**Acid-Induced Aggregation and Gelation of Bovine Sodium Caseinate-Carboxymethylcellulose Mixtures**

María Eugenia Hidalgo1, Bibiana D. Riquelme1,2, Estela M. Alvarez1, Jorge R. Wagner3 and Patricia H. Risso1,2,4 *1Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario 2Instituto de Física Rosario (IFIR), CONICET-UNR, (2000), Rosario, 3Departamento de Ciencia y Tecnología, 4Facultad de Ciencias Veterinarias,Universidad Nacional de Rosario, Rosario Universidad Nacional de Quilmes, Buenos Aires, Argentina* 

#### **1. Introduction**

74 Food Industrial Processes – Methods and Equipment

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The main protein fraction in bovine and ovine milk is represented by caseins (76-83% of total proteins). Caseins (CN) occur in milk as stable colloidal aggregates known as casein micelles, mainly composed by S1-, S2-, - and -CN (Walstra et al., 1984). Among different types of CN, there are some important characteristics that make the difference between them, based on their charge distribution and their sensitivity to be precipitated by Ca2+. - CN fraction, insensitive to Ca2+, acts as protection that attempts to prevent other CN from Ca2+-induced precipitation (Qi et al., 2001). From a nutritional point of view, caseins have all the essential aminoacids and play an important role in calcium and phosphate transport, representing an easily digestible source of nutrients, contributing to a carefully balanced diet (Linde, 1982). CN and their derived salts, the caseinates, are extensively used in food industry because of their physicochemical, nutritional and functional properties that make them valuable ingredients in complex food preparations.

Caseinates (CAS) are prepared by acid precipitation of milk casein at its isoelectric point (pH 4.6) and resolubilized by increasing the pH. If the increase in the pH is carried out by the addition of NaOH, it is possible to end up obtaining sodium caseinate (NaCAS), a more soluble form of CN. In these conditions, the micellar structure is destroyed and the NaCAS form aggregates or sub-micelles due to the high proportion of hydrophobic amino acid side chains that self-associate in aqueous solutions (Farrell et al., 1990). Further association of submicelles to form the large casein micelles present in milk is prevented by the removal of most of the calcium (Oakenfull et al., 1999). NaCAS is commonly employed as additive in a great variety of food products because of its high emulsifying, water-binding and gelation capabilities, its heat stability and its contribution to the food texture and juiciness. Waterholding capacity and gelling properties are used to improve rheological properties, texture, stability, and appearance of many food products such as processed meats, surimi, cheese,

Acid-Induced Aggregation and Gelation of

Bovine Sodium Caseinate-Carboxymethylcellulose Mixtures 77

Jong et al., 2009). The relative concentration of a biopolymer mixture is critical for the gelling process. Increasing the macromolecule concentration can improve the gelation process, since the macromolecules become closer to each other, facilitating aggregate formation and contributing to the strengthening of the structure (Picone & da Cunha, 2010; Yamamoto & Cunha, 2007). However, above a critical value, thermodynamic incompatibility takes place

Carboxymethylcellulose (CMC) is an anionic linear polysaccharide that comes from cellulose, and has been widely used as a stabilizer in food products, for example in acidified milk drinks (Du et al., 2009). CMC is generally used in aqueous solutions, where useful characteristics such as high viscosity at low concentrations, defoaming, surfactant, and bulking abilities are applicable (de Britto & Assis, 2009). Delhen and Stefancich reported the formation of soluble complexes at low pH values in -casein-CMC systems. The backbone of CMC seems to be too rigid for interacting appreciably with proteins. However, lowering the pH, which reduces the free charges on the polymer backbone and hence the stiffness of the macromolecule, enables CMC to interact with the protein (Delben & Stefancich, 1997). Yu et al. informed that the addition of CMC to calcium caseinate, enhance its aggregation and seem to prevent protein precipitation during storage (Yu et al., 2004). Du et al., investigated the interaction between CMC and casein micelles and the influence on the stability of acidified milk drinks (Du et al., 2007; Du et al., 2009). They found that at pH 6.7, there was no interaction between caseins and CMC due to charge repulsion and mixtures of casein and CMC were stable at low CMC concentrations. Above a certain CMC concentration, depletion flocculation occurred leading to phase separation. Electrosorption of CMC onto casein micelles took place below pH 5.2 and the adsorbed CMC layer on the surface of casein could prevent flocculation of casein micelles by steric repulsion. In addition, the non-adsorbed CMC increased the viscosity of serum and slowed down the sedimentation of casein particles. In the case of low CMC concentrations, CMC/casein micelles mixture was phase separated via bridging flocculation. With increasing CMC concentrations, the casein micelles were effectively coated

In previous work, we found that the compactness and average size of the aggregates formed at the end of the acidification process of ovine caseinate depend on the kinetics of the aggregation phenomena. As the aggregation process becomes slower, the more easily a polypeptide chain could acquire different orientations, leading to the formation of a more compact aggregates and gels with more elasticity and hardness (Nespolo et al., 2010). Therefore, given that CMC affects the stability of colloidal particles in solution such as NaCAS particles, this polysaccharide can affect the kinetics of acid aggregation and gelation processes, and thus control the microstructure of the aggregates and gels formed. The aim of this work was to investigate conformational, aggregating and gelling behaviours of NaCAS

Bovine sodium caseinate powder, CMC low viscosity (50-200 cP, 4 % in H2O, 25 °C), GDL, tris(hydroxymethyl)aminomethane (Tris), 8-anilino-1-naphthalenesulfonate (ANS) as an ammonium salt, and sodium azide were purchased from Sigma-Aldrich Co. (Steinheim, Germany). HCl, and NaOH were provided by Cicarelli SRL (San Lorenzo, Argentina). The

and phase separation is observed (de Jong & van de Velde, 2007).

and consequently electrostatic and sterically stabilized.

**2. Materials and methods** 

**2.1 Materials** 

aqueous solutions in the presence of different concentrations of CMC.

CMC stock solutions were also prepared in water and stored at 4 °C.

yogurt and confectionary products (Corzo-Martínez et al., 2010). Some of these properties make caseinates useful and desirable ingredients in the preparation of bakery and confectionery products, where they can be used as milk substitutes (Gaucheron, 1997).

Dissociation and a further aggregation step of casein fractions due to caseinate acidification results in the formation of a gel structure. A possible explanation to this effect is that as the pH is adjusted toward the isoelectric point it causes a decrease of the repulsive interactions, resulting in a destabilization of the colloidal aggregates as the pH drops slightly below 5 at a given temperature (Braga et al., 2006; Ruis et al., 2007). Nowadays, a process that has gained the attention of food industry is direct acidification by the addition of a lactone, such as glucono-δ-lactone (GDL), which slowly hydrolyzes to gluconic acid with a resulting reduction in pH. GDL allows us to overcome some of the difficulties associated with the traditional process of using bacteria. In fact, the final pH of the system is a function of the amount of GDL added, whereas starter bacteria produce acid until they inhibit their own growth as the pH becomes lower (de Kruif, 1997; Lucey et al., 1998). Gels made with the two types of acidifying precursors, bacterial cultures (lactic fermentation) or the addition of GDL, differ in their rheological properties partly as a function of the velocity of acidification. In GDL gels, the isoelectric point (pH 4.6) can be reached faster and remains stable, thus allowing longer aging near this point. This phenomenon contributes to the continuous fusion and rearrangement of casein particles (Ribeiro et al., 2004). Acid gel formation of NaCAS dispersions has been examined leading to quantitative structural information for testing ideas about the fractal properties of casein gels (Bremer et al., 1993).

The effect of different processing parameters (heat treatment, temperature and pH conditions), the presence of other ingredients or the GDL concentration on the microstructure of acid gels has been investigated (Belyakova et al., 2003; Braga et al., 2006; Lucey et al., 2001; Nespolo et al., 2010; Perrechil et al., 2009). Particularly, protein/polysaccharide/water mixtures are frequently used in the food industry as thickening agents for low or zero fat products (Semenova et al., 2009). However, the interaction of proteins with polysaccharides in solution could influence in a positive or negative way, depending on the colloidal system in question, the functionality of the protein and, therefore, the food properties, due to the balance of the protein–protein and protein–solvent interactions. The rheological and structural properties of protein–polysaccharide gels depend on biopolymer interactions that can be influenced by the concentration and molecular structure of biopolymers. Three different systems can result from the mixture of proteins and polysaccharides in aqueous solution: a) stable homogeneous solutions; b) associative phase separation or coacervation, in which both components are concentrated in the same phase due to the formation of a complex; c) segregative phase separation, where the two components are in different phases due to the limited thermodynamic compatibility (Tolstoguzov, 1991).

In the case of coacervation, phase more concentrated in colloid component is the coacervate and the other phase is the equilibrium solution. Associative phase separation of two polymers in water occurs if there is an electrostatic attraction. Complex coacervation is caused by the interaction of two oppositely charged colloids (de Kruif et al., 2004).

When both polymers have the same charge, repulsive interactions lead to incompatibility between proteins and polysaccharides as a result of differences in their molecular properties, such as shape, size or charge and may cause phase separation. In the case of gelation of the proteins and polysaccharides, the balance between phase separation and gelation process determines the micro-structure and the mechanical properties of gels (de

yogurt and confectionary products (Corzo-Martínez et al., 2010). Some of these properties make caseinates useful and desirable ingredients in the preparation of bakery and confectionery products, where they can be used as milk substitutes (Gaucheron, 1997). Dissociation and a further aggregation step of casein fractions due to caseinate acidification results in the formation of a gel structure. A possible explanation to this effect is that as the pH is adjusted toward the isoelectric point it causes a decrease of the repulsive interactions, resulting in a destabilization of the colloidal aggregates as the pH drops slightly below 5 at a given temperature (Braga et al., 2006; Ruis et al., 2007). Nowadays, a process that has gained the attention of food industry is direct acidification by the addition of a lactone, such as glucono-δ-lactone (GDL), which slowly hydrolyzes to gluconic acid with a resulting reduction in pH. GDL allows us to overcome some of the difficulties associated with the traditional process of using bacteria. In fact, the final pH of the system is a function of the amount of GDL added, whereas starter bacteria produce acid until they inhibit their own growth as the pH becomes lower (de Kruif, 1997; Lucey et al., 1998). Gels made with the two types of acidifying precursors, bacterial cultures (lactic fermentation) or the addition of GDL, differ in their rheological properties partly as a function of the velocity of acidification. In GDL gels, the isoelectric point (pH 4.6) can be reached faster and remains stable, thus allowing longer aging near this point. This phenomenon contributes to the continuous fusion and rearrangement of casein particles (Ribeiro et al., 2004). Acid gel formation of NaCAS dispersions has been examined leading to quantitative structural information for

testing ideas about the fractal properties of casein gels (Bremer et al., 1993).

thermodynamic compatibility (Tolstoguzov, 1991).

The effect of different processing parameters (heat treatment, temperature and pH conditions), the presence of other ingredients or the GDL concentration on the microstructure of acid gels has been investigated (Belyakova et al., 2003; Braga et al., 2006; Lucey et al., 2001; Nespolo et al., 2010; Perrechil et al., 2009). Particularly, protein/polysaccharide/water mixtures are frequently used in the food industry as thickening agents for low or zero fat products (Semenova et al., 2009). However, the interaction of proteins with polysaccharides in solution could influence in a positive or negative way, depending on the colloidal system in question, the functionality of the protein and, therefore, the food properties, due to the balance of the protein–protein and protein–solvent interactions. The rheological and structural properties of protein–polysaccharide gels depend on biopolymer interactions that can be influenced by the concentration and molecular structure of biopolymers. Three different systems can result from the mixture of proteins and polysaccharides in aqueous solution: a) stable homogeneous solutions; b) associative phase separation or coacervation, in which both components are concentrated in the same phase due to the formation of a complex; c) segregative phase separation, where the two components are in different phases due to the limited

In the case of coacervation, phase more concentrated in colloid component is the coacervate and the other phase is the equilibrium solution. Associative phase separation of two polymers in water occurs if there is an electrostatic attraction. Complex coacervation is

When both polymers have the same charge, repulsive interactions lead to incompatibility between proteins and polysaccharides as a result of differences in their molecular properties, such as shape, size or charge and may cause phase separation. In the case of gelation of the proteins and polysaccharides, the balance between phase separation and gelation process determines the micro-structure and the mechanical properties of gels (de

caused by the interaction of two oppositely charged colloids (de Kruif et al., 2004).

Jong et al., 2009). The relative concentration of a biopolymer mixture is critical for the gelling process. Increasing the macromolecule concentration can improve the gelation process, since the macromolecules become closer to each other, facilitating aggregate formation and contributing to the strengthening of the structure (Picone & da Cunha, 2010; Yamamoto & Cunha, 2007). However, above a critical value, thermodynamic incompatibility takes place and phase separation is observed (de Jong & van de Velde, 2007).

Carboxymethylcellulose (CMC) is an anionic linear polysaccharide that comes from cellulose, and has been widely used as a stabilizer in food products, for example in acidified milk drinks (Du et al., 2009). CMC is generally used in aqueous solutions, where useful characteristics such as high viscosity at low concentrations, defoaming, surfactant, and bulking abilities are applicable (de Britto & Assis, 2009). Delhen and Stefancich reported the formation of soluble complexes at low pH values in -casein-CMC systems. The backbone of CMC seems to be too rigid for interacting appreciably with proteins. However, lowering the pH, which reduces the free charges on the polymer backbone and hence the stiffness of the macromolecule, enables CMC to interact with the protein (Delben & Stefancich, 1997).

Yu et al. informed that the addition of CMC to calcium caseinate, enhance its aggregation and seem to prevent protein precipitation during storage (Yu et al., 2004). Du et al., investigated the interaction between CMC and casein micelles and the influence on the stability of acidified milk drinks (Du et al., 2007; Du et al., 2009). They found that at pH 6.7, there was no interaction between caseins and CMC due to charge repulsion and mixtures of casein and CMC were stable at low CMC concentrations. Above a certain CMC concentration, depletion flocculation occurred leading to phase separation. Electrosorption of CMC onto casein micelles took place below pH 5.2 and the adsorbed CMC layer on the surface of casein could prevent flocculation of casein micelles by steric repulsion. In addition, the non-adsorbed CMC increased the viscosity of serum and slowed down the sedimentation of casein particles. In the case of low CMC concentrations, CMC/casein micelles mixture was phase separated via bridging flocculation. With increasing CMC concentrations, the casein micelles were effectively coated and consequently electrostatic and sterically stabilized.

In previous work, we found that the compactness and average size of the aggregates formed at the end of the acidification process of ovine caseinate depend on the kinetics of the aggregation phenomena. As the aggregation process becomes slower, the more easily a polypeptide chain could acquire different orientations, leading to the formation of a more compact aggregates and gels with more elasticity and hardness (Nespolo et al., 2010). Therefore, given that CMC affects the stability of colloidal particles in solution such as NaCAS particles, this polysaccharide can affect the kinetics of acid aggregation and gelation processes, and thus control the microstructure of the aggregates and gels formed. The aim of this work was to investigate conformational, aggregating and gelling behaviours of NaCAS aqueous solutions in the presence of different concentrations of CMC.
