An exocellular polysaccharide and its interactions with proteins
In the food industry polysaccharides are used as thickening or gelling agents. Polysaccharides are usually extracted from plants. Micro-organisms are also capable of excreting polysaccharides: exocellular polysaccharides (EPSs). In some cases EPSs are produced in-situ in food products, notably in acidified milk products. These EPSs function effectively as food thickeners but do not need to be declared in the food label.Systematic physical analysis of an exocellular polysaccharide produced by a lactic acid bacterium has hardly been performed until now. In order to obtain a better understanding of the role of EPS in (acidified) milk products the physical properties of an EPS from the lactic acid bacterium strain Lactococcus lactis subsp. cremoris B40 were studied (Chapters 2-4) as well as its interactions with milk proteins (Chapters 5-8). The ionic strength of the EPS solutions was always set at 0.10 M, about the ionic strength in milk.In Chapter 2 the isolation, purification and analysis of the molecular properties of EPS from L. lactis B40, our 'model' EPS, are investigated. The polysaccharide was separated from most low molar mass compounds in the culture broth by filtration processes. Gel permeation chromatography (GPC) was used to size-fractionate the polysaccharide. Fractions were analyzed by multi-angle static light scattering in aqueous solutions from which a number- (M n ) and weight-averaged (M w ) molar mass of (1.47 ± 0.06)·10 3and (1.62 ± 0.07)·10 3kg/mol, respectively, were calculated so that M w /M n1.13. The number-averaged radius of gyration was found to be 86 ± 2 nm. The hydrodynamic radius as determined from dynamic light scattering was consistent with the radius of gyration.The viscosity of the EPS solutions was studied in simple shear flow as described in Chapter 3. Firstly, the zero-shear viscosity was determined as a function of the concentration. The intrinsic viscosity was determined from the data in the low concentration range. The intrinsic viscosity and the concentration dependence of the (zero-shear) viscosity of the B40 EPS could be predicted from the molar mass and the hydrodynamic radius. In addition the shear-thinning behavior was measured at several concentrations. The shear rate at which the viscosity starts to decrease scales with polymer concentration in accordance with the Rouse theory. By combining existing theories (Rouse and Bueche) it is possible to predict the intrinsic viscosity, concentration dependence of the viscosity, and shear-thinning behavior in terms of the molar mass and the hydrodynamic radius.The measurements and theoretical description of the dynamic rheological properties of the EPS are presented in Chapter 4. Dynamic rheological measurements were performed as a function of frequency and EPS concentration. The dynamic properties could be described by the bead-spring model of Rouse. Concentrated EPS solutions have a significant elasticity at high concentrations and high frequencies, which is indicative of the presence of significant normal stress differences. It is suggested that these normal stresses may explain the contribution of the EPSs to the ropy behavior of yogurts.Having characterized the EPS in aqueous solution, its interaction with the most relevant colloidal (protein) particles present in milk products was studied. As the polysaccharide studied in this thesis occurs in dairy products our focus was on the interactions and phase behavior of EPS with the colloidal components in milk. There are three distinctly different types of particles in the colloidal size range in milk: fat globules, casein micelles and whey proteins. Smaller molecular species (over 100,000 in milk) are considered as part of the continuous phase.In Chapter 5 the interactions with whey proteins are described. Native whey proteins and EPS were co-soluble; they could be mixed in all proportions. However, an effective attraction (a depletion interaction) is induced between aggregated-whey-protein colloid (AWC) particles when they are mixed with the EPS. This depletion interaction originates from a loss of conformational entropy of the EPSs near the surface of neighboring AWC particles and leads to a phase separation at high enough EPS and/or AWC concentrations. The effect of the depletion interaction on the properties of the mixtures of EPS and AWC particles was first studied in the stable, i.e. one-phase region. The strength of attractions was characterized by small-angle neutron scattering (SANS) and dynamic light scattering (DLS). The SANS results could be described quantitatively by the Vrij theory and integral theory (Ornstein-Zernike with HNC closure) in combination with the Schaink-Smit theory and allowed a determination of the position of the spinodal. The DLS results could be described reasonably well by using a theory of Dhont and Kawasaki.Furthermore, the experimental phase boundary was determined and compared with the Schaink-Smit theory, a mean-field theory which evaluates the free energy of a mixture of colloids and large non-adsorbing polymers. The spinodal so calculated was found to be consistent with the experimentally determined position of the phase boundary.Spinodal phase separation kinetics was investigated by small-angle light scattering (SALS). At low Q a scattering peak was detected which shifted to lower Q's with time, in agreement with other experimental data and theoretical predictions for spinodal decomposition. Both the scaling of the scattered intensity with Q and the scaling of the Q-position of the peak with time agree with theoretical predictions of Furukawa and Siggia.The interactions between EPS B40 and casein micelles are treated in in Chapter 6. Casein micelles become mutually attractive when the EPS is added to skim milk. The attraction can be explained as a depletion interaction between the casein micelles induced by the non-adsorbing EPS. We used three scattering techniques (SANS, turbidity measurements and DLS) to measure the attraction. The Vrij theory in combination with integral theory and all the experiments showed that casein micelles became more attractive upon increasing the EPS concentration.The phase separation arising from depletion interaction in mixtures of casein micelles and EPS is described in Chapter 7. We have determined a phase diagram that describes the separation of skim milk with EPS into a casein-micelle-rich phase and an EPS-rich phase. We compared the phase diagrams with those calculated from theories developed by Vrij, and by Lekkerkerker and co-workers, showing that the experimental phase boundary can be predicted quite well. From measurements of the self-diffusion of the casein micelles in the presence of EPS the spinodal was calculated, which corresponds to the visual observations.The effect of adding the EPS to an oil-in-water emulsion, stabilized with whey proteins, is reported in Chapter 8. Even at low EPS concentrations the emulsion phase separates. The phase line could be described by depletion interaction theory of Vrij. At high EPS concentrations and dispersed phase volume fractions above 10% we found a stable 'gel'-like region in the phase diagram. In that region the oil droplets attract one another so strongly that a space-filling network is formed at sufficient oil volume fractions.A kinetic study showed that the rate of creaming/demixing decreases with volume fraction of oil of the system (hydrodynamics) and strongly depends on the concentration of EPS (strength of depletion interaction and continuous-phase viscosity). At low EPS concentration the creaming rate strongly increased with EPS concentration since attractions enhance creaming. At higher EPS concentrations creaming was slowed down by the viscosity increase of the continuous phase and the particle network which was created. This network became so strong at high EPS concentrations that creaming was absent in the 'gel' region. The rheological behavior of the 'gel' was studied by measuring flow curves which could be interpreted by the Potanin model, which describes the rheology of a dispersion of weakly aggregating particles.In Chapter 9 the practical implications of this work are described. In order to understand the thickening effect of EPSs the molar mass, radius of gyration, and their interrelation are very important. It is indicated how the effectivity of a polysaccharide can be analyzed on the basis of the molar mass and the radius of gyration. The relation between the radius of gyration and the molar mass depends on the kind of monosaccharide residues, the linkage type, and the solvent. Further it is addressed how a fundamental understanding of the interactions between polysaccharides and proteins leads to predictions of the phase line and interpretation of the measured phase behavior. The unwanted effect of phase separation can then be suppressed by using only biopolymer concentrations at which the system is still stable. An understanding of the biopolymer interactions may thus make it possible to adjust the properties of food dispersions. Finally, some suggestions for further research are given.
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| Format: | Doctoral thesis biblioteca |
| Language: | English |
| Subjects: | lactococcus lactis subsp. cremoris, physical properties, polysaccharides, fysische eigenschappen, polysacchariden, |
| Online Access: | https://research.wur.nl/en/publications/an-exocellular-polysaccharide-and-its-interactions-with-proteins |
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