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Chapter 2TheAssemblyand Disassemblyof BiopolyelectrolyteMultilayersand Their Potential in theEncapsulation

and Controlled Releaseof ActiveIngredientsfromFoods

J . Moffat,R. Parker, T. R.Noel,D. Duta, and S. G . RingInstituteof Food Research,Norwich ResearchPark, Colney,

Norwich NR4 7UA, United Kingdom

The assembly of multilayers using layer-by-layer (LbL)deposition of oppositely charged biopolyelectrolytes has thepotential to form novel barriers with applications in controlleddelivery in the food and pharmaceutical industries. Multilayerassembly was studied using F T I R - A T R and a quartz crystalmicrobalance to give information on the massof the depositedlayers, their chemical characteristics and hydration. Thepolyanions examined were pectins of varying degrees ofesterification, while chitosan, poly-L-lysine and, at a pH belowits pI, -lactoglobulin were the polycations. Multilayerformation occurred at pH's when both anionic and cationicpolyelectrolytes carried a charge. The hydration of thestructures was sensitive to variations in pH and ionicstrength,resulting in environmentally responsive behavior. Disassembly could be triggered by change in pH, or pH history,resulting in an initial weakening of attractive interactions andsubsequent solubilisation of the components. A centrifugationtechnique for applying a chitosan/pectin multilayer coating toemulsion droplets is also described.

2009 American Chemical Society 35

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36Introduction

The structure and composition of foods affect the site and rateof releaseoftheir components and, if manipulated in a rational way, offers the opportunity forfoods with controlled or modified release functionalities. M ultiple phases arepresent in foods (/); both in foods themselves and when they are mixed withdigestive fluids and so interfacial processes assume a key role in the science ofthe release process. In pharmaceutical technology there are many types offunctional coatings and structures for achieving controlled and modified releaseof bioactive compounds (2). Designing foods as delivery systems, however,presentsa number of distinctlydifferent challenges.

Polyelectrolytemultilayerapproachesoffer ameansof structuring interfaceswith potentially useful functionalities (3). The majority of research has been onsynthetic polyelectrolyte multilayers(3, 4) but more recent studies have reportedresults for biopolyelectrolytes(5, 6) including food biopolymers (7, 8).

In previous work we have studied the assembly of biopolyelectrolytemultilayers comprising anionic polysaccharides with cationic polysaccharides,polyamino acids or globular proteins using arangeof complementary techniques(9, 10, 11). Surface plasmon resonancewas used to follow the sequential layerby layer deposition of pectin and chitosan in real-time under conditions ofcontinuous flow (9). The stoichiometry of pectin/poly-L-lysine multilayers wassuch that the polymers carry a net positive charge contrary to the commonassumption of intrinsic charge balance (//).

In the present work the aim was to identify the factors underlying thedisassembly of multilayers inresponseto changing environmental conditions. Inorder to test whether multilayer environmental responsiveness investigated uponplane surfaces can be related to release characteristics in three dimensionalsystems, the multilayers need to be deposited on disperse systems. Acentrifugation method for achieving this is also described.

Materialsand MethodsMaterials

Poly-L-lysine hydrobromide (PLL) with a mean degree of polymerizationof 70 and -lactoglobulin (BLG) were obtained from Sigma, low molecularweight chitosan (nominal degree of acetylation 75-85%) was obtained fromA ldrich, citrus pectins withdegreesof esterification 51% and 71% were obtainedfromC P K elco (Denmark) and citrus polygalacturonic acid ( P G A ) was obtainedfrom Fluka. D 2 0 (99.9%), hexadecane, L-a-phosphatidylcholine, sucrose andbuffer salts were also obtained from Sigma. Chemical and physicochemical

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37characterisation of the pectins has been reported previously (//, 12). Formultilayer deposition polymers were dissolved at a concentration of 0.6 mg mL "1in 10 mM buffer solutionwith 30 mM NaC l . Forbase layers P L L was dissolvedat a concentration of 0.08 mgmL " 1.

Multilayer-coatedemulsionpreparationFive g of hexadecane was blended for 3 min with 95 g of 1% w/w L-a-

phosphatidylcholine in 100 mM pH 3.0 acetatebuffer using an ultra Turrax T25fitted with an S-25N head. 1 mL of the emulsion was then dispersed in 40 mL of25% w/w sucrose. A gradient was formed in a 15 mL centrifuge tube in theorder: emulsion/25% w/w sucrose; chitosan/20% w/w sucrose; pectin (orPGA)/15% w/w sucrose; chitosan/10% w/w sucrose; pectin (or PGA) /5% w/wsucrose. The polymer concentration was 0.08 mg/mL in pH 3.6 acetate buffer.Each layer was separated by a layer of buffer containing sucrose of theappropriate concentration. After centrifugation at 2000 g for 30 min, emulsiondroplets were harvested from eachof the buffer layers.

Fourier Transform Infrared-Attenuated Total Reflection (FTIR-ATR)Spectroscopy

Infrared spectra were collected, over therange2000-1500 cm"1, on aNicolet860 F T I R spectrometer (Thermo Electron Corporation, Madison, USA) fittedwith a silicon cylindrical ATR crystal in a M icroC ircle liquid ATR cell(SpectraTech, Warrington,U K ) . Methods used were as detailed inKrzeminski etal. (11). The silicon crystal presented a silica surface for multilayer deposition.The multilayer was deposited by a layer-by-layer (LbL) procedure (3). A baselayer of the cationic polyaminoacid P L L is first deposited on the silica surfaceof the ATR crystal of the F T I R by injecting P L L solution into the sample cell.After an adsorption time of 3min, the excess solution is washed off by injectingbuffer solution (as used to dissolve the P L L ) . This is followed by injecting apolyanion solution, followed by a buffer wash, a polycation solution, a bufferwash and so on until the required number of layers has been deposited.Absorbances were measured relative to a baseline at 1800 cm"1 and quantitativeanalysis was based on methods of Sperlineet al. (13).

Quartz Crystal MicrobalanceMeasurements were made with a D300 quartz crystal microbalance with

dissipation monitoring( Q C M D , Q-Sense A B , Vstra Frlunda, Sweden) with a

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38Q A F C 302 axial flow measurements chamber as described inKrzeminski et al.,(11). The sensing element was a disc-shaped piezoelectric quartz crystalsandwiched between two gold electrodes which presented a gold surface formultilayer deposition. An approximate analysis of the frequency shift of themicrobalance can be made using the Sauerbrey approach (14) to give thehydrated mass of the adsorbed layer. A more refined analysis takes theviscoelasticity of the multilayer into account (15) and was performed usingQTools software (Q-SenseA B , as above).

Microelectrophoresis

Electrophoretic mobility of the emulsion droplets in pH 5.6 acetate bufferwas determined using a Zetasizer 3 (Malvern Instruments) fitted with a AZ4electrophoresis cell calibrated using a latex electrophoretic mobility standard.

ResultsThe disassembly of multilayers was studied by first assembling multilayers

directly on the surface of the sensing element of the spectrometer ormicrobalance and then following their behavior as the solvent conditions werechanged. Pectin/protein multilayers can be assembled at pH's below theisoelectric point of the protein under conditions under which the pectin carries anegative charge due to its ionized anhydrogalacturonic acid groups and theprotein carries a net positive charge because its positively charged residuesoutnumber its negatively charged residues. Figure 1 shows the result of a pHtitration on a 8 layer pectin/p-lactoglobulin multilayer probed using F T I R - A T R .

The spectrum of the multilayer in the frequency range 1500 - 1800 cm"1 isdominated by the amide I band (1635 cm"1) of the B L G with small absorptionbands arising from the - C O O D stretch vibrations of the anhydrogalacturonicacid groups (1730 cm"1), -C=0 uronic acid methyl ester and the asymmetricstretch o f - C O O " (1612 cm"1) of the pectin (16). The pK a of the uronic acid ofpectin vary in the range3.5 - 4.5 (17) and so both ionised and unionised speciescan be expected at pD 3.6. The carboxyl and carboxylate groups of the acidicamino acid residues (aspartic and glutamic acid) of B L G absorb in the region of1725 cm"1 and 1572 cm", respectively. Figure 1 shows spectra for the multilayeras it is rinsed with buffers of increasing pH over the range 3.6 - 5.4. Initially,over the range 3.6 - 4.8, the absorbance associated with carboxyl groups ofpectin and/or the protein (1725-1730 cm"1) shows a small decrease.Simultaneously there is an increase in the absorbance associated with theaspartateand glutamate residues of the protein (1572 cm"1). This indicates thatwhile there is an increasing number of negative charges on the protein and a

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