Patricia Hilda Risso

A study on bovine kappa-casein aggregation after the enzymatic action of chymosin

Kappa-casein (kappa-CN) is a milk phospho-glycoprotein that plays an important role in the electrostatic and steric stabilization of casein micelles suspensions. kappa-CN characteristics such as its aminoacidic composition and its high denaturalization temperature make this protein an excellent nutrient. The enzymatic destabilization of kappa-CN is the basis of cheese and yoghurt manufacture. In this paper, modifications on the kappa-CN aggregation process by hydrolysis with chymosin derived from the initial protein state and the presence of sucrose and lactose were studied. Our study shows that, during its self-association, kappa-CN opens its conformation by raising the exposition of hydrophobic regions to the medium. Hence, the initial polymerization or association state of kappa-CN affects on the aggregation stage after the enzymatic action of chymosin. The inhibition of aggregation when the protein was previously associated suggests that the region which participates in the aggregation of p-kappa-CN particles, would be the one involved in the self-association during the heating of kappa-CN samples. Sucrose and lactose also affect the aggregation of proteolized particles of kappa-CN. Furthermore, the nature and the concentration of the added sugars had significant influence on the protein aggregation process.

Study of the inhibitory effect of hydrophobic fluorescent markers on the enzymic coagulation of bovine casein micelles: action of TNS

The interaction of 2-(p-toluidinyl) naphthalene-6-sulfonate (TNS) with casein micelles (CM) was studied by fluorescence spectroscopy. Fluorescence emission spectra of the complex showed blue shift and intensity enhancement of TNS fluorescence, suggesting the insertion of the marker in low polarity regions of CM. An energy transfer process between the proteins and the marker was detected, showing that most of the TNS binding sites were in the proximity of CM fluorescent residues. TNS inhibited the aggregation step of CM enzymic coagulation, producing probably a decrease of size and amount of aggregates formed. This effect could not be related only to changes in CM net charge, but also possibly to the occupancy of surface hydrophobic regions by the marker. About a 20% decrease in the TNS fluorescence intensity was observed during the proteolytic step of coagulation which could be attributed to release of the marker from its binding sites located in the CM external layer. A bound TNS release was also observed during the initial time of the aggregation step, probably by removal of the bound marker from contact regions between aggregating particles upon their collisions. A further increase, related to the aggregation step, could indicate the uptake of the marker by new hydrophobic sites created in the complex structure of the clusters. The results pointed to the participation of surface hydrophobic regions of renneted CM in their aggregation process.

Acid-Induced Aggregation and Gelation of Sodium Caseinate-Guar Gum Mixtures

The aim of this work was to study the formation of bovine sodium caseinate (NaCAS) acid gels induced by addition of glucono-δ-lactone (GDL) in the presence of guar gum (GG). At low biopolymer’s concentrations, a one-phase system was observed, whereas at higher mixture concentrations two-phase systems were formed. Aggregation (at low NaCAS concentrations) and gelation (at high NaCAS concentrations) processes were analyzed through the use of full and fractional factorial experiment designs, using turbidimetric and rheological techniques. Finally, the gel images were obtained by confocal laser scanning microscopy and the images were analyzed. Results showed that at low NaCAS concentrations, the presence of GG affects the pH at which aggregation begins but was not significant for the time at which aggregation begins. On the other hand, at high NaCAS concentrations, the concentration of GG only affected significantly the elastic character of acid gels. As polysaccharide concentration increases, the gels obtained were weaker and with larger pores. Also, the formation of NaCAS droplet-shaped structures at certain biopolymer ratio was observed. The presence of GG affects both the rate of gelation and phase separation, which, in turn, determine the type of gel microstructure. Phase separation seems to occur prior to protein gelation because the protein gel network is discontinued, hindering the gel compactness and reducing gel strength. In summary, GG modifies NaCAS stabilization (self-association and phase separation) and the viscoelasticity and microstructure of NaCAS acid gels. The control of such processes and properties would allow obtaining mixture gels with different textures.

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

Maria Eugenia Hidalgo1, Bibiana D. Riquelme1,2, Estela M. Alvarez1, Jorge R. Wagner3 and Patricia H. Risso1,2,4 1Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario 2Instituto de Fisica Rosario (IFIR), CONICET-UNR, (2000), Rosario, 3Departamento de Ciencia y Tecnologia, 4Facultad de Ciencias Veterinarias,Universidad Nacional de Rosario, Rosario Universidad Nacional de Quilmes, Buenos Aires, Argentina

Comparative study of the action of anionic and non-ionic hydrophobic fluorescent markers on the enzymic coagulation of heated bovine casein micelles

Abstract The action of anionic (ANS, TNS) and non-ionic (NR) hydrophobic fluorescent markers on the enzymic coagulation of heated bovine casein micelles (HCM) was studied by spectrophotometric and spectrofluorimetric techniques. In the presence of the markers a conformational change following the κ-casein proteolysis was observed. Both ANS and TNS were partially released during this process, showing that a fraction of the markers was bound in the neighborhood of the site of action of chymosin. These anionic markers produced a decrease of the aggregation rate, probably by increase of the negative charge of the aggregating particles. Turbidity measurements suggested that the presence of ANS and TNS introduced changes in the aggregation mechanism, giving place to less compact aggregates. On the other hand, the binding of NR is not affected by the κ-casein proteolysis and tended to produce an increment of the aggregation rate, especially at high marker concentrations. Although turbidity measurements showed differences in the aggregation mechanism, the fractal dimension of the aggregates did not change by NR action. The effect of this non-ionic marker could be produced through an increment of the surface hydrophobicity and the number of regions able to give interparticle links, thereby increasing the number of effective collisions.

Milk protein suspensions enriched with three essential minerals: Physicochemical characterization and aggregation induced by a novel enzymatic pool

Structural changes of casein micelles and their aggregation induced by a novel enzymatic pool isolated from Bacillus spp. in the presence of calcium, magnesium or zinc were investigated. The effect of cations on milk protein structure was studied using fluorescence and dynamic light scattering. In the presence of cations, milk protein structure rearrangements and larger casein micelle size were observed. The interaction of milk proteins with zinc appears to be of a different nature than that with calcium or magnesium. Under the experimental conditions assayed, the affinity of each cation for some groups present in milk proteins seems to play an important role, besides electrostatic interaction. On the other hand, the lowest aggregation times were achieved at the highest calcium and zinc concentrations (15 mM and 0.25 mM, respectively). The study found that the faster the aggregation of casein micelles, the less compact the gel matrix obtained. Cation concentrations affected milk protein aggregation kinetics and the structure of the aggregates formed.

Mineral fortification modifies physical and microstructural characteristics of milk gels coagulated by a bacterial enzymatic pool

An enzymatic pool from the Amazonian bacterium Bacillus sp. P7 was used as milk coagulant. Discovery of novel coagulants is of great interest in dairy industry for the development of new textures in cheese. Color, mechanical and microstructural characterization of milk gels induced by the bacterial enzymatic pool was carried out. Effect of mineral fortification on these characteristics was studied. Whiter gels with smaller pore diameters were obtained in the presence of Ca2+ or Mg2+. These characteristics seemed to be influenced by the effect of ionic strength on casein structure which was also evidenced by digital texture features analysis. On the other hand, specific affinity of the assayed cations for milk proteins showed to be important in the development of the mechanical texture of the gels. Firmness and fracture force of milk gels obtained in the presence of Zn2+ or Ca2+ were higher than in the presence of Mg2+ and Na2+.

Glycosylation, denaturation, and aggregation of soy proteins in defatted soy flakes flour: Influence of thermal and homogenization treatments

ABSTRACT This article provides a systematic study of the impact of different thermal treatments (62 ± 2°C, without and with relative humidity control, 79%) on soy protein in defatted soy flour and their aqueous dispersions. The effect of dispersing treatments (magnetic stirring, high-speed, and high-pressure homogenization) on dispersions also was assessed. Changes in protein solubility (water and 0.2 g/100 g potassium hydroxide solution), apparent-reactive lysine content, urease and trypsin inhibitor activities, protein denaturation, and Fourier transform infrared spectra were studied. Glycosylation, aggregation, and denaturation of storage and biologically active soy proteins were observed in different degrees, being mainly promoted by the control of relative humidity and the dispersibility of the sample.

Physicochemical study of mixed systems composed by bovine caseinate and the galactomannan from Gleditsia amorphoides

Model systems formed by sodium caseinate (NaCAS) and espina corona gum (ECG) were studied. There was no evidence of attractive interactions between NaCAS and ECG macromolecules. Aqueous mixtures of NaCAS and ECG phase-separate segregatively over a wide range of concentrations. According to the images obtained by confocal laser scanning microscopy, NaCAS particles form larger protein aggregates when ECG is present in the system. An increase in the hydrodynamic diameter of NaCAS particles, as a result of ECG addition, was also observed by light scattering in diluted systems. A depletion-flocculation phenomenon, in which ECG is excluded from NaCAS surface, is proposed to occur in the concentrated mixed systems, resulting in NaCAS aggregation. ECG raises the viscosity of NaCAS dispersions without affecting the Newtonian flow behaviour of NaCAS. These results contribute to improve the knowledge of a barely-studied hydrocolloid which may be useful in the development of innovative food systems.

Effect of Xanthan Gum on the Rheological Behavior and Microstructure of Sodium Caseinate Acid Gels

The aim of this work was to study the effect of xanthan gum (XG) on the gelation process of bovine sodium caseinate (NaCAS) induced by acidification with glucono-δ-lactone (GDL) and on the mixed acid gel microstructure. Before GDL addition, segregative phase separation was observed in all the NaCAS-XG mixtures evaluated. The gelation process was analyzed by using a fractional factorial experimental design. The images of the microstructure of the mixed acid gels were obtained by conventional optical microscopy and the mean diameter of the interstices was determined. Both the elastic character and the microstructure of the gels depended on the concentrations of XG added. As XG concentration increased, the kinetics of the gelation process was modified and the degree of compactness and elasticity component of the gel network increased. The microstructure of gels depends on the balance among thermodynamic incompatibility, protein gelation and NaCAS-XG interactions.