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CHITOSAN -WHEY PROTEIN COMPLEX (CS-WP) AS DELIVERY SYSTEMS TO IMPROVE BIOAVAILABILITY OF IRON

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Scientific Journal Impact Factor: 1.205

International Journal of Applied And Pure Science and

Agriculture

www.ijapsa.com

CHITOSAN -WHEY PROTEIN COMPLEX (CS-WP) AS DELIVERY

SYSTEMS TO IMPROVE BIOAVAILABILITY OF IRON

El-Sayed M. M.1, Hassan Z.M.R2, Mervat I. Foda1, Awad R. A.2 and Heba H. Salama1

1

Dairy Science &Technology Department, National Research Center, Egypt.

2

Food Science Department; Faculity of Agriculture; Ain Shams University; Cairo; Egypt

Abstract

Use of whey proteins as a natural nano-capsular vehicle to carry and improve the bioavailability of iron was investigated in this study. Nanoparticles of whey protein concentrate-chitosan (CS-WPC) complex were prepared with the aim of developing a biocompatible carrier for the oral administration of iron as a nutraceuticals. Effects of pH, concentration of native CS-WPC and iron on the nanoparticles with sodium tripolyphosphate (TPP) prepared by ionic gelation were investigated. CS-WPC were loading with different iron concentration namely; ferrous sulphate. The surface charge of the particles was positive and negative that strongly pH dependent and showed positive charge after iron loading at low protein concentration and was negative at 8 and 12 % when the pH increased to 5.5. The association efficiency (AE) and loading efficiency (LE) of CS-WPC nanoparticles was highly sensitive to formulation pH. This adsorption can be mainly attributed to electrostatic, hydrophobic interactions and hydrogen bonding between WPC and CS. The iron release experiments showed that the nanoparticles prepared with native WPC had favorable properties to resist acid and pepsin degradation in simulated gastric conditions. When transferred to simulated intestinal conditions, the WPC shells of the nanoparticles were not degraded by pancreatin showing the same results with and without enzymes after 6 h. CS-WPC iron nanoparticles at level 0, 3, 6, 9, 12 mg/g protein showed very high bioavailability after evaluated in simulated gastric and intestinal fluids in the presence or absence of the enzymes.

Keywords: Nanoparticles; Whey proteins concentrate; Chitosan; Iron bioavailability

I. Introduction

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can hold large amount of water while maintaining a network structure (Qui and Park 2001). An important functional property of whey proteins is their ability to form cold-induced gel matrices by addition of cations to preheated denatured suspension, which results in the formation of a network hydrogel cross-linked via the added cation with carboxyl groups on denatured protein (Remondetto and Subirade 2003).

Iron is considered to be one of the essential minerals required by the human body. Although milk is a good source of minerals, its iron content is too low (0.2- 0.5 mg iron/L) to contribute significantly to daily dietary requirements (Flynn and Cashman, 1997). Fortification of milk or dairy products with iron has been considered as a potential approach to deliver this nutritionally important mineral in required quantities to the consumer. Therefore it can help in preventing iron deficiency in humans, which is a major nutritional problem worldwide (Hurrell and Cook, 1990). However, recurrent problems are associated with iron fortification, including variable bioavailability, formation of sediments, organoleptic defects and the effect of iron on lipid oxidation. The whey protein fraction is slightly modified by iron supplementation of milk (Hekmat and McMahon 1998) but the nature of whey proteins by iron is not determined precisely modified. Some model studies on the interaction between iron and purified α-LA and ß-lg indicated that these proteins bind iron and their binding abilities decreased with lowering pH value (Baumy and Brule 1988).

Therefore, the aims of the present study were to investigate the potential uses of whey proteins as a base of nanoparticles and prepare a natural nano-capsular vehicle of whey proteins to carry and improve the bioavilability of iron.

II. Materials and Methods

Whey Protein Concentrate (WPC)

Demineralised Whey Protein Concentrate (WPC) in a powder form was obtained from FRIESLAND Hiprotal, New York, USA.

Chitosan (CS)

Chitosan (C6H11NO4)n, molecular weight of 100,000 -300,000 D was provided by Acros Organics, New Jersey, USA.

Sodium tripolyphosphate (TPP)

Sodium tripolyphosphate (TPP), was purchased from Acros Organics, New Jersey; USA.

Ferrous sulphate

Ferrous sulphate salt (FeSO4.7H2O) was obtained from SISCO Research Laboratories PVT, Ltd. Mumbai, INDIA.

Pepsin and pancreatin

Pepsin enzyme 1:60000, from porcine stomach mucosa, in crystallized and lyophilized form and pancreatin (4X) from hog pancreas were obtained from Sigma-Aldrich Co., St Louis, USA.

Preparation of protein solutions Whey protein solutions (nanoparticles)

Stock solution of whey protein (WP) (about 15%) from whey protein concentrate (WPC) was prepared by hydrating WPC in deionized water with agitation at room temperature for 1 h. The solution was allowed to rest for 2 h. before further treatment in order to permit a good protein hydration as suggested by Beaulieu, et al., (2002). Protein concentration was measured by the absorbance at 280 nm, and then diluted to concentrations of 2, 4, 8 and 12 % final concentration.

Preparation of chitosan-whey protein nanoparticles (CS-WP)

Nanoparticles of CS-WP were prepared by the method adapted from that reported by Janes et al.,

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various concentrations (2, 4, 8 and 12 %) were adjusted to various pH values (4.5, 5.5, 6.5 and 7.5) with 1 mg/ml HCl and NaOH. These solutions were added to CS solutions (0.1%) in aqueous acetic acid to form CS-WP nano complexes with CS concentration of 2 mg/ml. Two ml of TPP solution (1 mg/ml) was added as drop wise to 5 ml of CS-WP nano complexes, opalescent suspension was formed spontaneously under stirring at room temperature, and further examined as nanoparticles. The final pH of the nanoparticle suspension was measured and the nanoparticles were characterized immediately. All experiments were performed in triplicates.

Immobilization of iron (Fe2+) at CS-WP nanoparticles

Ferrous sulphate solution (2%) was added to the prepared WPC solutions of various concentrations and pH values as described before to form whey protein iron complexes with iron concentration of 0, 3, 6, 9, 12 mg iron/g protein as suggested by Remondetto, et al., (2004). The WP-iron solutions were added to CS solution in aqueous acetic acid (0.1%) to form CS-WP-Iron complex with CS concentration of 2.0 mg/ml (Janes, et al., 2001). Two ml of TPP solution (1.0 mg/ml) was added as drop wise to 5 ml of the prepared CS-WP-Iron complexes; opalescent suspension was formed spontaneously under stirring at room temperature, and further examined as nanoparticles. The final pH of the nanoparticle suspension was measured, and the nanoparticles were characterized immediately. All experiments were performed in triplicates.

pH Value measurement:

The final pH value of the nanoparticle suspensions was measured and adjusted using a Laboratory pH meter, HANNA– instrument, 211 micro processor, USA.

Characterization of nanoparticles Particle size and morphology

Freshly-prepared nanoparticles were diluted with distilled water (1:10) and placed on a copper grid then coated with carbon (carrier powder) and left to dry at room temperature. Size and external morphology of nanoparticles were examined by transmission electron microscopy, (TEM, JEOL, JEM, 1230 Japan) working at 100 KVas described by Chen and Subirade (2005).

Surface charge of nanoparticles

Measurement of Zeta potential values of nanoparticles was performed using Laser Zeta meter (Malvern Instruments) model “Zeta Sizer 2000”. Sample of 0.01 gram was added to 50 ml double distilled water. The pH value was then adjusted to 4.5, 5.5, 6.5 and 7.5, using 1mg/ml HCl or NaOH. The sample was shaked for 30 min, followed by recording the pH value and, zeta potential measurments of the particles were performed using an aqueous dip cell in the automatic mode as described by Chen and Subirade (2005).

Coating properties of whey protein (WP)

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AE = WP of amount Total ernat in WP WP of amount

Total − sup

X 100 Eq. (1)

particles ered re of Weight ernat in WP WP of amount Total LE cov sup − =

X 100 Eq. (2)

Encapsulation capacity of iron (Fe2+)

The iron encapsulated nanoparticles were prepared by incorporating ferrous sulfate into the CS-WP complexes to a final concentration of 3, 6, 9 and 12 mg/g protein. For the determination of iron encapsulation capacity, the iron encapsulated CS-WP nanoparticles were separated from the aqueous suspension medium by ultracentrifugation at 30.000g, for 30 min. The amount of free iron in the clear supernatant was determined as mentioned by Shu and Zhu (2002) with 1, 10-Phenanthroline by measuring the absorbance at 510 nm using spectrophotometer (Nanodrop).Iron encapsulation capacity (EC) for iron was calculated using the following equation:

Fe of amount Total ernat the in Fe Fe of amount Total EC sup −

= X 100 Eq. (3)

Release of iron (Fe2+)

The iron encapsulated CS-WP nanoparticles which separated from 14 ml suspension were re-dispersed into test tubes with 4 ml HCl solution (pH 1.2) at 37°C under agitation for 30 min. The pH value of the suspension was then raised to 7.5 with concentrated NaOH (1 N), and 0.2 ml phosphate buffer (0.5M, pH 7.5) was added. The mixture was adjusted to a final volume of 5 ml with distilled water. The iron release in phosphate buffer (pH 7.5) at 37 °C was carried out under agitation for 6 h. At predetermined incubation time, samples were centrifuged at 30.000 g for 30 min and the iron released was determined by UV-Vis spectrophotometer (Nanodrop), as mentioned earlier. Following supernatant extraction and pellets were discarded (destructive sampling). The in vitro release of iron was also evaluated in simulated USP gastric and intestinal fluids in the presence of enzymes, using the method mentioned by Beaulieu et al., (2002). Iron encapsulated CS-WP nanoparticles separated from 14 ml suspension were re-dispersed in 4 ml of 0.1N HCl in a test tube and magnetically stirred for 10 min at 37°C. Pepsin solution, 0.05 ml (1 mg/ml, 0.1 N HCl), was added to initiate the hydrolysis. The digestion was carried out for 30 min and stopped by raising the pH to 7.5 with NaOH, and then phosphate buffer (0.2 ml; 0.5M, pH 7.5) was added. The reaction was initiated by adding 0.05 ml of pancreatin enzyme (10 mg/ml) prepared in phosphate buffer (0.02M, pH 7.5). The reaction mixture was adjusted to 5 ml with distilled water, and the digestion was carried out for 6 h. The amount of iron released was expressed as a percentage of the total iron encapsulated in the nanoparticles as calculated from the EC value. The iron release experiments were repeated three times.

III. Results and Discussion

Characterization of nanoparticles

Chitosan – whey protein (CS-WP) nanoparticles Morphology and size of nanoparticles

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transmission electron microscopy (TEM) and illustrated in Fig.s. 1. The CS-WP nanoparticles had a homogeneous structure of evenly sized particles and pores. Microscopic observation revealed that the structure of the complexes became more compact as the pH of the mixture decreased in complex (Fig.. 1A & B). The interior structure of CS-WP nanoparticles (the same sample) demonstrated a circular shape consisting of a dark core and light shell (Fig.1 B). The shells of the nanoparticles were destructed to some extent in the sample treatment process, showing an irregular surface.

Fig. (1): TEM photographs of CS-WP nanoparticles (2.0 mg/ml-2%) prepared at pH 5.5 of suspension containing 2% WP and 2 mg/ml CS. A (low magnification), B (high magnification).

In the preparation process, it was supposed that a CS-TPP core was initially generated as an ionic gelation involving positively charged amino groups on the CS molecular chains and negatively charged TPP in the case of dropping TPP into the mixture. Then WP molecules in the bulk phase approached the CS-TPP core, readily adsorbed onto interfaces and a membrane of WP was formed on the surface of the CS-TPP core through a combination of ionic, hydrophobic interactions and hydrogen bonding. Thus, the CS-WP nanoparticles with core-shell structure were formed (Leaver and Horne, 1993).

The size constancy may be the result of a better structured complex networks, due to enhanced display of positive groups by WP molecules, and hence binding to CS molecules with greater strength. This may also explain the compact structure observed on the TEM photographs. Image analysis of TEM photographs for CS-WP suspension shows that the particle size ranged between 13 and 70.6 nm, with an average size of 44.41nm indicating that the nanoparticles were formed in nanoscale particles. The formed nanoparticles were appeared spherical in shape with smooth surfaces.

Surface charge of nanoparticles

Surface charge of chitosan-whey protein (CS-WP) nano-particles

Zeta potential is a physical property which is exhibited by any particle in suspension. In aqueous media, the pH of the sample is one of the most important factor that affects its zeta potential. Therefore a zeta potential versus pH curve will be positive at low pH and negative at high pH. The point where the plot passes through zero zeta potential is called the isoelectric point and is very important from a practical consideration. It is normally the point where the colloidal system is least stable.

Zeta potential values (mV) of CS-WP nanoparticles as affected by whey protein concentrations at different pH values are shown in Fig. (2). It could be noticed that when the pH value increased from 4.5 to 7.5 the ζ-potential of CS-WP for protein concentration of (2, 4, 8 and 12 %) changed from positive (+16.5, +15.1, +14.2 and +10.5 mV) to negative charge (-1.1, -5.1, -11.6 and -14.5 mV). These results are agreement with Harnsilawat et al., (2006).

The ζ-potential of the CS-WP generally changed from positive to negative as the pH was increased from 4.5 to 7.5, with the point of net zero charge being expected to be around pH 5. The progressive loss of positive charge on CS-WP with increasing pH is due to deprotonation of amino groups (pKa-6.3). At pH 4.5, the ζ-potential of the mixed system was highly positive, which can be

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attributed to the fact that both WP and chitosan are positively charged at this pH. The measurements of the electrical properties of the WP and chitosan complex in aqueous solution indicated that the signs of their charges were opposite between pH 4.5 and 7.5, and hence, one would expect them to form complexes in this range.

This complex was formed between negatively charged groups on the protein (e.g., -COO-) and positively charged groups on the chitosan (e.g., -NH4+). At pH 4.5, most of the carboxyl groups on the protein would be protonated (-COO- + H + T -COOH, pKa - 4), and therefore little binding occurred. However, when the pH was increased, the pH dependence of the electrical characteristics (ζ -potential) of aqueous solutions containing WP (2 %), chitosan (2.0 mg/ml), would be an increasingly large number of negatively charged groups on the protein surface which could act as binding sites for the positively charged groups on chitosan. This would explain why complexation was observed at pH values below the isoelectric point of the protein (where both the protein and chitosan had a net positive charge).

Fig. (2): Zeta potential (mV) of CS-WP nanoparticles with different WP concentration at different pH values.

At relatively low pH (<6.5), chitosan was positively charged and tended to be soluble in dilute aqueous solutions, but at higher pH it tended to lose its charge and may precipitate from solution due to deprotonation of the amino groups. Chitosan can interact with proteins to form either soluble or insoluble complexes.

Ionic, hydrophilic-hydrophobic interaction or van der Waals forces are responsible for chitosan-protein interaction. These results confirmed that both chitosan and TPP took part in the formation of complexes with proteins as reported by Zubareva et al., (2011).

Surface charge of CS-WP-iron complex

Zeta potential (Surface electrical potential, mV) of (CS-WP-Fe) complex as a function of pH and WP concentration illustrated in Fig. (3). The effect of pH on the interaction between WP and chitosan indicated that at lower pH value (4.5) the net charge was positive on surface of particles. When loading the iron at different concentrations (3, 6, 9 and 12 mg/g protein), a positive charge was also occurred, and increased by increasing the iron concentration. Zeta potential at pH 4.5 with all of iron ratios at different protein concentrations showed positive charge but the positive charge differed depends on the concentration of iron and protein. It could be noticed that the net positive charge was increased with increasing the iron concentration. On the other hand, increasing the protein (WP) concentration and pH value led to an increase in negative charge and lowered positive charge on the particles. At pH 7.5 most of charges on surface of formed nanoparticles were negative especially with higher protein concentrations (8, 12%).

- 1 5 - 1 0 - 5 0 5 1 0 1 5 2 0

4 4 .5 5 5 .5 6 6 . 5 7 7 .5 8

2 % W P 4 % W P 8 % W P 1 2 % W P

Z

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The surface charge increased from +17.4 to +24.3 for nanoparticles of 2% WP at pH 4.5 by increasing the iron concentration into CS-WP nanoparticles from 3 to 12 mg/g WP respectively. At pH 5.5 the positive charge decreased and negative charge started to appear at 12% protein concentration while the positive charge increased with increasing the loaded iron.

The positive charge decreased and the negative charge increased when the iron concentration increased. The maximum positive was noted at pH 4.5 with 2% WP concentration and 12 mg/g WP (+24.3) while the maximum negative charge was appeared at pH 7.5, with 12% WP concentration (-14.5) and 0.0 mg iron/g loaded.

From the data presented in Fig. (3) it could be generally stated that the electrical charge appear on surface of nanoparticles formed of CS-WP-iron complex was positive at pH 4.5. The net positive charge increased by loading iron into the particles due to the positive charge on Fe molecule. At pH 4.5 the charge appeared to be positive with all WP concentrations but the net decreased by increasing WP ratio in the particles. At pH 5.5, the negative charge starts to appear with WP ratio of 12 while it appeared with 4 and 2% of WP at pH 6.5 and 7.5 respectively. It could be also noticed that at pH 7.5, all charge on CS-WP-iron nanoparticles were negative with higher WP ratios (8 and 12%). Increasing the pH value and protein (WP) ratio of nanoparticles helped to appear a negative charge on particle surface while lowering the pH value and loading Fe into particles led to positive charge into surface.

Fig. (3): Zeta potential (mv) of nanoparticles from CS-WP-iron complexes with various iron ratios as affected by pH value at different whey protein concentrations.

-5 0 5 1 0 1 5 2 0 2 5

4 4 .5 5 5 .5 6 6 .5 7 7 .5 8 (2% W P )

Z e ta p o te n ti a l (M V )

p H val ues

-10 -5 0 5 10 15 20 25

4 4.5 5 5.5 6 6.5 7 7.5 8 (4% WP) Z e ta p o te n ti a l (M v ) pH values -15 -10 -5 0 5 10 15 20 25

4 4.5 5 5.5 6 6.5 7 7.5 8

(8% WP) Z e ta p o te n ti a l (m v ) pH values -15 -10 -5 0 5 10 15 20 25

4 4.5 5 5.5 6 6.5 7 7.5 8

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It was reported that one of the major factors influencing the electrostatic interaction of charged biopolymers in aqueous solutions is the pH, since this affects both the sign (−/+) and magnitude of the charge of them (Guzey and McClements 2006).

Coating properties of CS-WP nanoparticles

Association efficiency (AE)

The association efficiency (AE) obtained as a function of pH for CS-WP nanoparticles prepared with different concentrations of WP is presented in Fig. (4). The AE obviously increased with increasing pH value until reach to the maximum at pH 7.5. A small amounts of WP were coated on CS-TPP core at pH 4.5, while the AE values began to increase sharply and steadily by increasing the pH value to maximum of 68.8 % at pH 7.5 and 2% WP concentration until pH value of 7.5. With pH value increasing, AE value increased sharply

AE decreased dramatically with increasing WP concentration and the maximum decrease was observed at 12% WP concentration and lower pH (4.5). The highest AE value was noted at pH 7.5 with 2% WP, while the minimum AE was at 12 % and pH 4.5 (36.6). From the data shown in Fig. (4) it was noted that AE values increased by increasing pH values from 4.5 to 7.5 while decreased by increasing WP concentration from 2 to 12 %.

The changes of the AE value as a function of pH exhibit three association dominations corresponding to three kinds of interactions between WP and CS. As a cationic polyelectrolyte, CS was positively charged due to protonation of amino groups on the molecular chain at pH below 9.0. While the net charge on a protein is dependant on pH and interaction of a protein with CS-TPP core surface will therefore vary with pH, which leads to different AE values.

Fig. (4): Association efficiency (AE) for chitosan-whey protein (CS-WP) nanoparticles as a function of pH and WP concentrations.

At pH value lower than 4.3, both CS and ß-lg were positively charged, and thus strong repulsion prevents association of ß-lg on CS-TPP core. When pH value increased in the range of 4.3 to 5.9, where the pI of ß-lg exists, intraionic attractions between COO- and NH3+ for example resulted in seldom residual ionic groups on ß-lg. In this pH range, hydrophobic interactions and hydrogen bondings between ß-lg and CS are supposed to dominate to explain the steadily increase of the AE value. In fact, it is revealed that the hydrophobic interactions are the most important aspect of protein adsorption onto the nanoparticle surfaces. With further increase of the pH value above 5.9, CS is

35 40 45 50 55 60 65 70

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(2% WP) (4%WP) (8%WP) (12%WP)

A

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positively charged, while ß-lg becomes negatively charged, the driving force for ß-lg association thus, changed from hydrophobic interactions gradually to electrostatic attractions. These findings are in agreement with Chen and Subirade (2005).

The AE value was also affected by WP concentration. From the data presented in Fig. (4), it could be noticed that the highest AE value was achieved with lower WP concentration, (2%) especially at pH 6.5. The AE showed lower value with increasing the WP% being lowest with 12% WP at lower pH (4.5).

Loading efficiency (LE)

The LE values which measure the amount of WP loaded on unit weight of nanoparticles were also strongly pH dependent and showed similar changing tendency of AE values as a function of pH, as demonstrated in Fig. (5). After centrifugation, each sample was vacuum dried and the pellet was weighted carefully. The pellet weight increased obviously with increasing pH value up to pH 6.5, indicating an increase in the number of nanoparticle formed at higher pH value. However, the maximum LE values were recorded at pH 6.5 and with further increase of the pH, LE values decreased. A reasonable explanation for this increase could be a conformation change of the absorbed WP.

As globular protein, near pI, native WP apparently adsorbs onto hydrophobic surface in an end on orientation. With increasing pH value, the surface charge of the WP increases accordingly, and the high charge density opposite that of the positively charged CS surface leads to switch of the orientation of the WP on the CS-TPP core surface to side-on in order to maximize electrostatic interactions, which is then followed by decrease of the LE value Chen and Subirade (2005). The LE value was enhanced dramatically by increasing the concentration of WP from 2 to 12 % as shown in Fig. (5), and reached to the maximum at concentration 12% WP. Regardless of the WP concentration, optimal LE value was achieved at pH 6.5. However, at low pH values (around pH 4.5), the adsorption equilibrium reached at concentration of 2 % WP, while less WP was coated on CS-TPP core above 2 % WP concentration due to strong electric repulsion between native WP and CS molecular chains, where both of them are strongly positively charged.

Fig. (5): Loading efficiency (LE) for chitosan-whey protein (CS-WP) nanoparticles as a function of pH and WP concentration.

Encapsulation capacity (EC) of CS-WP nanoparticles

The encapsulation capacity (EC) of iron into CS-WP nanoparticles prepared with different WP concentrations at various pH values was determined and displayed in Fig. (6). There were no

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2%WP 4%WP 8%WP 12%WP

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significant differences in encapsulation efficiency and it was approximately similar for all samples studied with different protein and iron concentrations at different pH values. From the data in Fig. (6) it can be noticed that EC was higher than 99.7% for all studied samples indicating a high iron encapsulation. The lowest EC was observed with 2% WP at pH 4.5 while the highest EC was 99.998% that obtained with many samples especially at high pH value. The EC% increased with increasing WP concentration from 2 to 12 % because there may be more binding sites on WP chain.

It could be generally observed from results in Fig. (6) that whey protein have high ability to bind iron or any other hydrophobic molecules The EC value of iron ranged between 99.746 to 99.998 % in all experiments. The values were slightly lower at low pH (4.5) and higher at higher pH (7.5). Regardless the pH value, the EC % of iron increased with increasing the concentration of loaded iron and WP % used.

Sugiarto and Singh (2009) demonstrated that WP have sites to bind iron (8 sites) and compact structure of whey proteins (globular proteins) may be the reason for their strong ability to bind added iron. The solubility of iron was considerably greater in the presence of whey protein than in the absence of protein but the solubility decreased gradually with increasing levels of ferrous in the mixture.It varied between 80% and 90% with concentrations of added iron.

Fig. (6): Encapsulation capacity (EC %) of iron (Fe2+) as a function of pH for chitosan-whey protein (CS-WP) nanoparticles prepared with different WP concentrations.

99.7 99.75 99.8 99.85 99.9 99.95 100

4 4.5 5 5.5 6 6.5 7 7.5 8

(2% WP)

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99.7 99.75 99.8 99.85 99.9 99.95 100

4 4.5 5 5.5 6 6.5 7 7.5 8

(4% WP)

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99.7 99.75 99.8 99.85 99.9 99.95 100

4 4.5 5 5.5 6 6.5 7 7.5 8 (8% WP)

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The acidification i.e. lower pH caused a marked decrease in the ability of whey protein to bind iron. Changes in pH generally affected the complex formation between metal ions and proteins as hydrogen ions compete with the metal ions for binding to protein. At low pH, the reactive side chains of amino groups tend to become protonated, which decreases their affinity for cations, thus reducing their complexation with the protein. The protonation of ionic amino acids at low pH reduced the ability of whey proteins to bind iron.

WP showed maximum binding of approximately 6 mol iron/mol protein (18 mg iron/g protein). This amount decreased markedly to approximately 1 mg iron/g protein as the pH decreased to 5.5 and was only approximately 1-2 mg iron/g protein at pH≤ 5.5.

It can be concluded from the obtained results that the added ratio of iron used in our experiments should be increased or the WP % decreased to observe the differences among all samples in iron encapsulation into CS-WP nanoparticles.

Iron release

In order to investigate the release profile of iron from CS-WP nanoparticles (in vitro), a simulated gastro-intestinal fluid in absence or the presence of digestive enzymes, (pepsin and pancreatin) was prepared and the results are demonstrated in Fig. (7).

In vitro release of iron into simulated gastric intestinal tract was evaluated for CS-WP nanoparticles. The release profile of iron in the presence or absence of digestive enzymes is presented in Fig. (7). In the simulated gastric medium at pH 1.2, less than 1% of the iron was released from nanoparticles after 6 h. of incubation at 37°C. The iron released after 6 h. from nanoparticles was very slow and may be needed more time to release more from CS-WP or use more iron concentration or decrease the whey protein concentration.The solid lines present iron release in the simulated gastric fluid (pH 1.2) with pepsin for 6 h and then transferred into the intestinal buffer (pH 7.5, without pancreatin) for 6 h.

Almost the same release profiles of iron from all kinds of nanoparticles in the simulated gastric fluid in the presence of the pepsin were observed compared to those in the absence of pepsin. The results obtained for iron release with or without enzyme didn't reveal any difference and after 6 h of agitation there was almost no iron release (less than 1%) for all studied CS-WP nanoparticles.

Fig. (7): Iron release from CS-WP nanoparticles after agitation for 6 h. in simulated gastric fluids at pH (1.2)/37°C with or without enzyme.

0 0.2 0.4 0.6 0.8 1

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It is interesting to notice that iron was very weakly released from CS-WP nanoparticles at different concentrations of protein suggesting a coating of protein with firmer structure on the surface of the CS-TPP core. As demonstrated in Fig. (7) no differences in release profile were observed for native WP, indicating that the WP shell could resist degradation of pepsin in the gastric tract. Pepsin is known to preferentially attack peptide bonds involving hydrophobic aromatic amino acids. In its native structure, WP is known to be resistant to hydrolysis by pepsin since its hydrophobic amino acids are located in the internal core of its calyx-like structure (Morr and Ha 1993). From above results, it seems that nanoparticles of CS-WP has potential to be applied as oral administration carriers for bioactive compounds such as iron due to desirable properties to resist both acid and pepsin effects in the gastric tract. Generally, iron release increased with increasing iron concentration while decreased with increasing WP concentration. Therefore it can be concluded that nanotechnology improved the bioavailability of iron and open the door to new functionalities and applications for nanoparticle delivery systems.

Acknowledgment

Great thanks to Dr. Daniel Otzen, Professor of Protein Biophysics at Aarhus University, Aarhus, Denmark, and his group members for helping and providing with different advises.

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Figure

Fig. (1): TEM photographs of CS-WP nanoparticles (2.0 mg/ml-2%) prepared at pH 5.5 of suspension containing 2% WP and 2 mg/ml CS
Fig. (2): Zeta potential (mV) of CS-WP nanoparticles with different WP concentration at different pH values
Fig. (3): Zeta potential (mv) of nanoparticles from CS-WP-iron complexes with various iron ratios as affected by pH value at different whey protein concentrations
Fig. (4): Association efficiency (AE) for chitosan-whey protein (CS-WP) nanoparticles as a function of pH and WP concentrations
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References

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