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

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

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 1:1.5 R 10.

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 fractal dimension of aggregates.

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

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 during the formation of aggregates.

Acid-Induced Aggregation and Gelation of

**3.7 Digital images of gels** 

2002).

NaCAS:CMC 4:1.

Bovine Sodium Caseinate-Carboxymethylcellulose Mixtures 85

same way as -casein at neutral pH. These can be the reason of the increase on the stability of NaCAS:CMC mixtures against acid aggregation and gelation. The degree of compactness and the elasticity of NaCAS aggregates and gels respectively were higher at low CMC proportion but underwent a sharp decrease when the polysaccharide amount rises. On the other hand, the degree of thermodynamic compatibility affected the final elasticity of mixed gels. At 0.375 wt% of CMC, the two biopolymers are in the same phase, but at higher proportions of CMC there is a thermodynamic incompatibility and phase separation occur.

The microstructure of protein gels can be characterized through optical analysis (Lucey,

Fig. 5 shows the transmission images of gels obtained for mixed gels at constant NaCAS concentration (3 wt%) and different CMC proportions. From the digital images of gels, it

Fig. 5. Images of gels obtained using a conventional inverted microscopy with an objective 100x and a digital camera with a zoom 7.1x, at NaCAS 3 wt%, 35 °C and R 1, for different NaCAS:CMC ratios: a) without CMC, b) NaCAS:CMC 8:1, c) NaCAS:CMC 6:1, and d)

This incompatibility appears to induce the formation of weaker gels.

was possible to observe differences in the internal microstructure of gels.

On the other hand, the degree of compactness of acid aggregates, estimated by Df, slightly diminishes as CMC:NaCAS ratio increases.

Fig. 4 shows, as an example, the average hydrodynamic diameters of NaCAS particles measured by DLS and variations during the acid aggregation at 35°C. These profiles confirm the existence of the two stages mentioned above. In addition, the average hydrodynamic diameters determined by DLS showed a good linear correlation (r=0.9082; p<0.0018) with the values, allowing us to corroborate that the parameter can be used to estimate the average size of the particles. Therefore the use of simple spectrophotometric techniques could produce reliable results in studying processes of aggregation or gelling of proteins as caseinates.

Fig. 4. Average hydrodynamic diameters (A) and parameter (B) of NaCAS particles during the acid aggregation of 0.5 wt% NaCAS, R 0.5, at 35°C.

#### **3.6 Rheological properties of acid gels**

Table 2 shows tg, pHg and the maximum G' (G'max) reached during protein gel formation after addition of GDL at 35°C. Gel times increase and the pHg decrease as CMC percentage becomes higher revealing a stabilizing effect of CMC.


Table 2. Values of tg, G'max and pHg of gels obtained from NaCAS:CMC mixtures at 35ºC and R 1.

As mentioned, it have been reported that an adsorbed CMC layer on the surface of casein micelles gives rise to a repulsive interaction between the casein micelles at low pH in the same way as -casein at neutral pH. These can be the reason of the increase on the stability of NaCAS:CMC mixtures against acid aggregation and gelation. The degree of compactness and the elasticity of NaCAS aggregates and gels respectively were higher at low CMC proportion but underwent a sharp decrease when the polysaccharide amount rises. On the other hand, the degree of thermodynamic compatibility affected the final elasticity of mixed gels. At 0.375 wt% of CMC, the two biopolymers are in the same phase, but at higher proportions of CMC there is a thermodynamic incompatibility and phase separation occur. This incompatibility appears to induce the formation of weaker gels.

#### **3.7 Digital images of gels**

84 Food Industrial Processes – Methods and Equipment

On the other hand, the degree of compactness of acid aggregates, estimated by Df, slightly

Fig. 4 shows, as an example, the average hydrodynamic diameters of NaCAS particles measured by DLS and variations during the acid aggregation at 35°C. These profiles confirm the existence of the two stages mentioned above. In addition, the average hydrodynamic diameters determined by DLS showed a good linear correlation (r=0.9082; p<0.0018) with the values, allowing us to corroborate that the parameter can be used to estimate the average size of the particles. Therefore the use of simple spectrophotometric techniques could produce reliable results in studying processes of aggregation or gelling of

Fig. 4. Average hydrodynamic diameters (A) and parameter (B) of NaCAS particles during

Table 2 shows tg, pHg and the maximum G' (G'max) reached during protein gel formation after addition of GDL at 35°C. Gel times increase and the pHg decrease as CMC percentage

NaCAS 3% 7.54 31.6 4.72 NaCAS 3%-CMC 0.375% (8:1) 17.73 74.4 4.49 NaCAS 3%-CMC 0.50% (6:1) 21.50 51.4 3.90 NaCAS 3%-CMC 0.75% (4:1) 24.99 26.3 3.77 NaCAS 3%-CMC 1.5% (2:1) 81.36 4.8 3.79

Table 2. Values of tg, G'max and pHg of gels obtained from NaCAS:CMC mixtures at 35ºC and

As mentioned, it have been reported that an adsorbed CMC layer on the surface of casein micelles gives rise to a repulsive interaction between the casein micelles at low pH in the

System tg (min 0.01) G´max ( 0.1) pHg ( 0.01)

the acid aggregation of 0.5 wt% NaCAS, R 0.5, at 35°C.

becomes higher revealing a stabilizing effect of CMC.

**3.6 Rheological properties of acid gels** 

R 1.

diminishes as CMC:NaCAS ratio increases.

proteins as caseinates.

The microstructure of protein gels can be characterized through optical analysis (Lucey, 2002).

Fig. 5 shows the transmission images of gels obtained for mixed gels at constant NaCAS concentration (3 wt%) and different CMC proportions. From the digital images of gels, it was possible to observe differences in the internal microstructure of gels.

Fig. 5. Images of gels obtained using a conventional inverted microscopy with an objective 100x and a digital camera with a zoom 7.1x, at NaCAS 3 wt%, 35 °C and R 1, for different NaCAS:CMC ratios: a) without CMC, b) NaCAS:CMC 8:1, c) NaCAS:CMC 6:1, and d) NaCAS:CMC 4:1.

Acid-Induced Aggregation and Gelation of

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ISSN 0268-005X

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Performing a qualitative analysis of these images, it is possible to observe different degrees of structure of the gels formed at different ratios of CMC. Table 3 shows the values of mean pore size of NaCAS gels in the absence and presence of CMC obtained from digital images. In the presence of lower concentration of CMC, the slower rate of gelation (higher tg) produced gels more structured, more compact and with smaller pores. This is due to, if the process is performed slowly, the gel mesh can be restructured by breaking of some interactions and formation of new ones, forming a tighter mesh and, therefore, progressively smaller pores. Other authors have also reported that processing speed can affect the hardness and elasticity of the gel formed (Cavallieri & da Cunha, 2008). But with increasing CMC concentration, there was an increase in the average pore diameter. Mixtures NaCAS:CMC 2:1 failed to gel consistency.


Table 3. Average pore sizes of NaCAS gels in the absence and presence of different concentrations of CMC, at 35ºC and R 1.

These results are consistent with the values of G'max (Table 2) obtained for the different mixtures. Gels with larger pores will be less elastic.
