Remco Tuinier

Polysaccharide protein interactions

Abstract The interaction between proteins and polysaccharides, as can be observed in food related systems, is systematically discussed by separating biopolymer interactions into respectively enthalpy- and entropy-dominated types. We present examples of typically enthalpy driven phase separations, such as biopolymer incompatibility, described by the classical Flory-Huggins theory. This behavior is for instance observed for the system gelatin–dextran. In mixed systems where excluded volume or depletion interaction plays an important role the phase separation is mainly driven by entropy. Here the interaction of (random coil) polysaccharides and protein (covered) particles such as casein micelles and emulsion droplets in dairy systems may lead to phase separation as well.

Stability of casein micelles in milk

Casein micelles in milk are proteinaceous colloidal particles and are essential for the production of flocculated and gelled products such as yogurt, cheese, and ice-cream. The colloidal stability of casein micelles is described here by a calculation of the pair potential, containing the essential contributions of brush repulsion, electrostatic repulsion, and van der Waals attraction. The parameters required are taken from the literature. The results are expressed by the second osmotic virial coefficient and are quite consistent with experimental findings. It appears that the stability is mainly attributable to a steric layer of κ-casein, which can be described as a salted polyelectrolyte brush.

Substructure of bovine casein micelles by small-angle x-ray and neutron scattering

The casein micelles of cow’s milk are polydisperse, more-or-less spherical, protein particles of up to several hundred nanometer in size, containing about 7% by dry weight of calcium phosphate. Small-angle neutron scattering with contrast variation and small-angle X-ray scattering were used in critical tests of models of casein micelle substructure. An inflexion in the neutron scattering curve near Q � /0.35 nm � 1 was observed in heavy water which became a more pronounced subsidiary maximum at the match point of the protein. In water-rich buffers, where the contrast between protein and calcium phosphate is small, the inflexion was less apparent. The position of the inflexion and its variation in shape and relative importance with contrast matching are explained poorly, if at all, by the submicelle models of casein micelle substructure. However, the observations are explained by a model in which a relatively uniform protein matrix contains a disordered array of calcium phosphate ion clusters. A notable achievement of the model is the prediction of the position of the subsidiary maximum from independent measurements of the intrinsic viscosity of micelles, their mass fraction of calcium phosphate and the mass of the core of a calcium phosphate nanocluster. # 2002 Elsevier Science B.V. All rights reserved.

Role of exopolysaccharides produced by Lactococcus lactis subsp. cremoris on the viscosity of fermented milks

Lactic acid bacteria strains that produce exopolysaccharide (EPS) are employed in the manufacture of fermented milk to improve its texture and viscosity. No clear correlation has been found between EPS concentration and the viscosity of the fermented milk. It is supposed that the molecular characteristics, i.e. radius of gyration and molar mass, of these biopolymers play a part. The subject of this study was to clarify whether the viscosifying properties of EPS in aqueous solution can be related to the viscosity of fermented milk products. Therefore, we aimed at relating the molecular characteristics of EPSs produced by several strains of Lactococcus lactis subsp. cremoris in milk at three incubation temperatures to the viscosity of the fermented products. For a given EPS concentration, a positive correlation between the EPS-viscosifying parameters (intrinsic viscosity, Kuhn segments length and thickening efficiency) and the Posthumus viscosity of fermented milks was found.

Exopolysaccharides produced by Lactococcus lactis: from genetic engineering to improved rheological properties?

Over the last years, important advances have been made in the study of the production of exopolysaccharides (EPS) by several lactic acid bacteria, including Lactococcus lactis. From different EPS-producing lactococcal strains the specific eps gene clusters have been characterised. They contain eps genes, which are involved in EPS repeating unit synthesis, export, polymerisation, and chain length determination. The function of the glycosyltransferase genes has been established and the availability of these genes opened the way to EPS engineering. In addition to the eps genes, biosynthesis of EPS requires a number of housekeeping genes that are involved in the metabolic pathways leading to the EPS-building blocks, the nucleotide sugars. The identification and characterisation of several of these housekeeping genes (galE, galU, rfbABCD) allows the design of metabolic engineering strategies that should lead to increased EPS production levels by L. lactis. Finally, model developme nt has been initiated in order to predict the physicochemical consequences of the addition of a EPS to a product.

A simple patchy colloid model for the phase behavior of lysozyme dispersions

We propose a minimal model for spherical proteins with aeolotopic pair interactions to describe the equilibrium phase behavior of lysozyme. The repulsive screened Coulomb interactions between the particles are taken into account assuming that the net charges are smeared out homogeneously over the spherical protein surfaces. We incorporate attractive surface patches, with the interactions between patches on different spheres modeled by an attractive Yukawa potential. The parameters entering the attractive Yukawa potential part are determined using information on the experimentally accessed gas-liquid-like critical point. The Helmholtz free energy of the fluid and solid phases is calculated using second-order thermodynamic perturbation theory. Our predictions for the solubility curve are in fair agreement with experimental data. In addition, we present new experimental data for the gas-liquid coexistence curves at various salt concentrations and compare these with our model calculations. In agreement with earlier findings, we observe that the strength and the range of the attractive potential part only weakly depend on the salt content.

Depletion interaction between spheres immersed in a solution of ideal polymer chains

The depletion interaction between two spheres due to nonadsorbing ideal polymers is calculated from the polymer concentration profile using the excess (negative) adsorption. Computer simulations show that the polymer concentration profiles around two spheres are well described by the product function of the concentration profile around a single sphere. From the interaction potential between two spheres the second osmotic virial coefficient, B2 , is calculated for various polymer-colloid size ratios. We find that when the polymers become smaller than the spheres, B2 remains positive in the dilute regime. This shows that the depletion interaction is ineffective for relatively small spheres.

Isolation and physical characterization of an exocellular polysaccharide.

The physical properties of a polysaccharide produced by the lactic acid bacterium Lactococcus lactis subsp. cremoris strain NIZO B40 were investigated. Separation of the polysaccharide from most low molar mass compounds in the culture broth was performed by filtration processes. Residual proteins and peptides were removed by washing with a mixture of formic acid, ethanol, and water. Gel permeation chromatography (GPC) was used to size fractionate the polysaccharide. Fractions were analyzed by multiangle static light scattering in aqueous 0.10 M NaNO3 solutions from which a number- (Mn) and weight-averaged (Mw) molar mass of (1.47 +/- 0.06).10(3) and (1.62 +/- 0.07).10(3) kg/mol, respectively, were calculated so that Mw/Mn approximately 1.13. The number-averaged radius of gyration was found to be 86 +/- 2 nm. From dynamic light scattering an apparent z-averaged diffusion coefficient was obtained. Upon correcting for the contributions from intramolecular modes by extrapolating to zero wave vector a hydrodynamic radius of 86 +/- 4 nm was calculated. Theoretical models for random coil polymers show that this z-averaged hydrodynamic radius is consistent with the z-averaged radius of gyration, 97 +/- 3 nm, as found with GPC.

Phase behaviour of mixtures of colloidal spheres and excluded-volume polymer chains

We study the phase behaviour of mixtures of colloidal spheres and polymers that have an excluded-volume interaction dispersed in a (background) solvent using the concept of free volume theory. The depletion layer thickness is calculated from the negative adsorption of polymer segments around a sphere. The correlation length and thermodynamic properties of the excluded-volume interacting polymer chains in solution are taken into account by using results from the renormalization group theory. For small polymer–colloid size ratios the difference from an ideal description of the polymers is small, while for larger size ratios the gas–liquid coexistence region shifts in the direction of higher polymer concentrations and at the same time the liquid–crystal coexistence region becomes more extended. Both the gas–liquid region and the gas– liquid–crystal region become less extended. These features are compared to experiment.

The effect of depolymerised guar gum on the stability of skim milk

We studied the interactions between casein micelles and (depolymerised) guar gum, degraded by heating its aqueous solutions at low pH. The guar samples were characterised by a combination of size-exclusion chromatography and light scattering, which yielded the distribution of molar mass and corresponding radius of gyration. The relation between molar mass and radius of gyration showed that guar can be described as a random coil. Mixing guar gum with casein micelles led to a phase separation. This phase separation originates from a depletion interaction leading to an effective attraction between the casein micelles by non-adsorbing guar. The polymer concentration at the phase boundary increases with decreasing guar chain length. Vrijs theory for depletion interaction was used to calculate the phase boundary from the size of the casein micelles and the size and molar mass of the guar samples. The theory of Vrij predicts the trends for the phase boundary with respect to the molar mass dependence as found experimentally.