- Email: [email protected]
Phase separation, rheology and structure of micellar casein-galactomannan mixtures
International Dairy Journal 9 (1999) 353}357
Phase separation, rheology and structure of micellar casein-galactomannan mixtures Sophie Bourriot, Catherine Garnier, Jean-Louis Doublier* Laboratoire de Physico-Chimie des Macromole& cules, INRA, BP 71627, 44316 Nantes Cedex 3, France
Abstract The properties of micellar casein-galactomannan (guar gum and locust bean gum) mixed systems have been investigated at 203C, pH 7 and in 0.25 M NaCl; in these conditions, the mixtures tended to phase separate probably due to depletion}#occulation mechanisms. The ternary phase diagram has been established for each galactomannan}casein system according to the biopolymer concentrations. The rheological properties of these mixed systems have been investigated in the monophasic domain, as well as in the biphasic one. Modi"cations of the #ow and the viscoelastic properties were clearly evidenced in the two-phase domain. The ultrastructure of the galactomannan}micellar casein mixtures has been described using confocal laser scanning microscopy. In the biphasic domain, observations of the mixed system at concentrations corresponding to those of rheological measurements clearly evidenced that the phase separation process has yielded a continuous network mostly composed of the aggregated micellar casein. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Casein; Galactomannan; Microscopy; Rheology
1. Introduction Biopolymer mixtures are widely used in the food industry because they impart a desirable texture to foodstu!s. They occur very often in dairy products in which casein constitutes indeed the major protein component (Swaisgood, 1982). Therefore, knowledge of the mechanisms occurring in casein}polysaccharide mixed systems is of great importance in order to develop speci"c properties in dairy products. Casein micelles possess a relatively large and rather complex structure (diameter 20}600 nm) (Schmidt, 1982). This colloidal assembly is a supramolecular association of individual casein subunits: a- , a- , b- and i-caseins. These fractions are organized within the micelle according to their hydrophobic and hydrophilic groups, yielding submicelles; i-casein is thought to be mainly present on the surface of the micelle, providing a sterically stabilizing outer layer (Visser, 1992). The submicelles are held together by colloidal calcium phosphate (Van Dijk, 1990). This implies that the micelle is in dynamic
equilibrium with its ionic environment (Visser, 1992; Holt, 1992). Galactomannans are seed polysaccharides. They have a linear backbone of (1-4) linked b-D-mannose residues substituted with side chains constituted by single (1-6)-aD-galactose residues (Dea & Morrison, 1975). Guar gum (GG) has a mannose-to-galactose ratio of about 1.8, while for locust bean gum (LBG), this ratio is about 3.5. Galactomannans are used in the food industry mainly as thickening agents. The aim of this study was (i) to understand the physico-chemical interactions between micellar casein and a neutral food polysaccharide and (ii) to characterize the behaviour of the mixtures. For this purpose, rheological measurements have been performed. In addition, the structure of the systems was observed by Confocal Laser Scanning Microscopy (CLSM) and the phase diagrams of the mixtures were established.
2. Materials and methods 2.1. Materials
* Corresponding author. Tel.: #33-2-40-67-50-55; fax: #33-2-4067-50-43. E-mail address: [email protected] (J. Doublier)
Micellar casein (MC) was a native calcium phosphocaseinate sample puri"ed by ultra"ltration and then
0958-6946/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 0 8 7 - 4
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S. Bourriot et al. / International Dairy Journal 9 (1999) 353}357
freeze-dried. This was kindly supplied by Laboratoire de Recherches et de Technologie Laitie`re (INRA Rennes, France) and was prepared by P. Schuck. It had the following characteristics: total protein content 90.7%; non-casein protein 5.0%; lactose 0.5%; salts 8.3%. Guar gum (GG) and locust bean gum (LBG) were provided by Systems Bio Industry (France). The intrinsic viscosity was 12.9 dl g\ in 0.25 M NaCl at 203C for the guar gum sample and 7.4 dl g\ for the locust bean gum sample. 2.1.1. Preparation Micellar casein (10%) was dispersed in 0.25 M NaCl, at 203C, pH 7, by stirring with a paddle at 1300 rpm for 5 min and then sonicated for 8 min at 50 W. The particle size distribution was checked using a Malvern Mastersizer IP laser granulometer. The average diameter was 0.32 lm, which was in agreement with literature data (Swaisgood, 1982; Schmidt, 1982; Visser, 1992). Galactomannan solutions (1%) were prepared at 203C in 0.25 M NaCl under magnetic stirring during 1 h and then heated at 803C during 30 min. To compare phase diagrams, the guar gum sample has been hydrolysed by means of ultrasonic waves (40 s at 10 W); by this method the intrinsic viscosity of the hydrolysed guar gum (HGG) was decreased to 8.5 dl g\. The mixtures were prepared at appropriate ratios in test tubes at 203C. 2.2. Methods 2.2.1. Rheology Rheological measurements were performed using a controlled strain rheometer (Rheometrics Fluid Spectrometer RFS II) in steady and oscillatory shear with cone-plate geometry (diameter 5 cm, angle 0.04 rad, gap 50 lm) at 203C after 5 h.
chosen to avoid casein micelle sedimentation. Phase separation boundary was then detected only by eye since the two phases were clearly separated when phase separation occurred. The upper phase was a galactomannan-rich phase while micellar casein was more concentrated in the lower phase. For the upper phase, the casein content was obtained by measuring absorbance at 277 nm, the galactomannan concentration being determined with a refractometer. The composition of the lower phase was obtained by calculating the concentrations from the volumes.
3. Results 3.1. Flow behaviour Fig. 1 shows the #ow curves displayed by a guar gum solution at 0.2%, a casein suspension at 3% and a mixture containing 0.2% of guar gum and 3% of casein. The casein suspension exhibited a Newtonian behaviour and was slightly viscous (6.0;10\ Pa s). The galactomannan solution at 0.2% was also Newtonian with a viscosity of 1.8;10\ Pa s. However, when micellar casein was added to the guar gum solution, the #ow behaviour was strongly modi"ed. A dramatic increase of the apparent viscosity of the system was observed. A synergistic e!ect was exhibited since the apparent viscosity of the mixture was much higher than the sum of the viscosities of the individual components of the system at the same concentrations. Moreover, the #ow behaviour of the mixture was slightly thixotropic. From a general point of view, it appeared that the viscosity, as well as the thixotropic loop, were much greater when the total biopolymer concentrations increased. Conversely, these e!ects were clearly reduced when the concentrations were lower.
2.2.2. Confocal microscopy Confocal Laser Scanning Microscopy was performed with a Zeiss LSM 410 Axiovert microscope. CLSM was used in #uorescence mode. Micellar casein was labelled with 8-anilino-1-naphtalene sulphonic acid (ANS) which is thought to be restricted to hydrophobic areas. In this state, the ANS is #uorescent. The excitation using the UV laser was performed at 364 nm and the emission was recorded between 450 and 497 nm. The samples were placed between a slide and a coverslip and sealed to prevent evaporation. Blends were examined with a water immersed ;40 objective. 2.2.3. Phase diagrams Phase diagrams were established according to the polymer concentrations in 0.25 M NaCl, at pH 7 and 203C. After aging for 24 h, the mixtures were centrifuged at 1500 g. These mild conditions of centrifugation were
Fig. 1. Flow behaviour of a guar gum solution (GG), a casein suspension (MC) and a guar}casein mixture (GG}MC) in 0.25 M NaCl.
S. Bourriot et al. / International Dairy Journal 9 (1999) 353}357
355
Fig. 2. Viscoelastic properties of guar gum solution (GG), casein suspension (MC) and a guar gum}casein mixture (GG}MC) in 0.25 M NaCl. G : "lled symbols; G : empty symbols.
3.2. Viscoelastic properties Fig. 2 illustrates the viscoelastic behaviour of the three previous systems. For guar gum solution, the strain was imposed at 40% and for casein suspension, it was at 20%, which was within the linear region. G and G were both strongly dependent on the frequency. These one-component systems behave like macromolecular solutions without any organization of the polymers in the medium. In contrast, casein}guar gum mixed system exhibited quite di!erent viscoelastic properties. G and G were less dependent on the frequency. In addition, the values of the moduli were slightly higher. It is clear that casein}guar gum mixture displayed the behaviour of a slightly structured system. Rheological measurements have also been performed in the case of casein}locust bean gum mixtures and the same main features have been observed: an increase of the viscosity, a thixotropic behaviour and a structuring of the systems appeared when casein and LBG were mixed together. According to the rheological measurements in steady shear as well as in oscillatory shear, it appears that mixing casein and galactomannans leads to the formation of a structure within the system, the higher the biopolymer concentrations, the more structured the mixtures. 3.3. Structure of the mixtures Labelling the micellar casein with ANS allowed us to localize the micellar casein in the mixture by #uorescence microscopy since guar gum is not #uorescent at the wavelength used (364 nm). Moreover, the confocal laser scanning microscopy (CLSM) is able to focus only on one plane of the sample. This means that the #uorescence
Plate 1. Guar gum 0.2%#casein 0.3%; scale bar"25 lm.
of the rest of the sample does not interfere with the #uorescence of the focal plane. Therefore, CLSM gives not only high-resolution information about one plane but can also indicate the three-dimensional structure of the sample by superimposing di!erent focal planes. Casein micelles appear in clear in the photograph owing to the #uorescence of the ANS while the dark areas correspond to zones devoid of casein, thus containing mostly guar gum. Plate 1 corresponds to a focal plane of a 0.2% guar gum#0.3% casein mixture. Fluorescence was regularly distributed in the photograph indicating that casein was spread all over the medium. The system is seen to be homogeneous. Plate 2 shows the result obtained by mixing 0.2% of guar gum and 3% of casein. In this case, the appearance of the #uorescence was not so uniform. The casein micelles were concentrated in areas which were very polydisperse in size and may originate from large coalescing droplets (10}100 lm). It should be recalled that the average size of one casein micelle is around 0.3 lm. Therefore, the micelles appear aggregated, constituting a continuous phase and hence, a structuring of the system. Two phases obviously coexisted in this system; one phase contained mainly casein micelles whereas the other one was a guar gum rich phase. In contrast, the former mixture (Plate 1) did not exhibit any phase separation. 3.4. Phase diagrams From the previous microscopic results, it appears that the mixtures phase separate beyond a certain concentration of biopolymers. Phase diagrams were therefore
356
S. Bourriot et al. / International Dairy Journal 9 (1999) 353}357
Fig. 4. Comparison of micellar casein}guar gum (䉭), micellar casein}hydrolysed guar gum (䊐) and micellar casein}locust bean gum (*) phase diagrams in 0.25 M NaCl, pH 7, 203C.
Plate 2. Guar gum 0.2%#casein 3%; scale bar"50 lm.
lowest polymer concentration (Cs) leading to phase separation (0.4%) (Tolstoguzov, 1992) they were 0.18% guar gum and 0.22% casein Fig. 4 shows the casein}guar gum phase diagram compared to the casein-HGG and casein-LBG phase diagrams. In the presence of the locust bean gum and the hydrolysed guar gum, the area of the two-phase region decreased compared to the result obtained with GG. However, the concentration of the yield point (Cs) was the same whatever the nature of the galactomannan (0.4%).
4. Discussion
Fig. 3. Casein}guar gum phase diagram in 0.25 M NaCl, pH 7, 203C.
established in order to map the state of the mixtures according to their composition. Fig. 3 illustrates the main features of the phase diagram of casein}guar gum mixtures in 0.25 M NaCl, pH 7 and at 203C. The binodal separates the one-phase region from the two-phase region and was built by direct observation in test tubes of the phase separation (points *). Then, for initial mixtures (points 䊐) which separated into two phases, the lower phase (points 䉱) and the upper phase (points 䉭) were analysed allowing the tie-lines to be drawn. The critical point represents the composition of systems separating into two phases of the same volumes and composition (Tolstoguzov, 1992). The coordinates of the critical point were determined to 0.8% casein and 0.09% guar gum. For the yield point, de"ned as the
The rheological data show a transition from the behaviour of a macromolecular solution to a slightly structured system when casein was added to galactomannan solutions, whether the polysaccharide was guar gum or locust bean gum. According to microscopic observations, this particular behaviour is undoubtedly related to phase separation phenomena. Demixing may be ascribed to the aggregation of casein micelles, probably by depletion} #occulation mechanisms. The micelles are actually spherical particles. Aggregation can be induced by the presence of a polymer, as "rst predicted by Asakura and Oosawa for the general case of hard spheres in the presence of macromolecules (Asakura & Oosawa, 1954; Asakura & Oosawa, 1958). This theoretical treatment explains how a dispersion of such particles can be #occulated by the exclusion of a polymer molecule from the space between close spheres. Since the osmotic pressure of the bulk polymer solution is greater than in the excluded zone containing pure solvent, the particles are pushed together and thus aggregated. In the same way, galactomannan can be excluded from the space between
S. Bourriot et al. / International Dairy Journal 9 (1999) 353}357
the micelles when the casein concentration increases. Since LBG or HGG have a lower intrinsic viscosity than the initial guar gum sample, they occupy a smaller volume in the medium than the guar gum chains. The exclusion of the polymer thus occurs to a lesser extent, resulting in a decrease of the aggregation of the particles at the same concentration. The two-phase domain was indeed reduced in the case of casein-LBG or casein-HGG mixtures as compared to systems containing guar gum (Fig. 4). A simple way to con"rm that the particles are weakly #occulated is to dilute an aggregated sample (Patel and Russel, 1989); this leads to a one phase system, indicating that the process is reversible which is consistent with the depletion}#occulation mechanism. The #ocs form the continuous phase of the system, increasing the viscosity of the medium. Furthermore, the network can be easily broken under shear because the interactions between the #ocs are not covalent, leading to the appearance of the thixotropic behaviour (Clark & RossMurphy, 1987). To summarize, it is clear that the modi"cations of the behaviour of individual components can be ascribed to casein #occulation induced by the presence of the galactomannan polymer. Acknowledgements Financial support to one of the authors (S. Bourriot) from CANDIA (France) is gratefully acknowledged.
357
References Asakura, S., & Oosawa, F. (1954). On interaction between two bodies immersed in a solution of macromolecules. Journal of Chemical Physics, 22, 1255}1256. Asakura, S., & Oosawa, F. (1958). Interaction between particles suspended in solutions of macromolecules. Journal of Polymer Science, 33, 183}192. Clark, A. H., & Ross Murphy, S. B. (1987). Structural and mechanical properties of biopolymer gels. Advances in Polymer Science, 83, 55}192. Dea, I. C. M., & Morrison, A. (1975). Chemistry and interactions of seed galactomannans. Advances in Carbohydrate Chemistry and Biochemistry, 31, 241}312. Holt, C. (1992). Structure and stability of bovine casein micelles. In C. B. An"sen, J. T. Edsall, F. M. Richards, & D. S. Eisenberg, Advances in protein chemistry~43 (pp. 63}151). London: Academic Press. Patel, P. D., & Russel, W. B. (1989). An experimental study of aqueous suspensions containing dissolved polymer. B. Rheology. Journal of Colloid and Interface Science, 131, 201}210. Schmidt, D. G. (1982). Association of casein and casein micelle structure. In P. F. Fox, Developments in dairy chemistry*1 (pp. 60}86). London: Applied Science Publishers. Swaisgood, H. E. (1982). Chemistry of milk protein. In P. F. Fox, Developments in dairy chemistry*1 (pp. 1}59). London: Applied Science Publishers. Tolstoguzov, V. B. (1992). The functional properties of foods proteins. In G. O. Phillips, P. A. Williams, & D. J. Wedlock, Gums and stabilizers for the food industry*6 (pp. 241}266). Oxford: IRL Press. Van Dijk, H. J. M. (1990). The properties of casein micelles*1. The nature of the micellar calcium phosphate. The Netherlands Milk and Dairy Journal, 44, 65}81. Visser, H. (1992). A new casein micelle model and its consequences for pH and temperature e!ects on the properties of milk. In H. Visser, Protein interactions (pp. 135}165). New York: VCH.
Phase separation, rheology and structure of micellar casein-galactomannan mixtures Sophie Bourriot, Catherine Garnier, Jean-Louis Doublier* Laboratoire de Physico-Chimie des Macromole& cules, INRA, BP 71627, 44316 Nantes Cedex 3, France
Abstract The properties of micellar casein-galactomannan (guar gum and locust bean gum) mixed systems have been investigated at 203C, pH 7 and in 0.25 M NaCl; in these conditions, the mixtures tended to phase separate probably due to depletion}#occulation mechanisms. The ternary phase diagram has been established for each galactomannan}casein system according to the biopolymer concentrations. The rheological properties of these mixed systems have been investigated in the monophasic domain, as well as in the biphasic one. Modi"cations of the #ow and the viscoelastic properties were clearly evidenced in the two-phase domain. The ultrastructure of the galactomannan}micellar casein mixtures has been described using confocal laser scanning microscopy. In the biphasic domain, observations of the mixed system at concentrations corresponding to those of rheological measurements clearly evidenced that the phase separation process has yielded a continuous network mostly composed of the aggregated micellar casein. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Casein; Galactomannan; Microscopy; Rheology
1. Introduction Biopolymer mixtures are widely used in the food industry because they impart a desirable texture to foodstu!s. They occur very often in dairy products in which casein constitutes indeed the major protein component (Swaisgood, 1982). Therefore, knowledge of the mechanisms occurring in casein}polysaccharide mixed systems is of great importance in order to develop speci"c properties in dairy products. Casein micelles possess a relatively large and rather complex structure (diameter 20}600 nm) (Schmidt, 1982). This colloidal assembly is a supramolecular association of individual casein subunits: a- , a- , b- and i-caseins. These fractions are organized within the micelle according to their hydrophobic and hydrophilic groups, yielding submicelles; i-casein is thought to be mainly present on the surface of the micelle, providing a sterically stabilizing outer layer (Visser, 1992). The submicelles are held together by colloidal calcium phosphate (Van Dijk, 1990). This implies that the micelle is in dynamic
equilibrium with its ionic environment (Visser, 1992; Holt, 1992). Galactomannans are seed polysaccharides. They have a linear backbone of (1-4) linked b-D-mannose residues substituted with side chains constituted by single (1-6)-aD-galactose residues (Dea & Morrison, 1975). Guar gum (GG) has a mannose-to-galactose ratio of about 1.8, while for locust bean gum (LBG), this ratio is about 3.5. Galactomannans are used in the food industry mainly as thickening agents. The aim of this study was (i) to understand the physico-chemical interactions between micellar casein and a neutral food polysaccharide and (ii) to characterize the behaviour of the mixtures. For this purpose, rheological measurements have been performed. In addition, the structure of the systems was observed by Confocal Laser Scanning Microscopy (CLSM) and the phase diagrams of the mixtures were established.
2. Materials and methods 2.1. Materials
* Corresponding author. Tel.: #33-2-40-67-50-55; fax: #33-2-4067-50-43. E-mail address: [email protected] (J. Doublier)
Micellar casein (MC) was a native calcium phosphocaseinate sample puri"ed by ultra"ltration and then
0958-6946/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 0 8 7 - 4
354
S. Bourriot et al. / International Dairy Journal 9 (1999) 353}357
freeze-dried. This was kindly supplied by Laboratoire de Recherches et de Technologie Laitie`re (INRA Rennes, France) and was prepared by P. Schuck. It had the following characteristics: total protein content 90.7%; non-casein protein 5.0%; lactose 0.5%; salts 8.3%. Guar gum (GG) and locust bean gum (LBG) were provided by Systems Bio Industry (France). The intrinsic viscosity was 12.9 dl g\ in 0.25 M NaCl at 203C for the guar gum sample and 7.4 dl g\ for the locust bean gum sample. 2.1.1. Preparation Micellar casein (10%) was dispersed in 0.25 M NaCl, at 203C, pH 7, by stirring with a paddle at 1300 rpm for 5 min and then sonicated for 8 min at 50 W. The particle size distribution was checked using a Malvern Mastersizer IP laser granulometer. The average diameter was 0.32 lm, which was in agreement with literature data (Swaisgood, 1982; Schmidt, 1982; Visser, 1992). Galactomannan solutions (1%) were prepared at 203C in 0.25 M NaCl under magnetic stirring during 1 h and then heated at 803C during 30 min. To compare phase diagrams, the guar gum sample has been hydrolysed by means of ultrasonic waves (40 s at 10 W); by this method the intrinsic viscosity of the hydrolysed guar gum (HGG) was decreased to 8.5 dl g\. The mixtures were prepared at appropriate ratios in test tubes at 203C. 2.2. Methods 2.2.1. Rheology Rheological measurements were performed using a controlled strain rheometer (Rheometrics Fluid Spectrometer RFS II) in steady and oscillatory shear with cone-plate geometry (diameter 5 cm, angle 0.04 rad, gap 50 lm) at 203C after 5 h.
chosen to avoid casein micelle sedimentation. Phase separation boundary was then detected only by eye since the two phases were clearly separated when phase separation occurred. The upper phase was a galactomannan-rich phase while micellar casein was more concentrated in the lower phase. For the upper phase, the casein content was obtained by measuring absorbance at 277 nm, the galactomannan concentration being determined with a refractometer. The composition of the lower phase was obtained by calculating the concentrations from the volumes.
3. Results 3.1. Flow behaviour Fig. 1 shows the #ow curves displayed by a guar gum solution at 0.2%, a casein suspension at 3% and a mixture containing 0.2% of guar gum and 3% of casein. The casein suspension exhibited a Newtonian behaviour and was slightly viscous (6.0;10\ Pa s). The galactomannan solution at 0.2% was also Newtonian with a viscosity of 1.8;10\ Pa s. However, when micellar casein was added to the guar gum solution, the #ow behaviour was strongly modi"ed. A dramatic increase of the apparent viscosity of the system was observed. A synergistic e!ect was exhibited since the apparent viscosity of the mixture was much higher than the sum of the viscosities of the individual components of the system at the same concentrations. Moreover, the #ow behaviour of the mixture was slightly thixotropic. From a general point of view, it appeared that the viscosity, as well as the thixotropic loop, were much greater when the total biopolymer concentrations increased. Conversely, these e!ects were clearly reduced when the concentrations were lower.
2.2.2. Confocal microscopy Confocal Laser Scanning Microscopy was performed with a Zeiss LSM 410 Axiovert microscope. CLSM was used in #uorescence mode. Micellar casein was labelled with 8-anilino-1-naphtalene sulphonic acid (ANS) which is thought to be restricted to hydrophobic areas. In this state, the ANS is #uorescent. The excitation using the UV laser was performed at 364 nm and the emission was recorded between 450 and 497 nm. The samples were placed between a slide and a coverslip and sealed to prevent evaporation. Blends were examined with a water immersed ;40 objective. 2.2.3. Phase diagrams Phase diagrams were established according to the polymer concentrations in 0.25 M NaCl, at pH 7 and 203C. After aging for 24 h, the mixtures were centrifuged at 1500 g. These mild conditions of centrifugation were
Fig. 1. Flow behaviour of a guar gum solution (GG), a casein suspension (MC) and a guar}casein mixture (GG}MC) in 0.25 M NaCl.
S. Bourriot et al. / International Dairy Journal 9 (1999) 353}357
355
Fig. 2. Viscoelastic properties of guar gum solution (GG), casein suspension (MC) and a guar gum}casein mixture (GG}MC) in 0.25 M NaCl. G : "lled symbols; G : empty symbols.
3.2. Viscoelastic properties Fig. 2 illustrates the viscoelastic behaviour of the three previous systems. For guar gum solution, the strain was imposed at 40% and for casein suspension, it was at 20%, which was within the linear region. G and G were both strongly dependent on the frequency. These one-component systems behave like macromolecular solutions without any organization of the polymers in the medium. In contrast, casein}guar gum mixed system exhibited quite di!erent viscoelastic properties. G and G were less dependent on the frequency. In addition, the values of the moduli were slightly higher. It is clear that casein}guar gum mixture displayed the behaviour of a slightly structured system. Rheological measurements have also been performed in the case of casein}locust bean gum mixtures and the same main features have been observed: an increase of the viscosity, a thixotropic behaviour and a structuring of the systems appeared when casein and LBG were mixed together. According to the rheological measurements in steady shear as well as in oscillatory shear, it appears that mixing casein and galactomannans leads to the formation of a structure within the system, the higher the biopolymer concentrations, the more structured the mixtures. 3.3. Structure of the mixtures Labelling the micellar casein with ANS allowed us to localize the micellar casein in the mixture by #uorescence microscopy since guar gum is not #uorescent at the wavelength used (364 nm). Moreover, the confocal laser scanning microscopy (CLSM) is able to focus only on one plane of the sample. This means that the #uorescence
Plate 1. Guar gum 0.2%#casein 0.3%; scale bar"25 lm.
of the rest of the sample does not interfere with the #uorescence of the focal plane. Therefore, CLSM gives not only high-resolution information about one plane but can also indicate the three-dimensional structure of the sample by superimposing di!erent focal planes. Casein micelles appear in clear in the photograph owing to the #uorescence of the ANS while the dark areas correspond to zones devoid of casein, thus containing mostly guar gum. Plate 1 corresponds to a focal plane of a 0.2% guar gum#0.3% casein mixture. Fluorescence was regularly distributed in the photograph indicating that casein was spread all over the medium. The system is seen to be homogeneous. Plate 2 shows the result obtained by mixing 0.2% of guar gum and 3% of casein. In this case, the appearance of the #uorescence was not so uniform. The casein micelles were concentrated in areas which were very polydisperse in size and may originate from large coalescing droplets (10}100 lm). It should be recalled that the average size of one casein micelle is around 0.3 lm. Therefore, the micelles appear aggregated, constituting a continuous phase and hence, a structuring of the system. Two phases obviously coexisted in this system; one phase contained mainly casein micelles whereas the other one was a guar gum rich phase. In contrast, the former mixture (Plate 1) did not exhibit any phase separation. 3.4. Phase diagrams From the previous microscopic results, it appears that the mixtures phase separate beyond a certain concentration of biopolymers. Phase diagrams were therefore
356
S. Bourriot et al. / International Dairy Journal 9 (1999) 353}357
Fig. 4. Comparison of micellar casein}guar gum (䉭), micellar casein}hydrolysed guar gum (䊐) and micellar casein}locust bean gum (*) phase diagrams in 0.25 M NaCl, pH 7, 203C.
Plate 2. Guar gum 0.2%#casein 3%; scale bar"50 lm.
lowest polymer concentration (Cs) leading to phase separation (0.4%) (Tolstoguzov, 1992) they were 0.18% guar gum and 0.22% casein Fig. 4 shows the casein}guar gum phase diagram compared to the casein-HGG and casein-LBG phase diagrams. In the presence of the locust bean gum and the hydrolysed guar gum, the area of the two-phase region decreased compared to the result obtained with GG. However, the concentration of the yield point (Cs) was the same whatever the nature of the galactomannan (0.4%).
4. Discussion
Fig. 3. Casein}guar gum phase diagram in 0.25 M NaCl, pH 7, 203C.
established in order to map the state of the mixtures according to their composition. Fig. 3 illustrates the main features of the phase diagram of casein}guar gum mixtures in 0.25 M NaCl, pH 7 and at 203C. The binodal separates the one-phase region from the two-phase region and was built by direct observation in test tubes of the phase separation (points *). Then, for initial mixtures (points 䊐) which separated into two phases, the lower phase (points 䉱) and the upper phase (points 䉭) were analysed allowing the tie-lines to be drawn. The critical point represents the composition of systems separating into two phases of the same volumes and composition (Tolstoguzov, 1992). The coordinates of the critical point were determined to 0.8% casein and 0.09% guar gum. For the yield point, de"ned as the
The rheological data show a transition from the behaviour of a macromolecular solution to a slightly structured system when casein was added to galactomannan solutions, whether the polysaccharide was guar gum or locust bean gum. According to microscopic observations, this particular behaviour is undoubtedly related to phase separation phenomena. Demixing may be ascribed to the aggregation of casein micelles, probably by depletion} #occulation mechanisms. The micelles are actually spherical particles. Aggregation can be induced by the presence of a polymer, as "rst predicted by Asakura and Oosawa for the general case of hard spheres in the presence of macromolecules (Asakura & Oosawa, 1954; Asakura & Oosawa, 1958). This theoretical treatment explains how a dispersion of such particles can be #occulated by the exclusion of a polymer molecule from the space between close spheres. Since the osmotic pressure of the bulk polymer solution is greater than in the excluded zone containing pure solvent, the particles are pushed together and thus aggregated. In the same way, galactomannan can be excluded from the space between
S. Bourriot et al. / International Dairy Journal 9 (1999) 353}357
the micelles when the casein concentration increases. Since LBG or HGG have a lower intrinsic viscosity than the initial guar gum sample, they occupy a smaller volume in the medium than the guar gum chains. The exclusion of the polymer thus occurs to a lesser extent, resulting in a decrease of the aggregation of the particles at the same concentration. The two-phase domain was indeed reduced in the case of casein-LBG or casein-HGG mixtures as compared to systems containing guar gum (Fig. 4). A simple way to con"rm that the particles are weakly #occulated is to dilute an aggregated sample (Patel and Russel, 1989); this leads to a one phase system, indicating that the process is reversible which is consistent with the depletion}#occulation mechanism. The #ocs form the continuous phase of the system, increasing the viscosity of the medium. Furthermore, the network can be easily broken under shear because the interactions between the #ocs are not covalent, leading to the appearance of the thixotropic behaviour (Clark & RossMurphy, 1987). To summarize, it is clear that the modi"cations of the behaviour of individual components can be ascribed to casein #occulation induced by the presence of the galactomannan polymer. Acknowledgements Financial support to one of the authors (S. Bourriot) from CANDIA (France) is gratefully acknowledged.
357
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