Complex coacervation of pea protein isolate and alginate polysaccharides

Complex coacervation between pea protein isolate (PPI) and alginate (AL) was investigated as a function of pH (1.50–7.00) and mixing ratio (1:1–20:1 PPI:AL) by turbidimetric analysis and electrophoretic mobility during an acid titration. Conformational changes to the secondary structures during coacervation were also studied by Raman spectroscopy. Critical structure-forming events associated with the formation of soluble (pH 5.00) and insoluble (pH 2.98) complexes were found for a 1:1 PPI–AL mixture, with optimal biopolymer interactions occurring at pH 2.10 (pH_opt). As mixing ratios increased between 4:1 and 8:1, critical pHs shifted towards higher pH. Maximum coacervate formation at pH_opt occurred at a mixing ratio of 4:1. Electrophoretic mobility measurements showed a shift in net neutrality from pH 4.00 in homogenous PPI solutions, to pH 1.55 for the 1:1 mixture. As biopolymer ratios increased towards 8:1 PPI:AL, net neutrality shifted to higher pHs (～3.80). Raman spectroscopy revealed minimal complexation-induced conformational changes. Findings could aid in the design of pH-sensitive biopolymer carriers for use in functional food and bio-material applications.     

Effect of pH on the functional behaviour of pea protein isolate–gum Arabic complexes

The functional behaviour (solubility, emulsifying and foaming properties) of pea protein isolate (PPI) and gum Arabic (GA) mixtures were investigated as a function of pH (4.30–2.40) within a region dominated by complex coacervation. Emulsion stability was also investigated using a one- and two-step emulsification approach. Complex coacervation was monitored by turbidimetric acid titration at a 2:1 PPI–GA ratio to reveal the formation of soluble (pH 4.23) and insoluble (pH 3.77) complexes, maximum biopolymer interactions (pH 3.60), and dissolution of complexes (pH 2.62). Emulsion stability was greater for mixed systems relative to PPI alone at pHs between 3.10 and 4.00, and in those prepared using the one-step method. Foam expansion was independent of both biopolymer content and pH, whereas foam stability was improved for the mixed system between pH 3.10 and 4.00. The pH-solubility minimum was broadened relative to PPI to more acidic pHs. Findings suggest that admixtures of PPI and GA under complexing conditions could represent a new blended food and/or biomaterial ingredient.     

Associative phase separation involving canola protein isolate with both sulphated and carboxylated polysaccharides

Associative phase separation within admixtures of canola protein isolate (CPI) and anionic polysaccharides (alginate and ι-carrageenan) was investigated as a function of pH (1.50–7.00) and biopolymer weight mixing ratio (1:1–50:1, w/w) by turbidimetric analysis. Solubility of formed complexes was also studied vs. protein alone as a function of pH. In both CPI–polysaccharide systems, critical pH, associated with the onset of soluble and insoluble complexes, shifted to higher pHs as the mixing ratios increased to a 20:1 CPI–polysaccharide ratio, and then became constant. Complexes formed primarily through electrostatic attractive forces with secondary stabilisation by hydrogen bonding. Solubility of the CPI–alginate system was significantly enhanced relative to CPI alone or CPI–ι-carrageenan.     

Effect of pH,biopolymer mixing ratio and salts on the formation and stability of electrostatic complexes formed within mixtures of lentil protein isolate and anionic polysaccharides (κ‐carrageenan and gellan gum)

Associative phase separation within lentil protein isolate (LPI), polysaccharide (κ‐carrageenan (κ‐CG) and gellan gum (GG)) mixtures was investigated as a function of pH (1.50–8.00) and mixing ratio (1:1–30:1; LPI/polysaccharide) by turbidity and electrophoretic mobility. Effects of salts (NaCl, KCl and CaCl₂) on complex stability were also studied as a function of ionic strength. Coacervation typically follows two pH‐dependent forming events associated with the formation of soluble and insoluble complexes. The addition of polysaccharides to a LPI system (at all ratios) resulted in a significant drop in turbidity over the entire pH range and a shift in net neutrality to lower pH relative to LPI alone; where LPI aggregation was inhibited by repulsive forces between neighbouring polysaccharide chains. As the biopolymer mixing ratio increased, structure formation was less inhibited. The addition of salts resulted in the disruption of formed LPI/polysaccharide complexes.     

Formation and functionality of whey protein isolate–(kappa-, iota-, and lambda-type) carrageenan electrostatic complexes

The formation of electrostatic complexes between whey protein isolate (WPI) and (κ-, ι-, λ-type) carrageenan (CG) was investigated by turbidimetric measurements as a function of pH (1.5–7.0), biopolymer weight-mixing ratio (1:1–75:1 WPI:CG) and NaCl addition (0–500 mM) to better elucidate underlying mechanisms of interaction. Emulsion stabilizing effects of formed complexes was also studied to assess their potential as emulsifiers. Complex formation followed two pH-dependent structure-forming events associated with the formation of soluble (pH_c) and insoluble (pH_?1) complexes. For both the WPI–κ-CG and WPI–ι-CG mixtures, pH_c and pH_?1 occurred at pH 5.5 and 5.3, respectively, whereas in the WPI–λ-CG mixture values were slightly higher (pH_c = 5.7; pH_?1 = 5.5). In all mixtures, maximum turbidity was found to occur near pH 4.5, before declining at lower pHs. Biopolymer mixing ratios corresponding to maximum OD was found to occur at the 12:1 ratio for both the WPI–κ-CG and WPI–λ-CG mixtures, and 20:1 ratio for WPI–ι-CG mixture. The addition of NaCl disrupted complexation within WPI–κ-CG mixtures as levels were raised, whereas when ι-CG and λ-CG was present, complexation was enhanced up to a critical Na⁺ concentration before declining. Adsorption of CG chains to the small WPI–WPI aggregates during complexation was proposed to be related to both the linear charge density and conformation of the CG molecules involved. Emulsion stability in the mixed systems (12:1 mixing ratio), regardless of the CG type (κ, ι, λ), was significantly higher than individual WPI solutions indicating enhanced ability to stabilize the oil-in-water interface.     

Formation and functional attributes of electrostatic complexes involving napin protein isolate and anionic polysaccharides

The formation of electrostatic complexes involving a napin protein isolate (NPI) and carboxylated alginate (AL) and sulfated (κ-, ι-, λ-type carrageenan) (CG) polysaccharides was investigated at a biopolymer mixing ratio of 10:1 (NPI/polysaccharide) as a function of pH (4.0–12.0) using turbidity and electrophoretic mobility. The functionality of the ensuing complexes was tested on the basis of their solubility, emulsion stability and foaming capacity and stability relative to NPI alone. Complexation follows two pH-dependent structure forming events associated with the formation of ‘soluble’ and ‘insoluble’ complexes. Soluble and insoluble complexes for NPI–AL, NPI–κ-CG, NPI–ι-CG and NPI–λ-CG mixtures occurred at pHs 7.1 and 6.2, 8.6 and 7.0, 9.5 and 9.3, and 9.0 and 8.7, respectively. Complexation resulted in a shift in net neutrality from 5.0 for NPI alone to pH 4.2, 3.7, 3.2 and 2.3 in the presence of κ-CG, ι-CG, λ-CG and AL, respectively. Solubility and foaming capacity of NPI were reduced with the addition of polysaccharide. Foaming stability was similar for NPI–κ-CG and NPI–λ-CG mixtures relative to NPI, but increased and decreased for NPI–ι-CG and NPI–AL mixtures, respectively. Emulsion stability was found to be similar for all mixtures relative to NPI, except for the NPI–ι-CG mixture which had reduced emulsion stability.     

Complex coacervation between β-lactoglobulin and acacia gum in aqueous medium

The compatibility of β-lactoglobulin (β-lg) and acacia gum in aqueous medium was investigated as a function of the pH (3.6–5.0), the protein to polysaccharide weight ratio (50:1–1:20) and the total biopolymer concentration (0.1–5 wt%). The ternary phase diagrams obtained at low ionic strengths (0.005–10.7 mM) typically accounted for phase separation through complex coacervation. Thus a drop-shaped two-phase region was anchored in the water-rich corner. The electrostatic nature of the interactions between the two biopolymers was pointed out according to the pH dependence of the two-phase region's breadth. Following the absorbance of the mixtures at 650 nm, the influence of the protein to polysaccharide ratio was also demonstrated. Electrophoretic mobility (μ_E) measurements and chemical analyses of separated phases revealed the formation of soluble and insoluble coacervates and complexes. A remarkable value of the protein to polysaccharide weight ratio (2:1) at pH 4.2 gave the same protein to polysaccharide (Pr:Ps) ratio in the two phases after 2 days, implying that electrostatic interactions are maximum between β-lg and acacia gum. The increase of the total biopolymer concentration reduced the influence of pH and protein to polysaccharide ratio. Also, the increase of the pH close to the β-lg IEP reduced the influence of the total biopolymer concentration and Pr:Ps ratio. As the biopolymer content was increased at pH 3.6 and 4.2, the relative β-lg solubility increased probably because of the self-suppression of complex coacervation.     

Complexation of pea protein isolate with dextran sulphate and interfacial adsorption behaviour and O/W emulsion stability at acidic conditions

This study was aimed at improving the emulsifying property and physical stability of pea protein isolate (PPI) stabilised emulsions at acidic conditions by complexation with dextran sulphate (DS). Soluble and insoluble complexes with different charge and particle size were formed depending on the phase separation behaviour. The surface adsorption of PPI became slower after complexation with DS, but the percentage of adsorbed proteins at the oil–water interface was not affected. The formation of PPI–DS soluble complexes at high content of DS (≥0.4%) significantly improved the negative net charges of PPI, prevented the aggregation of protein, which further improved the emulsifying property of PPI at acidic conditions through the strong electrostatic repulsion and steric hindrance effects. Insoluble complexes with relatively weak net charge and large particles were formed at low DS content (≤0.2%), resulting in the bridging flocculation of oil droplets at pH 5 and 4. Thus, the emulsifying ability of PPI under acidic conditions could be significantly improved by formation of soluble complexes with DS.     

Characterisation of whey protein isolate–gum tragacanth electrostatic interactions in aqueous solutions

The main objectives of this study were to measure molecular parameters of gum tragacanth by GPC‐MALLS system and investigate the complexation behaviour of whey protein isolate/gum tragacanth mixed dispersions (0.5 wt% total biopolymer concentration) as a function of pH (7.00–2.00) and the biopolymer mixing ratio (r = 0.1–10) using spectrophotometric, zeta potential and precipitate yield determination methods. GPC‐MALLS revealed that gum tragacanth contains relatively heterogeneous particles with high weight‐average and number‐average (M_w = 7.74 × 10⁵ g mol^?1 and M_n = 3.87 × 10⁵ g mol^?1) molecular mass and high dispersity index (~2.04 ± 0.3). Results of complexation displayed that as the biopolymer mixing ratio increases, the net neutrality shifts to the higher pHs. The critical values associated with the complex structure formation were found at r = 2 in which the charge density of the mixture was near zero at a wide range of pH (3.0–4.0). However, the highest precipitate yield achieved in pH 3.4.     

Complex coacervation between flaxseed protein isolate and flaxseed gum

Flaxseed protein isolate (FPI) and flaxseed gum (FG) were extracted, and the electrostatic complexation between these two biopolymers was studied as a function of pH and FPI-to-FG ratio using turbidimetric and electrophoretic mobility (zeta potential) tests. The zeta potential values of FPI, FG, and their mixtures at the FPI-to-FG ratios of 1:1, 3:1, 5:1, 10:1, 15:1 were measured over a pH range 8.0–1.5. The alteration of the secondary structure of FPI as a function of pH was studied using circular dichroism. The proportion of ɑ-helical structure decreased, whereas both β-sheet structure and random coil structure increased with the lowering of pH from 8.0 to 3.0. The acidic pH affected the secondary structure of FPI and the unfolding of helix conformation facilitated the complexation of FPI with FG. The optimum FPI-to-FG ratio for complex coacervation was found to be 3:1. The critical pH values associated with the formation of soluble (pHc) and insoluble (pH_ɸ1) complexes at the optimum FPI-to-FG ratio were found to be 6.0 and 4.5, respectively. The optimum pH (pH_opt) for the optimum complex coacervation was 3.1. The instability and dissolution of FPI–FG complex coacervates started (pHɸ₂) at pH 2.1. These findings contribute to the development of FPI–FG complex coacervates as delivery vehicles for unstable albeit valuable nutrients such as omega-3 fatty acids.     

Characterisation of interactions between fish gelatin and gum arabic in aqueous solutions

The interactions between fish gelatin (FG) and gum arabic (GA) in aqueous solutions were investigated by turbidimetry, methylene blue spectrophotometry, zeta potentiometry, dynamic light scattering, protein assay, and state diagram at 40 °C and a total biopolymer concentration (C(T)) of 0.05%. FG underwent complex coacervation with GA, possibly via its conformational change, depending on pH and FG to GA ratio (FG:GA). The formation of FG-GA complexes was the most intense when pH 3.55 and FG:GA=50:50 (6.6:1 M ratio), however, the coacervate phase was found to be composed of a much higher FG fraction. The pH range of complex formation shifted to a higher pH region with increasing FG:GA. Soluble and insoluble FG-GA complexes were formed even in a pH region where both biopolymers were net-negatively charged. Varying C(T) significantly influenced not only the formation of FG-GA complexes but also the development and composition of coacervate phase.     

Temperature-dependent complexation between sodium caseinate and gum arabic

Complex formation between sodium caseinate and gum arabic as a function of temperature was investigated using dynamic light scattering, fluorescence and NMR. At neutral pH, the turbidity and the particle size increased when sodium caseinate and gum arabic mixtures were heated in situ at temperatures above a critical temperature. The increases in turbidity and particle size were reversible. This effect was considered to be due to hydrophobic interactions, leading to the formation of a complex between sodium caseinate and gum arabic. ¹H NMR spectroscopy showed that ANS, which bound to caseinate at low temperatures in caseinate solution or a caseinate-gum arabic mixture, was released at high temperatures upon formation of a caseinate or caseinate-gum arabic complex. This supported changes observed in the fluorescence of 8-anilino-1-naphthalene sulfonate upon binding to caseinate, which decreased at high temperatures for caseinate alone or when sodium caseinate was mixed with gum arabic. Light-scattering (turbidity) and dynamic light-scattering studies show that the temperature-dependent complexation between sodium caseinate and gum arabic was sensitive to the mass ratio of protein to gum arabic (greater complexation at a 1:5 ratio than a 1:1 ratio) and the pH (maximum complexation at pH 6.5).     

Complex Gels of Proteins and Acidic Polysaccharides. Part II. The Effect of Electrostatic Interaction on Structure Formation in Complex Gels of Gelatin and Alginate

Complex gelatin-alginate gels prepared from insoluble (type I) and soluble (type II) complexes are studied by the thermomechanical technique and by polarimetry. Gels of both types are thermoirreversible at concentrations higher than 6% and at weight fractions of alginate of 0.3 or higher. Suppression of complexation between gelatin and alginate by introduction of a neutral salt or by increasing pH above pI_G leads to formation of thermoreversible gels the thermomechanical properties of which are similar to those of gelatin gels. Change of optical rotation on heating is less for complex gels than for gelatin gels of the same concentration.     

Complexation between milk proteins and polysaccharides via electrostatic interaction: principles and applications – a review

This article details recent research conducted on the complexation between milk proteins and polysaccharides and the properties of the complexes, and the application of such relationships to the food industry. Complexation between proteins and polysaccharides through electrostatic interactions gives either soluble complexes in a stable solution or insoluble complexes, leading to phase separation. The formation and the stability of these complexes are influenced by pH, ionic strength, ratio of protein to polysaccharide, charge density of protein and polysaccharide as well as processing conditions (temperature, shearing and time). The functional properties of milk proteins, such as solubility, surface activity, conformational stability, gel‐forming ability, emulsifying properties and foaming properties, are improved through the formation of complexes with polysaccharides. These changes in the functional properties provide opportunities to create new ingredients for the food industry.     

Complex coacervation of pea protein isolate and tragacanth gum: Comparative study with commercial polysaccharides

The ability of pea protein isolates (PPI) to form complex coacervates with tragacanth gum was investigated. The coacervate formation was structurally compared to three other PPI-polysaccharide interaction models: arabic gum and sodium alginate (known to form coacervates with PPI) and tara gum, a galactomannan. The effects of the pH and protein/polysaccharide ratio were mainly investigated using turbidity and zeta potential measurements. Regarding the pH of soluble complex formation, the pH of complex coacervates increased with the increase in protein-anionic polysaccharide mixture ratio. SEM images revealed the ability of the spray-drying process to form spherical particles of pea protein-polysaccharide complexes. The specificity of the microparticle surface was protein-dependent. FTIR analyses of coacervates showed the electrostatic interaction between the PPI and the polysaccharides. The results showed that tragacanth gum could be used as an alternative to gum arabic to form complex coacervates with PPI based on zeta potential measurements and coacervation yield studies.     

Effect of polysaccharide charge on formation and properties of biopolymer nanoparticles created by heat treatment of β-lactoglobulin–pectin complexes

Biopolymer nanoparticles can be formed by heating globular protein/polysaccharide mixtures above the thermal denaturation temperature of the protein under pH conditions where the two biopolymers are weakly electrically attracted to each other. In this study, the influence of polysaccharide linear charge density on the formation and properties of these biopolymer nanoparticles was examined. Mixed solutions of globular proteins (β-lactoglobulin) and anionic polysaccharides (high and low methoxyl pectin) were prepared. Micro-electrophoresis, dynamic light scattering, turbidity and atomic force microscopy (AFM) measurements were used to determine the influence of protein-to-polysaccharide mass ratio (r), solution pH, and heat treatment on biopolymer particle formation. Biopolymer nanoparticles (d < 500 nm) could be formed by heating protein–polysaccharide complexes at 83 °C for 15 min at pH 4.75 and r = 2:1 in the absence of added salt. The biopolymer particles formed were then subjected to pH and salt adjustment to determine their stability. The pH stability was greater for β-lactoglobulin-HMP complexes than for β-lactoglobulin-LMP complexes. The addition of 200 mM sodium chloride to heated complexes greatly improved the pH stability of HMP complexes, but decreased the pH stability of LMP complexes. The biopolymer particles formed consisted primarily of β-lactoglobulin, which was probably surrounded by a pectin coating at low pH values. AFM measurements indicated that the biopolymer nanoparticles formed were spheroid in shape. These biopolymer particles may be useful as delivery systems or fat mimetics.     

Impact of cosolvents on formation and properties of biopolymer nanoparticles formed by heat treatment of β-lactoglobulin–Pectin complexes

The purpose of this study was to determine the influence of neutral cosolvents on the formation and properties of biopolymer nanoparticles formed by thermal treatment of protein–polysaccharide electrostatic complexes. Biopolymer particles were formed by heating (85 °C, 20 min) an aqueous solution containing a globular protein (β-lactoglobulin) and an anionic polysaccharide (beet pectin) above the thermal denaturation temperature (T_m) of the protein under pH conditions where the biopolymers formed electrostatic complexes (pH 5). The impact of two neutral cosolvents (glycerol and sorbitol) on the self-association of β-lactoglobulin and on the formation of β-lactoglobulin–pectin complexes was examined as a function of solution pH (3–7) and temperature (30–95 °C). Glycerol had little impact on the pH-induced self-association or aggregation of the biopolymers, but it did increase the thermal aggregation temperature (T_a) of the protein–polysaccharide complexes, which was attributed to its ability to increase aqueous phase viscosity. Sorbitol decreased the pH where insoluble protein–polysaccharide complexes were formed, and greatly increased their T_a, which was attributed to its ability to increase T_m, alter biopolymer–biopolymer interactions, and increase aqueous phase viscosity. This study shows that neutral cosolvents can be used to modulate the properties of biopolymer nanoparticles prepared by thermal treatment of protein–polysaccharide electrostatic complexes.     

Formation of biopolymer particles by thermal treatment of β-lactoglobulin–pectin complexes

The purpose of this study was to prepare and characterize biopolymer particles based on thermal treatment of protein–polysaccharide electrostatic complexes formed from a globular protein (β-lactoglobulin) and an anionic polysaccharide (beet pectin). Initially, the optimum pH and pectin concentration for forming protein–polysaccharide complexes were established by mixing 0.5 wt% β-lactoglobulin solutions with beet pectin (0–0.5 wt%) at different pH values (3–7). Biopolymer complexes in the sub-micron size range (d = 100–300 nm) were formed at pH 5.0 and 0.1 wt% pectin. These particles were then subjected to a thermal treatment (30–90 °C at 0.8 °C min⁻¹). The presence of pectin increased the thermal aggregation temperature of the protein, although aggregate formation was still observed when the protein–polysaccharide systems were heated above about 70 °C. The impact of pH (3–7) on the properties of heat-treated biopolymer particles (83 °C, 15 min, pH 5) was then established. The biopolymer particles were stable to aggregation over a range of pH values, which increased as the amount of pectin was increased. The biopolymer particles prepared in this study may be useful for encapsulation and delivery of bioactive food components, or as substitutes for lipid droplets.     

Formation and dynamic interfacial adsorption of glycinin/chitosan soluble complex at acidic pH: Relationship to mixed emulsion stability

The formation and interfacial adsorption of glycinin/chitosan (CS) soluble complex were investigated at acidic pH. The stability of the mixed emulsion stabilized by the complex was also evaluated at pH 4.5. Turbidimetric analysis, isothermal titration calorimetry (ITC) and dynamic light scattering were used to characterize the dynamic formation of the complex. The results showed that soluble complexes were formed mainly at pHs between 4.0 and 6.0, depending on CS/Glycinin mixing ratio. At pH 4.5, soluble complex was formed and saturated at mixing ratio = 0.1, showing a maximum size distribution at 164.2 nm. We found that the glycinin/CS soluble complex showed improved interfacial adsorption than glycinin at pH 4.5. In detail, dynamic interfacial adsorption data showed the coefficient of diffusion (K_diff), unfolding (K₁) and rearrangement (K₂) for soluble complex (K_diff, K₁ and K₂: 0.58 mNm⁻¹ s^−0.5, 2.23 E−4 s⁻¹ and 5.78 E−4 s⁻¹) were higher than those of the glycinin (K_diff, K₁ and K₂: 0.32 mNm⁻¹ s^−0.5, 1.72 E−4 s⁻¹ and 4.63 E−4 s⁻¹). The droplet size and confocal observation of the mixed emulsion fabricated with glycinin/CS soluble complex displayed improved stability at mixing ratios of 0.1 to/and 0.2, suggesting the synergistic effect of the two molecules. We concluded that interfacial and emulsifying properties of glycinin could be improved by formation of glycinin/CS soluble complex at acidic pH.     

Thermal treatment to form a complex surface layer of sodium caseinate and gum arabic on oil–water interfaces

This study reported an alternative approach to electrostatic interaction to generate a biopolymer complex surface layer on emulsion droplets. By increasing the temperature, a complex surface layer of sodium caseinate (CN) and gum arabic (GA) on emulsion droplets could be formed either through the adsorption of complexes formed in the solution or through direct complexation of CN and GA with the surface. Mixtures of CN and GA were heated at pH 7 and then used to form oil-in-water emulsions at high temperatures. Changes in the average particle size and the ζ-potential of the emulsions indicated that complexes of GA and CN adsorbed to the interface at temperatures above 60 °C. A thick complex surface layer was also observed using confocal laser scanning microscopy. The addition of GA or CN to emulsions made with CN or GA resulted in an apparent binding of GA or CN to the emulsion droplets that depended on the sequence of addition. This temperature-induced formation of a complex surface layer was considered to be due to hydrophobically driven complexation between CN and the protein fraction of GA. The formation of the complex surface layer was dependent on the concentration, the temperature, and the addition order of the second component. This finding may imply an alternative option for the formation of biopolymer multilayers or complex surface layers on colloidal particles.