ORGANIZACIÓN Y FUNCIONES

Certain pectin structures (LM or HM blocky pectin) are highly sensitive to calcium and it has recently been shown that low DM pectins can be gelled with serum extracted from milk at increasing stages of acidification (Harte et al., 2007). In the literature, there are different milk protein systems in which the interaction of pectin with calcium has been eliminated as a complicating factor, allowing the investigation of the interaction of pectin and casein directly: these are using (1) acid sodium caseinate gel in presence of pectin, (2) oil in water emulsions stabilized by sodium caseinate and (3) pectin-casein mixtures.

4.3.1 Acidified sodium caseinate gels with pectin

In the presence of LM and HM pectin (1% for 2.5% sodium caseinate), proteins aggregates, obtained by breaking and homogenizing the primary acid sodium caseinate gel, are smaller; both LM and HM pectin fine structures are thus inhibiting the re-association of the protein network. However, sedimentation is observed after centrifugation for LM pectin and not HM pectin at low pH (3.8 and 4.8): HM pectin stabilized more effectively sodium caseinate dispersion. But it is worth mentioning that both, the degree of esterification and molecular weight were different for the two pectins investigated (Pereyra et al., 1997).

The addition of LMA pectin (0-1%w/v) to sodium caseinate (2%w/v) before acidification changed the evolution of the gel strength with pH (Matia- Merino et al., 2004). Indeed, without pectin, G’ during acidification reaches a maximum and decreased to a lower plateau: below pH 4.6, the proteins are negatively charged and the repulsive interactions weaken the network. When pectin is added (at 0.05% w/v or above), the maximum after the gelation point (taken as time at which G’ equal G’’) is not observed anymore: the repulsive interactions seem to be reduced. Furthermore, as the pectin concentration is increased, the final gel strength decreased and above 0.8%, the system doesn’t gel anymore. Calcium has been added to the same system with the effect that at low pectin concentration (below 0.2% w/v), caseinate gel with and without

42 calcium have similar rheological profile but at higher polymer concentration (0.2%w/v and above), syneresis with gel shrinkage is observed in the presence of calcium.

4.3.2 Thermodynamic compatibility of pectin / casein

mixtures

Four different regimes are observed on mixing protein with an anionic polysaccharide as a function of the pH and ionic strength (Weinbreck et al., 2003, de Kruif et al., 2004):

At pH above the isoelectric point of the protein and the polysaccharide and at low ionic strength, protein and polysaccharides are both negatively charged and cosoluble.

At pH close or below the isoelectric point of the protein and above the isoelectric point of the polysaccharide, the formation of soluble protein/polysaccharides complexes occurred.

By lowering the pH, the amount of positive net charge on the protein and negative charge on the polysaccharide are becoming similar. The soluble complexes could aggregate and form complex coacervates.

At pH values below the pKa of the polysaccharide, the acidic groups on the polysaccharides are less charged and the complexes can be dissolved.

However, pectin-caseinate mixtures as polysaccharide-protein mixtures are unstable and phase separation occurs above a certain polymer concentration. Depending on the pH, two different interactions lead to phase separation: repulsive (segregative) or attractive (associative) interactions (Tolztoguzov, 1991, Schmitt et al., 1998, Turgeon et al., 2003, Redigueri, 2007). At pH 6.8, pectin and caseinate charges are of opposite sign. Pectin doesn’t adsorb to the caseinate, and the non-absorbing polymer is depleted from the surrounding of the colloids. This depletion by segregation leads to phase separation (Rediguieri et al., 2007). The thermodynamic compatibility increases with increasing the pH from 6 to 8 (Einhorn-Stoll et al., 2001). This has been explained by an increasing of the repulsion force between the proteins

43 themselves and unfolding of these proteins which give more sites for weak interaction between pectin and caseinate involving opposites charged patches (Einhron-Stroll, 2001). Below pH 5.5, pectin and caseinate concentrate in a single phase which is characteristic for complexation (Redigueri, 2007). This complexation is reversible with pH but is resistant to high ionic strength (Redigueri, 2007). At low concentration of polymer and protein (0.9% each), the pectin caseinate system is phase separating but only at the microscopic scale (de Kruif and Tromp, 2008). Depending on the pH and the respective phase diagrams, caseinate droplets in pectin solution (at pH 5.38 and above) or droplets of pectin-caseinate complexes (at pH 5.22 and below) are observed (de Kruif and Tromp, 2008).

4.3.3 Oil in water emulsions stabilized by sodium

caseinate containing pectin

Pectin inhibits the aggregation of sodium caseinate-coated droplets at pH 5 and below (Dalgleish and Hollocou, 1997). At pH 7, pectin doesn’t adsorb onto the caseinate layer covering the oil droplet and at a certain concentration of polymer, flocculation and coalescence is observed resulting from depletion exerted by pectin (Dickinson et al., 1998, Surh et al., 2006). At pH 5.5, the zeta potential which was positive with only casein is negative in the presence of pectin: the polymer adsorbs to the surface of the oil droplets (Dickinson et al., 1998). The interaction of pectin with the caseinate covered oil droplet involves relatively weak and reversible interactions (Dickinson et al., 1998). Pectin reduces droplet aggregation but doesn’t eliminate it: unabsorbed polymer is still reported to induce destabilization by depletion. At pH 4 and below, extensive droplet flocculation is observed in presence of pectin, due to adsorption, which might reduce the magnitude of the zeta-potential and thus the electrostatic repulsion between the droplets and/or the polymer might bridge the droplets (Surh et al., 2006). For the stabilization of caseinate coated oil droplet by pectin, there seems to be little effect of the pectin fine structure (LM or HM).

44

5 Instrumentation

This section briefly reviews the techniques and instrumentation which will be used in this thesis. Further details of the different experimental methodologies are reported in the relevant chapters.

5.1 Method to study pectin fine structure: capillary