Spectrofluorometric Study on Surface Hydrophobicity of Bovine Casein Micelles in Suspension and during Enzymic Coagulation (original) (raw)
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Food Hydrocolloids, 2000
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 k-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 k-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.
Effect of temperature and pH on the aggregation and the surface hydrophobicity of bovine κ-casein
Colloid and Polymer Science, 2008
κ-Casein (κ-CN) aggregation by heating has been studied at pH 7.2 and 5.2 using UV-visible spectrophotometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis, spectrofluorometric study of the 1-8 aniline naphtalene sulfonate (ANS)-κ-CN binding and circular dichroism (CD) spectroscopy. The aggregation process to form aggregates like micelles or submicelles and the structural characteristics of these aggregates were pH dependent. Far-UV CD showed that the aggregates obtained by heating presented changes in the κ-CN secondary structure. Near-UV CD spectra showed a certain degree of tertiary organization in the Tyr environment for the protein heated or unheated, only at pH 5.2. ANS binding at both pH was quite different and depends on the self-association process. Heating produced exposition of hydrophobic binding sites only at pH 7.2, including those in the neighborhood of the κ-CN Trp residue.
Casein micelle size and composition related to the enzymatic coagulation process
Biochimica et Biophysica Acta (BBA) - General Subjects, 1980
Chymosin (EC 3.4.23.4) and rennet, the latter containing about 85% chymosin and 15% pepsin, have been compared according to their coagulation properties with native micelles of different sizes or monomeric caseins as substrate. The casein micelles were separated on columns of controlled-pore glass (CPG-10/3000), which fractionates particles of up to 300 nm diameter. The results show that the coagulation time varies with the micelle size. The effect, which is more pronounced with chymosin than with rennet, appears to be related to the availability of K-casein. Therefore the largest micelles, with a lower K-casein content, showed longer coagulation times than medium size micelles. In the region of the smallest micelles this time increases again, probably due to an increased /3-casein content. Addition of monomeric K-casein decreased the coagulation time with both rennet and chymosin, but as1-and fi-casein had the opposite effect. When isolated monomeric caseins were treated alone with rennet or chymosin, K-casein caused turbidity, but as 1-and fi-casein did not. Centrifugation experiments with micelles after monomeric casein addition showed that a limited amount of the added casein was able to join the micelle. This was confirmed by chromatographic studies.
Food Hydrocolloids, 2014
Cooling of milk causes the release of b-casein from the casein micelles. With subsequent washing steps, using milk UF permeate, it was possible to remove up to 60% of its initial concentration and study the interactions of b-casein depleted micelles with two model molecules, resveratrol and curcumin. With the release of b-casein there was an increase in the apparent diameter of the casein micelles, with no changes in the amount of colloidal calcium phosphate, or to the integrity of the protein particles when observed by Cryo-TEM. In addition, there was an increase in the affinity for both curcumin, a hydrophobic molecule and resveratrol, a hydrophilic molecule. Thus, with the removal of the b-casein, there appeared to be an overall loosening of the micellar structure, giving an increase in the accessibility to the inner hydrophobic regions.
Colloid and Polymer Science, 2007
The casein micelles of reconstituted nonfat milk that have been fractionated by controlled pore glass chromatography showed a relationship between their size and their proteic composition: The fractions containing the smaller particles were richer in κ-casein than the fractions containing the bigger ones, in accordance with the casein micelle model of submicelles. The initial aggregation rate of micelles of different sizes, partially proteolyzed with chymosin (para-casein micelles), was measured in conditions of enzyme excess in which aggregation is the ratelimiting step of enzymatic coagulation, showing higher rates for the smaller micelles with the production of less compact para-casein micelle networks. This behavior could be explained in terms of electrostatic and steric colloidal stabilization due to their lower negative net charge and size and to a higher surface density of hydrophobic "patches" of proteolyzed κ-casein related to a higher probability of effective collisions between particles. Differences in the βcasein content did not seem to affect the initial aggregation rate of the micelles. On the contrary, the modifications of the micelle surface by heating affected the colloidal stability of the hydrolyzed micelles in different ways. The denaturation of the whey proteins and the formation of covalent complexes with κ-casein modify the micelle sur-face, increasing specially the steric stabilization, and produces a diminution in the number of hydrophobic sites that could be able to give interparticle hydrophobic interactions.
The Journal of Physical Chemistry A, 2003
Absorption spectra and fluorescence properties of 2-hydroxy-substituted Nile Red (HONR) have been studied in various solvents and micelles. The solvent dependence of the rate constant of the internal conversion was found to exhibit two entirely distinct domains. Going from apolar toward polar solvents, first, the deceleration of the radiationless deactivation was observed due to the diminishing extent of the vibronic coupling between the neighboring S 1 and S 2 singlet excited states. After reaching a minimum, the rate of the transition to the ground state significantly increased in alcohols because of the efficient energy dissipation via intermolecular hydrogen bonding. The correlations of the excited-state energy, fluorescence lifetime, absorption, and fluorescence maxima with the E T (30) solvent polarity parameter were used to characterize the local environment of HONR in micelles. Fluorescence quenching studies provided further evidences for the differences in the binding site of the dye in cationic, anionic and nonionic micelles.
Composition, Structure, and Integrity of Casein Micelles: A Review
Journal of Dairy Science, 1984
This review is an attempt to bring what is known about casein micelles together into a coherent summary. Some of the earlier models and theories no longer appear workable, and much is left to be concluded on this subject. It is hoped with what we now know that those who already have contributed and others will continue to pursue the understanding of casein micelles. PHYSICAL PROPERTIES OF CASEIN MICELLES The biological function of bovine casein micelles is to provide efficient nutrition to the young calf. It does not require inherently a high degree of ordered structure but, rather, an effective mechanism for secretion of a highly concentrated solution of protein, calcium, and phosphate. During the past 20 yr research has been extensive to determine the composition and structure of casein micelles and to identify forces that maintain their integrity. The approximate composition of bovine casein micelles is in Table 1. In cows' milk, casein micelles occur in colloidal dispersion. Structure and properties of the casein group of proteins, which comprise over 90% of the mass of casein micelles, have been reviewed (11, 13, 76, 89, 90, 98). Casein micelles are highly hydrated and spongelike colloidal particles containing about 3.7 g H20/g protein (6, 47, 48). Relatively little of this water (.5 g H20/g protein) is bound to the protein. The remainder is occluded within the micelle and moves with the micelle during hydrodynamic experiments. Numerous models describing casein micelles have been
Rennet coagulation of casein micelles and heated casein micelles: action of Ca2+ and pH
Food Hydrocolloids, 1999
The effect of Ca 2ϩ and pH on the aggregation kinetics of heated para casein micelles (pHCM) was studied and compared with those of nonheated para casein micelles (pCM), using turbidimetry. Although it is known that pHCM have a higher stability than pCM, possibly because of steric stability, the increase of Ca 2ϩ concentration and acidification produced loss of stability in a similar way for both kinds of micelles. This behaviour could be associated with the neutralization of micelle negative charges, either by Ca 2ϩ and Ca 2ϩ complexes or by protonation. Variations in steric stability appeared, if they exist, less important than charge modifications. ᭧ Food Hydrocolloids 13 (1999) 235-238 0268-005X/99/$ -see front matter ᭧
Journal of Dairy Science, 2013
In this study, the surface tension, miscibility, and particle size distribution of a solution containing an α s -casein (CN)-rich CN fraction (54 wt % α s -CN, 32 wt % β-CN, and 15 wt % κ-CN) were determined at pH 6.6. The nondialyzed CN fraction was compared with a dialyzed one. In the nondialyzed sample, every charge on the protein was compensated by 0.3 charges coming from counterions, whereas in the dialyzed sample, only 0.2 charges could be assigned to each charge on the protein. This relation was determined by calculating the charges at the proteins, taking the measured mineral content into account. The surface tension was measured as a function of the protein concentration by the du Noüy ring method at room temperature. Results indicated alterations in the surface properties after reduction of counterions. The equilibrium surface tension above the critical micelle concentration increased from 40.1 × 10 −3 to 45 × 10 −3 N/m, the critical micelle concentration increased from 0.9 × 10 −4 to 2 × 10 −3 mol/L, and the minimal area occupied per molecule at the surface increased from 2.4 × 10 −18 to 4.6 × 10 −18 m 2 . Cloud points were determined by measuring the absorbance of CN solutions as a function of the temperature. The cloud points were found to be concentration dependent and had a minimum at 0.2 wt % at 34°C for nondialyzed CN and at 0.25 wt % at 28°C for dialyzed CN, again demonstrating the influence of counterion reduction. Below the cloud point, a micellar phase was found to exist. The hydrodynamic diameter of the micelles were characterized by dynamic light scattering in both auto-and cross-correlation mode. However, no influence of reduction in counterions could be observed, possibly due to the fact that dynamic light scattering is not a suitable method for this type of system. The presence of self-assembled structures was verified by freeze-fracture electron microscopy. The observed differences between dialyzed and nondialyzed samples were explained by changes in the counterion cloud surrounding the proteins. Consequently, the electrostatic interactions between as well as within the CN are altered by dialysis, which, in turn, affects the behavior at the surface as well as the properties in the solution.
Journal of Agricultural and Food Chemistry, 1997
The kinetics of aggregation of para-casein micelles (pCM) and of heated para-casein micelles (HpCM) were studied and compared by estimating the initial rate of aggregation from the initial rate of turbidity increase. Aggregation of both pCM and HpCM suspensions appeared as cases of slow Brownian flocculation, with a steric contribution to the suspension's stability which could explain their aggregation behavior, including the presence of the Berridge effect at low temperatures. Glycerol addition to the medium, besides the reduction in aggregation rate by increase of medium viscosity, produced an initial aggregation rate increase, probably by interaction with the proteins of the micelle external layer. The differences observed between pCM and HpCM aggregation rate could be related to the changes introduced by high heating in the surface structure of the micelles.