Complex coacervation between beta-lactoglobulin and Acacia gum: a nucleation and growth mechanism

Complex coacervation between proteins and polysaccharides is a demixing process mainly driven by electrostatic interactions. During this process many structural transitions occur, involving the formation of soluble complexes, aggregated complexes, and coacervates. The dynamic mechanism of complexation/coacervation was studied on beta-lactoglobulin (BLG)/Acacia gum (AG) mixed dispersions (0.1 wt% total concentration; BLG:AG ratio of 2:1) using small angle static light scattering (SALS). Acidification of BLG/AG dispersions was induced by dissolution of 0.11 wt% glucono-delta-lactone, allowing in situ SALS measurements. Time evolution of turbidity, scattered light intensity at 46 degrees scattering angle (I46) or slope of scattering functions at high q range revealed the existence of six pH-induced structural transitions. During BLG/AG complexation and before coacervation took place, scattering profiles displayed a monotonic decrease of I(q) as a function of q. A correlation peak in the scattering functions was only observed when coacervates appeared in the system. The wave vector q(max) corresponding to the maximum in scattered intensity first shifted toward larger q values, indicating an increasing number of coacervates, then shifted toward smaller q values, as a consequence of the system coarsening. The power laws q(max) approximately t(-alpha) and I(max) approximately t(-beta) gave values of 1.9 and 9.2, respectively, values much larger than those expected for intermediate and late stages of spinodal decomposition. From these results, it was concluded that complex coacervation between BLG and AG was a nucleation and growth type process. In addition, the temporal evolution of I46 followed power laws with two different exponents. First exponent corresponding to BLG/AG complexation was 3.0+/-0.3 and indicated a diffusion-controlled growth mechanism. Second exponent corresponding to the initiation of phase separation to the coacervation process was 6.5+/-0.3 and revealed an interfacially-controlled growth mechanism.     

Study of beta-lactoglobulin/acacia gum complex coacervation by diffusing-wave spectroscopy and confocal scanning laser microscopy

Complex coacervation has been investigated on mixtures of beta-lactoglobulin (beta-lg) and acacia gum (AG) at pH 4.2 where these two macromolecules interact electrostatically. Changes in beta-lg/AG complex coacervation induced by the presence of beta-lg aggregates were considered. The nature and structure of particles resulting from complex coacervation were determined by using confocal scanning laser microscopy (CSLM). CSLM revealed fundamental differences in the structure of each of the studied dispersions (at 1 wt.% total concentration). Spherical vesicular coacervates and precipitates (based on beta-lg aggregates) were the hallmark of BLG/AG dispersions (beta-lg dispersion containing insoluble aggregates). Only coacervates were visible in AF-BLG/AG dispersions (beta-lg dispersion free of insoluble aggregates). The latter were characterised by the presence of large foam-like coacervates induced by partial coalescence of single coacervates, especially at the 2:1 protein to polysaccharide (Pr:Ps) ratio. Diffusing wave spectroscopy (DWS) was used to study the stability of dispersions as a function of time. Depending on the Pr:Ps ratio and the presence of beta-lg aggregates, the intensity correlation function (g(2)(t)) shifted to lower correlation times rapidly after mixing of both macromolecules. This revealed the formation of a large number of small particles, characterised by faster Brownian motion. At 1 and 5 wt.% total concentration, the 8:1 Pr:Ps ratio exhibited a rapid decrease of the backscattered intensity in time, both for BLG/AG and AF-BLG/AG mixtures, revealing rapid sedimentation/coalescence of particles. This precluded the achievement of a stable correlation function. For the 2:1 Pr:Ps ratio, mixtures exhibited both coalescence and sedimentation phenomena as confirmed by shifts in the g(2)(t) towards larger correlation times and the decrease of the initial value of g(2)(t) with time. Mixtures obtained for the 1:1 Pr:Ps ratio were characterised by small variations in the DWS signal, emphasising the stability of produced particles. The increase of the total biopolymer concentration reduced the effect of both Pr:Ps ratio and presence of protein aggregates. From CSLM and DWS observations, possible differences in the complex coacervation mechanism in both types of mixtures were highlighted. The use of protein aggregates to control complex coacervation was underlined.     

Formation of concentrated biopolymer particles composed of oppositely charged WPI and pectin for food applications

The application of protein–polysaccharide complexes as potential structure modifier, fat replacer, or emulsifying agents in food dispersions has gained increasing interest amongst scientists and manufacturers. Based on associative complexation, low biopolymer concentrations are typically used to generate particulated complexes. The current study, however, presents results that focused on the formation of concentrated biopolymer dispersions. A simple heat treatment was applied to tailor the overall water content of the biopolymer dispersion. For that purpose, whey protein isolate (WPI) and citrus pectin (DE 71%) solutions were mixed at different pH and biopolymer ratios to induce complex coacervation and subsequently heat-treated (??=?90–95°C). Phase separation behavior, microstructural, rheological, and electrical properties of the complexes were investigated by surface charge, turbidity, particle size, rheometry, and light microscopy measurements. Results revealed that complexation was induced under acidic conditions, whereas high WPI:citrus pectin ratios led to positive surface charges, promoting the formation of large and dense particles. In addition, concentrated complex dispersions with water contents ≥80% could be manufactured and easily re-dispersed, whereas complexes maintained their particulate structures. Results are of importance for future studies where we intend to incorporate concentrated biopolymer particles as structuring agents in complex food matrices.     

Associative phase separation of beta-lactoglobulin/pectin solutions: a kinetic study by small angle static light scattering

Interpolymer complexation between beta-lactoglobulin (beta-lg) and pectin led to phase separation. Small angle static light scattering and phase contrast microscopy were used to monitor the phase separation of beta-lg/low-methoxyl or high-methoxyl-pectin (LM- or HM-pectin) dispersions as they were slowly acidified from pH 7 to 4 with glucono-delta-lactone (GDL). The monotonic decrease in scattered light intensity with the wave vector was associated with a nucleation and growth phase separation mechanism. Microscopic observations and turbidity measurements showed the increase of complex amounts with lower pH and at higher beta-lg/pectin ratios. The formation of intrapolymer complexes was initiated at pH 6.4 with the LM-pectin and at pH 5.0 with the HM-pectin. Local ordering with increasing amounts of small complexes was observed as scattered light intensity increased at intermediate q values. The beta-lg/LM-pectin complexes at the 5:1 and the 2:1 weight ratios and the beta-lg/HM-pectin complexes at 5:1 weight ratio have fractal structures. The formation of large amounts of small assemblies and sedimentation would be responsible for the decrease in the number and volume mean diameters and fractal dimension of beta-lg/LM-pectin complexes over time.     

Biopolymers phase separation monitored by a plasmonic sensor

We report here a real-time study of interactions induced phase separation between β-lactoglobulin (BLG) and Acacia gum (AG) by analyzing the localized surface plasmon resonance of silver nanoparticles. We showed that the binding of BLG to AG is accompanied by refractive index changes, in relation with optical properties and structural changes of the complexes formed.     

Synthesis and aqueous solution behavior of phosphonate-functionalized chitosans

A series of novel phosphonate-functionalized pH-responsive chitosans were directly synthesized via Michael addition of chitosan with mono-(2-acryloyloxyethyl) phosphonate. The conformational and phase transitions of these phosphonate-functionalized chitosans were investigated by turbidity and UV-vis spectroscopy studies. The results indicated that the inter- or intra-chain electrostatic interactions of the phosphonate-functionalized chitosans could be controlled via adjusting the solution pH, leading to the reversible conformational and phase transitions of these chitosans. These phosphonate-functionalized chitosans exhibited typical anti-polyelectrolyte effect and polyelectrolyte effect.     

Detection of the equilibrium folding intermediate of beta-lactoglobulin in the presence of trifluoroethanol by mass spectrometry

Nano-electrospray ionization mass spectrometry (nano-ESI-MS) was used to monitor the effect of trifluoroethanol (TFE) on the conformational properties of beta-lactoglobulin (BLG). TFE stabilizes protein secondary structure, particularly alpha-helices. However, it also acts as a denaturant above critical concentrations. In the case of BLG, TFE at low concentrations is known to induce formation of an equilibrium intermediate that contains non-native helical structure. Such an intermediate is thought to form also under physiological conditions, playing a role in BLG folding in vivo by preventing aggregation. This well-characterized system was chosen in order to test species distributions obtained by nano-ESI-MS. BLG spectra at increasing concentrations of TFE at pH 2 indicate transient accumulation of a conformer whose charge-state distribution (CSD) falls between that of the native and that of the denatured protein, indicating that the TFE-induced, partially folded form can be selectively monitored by this technique. The condition of its maximum accumulation corresponds to 16% TFE, in excellent agreement with results from solution experiments. In contrast, titrations with methanol or acetonitrile (ACN) reveal apparent two-state transitions from native to fully unfolded BLG. At 10% TFE, the protein appears to be still fully folded at room temperature but, if unfolding is elicited by the combination with other denaturing agents, e.g. heat or low concentrations of ACN, it proceeds via formation of the intermediate. Thus, TFE can also induce formation of the BLG intermediate in synergism with generic denaturing agents. This study indicates good agreement between ESI-MS and other biophysical methods monitoring protein conformational transitions in the presence of TFE.     

Comparison of bovine and porcine beta-lactoglobulin: a mass spectrometric analysis

Nano-electrospray-ionization mass spectrometry (nano-ESI-MS) is applied to comparison of bovine and porcine beta-lactoglobulin (BLG and PLG). The conformational and oligomeric properties of the two proteins under different solvent and experimental conditions are analyzed. The pH-dependence of dimerization is described for the pH range 2-11. The results indicate maximal dimer accumulation at pH 6 for BLG and pH 4 for PLG, as well as a lower stability of the PLG dimer at pH 4 compared to BLG at pH 6. Conformational stability appears to be higher for BLG at acidic pH, but higher for PLG at basic pH. The higher stability of BLG at low pH is revealed by means of either chemical or thermal denaturation. Equilibrium folding intermediates of both proteins are detected. Finally, conditions are found that promote dissociation of the BLG dimer at pH 6 into folded monomers.     

Complexation of bovine serum albumin and sugar beet pectin: structural transitions and phase diagram

The complexation between bovine serum albumin (BSA) and sugar beet pectin (SBP) was studied in situ by coupling glucono-δ-lactone (GDL) induced acidification with dynamic light scattering and turbidity measurements. Individual measurements at specific pHs and mixing ratios were also carried out using zeta potentiometry, gel permeation chromatography-multiangle laser light scattering (GPC-MALLS), and isothermal titration calorimetry (ITC). These investigations together enabled the establishment of a phase diagram of BSA/SBP and the identification of the molecular events during protein/polysaccharide complexation in relation to the phase diagram, which showed five regions: (I) a stable region of mixed individual soluble polymers, (II) a stable region of intramolecular soluble complexes, (III) a quasi-stable region of intermolecular soluble complexes, (IV) an unstable region of intermolecular insoluble complexes, and (V) a second stable region of mixed individual soluble polymers, on lowering pH. We found for the first time that the complexation could take place well above the critical pH(c), the value that most previous studies had regarded as the onset occurrence of complexation. A model of structural transitions between the regions was proposed. The borderline between region II and region III represents the BSA/SBP stoichiometry for intramolecular soluble complex at a specific pH, while that between region III and region IV identifies the composition of the intermolecular insoluble complex. Also studied was the effect of NaCl and CaCl(2) on the phase diagram and structural transitions.     

pH-thermosensitive behavior of N,N-dimethylaminoethyl methacrylate (Co)polymers with N-vinylcaprolactam

The effect of acidity of a medium on the phase separation temperature and the intensity of light scattering for dispersions produced by heating of aqueous solutions of N,N-dimethylaminoethyl methacrylate, N-vinylcaprolactam, and their copolymers has been studied. It has been demonstrated that the phase separation temperature and the turbidity of polyvinylcaprolactam (and vinylcaprolactam-enriched copolymers) solutions are pH-independent. Poly(N,N-dimethylaminoethyl methacrylate) (and N,N-dimethylaminoethyl methacrylateenriched copolymers) exhibits temperature sensitivity only in the alkaline region, and the phase separation temperature and turbidity versus pH plots are described by curves with maxima. The addition of sodium dodecyl sulfate to polymer solutions in the general case causes an increase in the phase separation temperature. However, if positive charges occur on macromolecules (in initial solutions of poly(N,N-dimethylaminoethyl methacrylate) or acidified solutions of polyvinylcaprolactam), the increase in the phase separation temperature is preceded by its decrease owing to the electrostatic interaction of surfactant anions with cationic centers. As acid is introduced into the H₂O-sodium dodecyl sulfate-polyvinylcaprolactam ternary system, the phase separation temperature of the polyvinylcaprolactam-dodecyl sulfate complex is decreased.     

Rheological properties of mixed gels made of microparticulated whey proteins and β-lactoglobulin

Microparticulated whey proteins (MWP) in suspension or included in a globular protein network revealed that the degree of flocculation was important in the final structure of the material. The MWP dispersions developed an antithixotropic character when low shear rate was applied to the system even at low temperature. Similar results were obtained on globular protein solutions and could be interpreted in terms of an orthokinetic aggregation. MWP participated to the formation and reinforcement of heat-induced β-lactoglobulin (BLG) gels. The mechanical properties of mixed gels demonstrated synergestic effects at low MWP massic fraction (BLG:MWP ratio higher than 2:1). A possible interpretation would be that both interaction of MWP with the network and flocculation of the MWP inside the pores could occur. However, increasing the fraction of MWP induced heterogeneities in the protein network. In particular, micro-phase separation due to steric hindrance effects or thermodynamic incompatibility between the two components could occur and decrease viscoelastic properties of mixed gels.     

Monitoring the Tanford transition in β‐lactoglobulin by 8‐anilino‐1‐naphthalene sulfonate and mass spectrometry

The fluorescent dye 8‐anilino‐1‐naphthalene sulfonate (ANS) is known to interact with proteins by conformation‐specific hydrophobic interactions and rather nonspecific electrostatic interactions. To which category the complexes detectable by mass spectrometry (MS) belong is still the subject of debate. Here, the Tanford transition in β‐lactoglobulin (BLG) is exploited as an experimental device to expose hydrophobic binding sites by an increase in pH, rather than, as usually done, by lowering the pH. Complex formation is monitored by electrospray ionization (ESI)‐MS and fluorescence spectroscopy. Both techniques reveal stronger ANS binding to BLG at pH 7.9 than at pH 5.9, suggesting that dye binding inside the calyx, which is known to be hydrophobically driven in solution, can contribute to the complexes detected by ESI‐MS. Electrostatic interactions between the protein and the ANS sulfonate group can only be weaker at pH 7.9 than at pH 5.9, supporting the interpretation of the results by the protein conformational change. Lysozyme is used as a negative control, which shows no variation in the interaction with ANS in the same range of pH, in the absence of conformational changes. However, comparison of MS and fluorescence data at variable pH for BLG and myoglobin (Mb) suggests that conformation‐specific ANS binding to proteins is detectable by ESI‐MS only inside well‐structured cavities of folded structures, like the BLG calyx and apoMb heme pocket. Indeed, ANS interactions with highly dynamic structures or molten globules, although detectable in solution, are easily lost in the gas phase. Copyright © 2008 John Wiley & Sons, Ltd.     

FTIR spectroscopic analyses of the temperature and pH influences on stratum corneum lipid phase behaviors and interactions

A thermotropic investigation of different lipid dispersions containing ceramide 3 (CER3) or sphingomyelin (SPM), perdeuterated palmitic acid (PA-d(31)) and cholesterol (CH) or cholesterol sulfate (CS) at pH 5.2 and 7.4 used as model membranes, has been carried out by Fourier transform infrared (FTIR) spectroscopy in order to estimate the importance of these lipids and of the temperature and pH for the maintenance of the structural organization of the stratum corneum (SC) lipid lamellae. The results obtained for the CER3 and SPM mixtures at pH 5.2 and 7.4 indicated that the little size of the polar headgroup of CER3 compared with that of SPM could permit a more closely packing in the CER3 acyl chains. Moreover, the CH and CS induced an increase of the order in the CER3 acyl chains over the physiological temperatures while a disordering was seen above 60 degrees C. In addition, the thermal phase behaviors observed for the CER3/PA-d(31) dispersion at pH 5.2 and 7.4, suggested a phase separation between the CER3 and PA-d(31) molecules in this mixture. Nevertheless, the miscibility between the CER3 and PA-d(31) was raised in the presence of CH or CS at pH 5.2. In particular, the incorporation of these sterols into the CER3/PA-d(31) dispersion at pH 5.2 appeared to result in an increase of the order in the acyl chains of CER3 and PA-d(31) at about 37 degrees C. In contrast, a phase separation was observed between the CER3 and PA-d(31) in the CER3/PAd(31)/CH and CER3/PA-d(31)/CS dispersions at pH 7.4. Interestingly, the pH change from 5.2 to 7.4 in these tertiary dispersions was also accompanied by a substantial deprotonation of the PA-d(31) molecules which seemed more pronounced in the presence of CH as compared with CS. Altogether, the results suggested that the ceramides, fatty acids and sterols could play an important structural role in the SC cohesion.     

pH-Dependent aggregation properties of mixtures of sugar-based gemini surfactants with phospholipids and single-tailed surfactants

Sugar-based gemini surfactants (GSs) display rich pH-dependent phase diagrams and are considered to be promising candidates as gene- and drug-delivery vehicles for biomedical applications. Several sugar-based GSs form vesicles around neutral pH. The vesicular dispersions undergo transitions toward wormlike micelles and spherical micelles at acidic pH, whereas flocculation followed by redispersion upon charge reversal is observed at basic pH. The influence of various amounts of the double-tailed phospholipids DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and of the single-tailed surfactants lyso-PC (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine) and OTAC (octadecyltrimethylammonium chloride) on the phase behavior of GS1 (1,8-bis(N-octadec-9-yl-1-deoxy-D-glucitol-1-ylamino)3,6-dioxaoctane) was determined as a function of pH, in water and in water at physiological ionic strength. The pH corresponding to the phase transitions and the characteristics of the aggregates were determined by means of a combination of physical techniques: static and dynamic light scattering (SLS and DLS), fluorescence spectroscopy, cryo-TEM and diffusion- and (31)P NMR. The results show that the additives affect the phase behavior of the GS1 dispersions in a pH-dependent fashion. In the presence of double-tailed phospholipids, a higher degree of protonation of GS1 must be reached to observe micelle formation, whereas single-tailed surfactants affect these transitions only slightly. In the presence of increasing amounts of lyso-PC, the pH range of flocculation becomes more narrow, indicating the increased hydration of the vesicles. The pH of redispersion after charge reversal is particularly sensitive to the presence of positively charged additives. It is suggested that the cationic headgroups disturb the hydrogen-bond structure of water at the vesicular surface, hampering OH(-) binding. The effect of an increase in ionic strength to physiological values is found to be modest, except for the dispersions containing the positively charged additives.     

pH-induced deswelling kinetics of sterically stabilized poly(2-vinylpyridine) microgels probed by stopped-flow light scattering

Near-monodisperse, sterically stabilized poly(2-vinylpyridine) (P2VP) microgels were synthesized by emulsion polymerization. These particles exhibited completely reversible pH-responsive swelling/deswelling behavior in aqueous solution. Stopped-flow light scattering was employed to investigate the kinetics of pH-induced deswelling in highly dilute dispersions. Upon a pH jump from 2 to various final solution pH values (>or=5.4), the scattered light intensity of an aqueous dispersion of a 1,960 nm microgel exhibited an abrupt initial increase, followed by a gradual decrease to the final equilibrium value. The whole microgel-to-latex deswelling process occurred over time scales of approximately 0.5-1.0 s, which is much slower than the kinetics for latex-to-microgel swelling. The microgel deswelling kinetics depends on the final pH, with a higher final pH leading to a faster rate of shrinkage. Close inspection of the deswelling kinetics during the early stages (<0.2 s) revealed that initial microgel collapse occurred within approximately 50 ms, with more rapid transitions being observed when higher final pH values were targeted. Addition of external salt significantly accelerates the kinetics of deswelling. Systematic studies of the microgel-to-latex transition for a series of six near-monodisperse P2VP particles (with swollen microgel diameters ranging from 1270 to 4230 nm) has also been investigated. The characteristic deswelling time for initial microgel collapse, tau deswell, correlated fairly well with the initial swollen microgel radius, R, in agreement with the Tanaka equation. Moreover, the collective diffusion coefficient of the gel network, D, calculated from the slope of the tau deswell- R (2) curve, was of the order of 10 (-7) cm (2) s (-1).     

Sedimentation stability of mixed dispersions of kaolin and synthetic latexes

Sedimentation stability of mixed dispersions of kaolin and synthetic latexes was studied in relation to the chemical nature of the phase boundary, particle size, pH of the dispersion medium, and concentration ratio of components of the dispersed phase.     

Study of pH-triggered heteroaggregation and gel formation within mixed dispersions

This work involves an investigation of pH-triggered heteroaggregation and gelation within mixed dispersions of polystyrene (PS) and pigment particles. The PS particles were stabilised by a carboxylated alkyl ethoxylate surfactant which is pH-responsive. The pigment used was beta-copper phthalocyanine. The pigment particles contained a co-surfactant system consisting of the carboxylated alkyl ethoxylate and a non-ionic surfactant. The latter was a beta-naphthol ethoxylate. The PS and pigment particles were characterised using SEM, TEM, photon correlation spectroscopy and electrophoretic mobility measurements. The PS dispersions exhibited pH-triggered aggregation when the pH was decreased to below a critical value (pH(crit)), which was 1.9. Concentrated PS dispersions formed stable particle gels at pH values less than or equal to pH(crit). Dilute pigment dispersions were found to have a pH(crit) of 3.45. However, concentrated pigment dispersions did not form gels when the pH was decreased to below pH(crit). A phase diagram for the mixed dispersions was constructed which showed a gel phase existed at pH values between 2.0 and 3.0, which corresponds to a pH region higher than pH(crit) for the PS particles. This implicates PS-pigment inter-particle bonds in the gel structure. The heteroaggregate gels were investigated using dynamic rheological measurements and it was apparent that the highest elastic modulus values were obtained in the pH range of approximately 2 to 3. SEM images provided evidence of heteroaggregates with diameters of a few micrometers. These primary heteroaggregates are suggested to be the network forming unit for the gels formed in mixed dispersions. The data from the study are used to propose a conceptual model for the structure of the heteroaggregate gels.     

Preparation and adsorption of refined polyelectrolyte complex nanoparticles

We report on bulk and surface properties of centrifuged nonstoichiometric polyelectrolyte complex (PEC) dispersions. PECs were prepared by mixing poly(diallyldimethylammonium chloride) (PDADMAC) and sodium poly(maleic acid-co-alpha-methylstyrene) (PMA-MS) at the monomolar mixing ratio of 0.6 and polymer concentration >/=1 mmol/l. Centrifugation of initial PEC dispersions revealed three phases: supernatant (SUP), coacervate (COAC), and an insoluble precipitate. Mass, turbidity, particle hydrodynamic radii (R(h)), and the titratable charge amount were determined for those phases. The turbid COAC phase consisted of 200-nm nanoparticles and carried 60% of the polymer mass and 20% of the titratable charge amount of the initial PEC dispersion. The SUP phase showed no turbidity and no such nanoparticles, but carried 80% of the initial titratable charge amount, presumably caused by excess polycations. Furthermore, linear dependences of turbidity and R(h) on COAC concentration was observed. COAC adsorption was studied at polyelectrolyte multilayer (PEM) modified silicon surfaces in dependence on both adsorption time and concentration using attenuated total-reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The adsorption data were fitted by the simple Langmuir model. Comparison of COAC particles and polystyrene latices revealed similar adsorption features. SEM and AFM measurements resulted in hemispherically shaped adsorbed COAC particles with coverages >/=25%, whose calculated volumes correlated well with those in dispersion obtained by PCS.     

Interactions of β-Lactoglobulin with Bovine Submaxillary Mucin vs. Porcine Gastric Mucin: The Role of Hydrophobic and Hydrophilic Residues as Studied by Fluorescence Spectroscopy

The aim of this study was to investigate binding interactions between β-lactoglobulin (BLG) and two different mucins, bovine submaxillary mucins (BSM) and porcine gastric mucin (PGM), using intrinsic and extrinsic fluorescence spectroscopies. Intrinsic fluorescence spectra showed an enhanced decrease of fluorescence intensity of BLG at all pH conditions when BLG was mixed with PGM rather than with BSM. We propose that, unlike BSM, the tertiary structure of PGM changes and the hydrophobic regions are exposed at pH 3 due to protonation of negatively charged residues. Results suggest that PGM also facilitated the structural unfolding of BLG and its binding with PGM by a hydrophobic interaction, especially at acidic pH, which was further supported by extrinsic fluorescence spectroscopy. Hydrophobic interaction is suggested as the dominant interaction mechanism between BLG and PGM at pH 3, whereas electrostatic interaction is the dominant one between BLG and BSM.     

Phase separation behavior of whey protein isolate particle dispersions in the presence of xanthan

In this study, phase separation of colloidal whey protein isolate (WPI) particle dispersions was studied using a rod-like polysaccharide xanthan. Effects of different xanthan concentration, particle volume fraction, and temperature were analyzed by visual observations, turbidity measurements, and particle mobility tracking method. Particle mobility was determined using a diffusing wave spectroscopy (DWS) set up. Xanthan concentration was kept low in order not to increase the viscosity of dispersions, so that the phase separation could be observed easily. Visual observations showed that there was a minimum concentration of xanthan to induce phase separation at a constant particle volume fraction, and xanthan concentration was found to have an important effect on the degree of phase separation. The temperature was also found to have an effect on depletion mechanism. Phase separation was mainly a result of different sizes of WPI particles, and xanthan induced the depletion interaction between WPI particles, as supported by the data obtained from DWS. The results of this study explained both the mechanism and the stability range of particle dispersions in the presence of xanthan, which is important for the design of stable systems, including colloidal particles.