Maltodextrin

Biomacromolecules 2001, 2, 812-823

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Phase Separation in Gelatin/Maltodextrin and Gelatin/ Maltodextrin/Gum Arabic Mixtures Studied Using Small-Angle Light Scattering, Turbidity, and Microscopy Michael F. Butler* and Mary Heppenstall-Butler Unilever Research, Colworth House, Sharnbrook, Bedfordshire, MK44 1LQ, U.K. Received January 5, 2001; Revised Manuscript Received June 5, 2001

The kinetics of phase separation were observed in the gelatin/maltodextrin and gelatin/maltodextrin/gum arabic systems, where gum arabic was added as a minority component, using small-angle light scattering, turbidity measurement, and confocal scanning laser microscopy. Phase separation occurred by spinodal decomposition for quenches both above and below the temperature at which gelatin gelled. Coarsening of the phase-separated microstructure was hindered by gelation, and a hydrodynamic mechanism, observed when the gelatin remained in the liquid state, was suppressed. Gum arabic, containing both polysaccharide and polypeptide components, was hypothesized to be potentially interfacially active in the gelatin/maltodextrin system, in analogy with synthetic block copolymer compatibilizers in demixed synthetic polymer systems. The hypothesis was experimentally refuted under the chosen experimental conditions, as no evidence was found to suggest that it altered the phase separation behavior. Introduction Phase Separation in Biopolymer Systems. In polymer systems, attention has largely been paid to phase separation in blends of immiscible synthetic polymers. In the context of the food industry, however, the study of mixed biopolymer solutions is important, because the morphologies and phase separation kinetics in these systems have an important influence on textural attributes and product stability. Fortunately, such ternary systems, containing two polymers and a common solvent, display qualitative features similar to binary ones, and the techniques and analysis employed in the study of binary systems are equally applicable. Additional complexity is introduced, however, in the situation where gelation can occur in one or both of the components. When gelation occurs, the microstructure is trapped at the early stages of phase separation and coarsening of the microstructure is inhibited.1-3 It is also possible that gelation may trigger phase separation via the formation of regions of enhanced concentration. Where the times required for gelation and phase separation to occur differ vastly, it is possible to separate their effects (as in the case of agarose4,5). Where they occur over similar time scales, it becomes more difficult, however, and the complex interplay between the phase separation and gelation kinetics enables a range of morphologies to be produced. In this paper, the gelatin (polypeptide)/maltodextrin (polysaccharide) system was chosen because the relative positions of the phase separation and gelation temperatures allow phase separation to be studied in the liquid and gel states. Studies of the closely * E-mail: [email protected]. Telephone: +44 (0)1234 22 2958. Fax: +44 (0)1234 22 2757.

related gelatin/dextran system have shown marked differences between the phase separation behavior in these two cases.1 In addition to the quench temperature, it is also known, from studies of synthetic polymer mixtures, that the addition of small amounts of copolymers containing portions compatible with each of the phases in the demixed system can affect the phase separation kinetics and final properties of the mixture (by altering, for example, domain size and coarsening rate).6 Using this principle the glycoprotein gum arabic, which is known to contain polypeptide portions and polysaccharide portions, was proposed as a potential biopolymer compatibilizer in the gelatin/maltodextrin system. To our knowledge, the principle of using a biocompatibilizer in a phase-separated biopolymer system has not been tested before. The work reported in this paper was therefore aimed at testing whether a biopolymer containing characteristics of each phase (in this case a glycoprotein), can act as a compatibilizer and affect the development of the microstructure in a phase-separated biopolymer system. To do this, the phase separation behavior of the gelatin/maltodextrin system was compared with the gelatin/maltodextrin/gum arabic system, where gum arabic was present in amounts where it would only have an influence on phase separation if it were active at the gelatin/maltodextrin interface. In addition, the paper also provides a comparison of three widely used, separate techniques (scattering, turbidity, and microscopy) for studying phase separation. Theory of Phase Separation. Phase separation occurs in a mixture of components when it is energetically favorable for the components to exist in separate phases rather than being mixed together.1 It is generally accepted that there are two possible mechanisms of phase separation, depending on

10.1021/bm015503r CCC: $20.00 © 2001 American Chemical Society Published on Web 08/23/2001

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Phase Separation in Biopolymer Mixtures

where the system is quenched to in the phase diagram. Quenches near the binodal result in the nucleation and growth mechanism whereas deeper quenches, such that the second derivative of the free energy with respect to concentration, ∂2f/∂c2, is negative, result in a mechanism known as spinodal decomposition. In this region the free energy decreases immediately, even for infinitesimally small concentration fluctuations, and there is no restoring force opposing the formation of a two-phase structure.7 The dynamics of phase separation by spinodal decomposition are characterized by three regimes: the early, intermediate, and late stages of growth. According to the simplest theory of spinodal decomposition that was developed for small molecular weight materials (Cahn’s linear theory8,9), in the early stages one particular wavelength in the concentration fluctuation will grow most rapidly, causing one characteristic length scale to be expressed in the morphology. After a while, in the intermediate growth stage, nonlinear growth terms cause the concentration fluctuations to move to a larger length scale. In polymer systems, the presence of chain entanglement makes viscoelastic effects important, especially when there is a large difference in the material properties of the components in the mixture, introducing a range of length and time scales. There is now no longer only one time or length scale that describes the system, and the viscoelastic model of phase separation, which differs from the linear theory in its description of the initial growth rates of the concentration fluctuations during the early stages of phase separation, is thought to be relevant.10,11 At the late stages, coarsening of the microstructure occurs, with the characteristic length scale of the concentration fluctuations being related to the time after initial phase separation by a power law. Spinodal decomposition is often studied using small-angle light scattering, because the scattering pattern is the spatial Fourier transform of the real-space concentration fluctuations, which is the most convenient way to quantify the concentration fluctuation distribution. The scattering intensity can be written as I(q) ) I(q,0)e2R(q)t

(

)

1 - q2/2qm2 1 + ξve2q2

(3)

where ξve is known as the “viscoelastic length”, and gives the length scale above which the dynamics are dominated by diffusion and below which are determined by viscoelastic effects. The Cahn-Hilliard plot should therefore be nonlinear, although fitting with the function in eq 3 will yield the parameters Deff, qm and ξve. As for the linear theory, there is a maximum in the amplification factor and scattering intensity, the position of which yields the characteristic length scale, ξ, that is observed in the structure, given by ξ)

2π qm

(4)

Additionally, information on the interface between the different phases can be obtained by measuring the asymptotic shape of the scattering intensity, Iasympt, at high q values. For the case of sharp interfaces the following relation, known as Porod’s law,12 gives the intensity, Iasympt (provided that the length scales in which there are inhomogeneities in refractive index are much larger than 1/q): A Iasynpt(q) ∝ 〈δn2〉 4 q

(5)

δn is the refractive index difference between the two phases and A is the amount of interfacial area between the two phases. A plot of ln(I) vs ln(q), known as a Porod plot, should yield a straight line with a gradient of -4, if the interfaces are sharp compared to the wavelength of light. If it is assumed that during the late stage of phase separation the compositions of the two phases are close to the equilibrium coexistence values and that there is a pattern of domains separated by equilibrium interfaces, then the presence of only one relevant length scale and time scale causes the structure to exhibit self-similarity.10,13 The scattering patterns should therefore collapse onto a master curve, scaled by the mean domain size. Assessing the validity of the following relation can identify dynamic scaling

(1)

where the scattering vector, q (which is related to the scattering angle, θ, and the wavelength, λ, by the relation q ) (4π/λ) sin(θ/2)) is related to the real-space length, d, by q ) 2π/d, and R(q), known as the amplification factor, describes the growth in concentration fluctuations at scattering vectors q corresponding to real-space lengths, d. In the linear theory of spinodal decomposition, R(q) (and therefore the scattering intensity) has a maximum at a q value denoted by qm, and is given by R(q) ) Deff(q)q2 1 -

(

Rve(q) ) Deffq2

)

q2 2qm2

(2)

where Deff is the effective diffusion coefficient of the solutes. A plot of R(q)/q2 vs q2 (known as a Cahn-Hilliard plot) should be linear if the linear theory is applicable. In the presence of viscoelasticity, however, eq 2 is modified to give a different expression for the growth rate

qm3(t)Im(t) q qm I(q,t)q2 dq ∫q〈〈q

) constant

(6)

m

where Im is the peak intensity and qm the position of the peak. The absence of dynamic scaling may indicate that the compositions of the phases have not reached their equilibrium values. The power law relationships between characteristic size and time that are observed in the late stage can be separated into three distinct mechanisms, depending on the means of mass transport. The first two mechanisms, which both give an exponent of 1/3, are the growth of larger droplets at the expense of smaller droplets (Ostwald ripening) with mass transport occurring by diffusion through the matrix14 and movement of the droplets toward each other leading to coalescence.12 In concentrated systems, the coalescence mechanism is expected to be dominant, whereas in dilute systems, where the droplets are less likely to meet, or in

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gelled systems, where the droplets are less likely to be mobile enough to move toward each other, the Ostwald ripening mechanism will be more important. The third mechanism, which gives an exponent of 1, occurs when hydrodynamic flow is the cause of mass transport.15 This mechanism occurs when there is sufficient material within the coarsening phase for flow fields to become established inside the coarsening phase which are coupled to flow fields in the matrix via the interface. Because it requires fluid flow, it is only observed in liquid systems, and will not be observed if gelation occurs.1 Experimental Section Materials. The gelatin was a lime-treated gelatin (LH1e) supplied by SKW. Using size-exclusion chromatography (SEC) coupled with light scattering the weight and numberaverage molecular weights were found to be as follows: Mw, 146 000; Mn, 83 300. The maltodextrin (SA2, supplied by Avebe) possessed a broad molecular weight distribution; the DE value of 2 allowed an estimate of 9000 to be made for its value of Mn, however. Moisture contents of 12.4% and 10.0% for gelatin and maltodextrin respectively were taken into account when making solutions. The gum arabic (gum acacia, GA) was supplied by Fisher Scientific. Molecular weights were not known for this material, although from the literature a value of Mw between 250 000 and 1 000 000 is expected.16 To prevent biological degradation, all solutions were used only on the day that they were made. When gum arabic was present, it was as a minor additive and always at a fixed concentration. Care was taken throughout sample preparation to prevent dehydration. Solutions for all the experiments except for the dynamic light scattering were prepared as follows: SA2 powder was first dispersed in cold de-ionized water then mildly stirred at 95 °C for 30 min to form a solution. Gelatin solutions were prepared similarly, but were not heated above 60 °C to prevent any thermal degradation. Before heating, sodium chloride was added to the gelatin solution to increase the total ionic strength of the final mixed solution to 0.3 M (this figure includes the ionic contributions from the SA2 and the gelatin). After 30 min at 95 °C the SA2 was cooled to 60 °C for 5 min and then mixed with the gelatin solution, to form a final mixture containing 5 wt % gelatin and 3 wt % maltodextrin. The mix was then kept at 60 °C and stirred continuously. After mixing and while still at 60 °C, the solution had a pH (measured using a Jenway 3071 automatic temperature compensated pH meter) of approximately 5.6; this was increased to pH 7 by the dropwise addition of 1 M sodium hydroxide. A 10 wt % solution of gum arabic (GA) was prepared and heated to 60 °C for half an hour while being stirred. The gum arabic was added to the gelatin/SA2/0.3M salt mix (at 60 °C and pH 7). The conditions of salt concentration 0.3 M and pH 7.0 were chosen to prevent the formation of insoluble complex coacervates between the oppositely charged gelatin and gum arabic molecules.17 Using a Gilson pipet, 100 µL of the gum arabic solution was added to 15 mL of the gelatin/SA2 mix, making the gum arabic content approximately 0.07 wt % of the final mix. Samples either had zero or 0.07% gum arabic in them.

Butler and Heppenstall-Butler

Small-Angle Light Scattering (SALS). Samples for study by SALS were made by placing a drop of the mixed solution at 60 °C onto a glass coverslip (thickness 0.17 mm, diameter 22 mm) in the center of a 75 µm stainless steel spacer (diameter 14 mm), which was heated to 65 °C on the heating/ cooling stage (Linkam THMS600). Another, identical, glass coverslip was placed on top of the sample to ensure that it was a constant thickness of 75 µm. SALS patterns were formed via the interaction of a collimated, spatially filtered, laser beam (Spectra-Physics model 127, 35 mW, wavelength 632.8 nm, beam diameter approximately 1 mm) with the sample. The sample was mounted on the heating/cooling stage set to cool the sample at a rate of 50 °C/min by a flow of liquid nitrogen passed through the block using a pump. The aperture of the heating/ cooling block on which the sample was mounted was approximately 2.5 mm. Light transmitted through and scattered by the sample was collected by a bi-convex lens and projected onto a grayed glass screen on which a small circular black beam-stop was placed to obscure the directly transmitted beam. A CCD camera equipped with a variable zoom lens focused on the screen imaged the scattering pattern. Scattering patterns were recorded every 2 s to investigate the early-stage phase separation and every 30 s to investigate the late-stage behavior. Data collection began at the start of the temperature profile, beginning with an isotherm at 65 °C for 2 min followed by a rapid quench at 50 °C/min to 15, 25, or 35 °C. The scattering patterns collected during the first 2 min at 65 °C were averaged, and this average was subtracted from the subsequently collected scattering patterns. The 2D scattering patterns were radially averaged prior to analysis. Dynamic Light Scattering (DLS). Dynamic light scattering (DLS) was performed using a Malvern Autosizer 4700. The 0.05 wt % solutions were made of gelatin, SA2, and gum arabic. All solutions were made up to have 0.3M salt content and to be pH 7. The SA2 solution was heated and stirred at 95 °C for 30 min before being cooled to 60 °C. The gelatin and the gum arabic solutions were heated to 60 °C and stirred for 30 min before being used. A 0.025 wt % gelatin/0.025 wt % gum arabic mixed solution was made by weighing out the gelatin, gum arabic, sodium chloride and water into a bottle before being stirred for 30 min at 60 °C. The 0.025 wt %/0.025 wt % gelatin/SA2 and 0.025 wt %/0.025 wt % gum arabic/SA2 mixed solutions were made by mixing equal volumes of premade single ingredient 0.05% solutions at 60 °C. All the solutions were filtered using a 0.45 µm pore size sterile filter. About 2 mL of solution was placed in a cylindrical glass cuvette which was placed in a temperature controlled sample environment in the DLS apparatus. The correlation function was measured at angles of 60, 90, and 120°, at temperatures of 15, 25, 35, and 60 °C. Three measurements, consisting of 10 subruns each, were made for each sample at each angle and temperature to ensure reproducibility of results. Analysis of the correlation function to yield a particle size distribution was made using the CONTIN analysis.



Turbidity. Turbidity was used to measure the droplet sizes in the phase-separated mixtures, for comparison with the SALS results, and to measure the cloud point temperature, used as a measure of the phase separation temperature, for gelatin/maltodextrin mixtures with and without gum arabic. Phase separation temperatures were measured by monitoring the turbidity of a solution cooled at 1 °C/min at 800 nm. For the size measurements, 3 mL aliquots of the final 0.3M salt, pH 7, gelatin/SA2 mix as used in the SALS experiments, with or without added gum arabic, were transferred to 10 mm PMMA spectrophotometer grade cuvettes using a prewarmed pipet and transferred immediately to a single sample cell prewarmed to 60 °C. Turbidity spectra were measured using a Perkin-Elmer Lambda 40 spectrophotometer connected to a programmable cooler. Spectra were recorded over wavelength range between 600 and 800 nm. Two types of cooling profiles were used; in the first the sample was retained at 60 °C for 2 min then cooled at 1 °C/min to 10 °C, where it remained for a further 2 min. In the second, chosen to be as close as possible to the quenches used in the SALS experiments, the sample started at 60 °C for 2 min followed by a fast cool to 35, 25, or 15 °C where it remained for up to an hour and a half. The temperature profile of the sample during the quench was recorded by the controlling PC. Spectra were taken every 10 s during the temperature profile. The analysis of the turbidity data has been reported previously where it is explained in detail.18 It is based on a Rayleigh-Gans-Debye treatment of the wavelength dependence of turbidity, taking into account the finite acceptance angle of the spectrophotometer. The analysis is only valid for modeling the growth kinetics of spherical droplets below 5 µm in diameter; above this size reasonable fits to the data to extract the droplet size could not be obtained. Confocal Laser Scanning Microscopy (CLSM). Solutions for use on the CLSM were prepared as for the SALS and turbidity samples, except that a pinch of rhodamine B powder was added to the hot mix and stirred in after the pH had been altered and the gum arabic added. The samples were made in the same way as those for SALS. The sample was placed on a temperature-controlled stage (Linkam THMS600). The temperature profile used was the same as for the SALS experiments. Micrographs of the mixtures were acquired using a Biorad MRC 600 CLSM. A 488 nm Argon laser excited the rhodamine B, which provided the contrast between the gelatin (which appeared dark) and SA2 (which appeared pale) phases. Micrographs were captured using COMOS software. A ×20 lens was used with a digital zoom of ×2.5, ×5, or ×10. The micrographs taken using the ×2.5 zoom were suitable for image analysis. A fast Fourier transform (FFT) was taken of each micrograph and then radially averaged. In the cases where a peak was present in the FFT of the image, the position of maximum intensity was taken as the peak position. Both real and reciprocal space were calibrated using a square array of circles and a graticule.

Figure 1. Evolution of the SALS intensity profiles for gelatin/ maltodextrin/gum arabic quenched to 15 °C.


Results Small-Angle Light Scattering. General Observations. In all cases, a quench into the two-phase region resulted in an isotropic ring in the 2D scattering pattern measured by the CCD camera, which translated into a broad peak in the radially averaged scattering intensity. Figures 1-3 show the development with time of the radially averaged scattering intensity for the gelatin/maltodextrin/gum arabic system quenched to 15, 25, and 35 °C, respectively. Similar results were obtained for the system without gum arabic. For all of the temperatures an initial peak formed that remained in roughly the same position for a few seconds after the onset of phase separation but rapidly began to move to lower q values (representing larger length scales). Evolution of the structure was rapidly arrested in the sample quenched to 15 °C. Coarsening of the microstructure occurred to greater extents as the quench temperature increased. The initial peak appeared at lower q values (larger length scales) as the quench temperature increased. For the same quench temperature, no significant differences in the initial peak position were measured between the samples with gum arabic and those without (from the average of five measurements).

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Figure 4. Evolution of the peak position at early times for the samples quenched to 15 °C.


Early-Stage Phase Separation. Figures 4-6 show the evolution of the peak position with time during early-stage phase separation for quenches to 15, 25, and 35 °C, respectively. The initial quasi-stationary peak position can be seen in the first few seconds leading to coarsening and transition to late-stage behavior. There was no difference, within the limits of experimental reproducibility, between



the initial peak position and subsequent behavior for the samples containing gum arabic and those without. As the quench temperature decreased, the time before coarsening began became smaller. In the samples quenched to 15 °C, a coarsening regime with an exponent of -1/3 was never reached, although the initial 10-20 s of coarsening was more rapid than the late-stage behavior. For the samples quenched to 25 and 35 °C, a coarsening regime with a gradient of about -1/3 was achieved. The rate of coarsening subsequently decreased and then remained constant for the sample quenched to 25 °C. For the sample quenched to 35 °C the rate of coarsening established after the initial stages of phase separation did not slow. From the early stages of phase separation, when the peak was stationary or quasi-stationary, plots were made of the evolution of scattering intensity with time for all of the q values. From these plots the amplification rate was calculated for each q value and Cahn-Hilliard plots were constructed, shown in Figure 7, parts a and b, for the samples with and without gum arabic, respectively. These plots were not linear, as predicted by the fluid model of phase separation, although the equation predicted by the viscoelastic model was found to yield reasonable fits to the data for the samples with gum arabic, and excellent fits for the samples without gum arabic. The values of Deff, initial qm and ξve obtained from fitting the viscoelastic model to the Cahn-Hilliard plots are shown in Table 1. The values obtained from fitting the viscoelastic theory were fairly reasonable, with the exception of the initial qm for the sample quenched to 35 °C with gum arabic. Late-Stage Phase Separation. The presence of gum arabic did not affect the late-stage coarsening behavior, which was described in all cases by a power law. The major influence was the quench temperature, which determined the exponent of the power law. To demonstrate this, a comparison of the peak positions for the samples containing gum arabic and quenched to the different temperatures is shown in Figure 8 as an example. In the gelled samples the latestage coarsening was less rapid than in the nongelled sample, and the rate of coarsening decreased with decreasing quench temperature. The actual values of the coarsening power law exponents were -0.01, -0.18, and -0.27 (with an uncertainty of about 10% calculated from the repeat measure-



Figure 8. Comparison of the evolution of the peak position for the samples containing gum arabic quenched to different temperatures.

Figure 7. Cahn-Hilliard plot for (a) gelatin/maltodextrin/gum arabic mixtures and (b) gelatin/maltodextrin mixtures fitted with an expression (solid line) from the viscoelastic theory of phase separation. Table 1. Parameters Obtained from Fitting the Cahn-Hilliard Plots to the Viscoelastic Model of Phase Separation sample

Deff (µm2s-1)

qm (µm-1)

ξve (µm)

15, without GA 15, with GA 25, without GA 25, with GA 35, without GA 35, with GA

0.02 0.03 0.01 0.06 0.03 0.04

3.52 2.03 3.22 2.04 4.16 22.44

0.33 1.85 0.24 4 4.43 4.08

ments) for the samples quenched to 15, 25, and 35 °C, respectively. In the samples that were quenched to 35 °C and remained fluid, the power law behavior with a gradient of about -1/3 was followed by a broad transition, occurring over several hundred seconds, to a power law with a lower exponent (i.e., the rate of coarsening increased), measured to be -0.84 ( 0.08. Parts a and b of Figure 9 show the evolution of the peak intensity with time for the samples with and without gum arabic, respectively. For the quench to 25 °C the intensity continued to increase throughout the experiment whereas for quenches to 15 and 35 °C the peak height reached a maximum value and then decreased slightly. The time at which this maximum occurred in the latter case coincided with the transition of the gradient of the plot of qm vs time from -1/3 to -1.

Figure 9. Evolution of the peak height for (a) gelatin/maltodextrin/ gum arabic and (b) gelatin/maltodextrin samples quenched to different temperatures.

Figure 10 shows plots of eq 6 to test for dynamic scaling in the late-stages of phase separation for the samples quenched to 25 and 35 °C without gum arabic. Dynamic scaling was not tested for the samples quenched to 15 °C because at that temperature the structure had become trapped by gelation and the scattering curves were virtually unchanged with time anyway. The dynamically scaled amplitude was not constant at all for the samples quenched to 25

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Figure 10. Plot to test for dynamic scaling in samples without gum arabic.

Figure 12. CLSM images of the microstructure formed after quenches to (a) 15, (b) 25, and (c) 35 °C in gelatin/maltodextrin/gum arabic samples. The times are the time after the quench temperature was reached. Image width 72 µm.

Figure 11. Porod plot for the sample with gum arabic quenched to (a) 25 °C, (b) 35 °C.

°C. However, the samples quenched to 35 °C appeared to show dynamic scaling for part of the first coarsening regime. The breakdown of dynamic scaling occurred during the transition from the first coarsening regime to the second, more rapid, one. Parts a and b of Figure 11 show Porod plots for the sample containing gum arabic quenched to 25 and 35 °C, respectively. Similar plots were obtained in the samples without gum arabic. The Porod region was not accessible for the samples quenched to 15 °C. A line with a slope of -4 is

superimposed on the plot for comparison. For 35 °C the slope of the high q region of the scattering intensity gradually decreased until a value of -4 was obtained. For samples quenched to 25 °C, the slope did not reach -4 over the time scale studied and the intensity of the high q region of the scattering curve remained approximately the same. Dynamic Light Scattering. Under the conditions used, gelatin had a diameter of approximately 22 nm, increasing slightly with decreasing temperature. Gum arabic had a size of approximately 38 nm, also decreasing slightly with increasing temperature. Maltodextrin had a bimodal size distribution, with sizes of 2 nm and 50 nm. The size distributions of the mixed systems were consistent with them being simple mixtures, and no species with sizes greater than any of the individual components were found. Confocal Laser Scanning Microscopy. To demonstrate the development of the morphology of the samples quenched to different temperatures, micrographs of quenches to 15, 25, and 35 °C, in the samples containing gum arabic, are shown in Figure 12, parts a-c, respectively. The morphologies observed for the samples without gum arabic were identical to those with it, within error. For the lower two quench temperatures a visible microstructure was observable by the time the quench temperature was attained. At 35 °C, however, the microstructure took longer to appear and was only observed at some time after the quench temperature was reached. The samples quenched to 15 and 25 °C initially possessed a mottled texture that, for the case of the 15 °C sample, was retained during coarsening. The initial microstructure of the samples quenched to 35 °C was too indistinct to determine whether it was mottled or consisted of separate


Figure 13. CLSM images of a gelatin/maltodextrin/gum arabic sample showing the occurrence of sedimentation in the type of sample used for CLSM and SALS. The sample was quenched to 35 °C and the images taken after 2700s. Images taken at the top and bottom of a 75 µm thick sample. Image width: 144 µm.

spheres. However, after a while separate spheres were definitely present, eventually reaching diameters up to 20 µm. The extent of sedimentation in the samples was measured by moving the imaging plane vertically through the sample. Figure 13 shows two images taken at different depths in a sample quenched to 35 °C and shows that after about 2000 s a significant amount of sedimentation had occurred. By taking long exposure images at a fixed position within the sample, the extent of movement of the particles within the field of view was also observed. Figure 14 shows some long time exposures for a sample quenched to 35 °C. Significant lateral motion was observed as shown by the rapid change in the number of droplets present in the field of view after 1 min in Figure 14. Figure 15 shows a sequence of Fourier transforms obtained from confocal images taken at different times after the quench to 35 °C for the sample with gum arabic. In this, as for all of the samples at all of the quench temperatures, the Fourier transform consisted of a broad peak, similar to the SALS patterns, that moved to lower q values with increasing time. The position of the peak measured by SALS was approximately the same as the peak position from the FFT of the images. Furthermore, the real-space distance represented by this peak corresponded roughly to the interparticle distance on the images. Figure 16 shows the change in the peak position for the samples quenched to 15, 25, and 35 °C. As for the SALS data, the peak position did not change significantly for the sample quenched to 15 °C. A gradual decrease in peak position was observed for the 25 °C quench and for the 35


Figure 14. CLSM images of a gelatin/maltodextrin/gum arabic sample demonstrating the mobility of the particles during quenches to 35 °C. The top micrograph is a 60 s average taken 5940 s after reaching the quench temperature. The bottom micrograph is a 3 s average taken immediately after the top micrograph. Image width: 72 µm.

Figure 15. Evolution of the FFT of the CLSM image with time for gelatin/maltodextrin/gum arabic quenched to 35 °C.

°C quench samples the more rapid decrease in peak position with time followed a power law with an exponent of approximately -1/3. No significant difference was measured between the samples containing gum arabic and those that did not. Turbidity. Figure 17 shows the phase separation temperature for the gelatin and maltodextrin continuous samples, with and without gum arabic, measured by turbidity. The addition of 0.07% gum arabic had no noticeable effect on the phase transition temperatures measured using turbidity at a cooling rate of 1 °C/min, at any bulk concentration. Moreover, the actual turbidity profile during cooling was not significantly affected by the presence of 0.07% gum arabic.

820


Figure 16. Evolution of the peak position in the FFT of the images for samples quenched to 15 (circles), 25 (triangles), and 35 °C (squares). The open symbols represent samples with gum arabic, and the closed symbols represent samples without gum arabic.


Figure 18. Evolution of the droplet sizes from turbidity measurements for samples quenched to 35 °C. The open symbols represent samples with gum arabic and the closed symbols represent samples without gum arabic.

Discussion

Figure 17. Phase separation temperature, TPS, measured for 2% and 5% gelatin solutions, with and without gum arabic, for a range of maltodextrin concentrations.

For the quenched samples for which turbidity measurements at a range of wavelengths were used to calculate droplet sizes, the following observations were made. Greater turbidities were achieved, and the turbidity increased most rapidly, in the samples quenched to the lowest temperatures. The samples quenched to 15 °C achieved their maximum turbidity before the final quench temperature was reached whereas for the samples quenched to 25 and 35 °C it reached a maximum just after the quench temperature was reached. The presence of gum arabic did not significantly alter the turbidity profiles. Figure 18 shows the results of the size analysis of the turbidity spectra for the samples quenched to 35 °C, because these were the only samples that were shown by the microscope images to contain spherical droplets. For these samples, there was a significant region after the quench temperature was reached in which the size increased with time by a power law with an exponent of 1/3, as observed by both SALS and image analysis. The sizes measured by turbidity were, however, significantly lower than the characteristic size in the structure measured by SALS and image analysis. They were also lower than the sizes of the droplets that could be measured directly from the micrographs.

Early Stages of Phase Separation. The broad peak in the SALS intensity profiles (and in the FFT of the confocal microscope image) that formed at the onset of phase separation, and initially remained in roughly the same position while increasing in amplitude exponentially, is indicative of a spinodal decomposition phase separation mechanism.7 The mottled microstructures that were observed in the quenches to 15 and 25 °C are also characteristic of a spinodal decomposition mechanism. The differences between the structures produced by quenching to different temperatures may be explained simply in terms of the linear theory of phase separation.1 The growth of structures at larger length scales for quenches to lower temperatures is because the second derivative of the free energy becomes more negative at lower temperatures, causing a greater drive for phase separation. The competition between phase separation and gelation was demonstrated by the initial stages of phase separation and coarsening. In the gelled samples, the time during which the peak remained quasi-stationary was smaller for lower temperatures because the phase separation kinetics were more rapid. However, the greater reduction in duration, and smaller coarsening exponent, of the initial coarsening regime for the 15 °C quench temperature was a consequence of gelation rapidly slowing down the phase separation kinetics.1-3 When the samples were quenched to 25 °C, the phase separation kinetics were impeded to a lesser extent, although significantly more compared to the nongelling samples. Loren and Hermansson3 found that by altering the gelatin and maltodextrin concentrations it was possible to change the relative rates of gelation and phase separation. In some systems (5% gelatin/5% maltodextrin), phase separation was observed to occur before gelation trapped the microstructure (although their system was slightly different to the one used in the current report since no salt was added). The nonlinearity of the Cahn-Hilliard plots demonstrates that the linear model does not provide a completely accurate description of the phase separation mechanism. Nevertheless,


the generally correct predictions of the observed trends for the values of the characteristic length scale appear to show that it is not completely inaccurate. The reasonable fits of the Cahn-Hilliard plots provided by the viscoelastic theory suggest at first glance that this may be a more accurate description of the phase separation mechanism. However, although the values for the characteristic length scale and viscoelastic length, and the relation between them, are roughly as expected10 and generally reasonable, they do not provide sufficient evidence to either confirm or discount the viscoelastic model. Curvature of the Cahn-Hilliard plot has been explained by the presence of different time scales in the phase separation process,19 and it has been suggested that the conformation change of the gelatin molecules at the onset of gelation may be one of the responsible factors.2,3 However, because the Cahn-Hilliard plot is curved even in the absence of ordering of the gelatin, it is more likely that the curvature is a general feature of viscoelastic systems. Late Stages of Phase Separation. The coarsening power law exponent from SALS of approximately 1/3 measured for the fluid samples combined with the confocal microscope images demonstrating the mobility of the droplets suggests that droplet coalescence was the dominant coarsening mechanism when gelation did not occur. The transition to a region with a more rapid rate of coarsening (i.e., approximately -1) once the droplets had reached a certain size is interpreted as the onset of hydrodynamic flow becoming an important mechanism for the re-shaping and coalescence of droplets.1 The CLSM images support this hypothesis via the demonstration of the highest droplet mobility at the later times at which SALS suggested that the hydrodynamic mechanism was operative. The transition was not sharp, however, showing that the hydrodynamic mechanism coexisted with the other ones from its onset. Coexistence of coarsening mechanisms is the most likely explanation for the measured power law, after the transition, being slightly (but significantly) lower than the value of 1 that is predicted for the hydrodynamic mechanism. Coexistence of mechanisms is supported by the droplet size analysis obtained from turbidity measurements, which appeared to show that the smaller droplets continued to coarsen via coalescence. It should be noted, however, that the turbidity size analysis assumes, incorrectly, that the scattering structure factor is equal to unity. Simulations of the scattering patterns from distributions of spheres show, however, that the amount of light scattered in the region of the SALS pattern above the acceptance angle of the spectrophotometer, which is the quantity used by the turbidity technique to calculate droplet sizes, may be very similar to the amount in the case where the scattering pattern displays a maximum.20 If this is the case, then it is reasonable to assume that the sizes calculated from the turbidity technique really do represent the smaller droplets in the mixture. In the cases where gelation occurred, coarsening occurred via diffusion of molecules through the matrix rather than by coalescence of droplets. The hydrodynamic mechanism, with a power law exponent of 1, was not observed in any of the gelled systems because flow was suppressed by gelation. The Porod plots provide information about the develop-


ment of the interface in the phase separating mixtures. They show that in the sample quenched to 35 °C the interface was sharper (i.e., the thickness of the interface was negligible compared to the size of the microstructure) than in the 25 °C gelled sample. Inspection of the confocal micrographs also reveals apparently sharper droplets in the nongelled sample. It is likely that gelation arrested the development of the microstructure and prevented the interface becoming sharp in the gelled sample. The shift of the curve to lower intensity values for all samples, gelling and nongelling, is in contrast to previously reported work on the similar gelatin/ dextran system.1 In that work, a decrease in intensity was measured for the nongelling sample but the curves overlapped in the gelling sample, interpreted as droplet coalescence causing domain growth in the former case but droplet aggregation and not coalescence in the latter. However, in that study the gelatin phase was the droplet phase, whereas in the present study it is the continuous phase. It is therefore apparent that inversion of the phases causes qualitatively different coarsening behavior. In the present study, the much slower rate of decrease of intensity of the curves in the gelling sample supports the much slower rate of coarsening of the microstructure. No definite information is revealed on the precise mechanism (i.e., evaporation-condensation or droplet coalescence), although the faster rate of change of interfacial area in the nongelling sample is consistent with the previous assertion that droplet coalescence is the dominant process in that sample. For dynamic scaling to apply, the system must have attained the equilibrium phase composition and the phase separation must be described by only one length and time scale. The absence of dynamic scaling in the gelled samples may be evidence that phase separation is more accurately described for these samples by the viscoelastic theory, where a range of length scales affect the phase separation process. As gelation proceeds, it is necessary to introduce an elastic energy term into the free energy of mixing because a weakly cross-linked network is formed.21 Because this term changes as the extent of gelation increases, the free energy term constantly changes, raising the spinodal temperature and consequently continually reducing the characteristic length scale of the microstructure. Also, it should be noted that because the interface between the phases was not sharp in the gelled samples, there could be more than one length scale in the system anyway (namely the characteristic length scale determined by spinodal decomposition and the interfacial length scale). The presence of a significant interfacial length scale has been postulated as a cause of the absence in scaling.22 In the nongelling system viscoelastic effects were not important during the late stages of phase separation, since dynamic scaling was observed in both the gelatin/maltodextrin system reported in the present study and the gelatin/ dextran system reported previously. Although most theoretical work has concentrated on the scaling behavior during the 1/3 power law regime, a change of scaling, possibly leading to the breakdown that was observed in the fluid gelatin/maltodextrin and gelatin/dextran systems, with the onset of hydrodynamic coarsening has been suggested theoretically.23-25 In the gelatin/dextran system, it was

822


postulated that changes in phase composition, possibly related to the polydispersity of the components, were responsible for the breakdown of dynamic scaling at the crossover between the different coarsening regimes.1 This explanation appears to be supported by the observation in the present study of the changing peak height in the SALS patterns (which is related to the compositions of the two phases) during hydrodynamic coarsening. That the peak height also decreased after reaching a maximum value for the gelled sample formed by the quench to 15 °C indicates that gradual changes in phase composition occur generally and that the equilibrium values are not rapidly attained. The Influence of Gum Arabic on the Phase-Separated Microstructure. Depending on whether phase separation is described by the linear or the viscoelastic model, the equations that describe the development of the structure at different length scales are determined by either two or three considerations. In the linear theory, the important factors are the second differential of the free energy of mixing and the mobility of the components. The viscoelastic theory introduces an additional variable, the “viscoelastic length”, which is determined by the material properties of the components. First, it is important to note that the amount of gum arabic added was small and therefore not sufficient to alter the phase diagram by altering the concentration of the gelatin and maltodextrin components. It also had no effect on the pH of the mixture. Therefore, any differences in the phase separation behavior in the presence of gum arabic were expected to result from changes to the mechanism or kinetics of phase separation itself. The results showed that none of the above considerations were affected by gum arabic (in the conditions that no coacervates were formed). There was direct evidence from the turbidity data that the free energy of mixing was unaffected by the gum arabic, since the phase separation temperatures and turbidity values during cooling remained unchanged. Dynamic light scattering showed that the mobility of the components was unlikely to be drastically altered, since no association between gum arabic and any of the components was measured in dilute solution. It would appear that these results were also valid in concentrated solution, since there was no difference between the samples with and without gum arabic. That no difference in the initial rate of coarsening was observed when the system was fluid, at 35 °C, suggests that the gum arabic did not influence the continuous phase viscosity. Therefore, the terms involved in the viscoelastic theory of phase separation were not affected by the presence of gum arabic. Furthermore, if the gum arabic resided at the interface between the matrix and droplets, it did not affect the frequency of successful collisions and cannot have altered the interfacial tension significantly. The latter effect would be expected to have caused a difference in initial characteristic size in the structure, which was not seen. Conclusions Phase separation was observed using time-resolved smallangle light scattering, confocal laser scanning microscopy,


and turbidity measurements in the gelatin/maltodextrin and gelatin/maltodextrin/gum arabic systems in which the gelatinrich component was the continuous phase. All of the techniques provided complementary information on aspects of structure development during phase separation, although only SALS was able to provide information on the early stages of phase separation. SALS showed that phase separation occurred by spinodal decomposition. Under conditions in which the gelatin formed a gel, phase separation occurred over the same time scale as gelation. As the quench temperature was decreased the amount of coarsening of the phase-separated microstructure was hindered to a greater extent; at 15 °C the structure was trapped about 1 min after the onset of phase separation, whereas at 25 °C coarsening continued. In the trapped microstructure, the interface between the two phases remained relatively diffuse although the phase compositions in the separated phases continued to change slowly. The coarsening of the gelled samples proceeded in a different manner than previously reported in the phase-inverted system (i.e., where the gelatin-rich phase formed droplets). In the nongelling sample, coarsening occurred by droplet coalescence. Once the droplets had reached a certain size, there was a transition to a hydrodynamic regime where the droplets were highly mobile and convective effects accelerated coarsening. During this process the compositions of the two phases continued to change, and the interface between them became sharp. When gum arabic did not form coacervates with gelatin, but when enough was added to have had an effect at the gelatin/maltodextrin interface if it was interfacially active, no major difference was measured between the microstructure and phase separation kinetics with and without gum arabic. Therefore, it is concluded that under the conditions used, gum arabic was not interfacially active in the gelatin/ maltodextrin system and cannot therefore be used as a “biocompatibilizer” in the phase-separated system. Acknowledgment. Helpful discussions with Ian Norton, Allan Clark, and Bill Williams, of Unilever Research, Colworth, England, are gratefully acknowledged. References and Notes (1) Tromp, R. H.; Rennie, A. R.; Jones, R. A. L. Macromolecules 1995, 28, 4129. (2) Bansil, R.; Lal, J.; Carvalho, B. L. Polymer 1992, 33, 2961. (3) Loren, N.; Hermansson, A.-M. Int. J. Biol. Macromol. 2000, 27, 249. (4) Emanuele, A.; Distefano, L.; Giacomezza, D.; Trapanese, M.; PalmaVitorelli, M. B.; Palma, M. U. Biopolymers 1991, 31, 859. (5) Leone, M.; Sciortino, F.; Migliore, M.; Fornili, S. L.; Palma-Vitorelli, M. B. Biopolymers 1987, 26, 743. (6) Shull, K. R.; Kramer, E. J. Macromolecules 1990, 23, 476. (7) Strobl, G. in The Physics of Polymers; Springer-Verlag: Berlin, 1996. (8) Cahn, J. W.; Hilliard, J. E. J. Chem. Phys. 1958, 92, 258. (9) Cahn, J. W. J. Chem. Phys. 1965, 42, 93. (10) Tanaka, H. J. Phys: Condens. Matter 2000, 12, 207. (11) Hohenberg, P. C.; Halperin, B. I. ReV. Mod. Phys. 1977, 49, 435. (12) Porod, G. in Small-Angle X-ray Scattering; Glatter, O., Kratky, O. Eds.; Academic Press: London, 1982. (13) Binder, K.; Stauffer, D. Phys. ReV. Lett. 1974, 33, 1006. (14) Lifshitz, I. M.; Slyozov, V. V. J. Phys. Chem. Solids 1961, 19, 35. (15) Siggia, E. D. Phys. ReV. A 1979, 20, 595.

Phase Separation in Biopolymer Mixtures (16) Whistler, R. L.; Miller, J. N. in Industrial Gums, Polysaccharides and their DeriVatiVes; Academic Press: London, 1959. (17) Philipp, B.; Dautzenberg, H.; Linow, K.-J.; Ko¨tz, J.; Dawydoff, W. Prog. Polym. Sci. 1989, 14, 91. (18) Williams, M. A. K.; Aymard, P.; Clark, A. H.; Norton, I. T. Langmuir 2000, 16, 7383. (19) Binder, K.; Frisch, H. L.; Ja¨ckle, J. J. Chem. Phys. 1986, 85, 1505. (20) Elic¸ abe, G. E.; Larrondo, H. A.; Williams, R. J. J. Macromolecules 1998, 31, 8173.

Biomacromolecules, Vol. 2, No. 3, 2001 823 (21) Binder, K.; Frisch, H. L. J. Chem. Phys. 1984, 81, 2126. (22) Rogers, T. M.; Elder, K. R.; Desai, R. C. Phys. ReV. B 1988, 37, 9638. (23) Mazenko, G. F. Phys. ReV. B 1991, 43, 8204. (24) Farrell, J. E.; Valls, O. T. Phys. ReV. B 1989, 40, 7027. (25) Farrell, J. E.; Valls, O. T. Phys. ReV. B 1990, 42, 2353.

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