Structure Of Coacervates Research Articles

Encapsulation of different spice essential oils in quinoa protein isolate-gum Arabic coacervates for improved stability

A fully plant-based wall material, quinoa protein isolate-gum Arabic (QPI-GA) complex coacervate, was developed for stability improvement of different spice essential oils (EOs): Sichuan pepper EO (SPEO), ginger EO (GEO), and star anise EO (SAEO). The optimum pH and QPI-to-GA ratio to form QPI-GA coacervates were experimentally confirmed to be 3.6 and 4:1. IF, FTIR, and XRD analyses (shown in supplementary information) indicated that complex coacervation generated electrostatic interactions between QPI and GA molecules and caused a more ordered structure of QPI-GA coacervates. The QPI-GA coacervates exhibited high encapsulation efficiency for SPEO (82.53 ± 1.59 %), GEO (88.19 ± 2.37 %), and SAEO (84.68 ± 1.51 %). The encapsulated EOs showed substantially higher stability in comparison to free EOs, while some differences in each type of stability among encapsulated EOs were observed. Additionally, the QPI-GA coacervate wall materials made EOs stable in oral and gastric stages and sustainably release in intestinal stage, which contributed to the excellent absorption of EOs in simulated digestion.

Heteroprotein complex coacervation of lactoferrin and osteopontin: Phase behaviour and thermodynamics of formation

This research describes the formation of reversible heteroprotein complex coacervates between the bioactive milk proteins lactoferrin (LF) and osteopontin (OPN) within specific combinations of pH (∼4–6), ionic strength (≤30 mM added NaCl), protein stoichiometry (LF:OPN mass mixing ratios of ∼ 2–8) and total protein concentration (≤∼8% w/v). Electrostatic attraction between these proteins is possible across a wide range of pH values due to the acidic isoelectric point of OPN (∼ pH 3.5) and the basic isoelectric point of LF (∼ pH 8.7). Thermodynamic analysis of the OPN-LF interaction indicated that the protein-protein binding had enthalpic and entropic contributions which resulted in liquid-liquid phase separation given the appropriate combination of intrinsic and extrinsic conditions. The complex coacervates were found to be a concentrated protein phase (∼27% protein, w/w) which was highly hydrated (>70% moisture, w/w) and occurred spontaneously within LF: OPN ratios naturally found in human milk. The structure, biological functionality, and applications of heteroprotein complexes and/or complex coacervates between LF and OPN will be explored in separate work.

Insights into Structure and Aggregation Behavior of Elastin-like Polypeptide Coacervates: All-Atom Molecular Dynamics Simulations.

The stimuli-responsive character of elastin-like polypeptides (ELP) has led to their use in a wide range of applications. The temperature-triggered aggregation, or LCST behavior, of ELPs is a complex and multistep phenomenon, which proposed to include the structural transitions, loss of hydrophobic hydration, expulsion of water molecules and physical association of chains. Thus, the origin and detailed mechanism of LCST in ELPs is difficult to elucidate. Here, to gain insights into structure and dynamics of coacervates, we performed all-atom molecular dynamics simulations of 27 90-mer ELPs in explicit water at 350 K. Two sequences, poly(VGPVG)18 and poly(VPGVG)18, were examined due to their experimentally observed differences in thermal hysteresis albeit identical overall composition but different arrangement of amino acids. The simulation results indicate that surface hydrophobicity of poly(VGPVG) aggregate is less than that of the poly(VPGVG) aggregate, and there are marked changes in torsion angles and the propensities of secondary structural motifs during the aggregation process. Moreover, there are significant differences between structure of a single polypeptide in water and structure within the aggregate. Overall, the aggregation process is driven by the formation of peptide-peptide interactions whereas the average hydration of peptides remains almost the same between dissolved and aggregated states. Even though the aggregation is driven by the hydrophobic interactions, ELP coacervate has no hydrophobic core and contains many water molecules. Overall, our findings provide an insight into the sequence-dependent structure of coacervates and molecular behavior of individual peptides during aggregation.

Dynamic Coupling in Unentangled Liquid Coacervates Formed by Oppositely Charged Polyelectrolytes.

We develop a scaling theory that predicts the dynamics of symmetric and asymmetric unentangled liquid coacervates formed by solutions of oppositely-charged polyelectrolytes. Symmetric coacervates made from oppositely-charged polyelectrolytes consist of polycations and polyanions with equal and opposite charge densities along their backbones. These symmetric coacervates can be described as mixtures of polyelectrolytes in the quasi-neutral regime with a single correlation length. Asymmetric coacervates are made from polycations and polyanions with unequal charge densities. The difference in charge densities results in a double semidilute structure of asymmetric coacervates with two correlation lengths, one for the high-charge-density and the other for the low-charge-density polyelectrolytes. We predict that the double-semidilute structure in asymmetric coacervates results in a dynamic coupling which increases the friction of the high-charge-density polyelectrolyte. This dynamic coupling increases the contribution to the zero-shear viscosity of the high-charge-density polyelectrolyte. The diffusion coefficient of the high-charge-density polyelectrolyte is predicted to depend on the concentration and degree of polymerization of the low-charge-density polyelectrolyte in the coacervate if the size of the low-charge-density polymer is smaller than the correlation length of the high-charge-density polymer. We also predict a non-monotonic salt concentration dependence of the zero-shear viscosity of asymmetric coacervates.

Ion Pairing and the Structure of Gel Coacervates.

A scaling model for the structure of coacervates is presented for mixtures of oppositely-charged polyelectrolytes of both symmetric and asymmetric charge-densities for different degrees of electrostatic strength and levels of added salt. At low electrostatic strengths, weak coacervates, with the energy of electrostatic interactions between charges less than the thermal energy, k B T, are liquid. At higher electrostatic strengths, strong coacervates are gels with crosslinks formed by ion pairs of opposite charges bound to each other with energy higher than k B T. Charge-symmetric coacervates are formed for mixtures of oppositely-charged polyelectrolytes with equal and opposite charge-densities. While charge-symmetric weak coacervates form a semidilute polymer solution with a correlation length equal to the electrostatic blob size, charge-symmetric strong coacervates form reversible gels with a correlation length on the order of the distance between bound ion pairs. Charge-asymmetric coacervates are formed from mixtures of oppositely-charged polyelectrolytes with different charge-densities. While charge-asymmetric weak coacervates form double solutions with two correlation lengths and qualitatively different chain conformations of polycations and polyanions, charge-asymmetric strong coacervates form bottlebrush and star-like gels. Unlike liquid coacervates, for which an increase in the concentration of added salt screens electrostatic interactions, causing structural rearrangement and eventually leads to their dissolution, the salt does not affect the structure of strong coacervates until ion pairs dissociate and the gel disperses.

Ionic strength and hydrogen bonding effects on whey protein isolate-flaxseed gum coacervate rheology.

Whey protein isolate (WPI) was mixed with anionic flaxseed (Linum usitatissimum L.) gum (FG), and phase transition during coacervate formation was monitored. Effects of ionic strength and hydrogen bonding on coacervation of WPI‐FG system and corresponding rheological properties were investigated. During coacervate formation, structural transitions were confirmed by both turbidimetry and confocal laser scanning microscopy. Increasing ionic strength with sodium chloride (50 mM) decreased optical density (600 nm) at pHmax. Correspondingly, pHc and pHϕ1 decreased from pH 5.4 to 4.8 and from 5.0 to 4.6, respectively, while pHϕ2 increased from pH 1.8 to 2.4. Sodium chloride suppressed biopolymer electrostatic interactions and reduced coacervate formation. Adding urea (100 mM) shifted pHϕ1, pHmax, and pHϕ2 from 4.8, 3.8, and 1.8 to 5.0, 4.0, and 2.2, respectively, while pHc was unaffected. Optical density (600 nm) at pHmax (0.536) was lower than that of control in the absence of urea (0.617). This confirmed the role of hydrogen bonding during coacervate formation in the biopolymer system composed of WPI and FG. Dynamic shear behavior and viscoelasticity of collected coacervates were measured, and both shear‐thinning behavior and gel‐like properties were observed. Addition of sodium chloride and urea reduced ionic strength and hydrogen bonding, resulting in decreased WPI‐FG coacervate dynamic viscosity and viscoelasticity. The disturbed charge balance contributed to a loosely packed structure of coacervates which were less affected by altered hydrogen bonding. Findings obtained here will help to predict flaxseed gum behavior in protein‐based foods.

Coacervation in Cationic Polyelectrolyte Solutions with Anionic Amino Acid Surfactants.

Coacervates formed by cationic polyelectrolytes and anionic surfactants are utilized to improve the user's tactile perception of shampooing hair during washing and after drying. In this study, we investigated the formation and structure of coacervates in aqueous systems containing anionic amino acid surfactants. The phase behaviors at constant temperature were investigated in aqueous systems combining cationic polyelectrolyte JR-400 with potassium cocoyl glutamate (CoGluK) or potassium cocoyl glycinate (CoGlyK) for a qualitative depiction of coacervate formation. The composition range of coacervate formation varied with the hydrophilic group of the surfactant. The surface tension was measured at different surfactant concentrations and constant polyelectrolyte concentration. The surface tension behavior revealed the critical association concentrations and critical micelle concentrations, indicating that coacervate was produced via complex formation through electrostatic interaction between opposite charges. Optical microscopy and small-angle X-ray scattering measurements revealed that the coacervates were composed of fibrous aggregates, a few microns thick, and those formed in the CoGlyK system had thicker fibers.

Complex Coacervation in Asymmetric Solutions of Polycation and Polyanion.

We study coacervation upon mixing two aqueous solutions of polyelectrolytes (PEs) with opposite charge, by considering asymmetric effects of PE composition and charge valency. The phase behavior, interfacial structure, and coacervate composition are investigated by a classical density-functional theory. We find two types of coacervation that are different in their density. Supernatant phase in low-density coacervation (LDCA) fully consists of small ions, while in high-density coacervation (HDCA) it contains a considerable amount of PE chains. Asymmetric PE composition could generate an electric double layer at the interface of coacervate. For HDCA, ion density changes monotonically, while for LDCA it shows a global minimum at the double layer, giving a low tension value. Charged species of high charge valency enhance the existence of double layer. Our results explained the coacervate structure of low interfacial tension, which is important for experiments and industrial applications.

Rheological and Microstructural Characteristics of Canola Protein Isolate-Chitosan Complex Coacervates.

Wastage of byproducts such as canola meal is a pressing environmental concern, and canola protein isolate (CPI)-chitosan (Ch) coacervates have a good potential to utilize and convert the wastes into a high value added product. Yet so far, there is very limited rheological and microstructural information to assist in proper utilization of CPI -Ch complex coacervates. The rheological and microstructural properties of the complex coacervates formed from CPI and chitosan Ch at various CPI-to-Ch mixing ratios (1:1, 16:1, 20:1, and 30:1) and pH values (5.0, 6.0, and 7.0) were therefore investigated. These CPI-Ch complex coacervate phases were found to exhibit elastic behavior as evidenced by significantly higher elastic modulus (G') compared to viscous modulus (G″) in all the tested ratios and pH ranges. They also exhibited shear-thinning behavior during viscous flow. The complex coacervates formed at the optimum CPI-to-Ch ratio of 16:1 and pH of 6.0 demonstrated the highest G', G″, and shear viscosity, which correlated well with the high strength of electrostatic interaction and thick-walled, sponge-like, less-porous microstructure at this condition. The higher shear viscosity of the coacervate at pH 6.0 was most likely induced by stronger attractive electrostatic interactions between CPI and Ch molecules, due to the formation of a rather densely packed complex coacervate structure. Hence, it can be concluded that the microstructural observations of denser structure correlated well with the rheological findings of stronger intermolecular bonds at the optimum CPI-to-Ch ratio of 16:1 and pH of 6.0. The complex coacervate phase formed at a CPI-to-Ch ratio of 16:1 and pH of 6.0 also showed glassy consistency at low temperatures and rubbery consistency above its glass-transition temperature. This study identified the potential for the newly developed CPI-Ch complex coacervate to be used as an encapsulating material due to its favorable strength. This would drastically reduce the wastage of byproducts, provide a solution to tackle the pressing global issue of wastage of byproducts, and bring about a more environmentally friendly paradigm.

Rheological and structural properties of rice bran protein-flaxseed (Linum usitatissimum L.) gum complex coacervates

Complex coacervation of protein/polysaccharide mixtures has obtained considerable research in the encapsulation of bioactive materials. The rheological properties of the coacervates of rice bran protein/flaxseed gum (RBP/FG) at different pH values including 3.3, 4.0 and 5.3 and varying protein to polysaccharide ratios 3:1, 6:1 and 9:1 were investigated using small amplitude oscillatory shear (SAOS). Moreover, the interrelationship between the ζ-potential, FTIR and viscoelastic properties of the samples was studied. The coacervates showed shear-thinning phenomenon due to the linear reduction of complex viscosity (η*) by increasing frequency. Furthermore, the highest complex modulus (G*) and more compact coacervate structure were obtained at pH = 4.0, revealing less deformability and flow behavior. All the coacervates showed higher storage modulus (G′) than loss modulus (G″) indicating the formation of highly interconnected gel-like structure and were fitted by the Power law model. The maximum fracture stress was obtained at pH 4.0 revealing the highest intermolecular interactions between RBP and FG. The absolute ζ-potential of the coacervate at pH 4.0 and R = 9:1 was close to 0 and less than the other samples in which the highest fracturability and gel strength of the complex coacervate was achieved. It seems this property of the coacervate would be suitable for encapsulation of bioactive materials. The high aggregation of coacervates at pH 4.0 makes them more readily neutralized by protein binding. FTIR results showed that the spectrum of the coacervate was different from each individual biopolymer, related to their compatibility and intermolecular interactions between the functional groups of RBP and FG. Consequently, RBP/FG coacervates can be managed to some extent to achieve proper textural properties in dairy formula through manipulating pH and biopolymer mixing ratio.

Genipin-crosslinked O-carboxymethyl chitosan-gum Arabic coacervate as a pH-sensitive delivery system and microstructure characterization.

The possibility of genipin-crosslinked O-carboxymethyl chitosan-gum Arabic coacervate as a pH-sensitive delivery vehicle was investigated. O-carboxymethyl chitosan-gum Arabic coacervates separated in pH 3.0, 4.5, and 6.0 were crosslinked by genipin for different durations and the crosslinked products were subjected to crosslinking degree, swelling behavior, bovine serum albumin release profile, and microstructure characterization. Genipin-crosslinking greatly improved the stability of the coacervates against the simulated gastric solution and created certain pH-sensitivity. The coacervates displayed higher swelling ratios in the simulated gastric solution than in the simulated intestine and colon solutions; meanwhile, the coacervates prepared in pH 4.5 and 6.0 swelled more severely than the complex separated in pH 3.0. Nevertheless, the bovine serum albumin release in the simulated gastric solution from the microcapsules prepared in pH 6.0 was much lower than those prepared in pH 4.5 and 3.0, whose cumulative release percentages in the three simulated solutions were 17.14%, 55.23%, and 79.79%, respectively, in crosslinking duration 2 h. X-ray diffraction, scanning electron microscopy, and transmission electron microscopy analysis revealed that genipin-crosslinking improved the regularity and compactness of coacervate structure, whereas confocal laser scanning microscopy observation indicated that O-carboxymethyl chitosan content was possibly the major reason for the different swelling and bovine serum albumin release behavior of the coacervates. It was concluded that the genipin-crosslinked O-carboxymethyl chitosan-gum Arabic coacervate was a potential intestine-targeted delivery system and its delivery performance could be tailored by varying the crosslinking degree and coacervation acidity.

Complex Coacervation as a Novel Microencapsulation Technique to Improve Viability of Probiotics Under Different Stresses

A physicochemical approach has been undertaken to develop polymeric microcapsules for delivering probiotic bacteria with improved viability in functional food products. Two probiotic strains of Lactobacillus paracasei subsp. paracasei (E6) and Lactobacillus paraplantarum (B1), isolated from traditional Greek dairy products, were microencapsulated by complex coacervation using whey protein isolate (WPI, 3 % w/v) and gum arabic (GA, 3 % w/v) solutions mixed at 2:1 weight ratio. The viability of the bacterial cells during processing (heat treatment and high salt concentrations), under simulated gut conditions (low pH and high bile concentrations) and upon storage, was evaluated. Entrapment of lactobacilli in the complex coacervate structure enhanced the viability of the microorganisms when exposed to a low pH environment (pH 2.0). Both encapsulated strains retained high viability in simulated gastric juice (>73 %; log scale), especially in comparison with non-encapsulated (free) cells (<19 %). Moreover, after 60 days of refrigerated storage at pH 4.0, the viability of microencapsulated cells was more than 86 %, implying improved protection in comparison with the free cells (<59 %). Complex coacervation with WPI/GA has the potential to deliver live probiotics in low pH foods or fermented products; it is also important to note that the complexes can dissolve at pH 7.0 (gut environment) releasing the microbial cells (desired feature of target delivery systems).

Coacervation in Biopolymers

In this review, a detailed discussion on salient features of intermolecular interactions leading to phase separation and coacervation is discussed. Biomolecular solutions exist as gels, coacervates and melts with each of these phases having its signature physico-chemical properties which is discussed in this review. The discussions are supported by robust experimental data obtained from an array of methods like turbidimetry, electrophoresis, viscosity, light scattering etc. The inevitability of the phenomenon of self-organization in biopolymers results in generation of a variety of soft matter phases which do not, however, make it predictable. For instance the associate aggregation is a process which remains obscure, as every protein aggregates in a different manner under different conditions. One known feature to the aggregation of proteins is the strong dependence upon pH, salt concentration, and temperature. Beyond the influence of these factors and their effects on aggregation, the process is not well understood. In summary, a comprehensive account of biomolecular phase states and their inherent attributes are presented in this review. Potential applications of coacervates are many starting from protein purification, drug encapsulation to treatment of organic plumes. This calls for better understanding of the coacervate structure and the transport of biomolecules inside this phase. Several questions pertaining to the structure of coacervates can arise. The foremost of these is, is it a gel-like or a solution-like phase? Though presence of hydrogen bonding and hydrophobic sites on the polyions influence biomolecular binding, they hardly play any role in deciding the persistence length of the polyion unlike the surface charge. Interestingly, we observed that the differential binding (SPB versus EB) was found to be a function of intrinsic persistence length only which we conclude as a significant observation.

Fish gelatin/Laponite biohybrid elastic coacervates: A complexation kinetics–structure relationship study

Complex coacervation in exfoliated Laponite nanoplatelets and fish gelatin mixtures was studied as a function of four key parameters: pH, ionic strength, gelatin/Laponite weight ratio, and total weight. The aim was to understand how these parameters influence phase separation kinetics, composition, internal structure, and viscoelastic properties of coacervates. By careful experimental design and turbidity measurements, the optimum conditions for coacervation were obtained. Thermogravimetric analysis revealed an outstanding heat-resistance for gelatin/nanoclay coacervates. Finally, structure of the coacervate phase was characterized by oscillatory shear experiments. The storage modulus data was observed to follow a power-law behavior and it was confirmed that under the optimum conditions, the coacervate phase was dense and structured with a characteristic length scale (ξrheol) of ∼8.25nm. Regardless of the physicochemical condition at which complexation occurred, it was shown that the equilibrium structure of the coacervates is related to the kinetics of intermediate and late stages of phase separation; as the new defined kinetics parameter K was observed to be inversely proportional to ξrheol that quantifies the compactness of the coacervate networks.

Asymmetric Collapse in Biomimetic Complex Coacervates Revealed by Local Polymer and Water Dynamics

Complex coacervation is a phenomenon characterized by the association of oppositely charged polyelectrolytes into micrometer-scale liquid condensates. This process is the purported first step in the formation of underwater adhesives by sessile marine organisms, as well as the process harnessed for the formation of new synthetic and protein-based contemporary materials. Efforts to understand the physical nature of complex coacervates are important for developing robust adhesives, injectable materials, or novel drug delivery vehicles for biomedical applications; however, their internal fluidity necessitates the use of in situ characterization strategies of their local dynamic properties, capabilities not offered by conventional techniques such as X-ray scattering, microscopy, or bulk rheological measurements. Herein, we employ the novel magnetic resonance technique Overhauser dynamic nuclear polarization enhanced nuclear magnetic resonance (DNP), together with electron paramagnetic resonance (EPR) line shape analysis, to concurrently quantify local molecular and hydration dynamics, with species- and site-specificity. We observe striking differences in the structure and dynamics of the protein-based biomimetic complex coacervates from their synthetic analogues, which is an asymmetric collapse of the polyelectrolyte constituents. From this study we suggest charge heterogeneity within a given polyelectrolyte chain to be an important parameter by which the internal structure of complex coacervates may be tuned. Acquiring molecular-level insight to the internal structure and dynamics of dynamic polymer complexes in water through the in situ characterization of site- and species-specific local polymer and hydration dynamics should be a promising general approach that has not been widely employed for materials characterization.

Characterization of Fish Gelatin at Nanoscale Using Atomic Force Microscopy

Atomic force microscopy (AFM) was used as a meaningful tool to characterize the nanostructure of gelatin from catfish (Ictalurus punctatus) skin. The gelatins extracted with pretreatments including acid pretreatment, alkaline pretreatment, and alkaline followed by acid pretreatment (optimized extraction conditions). The resulting gelatins were imaged using AFM and their nanostructure was studied. The AFM images showed that gelatin extracted with acid pretreatment had a coacervate structure while with alkaline pretreatment there were separate aggregates. Spherical aggregates and annular pores were observed in AFM images of gelatin with the optimized extraction conditions. AFM imaging of gelatin with a relative high concentration (0.5%) was successfully done and the results help researchers to understand gelatin structures at the nanoscale.

Microstructure of β-Lactoglobulin/Pectin Coacervates Studied by Small-Angle Neutron Scattering

Small-angle neutron scattering (SANS) has been used to investigate the microstructure of beta-lactoglobulin/pectin coacervates prepared by different initial protein/polysaccharide weight ratio (r), sodium chloride concentration (C(NaCl)), and pectin charge density. The higher r and higher pectin charge density lead to higher scattering intensity at small q range (0.007 Angstrom(-1) < q < 0.02 Angstrom(-1)), suggesting that the charges of pectin chains are screened significantly by the binding of oppositely charged protein molecules, leading to a tighter aggregation of pectin chains. On the other hand, the appearance of a shoulder peak at intermediate q range (0.04 Angstrom(-1) < q < 0.2 Angstrom(-1)) is used to interpret the formation of protein domains in beta-lactoglobulin/pectin coacervates. At C(NaCl) = 0.1 M, the coacervate of beta-lactoglobulin and pectin A does not show a shoulder peak at intermediate q range at r = 10:1, suggesting that protein molecules are separately bound on pectin chains. However, a shoulder peak appears at intermediate q range at r = 20:1 and 30:1, and the average protein domain size estimated from the shoulder peak position is 7.2 and 8.5 nm, respectively, for these two coacervates. When C(NaCl) increases from 0.05 to 0.2 M, the shoulder peak shifts toward smaller q and becomes broader, indicating that the addition of a higher amount of salt leads to a more heterogeneous coacervate structure. Pectin B with a lower linear charge density favors the formation of larger protein domains. The formation of protein domains in beta-lactoglobulin/pectin coacervates is partially ascribed to the self-aggregation of beta-lactoglobulin molecules. Two kinds of microstructures of beta-lactoglobulin/pectin coacervates with and without observable protein domains have been proposed.

Microscopic structure of gelatin coacervates

Microscopic structure of simple coacervates of gelatin having concentration ∼130 g/l were studied at 25 °C by atomic force microscopy (AFM), rheology, small angle neutron scattering (SANS), UV absorption and circular dichroism (CD) techniques. The behavior of viscoelastic exponents Δ′ and Δ″ of storage and loss modulii ( G′( ω) ∼ ω Δ ′ , G″( ω) ∼ ω Δ ″ ) revealed that, Δ′ = 0.25 ± 0.01 and Δ″ = 0.78 ± 0.1 for coacervates. The mass fractal dimension ‘ d f’ for coacervate was found to be 2.27, which attributed a compact heterogeneous network structure to the coacervates. This is supported by AFM pictures. The CD and UV absorption data indicated presence of helical structures inside the coacervates phase. SANS results showed the existence of a single length scale associated with this system identified as gelatin persistence length, ζ = 27 ± 2 Å. These studies indicate that the coacervate phase is a low dimensional dense heterogeneous material comprised of strongly interconnected triple helices which imparts a large storage modulus to this material.

Diffusivity of whey protein and gum arabic in their coacervates.

Structural properties of whey protein (WP)/gum arabic (GA) coacervates were investigated by measuring the diffusivity of WP and GA in their coacervate phase as a function of pH by means of three different complementary techniques. The combination of these measurements revealed new insights into the structure of coacervates. Nuclear magnetic resonance (NMR) measured the self-diffusion coefficient of the GA in the coacervate phase prepared at various pH values. Fluorescence recovery after photobleaching (FRAP) was measured using a confocal scanning laser microscope. The WP and GA were covalently labeled with two different dyes. The time of fluorescence recovery, related to the inverse of the diffusion coefficient, was evaluated from the measurements, and the diffusivity of the WP and GA on a long time scale could be individually estimated at each pH value. Diffusing wave spectroscopy (DWS) combined with transmission measurement was carried out in the coacervate phase, and the diffusion coefficient, corresponding to the averaged diffusion of all particles that scattered in the system, was calculated as a function of pH. Independently of the technique used, the results showed that the diffusion of the WP and GA within the coacervate phase was reduced as compared to a diluted biopolymer mixture. NMR, DWS, and FRAP measurements gave similar results, indicating that the biopolymers moved the slowest in the coacervate matrix at pH 4.0-4.2. It is assumed that the diffusion of the WP and GA is reduced because of a higher electrostatic interaction between the biopolymers. Furthermore, FRAP results showed that in the coacervate phase WP molecules diffused 10 times faster than GA molecules. This result is very relevant because it shows that WP and GA move independently in the liquid coacervate phase. Finally, DWS measurements revealed that the coacervate phase rearranged with time, as evidenced by a decrease of the diffusion coefficient and a loss of the turbidity of the sample. A more homogeneous transparent coacervate phase was obtained after a few days/weeks. Faster rearrangement was obtained at pH 3.0 and 3.5 than at higher pH values.

Composition and structure of whey protein/gum arabic coacervates.

Complex coacervation in whey protein/gum arabic (WP/GA) mixtures was studied as a function of three main key parameters: pH, initial protein to polysaccharide mixing ratio (Pr:Ps)(ini), and ionic strength. Previous studies had already revealed under which conditions a coacervate phase was obtained. This study is aimed at understanding how these parameters influence the phase separation kinetics, the coacervate composition, and the internal coacervate structure. At a defined (Pr:Ps)(ini), an optimum pH of complex coacervation was found (pH(opt)), at which the strength of electrostatic interaction was maximum. For (Pr:Ps)(ini) = 2:1, the phase separation occurred the fastest and the final coacervate volume was the largest at pH(opt) = 4.0. The composition of the coacervate phase was determined after 48 h of phase separation and revealed that, at pH(opt), the coacervate phase was the most concentrated. Varying the (Pr:Ps)(ini) shifted the pH(opt) to higher values when (Pr:Ps)(ini) was increased and to lower values when (Pr:Ps)(ini) was decreased. This phenomenon was due to the level of charge compensation of the WP/GA complexes. Finally, the structure of the coacervate phase was studied with small-angle X-ray scattering (SAXS). SAXS data confirmed that at pH(opt) the coacervate phase was dense and structured. Model calculations revealed that the structure factor of WP induced a peak at Q = 0.7 nm(-1), illustrating that the coacervate phase was more structured, inducing the stronger correlation length of WP molecules. When the pH was changed to more acidic values, the correlation peak faded away, due to a more open structure of the coacervate. A shoulder in the scattering pattern of the coacervates was visible at small Q. This peak was attributed to the presence of residual charges on the GA. The peak intensity was reduced when the strength of interaction was increased, highlighting a greater charge compensation of the polyelectrolyte. Finally, increasing the ionic strength led to a less concentrated, a more heterogeneous, and a less structured coacervate phase, induced by the screening of the electrostatic interactions.