Kappa-Carrageenan : a study on its physico-chemical properties, sol-gel transition and interaction with milk proteins

In this thesis a characterization of the milkreactive polysaccharide κ-carrageenan by means of light scattering, viscosimetry and sedimentation experiments is presented. The disorder/order transitions occurring at the gelpoint of these substances are discussed in terms of a coil/double helix transition. Further the so-called milk reactivity, i.e. its ability to stabilize dairy products and the influence of molecular weight on its stabilizing properties are studied.In Chapter I a short description is given of the different types of carrageenans known and of the casein components, the main protein constituents of milk.In Chapter 11 samples of κ-carrageenan varying in molecular weights from 17500 up to 836000 are studied by different physical methods such as light scattering, viscosimetry and ultracentrifugation. It has been found that in aqueous solutions κ-carrageenan molecules can best be described as expanded coils. This expansion is due to long-range interactions such as excluded volume effects and electrostatic interactions. The expansion varies with the ionic strength as might be expected for polyelectrolytes with partly screened electrical charges.In the unperturbed state, the κ-carrageenan molecule can be described as a flexible coil with a statistical segment length of 0.83 rim. From intrinsic viscosity and light scattering data the Flory-Fox viscosity constant Φcalculated for κ-carrageenan solutions is found to be 0.45 x 10 21-0.75 x 10 21at an ionic strength of 0.1175, which is considerably lower than in the case of uncharged polymers in good solvents (2.5 x 10 21).It is further demonstrated in Chapter II that the expressions for molecular parameters such as radius of gyration, intrinsic viscosity and sedimentation coefficient are severely influenced by the polydispersity of the κ-carrageenan samples, which is corrected for by the introduction of a Schulz-Zimm distribution function.The disorder-order transitions taking place at the gelpoint of κ-carrageenan solutions are studied by optical rotation and light scattering (Chapter III). The coincidence of both sets of experimental data affords good evidence that the sol-gel transition is accompanied by a conformational change. Such conformational changes are also observed in solutions of ι-carrageenan but not in those of λ-carrageenan. In solutions of κ- and ι-carrageenan the transition temperatures are linearly dependent on the logarithm of the salt concentration which is explained by the formation of double helices at the gelpoint.The midpoint temperature and the abruptness of the transitions as observed by optical rotation decrease with decreasing molecular weight of the κ-carrageenan samples.Heats of gelation are measured by differential scanning calorimetry. For κ-carrageenan the enthalpy increases with ionic strength, which has been ascribed to the occurrence of a secondary process in which double helices are assembled into larger aggregates. The enthalpy changes observed for the coildouble helix transition of ι-carrageenan are similar to that observed with κ-carrageenan.In Chapter IV it is shown that the milk reactivity of carrageenan is due to an electrostatic interaction between carrageenan and κ-casein. The specificity of κ-casein for such interactions has been ascribed to a cluster of positive electrical charges in its amino acid sequence, which is absent in the other main components of casein i.e. α s 1 and β-casein. This electrostatic complex formation with κ-casein takes place with all types of carrageenan and is maximal at an ionic strength of about 0.2.The setting point temperatures observed in mixtures of κ- or ι-carrageenan with κ-casein are comparable to those of the corresponding pure carrageenan solutions. This demonstrates that the sol-gel transition in carrageenan-κ-casein mixtures is due to the interactions of free carrageenan loops or tails proper and that κ-casein is not involved in this process. The fact that pure λ-carrageenan does not gel also offers an explanation for its lack of milk reactivity.In the Appendix to Chapter IV it is demonstrated that besides κ-casein also the hitherto less well-known α s3 - and α s4 -caseins interact electrostatically with x- carrageenan. By electrostatic affinity chromatography using κ-carrageenan as column material the total content of κ-, α s3 - and α s4 -casein isolated has been found to be about 20% of the amount of whole casein.Since gel formation in κ-carrageenan-milk systems is primarily due to interaction of κ-carrageenan chains, the chain length (and thus the molecular weight) is an important parameter with the milk reactivity. In Chapter V the relationship between milk reactivity and molecular weight of the carrageenan has been investigated. The results offer explanations for the differing retarding influence of high molecular weight κ-carrageenan (< M >w >10 5) on the sedimentation of cocoa particles in chocolate milk and the creaming of fat globules in evaporated milk (and for the ineffectiveness in this respect of low molecular weight κ-carrageenan (< M >w >10 5)

Calcium-binding capacity of organic and inorganic ortho- and polyphosphates

The aim of this research was to determine the calcium-binding capacity of inorganic and organic ortho- and polyphosphates. This calcium-binding capacity can be used to influence the stability of, for example, casein micelles in dairy systems. Four phosphates were selected: disodium uridine monophosphate (Na2UMP, organic orthophosphate), disodium hydrogen phosphate (Na2HPO4, inorganic orthophosphate), sodium phytate (SP, organic polyphosphate), and sodium hexametaphosphate (SHMP, inorganic polyphosphate). Concentrations of up to 100 mmolL-1 phosphate were added to a 50 mmolL-1 CaCl2 solution. The samples were prepared at pH 8.0 and were analyzed before and after sterilization for calcium-ion activity, conductivity, pH, sediment, and turbidity. Both SHMP and SP are strong chelators, as calcium ions bind to these phosphates in the ratio of 3:1 and 6:1, respectively. Calcium ions also strongly bind to Na2HPO4, but in a ratio of 3:2 with insoluble Ca3(PO4)2 complexes as result. The equilibrium position of Na2UMP is not strong towards the chelated complex, and significant levels of free calcium and free phosphate can exist. An equilibrium constant of Lmol-1 was determined for calcium uridine monophosphate (CaUMP) complexes. Both calculation of the equilibrium constant and analysis on the CaUMP precipitate confirmed a reactivity of 1:1 between calcium and Na2UMP. The CaUMP complexes are well soluble at ambient temperature, and insoluble complexes appear after sterilization, because the solubility of CaUMP decreases during heating. Finally, we concluded that the structure of phosphate molecules determines their calcium-binding capacity rather than organic or inorganic origin of phosphate

Effect of calcium chelators on physical changes in casein micelles in concentrated micellar casein solutions

The effect of calcium chelators on physical changes of casein micelles in concentrated micellar casein solutions was investigated by measuring calcium-ion activity, viscosity and turbidity, and performing ultracentrifugation. The highest viscosities were measured on addition of sodium hexametaphosphate (SHMP), because it cross-linked the caseins. For the weak calcium chelator disodium uridine monophosphate (Na2UMP), physical changes in the solutions were negligible. Disodium hydrogen phosphate (Na2HPO4), trisodium citrate (TSC), and sodium phytate (SP) caused similar increases in viscosity, but had different effects on turbidity. The increase in viscosity was attributed to swelling of the casein micelles (i.e., increased voluminosity) at decreasing calcium-ion activity. The major decrease in turbidity was due to dissociation of the casein micelles. The extent of micellar dissociation was dependent on the type and concentration of calcium chelator. It seems that the micelles were dissociated in the order of SHMP = SP > TSC > Na2HPO4 > Na2UM

Effect of calcium chelators on physical changes in casein micelles in concentrated micellar casein solutions

The effect of calcium chelators on physical changes of casein micelles in concentrated micellar casein solutions was investigated by measuring calcium-ion activity, viscosity and turbidity, and performing ultracentrifugation. The highest viscosities were measured on addition of sodium hexametaphosphate (SHMP), because it cross-linked the caseins. For the weak calcium chelator disodium uridine monophosphate (Na2UMP), physical changes in the solutions were negligible. Disodium hydrogen phosphate (Na2HPO4), trisodium citrate (TSC), and sodium phytate (SP) caused similar increases in viscosity, but had different effects on turbidity. The increase in viscosity was attributed to swelling of the casein micelles (i.e., increased voluminosity) at decreasing calcium-ion activity. The major decrease in turbidity was due to dissociation of the casein micelles. The extent of micellar dissociation was dependent on the type and concentration of calcium chelator. It seems that the micelles were dissociated in the order of SHMP = SP > TSC > Na2HPO4 > Na2UM