommarine processing waste and shellfish: A review. Journal ofFunctional Foods, 4, 6–24.

Haug, A., & Hostmark, A. T. (1987). Lipoprotein lipases,lipoproteins and tissue lipids in rats fed fish oil or coconut oil.Journal of Nutrition, 117, 1011–1017.

Heymsfield, S. B. (2010). Meal replacements and energy balance.Physiology & Behavior, 100, 90–94.

Heymsfield, S. B., van Mierlo, C. A., van der Knaap, H. C., Heo, M.,& Frier, H. I. (2003). Weight management using a mealreplacement strategy: Meta and pooling analysis from sixstudies. International Journal of Obesity and Related MetabolicDisorders, 27, 537–549.

Hill, J. O., & Peters, J. C. (2002). Biomarkers and functional foods forobesity and diabetes. British Journal of Nutrition, 88(Suppl. 2),S213–S218.

Jull, A. B., Ni Mhurchu, C., Bennett, D. A., Dunshea-Mooij, C. A., &Rodgers, A. (2008). Chitosan for overweight or obesity. CochraneDatabase of Systematic Reviews, 3. CD003892.

Kubota, N., Tatsumoto, N., Sano, T., & Toya, K. (2000). A simplepreparation of half N-acetylated chitosan highly soluble inwater and aqueous organic solvents. Carbohydrate Research,324, 268–274.

Kanauchi, O., Deuchi, K., Imasato, Y., Shizukuishi, M., &Kobayashi, E. (1995). Mechanism for the inhibition of fatdigestion by chitosan and for the synergistic effect ofascorbate. Bioscience, Biotechnology & Biochemistry, 59, 786–790.

Livesey, G. (2003). Health potential of polyols as sugar replacers,with emphasis on low glycaemic properties. Nutrition ResearchReviews, 16, 163–191.

Maljaars, P. W., Peters, H. P., Mela, D. J., & Masclee, A. A. (2008).Ileal brake: A sensible food target for appetite control. Areview. Physiology & Behavior, 95, 271–281.

Mhurchu, C. N., Dunshea-Mooij, C., Bennett, D., & Rodgers, A.(2005). Effect of chitosan on weight loss in overweight andobese individuals: A systematic review of randomizedcontrolled trials. Obesity Reviews, 6, 35–42.

Ngo, D. N., Lee, S. H., Kim, M. M., & Kim, S. K. (2009). Production ofchitin oligosaccharides with different molecular weights andtheir antioxidant effect in RAW 264.7 cells. Journal of FunctionalFoods, 1, 188–198.

Nicklas, T. A., Hampl, J. S., Taylor, C. A., Thompson, V. J., & Heird,W. C. (2004). Monounsaturated fatty acid intake by childrenand adults: Temporal trends and demographic differences.Nutrition Reviews, 62, 132–141.

Ogden, C. L., Carroll, M. D., Curtin, L. R., McDowell, M. A., Tabak, C.J., & Flegal, K. M. (2006). Prevalence of overweight and obesityin the United States, 1999–2004. The Journal of the AmericanMedical Association, 295, 1549–1555.

Ogden, J., & Flanagan, Z. (2008). Beliefs about the causes andsolutions to obesity: A comparison of GPs and lay people.Patient Education and Counseling, 71, 72–78.

Olsson, J., Sundberg, B., Viberg, A., & Haenni, A. (2010). Effect of avegetable-oil emulsion on body composition; a 12-week studyin overweight women on a meal replacement therapy after aninitial weight loss: A randomized controlled trial. EuropeanJournal of Nutrition, 50, 235–242.

Pangestuti, R., & Kim, S. K. (2011). Biological activities and healthbenefit effects of natural pigments derived from marine algae.Journal of Functional Foods, 3, 255–266.

Rolls, B. J., Castellanos, V. H., Halford, J. C., Kilara, A., Panyam, D.,Pelkman, C. L., Smith, G. P., & Thorwart, M. L. (1998). Volume offood consumed affects satiety in men. American Journal ofClinical Nutrition, 67, 1170–1177.

Sajomsang, W. (2010). Synthetic methods and applications ofchitosan containing pyridylmethyl moiety and its quaternizedderivatives: A review. Carbohydrate Polymers, 80, 631–647.

Schellekens, H., Dinan, T. G., & Cryan, J. F. (2009). Lean mean fatreducing ‘‘ghrelin’’ machine: Hypothalamic ghrelin andghrelin receptors as therapeutic targets in obesity.Neuropharmacology, 58, 2–16.

Shahidi, F., Arachchi, J. K. V., & Jeon, Y. J. (1999). Food applicationsof chitin and chitosans. Trends in Food Science and Technology, 10,37–51.

Skoog, S. M., Bharucha, A. E., Camilleri, M., Burton, D. D., &Zinsmeister, A. R. (2006). Effects of an osmotically active agent

1134 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

on colonic transit. Neurogastroenterology and Motility, 18,300–306.

Sorensen, I., Pedersen, H. L., & Willats, W. G. (2009). An array ofpossibilities for pectin. Carbohydrate Research, 344, 1872–1878.

Stewart, L., Reilly, J. J., & Hughes, A. R. (2009). Evidence-basedbehavioral treatment of obesity in children and adolescents.Child & Adolescent Psychiatric Clinics of North America, 18,189–198.

Sumiyoshi, M., & Kimura, Y. (2006). Low molecular weightchitosan inhibits obesity induced by feeding a high-fat dietlong-term in mice. Journal of Pharmacy and Pharmacology, 58,201–207.

Swartz, T. D., Duca, F. A., & Covasa, M. (2009). Differential feedingbehavior and neuronal responses to CCK in obesity-prone and-resistant rats. Brain Research, 1308, 79–86.

Tripoli, E., Giammanco, M., Tabacchi, G., Di Majo, D., Giammanco,S., & La Guardia, M. (2005). The phenolic compounds of oliveoil: Structure, biological activity and beneficial effects onhuman health. Nutrition Research Reviews, 18, 98–112.

Wang, Y. M., & van Eys, J. (1981). Nutritional significance offructose and sugar alcohols. Annual Review of Nutrition, 1,437–475.

Yun, J. W. (2010). Possible anti-obesity therapeutics from nature: Areview. Phytochemistry, 71, 1625–1641.

Zeng, L., Qin, C., Wang, W., Chi, W., & Li, W. (2008). Absorption anddistribution of chitosan in mice after oral administration.Carbohydrate Polymers, 71, 435–440.

Zhang, J., Liu, J., Li, L., & Xia, W. (2008). Dietary chitosan improveshypercholesterolemia in rats fed high-fat diets. NutritionResearch, 28, 383–390.