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Evidence and characterization of complex coacervates containing plant proteins: application to the microencapsulation of oil droplets
Colloids and Surfaces A: Physicochem. Eng. Aspects 232 (2004) 239–247
Evidence and characterization of complex coacervates containing plant proteins: application to the microencapsulation of oil droplets V. Ducel a , J. Richard b , P. Saulnier a , Y. Popineau c , F. Boury a,∗ a
c
INSERM ERIT-M 0104, “Ingéniérie de la Vectorisation Particulaire”, 10 rue A. Boquel, 49100 Angers, France b Ethypharm, 194 bureaux de la colline, 92213 Saint Cloud, France INRA, URPVI “Protéines végétales et leurs interactions”, rue de la Géraudière, BP 71627, 44316 Nantes Cedex 3, France Received 7 July 2003
Abstract The potentiality of using vegetal proteins in complex coacervation was studied through zeta potential and turbidity measurements. Two model proteins were tested and compared: a cereal protein (alpha gliadin) and a leguminous protein (pea globulin). The effect of pH and value of the protein–anionic compound ratio were mainly investigated. The morphology of the coacervate was studied under different conditions and encapsulation of oil was achieved from the two proteins. Optimum coacervation was obtained with arabic gum at pH 2.75 for pea globulins with a protein–polysaccharide ratio of 30:70, and at pH 3 for alpha gliadins with a protein–polysaccharide ratio of 50:50. In addition to their charge density profile, this study showed that the steric conformation of both macromolecules forming the complex was a key parameter determining the ability of the coacervates to encapsulate oil droplets. © 2003 Elsevier B.V. All rights reserved. Keywords: Complex coacervation; Vegetal proteins; Zeta potential; Aggregation; Encapsulation
1. Introduction Complex coacervation is a phase separation process based on the simultaneous desolvation of oppositely charged polyelectrolytes induced by media modifications. The emergence of a dispersed phase of dense coacervates made of concentrated polyelectrolytes and a dilute equilibrium phase [1] is dependent on the pH, ionic strength and polyion concentrations. Gelatin, a collagen hydrolysis product, is widely used in complex coacervation. It is associated with different polysaccharides to neutralize its charge and thereby form a complex. Usually, the gelatin is positively charged and the coacervation is induced by anionic colloids like pectin, alginate, arabic gum, carboxymethylcellulose [2–5]. Polysaccharides positively charged like chitosan can be used if the protein is negatively charged [6]. Complex coacervation is used to protect sensitive products like vitamin from oxidation [7]. However, the use of gelatin generates a safety problem due to the emergence of the prion diseases. Albumin (bovine serum ∗
Corresponding author. Tel.: +33-2-41735907; fax: +33-2-41735853. E-mail address: [email protected] (F. Boury).
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albumin, BSA) was proposed as an alternative to gelatin because this protein was more biocompatible than gelatin [8,9], but the bovine origin of the protein makes it still suspected. The aim of this work was to study the ability of vegetal proteins to form coacervates in the presence of polysaccharides, so as to propose an alternative to the use of bovine proteins. The aim would be to develop biodegradable and biocompatible microcapsule systems suitable for oral administration. The use of vegetal proteins as vicilin [10] or gliadin [11] in simple coacervation has been reported in previous papers. Conversely, no previously reported work deals with the use of these vegetal proteins in a complex coacervation process.
2. Materials and methods 2.1. Materials Pea globulins were extracted at pilot scale from flour [12]. Globulins represent around 50% of the total proteins. Globulins are globular proteins composed of two fractions: 11S and 7S. 11S globulins are legumins, their structure is
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hexameric and their molecular weight is included between 350 and 400 kDa. These proteins have a beta sheet-rich structure and have also disulfure bridges. 7S is a glycoprotein called vicilin. It is a trimer with a molecular weight of 150 kDa. Alpha gliadin is a wheat protein, purified from a whole gliadin fraction by chromatography [13]. It is a monomeric protein, whose molecular weight is around 30 kDa, and contains six cysteins per molecule forming three intramolecular disulfide bonds [14]. These proteins were chosen to obtain concentrated preparations in the range usually used in the preparation processes of microparticles. Arabic gum was obtained from Cooper. A low viscosity grade of sodium alginate (5% (w/v) solution viscosity: 205 mPa/s) was provided by Danisco (Grinsted Alginate LFD 1505). The carboxymethylcellulose (Blanose 7 LF) was purchased from Aqualon and was a low viscosity grade (a viscosity of 25–50 mPa/s for a 2% (w/v) solution at 25 ◦ C). The polyphosphates were supplied by Giulini Chemie. Miglyol 812N, a mixture of medium chain triglycerides, was the model oil encapsulated and was kindly given by Sasol. 2.2. Coacervate preparation Vegetal proteins were dissolved in 1% (v/v) acetic acid solution (pH 2.9) because the solubilization of the protein was easier when the pH was far from the isoelectric point. This condition allowed to obtain a concentration of 10 g/l. Polysaccharides were solubilized in distilled water at 40 ◦ C under stirring (250 rpm until complete dissolution). The solutions were adjusted to the desired pH with a 25% (v/v) acetic acid solution and a 0.1 M sodium hydroxide solution. Coacervates were prepared at 30 ◦ C by adding the polysaccharide solution to protein solution under stirring. The 20 ml batches were prepared in 50 ml beakers and the mixtures stirred at 500 rpm using a mechanical stirrer (three blades of 2 cm diameter). 2.3. Zeta potential determination A 5 g/l polyion concentration was used to measure the zeta potential. The measurements were made using a Zetasizer 2000 (Malvern Instruments). Acid (50% acetic acid) and base (0.1 M sodium hydroxide) were automatically added by a titrator and the zeta potential was determined for each pH value. In order to measure the coacervate zeta potential, 100 l of coacervate is diluted in 10 ml of distilled water at the same pH value so as to allow the measurement of electrokinetic mobilities. All the zeta potential values were measured with an accuracy of ±2 mV. 2.4. Turbidity measurement Carboxymethylcellulose, alginate, arabic gum and protein solutions (0.05% w/v) were used for complex coacervation. The protein solution was kept under stirring at 500 rpm for
10 min at 30 ◦ C before adding the anionic polymer solution. After stirring for 1 min at 500 rpm, the turbidity of the mixture was determined from the absorbance at 600 nm (Uvikon 922, Kontron instruments spectrophotometer) in 1 cm thick optical cells against distilled water. In order to detect a possible precipitation of the components followed by sedimentation, two measurements were performed successively. Only the results of the first measurement was presented in the figures. 2.5. Preparation of microcapsules Encapsulation experiments have been carried out at different pH values and values of the protein/polysaccharide ratio that were chosen on the basis of the turbidity measurements. The solutions were kept under stirring for 10 min at 30 ◦ C, which is the temperature at the beginning of the encapsulation process. Miglyol 812N was emulsified in a solution of 10 g/l protein (o/w emulsion), the pH of the solution of polyions is adjusted using acetic acid and sodium hydroxide before the emulsion. The core/shell ratio was 70:30 (it corresponds to the weight ratio of the oil coated and the coating polymers). The emulsion was stirred at 500 rpm for 1 min before adding the polysaccharide with the same system used before for the coacervate preparation. After 1 min, the water bath was drained and the system was cooled to 10 ◦ C while maintaining agitation. 2.6. Particle size determination The morphology and surface structure of the coacervate droplets and microcapsules were observed using an optical microscope (Nikon, eclipse E600, Japan). The particle size was also estimated using the microscope. Laser granulometry (Mastersizer, Malvern) was used to complete the information obtained by means of optical microscopy. The measurement was done 1 min after having stopped the stirring. Some droplets of the coacervate preparation were added to a constant volume of water for which the pH was adjusted at the same value as for the coacervation process. The droplets were added until to obtain an obscuration that allowed the measurement. The stirring speed during the measurement was maintained constant between the different samples. D [4, 3] is the mean diameter in volume whereas D [3, 2] is the mean diameter in surface called “Sauter diameter”. D (v, 0.5) is the size for which 50% of the sample particles has a lower size and 50% has a upper size. D (v, 0.1) is the size for which 10% of the sample particles has a lower size and D (v, 0.9) is the particle size for which 90% of the sample particles has a lower size. The particle size distribution width is characterized by the relation: [D(0.9) − D(0.1)] which defines the Span number. D(0.5)
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3. Results 3.1. Zeta potential determination Complex coacervation between two polyions is dependent on many parameters. The most important condition to fulfill is to bring oppositely charged polyions close enough to each other to make them interact. This condition implies that the process is strongly pH sensitive, as the charges of the polyions change with pH values. To determine the pH value for which the charges interact and neutralize each other, many authors have measured electrophoretic mobilities in order to determine the zeta potential of the compounds implied. In fact, zeta potential corresponds to the electrokinetic potential of the particle, measured at the surface shear between the particle with its own ionic corona and the surrounding medium. Conversion of electrophoretic mobility values to zeta potential values is obtained by multiplying the electrophoretic mobility value by a factor depending on permittivity and the nature of the dispersion medium, as described by the Smoluckowski equation: εζ V = , (1) 4πη where ε is the permittivity and η the viscosity of the dispersion medium. The zeta potential measurement makes it possible to determine the appropriate pH range for coacervation. For instance, the gelatin–arabic gum [15,16], BSA–alginic acid [8] and BSA–arabic gum [9] systems have been investigated using this method. These different polyions have been previously adsorbed onto silica particles prior to measurement. In these studies, the authors determined the conditions where the attracting forces between the oppositely charged components have a maximum value. In the present study, we have measured the electrokinetic potential (ζ potential) versus pH of the polyanions (arabic gum, carboxymethylcellulose, polyphosphate and sodium alginate) and proteins (pea globulins and alpha gliadins) in suspension in aqueous medium. Proteins and polyions were not adsorbed on silica particles in order to approach as closer as possible the coacervation conditions. The related results are presented in Fig. 1. Carboxymethylcellulose is a cellulosic ether obtained by a reaction between alkali cellulose and sodium monochloroacetate. Hydroxyl functions are substituted by carboxymethyl groups and the number of hydroxyl groups substituted on anhydroglucose unit determines the subsitution degree. The viscosity of the solution increases with the chain length and the substitution degree. The 7LF carboxymethylcellulose (Blanose) used has a low viscosity. Alginates are composed of  d-mannuronic and ␣ l-guluronic acids. Arabic gum has a skeleton mainly composed of d-galactose units; glucuronic acid groups give it an anionic character.
Fig. 1. Zeta potential measurements of different proteins (a: (䉬) pea globulin, b: (䊉) alpha gliadin) and polyanions (c: (䊐) arabic gum, d: () carboxymethylcellulose, e: (×) polyphosphates, f: (+) sodium alginate) vs. pH.
Arabic gum, carboxymethylcellulose and alginate were found to exhibit a negative zeta potential, whose absolute value increased when pH was increased in a typical range characteristic of each molecule (typically between 2.2 and 4.5, Fig. 1). In this pH range, ionisation of carboxylic acid groups occurs. For higher values of pH, the zeta potential did not changed anymore. For pH values higher than 3, the absolute value of zeta potential increased more rapidly for carboxymethylcellulose, polyphosphates and alginate than for arabic gum. Sodium alginate showed the lowest negative value (highest absolute value) of zeta potential. For pea globulins, it was not possible to measure the zeta potential above a pH 4 value, because of the precipitation of the proteins. Furthermore, it is known that the isoelectric point of pea globulins is in the pH 4.5–5 range [17] which is consistent with our results. For lower pH values, the zeta potential is positive and its absolute value increased when the pH decreased. It appears that the zeta potential of the protein is exactly opposed to the zeta potential of the polyanions at pH 3.5 (for arabic gum, carboxymethylcellulose and polyphosphates) and pH 3 for alginate (Fig. 1). Under these conditions, an exact electric charge neutralization can be assumed. Concerning alpha gliadin, the zeta potential value remained constant and low (between 5 and 10 mV) in the pH range studied. This result is consistent with the literature data, since gliadin is known to be a slightly charged protein due to the nature of its amino-acids. The theoretical titration curves show that the charge number is only 13 at pH around 1 for alpha gliadin whereas it is 498 for legumin. Moreover, the decrease of the charge of gliadin is around ten times lower than the decrease of the charge of legumin in the studied pH range (pH 3–4) which was used for the determination of the zeta potentials of these proteins. The theoretical titration curves were obtained from the amino-acid composition of proteins thanks to MW/PI/titration curve software program available on Ex Pa Sy server.
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Fig. 2. Effect of colloid mixing ratio on the absorbance (600 nm) of different anionic compounds associated with pea globulin at pH 3.5 ((䉬) arabic gum, (䊐) carboxymethylcellulose, () sodium alginate). The maximum of absorbance corresponds to the maximum of coacervate yield.
3.2. Turbidity measurements
Fig. 4. Turbidity of protein/polyanion mixture vs. polyanion concentration (w/w) at pH 3.5 for the system gliadin/polyanion ((䉬) arabic gum, (䊐) carboxymethylcellulose).
Turbidity measurements were carried out to optimize the coacervation system. Low colloid concentrations of 0.05% (w/v) were used and the absorbance was measured at 600 nm. The appearance of turbidity is usually attributed to appearance of scattering particles or droplets in the medium, related to coacervate formation. Light scattering is dependent on the size of the dispersed particles, the wavelength of the light, the refractive index of the particles relative to that of the medium and the particle concentration. This method was used to monitor the complex coacervate formation from vegetal proteins (pea globulins and alpha gliadins) and different anionic compounds like arabic gum, carboxymethylcellulose and sodium alginate. The total volume of the mixture was kept constant; conversely the relative volumes of the two colloidal suspensions varied. In Figs. 2–5, the absorbance of the protein/polyanion mixture has been plotted versus the anionic compound fraction. First, the use of different anionic colloids was compared. The pH of each solution was set at 3.5 because for this pH value, both proteins and polysaccharides were soluble. Pea globulin was found to be complexed by arabic gum to form
coacervates (the protein/polysaccharide ratio was 50:50), whereas the other anionic compounds did not give rise to coacervation (Fig. 2). The maximum of absorbance observed for 20% of the other anionic colloids (carboxymethylcellulose or alginate) was a consequence of the precipitation of the system since we observed a decrease of the turbidity with time (results of the second measurement). This could be related to the formation of dense aggregates which precipitate. This precipitation was checked by microscopy. A complete characterization of the system pea globulin/arabic gum was carried out by varying pH. When the pH value increased, the arabic gum fraction necessary to form coacervates decreased (Fig. 3). At pH 3.5, the optimal coacervation condition indicated by the maximum of turbidity, was obtained for a protein/polysaccharide ratio of 50:50. This result is consistent with the observation that under these conditions, the zeta potential experiment showed the total neutralization of the charge of the protein and the polyanions (see Fig. 1). It makes it possible to propose a relationship between the charge neutralization and the formation of coacervate droplets. Furthermore, for the system pea globulin/arabic gum, the zeta potential of the two polymers decreased at the same rate with pH. This finding could
Fig. 3. Effect of protein/polysaccharide ratio and pH on the absorbance (600 nm) for the system pea globulin/arabic gum: determination of the optimal ratio for different pH ((䉬) pH 2.5; (+) pH 2.75; (䊏) pH 3, () pH 3.5; (×) pH 4).
Fig. 5. Effect of protein/polysaccharide ratio and pH on the absorbance (600 nm) for the system alpha gliadin–arabic gum: determination of the optimal ratio for different pH ((䉬) pH 2.5; (䊐) pH 3; () pH 3.5; (×) pH 4).
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Table 1 Effect of concentration and pH on the coacervate particle size obtained from the system pea globulin/arabic gum as determined by using laser granulometry 1 g/l
Fig. 6. Quantity of arabic gum necessary to add in a mixing pea globulin/arabic gum to reach coacervation. The percentage of arabic gum, which corresponds with the optimum determined on Fig. 3, is plotted vs. pH.
explain the linearity observed in the plot of the variations of the arabic gum fraction involved in coacervation versus pH (Fig. 6). We have studied the effect of ionic strength on the coacervation process for the system pea globulin/arabic gum, at pH 3.5, for a protein/polysaccharide ratio of 50:50. The ionic strength did not alter the process for a value up to 50 mM (results not shown). It is known that the addition of salt classically causes shielding of the charges on the polyions resulting in a weaker attraction and the disappearance of the formation of coacervates at a critical salt concentration [5]. In our case, it was not possible to observe this effect because the protein began to precipitate before. For the system pea globulin/carboxymethylcellulose, the system precipitated whatever the ionic strength (10 and 50 mM). The ability of alpha gliadins to form coacervates with different anionic compounds has also been evaluated by the same method. The protein showed the same ability to form cocervates with arabic gum as the previous protein studied (Fig. 4). The ratio protein/polysaccharide necessary to obtain coacervation was found to be less sensitive to pH variation than for globulins (Fig. 5). For a given pH value, different ratios were found to induce coacervation. This could be attributed to the small variation of zeta potential of gliadins with pH. The amount of arabic gum needed to form coacervates was found to be smaller for gliadins than for globulins. This is consistent with the fact that the latter proteins bear a higher charge. In addition, gliadins were found to be able to form aggregates in the presence of carboxymethylcellulose. However, this system did not look like the well-defined coacervate droplets we obtained with arabic gum and the continuous phase remained turbid after formation of the aggregates. In order to characterize the size and the morphology of the coacervates for different concentrations and pH values, we have used optical microscopy. 3.3. Coacervate observation Optical microscopy was used to study how the morphology and the size of coacervates changed with poly-
10 g/l
pH 2.75
pH 3.5
pH 2.75
pH 3.5
D [4, 3] D [3, 2] D (v, 0.5)
7.68 m 5.63 m 7.23 m
28.01 m 8.3 m 24.97 m
20.71 m 18.25 m 20.03 m
97.03 m 22.80 m 76.65 m
Span
1.37
2.18
0.93
2.35
mer concentration and pH. The coacervates were prepared from the system pea globulin/arabic gum at the optimal conditions determined previously. At pH 3.5, the protein/polysaccharide ratio used was 50:50, and at pH 2.75, the protein/polysaccharide ratio was 30:70. At pH 3.5, for a polymer concentration of 1 g/l, small spherical droplets of coacervates were obtained. The mean size was around 10 m (Fig. 7a). At the same pH but for a polymer concentration of 10 g/l, the size of the coacervates increased (more than 50 m). Some particles agglomerate and coalesce which give rise to various morphologies (Fig. 7b). At pH 2.75, small droplets, similar to those obtained at pH 3.5, were formed for a concentration of 1 g/l (results not shown). For a concentration of 10 g/l, the coacervate showed a more regular shape than at pH 3.5 and the size ranged ∼30 m (Fig. 7c). These results were confirmed by laser granulometry. We can see (Table 1) that D [4, 3] is close to D (v, 0.5) and these last parameters are in accordance with measurements performed by optical microscopy. On one hand, laser granulometry confirms the increase of particle size with pH and concentration. On the other hand, at pH 2.75, the size distribution of the particles exhibits a log-normal profile so that D [4, 3] is close to D [3, 2]. The polydispersity of the particle sizes is more pronounced at pH 3.5 that at pH 2.75 since the Span number is higher at pH 3.5. Moreover, the higher value of D [4, 3] than D [3, 2] confirms this polydispersity since D [3, 2] is a data influenced by the proportion of small particles. The coacervate obtained from alpha gliadin/arabic gum system was also studied. For a polymer concentration of 1 g/l, with a gliadin/arabic gum ratio of 70:30 and of 50:50 respectively at pH 3.5 and 3, the coacervates consisted of small droplets for the two pH values studied (Fig. 8a and b) and the size of the droplets was found to be bigger (10 m) at pH 3.5 (Fig. 8b). For a polymer concentration of 10 g/l, at pH 3, the coacervate droplets kept their spherical shape and their size ranged ∼25 m (Fig. 8c). The coacervates obtained at pH 3.5 showed some droplets with an ovoid shape, whose size could reach 50 m (Fig. 8d). The zeta potential of the different coacervates obtained had been measured. It was found to be close to zero for all the optimal coacervation pH values, for the coacervates
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Fig. 7. Photographs of coacervate particles obtained from the system pea globulin/arabic gum: (a) polymers concentration of 1 g/l, pH 3.5, the bar is 50 m; (b) polymers concentration of 10 g/l, pH 3.5, the bar is 50 m; (c) polymers concentration of 10 g/l, pH 2.75, the bar is 100 m.
obtained from pea globulins, and slightly negative in the case of alpha gliadins at pH 3 (−4 mV). This striking result likely means that the mechanism of coacervate formation is mainly electrostatic in the case of pea globulins. In the case of alpha gliadins, the net charge of coacervates points out an excess of charges due to polyanions. As a conclusion, the coacervate morphology was dependent on the nature of the protein used, the pH and the polymer concentration. For this reason, the coacervate ability to be included in a membrane to encapsulate oil has to be studied. 3.4. Microcapsule formulation The encapsulation experiments were carried out at different pH values corresponding to the values used for the study of the coacervate morphology. For the pea globulin/arabic gum system, at pH 3.5, the coacervate droplets were found to deposit around the oil droplets, but they did not actually coalesce, as evidenced from microcapsule shape, which
showed a very irregular surface (Fig. 9a). At pH 2.75, the deposition of the coacervates on the oil droplets was more regular than at pH 3.5 and the oil was completely encapsulated (Fig. 9b). For the system alpha gliadin/arabic gum, at pH 3, the oil droplets were totally wrapped in the coacervate phase which formed a layer with a constant thick (Fig. 10a). No free oil could be observed in the continuous phase. At pH 3.5, the coacervate layer was less homogeneous than at pH 3 (Fig. 10b). This study showed the ability of the coacervate formed from vegetal proteins to encapsulate oil. This process is nevertheless dependent on the pH and the nature of the protein. For a complete encapsulation of oil, it seems that the aggregation or coalescence of the coacervates has to be prevented. This is evidenced by comparison of Figs. 7b and 9a, where the wall of the capsules is made of a discontinuous arrangement of coacervates, and well organized coacervate phases without aggregation (Fig. 7c), leading to smooth walls at the surface of the droplets (Fig. 9b).
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Fig. 8. Photographs of coacervate particles obtained from the system alpha gliadin/arabic gum: (a) polymers concentration of 1 g/l, pH 3; (b) polymers concentration of 1 g/l, pH 3.5; (c) polymers concentration of 10 g/l, pH 3; (d) polymers concentration of 10 g/l, pH 3.5. The bar is 50 m.
Fig. 9. Photographs of microcapsules obtained from the system pea globulin/arabic gum: (a) coacervation at pH 3.5, the bar is 100 m; (b) coacervation at pH 2.75, the bar is 100 m.
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Fig. 10. Photographs of microcapsules obtained from the system alpha gliadin/arabic gum: (a) coacervation at pH 3, the bar is 100 m; (b) coacervation at pH 3.5, the bar is 100 m.
It means that formation of coacervate droplets must result from restricted and controlled precipitation of protein chains onto oil droplets.
4. Discussion Many theories have been proposed for the interpretation of the coacervation process. Bungenberg de Jong [1], who carried an extensive characterization of complex coacervation between gelatin and acacia, showed that coacervation was dependent on molecular weights, concentration and ratio of the two interacting polyions. The influence of the ionic strength, the temperature and the random coil configuration of both polyions used was also quoted. The Vies–Aranyi theory states that coacervation occurs in two steps: spontaneous aggregation and rearrangement, contrary to Overbeek and Voorn theory that supported a spontaneous process [20,21]. The latter theory assumes that solvent–solute interactions are negligible. Carboxymethylcellulose shows an extended structure in solution, whereas arabic gum is globular. The hydrodynamic volume and viscosity are higher in the case of an extended structure and the charges are more accessible. The random coil type structure of arabic gum allows a considerable quantity of water to be occluded between the chains of the macromolecules [8]. By studying the potentiality of use of BSA in complex coacervation, Singh and Burgess [8] had remarked that precipitation was favoured with alginic acid. This behaviour was related to alginic acid structure, which has an extended coil configuration in solution and a stiff ribbon-type structure in the condensed phase. Contrary to arabic gum, alginic acid has a tendency to crosslink chains packed together which minimizes the occluding of water. The penetration of water through the molecules was nevertheless a key factor to promote coacervation rather than precipitation.
In our conditions, the coacervation process needs some charges not to be neutralized to avoid a too extended desolvation. It is known in literature that at acidic pH, pea globulins loose their hexameric structure, their chains become unfold and the charges become more accessible [18]. In this case, it seemed necessary to preserve charges carried by the anionic compound. The arabic gum, which is composed of six carbohydrate moieties in addition to a small proportion of protein [19] and has a coiled configuration, can preserve charges. This could be at the origin of the efficiency of this compound in complex coacervation. This hypothesis seems to be confirmed with the complex coacervation process using gelatin, for which many anionic colloids lead to coacervation. In this case, gelatin which has a coiled structure, keeps a sufficient density of charges on its chains to avoid precipitation, whatever the anionic compound used. Only the protein/anionic polymer ratio has to be adjusted, so that electrostatic interactions can control the coacervation process. For a protein to be used in a coacervation process, it must have a particular conformation which is both related to its amino-acid nature and its charge density profile. A given protein can not be replaced by another one just based on molecular weight considerations. For example, the BSA/arabic gum system was proposed [9] to replace gelatin since the molecular weights and the charge density profiles were very close. However, the high viscosity of the coacervates obtained from BSA made the system completely unsuitable for microencapsulation. The viscosity was related to the polar amino-acid groups of BSA.
5. Conclusion This work clearly establishes that pea globulins and alpha gliadins can be used in a complex coacervation process. For both proteins, arabic gum was shown to be the best anionic compound to form a complex coacervate. The conditions for
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coacervation (protein/anionic compound ratio) were found to be less restrictive for gliadins. On the other hand, the advantage of using pea globulins is that they are not allergen. The zeta potential measurements allowed us to determine the pH range where the protein is positively charged and the polysaccharide negatively charged. This method was used to optimize the pH conditions for coacervation and make easier the determination of the other parameters. Zeta potential determinations associated with turbidity measurements were shown to be an appropriate, smart method to carry out the preformulation study. From a prospective viewpoint, it would now be interesting to study the protein conformation under different conditions and when mixed with different anionic compounds. This study should help to predict the more adapted anionic polymer to induce formation of stable, individual complex coacervates for each protein.
Acknowledgements The authors wish to thank O. Thomas (MAINELAB SA) and C. Blassel (INRA, Nantes) for technical assistance.
References [1] M.G. Bungenberg de Jong, in: G.R. Kruyt (Ed.), Colloid Science, vol. II, Reversible Systems, Elsevier, New York, 1949, pp. 335–432.
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[2] J.N. McMullen, D.W. Newton, C.H. Becker, J. Pharm. Sci. 71 (1982) 628–632. [3] J.N. McMullen, D.W. Newton, C.H. Becker, J. Pharm. Sci. 73 (1984) 1799–1803. [4] I. Joseph, S. Venkataram, J. Pharm. 126 (1995) 161–168. [5] G.L. Koh, I.G. Tucker, J. Pharm. Pharmacol. 40 (1987) 233–236. [6] C. Lopez, R. Bodmeier, Int. J. Pharm. 135 (1996) 63–72. [7] V.B. Junyaprasert, A. Mitrevej, N. Sinchaipanid, P. Boonme, D.E. Wurster, Drug Dev. Ind. Pharm. 27 (6) (2001) 561–566. [8] O.N. Singh, D.J. Burgess, J. Pharm. Pharmacol. 41 (1989) 670–673. [9] D.J. Burgess, K.K. Kwok, P.T. Megremis, J. Pharm. Pharmacol. 43 (1991) 232–236. [10] I. Ezpeleta, J.M. Irache, S. Stainmesse, J. Gueguen, A.M. Orecchionin, Eur. J. Pharm. Biopharm. 42 (1996) 36–41. [11] M.C. Mauguet, J. Legrand, L. Brujes, G. Carnelle, C. Larré, Y. Popineau, J. Microencapsulation 19 (2002) 377–384. [12] I. Crévieu, S. Bérot, J. Guéguen, Nahrung 40 (5) (1996) 237–244. [13] C. Larré, Y. Popineau, W. Loisel, J. Cereal Sci. 14 (1991) 231–241. [14] M.C. Gianibelli, O.R. Larroque, F. MacRitchie, C.W. Wrigley, American Association of Cereal Chemists, 2001, no. C-2001-0926-01O, pp. 1–20. [15] D.J. Burgess, J.E. Carless, J. Colloid Interface Sci. 98 (1) (1984) 1–8. [16] H.J.W. Peters, E.M.G. van Bommel, J.G. Fokkens, Drug Dev. Ind. Pharm. 18 (1992) 123–134. [17] J. Gueguen, M. Chevalier, J. Barbot, F. Schaeffer, J. Sci. Food Agric. 44 (1988) 167–182. [18] M. Subirade, J. Gueguen, K.D. Schwenke, J. Colloid Interface Sci. 152 (2) (1992) 442–454. [19] A.M. Islam, G.O. Phillips, A. Sljivo, M.J. Snowden, P.A. Williams, Food Hydrocolloids 11 (4) (1997) 493–505. [20] J.T.G. Overbeek, M.J. Voorn, J. Cell. Comp. Physiol. 49 (Suppl. 1) (1957) 7–26. [21] A. Vies, C.J. Aranyi, J. Phys. Chem. 64 (1960) 1203–1210.
Evidence and characterization of complex coacervates containing plant proteins: application to the microencapsulation of oil droplets V. Ducel a , J. Richard b , P. Saulnier a , Y. Popineau c , F. Boury a,∗ a
c
INSERM ERIT-M 0104, “Ingéniérie de la Vectorisation Particulaire”, 10 rue A. Boquel, 49100 Angers, France b Ethypharm, 194 bureaux de la colline, 92213 Saint Cloud, France INRA, URPVI “Protéines végétales et leurs interactions”, rue de la Géraudière, BP 71627, 44316 Nantes Cedex 3, France Received 7 July 2003
Abstract The potentiality of using vegetal proteins in complex coacervation was studied through zeta potential and turbidity measurements. Two model proteins were tested and compared: a cereal protein (alpha gliadin) and a leguminous protein (pea globulin). The effect of pH and value of the protein–anionic compound ratio were mainly investigated. The morphology of the coacervate was studied under different conditions and encapsulation of oil was achieved from the two proteins. Optimum coacervation was obtained with arabic gum at pH 2.75 for pea globulins with a protein–polysaccharide ratio of 30:70, and at pH 3 for alpha gliadins with a protein–polysaccharide ratio of 50:50. In addition to their charge density profile, this study showed that the steric conformation of both macromolecules forming the complex was a key parameter determining the ability of the coacervates to encapsulate oil droplets. © 2003 Elsevier B.V. All rights reserved. Keywords: Complex coacervation; Vegetal proteins; Zeta potential; Aggregation; Encapsulation
1. Introduction Complex coacervation is a phase separation process based on the simultaneous desolvation of oppositely charged polyelectrolytes induced by media modifications. The emergence of a dispersed phase of dense coacervates made of concentrated polyelectrolytes and a dilute equilibrium phase [1] is dependent on the pH, ionic strength and polyion concentrations. Gelatin, a collagen hydrolysis product, is widely used in complex coacervation. It is associated with different polysaccharides to neutralize its charge and thereby form a complex. Usually, the gelatin is positively charged and the coacervation is induced by anionic colloids like pectin, alginate, arabic gum, carboxymethylcellulose [2–5]. Polysaccharides positively charged like chitosan can be used if the protein is negatively charged [6]. Complex coacervation is used to protect sensitive products like vitamin from oxidation [7]. However, the use of gelatin generates a safety problem due to the emergence of the prion diseases. Albumin (bovine serum ∗
Corresponding author. Tel.: +33-2-41735907; fax: +33-2-41735853. E-mail address: [email protected] (F. Boury).
0927-7757/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2003.11.001
albumin, BSA) was proposed as an alternative to gelatin because this protein was more biocompatible than gelatin [8,9], but the bovine origin of the protein makes it still suspected. The aim of this work was to study the ability of vegetal proteins to form coacervates in the presence of polysaccharides, so as to propose an alternative to the use of bovine proteins. The aim would be to develop biodegradable and biocompatible microcapsule systems suitable for oral administration. The use of vegetal proteins as vicilin [10] or gliadin [11] in simple coacervation has been reported in previous papers. Conversely, no previously reported work deals with the use of these vegetal proteins in a complex coacervation process.
2. Materials and methods 2.1. Materials Pea globulins were extracted at pilot scale from flour [12]. Globulins represent around 50% of the total proteins. Globulins are globular proteins composed of two fractions: 11S and 7S. 11S globulins are legumins, their structure is
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hexameric and their molecular weight is included between 350 and 400 kDa. These proteins have a beta sheet-rich structure and have also disulfure bridges. 7S is a glycoprotein called vicilin. It is a trimer with a molecular weight of 150 kDa. Alpha gliadin is a wheat protein, purified from a whole gliadin fraction by chromatography [13]. It is a monomeric protein, whose molecular weight is around 30 kDa, and contains six cysteins per molecule forming three intramolecular disulfide bonds [14]. These proteins were chosen to obtain concentrated preparations in the range usually used in the preparation processes of microparticles. Arabic gum was obtained from Cooper. A low viscosity grade of sodium alginate (5% (w/v) solution viscosity: 205 mPa/s) was provided by Danisco (Grinsted Alginate LFD 1505). The carboxymethylcellulose (Blanose 7 LF) was purchased from Aqualon and was a low viscosity grade (a viscosity of 25–50 mPa/s for a 2% (w/v) solution at 25 ◦ C). The polyphosphates were supplied by Giulini Chemie. Miglyol 812N, a mixture of medium chain triglycerides, was the model oil encapsulated and was kindly given by Sasol. 2.2. Coacervate preparation Vegetal proteins were dissolved in 1% (v/v) acetic acid solution (pH 2.9) because the solubilization of the protein was easier when the pH was far from the isoelectric point. This condition allowed to obtain a concentration of 10 g/l. Polysaccharides were solubilized in distilled water at 40 ◦ C under stirring (250 rpm until complete dissolution). The solutions were adjusted to the desired pH with a 25% (v/v) acetic acid solution and a 0.1 M sodium hydroxide solution. Coacervates were prepared at 30 ◦ C by adding the polysaccharide solution to protein solution under stirring. The 20 ml batches were prepared in 50 ml beakers and the mixtures stirred at 500 rpm using a mechanical stirrer (three blades of 2 cm diameter). 2.3. Zeta potential determination A 5 g/l polyion concentration was used to measure the zeta potential. The measurements were made using a Zetasizer 2000 (Malvern Instruments). Acid (50% acetic acid) and base (0.1 M sodium hydroxide) were automatically added by a titrator and the zeta potential was determined for each pH value. In order to measure the coacervate zeta potential, 100 l of coacervate is diluted in 10 ml of distilled water at the same pH value so as to allow the measurement of electrokinetic mobilities. All the zeta potential values were measured with an accuracy of ±2 mV. 2.4. Turbidity measurement Carboxymethylcellulose, alginate, arabic gum and protein solutions (0.05% w/v) were used for complex coacervation. The protein solution was kept under stirring at 500 rpm for
10 min at 30 ◦ C before adding the anionic polymer solution. After stirring for 1 min at 500 rpm, the turbidity of the mixture was determined from the absorbance at 600 nm (Uvikon 922, Kontron instruments spectrophotometer) in 1 cm thick optical cells against distilled water. In order to detect a possible precipitation of the components followed by sedimentation, two measurements were performed successively. Only the results of the first measurement was presented in the figures. 2.5. Preparation of microcapsules Encapsulation experiments have been carried out at different pH values and values of the protein/polysaccharide ratio that were chosen on the basis of the turbidity measurements. The solutions were kept under stirring for 10 min at 30 ◦ C, which is the temperature at the beginning of the encapsulation process. Miglyol 812N was emulsified in a solution of 10 g/l protein (o/w emulsion), the pH of the solution of polyions is adjusted using acetic acid and sodium hydroxide before the emulsion. The core/shell ratio was 70:30 (it corresponds to the weight ratio of the oil coated and the coating polymers). The emulsion was stirred at 500 rpm for 1 min before adding the polysaccharide with the same system used before for the coacervate preparation. After 1 min, the water bath was drained and the system was cooled to 10 ◦ C while maintaining agitation. 2.6. Particle size determination The morphology and surface structure of the coacervate droplets and microcapsules were observed using an optical microscope (Nikon, eclipse E600, Japan). The particle size was also estimated using the microscope. Laser granulometry (Mastersizer, Malvern) was used to complete the information obtained by means of optical microscopy. The measurement was done 1 min after having stopped the stirring. Some droplets of the coacervate preparation were added to a constant volume of water for which the pH was adjusted at the same value as for the coacervation process. The droplets were added until to obtain an obscuration that allowed the measurement. The stirring speed during the measurement was maintained constant between the different samples. D [4, 3] is the mean diameter in volume whereas D [3, 2] is the mean diameter in surface called “Sauter diameter”. D (v, 0.5) is the size for which 50% of the sample particles has a lower size and 50% has a upper size. D (v, 0.1) is the size for which 10% of the sample particles has a lower size and D (v, 0.9) is the particle size for which 90% of the sample particles has a lower size. The particle size distribution width is characterized by the relation: [D(0.9) − D(0.1)] which defines the Span number. D(0.5)
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3. Results 3.1. Zeta potential determination Complex coacervation between two polyions is dependent on many parameters. The most important condition to fulfill is to bring oppositely charged polyions close enough to each other to make them interact. This condition implies that the process is strongly pH sensitive, as the charges of the polyions change with pH values. To determine the pH value for which the charges interact and neutralize each other, many authors have measured electrophoretic mobilities in order to determine the zeta potential of the compounds implied. In fact, zeta potential corresponds to the electrokinetic potential of the particle, measured at the surface shear between the particle with its own ionic corona and the surrounding medium. Conversion of electrophoretic mobility values to zeta potential values is obtained by multiplying the electrophoretic mobility value by a factor depending on permittivity and the nature of the dispersion medium, as described by the Smoluckowski equation: εζ V = , (1) 4πη where ε is the permittivity and η the viscosity of the dispersion medium. The zeta potential measurement makes it possible to determine the appropriate pH range for coacervation. For instance, the gelatin–arabic gum [15,16], BSA–alginic acid [8] and BSA–arabic gum [9] systems have been investigated using this method. These different polyions have been previously adsorbed onto silica particles prior to measurement. In these studies, the authors determined the conditions where the attracting forces between the oppositely charged components have a maximum value. In the present study, we have measured the electrokinetic potential (ζ potential) versus pH of the polyanions (arabic gum, carboxymethylcellulose, polyphosphate and sodium alginate) and proteins (pea globulins and alpha gliadins) in suspension in aqueous medium. Proteins and polyions were not adsorbed on silica particles in order to approach as closer as possible the coacervation conditions. The related results are presented in Fig. 1. Carboxymethylcellulose is a cellulosic ether obtained by a reaction between alkali cellulose and sodium monochloroacetate. Hydroxyl functions are substituted by carboxymethyl groups and the number of hydroxyl groups substituted on anhydroglucose unit determines the subsitution degree. The viscosity of the solution increases with the chain length and the substitution degree. The 7LF carboxymethylcellulose (Blanose) used has a low viscosity. Alginates are composed of  d-mannuronic and ␣ l-guluronic acids. Arabic gum has a skeleton mainly composed of d-galactose units; glucuronic acid groups give it an anionic character.
Fig. 1. Zeta potential measurements of different proteins (a: (䉬) pea globulin, b: (䊉) alpha gliadin) and polyanions (c: (䊐) arabic gum, d: () carboxymethylcellulose, e: (×) polyphosphates, f: (+) sodium alginate) vs. pH.
Arabic gum, carboxymethylcellulose and alginate were found to exhibit a negative zeta potential, whose absolute value increased when pH was increased in a typical range characteristic of each molecule (typically between 2.2 and 4.5, Fig. 1). In this pH range, ionisation of carboxylic acid groups occurs. For higher values of pH, the zeta potential did not changed anymore. For pH values higher than 3, the absolute value of zeta potential increased more rapidly for carboxymethylcellulose, polyphosphates and alginate than for arabic gum. Sodium alginate showed the lowest negative value (highest absolute value) of zeta potential. For pea globulins, it was not possible to measure the zeta potential above a pH 4 value, because of the precipitation of the proteins. Furthermore, it is known that the isoelectric point of pea globulins is in the pH 4.5–5 range [17] which is consistent with our results. For lower pH values, the zeta potential is positive and its absolute value increased when the pH decreased. It appears that the zeta potential of the protein is exactly opposed to the zeta potential of the polyanions at pH 3.5 (for arabic gum, carboxymethylcellulose and polyphosphates) and pH 3 for alginate (Fig. 1). Under these conditions, an exact electric charge neutralization can be assumed. Concerning alpha gliadin, the zeta potential value remained constant and low (between 5 and 10 mV) in the pH range studied. This result is consistent with the literature data, since gliadin is known to be a slightly charged protein due to the nature of its amino-acids. The theoretical titration curves show that the charge number is only 13 at pH around 1 for alpha gliadin whereas it is 498 for legumin. Moreover, the decrease of the charge of gliadin is around ten times lower than the decrease of the charge of legumin in the studied pH range (pH 3–4) which was used for the determination of the zeta potentials of these proteins. The theoretical titration curves were obtained from the amino-acid composition of proteins thanks to MW/PI/titration curve software program available on Ex Pa Sy server.
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Fig. 2. Effect of colloid mixing ratio on the absorbance (600 nm) of different anionic compounds associated with pea globulin at pH 3.5 ((䉬) arabic gum, (䊐) carboxymethylcellulose, () sodium alginate). The maximum of absorbance corresponds to the maximum of coacervate yield.
3.2. Turbidity measurements
Fig. 4. Turbidity of protein/polyanion mixture vs. polyanion concentration (w/w) at pH 3.5 for the system gliadin/polyanion ((䉬) arabic gum, (䊐) carboxymethylcellulose).
Turbidity measurements were carried out to optimize the coacervation system. Low colloid concentrations of 0.05% (w/v) were used and the absorbance was measured at 600 nm. The appearance of turbidity is usually attributed to appearance of scattering particles or droplets in the medium, related to coacervate formation. Light scattering is dependent on the size of the dispersed particles, the wavelength of the light, the refractive index of the particles relative to that of the medium and the particle concentration. This method was used to monitor the complex coacervate formation from vegetal proteins (pea globulins and alpha gliadins) and different anionic compounds like arabic gum, carboxymethylcellulose and sodium alginate. The total volume of the mixture was kept constant; conversely the relative volumes of the two colloidal suspensions varied. In Figs. 2–5, the absorbance of the protein/polyanion mixture has been plotted versus the anionic compound fraction. First, the use of different anionic colloids was compared. The pH of each solution was set at 3.5 because for this pH value, both proteins and polysaccharides were soluble. Pea globulin was found to be complexed by arabic gum to form
coacervates (the protein/polysaccharide ratio was 50:50), whereas the other anionic compounds did not give rise to coacervation (Fig. 2). The maximum of absorbance observed for 20% of the other anionic colloids (carboxymethylcellulose or alginate) was a consequence of the precipitation of the system since we observed a decrease of the turbidity with time (results of the second measurement). This could be related to the formation of dense aggregates which precipitate. This precipitation was checked by microscopy. A complete characterization of the system pea globulin/arabic gum was carried out by varying pH. When the pH value increased, the arabic gum fraction necessary to form coacervates decreased (Fig. 3). At pH 3.5, the optimal coacervation condition indicated by the maximum of turbidity, was obtained for a protein/polysaccharide ratio of 50:50. This result is consistent with the observation that under these conditions, the zeta potential experiment showed the total neutralization of the charge of the protein and the polyanions (see Fig. 1). It makes it possible to propose a relationship between the charge neutralization and the formation of coacervate droplets. Furthermore, for the system pea globulin/arabic gum, the zeta potential of the two polymers decreased at the same rate with pH. This finding could
Fig. 3. Effect of protein/polysaccharide ratio and pH on the absorbance (600 nm) for the system pea globulin/arabic gum: determination of the optimal ratio for different pH ((䉬) pH 2.5; (+) pH 2.75; (䊏) pH 3, () pH 3.5; (×) pH 4).
Fig. 5. Effect of protein/polysaccharide ratio and pH on the absorbance (600 nm) for the system alpha gliadin–arabic gum: determination of the optimal ratio for different pH ((䉬) pH 2.5; (䊐) pH 3; () pH 3.5; (×) pH 4).
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Table 1 Effect of concentration and pH on the coacervate particle size obtained from the system pea globulin/arabic gum as determined by using laser granulometry 1 g/l
Fig. 6. Quantity of arabic gum necessary to add in a mixing pea globulin/arabic gum to reach coacervation. The percentage of arabic gum, which corresponds with the optimum determined on Fig. 3, is plotted vs. pH.
explain the linearity observed in the plot of the variations of the arabic gum fraction involved in coacervation versus pH (Fig. 6). We have studied the effect of ionic strength on the coacervation process for the system pea globulin/arabic gum, at pH 3.5, for a protein/polysaccharide ratio of 50:50. The ionic strength did not alter the process for a value up to 50 mM (results not shown). It is known that the addition of salt classically causes shielding of the charges on the polyions resulting in a weaker attraction and the disappearance of the formation of coacervates at a critical salt concentration [5]. In our case, it was not possible to observe this effect because the protein began to precipitate before. For the system pea globulin/carboxymethylcellulose, the system precipitated whatever the ionic strength (10 and 50 mM). The ability of alpha gliadins to form coacervates with different anionic compounds has also been evaluated by the same method. The protein showed the same ability to form cocervates with arabic gum as the previous protein studied (Fig. 4). The ratio protein/polysaccharide necessary to obtain coacervation was found to be less sensitive to pH variation than for globulins (Fig. 5). For a given pH value, different ratios were found to induce coacervation. This could be attributed to the small variation of zeta potential of gliadins with pH. The amount of arabic gum needed to form coacervates was found to be smaller for gliadins than for globulins. This is consistent with the fact that the latter proteins bear a higher charge. In addition, gliadins were found to be able to form aggregates in the presence of carboxymethylcellulose. However, this system did not look like the well-defined coacervate droplets we obtained with arabic gum and the continuous phase remained turbid after formation of the aggregates. In order to characterize the size and the morphology of the coacervates for different concentrations and pH values, we have used optical microscopy. 3.3. Coacervate observation Optical microscopy was used to study how the morphology and the size of coacervates changed with poly-
10 g/l
pH 2.75
pH 3.5
pH 2.75
pH 3.5
D [4, 3] D [3, 2] D (v, 0.5)
7.68 m 5.63 m 7.23 m
28.01 m 8.3 m 24.97 m
20.71 m 18.25 m 20.03 m
97.03 m 22.80 m 76.65 m
Span
1.37
2.18
0.93
2.35
mer concentration and pH. The coacervates were prepared from the system pea globulin/arabic gum at the optimal conditions determined previously. At pH 3.5, the protein/polysaccharide ratio used was 50:50, and at pH 2.75, the protein/polysaccharide ratio was 30:70. At pH 3.5, for a polymer concentration of 1 g/l, small spherical droplets of coacervates were obtained. The mean size was around 10 m (Fig. 7a). At the same pH but for a polymer concentration of 10 g/l, the size of the coacervates increased (more than 50 m). Some particles agglomerate and coalesce which give rise to various morphologies (Fig. 7b). At pH 2.75, small droplets, similar to those obtained at pH 3.5, were formed for a concentration of 1 g/l (results not shown). For a concentration of 10 g/l, the coacervate showed a more regular shape than at pH 3.5 and the size ranged ∼30 m (Fig. 7c). These results were confirmed by laser granulometry. We can see (Table 1) that D [4, 3] is close to D (v, 0.5) and these last parameters are in accordance with measurements performed by optical microscopy. On one hand, laser granulometry confirms the increase of particle size with pH and concentration. On the other hand, at pH 2.75, the size distribution of the particles exhibits a log-normal profile so that D [4, 3] is close to D [3, 2]. The polydispersity of the particle sizes is more pronounced at pH 3.5 that at pH 2.75 since the Span number is higher at pH 3.5. Moreover, the higher value of D [4, 3] than D [3, 2] confirms this polydispersity since D [3, 2] is a data influenced by the proportion of small particles. The coacervate obtained from alpha gliadin/arabic gum system was also studied. For a polymer concentration of 1 g/l, with a gliadin/arabic gum ratio of 70:30 and of 50:50 respectively at pH 3.5 and 3, the coacervates consisted of small droplets for the two pH values studied (Fig. 8a and b) and the size of the droplets was found to be bigger (10 m) at pH 3.5 (Fig. 8b). For a polymer concentration of 10 g/l, at pH 3, the coacervate droplets kept their spherical shape and their size ranged ∼25 m (Fig. 8c). The coacervates obtained at pH 3.5 showed some droplets with an ovoid shape, whose size could reach 50 m (Fig. 8d). The zeta potential of the different coacervates obtained had been measured. It was found to be close to zero for all the optimal coacervation pH values, for the coacervates
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Fig. 7. Photographs of coacervate particles obtained from the system pea globulin/arabic gum: (a) polymers concentration of 1 g/l, pH 3.5, the bar is 50 m; (b) polymers concentration of 10 g/l, pH 3.5, the bar is 50 m; (c) polymers concentration of 10 g/l, pH 2.75, the bar is 100 m.
obtained from pea globulins, and slightly negative in the case of alpha gliadins at pH 3 (−4 mV). This striking result likely means that the mechanism of coacervate formation is mainly electrostatic in the case of pea globulins. In the case of alpha gliadins, the net charge of coacervates points out an excess of charges due to polyanions. As a conclusion, the coacervate morphology was dependent on the nature of the protein used, the pH and the polymer concentration. For this reason, the coacervate ability to be included in a membrane to encapsulate oil has to be studied. 3.4. Microcapsule formulation The encapsulation experiments were carried out at different pH values corresponding to the values used for the study of the coacervate morphology. For the pea globulin/arabic gum system, at pH 3.5, the coacervate droplets were found to deposit around the oil droplets, but they did not actually coalesce, as evidenced from microcapsule shape, which
showed a very irregular surface (Fig. 9a). At pH 2.75, the deposition of the coacervates on the oil droplets was more regular than at pH 3.5 and the oil was completely encapsulated (Fig. 9b). For the system alpha gliadin/arabic gum, at pH 3, the oil droplets were totally wrapped in the coacervate phase which formed a layer with a constant thick (Fig. 10a). No free oil could be observed in the continuous phase. At pH 3.5, the coacervate layer was less homogeneous than at pH 3 (Fig. 10b). This study showed the ability of the coacervate formed from vegetal proteins to encapsulate oil. This process is nevertheless dependent on the pH and the nature of the protein. For a complete encapsulation of oil, it seems that the aggregation or coalescence of the coacervates has to be prevented. This is evidenced by comparison of Figs. 7b and 9a, where the wall of the capsules is made of a discontinuous arrangement of coacervates, and well organized coacervate phases without aggregation (Fig. 7c), leading to smooth walls at the surface of the droplets (Fig. 9b).
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Fig. 8. Photographs of coacervate particles obtained from the system alpha gliadin/arabic gum: (a) polymers concentration of 1 g/l, pH 3; (b) polymers concentration of 1 g/l, pH 3.5; (c) polymers concentration of 10 g/l, pH 3; (d) polymers concentration of 10 g/l, pH 3.5. The bar is 50 m.
Fig. 9. Photographs of microcapsules obtained from the system pea globulin/arabic gum: (a) coacervation at pH 3.5, the bar is 100 m; (b) coacervation at pH 2.75, the bar is 100 m.
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Fig. 10. Photographs of microcapsules obtained from the system alpha gliadin/arabic gum: (a) coacervation at pH 3, the bar is 100 m; (b) coacervation at pH 3.5, the bar is 100 m.
It means that formation of coacervate droplets must result from restricted and controlled precipitation of protein chains onto oil droplets.
4. Discussion Many theories have been proposed for the interpretation of the coacervation process. Bungenberg de Jong [1], who carried an extensive characterization of complex coacervation between gelatin and acacia, showed that coacervation was dependent on molecular weights, concentration and ratio of the two interacting polyions. The influence of the ionic strength, the temperature and the random coil configuration of both polyions used was also quoted. The Vies–Aranyi theory states that coacervation occurs in two steps: spontaneous aggregation and rearrangement, contrary to Overbeek and Voorn theory that supported a spontaneous process [20,21]. The latter theory assumes that solvent–solute interactions are negligible. Carboxymethylcellulose shows an extended structure in solution, whereas arabic gum is globular. The hydrodynamic volume and viscosity are higher in the case of an extended structure and the charges are more accessible. The random coil type structure of arabic gum allows a considerable quantity of water to be occluded between the chains of the macromolecules [8]. By studying the potentiality of use of BSA in complex coacervation, Singh and Burgess [8] had remarked that precipitation was favoured with alginic acid. This behaviour was related to alginic acid structure, which has an extended coil configuration in solution and a stiff ribbon-type structure in the condensed phase. Contrary to arabic gum, alginic acid has a tendency to crosslink chains packed together which minimizes the occluding of water. The penetration of water through the molecules was nevertheless a key factor to promote coacervation rather than precipitation.
In our conditions, the coacervation process needs some charges not to be neutralized to avoid a too extended desolvation. It is known in literature that at acidic pH, pea globulins loose their hexameric structure, their chains become unfold and the charges become more accessible [18]. In this case, it seemed necessary to preserve charges carried by the anionic compound. The arabic gum, which is composed of six carbohydrate moieties in addition to a small proportion of protein [19] and has a coiled configuration, can preserve charges. This could be at the origin of the efficiency of this compound in complex coacervation. This hypothesis seems to be confirmed with the complex coacervation process using gelatin, for which many anionic colloids lead to coacervation. In this case, gelatin which has a coiled structure, keeps a sufficient density of charges on its chains to avoid precipitation, whatever the anionic compound used. Only the protein/anionic polymer ratio has to be adjusted, so that electrostatic interactions can control the coacervation process. For a protein to be used in a coacervation process, it must have a particular conformation which is both related to its amino-acid nature and its charge density profile. A given protein can not be replaced by another one just based on molecular weight considerations. For example, the BSA/arabic gum system was proposed [9] to replace gelatin since the molecular weights and the charge density profiles were very close. However, the high viscosity of the coacervates obtained from BSA made the system completely unsuitable for microencapsulation. The viscosity was related to the polar amino-acid groups of BSA.
5. Conclusion This work clearly establishes that pea globulins and alpha gliadins can be used in a complex coacervation process. For both proteins, arabic gum was shown to be the best anionic compound to form a complex coacervate. The conditions for
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coacervation (protein/anionic compound ratio) were found to be less restrictive for gliadins. On the other hand, the advantage of using pea globulins is that they are not allergen. The zeta potential measurements allowed us to determine the pH range where the protein is positively charged and the polysaccharide negatively charged. This method was used to optimize the pH conditions for coacervation and make easier the determination of the other parameters. Zeta potential determinations associated with turbidity measurements were shown to be an appropriate, smart method to carry out the preformulation study. From a prospective viewpoint, it would now be interesting to study the protein conformation under different conditions and when mixed with different anionic compounds. This study should help to predict the more adapted anionic polymer to induce formation of stable, individual complex coacervates for each protein.
Acknowledgements The authors wish to thank O. Thomas (MAINELAB SA) and C. Blassel (INRA, Nantes) for technical assistance.
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