Effect of sugars on the phase behaviour, flow and interfacial properties of
protein/polysaccharide aqueous two-phase systems
Asja Portsch, Fotios Spyropoulos, Ian Norton.
Department of Chemical Engineering, University of Birmingham, Birmingham, United Kingdom
([email protected]; [email protected]; [email protected])
ABSTRACT
The paper reviews the phase behaviour, microstructure, flow behaviour and interfacial properties of four
sodium caseinate (NaCAS)/galactomannan (GM) model systems with sugar (i.e. glucose, fructose, sucrose
and trehalose) dissolved in citrate buffer (pH 5.8) at 20ºC.
The established phase diagrams showed that sugar type and sugar concentration affect the phase behaviour of
all systems. Below 15 wt% sugar, the miscibility of biopolymers improved and the increment of the sugar
concentration decreased the miscibility. Glucose, fructose and sucrose improved the compatibility of the
biopolymers, whereas trehalose had the opposite effect. Galactomannans with a mannose-to-galactose ratio
< 2 (i.e. guar gum (GG) and fenugreek gum (FG)) were more miscible than galactomannans with a mannoseto-galactose ratio > 2 (i.e. locust bean gum (LBG) and tara gum (TG)). The effect was pronounced only in
systems with the sugar present.
Quiescent microstructure and flow were largely unaffected by sugar type, but changed with its concentration.
Sugar increased the overall viscosity and enhanced the non-Newtonian behaviour of the model systems.
Phase sense (for blends of < 20 wt% sugar) depended on the volume fraction of each phase and the applied
shear rate. Under quiescent conditions, phase-inversion took place for equal phase volumes. However, at
higher shear regimes phase-inversion was observed for the initially polysaccharide-continuous blends. In the
presence of > 20 wt% sugar, phase sense depended on the volume fraction only; no shear-induced phaseinversion was observed.
Similarly, the interfacial properties of the systems were also affected by sugar type and concentration and
correlated well with their position in the phase diagram. The closer the systems were to the critical point, the
shorter the tile-line length they were positioned on, and the lower their interfacial tension was. The lowest
interfacial tension was measured for the systems with 30 wt% sugar (disaccharides especially).
Keywords: polysaccharide-protein aqueous two phase system; sugars; phase equlibria; optical rheology; interfacial
tension.
INTRODUCTION
In dairy emulsions, structure is formed by the process of phase separation that occurs due to the
thermodynamic incompatibility of its major components, proteins and polysaccharides. Depending on the
concentration of the proteins and polysaccharides in the mixture, two morphologies can arise: a colloidal
emulsion like dispersion (at low biopolymer concentrations) and a segregated one (at higher biopolymer
concentrations), which, if not stabilised, ends up in the formation of two phases of different densities, each
rich in one of the biopolymers [1, 2].
In reality, however, proteins and polysaccharides are accompanied with other low molecular ingredients,
such as salts and sugars, commonly added to foods to impart taste or texture [3, 4]. The addition of sugars
affects the functionality of the macromolecules and in turn changes their phase behaviour, rheological
behaviour and their interfacial properties. Quantifying and understanding the latter phenomena helps us better
understand low fat dairy emulsion-based foods [5, 6].
The aim of this work was to understand the effect of sugar concentration and sugar type on: (i) the phase
behaviour of NaCAS/LBG, NaCAS/TG, NaCAS/GG, and NaCAS/FG aqueous two-phase model systems
(atps), (ii) their quiescent microstructure, (iii) the effect of the microstructure on flow behaviour and (iv) the
interfacial properties of the systems. Finally, we looked at how common fermentable carbohydrates, such as
fructose, glucose and sucrose, the culprits of tooth decay and obesity, compare with an unfermentable and
significantly less digestible sugar such as trehalose.
MATERIALS & METHODS
Galactomananns (LBG: Danisco, Denmark; TG: Roeper, Germany; GG: Sigma-Aldrich, UK; FG, Emerald
Seed, Canada) and NaCAS (DMV International, the Netherlands) were dissolved in citrate buffer and
preserved with potassium sorbate (Fisher-Scientific, UK). Sugars used in experiments were commercially
available sucrose, trehalose, D-glucose and D-fructose (courtesy of Cargill, UK).
The phase behaviour of the systems was assessed by the volume-ratio method introduced by Polyakov et
al. [7]. The data points were fitted with the exponential decay function
in order to obtain
a mathematical expression for the binodals; [GM] and [NaCAS] are the individual concentrations of the
galactomannan and sodium caseinate, “a” and “b” are constants). Slopes of the tie-lines were calculated from
volume fraction data according to Spyropoulos et al. [8]. A Gemini HR Nano stress-controlled rheometer
(Malvern Instruments, UK) with cone and plate geometry (40 mm diameter, 4º truncation, 150 μm gap) was
used to measure viscosities of the equilibrium phases and their blends. The microstructures under shear and
at quiescent conditions were visualised with light microscopy, and the interfacial tensions of the systems
were measured by the modified drop retraction method [9, 10] by means of optical rheometry. For the
purpose of being able to see our systems under shear, a glass plate and plate (40 mm diameter) geometry was
used.
RESULTS & DISCUSSION
The effects of sugar on phase behaviour
Figure 1 illustrates the effect of sugar concentration (a), sugar type (b) and galactomannan type (c) on the
phase behaviour of the studied NaCAS/GM systems at pH 5.8 and at a temperature of 20 ºC. The binodals
best fit to experimental data (R2=0.90-0.99) and represent borders of miscibility for each system. Data show
that sugar concentration, sugar type and galactomannan type change the position of the binodals in the phase
diagram.
Figure 1. Phase diagrams for sodium-caseinate/galactomannan systems illustrating the effects of: (a) sugar concentration;
(b) sugar type; and (c) galactomannan type on the miscibility of macromolecules at 20ºC and pH 5.8.
The example of the NaCAS/LBG atps with sucrose in Figure 1a shows that moderate addition of sugar to the
systems increases the miscibility region, but further addition decreases it. In comparison to the reference atps
without sugar, only trehalose (Figure 1b) causes a reduction in the miscibility region for the NaCAS/GG atps.
On the other hand, sucrose, fructose and glucose improve miscibility. Figure 1c is a phase diagram of four
NaCAS/GM atps with 15 % glucose where a larger phase region of the phase diagram was observed for the
GM with a lower mannose:galactose ratio (i.e. GG and FG) compared to the GM with a higher
mannose:galactose ratio, like LBG and TG. The latter effect was not evident in a pure aqueous environment.
Studies of dilute systems of GM and NaCAS in the presence and absence of sucrose [4, 11] provide an
explanation for the behaviour. The presence of moderate sucrose concentrations causes swelling of GM
molecules and increases hydrophilicity of NaCAS molecules. However, higher sucrose concentrations
(i.e. > 20 %) concentrate the bulk and cause the macromolecules to compete for hydrodynamic space. The
differences in the phase behaviour between the systems containing various galactomannans are likely to be
influenced by the molecular structure (i.e. flexibility, galactose:mannose ratio and proneness to selfassociation), as well as the origins of the sample (i.e. method of extraction and resulting polydispersity).
The effects of sugar on microstructure and flow
The microstructure of NaCAS/GM systems was inspected in terms of the effects of volume fraction (Φ),
sugar concentration and sugar type under quiescent conditions and under shear. Under quiescent conditions,
the microstructure of all atps depended on the Φ of the individual phases only and stayed unchanged in the
presence of the sugar of any amount or type.
The micrographs (a-c) in Figure 2 show three distinct microstructures after shear. The positions of the blends
in the phase diagram are shown as full squares (). Depending on Φ, three microstructures formed: (i)
protein-continuous, (ii) bi-continuous and (iii) GM-continuous. Under quiescent conditions, the phase with
the highest Φ was by rule a continuous phase, which was not always true when atps were sheared.
Figure 2. Phase diagram of sodium-caseinate/locust bean gum systems with 0-40 wt% sucrose with light micrographs of
1.7 mm width showing blends of: (a) 10 %/ 90 %; (b) 50 %/50 % and (c) 70 %/30 % of the LBG-rich/NaCAS-rich phase
after cessation of shear. Systems from which the equilibrium phases were used to prepare blends are shown as , and the
blends are shown as .
When atps were sheared below the critical shear rate (~ 100 s-1), bi-continuous systems formed in the narrow
volume ratio of approximately 1:1 of the GM-rich phase:NaCAS-rich phase, regardless of sugar
concentration and type. In such systems, optical observation in a rotating rheometer confirmed that bicontinuous bands (Figure 2b) formed by side coalescence of extended droplets.
Figure 3 shows viscosity/composition (Φ) dependence on the example of NaCAS/LBG atps. Figure 3a
represents flow behaviour in a low sugar environment, whereas Figure 3b illustrates the behaviour in high
sugar environment.
While sugar type did not have a pronounced effect on the flow behaviour of atps, Φ and sugar concentration
were decisive for the occurrence of shear-induced phase-inversion. At the critical shear rate and low sugar
environment (< 20 wt%) GM-continuous systems, and/or systems with high Φ of the GM-rich dispersed
phase, phase inverted [1]. Under shear previously dispersed phase now became continuous. The event was
facilitated by a favourable viscosity ratio λ (eq. 4 in Figure 4) between the coexisting phases (see λ in
Figure 3). The systems with > 20 wt% behaved similarly to pure NaCAS and GM solutions. The viscosity
differences between the top and bottom phases were reduced and shear-induced phase-inversion was absent.
Figure 3. Semi-logarithmic plots of shear viscosity (at 1-1000 s-1) vs. mass fractions of the LBG-rich phase for the 3 wt%
sodium-caseinate/0.6 wt% locust bean gum system in the presence of (a) 0 % sucrose and (b) 60 % sucrose. The dashed
line divides regime where sodium-caseinate-rich phase is a dispersed phase (left) from the regime where locust bean gum
is a dispersed phase (right). The positions of the dashed lines were inferred from raw data.
In Figure 3, phase-inversion (25 % < Φ < 75 %) is reflected in a sudden change in dynamics of apparent
viscosity values. Depending on the amount of sucrose present, the rise in the apparent viscosity values in
Figures 3a and 3b differs in their patterns. The viscosity was less shear-dependant in low sucrose
environment (viscosity values for the same Φ are closer together) as for systems where a high amount of
sugar was present (the values are further apart).
The effects of sugar on interfacial tension
The effects of composition and Φ (morphology) were tested on the systems marked as open circles () in the
phase diagram in Figure 2. The effects of the sugar concentration and sugar type were, however, further
reviewed for 4 wt% NaCAS/0.2 wt% LBG; 5, 15, 20, and 30 wt% sucrose and 15 wt% glucose, 15 wt%
fructose, and 15 wt% trehalose were added to the system at one time.
The drop deformation method, which enabled us to measure interfacial tensions (σ) up to few μm/N
(Figure 4), was applied in the deformation parameter range of 0.04 < lnD < 0.25 [9]. The method assumed:
(i) ellipsoid/spherical shapes of deformed/retracted droplets and (ii) Newtonian behaviour of the equilibrium
phases at very low shear rates. Two dispersions with different phase sense were inspected on their σ. The
results (not shown) confirm σ stayed the same regardless of which phase was included and which one
dispersed.
Figure 4. Drop deformation method in three steps: (a) micrographs of LBG-rich dispersed phase in NaCAS-rich
continuous phase under shear (upper micrograph) and under quiescent conditions (bottom micrograph) enable calculation
of deformation parameter (D) in eq. 1; (b) semi logarithmic plot with linear regression (eq. 2) of D vs. time; and (c)
logarithmic plots of the shear viscosity of NaCAS-rich and LBG-rich equilibrium phases. ; L=drop length, W=drop
width, λ=viscosity ratio, μc=viscosity of the continuous phase, μd=viscosity of the dispersed phase, σ=interfacial tension,
τ=characteristic time.
Table 1 shows σ for systems with and without sucrose.
Table 1. Interfacial tensions (σ) in sodium-caseinate/locust bean gum aqueous two phase systems, average of five drops.
LBG
[wt%]
NaCAS
[wt%]
Sucrose
[wt%]
Average
interfacial tension
(σ) [μN/m]
0.25
2
0
6±2
0.2
3
0
19±4
0.2
4
0
803±418
0.2
4
5
430±165
0.2
4
15
260±22
0.2
4
20
67±13
0.2
4
30
113±42
0.15
5
0
5945±3516
0.2
5
0
3744±971
0.2
6
0
8139±4148
The data for atps of varied composition and no sugar in Table 1 show that the further atps is located from the
critical point (i.e. the point on the crossing of the straight line going through the points on the phase diagram
where the system consists of the same volume fractions of the both biopolymers, in Figure 2 marked as cross
()) the higher the σ it has. The systems furthest from the critical point (σ=0 μN/m) were also located on the
longest tie-lines (parallel to the tie-line shown in Figure 2), hence the correlation with tie-line length.
Higher concentration of NaCAS and lower concentration of GM in atps increase the σ of the systems due to
the amount of polar groups capable to react with water (NaCAS) and the amount of water needed for their
dissolution. In atps with high σ the driving force for the reduction of the interfacial area (i.e. drop formation)
is increased and hence they readily phase separate. On the other hand systems with lower σ are easily
dispersed and, if the σ is sufficiently low, form a single phase.
Table 1 also reviews interfacial tension values σ for atps with various amounts of sucrose. The presence of
sucrose in amounts of 5-30 wt% affects σ as follows: 20 wt% < 30 wt% < 15 wt% < 5 wt%. We attribute this
behaviour to the effects of sucrose on λ, and important parameter in the calculation of σ (eq. 4 in Figure 4).
Table 2. Interfacial tensions (σ) in sodium-caseinate/locust bean gum aqueous two phase systems, average of five drops.
LBG
[wt%]
NaCAS
[wt%]
Sugar
[wt%]
Sugar
type
Average
interfacial tension
(σ) [μN/m]
0.2
4
0
trehalose
182±56
0.2
4
15
sucrose
260±22
0.2
4
5
fructose
911±364
0.2
4
15
glucose
2077±310
Sugar type (Table 2) also affects the σ of our systems. Non-reducing disaccharides, such as trehalose and
sucrose, give atps a smaller σ compared to reducing monosaccharides like fructose and glucose. In these
systems with disaccharides, the movement of the solute from one phase to another is likely facilitated and
biopolymers are capable of interacting with water. This might not be the case with reducing monosaccharides
that are prone to spontaneously react with proteins. Due to their low σ, the emulsions with disaccharides were
easier to disperse than those containing monosaccharides.
CONCLUSION
The study compared the effects of commonly used sugars, sucrose, fructose and glucose, to less commonly
used trehalose, on the phase behaviour, flow behaviour and interfacial properties of a sodiumcaseinate/galactomannan (NaCAS/GM) atps at pH 5.8 and at a temperature of 20ºC.
These results demonstrate the incompatibility between biopolymers regardless of the sugar concentration and
type. In comparison to systems without sugar, the presence of a moderate amount of sugar (i.e. sucrose,
fructose and glucose) slightly improved the solvent quality, but any increment of sugar above the optimal
amount of 15 wt% caused miscibility to decrease. Trehalose caused a decrease in the miscibility of the
biopolymers.
Under quiescent conditions, phase-inversion was observed at a volume fraction (Φ) of about 50 % for all
sugar concentrations and types. However, at critical shear rates, the shear-induced phase-inversion observed
in a low sugar environment was absent in a high (>20 wt%) sugar environment, where the morphology of the
system depended solely on the Φ. Optical observations together with viscometry indicated a strong link
between the microstructure and rheology of the systems.
Interfacial tension (σ) was affected by sugar concentration and correlated well with the position of systems in
the phase diagram. Interfacial tension depended on the concentration of NaCAS and GM and increased with
the distance from the critical point of the phase diagram. Disaccharides, especially when in concentrations
> 20 wt%, caused a greater reduction in σ compared to monosaccharides.
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