Food Biophysics (2009) 4:240–247 DOI 10.1007/s11483-009-9121-z
ORIGINAL ARTICLE
Kinetics of Phase Separation of Oat β-Glucan/Whey Protein Isolate Binary Mixtures Vassilis Kontogiorgos & Susan M. Tosh & Peter J. Wood
Received: 27 November 2008 / Accepted: 18 June 2009 / Published online: 16 July 2009 # Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada 2009
Abstract The kinetics of phase separation and microstructure of oat β-glucan/whey protein binary mixtures varying in concentration (4–16% w/v protein, 0.3–1.2% w/v βglucan) and β-glucan molecular weight (1.3×106, 640× 103, 180×103, and 120×103 g/mol) was investigated by turbidimetry and fluorescent microscopy. The phase separation of the mixed systems was followed at pH 7.0 and at room temperature under quiescent conditions. Application of first principles revealed that phase separation of the systems follows first-order kinetics. Acceleration of the phase-separation process was observed with increase of βglucan concentration for the three lowest-MW samples but the highest molecular weight (1.3×106 g/mol) exhibited the opposite trend. Changes in the polysaccharide molecular weight resulted in considerable differences in β-glucan aggregate morphology in the mixed systems. The change in the continuity of the mixed system from polysaccharide-, to bi-, to protein-continuous was confirmed for a wide range of mixed systems differing in biopolymer concentration, and β-glucan molecular weight. Keywords Oat β-glucan . Whey protein . Phase behavior . Kinetics . Phase separation
S. M. Tosh (*) : P. J. Wood Agriculture and Agri-Food Canada, Food Research Centre, Guelph, Ontario, Canada e-mail:
[email protected] V. Kontogiorgos Department of Chemical and Biological Sciences, The University of Huddersfield, Queensgate, Huddersfield HD1 3DH, UK
Introduction The mixed linkage (1 → 3)(1 → 4)-β- D-glucans found principally in cereals, have received considerable research attention because of their physiological benefits. βGlucans are linear homopolysaccharides of consecutively linked (1→4)-β-D-glucosyl residues that are separated by single (1→3) linkages and their physical properties as well as their physiological aspects have been recently reviewed1,2. β-Glucans can be used as functional bioactive ingredients because they attenuate postprandial blood glucose and lower serum cholesterol levels 2–7 . The relatively high intake (3 g/day) recommended8 to achieve lower serum cholesterol levels presents some challenges in development of food formulations and understanding of the phase behavior and phase-separation kinetics of βglucans with food proteins will assist development of palatable formulations. β-Lactoglobulin (β-Lg) and α-lactalbumin (α-La) are the two major proteins in whey and have globular conformations and relatively low molecular weights (β-Lg ∼18 kDa, α-La ∼14 kDa). Whey proteins are expected to exhibit greater compatibility with a neutral polysaccharide as the pH moves away from their pI (β-Lg ∼5.1, α-La ∼4.3) in other words, at neutral or basic conditions. Because these two proteins make up ∼70% of protein in whey protein isolate (WPI), they determine its physicochemical properties whereas the remaining proteins (bovine serum albumin (BSA), immunoglobulins (Ig), and proteose-peptones) are expected to have little effect because of their low concentration9. Fundamental studies of the behavior of β-glucan in various model food systems have already been carried out10–14. In these studies, the functionality of β-glucans has been found to depend highly on polysaccharide structure
Food Biophysics (2009) 4:240–247
and concentration that determine the mechanical, thermal, gelation, and other physicochemical characteristics of the systems. The phase behavior of β-glucan/milk protein binary mixtures in the liquid state has already been investigated in different solvent environments and β-glucan molecular weight10,13. In these studies, the mixed systems exhibited phase separation under various conditions and this was attributed to thermodynamic incompatibility of the biopolymers. However, investigations of the rate at which phase separation occurs in binary β-glucan–protein systems in the liquid state have not yet been reported. Therefore, following up our previous investigation13, the objective of the present study was to examine the kinetics of phase separation of whey protein isolate/β-glucan binary mixtures varying in concentration and β-glucan molecular weight.
Materials and Methods Materials WPI was purchased from Davisco (BiPRO, Davisco Foods International, Inc., MN) with the following chemical characteristics: 97.8% w/w d.b protein, 4.5% w/w moisture, and 1.9% w/w ash. High-molecular-weight β-glucan (denoted as HMW) was isolated from oat at the POS Pilot Plant (Saskatoon, SA, Canada) essentially as described previously15. The isolate contained ∼80% dry basis βglucan, ∼4% w/w protein, ∼10% w/w moisture. The remainder was comprised of some remaining starch (∼2% w/w) and pentosans (∼4% w/w). Molecular weight of βglucan was analyzed using high-performance size-exclusion chromatography with post-column calcofluor addition as described elsewhere in detail7,16 and a typical unimodal distribution (peak MW, Mp) was obtained for all β-glucan preparations. The concentration of β-Glucan in solution was measured using flow-injection analysis (FIAstar 5010 Analyzer, Foss Analytical, Denmark) equipped with a fluorescent detector essentially as described previously17. Acid Hydrolysis From the HMW isolate, three more samples were obtained by controlled acid hydrolysis. HMW (1.3×106 g/mol) was dispersed in double-distilled water (1.5% w/v) at 90°C under continuous stirring in a sealed vial. When the polysaccharide was fully dispersed, the temperature was lowered to 70°C and concentrated HCl was added to bring the final HCl concentration to 0.1 M. The polysaccharide was hydrolysed for 0.5, 1, and 1.5 h and immediately after the end of hydrolysis the vials were cooled with running water to room temperature, and the pH was adjusted to 7.0
241
with 5 M NaOH. The hydrolysates were precipitated with three volumes of 95% v/v ethanol, and left standing for 1 h at 4°C. The precipitate was collected by filtration, washed with isopropyl alcohol, dried at room temperature overnight, and milled to fine powder. Some lowmolecular-weight polysaccharide is lost after precipitation, as it remains soluble. However, because the objective was to obtain samples differing in molecular weight and not to optimize the yield this approach is considered adequate for the purposes of the present investigation. The peak molecular weights of the hydrolysates were 640×103 (MHMW), 180×103 (MLMW), and 120 × 10 3 (LMW) g/mol for the 0.5, 1, and 1.5 h hydrolysis periods, respectively. Sample Preparation β-Glucan preparations were dispersed in 0.1 M Sorensen’s phosphate buffer (pH 7.0) with 0.02% w/v NaN3 as preservative at 90°C in a sealed vial under continuous stirring. The maximum concentration of HMW β-glucan solution that could be prepared was 1.5% w/v. Higher concentrations yielded insoluble β-glucan agglomerates resulting in inhomogeneous dispersions, and therefore, the concentration of all β-glucan stock solutions (MHMW, MLMW, LMW) was standardized to 1.5% w/v. Whey protein was dispersed (20% w/v) in the same buffer at room temperature until complete solubilization. All dispersions were left to hydrate overnight at 5°C followed by centrifugation at 15,000×g (5°C, 15 min) to remove any insoluble particles that may interfere with the turbidimetric measurements. It must be stressed that the stock solution concentrations refer to their concentrations after the centrifugation step. Stock solutions were mixed at five different volume ratios (20/80, (protein/polysaccharide), 35/65, 50/50, 65/35) in vials at constant volume (5 mL) yielding binary mixtures of different nominal concentrations (4/1.2 (%protein/% polysaccharide), 7/0.97, 10/0.75, 13/0.52, 16/0.3). Subsequently, the mixtures were homogenized (Polytron, Brinkmann Instruments Co, Switzerland) for 1 min at the lowest speed, immediately sonicated for 30 s to degas, and were placed in a cuvette for the turbidity measurements. Turbidity Measurements Changes in transmittance of the samples were followed for 20 min at room temperature with a spectrophotometer (Cary 3C, Varian Inc. Scientific Instruments, NC) at 600 nm using water as reference. The data were expressed as turbidity (m−1) using the following relationship: t¼
1 lnðT Þ L
242
Food Biophysics (2009) 4:240–247
where t is the turbidity, L is the cuvette pathlength (0.01 m), and T the transmittance. Following turbidity measurements, non-linear regression was performed on the experimental data using GraphPad Prism v. 4.00 (GraphPad Software, San Diego, USA). Measurements for each mixture were performed at least in triplicate and data are reported as mean with standard deviation. Fluorescence Microscopy Microscopy observations were carried out using an Olympus BX-FLA epifluorescent microscope equipped with a mercury burner and an Olympus filter cube with wide-band UV-excitation filter (330–385 nm) and emission barrier filter (420 nm). A Sensys digital camera and the Image-Pro Plus software (Media Cybernetics Inc.) were used to capture the images. Polysaccharide solution, pre-stained with the fluorescent dye calcofluor (0.01% w/v), was mixed with protein solution at different ratios as described previously, and sonicated to remove air bubbles. A drop of freshly prepared mixture was placed on a slide, covered with a cover slip and images were collected at room temperature. Preliminary viscosity measurements in the presence or absence of the fluorescent dye revealed that the stained mixtures exhibit the same viscosity as the unstained ones. Therefore, addition of the fluorescent probe at the levels described does not measurably alter the flow properties of the systems, and phase behavior of stained mixtures can be considered to represent that of unstained counterparts.
Kinetic Analysis of Phase Separation All samples studied, phase separated in time resulting in two macroscopically distinct phases, one rich in polysaccharide and another rich in protein. The phase-separation kinetics of the mixtures was followed with turbidimetric measurements under quiescent conditions at room temperature (Figure 1). It must be noted, that at low concentrations (<2% w/v) for the molecular weights used, β-glucan will not gel in the time-scale of the present investigation and, therefore, the mixtures will remain in the liquid state18,19. Furthermore, the turbidity of the stock solutions (protein and polysaccharide) remained constant during the course of the experiment (data not shown). Thus, changes in turbidity of the mixed systems can be attributed to the phase separation of the constituent biopolymer species. An important detail that must be pointed out is that the initial measurement (t=0 s) represents the turbidity immediately after the sonication step (t=30 s). Therefore, the fitting curves (Figure 1) lack the first seconds, which in exponential decay fitting are usually important. However, as it will become clearer later, the unavoidable sonication time was kept constant for all samples and the starting point is not variable. Therefore, the present methodology is sufficient to illustrate the dependence of phase separation on molecular weight and concentration of the biopolymer mixtures. Interestingly, turbidity either grows or decays exponentially during the course of measurement (Figure 1), 4% pr-1.2% ps 7% pr-0.97% ps 10% pr-0.75% ps 13% pr-0.52% ps 16% pr-0.3% ps
a.
b.
400
Turbidity (m-1)
Turbidity (m-1)
600
500
400
300
300
200 0
5
10
15
20
0
5
Time (min)
10
15
20
Time (min)
c.
300
Turbidity (m-1)
300
Turbidity (m-1)
Fig. 1 Turbidity evolution with changes in biopolymer concentration in the mixtures for a HMW, b MHMW, c MLMW, and d LMW β-glucan preparations (room temperature, 600 nm). Solid lines represent the non-linear regression fits and bars the standard deviation
Results and Discussion
200
100
d.
200
100 0
5
10
Time (min)
15
20
0
5
10
Time (min)
15
20
Food Biophysics (2009) 4:240–247
243
a.
k
1
0.1
LMW MLMW MHMW HMW
0.01 0.1
1
10
β -Glucan (%)
b. 1
0.8
0.6
k
a kinetic behavior that has been previously observed in other mixed biopolymer systems using turbidimetric measurements20–23. Turbidity monitoring beyond 20 min results in small signal to noise ratio (large noise) owing to density fluctuations induced by the phase separation of macroscopically visible polysaccharide or protein domain formation and therefore the measurement had to be terminated. The two different kinetic paths cannot be easily rationalized and the changes in turbidity could be attributed to changes in phase continuity of the system that occurs with changes in the biopolymer ratios in the mix. Changes in the continuity result in different light-scattering patterns in the system and depending on the continuous phase the turbidity either grows or decays exponentially. Such changes as the biopolymer ratio varies, i.e., a shift from polysaccharide-, to bi-, to protein-continuous system is commonly observed in mixed protein/polysaccharide systems24–28 and have been also confirmed for β-glucan/whey protein mixtures13. The nature of the phase-separation process can be understood by applying first principles of kinetic processes by considering the phase separation as a one-step irreversible process:
0.4
ONE
k TWO PHASE !
PHASES 0
W
W
LM
W
W
M
16-0.3
M
Mixture composition (%)
H
13-0.52
M
10-0.75
M
7-0.97
LM
4-1.2
H
where the initial one-phase system separates irreversibly into a two-phase system. The rate constant k gives information on the rate of turbidity change that corresponds to the rate of phase separation, with higher k values indicating faster phase-separation processes. Turbidimetric data were, therefore, collected and non-linear regression was performed using two empirical exponential models. For the fitting procedure, the functions f ðxÞ ¼ Að1 exp ðkxÞÞ þ B or f ðxÞ ¼ A expðkxÞ þ B were used for the exponentially growing or decaying turbidity, respectively. Pre-exponential parameter A is the amplitude of the decay or growth and corresponds to the initial turbidity, B is the equilibrium turbidity (constant number), and k is the rate constant29,30. Rate constant values were plotted against βglucan concentration in double-logarithmic plots revealing a straight-line relationship typical of first-order kinetics processes (Figure 2a). Furthermore, the kinetic parameters of the models as well as the continuous phase under each different combination are presented in Table 1. The rate of phase separation increases with increase of polysaccharide concentration for the low and intermediate molecular weight samples (LMW, MLMW, and MHMW) with the high-molecular-weight sample (HMW) exhibiting the opposite trend. Although the relationship between MW and rate of phase separation (for the same β-glucan concentration) does not appear to be straightforward it seems that low-molecular-weight β-glucans (LMW, MLMW) expedite phase separation compared to the higher-molecular-weight
0.2
Molecular weight
Fig. 2 a Double-logarithmic plots of the rate constant parameter k vs. β-glucan concentration varying in molecular weight, and b changes of the rate constant parameter with biopolymer concentration in the mixed system and β-glucan molecular weight. On the “mixture composition” axis the first number is the protein whereas the second the β-glucan nominal concentration in the mixture
samples (MHMW, HMW). This is more clearly depicted in Figure 2b where the rate constant is plotted against both mixture concentration and Mp of β-glucans. As is evident, the rate constant of the low-MW (MLMW, LMW) is almost double that of the high-MW (MHMW, HMW) samples showing that phase separation is expedited as the β-glucan molecular weight decreases. A possible relationship may exist between the coil overlap concentration (c*) and the phase-separation kinetics. Calculation of c* using Mark–Houwink equations for oat β-glucans31 showed that most of the β-glucan concentrations used in the present investigation were above c* (except two, Table 1). Coil overlap concentration demarcates the concentration region where the individual polymer chains start to entangle and interpenetrate. Beyond this
244 Table 1 Kinetic parameters derived from the empirical models that were used to follow the phase separation of whey protein/β-glucan binary mixtures varying in biopolymer concentration and β-glucan molecular weight
Food Biophysics (2009) 4:240–247
HMW
MHMW
MLMW
LMW The penultimate column shows the coil overlap concentration and the last column indicates the continuous phase of each system
A
B
k
r2
4–1.2 7–0.97 10–0.75 13–0.52 16–0.3 4–1.2 7–0.97 10–0.75 13–0.52 16–0.3 4–1.2 7–0.97 10–0.75 13–0.52 16–0.3
48.8 12.5 9.3 27.1 45.4 17.9 68.9 119.9 69.2 61.6 22.4 23.5 112 86.9 55.5
453 447 430 423 189 294 246 213 281 246 157 215 118 131 156
0.095 0.155 0.180 0.192 0.233 0.343 0.169 0.099 0.071 0.035 0.888 0.975 0.701 0.405 0.274
0.99 0.98 0.78 0.95 0.90 0.93 0.98 0.94 0.97 0.99 0.99 0.96 0.94 0.89 0.96
4–1.2 7–0.97 10–0.75 13–0.52 16–0.3
8.5 15.5 29.9 35.7 162
206 259 195 203 162
0.993 0.591 0.383 0.272 0.181
0.82 0.99 0.96 0.96 0.95
Concentration (% pr-% ps)
point, small changes in concentration result in dramatic changes in zero shear viscosity of the solution, which could decelerate phase separation. High-molecular-weight sample (HMW) seems to comply with the above reasoning and increasing concentration, therefore viscosity, delays the phase-separation process due to kinetic arrest of the system. Kinetic arrest of phase separation of biopolymer systems is a common phenomenon in mixed biopolymer systems when the one polymer has the ability to gel or high viscosity10,32–34. Furthermore, density fluctuations during phase separation may lead to local increase of β-glucan concentration that causes self-association of the biopolymer due to the enhanced interactions between the macromolecular chains. This may induce phenomena with longer relaxation times than simple viscosity-related relaxations that further delays the phase-separation events. However, a departure from this behavior was observed for the rest of the samples (LMW, MLMW, MHMW) as the rate of phase separation increased with increase in concentration. Therefore, another approach must be sought to interpret the results for the intermediate and lowmolecular-weight samples. Segregative phase separation, as in the present investigation, can be related to the Flory– Huggins approach or to depletion interaction theories depending on the conformation of the protein28,35. Usually, when the protein displays “polymer characteristics” (e.g., gelatin) the second virial coefficient treatment can be used to interpret the observations35–37 whereas depletion interaction theories are usually observed when the proteins are
c* (g/dL) 0.11
0.18
0.37
0.48
Continuous phase Polysaccharide Bi-continuous Bi-continuous Protein Protein Polysaccharide Bi-continuous Bi-continuous Protein Protein Polysaccharide Polysaccharide Protein Protein Protein Polysaccharide Polysaccharide Protein Protein Protein
particle-like, such as micellar casein or large aggregates of heat-denaturated proteins22. The majority of the work on whey protein involves investigations near the isoelectric point or after heat denaturation of the protein where the particulate nature of the proteins is suitable for treatment using depletion theories37–41. However, in the absence of whey protein particles, as in the present investigation, the excluded volume approach seems to be more appropriate. Following this line of thought, as the β-glucan concentration increases and/or MW decreases, the excluded volume of the polysaccharide in the mixture decreases42,43. This is accompanied with an increase of phase-separation rate suggesting a relationship between the excluded volume of the polysaccharide and the rate of phase separation for the low and intermediate-molecular-weight samples. It must be mentioned that a generalized relationship between β-glucan molecular weight, biopolymer concentration in the mixtures, and phase-separation kinetics is rather difficult to be extracted from the present investigation. It would seem that the factor that determines the rate of phase separation for the high-MW systems is different than that for the lower-MW polymeric mixtures. However, the kinetic treatment employed can identify the differences in the kinetics of phase separation between samples and clearly suggests that there is a tendency for the low-molecular-weight samples to accelerate phase separation. This kinetic approach presented also revealed that the phase separation follows first-order kinetics and that the differences in the kinetic behavior of the
Food Biophysics (2009) 4:240–247 Fig. 3 Fluorescent images of fresh whey protein/β-glucan binary mixtures varying in molecular weight, a LMW, b MLMW, c MHMW, and d HMW. The mixtures have the same biopolymer concentration (16–0.3 protein/β-glucan (w/v %)). Changes in domain morphology are evident with increasing the MW of the polysaccharide
245
a.
b.
100 µm
100 µm
d.
c.
100 µm
100 µm
Fig. 4 Fluorescent images of fresh whey protein/β-glucan binary mixtures (MHMW) varying in concentration, a 4–1.2, b 10–0.75, c 16–0.3, d 19–0.075 (protein/β-glucan (w/v %)). A transition from polysaccharide-, to bi-, to protein-continuous system is evident with change in β-glucan concentration. Dom designates β-glucan domains
a.
b.
Dom
Dom 100 µm
100 µm
d.
c.
Dom
Dom 100 µm
100 µm
246
mixtures may be also related to the micro-morphology of the mixtures. Such morphological characteristics can be probed using microscopy. Mixture Micro-Morphology Fluorescence microscopy was used to investigate the spatial arrangement and morphology of β-glucan as the concentration and MW varies. Whey proteins in the absence of βglucan cannot be visualized at this length-scale with the present technique and appear as a featureless background (Figure 3)13. On the other hand, calcofluor-stained polysaccharide fluoresces under the conditions employed giving bright patterns. Molecular-weight variation had a striking effect on the morphology of β-glucan domains (Figure 3). Low-MW samples (LMW, MLMW) create spherical droplets (Figure 3a, b) whereas in the high-MW counterparts (MHMW, HMW) the domains become irregular and apparently increase in size (Figure 3c, d). The formation of spherical β-glucan domains reflects the mobility of the polymers within the droplets, since spherical droplets have the lowest surface free energy. The formation of lobate polysaccharide domains in the systems with higher Mp (Figure 3c, d) indicates that the polymers in these systems have restricted mobility. Restricted mobility and stiffness of chain conformations cause decrease in solubility of the polysaccharide18,44,45 something that is evidenced by the intense clear dots (Figure 3c, d). This implies that aggregation may also occur in addition to phase separation for the HMW samples. Such a phenomenon that is generally induced by incomplete hydration, results from the presence of higher amount of cellulose-like moieties of β-glucan as the Mp increases18,44,45. Implications of molecular weight have been also observed in the gelation behavior of β-glucan that was found to depend on chain conformations, amount of cellotriosyl fragments as well as on the degree of the intrachain interactions44,45. Turbidimetry in combination with fluorescent microscopy identifies the dramatic changes in the behavior of β-glucan suggesting that as the molecular weight increases precipitation may be also involved in the observed phenomena. In order to examine how the continuity of the systems varies with changes in biopolymer concentration, images were taken from all different mixtures (Figure 4, Table 1). For the high-MW samples and at high polysaccharide concentrations, only β-glucan domains (Dom) can be observed, as the protein component cannot be resolved (Figure 4a). As the protein concentration increases, there is a transition in the continuity of the mixture from polysaccharide- to protein-continuous (Figure 4b–c, Table 1). The transition region (bi-continuous system) was not observed microscopically for the MLMW and LMW samples. It is possible that due to the low MW of those
Food Biophysics (2009) 4:240–247
samples the system passes from the one state (polysaccharide-continuous) to the other (protein-continuous) within a very small range of concentrations because of the increased mobility of the polymers within the droplets. Transition of morphological characteristics of biopolymer mixtures is commonly observed in microscopic investigations of binary biopolymer mixtures24,26,27 including β-glucan/whey protein mixtures13. The change of β-glucan domain from under nongelling conditions is a clear manifestation of the polysaccharide chain-length and the unusual relationship between the molecular weight and phase-separation kinetics may be influenced by the distinct micro-morphology of β-glucan aggregates, thus adding an extra dimension to the complexity of the system.
Conclusions In the present study, the kinetics of phase separation and microstructure of oat β-glucan/whey protein binary mixtures varying in concentration and molecular weight was investigated. Phase separation can be described as a firstorder kinetics process for all the systems studied. Increase of β-glucan concentration speeds up phase separation for the low- and intermediate-MW samples (640×103, 180× 103, and 120×103 g/mol) but the highest-molecular-weight sample (1.3 ×106 g/mol) exhibited an opposite trend. Furthermore, β-glucan molecular weight is linked to the rate of phase separation with the lower-MW samples accelerating the phase-separation process. Changes in polysaccharide MW resulted in dramatic differences in the β-glucan aggregate morphology in the mixed systems and the transition in the continuity of the mixed system from polysaccharide-, to bi-, to protein-continuous was established for a wide range of mixed systems differing in concentration and β-glucan molecular weight. Acknowledgments The authors wish to thank CreaNutrition AG (Switzerland), Swedish Oat Fiber AB (Sweden), and VINNOVA (Swedish Governmental Agency for Innovation Systems) for their generous contribution in providing financial support to this work.
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