Layer-by-Layer Assembly of Food-Grade Alginate/Chitosan Nanolaminates: Formation and Physicochemical Characterization

The alternate deposition of oppositely charged materials (layer-by-layer technique) is an effective approach to functionalize materials. Biopolymer-based nanolaminates obtained by the layer-by-layer technique can also be used to change the surface properties of food products or food contact materials. However, the final properties of nanolaminates may be affected by the conditions of the adsorbing solutions. The objective of this study was to form and characterize the physicochemical properties of nanolaminates assembled from alginate and chitosan solutions. The effect of pH, ionic strength and polysaccharide concentration on the properties of the adsorbing solutions was also evaluated. The ζ-potential, viscosity and whiteness index of the solutions were assessed before the assembly. Alginate/chitosan nanolaminates were characterized in terms of UV-visible spectroscopy, surface ζ-potential, contact angle, DSC analysis and SEM. The absorbance increased as a function of the number of polysaccharide layers on the substrate, suggesting an increase in the mass adsorbed. The surface ζ-potential of nanolaminates changed depending on the last polysaccharide deposited. Alginate layers were negatively charged, whereas chitosan layers were positively charged. Contact angles obtained in alginate layers were ≈ 10°, being mostly hydrophilic. Chitosan layers showed higher contact angle values (80°), indicating a more hydrophobic behavior. Microscopic examinations revealed the presence densely packed structures that corresponded to alginate/chitosan nanolaminates, having an estimated thickness of 700 nm. The results obtained in this work lay the basis for the rational design of polysaccharide-based nanolaminates in the food sector.     

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.     

Composition and rheological properties of beta-Lactoglobulin/pectin coacervates: effects of salt concentration and initial protein/polysaccharide ratio

The composition and rheological properties of beta-lactoglobulin/pectin coacervates have shown significant correlations with sodium chloride concentration (C(NaCl)) and initial protein/polysaccharide ratio (r). An increase of C(NaCl) from 0.01 to 0.21 M at r = 5:1 leads to the increase in both beta-lactoglobulin and pectin contents in the coacervates, which can be explained in terms of salt-enhanced effect at lower salt concentrations. Further increase of C(NaCl) from 0.21 to 0.41 M decreases the proportions of these two biopolymers in the coacervates, exhibiting salt-reduced effect at higher salt concentrations. Moreover, the stronger self-aggregation of beta-lactoglobulin with increasing salt concentration gives rise to a decreasing actual protein/polysaccharide ratio in the coacervates at 0.01-0.21 M C(NaCl) and r = 5:1. An increase of r from 5:1 to 40:1 often increases the actual amount of pectin chains in beta-lactoglobulin/pectin coacervates, but it exhibits a maximum in beta-lactoglobulin content at r = 20:1. A much higher storage modulus (G') than loss modulus (G' ') for all beta-lactoglobulin/pectin coacervates suggests the formation of highly interconnected gel-like structure. The values of G' increase as C(NaCl) increases from 0.01 to 0.21 M, whereas a further increase of C(NaCl) from 0.21 to 0.41 M causes G' values to decrease to much lower values. These results further disclose the salt-enhanced effect and the salt-reduced effect at low and high salt concentrations, respectively. On the other hand, increasing r from 5:1 to 40:1 favors the formation of stronger gel-like beta-lactoglobulin/pectin coacervates, which mainly originates from the higher actual amount of pectin chains in beta-lactoglobulin/pectin coacervates at higher r values.     

Rheological and Physicochemical Studies on Emulsions Formulated with Chitosan Previously Dispersed in Aqueous Solutions of Lactic Acid

Chitosan, a natural, cationic polysaccharide, may be a hydrocolloid strategic to formulate acidic food products, as it can act as both bio-functional and technofunctional constituent. Typically, acetic acid is used to disperse chitosan in aqueous media, but the use of this acid is limited in food formulations due to its flavor. In this study, chitosan was firstly dispersed (0.1% m/V) in lactic acid aqueous solutions (pH 3.0, 3.5 or 4.0), and then evaluated regarding its thickener and emulsion stabilizer properties. O/W emulsions were prepared and characterized in terms of rheological properties, droplets average diameters and droplets ζ-potential. Emulsions containing chitosan were 3 times more viscous than controls without chitosan, and presented storage modulus (G’) higher than loss modulus (G”). Furthermore, they displayed two different populations of droplets (average diameters of 44 and 365 nm) and positive ζ-potential values (+50 mV). Droplets average diameters and ζ-potential did not present significant changes (p > 0.05) after storage at 25 °C during 7 days. This study showed that i) food organic acids other than acetic acetic acid can be used to disperse chitosan for technological purposes, and ii) chitosan dispersed at very low concentrations (0.1 m/V %) had relevant effects on rheological and physicochemical aspects of food-grade emulsions.     

Flocculation performance of a cationic biopolymer derived from a cellulosic source in mild aqueous solution

The flocculation behavior of cationic, quaternary ammonium groups containing cellulosic biopolymers, CDACs, synthesized by cationizing dialdehyde cellulose in mild aqueous solution was studied in a kaolin suspension. In particular, the role of CDAC dosage and solution pH, NaCl concentration, and temperature were clarified. In addition, the initial apparent charge densities (CDs), particle sizes, ζ-potential, and stability of CDs were determined. CDACs possessed a high flocculation activity in neutral and acidic solutions, but a significant decrease was observed in alkaline solutions (pH >9). This was also seen as a decline in the apparent CD and particle size of the CDACs in alkaline conditions. The measurements also indicated that the apparent CD decreased to a constant level of 3 mmol/g in aqueous solutions. However, no notable decrease in flocculation performance was obtained after several days of storage. Moreover, the variation of NaCl concentration and temperature did not affect the flocculation activity.     

Study of electrokinetic potential of drug micro-carriers prepared from resorbable polymers bioplastotan

The study investigates electrokinetic potentials of microparticles prepared from biodegradable poly-3-hydroxybutyrate depending upon the method of preparation employed and taking into account the size of particles maintained in liquid media. The ζ-potential of microparticles prepared from emulsion by solvent evaporation method was ?20 mV; the ζ-potential of microparticles prepared by spray drying was reduced to ?95 mV. The value of ζ-potential was influenced by drug loading into microparticles; the drug-loaded microparticles maintained in balanced phosphate buffer for 30 days had higher physical stability than those without drug loading.     

Self-healing fish gelatin/sodium montmorillonite biohybrid coacervates: structural and rheological characterization

Complex coacervation driven by associative electrostatic interactions was studied in mixtures of exfoliated sodium-montmorillonite (Na(+)-MMT) nanoplatelets and fish gelatin, at a specific mixing ratio and room temperature. Structural and viscoelastic properties of the coacervate phase were investigated as a function of pH by means of different complementary techniques. Independent of the technique used, the results consistently showed that there is an optimum pH value at which the coacervate phase shows the tightest structure with highest elasticity. The solid-like coacervates showed an obvious shear-thinning behavior and network fracture but immediately recovered back into their original elastic character upon removal of the shear strain. The nonlinear mechanical response characterized by single step stress relaxation experiments revealed the same trend for the yield stress and isochronal shear modulus of the coacervates as a function of pH with a maximum at pH 3.0 and lower values at 2.5 and 3.5 pHs, followed by a very sharp drop at pH 4.0. Finally, small-angle X-ray scattering (SAXS) data confirmed that at pHs lower than 4.0 the coacervate phases were dense and structured with a characteristic length scale (ξ(SAXS)) of ~7-9 nm. Comparing the ξ(SAXS) with rheological characteristic length (ξ(rheol)) estimated from low-frequency linear viscoelastic data and network theory, it was concluded that both the strength of the electrostatic interactions and the conformation of the gelatin chains before and during of the coacervation process are responsible for the structure and rigidity of the coacervates.     

Preparation and Characterization of Zein/Sodium Caseinate/Xanthan Gum Complex for Encapsulation of Piperine and its In Vitro Release Study

To encapsulate piperine (Pip), as a poor water-soluble bioactive compound, zein-sodium caseinate-xanthan gum (Z-SG-XG) nanocomplex was prepared as a colloidal delivery system. The effect of different parameters involved in complexation process, including concentration of proteins, polysaccharide, and Pip on the encapsulation efficiency of Pip, particle size and stability of the nanocomplexes was investigated. Powders obtained by freeze-drying of the colloidal solution had relatively uniform particles compared to those obtained from conventional drying system and showed well redispersibility in water. At the optimal condition, a stable and homogeneous nanocomplex with a mean particle size of 145.9 ± 2.7 nm, PDI of 0.27 ± 0.01, and ζ-potential of −39.7 ± 1.3 mV was obtained. The antioxidant activity of Pip was significantly improved by encapsulation into the Z-SC-XG nanocomplex. Also, the in vitro release of Pip from the synthesized nanocomplexes in phosphate-buffer saline (PBS) solution and simulated gastrointestinal fluids (SGIF) was investigated and the release kinetic was studied as well. The Pip/Z-SG-XG nanocomplex showed a slower release in SGIF compared to the free Pip and nanoparticles without XG and SC, while its antioxidant activity was remarkable. Results suggested a possible utilization of Z-SC-XG nanocomplex for improving the water solubility, bioavailability and storage stability of Pip.

     

Rheological interfacial properties of plant protein-arabic gum coacervates at the oil-water interface

This study concerns the interfacial properties of the plant proteins-arabic gum coacervates, which are involved in encapsulation processes based on complex coacervation. The results make it possible to deduce the prerequisite characteristics of the protein, which are involved in the coacervate interfacial properties. The influence of pH and concentration on protein interfacial properties was also studied so as to enable us to predict the best conditions to achieve encapsulation. It has been established that, to obtain a good encapsulation yield, the coacervate must show high surface-active properties and its adsorption on the oil droplets must be favored compared to the free protein adsorption. On the other hand, mechanical properties of the interfacial film made of the coacervate, appear to be a key parameter, as reflected by the dilational viscoelasticity measurements. When compared to the properties of the proteins films, an increase of the rigidity of the interfacial film was shown with the coacervates. It was also observed that viscoelastic properties of the coacervate film were strongly reduced, as well as the associated relaxation times. In acidic conditions, the coacervates containing alpha-gliadin are characterized by an interfacial viscoelastic behavior. This behavior reflects the softness of the interfacial film. This viscoelasticity allows also the formation of a continuous layer around the oil droplets to be encapsulated. Drop tensiometry is shown to be a method that could allow the most adapted protein to be selected and the conditions of the coacervation process to be optimized with regard to concentration and pH.     

Synthesis of silver nanoparticles by solar irradiation of cell-free Bacillus amyloliquefaciens extracts and AgNO3

Silver nanoparticles (AgNPs) were obtained by solar irradiation of cell-free extracts of Bacillus amyloliquefaciens and AgNO₃. Light intensity, extract concentration, and NaCl addition influenced the synthesis of AgNPs. Under optimized conditions (solar intensity 70,000 lx, extract concentration 3 mg/mL, and NaCl content 2 mM), 98.23 ± 0.06% of the Ag⁺ (1 mM) was reduced to AgNPs within 80 min, and the ζ-potential of AgNPs reached −70.84 ± 0.66 mV. TEM (Transmission electron microscopy) and XRD (X-ray diffraction) analysis confirmed that circular and triangular crystalline AgNPs with mean diameter of 14.6 nm were synthesized. Since heat-inactivated extracts also mediated the formation of AgNPs, enzymatic reactions are likely not involved in AgNPs formation. A high absolute ζ-potential value of the AgNPs, possibly caused by interaction with proteins likely explains the high stability of AgNPs suspensions. AgNPs showed antimicrobial activity against Bacillus subtilis and Escherichia coli in liquid and solid medium.     

Physicochemical Aspects of Chitosan Dispersibility in Acidic Aqueous Media: Effects of the Food Acid Counter-Anion

Differences in formation of colloidal dispersions of chitosan in aqueous solutions of citric acid or lactic acid (25, 50 or 100 mM) were quantitatively studied. Protonation enthalpies, electrical conductivity and ζ-potential measurements were additionally undertaken, aiming at better understanding these differences at a molecular level. In dispersion kinetics assays, experimental data were well fitted (R²?>?0.9; MAPE?<?4 %) by a first-order kinetics model with two terms - one accounting for the fast, direct dispersion of biopolymers chains and another accounting for the slow dispersion of chains from lumps. In all cases, maximal dispersibility was reached after about 20?30 min of stirring. For both acids, the higher the acid concentration in the medium, the higher was the chitosan dispersibility. At a given acid concentration, chitosan showed higher dispersibility in lactic acid than in citric acid solutions. Protonation of chitosan -NH₂ groups was strongly exothermic, with ΔH values three times higher for citric acid (triprotic) than lactic acid (monoprotic) (ΔH?=??120 kJ?mol^{- 1} and ΔH?=??40 kJ?mol^{- 1}, respectively), indicating that chitosan -NH₂ protonation itself was not dependent on the type of acid. However, the electrical conductivity of suspensions of powdered chitosan in water evolved differently as these systems were titrated with citric or acid lactic. With citric acid, electrical conductivity remained virtually constant for acid concentration?<?of 15 mM, and then increased linearly as the acid concentration increased until 75 mM. Instead, with lactic acid, electrical conductivity progressively increased with increasing of acid concentration from 0 to 75 mM. The ζ-potential of chitosan dispersed particles was +28.5 mV and +52.1 mV in dispersions containing 10 mM of citric and lactic acids, respectively. The conjoint analysis of data from physicochemical analyses suggested that, contrarily to lactate anions, citrate anions bind more strongly on the electrical double layer of protonated, positively charged chains of chitosan, diminishing the inter-chains electrostatic repulsion, thus leading to a lower dispersibility of this polysaccharide in aqueous solutions of citric acid, compared to equimolar solutions of lactic acid.     

Polyelectrolyte complexes of lactoferrin and pH-sensitive microparticles on their basis

Suspensions of insoluble polyelectrolyte complexes of dextran sulfate (DS) of different molecular masses with lactoferrin (LF) have been fabricated and characterized. The encapsulation efficiency of LF and DS in a complex at pH 3.0 and 4.0 was assessed, and particles were characterized by their sizes and ζ-potential. The complexes formed at pH 3.0 differed by a higher stability level. The interaction with DS resulted in a twofold decrease in the antioxidant activity of LF, although the formation of complexes was not accompanied by conformational changes in LF molecules according to IR-spectrometry data. Microencapsulation was carried out by treating the suspensions with negatively charged LF-DS complexes with protamine and chitosane solutions with different molecular masses. The composition, size, and the ζ-potential of interaction products were assessed which allowed us to select the conditions for the preparation of pH-sensitive polyelectrolyte microparticles loaded with LF which would be able to gradually release glycoprotein under conditions that model the passage through the gastrointestinal tract of humans. These data indicate that this approach is promising for the creation of pH-sensitive biopolyelectrolytes suitable for oral administration of LF to target cells.     

Partitioning and Enhanced Self-Assembly of Actin in Polypeptide Coacervates

Biomolecules exist and function in cellular microenvironments that control their spatial organization, local concentration, and biochemical reactivity. Due to the complexity of native cytoplasm, the development of artificial bioreactors and cellular mimics to compartmentalize, concentrate, and control the local physico-chemical properties is of great interest. Here, we employ self-assembling polypeptide coacervates to explore the partitioning of the ubiquitous cytoskeletal protein actin into liquid polymer-rich droplets. We find that actin spontaneously partitions into coacervate droplets and is enriched by up to ～30-fold. Actin polymerizes into micrometer-long filaments and, in contrast to the globular protein BSA, these filaments localize predominately to the droplet periphery. We observe up to a 50-fold enhancement in the actin filament assembly rate inside coacervate droplets, consistent with the enrichment of actin within the coacervate phase. Together these results suggest that coacervates can serve as a versatile platform in which to localize and enrich biomolecules to study their reactivity in physiological environments.     

ζ-Potential and surface charge of Thermoplasma acidophila

The surface charge density and the ζ-potential of Thermoplasma acidophila was estimated from microscopic electrophoresis experiments. The cells moved towards the positive electrode. The mobility remained constant from pH 2 to 5, and increased for pH values higher than 6. The mobility at pH 6 decreased dramatically with increased external Ca²⁺ concentration. At pH 2 and an ionic strength similar to that of the growth medium, the ζ-potential was about 8 mV, negative relative to the bulk medium; the surface charge density was 1360esu/cm^-2 which corresponds to one elementary charge per 3500 A².     

Interactions and compatibility of 11 S globulin from Vicia faba seeds and sodium salt of carboxymethylcellulose in an aqueous medium

This work studies specific interactions and compatibility between a legumin and a linear carboxylated polysaccharide using gel permeation chromatography, sedimentation analysis, SDS gel electrophoresis, viscometry and phase analysis measurements. It uses the system water/11 S globulin/CMC as a model. Carboxymethylcellulose (CMC) molecules are able to cause a partial dissociation of the protein, subsequent formation of soluble interbiopolymeric complexes and partial aggregation of the free non combined protein at room temperature and pH 6.0-6.5. The maximal binding of biopolymers is observed at their equimolar ratio. The decrease in temperature of the mixture from 293 to 277 K leads to formation of the complex coacervate. The increase in pH from 6.0 to 7.6 results in suppression of complex formation and manifestation of the phenomenon of thermodynamic incompatibility if the total concentration of biopolymers in the system exceeds the critical concentration of segregative phase separation.     

Uptake of Fluorescent Iron Oxide Nanoparticles by Oligodendroglial OLN-93 Cells

To investigate the cellular accumulation and intracellular localization of dimercaptosuccinate-coated iron oxide nanoparticles (D-IONPs) in oligodendroglial cells, we have synthesized IONPs that contain the fluorescent dye BODIPY (BP) in their coat (BP-D-IONPs) and have investigated the potential effects of the absence or presence of this dye on the particle uptake by oligodendroglial OLN-93 cells. Fluorescent BP-D-IONPs and non-fluorescent D-IONPs had similar hydrodynamic diameters and ζ-potentials of around 60 nm and ?58 mV, respectively, and showed identical colloidal stability in physiological media with increasing particle size and positivation of the ζ-potential in presence of serum. After exposure of oligodendroglial OLN-93 cells to BP-D-IONPs or D-IONPs in the absence of serum, the specific cellular iron content increased strongly to around 1,800 nmol/mg. This strong iron accumulation was lowered for both types of IONPs by around 50 % on exposure of the cells at 4 °C and by around 90 % on incubation in presence of 10 % serum. The accumulation of both D-IONPs and BP-D-IONPs in the absence of serum was not affected by endocytosis inhibitors, whereas in the presence of serum inhibitors of clathrin-dependent endocytosis lowered the particle accumulation by around 50 %. These data demonstrate that oligodendroglial cells efficiently accumulate IONPs by an endocytotic process which is strongly affected by the temperature and the presence of serum and that BP-D-IONPs are a reliable tool to monitor by fluorescence microscopy the uptake and cellular fate of D-IONPs.     

Interaction of N-acylated and N-alkylated chitosans included in liposomes with lipopolysaccharide of gram-negative bacteria

The interactions of lipopolysaccharide (LPS) with the polycation chitosan and its derivatives — high molecular weight chitosans (300 kDa) with different degree of N-alkylation, its quaternized derivatives, N-monoacylated low molecular weight chitosans (5.5 kDa) — entrapped in anionic liposomes were studied. It was found that the addition of chitosans changes the surface potential and size of negatively charged liposomes, the magnitudes of which depend on the chitosan concentration. Acylated low molecular weight chitosan interacts with liposomes most effectively. The binding of alkylated high molecular weight chitosan with liposomes increases with the degree of its alkylation. The analysis of interaction of LPS with chitoliposomes has shown that LPS-binding activity decreased in the following order: liposomes coated with a hydrophobic chitosan derivatives > coated with chitosan > free liposomes. Liposomes with N-acylated low molecular weight chitosan bind LPS more effectively than liposomes coated with N-alkylated high molecular weight chitosans. The increase in positive charge on the molecules of N-alkylated high molecular weight chitosans at the cost of quaternization does not lead to useful increase in efficiency of binding chitosan with LPS. It was found that increase in LPS concentration leads to a change in surface ζ-potential of liposomes, an increase in average hydrodynamic diameter, and polydispersity of liposomes coated with N-acylated low molecular weight chitosan. The affinity of the interaction of LPS with a liposomal form of N-acylated chitosan increases in comparison with free liposomes. Computer simulation showed that the modification of the lipid bilayer of liposomes with N-acylated low molecular weight chitosan increases the binding of lipopolysaccharide without an O-specific polysaccharide with liposomes due to the formation of additional hydrogen and ionic bonds between the molecules of chitosan and LPS.     

Functional properties of hemoglobin immobilized in coacervates prepared from gelatin A and polyanionic carbohydrates

Complex coacervation is a phenomenon of phase separation that may occur in a solution of positively and negatively charged polyions. The resulting two phases are distinguished by the total concentration of both polyions, with the concentrated phase often containing vesicular structures composed of the two polyelectrolytes. We have used this phenomenon in an attempt to-prepare a hemoglobin-based red blood cell analog. Hemoglobin-containing coacervate vesicles have been prepared from gelatin A and the polyanionic carbohydrates acacia, pectin, or dextranstilfate. Hemoglobin seems to be anchored into the vesicle walls through interaction of its polyanion binding site with the negatively charged residues on the carbohydrates. Oxygen binding by the immobilized HbA is reversible and cooperative, with p50 values at 20 degrees C of 2.8, 6, and 24 mm Hg for the acacia- (pH 7.5), pectin- (pH 6.6), and dextransulfate-(pH 6.6) derived coacervates. Kinetic studies on CO binding show that the rate of CO uptake by the coacervates (t((1/2)) = 13-27 ms at 0.5 mM CO) is similar to that of human erythrocytes.The HbA-containing coacervates slowly dissolve in isotonic salt solutions (145 mM NaCl, pH 7.4), but they can be stabilized by treatment with glutaraldehyde. Oxygen binding by HbA incorporated into the stabilized coacervates derived from dextran sulfate is very similar to oxy gen binding by human red blood cells: p50 = 26 mm Hg and n = 1.89 at 37 degrees C in isotonic salt. These results show how a novel approach, based on an old concept, has led to the preparation of immobilized HbA, with functional properties similar to those of intraerythrocytic HbA.     

Gum arabic-chitosan complex coacervation

The formation of electrostatic complexes of gum Arabic (GA) with chitosan (Ch), two oppositely charged polysaccharides, as a function of the biopolymers ratio (RGA/Ch), total biopolymers concentration (TBconc), pH, and ionic strength, was investigated. The conditions under which inter-biopolymer complexes form were determined by using turbidimetric and electrophoretic mobility measurements in the equilibrium phase and by quantifying mass in the precipitated phase. Results indicated that optimum coacervate yield was achieved at RGA/Ch = 5, independently of TBconc at the resulting pH of solutions under mixing conditions. High coacervate yields occurred in a pH range from 3.5 to 5.0 for RGA/Ch = 5. Coacervate yield was drastically diminished at pH values below 3.5 due to a low degree of ionization of GA molecules, and at pH values above 5 due to a low solubility of chitosan. Increasing ionic strength decreased coacervate yield due to shielding of ionized groups.