**3. Results and discussions**

### **3.1 Thermodynamic compatibility of NaCAS:CMC mixtures**

The results obtained for mixtures of NACAS and CMC are shown in Fig. 1. The polysaccharide and protein concentrations, in each of the prepared binary solutions, correspond to a single point on the phase diagram. This approach provides a "map'' of the transition from the single-phase to the two-phase region of the phase diagram.

Fig. 1. Approach used for the determination of the phase diagrams for NaCAS:CMC systems after 24 h at 35 °C. Key: (○) one-phase clear solution, (∆) one-phase turbid solution, (▲) twophase samples, (●) two-phase gel-like systems

#### **3.2 Thermal stability of NACAS: CMC mixtures**

Both the CMC as all mixtures NaCAS:CMC in all relations tested were not affected by rising temperature within the temperature range studied (10-100 °C). This would indicate that the polysaccharide is thermally stable in this range, and that the addition of CMC to NACAS increases its thermal stability, since the NaCAS starts aggregating at about 60 °C in the absence of the polysaccharide.

Acid-Induced Aggregation and Gelation of

1:1.5 R 10.

fractal dimension of aggregates.

during the formation of aggregates.

**3.5 Effect of CMC on the NaCAS acid aggregation** 

Bovine Sodium Caseinate-Carboxymethylcellulose Mixtures 83

After addition of GDL, NaCAS solutions start a number of changes that lead to protein aggregation. The influence of CMC on this acid aggregation at 35°C, in conditions that no

Fig. 3. Variations of parameter as function of time (a) and pH (b) after GDL addition, at 35ºC. NaCAS concentration: 0.5 wt%; (●) NaCAS R 0.7; (▼) NaCAS:CMC 8:1 R 1; () NaCAS:CMC 4:1 R 1; (■) NaCAS:CMC 2:1 R 3; (▲) NaCAS:CMC 1:1 R 6; (○) NaCAS:CMC

The acid aggregation, induced by addition of GDL, showed two well-defined steps. At the beginning, a slow phase with a decrease of average size of protein particles is observed. The second step presents a sharp increase in the average size of particles due to formation of colloidal aggregates (aggregation time, tag) that grow until they reach a limit value, i.e., a

It is known that bovine sodium caseinate in aqueous solution has a considerable level of self-association, like sub-micelles or micelles (Farrell HM, 1996; Fox PF, 1983). Other authors have suggested that bovine sodium caseinate associates into small well-defined aggregates with an aggregation number that depends on the environmental conditions such as temperature, pH, or ionic strength. Probably star-like aggregates are formed with a hydrophobic centre and a hydrophilic (charged) corona (Pitkowski et al., 2008). The profiles in Fig. 3 suggest a slow dissociation of original caseinate aggregates or sub-micelles to form

These results show that tag increases as CMC proportion rises, partially due to a decrease in aggregation pH (pHag). Because the colloidal particles of NaCAS in suspension have a negative net charge, the addition of CMC would increase its electrostatic stability hindering their aggregation by a consequent increment of the net charge of the soluble particles. On the other hand, this effect can be related to an increase of the viscosity in the medium and a decrease of S0 in the presence of the polysaccharide. Since the rate of aggregation is limited by the diffusion of particles, an increment of generates a slower movement giving rise to an increase of tag. A decrease of S0 diminishes the participation of hydrophobic interactions

a large number of small particles, which finally aggregate to form bigger particles.

significantly changes on the rate at which pH becomes lower, is shown in Fig. 3.

#### **3.3 Analysis of conformational changes and surface hydrophobicity of NaCAS**

Emission spectra of intrinsic fluorescence of NaCAS and mixtures at different NaCAS:CMC ratios were analyzed. In the presence of CMC, a slightly decrease in the fluorescence intensity without changes in emission peaks was observed (data no shown). This would indicate no significant changes in the environment of the intrinsic protein fluorophores when the protein is in the presence of the polysaccharide.

S0 of the NaCAS was determined in the presence of different CMC concentrations, and it is listed in Table 1.


Table 1. S0 values of NaCAS in the presence of different concentrations of CMC, at 35ºC.

S0 decreased as CMC concentration increased, which would indicate a higher exposure of hydrophilic groups in the protein surface that protrude towards the aqueous environment. These results point to the adsorption of CMC on the surface of the protein.

#### **3.4 Effect of CMC on the viscosity of media**

Due to the fact that aggregation is limited by particles diffusion, it was determined the effect on the viscosity caused by the addition of CMC. An increment of r with the concentration of the polysaccharide is shown in Fig. 2, especially at CMC concentrations higher than 1.5 wt%.

Fig. 2. Relative viscosity (r) variations of the medium in the presence of different concentrations of CMC, T 35ºC.

Emission spectra of intrinsic fluorescence of NaCAS and mixtures at different NaCAS:CMC ratios were analyzed. In the presence of CMC, a slightly decrease in the fluorescence intensity without changes in emission peaks was observed (data no shown). This would indicate no significant changes in the environment of the intrinsic protein fluorophores

S0 of the NaCAS was determined in the presence of different CMC concentrations, and it is

S0 decreased as CMC concentration increased, which would indicate a higher exposure of hydrophilic groups in the protein surface that protrude towards the aqueous environment.

Due to the fact that aggregation is limited by particles diffusion, it was determined the effect on the viscosity caused by the addition of CMC. An increment of r with the concentration of the polysaccharide is shown in Fig. 2, especially at CMC concentrations higher than 1.5 wt%.

These results point to the adsorption of CMC on the surface of the protein.

Fig. 2. Relative viscosity (r) variations of the medium in the presence of different

**3.4 Effect of CMC on the viscosity of media** 

concentrations of CMC, T 35ºC.

0 64.3 0.0625 59.1 0.1250 54.2 0.2500 45.8 0.5000 26.6 0.7500 16.1 Table 1. S0 values of NaCAS in the presence of different concentrations of CMC, at 35ºC.

S0 (wt%-1) 0.2

**3.3 Analysis of conformational changes and surface hydrophobicity of NaCAS** 

when the protein is in the presence of the polysaccharide.

CMC (wt%)

listed in Table 1.
