Soy glycinin microcapsules by simple coacervation method

Colloids and Surfaces B: Biointerfaces 37 (2004) 1–8

Soy glycinin microcapsules by simple coacervation method J. Lazko a , Y. Popineau b , J. Legrand a,∗ a

GEPEA, UMR-CNRS 6144, BP 406, 44602 St-Nazaire Cedex, France b URPVI, INRA, BP 71627, 44316 Nantes Cedex 3, France Received 23 December 2003; accepted 3 June 2004 Available online 20 July 2004

Abstract Encapsulation of a dispersed oil phase (hexadecane) was realized by simple coacervation method using soy glycinin as the wall forming material. Suitable emulsification and coacervation conditions, that favor the formation of microcapsules wall, were identified and investigated. Mild acid (pH 2.0) and heat (55 ◦ C) treatments of the reaction medium during the emulsification step enhanced significantly the deposition of coacervated glycinin around oil droplets. A pronounced correlation between glycinin concentration in the continuous phase, specific surface of the dispersed phase and the microencapsulation efficiency was also observed. Coacervation step study concerned the morphology and the stability of microcapsules. Controlled initiation of the coacervation, by slow readjustment of the pH, allowed a homogeneous precipitation of glycinin around oil droplets as well as the absence of aggregation phenomena. Since the morphology of microcapsules was considerably affected by a prolonged stirring of the reaction medium, the coacervation and reticulation time were optimized in order to preserve the homogeneity of the microcapsules size distribution and the microencapsulation efficiency. © 2004 Elsevier B.V. All rights reserved. Keywords: Microcapsules; Soy glycinin; Simple coacervation; Protein denaturation

1. Introduction Biopolymers, such as proteins, are commonly used to encapsulate oil-in-water emulsions [1,2]. Simple and complex coacervation, spray drying and heat denaturation represent three major microencapsulation techniques based on proteins. Their principles are quite similar: emulsification of the core material (oil) is followed by microcapsules wall formation induced by environmental conditions changing. Concerning simple coacervation method, the protein precipitation around oil droplets is obtained by changing pH and temperature or by the “salting-out” technique. Widespread presence of microcapsules based on animal proteins such as gelatin, casein or albumin contrasts with a very limited use of plant proteins. Wheat gliadin was one of the rare plant storage proteins used for encapsulation of dispersed oil phase by simple coacervation method [3]. Chemical, enzymatic or physico-chemical modifications often

∗ Corresponding author. Tel.: +33-2-40-17-26-33; fax: +33-2-40-17-26-18. E-mail address: [email protected] (J. Legrand).

0927-7765/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2004.06.004

significantly improve functional properties of plant proteins, and could make these exceptional low cost, biocompatible and biodegradable raw materials, more appropriate to the current microencapsulation techniques [4,5]. Glycinin belongs to the 11S type storage proteins and represents the major globulin fraction of soybean seeds. It has a molecular mass around 360 kDa, a characteristic hexameric structure (AB)6 that is likely to undergo a dissociation in monomers: 11S ↔ 7S → 3S → 2S. The functional properties of this protein, in native or dissociated form, have been extensively studied [6–8]. Chemical modifications, thermally induced structural changes as well as acid treatments were used to enhance glycinin surface hydrophobicity, solubility, emulsifying and foaming properties, offering a range of possibilities for the development of new applications [9–13]. Concerning microencapsulation domain, microcapsules based on soy glycinin as the main wall forming material were already prepared, but only by complex coacervation technique [14]. This study underlined the essential role of SDS which, by the way of [glycinin+ -SDS− ] insoluble complex formation, allowed the precipitation of proteins around oil droplets.

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In the present work, the objectives were to study the encapsulability of oil by simple coacervation method using soy glycinin as wall forming material. The main problem consisted in finding suitable conditions of emulsification and coacervation that favor formation of protein wall around oil droplets. The first step of the proposed microencapsulation technique concerns protein-stabilized oil-in-water emulsion formation. A particular attention was given to different treatments of the reaction medium (acid conditions, heating) and to the initial protein concentration. Concerning the coacervation step, protein precipitation was induced by pH changing of the reaction medium. We investigated particularly the influence of pH adjustment kinetics and the effects of stirring time on the morphology of microcapsules and on the overall efficiency of microencapsulation.

2.3. Microscopy Emulsions and microcapsules were analyzed using a light microscope (Nikon Epiphot TME) connected to a color digital video camera (SONY XC-007). The dilution of the analyzed samples was made using pH 5.0 acetic buffer or distilled water. 2.4. Particle-size distribution

2. Experimental

The size distribution of the microcapsules was obtained by a Laser Diffraction Particle Size Analyzer LS 230 (Beckman-Coulter). Measurements were performed in pH 5.0 acetic buffer using a 15 ml module. Results were completed using a home-made software for image analysis, which allowed the size distribution determination by measuring diameters of more than 200 emulsion droplets and microcapsules on light microscope pictures.

2.1. Materials

2.5. Turbidimetric analysis

Purified glycinin 11S fraction was prepared by pilot scale extraction of defatted soy flour (variety Sogiflor G) at INRA laboratories (Nantes, France). Glutaraldehyde 25% aqueous solution was purchased from Prolabo (France), acetic acid 99% was from Fluka (Buchs, Switzerland). Bradford reagent and n-hexadecane were obtained from Sigma (St. Louis, USA). Fat red 7B dye purchased from Sigma (St. Louis, USA) was used to color the oil phase.

The turbidity measurements were carried out to evaluate suitable conditions of glycinin coacervation. Turbidity of the reaction medium was measured after 30 s and 20 min decantation using a HACH 2100AN IS turbidimeter.

2.2. Preparation of microcapsules Glycinin microcapsules containing hexadecane were prepared by a simple coacervation method. About 10 g of hexadecane were added to 400 ml of the glycinin aqueous solution (5 g/l) and the reaction medium was acidified (pH 2.0) by addition of HCl 1 M. The emulsification was carried out under 600 rpm magnetic stirring, at 55 ◦ C and pH 2.0 during 120 min. Protein coacervation was induced by a slow addition of NaOH 1 M up to pH 5.0 and the stirring was continued for a further 60 min at 25 ◦ C. Cross-linking of the glycinin precipitated around oil droplets was performed at 25 ◦ C by the addition of 10 ml of glutaraldehyde 25% aqueous solution. The pH was constantly adjusted to pH 5.0 with NaOH 1 M and stirring was maintained at the appropriate speed for further 30 min. The final product was collected after a decantation of the reaction medium, and then freeze-dried. In order to compare the influence of various treatments, the emulsification was also performed at 25 or 55 ◦ C, at pH 8.0 or 2.0 and during 2 or 24 h. In all cases, the coacervation was carried out at pH 5.0 and at 25 ◦ C. The microencapsulation efficiency related to the protein deposition around oil droplets was determined by the analysis of each phase of the reaction medium at the end of the microencapsulation process.

2.6. Protein concentration Protein concentration in aqueous solution was determined by colorimetric assay [15] using Bradford reagent (brilliant blue G in phosphoric acid and methanol). The protein–dye complex absorption was measured at 595 nm on a Lambda 10 Perkin-Elmer UV–vis spectrophotometer. Calibration was performed using purified soy glycinin as standard protein. 2.7. Microcapsules composition The composition of the microcapsules was determined by thermo-gravimetric analysis (TGA) using a NETZSCH Thermo-Microbalance TG 209. Samples were heated up to 600 ◦ C in several steps: from 30 to 100 ◦ C at 60 ◦ C/min, from 100 to 110 ◦ C at 1 ◦ C/min and from 110 to 600 ◦ C at 20 ◦ C/min heating rate. Calibration was performed using pure hexadecane, freeze-dried glycinin and freeze-dried cross-linked with glutaraldehyde glycinin.

3. Results and discussion 3.1. Glycinin solubility The preliminary glycinin solubility study was realized in order to define emulsification and coacervation pH domains. An optimum protein solubility was required for the emulsification step. Thus, according to the solubility and turbid-

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Fig. 1. Influence of pH on glycinin solubility (a) and on relative turbidity (b) of a 5 g/l glycinin solution.

ity measurements of the glycinin aqueous solution (5 g/l) (Fig. 1a and b), the emulsification was investigated under conditions corresponding to the native (pH 8) and dissociated (pH 2) glycinin. On the other hand, pH 5.0, close to the glycinin isoelectric pH region, was chosen as suitable coacervation condition. 3.2. Emulsification conditions 3.2.1. Effect of emulsification pH, time and temperature Emulsification is the first major step of the microencapsulation process. Firstly, the dispersion of the oil phase allows the formation of the oil droplets which represent the core of microcapsules. During the emulsification, the mean diameter of the oil droplets decreases with the time and converges progressively to an equilibrium value. After 2 h of stirring, the mean diameter remains constant around 90 ␮m in the case of 5 g/l glycinin aqueous solution, 5/1 (g/g) hexadecane/glycinin ratio, at pH 2 and 600 rpm stirring speed. Secondly, various treatments of proteins during the emulsification step could increase considerably the efficiency of the protein deposition around oil droplets during the coacervation step. We investigated the influence of acid treatment

(pH 2.0), mild heating (55 ◦ C) and prolonged stirring (24 h) during the emulsification on the efficiency of the microencapsulation and on the composition of microcapsules after coacervation and cross-linking (Table 1). We called standard emulsification conditions (pH 8, 2 h, 25 ◦ C) corresponding to the less denaturating conditions for proteins. The emulsification performed under standard conditions (pH 8, 2 h, 25 ◦ C) does not allow the precipitation of the initially introduced proteins around oil droplets. After decantation at the end of the microencapsulation process, the reaction medium consists of three phases: the light phase composed mainly of stabilized oil-in-water emulsion, the limpid aqueous phase and the precipitate composed of the major fraction of coacervated proteins which do not contribute to the encapsulation. Although some protein aggregates are present in the light phase (Fig. 2a), they do not cover totally the surface of the oil droplets. Their presence in the light phase is probably due to more entrapment of proteins in the droplets network than to the affinity between oil and aggregates. The acid treatment coupled with the mild heating at 55 ◦ C during the emulsification allows the precipitation of all of the proteins around oil droplets, and thus the formation of microcapsules. After decantation of the reaction medium,

Table 1 Influence of emulsification conditions (pH, time and temperature) on the microcapsules composition and on the encapsulation efficiency Emulsification conditions

Light phase composition (%) Hexadecane

pH pH pH pH pH pH pH pH

8, 8, 8, 8, 2, 2, 2, 2,

2 h, 25 ◦ C 2 h, 55 ◦ C 24 h, 25 ◦ C 24 h, 55 ◦ C 2 h, 25 ◦ C 2 h, 55 ◦ C 24 h, 25 ◦ C 24 h, 55 ◦ C

The results based on two replicates.

98.5 97.8 98.4 94.3 84.6 78.3 77.6 77.1

± ± ± ± ± ± ± ±

0.5 0.1 0.4 1.5 0.6 0.6 0.1 1.1

Coacervate 1.5 2.2 1.6 5.7 15.4 21.7 22.4 22.9

± ± ± ± ± ± ± ±

0.5 0.1 0.4 1.5 0.6 0.6 0.1 1.1

Efficiency of coacervate deposition on the oil/water interface (%) 6.5 9.6 6.9 24.9 67.1 94.7 97.5 100.0

± ± ± ± ± ± ± ±

2.4 0.5 1.9 6.6 2.6 2.7 0.3 4.8 (reference)

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Fig. 2. View of the light phase at the end of the microencapsulation process. Emulsification step performed under standard conditions (pH 8, 2 h, 25 ◦ C) (a) and under acid and thermal treatment (pH 2, 2 h, 55 ◦ C) (b).

only two phases are present: light phase composed of microcapsules and aqueous solution. The absence of precipitate and the analysis of the aqueous phase indicate that the totality of the initially introduced proteins participate to the formation of the wall of microcapsules (Fig. 2b). The significant increase of the microencapsulation efficiency would be a consequence of an improved affinity between oil and proteins. The acid denaturation of the 11S type globular proteins is characterized by alteration of their hexameric quaternary structure, dissociation in subunits and unfolding of protein chains [5,6]. An improved accessibility of the non-polar residues, initially located in the interior of the native globular form, would consequently enhance the hydrophobicity of the treated proteins. The mild heating at 55 ◦ C could also improve the denaturation and flexibility of the protein chains in the aqueous solution. Moreover, it could favor the protein adsorption and the surface denaturation at the oil–water interface. The results (Table 1) show a systematic increase of protein precipitation around oil droplets when the reaction medium is heated during the emulsification step. Even if, compared to the acid treatment, the mild heating has less impact, it allows the significant increase of the microencapsulation efficiency and an important decrease of the emulsification time. Thus, pH 2 and heating at 55 ◦ C during 2 h were chosen as suitable conditions of emulsification for the general microencapsulation process. In order to compare the influence of protein denaturation in the aqueous solution and protein adsorption on the oil surface, the reaction medium was divided in two parts:

one containing the totality of oil and only 10% of protein solution and another part, containing no oil and the remaining 90% of proteins. It was possible to apply the treatment (pH 2, 55 ◦ C) to each part separately, then to mix them together. Immediately after the addition of the glycinin solution to the emulsion, the reaction medium was cooled down to 25 ◦ C and adjusted to pH 5.0 and the microencapsulation efficiency was evaluated after coacervation step. Data shown on Table 2 underline the importance of the presence of the oil phase during the protein solution treatment. If the emulsion is prepared under standard conditions (pH 8, 25 ◦ C), the addition of the remaining 90% denaturated proteins (pH 2, 55 ◦ C) does not improve the microencapsulation efficiency. On the contrary, acid and thermal treatments of the protein solution during the emulsification allow to increase significantly the encapsulation rate, even if 90% of introduced proteins were not denaturated. However, the treatment of both emulsion and added proteins allows to get the optimum results. In conclusion, the acid and thermal protein denaturation should not be considered as the only reason that caused an improvement of the microcapsule wall formation. The increase of the microencapsulation efficiency would be essentially a consequence of various modifications of the interactions between proteins in aqueous solution and oil, induced by treatments during the emulsification step. 3.2.2. Effect of protein concentration The following study, concerning protein concentration during the emulsification, describes another effect of the

Table 2 Influence of combined acid and heat treatment of emulsion and/or glycinin solution on the microcapsules composition and on the encapsulation efficiency Operational conditions

Light phase composition (%)

Emulsion

Glycinin solution

Hexadecane

Coacervate

pH 8, 25 ◦ C pH 2, 55 ◦ C pH 2, 55 ◦ C

pH 2, 55 ◦ C pH 8, 25 ◦ C pH 2, 55 ◦ C

98.8 94.6 88.7

1.2 5.4 11.3

Microencapsulation efficiency (%)

6.4 27.8 58.1

J. Lazko et al. / Colloids and Surfaces B: Biointerfaces 37 (2004) 1–8 Table 3 Influence of glycinin concentration in emulsion on characteristics of the dispersed phase and microencapsulation efficiency Glycinin concentration in emulsion (g/l) Mean diameter (␮m) Specific surface (m2 /g) S/Smax (%) Coacervate fraction of the light phase (%) Volume occupied by the unused coacervate (%) Microencapsulation efficiency (%)

0.50 153.2 0.20 57

1.25 125.0 0.25 70

5.00 87.6 0.35 100

11 13

13 11

20 0

58

65

100

55 ◦ C

Emulsification was carried out at pH 2.0, during 2 h. Smax : maximum specific surface of the dispersed phase corresponding to the 5 g/l initial glycinin concentration in emulsion.

protein–oil interactions on the overall microencapsulation efficiency (Table 3). Emulsions were prepared at pH 2, under heating at 55 ◦ C, with the same quantity of oil but different protein concentrations, varying from 0.5 to 5 g/l. At the end of the 2 h emulsification, the reaction media were completed with other treated protein solutions (pH 2, 55 ◦ C) in order to adjust protein concentration to 5 g/l for all samples. Thus, the quantity of the available wall forming material was finally the same in all experiments. The microcapsules composition and the volume of unused proteins were quantified after coacervation and cross-linking steps. A strong correlation was observed between protein concentration during the emulsification and the quantity of precipitated proteins around oil droplets after the coacervation step. This relation could be explained by the introduction of another parameter: the specific surface of the oil dispersion, which is inversely proportional to the mean diameter of the oil droplets. Proteins, as tensio-active agents, stabilize oil-in-water emulsions. Thus, higher concentration of the surface-active agent increases the coalescence resistance of the oil droplets, and under the same stirring conditions, causes the decrease of the mean diameter of the emulsion (Table 3). The increase of the specific surface of the core material would therefore, rise the rate of proteins adsorbed on the oil–water interface,

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as well as the quantity of wall forming material after the coacervation. The evolution of S/Smax with glycinin concentration is quasi-similar to the one of microencapsulation efficiency. This shows that the microcapsules wall thickness is independent of protein concentration in our experimental conditions. 3.2.3. Conclusion Concerning the majority of microencapsulation techniques, studies of the emulsification step are often limited only to the dispersion of the substance to be encapsulated. However, in this work concerning the glycinin–hexadecane couple microencapsulation feasibility, the appropriate choice of the emulsification parameters allowed a significant improvement of the protein deposition around oil droplets during the coacervation. A more exhaustive investigation of protein–oil interactions and microcapsule wall formation should be realized in order to establish links between emulsification and coacervation, two main microencapsulation process steps. 3.3. Coacervation conditions 3.3.1. Effect of pH adjustment kinetics The coacervation step was carried out at pH 5.0. As the reaction medium was treated during the emulsification step, the pH was adjusted from pH 2.0 to 5.0 by addition of NaOH 1 M (5.5 ml of NaOH, 1 M for 400 ml of the reaction medium), and the microcapsules were finally cross-linked after the coacervation step. An important difference was noticed in the microcapsules morphology according to the pH adjustment kinetics (Fig. 3a and b). A slow NaOH addition (400 ␮l/min from pH 2.0 to 3.7 during 12.5 min and 200 ␮l/min from pH 3.7 to 5.0 during 2 min) allowed a homogeneous protein precipitation around oil droplets and perfect-spherical microcapsules. On the other hand, a significant agglomeration phenomenon was observed when pH adjustment was realized quickly (5.5 ml/min). The granulometric analysis by laser diffraction confirms previous observations and indicates an overall

Fig. 3. View of microcapsules obtained with slow (a) and fast (b) pH adjustment.

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Fig. 4. Evolution of the size distribution (frequency given on a volume basis) of unreticulated microcapsules during stirring.

increase of the mean diameter of particles from 101.7 ␮m (Fig. 4a) to 157.7 ␮m (Fig. 4b). 3.3.2. Coacervation step time and microcapsules resistance to stirring How long a coacervation step lasts is another important parameter influencing the morphology of microcapsules, as well as the efficiency of the microencapsulation. In order to find the optimal coacervation time, the reaction medium was analyzed during 24 h which followed the initiation of the protein coacervation, without the cross-linking. The evolution of the microcapsules morphology was observed by the optical microscope, the size distribution measurements were performed by image analysis, while the microcapsules composition and the microencapsulation efficiency were quantified by thermo-gravimetric and colorimetric analysis of the light phase, aqueous phase and precipitate after decantation of the reaction medium. Data shown in Table 4 indicate a drastic deterioration of the microcapsules during a prolonged stirring. The mean diameter systematically increases with the time while the quantity of proteins participating in the microcapsules wall formation decreases. Size repartitions (Fig. 4) and microscopy observations (Fig. 5) indicate a progressive evolution of small microcapsules to much bigger entities. Table 4 Influence of stirring after coacervation pH adjustment on microencapsulation efficiency Coacervation time (h)

0.2

Mean diameter (␮m) Specific surface (m2 /g)

100.5 0.324

107.7 0.297

146.9 0.250

285.5 0.137

21.1

19.8

15.8

5.3

87.6

78.0

57.2

7.0

16.7

13.2

12.0

5.2

Coacervate fraction of the light phase (%) Glycinin precipitated around oil droplets (%) Volume occupied by the light phase (%)

1

5

24

The thickness of the protein wall remains quite unchanged corresponding to the drastic decrease of the global wall forming material and also of the volume of the light phase. A linear dependence between specific surface of the core material and the rate of proteins participating in the microencapsulation has been observed. This result confirms the effect of protein concentration during the emulsification step on the microencapsulation efficiency (Table 3). It proves also that the wall thickness is quasi-independent on protein concentration. This phenomenon would probably require the consideration that proteins, which precipitate around oil droplets, form a flexible membrane and not a rigid wall. Thus, the size of microcapsules could evolve, especially in the stirred reaction medium, if the equilibrium between mechanical stress and the oil droplets stabilization has been disrupted. The flexible protein membranes would confer an additional mechanical resistance and elasticity and could prevent the shearing of the oil droplets. On the other hand, shocks between microcapsules in the agitated reaction medium could lead to the coalescence of their membranes and eventually to the fusion of their core materials. Some non-spherical forms are often observed (Fig. 6) and could represent an intermediate step of the microcapsule coalescence. 3.3.3. Effect of stirring on cross-linked microcapsules The comparative study was performed with the cross-linked microcapsules. The cross-linking reaction was realized only 10 min after the beginning of the coacervation, and the reaction medium was stirred for 24 h. Table 5 shows the evolution of the mean diameter of the cross-linked microcapsules compared to the unreticulated microcapsules. Data concerning non-stirred emulsion, unreticulated and reticulated microcapsules were joined to be used as a reference. No particular difference has been observed between the evolution of the mean diameter during the time for all samples, except for unreticulated microcapsules. Results indicate that the drastic deterioration of unreticulated capsules,

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Fig. 5. (a–d) Evolution of unreticulated microcapsules morphology during stirring.

described above, is a consequence of a mechanical stress only. Concerning cross-linked microcapsules, stirring has no significant impact on their mean diameter. The cross-linking of the coacervated proteins confers a particular rigidity to the microcapsules wall, but also seems to reduce its elasticity. The coalescence between cross-linked microcapsules does not occur, confirming the previous hypothesis. Reticulated microcapsules have higher resistance to the stirring than unreticulated capsules. However, they break under prolonged mechanical stress, releasing their core mate-

rial. A significant free oil layer (up to 30% of the initially introduced oil) had been observed when the reaction medium was allowed to settle after 40 h stirring. The absence of the dispersion of the released oil indicates the complete loss of emulsifying properties of the cross-linked protein network. 3.3.4. Conclusion According to the previous results, the coacervation conditions have a direct effect on the microcapsules morphology. Controlled initiation of the coacervation, in this particular case by slow readjustment of the pH, limits the aggregation phenomena and leads to a homogeneous precipitation of proteins around oil droplets. Since the coacervation disturbs the equilibrium between coalescence and break-up established during the emulsification, the coacervation step time should be reduced as much as possible. A prolonged stirring of the reaction medium after coacervation could significantly increase the heterogeneity of the microcapsules size repartiTable 5 Influence of stirring on the mean diameter (␮m) of emulsion, unreticulated and cross-linked microcapsules

Fig. 6. View of an intermediate step of microcapsules coalescence.

Time (h) Emulsion at rest Unreticulated microcapsules (at rest) Unreticulated microcapsules (stirred) Cross-linked microcapsules (at rest) Cross-linked microcapsules (stirred)

1 87.0 91.1 100.5 90.9 90.9

13 87.8 88.2 202.0 86.4 89.6

26 85.2 83.9 285.5 85.0 81.4

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