6 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


Introduction
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 Ca 2+ .-CN fraction, insensitive to Ca 2+ , acts as protection that attempts to prevent other CN from Ca 2+ -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, NaCAS suspensions were prepared from dissolution of commercial drug in distilled water (isoionic pH) at room temperature.After concentration measurements, 0.15 g.L -1 sodium azide was added as a bacteriostatic agent, and the solutions were stored at 4 °C.Protein concentration was determined by the Kuaye's method which is based on the ability of strong alkaline solutions to shift the spectrum of the amino acid tyrosine to higher wavelength values in the UV region (Kuaye, 1994).Stock solutions nearly 6 mM for ANS were prepared in distilled water and stored in the dark at 4 °C; the concentration was determined by absorbance measurements, using a molar extinction coefficient (of 4,950 M -1 cm -1 at 350 nm.

NaCAS-CMC phase diagram
Phase diagram was established at pH 6.8 (buffer Tris-HCl 10 mM) and 35 °C, and it was constructed by determining the transition from single to two-phase systems.The thermodynamic compatibility study was carried out using the method proposed by Spyropoulos et al.They propose to carefully prepare a series of polysaccharide/protein aqueous solutions to give binary systems and incubate them a certain time at a given temperature, and then evaluate a single or two-phase systems formation (Spyropoulos et al., 2010).These binary systems were prepared with the same CMC concentration but with NaCAS concentrations ranging from 0 to 4 wt% in one case, and with the same protein concentration but with polysaccharide concentrations ranging from 0 to 4.5 wt% in the other.A total of three samples were taken from each of these binary solutions and kept in sealed tubes at 35 °C for at least 24 h, after which the occurrence of phase separation (or not) was verified by visual inspection.

Thermal stability of NACAS in the presence or absence of CMC
The effects of thermal treatments on NaCAS, CMC and their mixtures were monitored through spectrophotometry with the aim of evaluating the biopolymer aggregation by heating at different temperatures.Measurements at increasing temperature were made at 650 nm from 10 to 100 °C with a heating rate of 0.5 °C per minute.The equipment used was a Jasco V-550 doublebean spectrophotometer equipped with a cell holder heated by Peltier effect and controlled by a programmable unit.The cell was filled with a 0.02 wt% NaCAS solution in buffer Tris-HCl 10mM pH 6.8 up to a final volume of 2.5 mL and sealed with a teflon stopper to avoid evaporation during each experiment.The spectrophotometer compartment was continuously purged with nitrogen to prevent the condensation of water vapor on the cell walls.This method was also performed in the presence of CMC at NaCAS:CMC proportion of 8:1, 4:1, 2:1, 1:1 and 1:1.5.

Spectrofluorimetric determinations
Fluorescence excitation and emission spectra of the NaCAS (0.1 wt%) were obtained using a spectrofluorometer Ami n c o B o w m a n S e r i e s 2 .M e a s u r e m e n t s w e r e c a r r i e d o u t i n t h e presence and absence of CMC, in order to detect any spectral shifts and/or changes in the relative intensity of fluorescence (FI).Previously, the excitation wavelength ( ex ) and the range of concentration with a non significant internal filter effect were determined.The samples (3 mL) used for the spectral analysis and FI measures were transferred into a fluorescence cell with a light path length of 1 cm and placed into a cell holder keeping the temperature constant at the fixed values desired.Values of FI were registered within the range of 300-400 nm at 35ºC using a ex of 286 nm.
Acid-Induced Aggregation and Gelation of Bovine Sodium Caseinate-Carboxymethylcellulose Mixtures 79 The surface hydrophobicity (S 0 ) was estimated according to the method of Kato and Nakai (Haskard & Li-Chan, 1998;Kato & Nakai, 1980), using ANS as an hydrophobic fluorescent marker.The measurements were carried out in an Aminco Bowman Series 2 spectrofluorometer, using an ex of 396 nm and an emission wavelength ( em ) of 489 nm, previously determined from excitation and emission spectra of protein-ANS complex, at a constant temperature of 35ºC.The FI was measured in samples containing ANS 0.04 mM and consecutive aggregates of 0.1 wt% NaCAS with or without CMC (FI b ).The FI was also determined in samples containing only protein (i.e.without the addition of the fluorescent probe) in the presence or absence of CMC at the same concentrations (FI p ).The difference between FI b and FI p (F) was calculated and S 0 was determined as the initial slope in the F vs. NaCAS concentration (wt%) curve.

Acid aggregation
Kinetics of NaCAS aggregation induced by the acidification with GDL, in the presence or absence of CMC, was analyzed by measuring turbidity (τ) in the range of 450 to 650 nm, in a Spekol 1200 spectrophotometer with a diode arrangement and a thermostatized cell.The amount of GDL added was calculated using the following relation: R used for this experiment was 0.5, at a temperature of 35 °C.Acidification was initiated by the addition of solid GDL to 10 g of NaCAS suspension (0.5 wt%).

Changes in size and compaction of particles
Changes in the protein average size were followed by the dependence of τ on wavelength ( ) of the suspensions, determined according to:  is a parameter that has a direct relationship with the average size of the particles, can be used to easily detect and follow rapid size changes, and was obtained from the slope of log τ versus log plots, in the 450 to 650 nm range, where the absorption due to the protein chromophores is negligible allowing the estimation of τ as absorbance (Camerini-Otero & Day, 1978;Risso et al., 2007).Absorption spectra and absorbance at 650 nm (A 650 ) were registered as a function of time until a maximum and constant value of A 650 was reached; simultaneously the pH decrease was measured.The measurements of pH were carried out on digital pH meter Orion SA 720, equipped with proton-selective glass membrane electrode combined with saturated calomel reference electrode.On the other hand, it has been shown that , for a system of aggregating particles of the characteristics of caseinates, tends, upon aggregation, toward an asymptotic value that can be considered as a fractal dimension (D f ) of the aggregates (Horne, 1987;Risso et al., 2007).
To verify if β was actually related to the average size of the particles, the size distribution functions and the hydrodynamic diameters of NaCAS particles were determined by dynamic light scattering (DLS) using a Brookhaven 9863 Model equipment with a He-Ne laser ( 0 = 632.8nm) with a maximal power of 15 mV, and using 90° as the measuring angle.Hydrodynamic diameters were calculated using the BI9000AT 6,5 Version software processing.To carry out this determination an amount of solid GDL was added to 8 mL of a 0.5 wt% NaCAS solution in order to obtain a GDL/protein relation of 0.5.Measurements at different times were performed until the maximum of  allowed by the instrument was reached while pH was simultaneously monitored.

Effect of CMC on the viscosity of media
The aggregation process is limited by diffusion, which depends on the medium viscosity ().Therefore, is important to determine the effect that the presence of the polysaccharide exerts on that property.The  was measured in triplicate, using a rotational viscosimeter Brookfield LV Master (LVDV-III) with cone/plate geometry and thermostatically controlled at a temperature of 35.00 ± 0.05ºC.The relative viscosity ( r ) was calculated as: where  sol is the solution viscosity and  0 is the water ones.

Acid gelation
Above a certain protein concentration, the loss of electrostatic stability by acidification results in the formation of a three-dimensional gel network.Effect of CMC concentration on the kinetic of gelation, rheological properties and microstructure of gels were investigated.

Rheological properties of acid gels
Rheological properties of NaCAS samples (3 wt%), in the absence or presence of CMC, were determined in a stress and strain controlled rheometer AR G2 model using a cone geometry (diameter: 40 mm, cone angle: 2°, cone truncation: 55 mm) and a system of temperature control with a recirculating bath (Julabo model ACW 100) connected to a Peltier plate.An amount of solid GDL according to a certain R was added to initiate the acid gelation.
Measurements were performed each 20 sec with a constant oscillation stress of 0.1 Pa and a frequency of 0.1 Hz.The Lissajous figures at various times were plotted to ensure that the measurements of storage or elastic modulus (G') and loss or viscous modulus (G'') were always obtained within the linear viscoelastic region.The G'-G'' crossover times (t g ) of acidified caseinate systems were considered here as the gel times, since most studies of milk/caseinate gelation have adopted this criterion (Braga et al., 2006;Curcio et al., 2001).pH at t g was also determined considering the pH value at the G'-G'' crossover (pH g ).

Conventional inverted microscopy
The degree of compactness of gels was evaluated through digital image analysis.For this, bottom surface image of gel were obtained by conventional inverted microscopy.To obtain the microscopic images, 90 L of each sample were placed in compartments of the LAB-TEK II cells.The samples were obtained by duplicate under a constant temperature set at 35ºC.Transmission images of gels were obtained using a conventional inverted microscopy (Union Optical) with an objective 100x and a digital camera (Canon PowershotA640) with a zoom 7.1x and microscope adapter of 52 mm.The average pore diameters of gels were determined using the program Image J. To do this, straight lines were drawn on the digital images and values of pore size were measured in pixels.These values were averaged (n=5) and obtained the average pore size.

Statistical analysis
The data are reported as the average values ± their standard deviations.Statistical analysis was performed with Sigma Plot 10.0 and Image J softwares.Relationship between variables was statistically analyzed by correlation analysis using Pearson correlation coefficient (r).
The differences were considered statistically significant at p < 0.05 values.

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.

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.

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Food Industrial Processes -Methods and Equipment 82

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.S 0 of the NaCAS was determined in the presence of different CMC concentrations, and it is listed in Table 1.

CMC (wt%)
S 0 (wt% S 0 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.

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%.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, t ag ) 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 t ag increases as CMC proportion rises, partially due to a decrease in aggregation pH (pH ag ).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 S 0 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 t ag .A decrease of S 0 diminishes the participation of hydrophobic interactions during the formation of aggregates.
On the other hand, the degree of compactness of acid aggregates, estimated by D f , slightly diminishes as CMC:NaCAS ratio increases.

Rheological properties of acid gels
Table 2 shows t g , pH g and the maximum G' (G' max ) reached during protein gel formation after addition of GDL at 35°C.Gel times increase and the pH g decrease as CMC percentage becomes higher revealing a stabilizing effect of CMC.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 www.intechopen.com Acid-Induced Aggregation and Gelation of 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, 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.

Digital images of gels
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.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 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 t g ) 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.
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.

Conclusion
As CMC proportion rises, the aggregation and gel times of NaCAS:CMC mixtures increased and the pH at which these processes begin decreased, revealing a stabilizing effect of CMC.
The degree of compactness diminished when the CMC proportion increased.This effect can be linked to protein conformational changes in the presence of CMC that lead to a decrease of surface hydrophobicity, which difficult the establishment of hydrophobic interactions.The gels also showed lower elasticity at CMC:NaCAS high ratios.Therefore, it is possible to obtain acid gels with different textures varying the protein:polysaccharide proportions due to surface hydrophobicity and electrostatic stability modification of NaCAS particles and due to changes on the kinetic of aggregation and gelation processes.

Acknowledgment
This work was supported by grants from the Universidad Nacional de Rosario.Thanks to MSc.Luis Martínez of the National University of Quilmes for their advice in the rheological measurements.Thanks to Prof. Mirta Armendariz of the National University of Rosario for their advice in the statistical analysis.The authors would like to thank Lic. Romina Ingrassia for the English revision.

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

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  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.
To determine

Table 1 .
S 0 values of NaCAS in the presence of different concentrations of CMC, at 35ºC.