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Romanian Journal of Food Science

Official Journal of the Romanian Association of Food Professionals

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Romanian Journal of Food Science 2011, 1(1): 7073 70

Formation of polysaccharide multilayer coatings on emulsion droplets Denisa E. DU 1,*, Timothy R. NOEL 2 and Stephen G. RING 2 1 National Institute of Research&Development for Food Bioresources, 6 Dinu Vintila street, 021102 Bucharest, Romania 2 Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK Received 9 November 2010; received in revised form 12 January 2011; accepted 14 January 2011

Abstract

The sequential deposition of chitosan and pectin on a supported phospolipid bilayer was examined using a quartz crystal microbalance. Multilayer formation was observed. The coating of phospholipid-stabilized emulsion droplets through centrifugation in a stepped sucrose gradient, containing successive layers of pectin and chitosan, was then investigated. Microelectrophoresis of the emulsion droplets at different stages in the centrifugation revealed that the sign of the surface charge reversed on passing through each polymer layer, and was indicative of multilayer formation.

Keywords: pectin, chitosan, emulsion, multilayer.

1. Introduction

Polyelectrolyte multilayers may be formed by the sequential deposition of oppositely charged polymers (Decher, 1997). Typically they may be formed by sequential dipping of a solid substrate into solutions of the polyelectrolyte, followed by rinsing in buffer. Most studies have used synthetic polyelectrolytes, but these structures may also be formed from biopolymers (Richert et al., 2004). One of the potential applications of this approach is for the encapsulation and controlled release of active ingredients (Shi & Caruso, 2001). The coating functions as a barrier under one set of environmental conditions, and respond to other conditions by losing its barrier properties enabling release of an active ingredient.

In a food context the active ingredients could include flavour molecules (both desirable and unde-sirable), nutrients and bioactive phytochemicals. * Corresponding author: Tel./Fax: +40 21 2109128 E-mail address: denisa.duta@bioresurse.ro

Many of these molecular species are hydrophobic and easily formulated in emulsions.

There is current interest in engineering the surface of emulsion droplets, including the formation of multilayers at the droplet surface (McClements, 2005), to improve their stability, or affect release properties (Klinkesorn et al., 2005; Klinkesorn et al., 2006; Thanasukarn et al., 2006). In order to permit the evaluation of multilayers as coatings we examine the use of gradient centrifugation to form the coating, from oppositely charged biopolymers, on emulsion droplets. 2. Materials and methods 2.1. Materials

Low molecular weight chitosan, with a reported degree of deacetylation of 75-85% and L--phosphatidylcholine was obtained from Sigma. Citrus pectin with a degree of esterification (DE) of 71% was obtained from CP Kelco (Denmark). Polygalacturonic acid was obtained from Fluka.

Formation of polysaccharide multilayer coatings on emulsion droplets

Romanian Journal of Food Science 2011, 1(1): 7073 71

2.2. Vesicle preparation Small unilamellar vesicles (SUVs) were prepared

using the method outlined by Barenholz et al. (1977). Dissolved phospholipid was dried under N2 onto the walls of a rotating round-bottomed flask; suspended in 10 mM Tris buffer pH 8.0 and sonicated under N2 using a Bandelin Sonopuls HD2200 sonicator fitted with a 13 mm diameter titanium flat tipped probe. Titanium particles and larger phospholipid structures were removed by centrifugation at 150,000 g for 4 hours. The supernatant (top 2/3) was stored under N2 at 4C until required. 2.3. Microelectrophoresis

Electrophoretic mobility of the emulsion droplets in acetate buffer pH 5.6 was determined using a Zetasizer 3 (Malvern Instruments) fitted with an AZ4 electrophoresis cell. The calibration of the instrument was verified using a latex standard with an electrophoretic mobility of 3.892 0.124 mcm/Vs with a corresponding -potential of 49.5 1.6 mV (Malvern). 2.4. Preparation and coating of emulsion droplets

A quantity of 5 g of hexadecane was blended for 3 min with 95 g of 1% w/w L--phosphatidylcholine in 100 mM acetate buffer pH 3 using an ultra Turrax T25 fitted with an S-25N head. 1 mL of the emulsion was then dispersed in 40 mL of 25% sucrose. A gradient was formed in a 15 mL centrifuge tube in the order: emulsion in 25% w/w sucrose; chitosan 20% w/w sucrose; polygalactu-ronic acid (or pectin) 15% w/w sucrose; chitosan 10% w/w sucrose; polygalacturonic acid (or pectin) 5% w/w sucrose.

The polymer concentration was 0.08 mg/mL in acetate buffer pH 3.6. Each layer was separated by a layer of buffer containing sucrose of the appropriate concentration. After centrifugation at 2000 g for 30 min, emulsion droplets were harvested from the buffer layers and subjected to microelectrophoresis. 2.5. Quartz Crystal Microbalance

Measurements were carried out using a D300 quartz crystal microbalance with dissipation monitoring (QCMD) (Q-Sense AB, Vstra Frlunda, Sweden) with a QAFC 302 axial flow measurement chamber as described above (Krzeminski et al., 2006). The sensing element is a disc-shaped, AT-cut piezoelectric quartz crystal sandwiched between two gold electrodes. The crystal is excited to oscillation at its fundamental resonant frequency (~5 MHz). A

small mass deposited (m) on the gold sensing surface will cause a decrease in resonant frequency (fn). If the mass is deposited evenly and is sufficiently rigid then the mass adsorbed is directly proportional to the change in frequency according to the Sauerbrey equation (Sauerbrey, 1959):

n

fCm n= (1) where: C = 17.7 ng/(cm2 Hz) is the mass sensitivity constant; n the overtone number (n = 1, 3, 5) (Hook et al., 1998; Hook et al., 2001).

For multilayer fabrication 0.08 mg /mL solutions of chitosan and pectin in acetate buffer pH 5.6 containing 50 mM NaCl were prepared. Polymer solution was flowed into the sample chamber, left for 5 min to allow deposition, and then buffer was flowed in, left for 2 min before being replaced with the oppositely charged polymer solution. The process was repeated to allow formation of the multilayer. 2.6. Primary emulsion preparation

The following solutions are necessary for emulsion preparation: 100 mM acetic acid stock solution in water with pH 3; 1 % w/v phospholipid in acetic acid 100 mM; 5 %, 10 %, 15 %, 20 % and 25 % sucrose solutions in acetic acid 100 mM; 0.08 mg/ml chitosan in sucrose solution 20 % in acetic acid; 0.08 mg/ml pectin or polygalacturonic acid in 15 % sucrose solution in acetic acid; 0.08 mg/ml chitosan in 10% sucrose solution in acetic acid; 0.08 mg/ml pectin or polygalacturonic acid in sucrose solution 5 % in acetic acid; 0.1 % v/v primary emulsion in 25% sucrose solution in acetic acid.

For a complete homogenisation of chitosan in sucrose solution it is necessary to ultrasonate the solution and to vortex for 1 hour, than to maintain it for 1224 hours at 25C.

A value of 5 % w/v n-hexadecan are homoge-neised with trans-2-nonenal (50 % from its amount) with 95 % 1% phospholipid solution in acetic acid 100 mM. The flavour compounds trans-2-nonenal is soluble in n-hexadecan and insoluble in water.

The homogenisations is done with a mixer (Ultra-Turax 25 T, Janke & Kunkle IKA Labortech, UK), with a high speed 13.500 20.500 /min for 3 minutes. The final aspect of emulsion is as a milky liquid, homogenous, which is maintained at room temperature and quickly analysed. For further experiments, the primary emulsion is diluted to 0.1% in sucrose solution 25 % in acetic acid 100 mM.

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Romanian Journal of Food Science 2011, 1(1): 7073 72

3. Results and discussion

Initial experiments examined multilayer deposition on a solid-supported phospolipid bilayer. This was followed by investigation of the coating of phospholipid-stabilized emulsion droplets.

The sequential deposition of chitosan (polycation) and pectin (polyanion) on a L--phosphatidyl choline surface was examined using a quartz crystal microbalance. Unilamellar vesicles were initially deposited on the silicon sensor surface.

The vesicles rapidly collapsed, as shown in Figure 1, to form a solid-supported lipid bilayer, with a characteristic negative frequency change (Richter et al., 2006; Zhdanov & Kasemo, 2001).

The subsequent sequential deposition of chitosan and pectin lead to a series of step-wise changes in frequency response indicative of multilayer formation (Marudova et al., 2005). After each deposition, on rinsing with buffer, the frequency response was maintained indicating the essentially irreversible nature of the polyelectrolyte binding.

The observed frequency response increases with increasing layer number, an effect attributed to the mobility of the lower molecular size polyelectrolyte allowing its diffusion, but not escape from, the growing surface, with a result it that it can capture progressively more oppositely charged polyelec-trolyte (Hubsch et al., 2004; Lavalle et al., 2004). Multilayer formation is comparable at pH 5.6 and 3.6 as shown in Figure 2. The observed frequency change after deposition of 6 polymer layers is equivalent to the deposition of a hydrated mass of ~ 860 ng/cm2.

0 10 20 30 40 50-80

-70

-60

-50

-40

-30

-20

-10

0

10

Freq

uenc

y ch

ange

f3 (

Hz)

Time (mins)

Vesicles

ChitosanPectin

ChitosanPectin

ChitosanPectin

Figure 1. Negative frequency change as a function of

time for the deposition of a chitosan/pectin multilayer on a supported lipid bilayer.

Hexadecane emulsion droplets stabilised with L--phosphatidyl choline were then subjected to centrifugation in a stepped sucrose density gradient consisting of alternating layers of aqueous sucrose solutions containing 0.08 mg/mL of either chitosan or pectin.

A dense sucrose solution, containing the emulsion droplets was introduced into the bottom of the gradient, and on centrifugation, the less dense emulsion droplets creamed to the surface. The electrophoretic mobility of the emulsion droplets at each stage of the creaming process was examined.

0 1 2 3 4 5 6 7

0

10

20

30

40

50

60

-ve

Freq

uenc

y ch

ange

(f3,

Hz)

Layer number Figure 2. Negative frequency change as a function of layer number for the formation of a chitosan/pectin

multilayer at pH 5.6 () and 3.6 ().

Figure 3 shows the change in zeta potential of the droplets after passing through alternating layer of chitosan and pectin or polygalacturonic acid. Initially the droplets had a small -ve potential, and subsequently oscillated between +ve and ve potentials as the droplets passed through pectin or polygalacturonic acid solutions.

The observed charge reversal at each stage is indicative of multilayer formation on the emulsion droplet, with an oscillating change in charge at the multilayer surface. 4. Conclusions

Centrifugation in a stepped sucrose gradient is a convenient procedure for forming a biopolymer multilayer coating on emulsion droplets so that the potential functionality of these coatings for the controlled release of active food components may be evaluated.

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Formation of polysaccharide multilayer coatings on emulsion droplets

Romanian Journal of Food Science 2011, 1(1): 7073 73

-1 0 1 2 3 4 5

-8

-4

0

4

8

12

Zeta

Pot

entia

l (m

V)

Layer Number Figure 3. Zeta potential of emulsion droplets

as a function of layer number during their coating with chitosan and pectin (), or polygalacturonic

acid () layers.

Acknowledgments

The authors thank the BBSRC core strategic grant for support; and the EC commission for the award of a Marie Curie fellowship to Denisa Eglantina DU (contract number QLK-1999-50512).

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