Simple and complex coacervation in systems involving plant proteins.

Plant-based foods are gaining popularity as alternatives to meat and dairy products due to sustainability and health concerns. As a consequence, there is a renewed interest in the phase behaviour of plant proteins and of mixtures of plant proteins and polysaccharides, in particular in the cases where coacervation is found to occur, i.e., liquid-liquid phase separation (LLPS) into two phases, one of which is rich in biopolymers and one of which is poor in biopolymer. Here we review recent research into both simple and complex coacervation in systems involving plant proteins, and their applications in food- as well as other technologies, such as microencapsulation, microgel production, adhesives, biopolymer films, and more.

High Field MicroMRI Velocimetric Measurement of Quantitative Local Flow Curves.

Performing rheo-microMRI velocimetry at a high magnetic field with strong pulsed field gradients has clear advantages in terms of (chemical) sensitivity and resolution in velocities, time, and space. To benefit from these advantages, some artifacts need to be minimized. Significant sources of such artifacts are chemical shift dispersion due to the high magnetic field, eddy currents caused by the pulsed magnetic field gradients, and possible mechanical instabilities in concentric cylinder (CC) rheo-cells. These, in particular, hamper quantitative assessment of spatially resolved velocity profiles needed to construct local flow curves (LFCs) in CC geometries with millimeter gap sizes. A major improvement was achieved by chemical shift selective suppression of signals that are spectroscopically different from the signal of interest. By also accounting for imperfections in pulsed field gradients, LFCs were obtained that were virtually free of artifacts. The approach to obtain quantitative LFCs in millimeter gap CC rheo-MRI cells was validated for Newtonian and simple yield stress fluids, which both showed quantitative agreement between local and global flow curves. No systematic effects of gap size and rotational velocity on the viscosity of a Newtonian fluid and yield stress of a complex fluid could be observed. The acquisition of LFCs during heterogeneous and transient flow of fat crystal dispersion demonstrated that local constitutive laws can be assessed by rheo-microMRI at a high magnetic field in a noninvasive, quantitative, and real-time manner.

Film Formation of High Tg Latex Using Hydroplasticization: Explanations from NMR Relaxometry.

The film formation of acrylic latex dispersions, containing different amounts of carboxylic acid functional groups by the incorporation of methacrylic acid (MAA), was studied with GARField 1H NMR at various relative humidities (RH). Polymer particles with glass-transition temperatures in the range from 26 to 50 °C formed films at room temperature because of hydroplasticization. It was found that with an increased drying rate due to lower RH, the evaporation flux of water was limited by the latex polymer. Only in the second stage of drying this phenomenon was more obvious with increasing MAA content. 1H NMR relaxometry was used to study the change of hydrogen mobilities during film formation and hardening of the films. This showed that the drying rate itself had no impact on the hydrogen mobility in the latex films as measured via the T2 relaxation time. Hydrogen mobilities of water and the mobile polymer phase only significantly decrease after most water has evaporated. This implies that the rigidity of the polymers increases with the evaporation of water that otherwise plasticizes the polymer through hydrogen bonding with the carboxylic acid groups. This hardening of the polymer phase is essential for applications in a coating. The hydrogen mobilities were affected by the MAA concentration. Densities of mobile hydrogens increase with increasing MAA content. This is expected if the mobile protons are contained in the MAA groups. The result thus confirms the role of carboxylic acid groups in hydrogen bonding and plasticization of the copolymers. Hydrogen mobilities, however, decrease with increasing MAA content, which is hypothesized to be caused by the formation of dimers of carboxylic acid groups that still hold water. They still enable short-range polymer hydrogen mobility due to hydroplasticization but limit long-range polymer mobility due to interaction between the carboxylic acid groups.

Protein Fibrils Induce Emulsion Stabilization.

The behavior of an oil-in-water emulsion was studied in the presence of protein fibrils for a wide range of fibril concentrations by using rheology, diffusing wave spectroscopy, and confocal laser scanning microscopy. Results showed that above a minimum fibril concentration depletion flocculation occurred, leading to oil droplet aggregation and enhanced creaming of the emulsion. Upon further increasing the concentration of the protein fibrils, the emulsions were stabilized. In this stable regime both aggregates of droplets and single droplets are present, and these aggregates are smaller than the aggregates in the flocculated emulsion samples at the lower fibril concentrations. The size of the droplet aggregates in the stabilized emulsions is independent of fibril concentration. In addition, the droplet aggregation was reversible upon dilution both by a pH 2 HCl solution and by a fibril solution at the same concentration. The viscosity of the emulsions containing fibrils was comparable to that of the pure fibril solution. Neither fibril networks nor droplet gel networks were observed in our study. The stabilization mechanism of emulsions containing long protein fibrils at high protein fibril concentrations points toward the mechanism of a kinetic stabilization.

Stability of colloidal dispersions in the presence of protein fibrils.

We studied the stability of monodispersed polystyrene latex dispersions with protein fibrils at different concentrations at pH 2 using microscopy and diffusing wave spectroscopy. At low fibril concentrations, fibrils induced bridging flocculation due to the opposite charges between fibrils and the latex particles. At higher fibril concentration the dispersions were stabilized due to steric and/or electrostatic repulsion. Upon further increasing fibril concentration, we find that the dispersion is destabilized again by depletion interaction. At even higher fibril concentration, the dispersions are stabilized again. These dispersions have a higher stability compared to the dispersions without fibrils. Interestingly, these dispersions contain single particles and small clusters of particles that do not grow beyond a certain size. Although the stabilization mechanism is not clear yet, the results from microscopy and diffusing wave spectroscopy point in the direction of a kinetic barrier that depends on fibril concentration.

Controlled Release from Zein Matrices: Interplay of Drug Hydrophobicity and pH.

PURPOSE: In earlier studies, the corn protein zein is found to be suitable as a sustained release agent, yet the range of drugs for which zein has been studied remains small. Here, zein is used as a sole excipient for drugs differing in hydrophobicity and isoelectric point: indomethacin, paracetamol and ranitidine. METHODS: Caplets were prepared by hot-melt extrusion (HME) and injection moulding (IM). Each of the three model drugs were tested on two drug loadings in various dissolution media. The physical state of the drug, microstructure and hydration behaviour were investigated to build up understanding for the release behaviour from a zein based matrix for drug delivery. RESULTS: Drug crystallinity of the caplets increases with drug hydrophobicity. For ranitidine and indomethacin, swelling rates, swelling capacity and release rates were pH dependent as a consequence of the presence of charged groups on the drug molecules. Both hydration rates and release rates could be approached by existing models. CONCLUSION: The drug state and pH dependant electrostatic interactions are hypothesised to influence release kinetics. Both factors can potentially be used to influence release kinetics release, thereby broadening the horizon for zein as a tuneable release agent.

Coating formation during drying of ß-lactoglobulin: gradual and sudden changes.

The drying dynamics of protein coatings is of importance for many applications. The main focus of research so far was to investigate macroscopic properties of protein coatings, leaving drying dynamics virtually unexplored. A unique combination of techniques is used to monitor drying of a coating containing the protein ß-lactoglobulin. The techniques used cover both macroscopic and microscopic aspects of the drying process. For all water fractions amenable to diffusing wave spectroscopy analysis (xw > 0.2 w/w), the tracer particles diffuse in the coating as in a Newtonian viscous medium. Magnetic resonance imaging shows both protein and water are distributed homogeneously over the coating during drying, up to water fractions above 0.2 w/w. When drying continues to lower water fractions, sudden transitions in drying behavior are observed by both dynamic vapor sorption and IR spectroscopy, which we suggest are due to changes in molecular interactions caused by dehydration of the protein backbone.

The Development of Direct Extrusion-Injection Moulded Zein Matrices as Novel Oral Controlled Drug Delivery Systems.

PURPOSE: To evaluate the potential of zein as a sole excipient for controlled release formulations prepared by hot melt extrusion. METHODS: Physical mixtures of zein, water and crystalline paracetamol were hot melt extruded (HME) at 80°C and injection moulded (IM) into caplet forms. HME-IM Caplets were characterised using differential scanning calorimetry, ATR-FTIR spectroscopy, scanning electron microscopy and powder X-ray diffraction. Hydration and drug release kinetics of the caplets were investigated and fitted to a diffusion model. RESULTS: For the formulations with lower drug loadings, the drug was found to be in the non-crystalline state, while for the ones with higher drug loadings paracetamol is mostly crystalline. Release was found to be largely independent of drug loading but strongly dependent upon device dimensions, and predominately governed by a Fickian diffusion mechanism, while the hydration kinetics shows the features of Case II diffusion. CONCLUSIONS: In this study a prototype controlled release caplet formulation using zein as the sole excipient was successfully prepared using direct HME-IM processing. The results demonstrated the unique advantage of the hot melt extruded zein formulations on the tuneability of drug release rate by alternating the device dimensions.

The Phase Behavior of γ-Oryzanol and ß-Sitosterol in Edible Oil.

The phase behavior of binary mixtures of γ-oryzanol and ß-sitosterol and ternary mixtures of γ-oryzanol and ß-sitosterol in sunflower oil was studied. Binary mixtures of γ-oryzanol and ß-sitosterol show double-eutectic behavior. Complex phase behavior with two intermediate mixed solid phases was derived from differential scanning calorimetry (DSC) and small-angle X-ray scattering (SAXS) data, in which a compound that consists of γ-oryzanol and ß-sitosterol molecules at a specific ratio can be formed. SAXS shows that the organization of γ-oryzanol and ß-sitosterol in the mixed phases is different from the structure of tubules in ternary systems. Ternary mixtures including sunflower oil do not show a sudden structural transition from the compound to a tubule, but a gradual transition occurs as γ-oryzanol and ß-sitosterol are diluted in edible oil. The same behavior is observed when melting binary mixtures of γ-oryzanol and ß-sitosterol at higher temperatures. This indicates the feasibility of having an organogelling agent in dynamic exchange between solid and liquid phase, which is an essential feature of triglyceride networks.

Phase Separation in Complex Mixtures with Many Components: Analytical Expressions for Spinodal Manifolds.

The phase behavior is investigated for systems composed of a large number of macromolecular components N, with N ≥ 2. Liquid-liquid phase separation is modeled using a virial expansion up to the second order of the concentrations of the components. Formal analytical expressions for the spinodal manifolds in N dimensions are derived, which simplify their calculation (by transforming the original problem into inequalities that can be evaluated numerically using linear programming techniques). In addition, a new expression is obtained to calculate the critical manifold and composition of the coexisting phases. The present analytical procedure complements previous attempts to handle spinodal decomposition for many components using a statistical approach based on random matrix theory. The results are relevant for predicting the effects of polydispersity on phase behavior in fields like polymer or food science and liquid-liquid phase separation in the cytosol of living cells.

Phase Behavior of Ternary Polymer Mixtures in a Common Solvent.

The Edmond-Ogston model for phase separation is extended to ternary polymer mixtures in a common solvent (de facto a quaternary mixture). The model assumes a truncated virial expansion of the Helmholtz free energy up to the second-order terms in the concentration of the polymers, and the second virial coefficients (B11, B22, B33, B12, B13, B23) are the six parameters of the model. New results from this model are presented in relation to earlier work on binary mixtures: a necessary condition for the virial coefficients for the occurrence of phase separation in two or three phases, an analysis of the different regions of (local) thermodynamic instability using the Descartes sign rule, an expression for the critical curves, a relation between the tangents in points along the critical curve, a relation between the concentration of components in the different phases according to the so-called Lambert-W function, and a consistency check for the composition of coexisting phases in ternary mixtures. The obtained results are evaluated in the maximally symmetric version of the model, where (B11, B22, B33) are equal and (B12, B13, B23) are equal, which leads to two remarkable observations: the concentration range over which two phases are formed is relatively narrow; not all phase separation occurs within a Gibbs triangle, but also, "out-of-Gibbs-triangle" binodals are observed. These results lead to a deeper insight into the phase behavior of ternary mixtures and show promise as a stepping stone toward modeling phase separation in mixtures with many components.

Relation between gelation conditions and the physical properties of whey protein particles.

Whey protein particles have several applications in modulating food structure and for encapsulation, but there is a lack of methods to prepare particles with a very high internal protein content. In this study whey protein particles with high internal protein content were prepared through emulsification and heat gelation of 25% (w/w) whey protein isolate solution at different pH (6.8 or 5.5) and NaCl concentrations (50, 200, or 400 mM). Particles formed at pH 6.8 were spherical, whereas those formed at pH 5.5 were irregular and had a cauliflower-like appearance. Both particles had an average size of few micrometers, and the particles formed at pH 5.5 had higher protein content (∼39% w/v) than the particles formed at pH 6.8 (∼18% w/v). Similarly, particle morphology and protein density were also affected by initial NaCl concentration: particles formed at 50 mM NaCl (pH 6.8) were spherical, whereas particles formed at either 200 mM NaCl (pH 6.7) or 400 mM NaCl (pH 6.6) were irregular and protein density of the particles increased with increasing initial NaCl concentration. Whey protein particles formed at pH 5.5 showed an excellent heat stability: viscosity of the suspensions containing approximately 30% of protein particles formed at pH 5.5 did not show any change after heating at 90 °C for 30 min while the viscosity of suspensions containing protein particles prepared at other conditions increased after heating. In summary, whey protein particles with varying microstructure, shape, internal protein density, and heat stability can be formed by using heat-induced gelation of whey protein isolate at different gelling conditions.

Influence of protein hydrolysis on the growth kinetics of ß-lg fibrils.

Recently it was found that protein hydrolysis is an important step in the formation of ß-lactoglobulin fibrils at pH 2 and elevated temperatures. The objective of the present study was to further investigate the influence of hydrolysis on the kinetics of fibril formation. Both the hydrolysis of ß-lactoglobulin and the growth of the fibrils were followed as a function of time and temperature, using SDS polyacrylamide gel electrophoresis and a Thioflavin T fluorescence assay. As an essential extension to existing models, the quantification of the effect of the hydrolysis on the fibrillar growth was established by a simple polymerization model including a hydrolysis step.

Phase-Separating Binary Polymer Mixtures: The Degeneracy of the Virial Coefficients and Their Extraction from Phase Diagrams.

The Edmond-Ogston model for phase separation in binary polymer mixtures is based on a truncated virial expansion of the Helmholtz free energy up to the second-order terms in the concentration of the polymers. The second virial coefficients (B 11, B 12, B 22) are the three parameters of the model. Analytical solutions are presented for the critical point and the spinodal in terms of molar concentrations. The calculation of the binodal is simplified by splitting the problem into a part that can be solved analytically and a (two-dimensional) problem that generally needs to be solved numerically, except in some specific cases. The slope of the tie-lines is identified as a suitable parameter that can be varied between two well-defined limits (close to and far away from the critical point) to perform the numerical part of the calculation systematically. Surprisingly, the analysis reveals a degenerate behavior within the model in the sense that a critical point or tie-line corresponds to an infinite set of triplets of second virial coefficients (B 11, B 12, B 22). Since the Edmond-Ogston model is equivalent to the Flory-Huggins model up to the second order of the expansion in the concentrations, this degeneracy is also present in the Flory-Huggins model. However, as long as the virial coefficients predict the correct critical point, the shape of the binodal is relatively insensitive to the specific choice of the virial coefficients, except in a narrow range of values for the cross-virial coefficient B 12.

Erratum: Addition to "Phase-Separating Binary Polymer Mixtures: The Degeneracy of the Virial Coefficients and Their Extraction from Phase Diagrams".

[This corrects the article DOI: 10.1021/acsomega.1c00450.].

Fracture of protein fibrils as induced by elongational flow.

The length distribution of whey protein fibrils is important for application purposes. However, it is hard to influence the length distribution of whey protein fibrils during production. One way of influencing the length distribution of the mature fibrils is exposing them to an external field, like a flow field. In this research whey protein fibrils were exposed to elongational flow to fracture the fibrils. A simple experimental setup was used to establish a range of elongational strain rates. The length distribution of the fractured fibrils was determined using transmission electron microscopy and was shown to be controllable at relatively low strain rates.

Physicochemical properties and gelling behaviour of Bambara groundnut protein isolates and protein-enriched fractions.

Plant proteins, and specifically those from legume crops, are increasingly recognised as sustainable and functional food ingredients. In this study, we expand on the knowledge of Bambara groundnut (Vigna subterranea (L.) Verdc.) [BGN] proteins, by characterising the composition, microstructure and rheological properties of BGN protein isolates obtained via wet extraction and protein-enriched fractions obtained via dry fractionation. The BGN protein isolates were compared in the context of the major storage protein, vicilin, as previously identified. Molecular weight analysis performed with gel electrophoresis and size-exclusion chromatography coupled to light-scattering, revealed some major bands (190 kDa) and elution patterns with molecular weights (205.6-274.1 kDa) corresponding to that of BGN vicilin, whilst the thermal denaturation temperature (Tp 91.1 °C, pH 7) of BGN protein isolates also coincided to that of the vicilin fraction. Furthermore, the concentration dependence of the elastic modulus G' of the BGN protein isolates, closely resembled that of BGN vicilin (both upon NaCl addition); suggesting that vicilin is the main component responsible for gelation. Confocal laser scanning and scanning electron micrographs revealed inhomogeneous aggregate structures, which implies that fractal scaling were better suited for description of the BGN protein isolate gel networks. Concerning the BGN protein-enriched fractions, both rotor and impact milling with air jet sieving and air classification, respectively, were successfully applied to separate these fractions from those high in starch; as evident from compositional analysis, particle size distributions and microscopic imaging. When considering sustainability aspects, dry fractionation could thus be a viable alternative for producing BGN protein-enriched fractions.

Thioflavin T fluorescence assay for beta-lactoglobulin fibrils hindered by DAPH.

The molecule 4,5-dianilinophthalimide was recently found to be an efficient compound in disaggregating amyloid fibrils involved in the Alzheimer's disease. In this study we have investigated whether the compound 4,5-dianilinophthalimide was able to disaggregate fibrils derived from beta-lactoglobulin. In addition to a Thioflavin T fluorescence assay, flow-induced birefringence was used as an independent technique to measure the total length concentration of the fibrils. An additional advantage of the latter technique is that not only the total length concentration, but also the length distribution of the fibrils can be measured. The results from flow-induced birefringence showed that the total amount of fibrils and also the length distribution of the fibrils was not influenced by the addition of 4,5-dianilinophthalimide, even though this was suggested by the results of the Thioflavin T assay. The results of flow-induced birefringence were confirmed by rheological measurements and transmission electron microscopy. Our findings show that the use of a Thioflavin T assay in order to probe the possible disaggregating effect of certain compounds can give misleading results.

Surface characterization of drying acrylic latex dispersions with variable methacrylic acid content using surface dilatational rheology.

HYPOTHESIS: Drying of latex dispersions often results in particle gradients at the latex-air interface. We expect that, by increasing the carboxylic acid content of latex particles, inter-particle interactions at the interface change. With dilatational rheology one could detect particle-particle interactions in an early stage of the drying process and elucidate the nature of these interactions. EXPERIMENTS: Acrylic latex dispersions were prepared with different amounts of methacrylic acid (MAA), ranging from 2 to 10 wt% on dry mass. Dilatational rheology studies during drying at different relative humidities RH were performed using profile analysis tensiometry. Visco-elastic properties of latex surfaces were used to identify inter-particle interactions at the surfaces depending on the drying rate and particle composition. FINDINGS: Drying at 85% RH did not show significant changes of the mechanical properties of the latex surfaces. Drying at 65 and 53% RH resulted in a change of the mechanical properties, ultimately showing non-linear visco-elastic behavior. This indicates that capillary and/or Van der Waals forces were operating between particles at the surface. With increasing MAA content the viscous contribution decreased, possibly due to the formation of more gel-like structures at the particle surface due to higher solubility of polymer segments near to the surface.

Composite Gels Containing Whey Protein Fibrils and Bacterial Cellulose Microfibrils.

In this study, we investigated the gelation of WPI fibrils in the presence of bacterial cellulose (BC) microfibrils at pH 2 upon prolonged heating. Rheology and microstructure were investigated as a function of BC microfibril concentration. The presence of BC microfibrils did not influence the gelation dynamics and resulting overall structure of the WPI fibrillar gel. The storage modulus and loss modulus of the mixed WPI-BC microfibril gels increased with increasing BC microfibril concentration, whereas the ratio between loss modulus and storage modulus remained constant. The WPI fibrils and BC microfibrils independently form two coexisting gel networks. Interestingly, near to the BC microfibrils more aligned WPI fibrils seemed to be formed, with individual WPI fibrils clearly distinguishable. The level of alignment of the WPI fibrils seemed to be dependent on the distance between BC microfibrils and WPI fibrils. This also is in line with our observation that with more BC microfibrils present, WPI fibrils are more aligned than in a WPI fibrillar gel without BC microfibrils. The large deformation response of the gels at different BC microfibril concentration and NaCl concentration is mainly influenced by the concentration of NaCl, which affects the WPI fibrillar gel structures, changing form linear fibrillar to a particulate gel. The WPI fibrillar gel yields the dominant contribution to the gel strength.