RESUMEN
The majority of the protein in cow's milk is contained in the particles known as casein micelles. This review describes the main structural features of these particles and the different models that have been used to define the interior structures. The reactions of the micelles during processing operations are described in terms of the structural models.
Asunto(s)
Caseínas/química , Manipulación de Alimentos , Leche/química , Animales , Micelas , Modelos Químicos , PasteurizaciónRESUMEN
Casein micelles were separated from unheated reconstituted skim milk powder (RSMP) and were resuspended in the serum of RSMP that had been heated, with and without dialysis of this serum against unheated RSMP. Using size-exclusion chromatography, it was found that the soluble complexes of whey protein (WP) with κ-casein in the serum of the heated milk bind progressively to unheated casein micelles during renneting, even prior to the onset of clotting. Similar trends were noted when casein micelles from RSMP heated at pH values of 6.7, 7.1, or 6.3, each with different amounts of WP coating the micelles, were renneted in the presence of soluble WP/κ-casein complexes. No matter what was the initial load of micelle-bound WP complexes, all micelle types were capable of binding additional serum protein complexes during renneting. However, it is not clear that this binding of WP/κ-casein complexes to the micellar surface is a direct cause of the impaired rennet clotting of the RSMP.
Asunto(s)
Caseínas/metabolismo , Quimosina/metabolismo , Calor , Micelas , Proteínas de la Leche/metabolismo , Leche/química , Animales , Alimentos en Conserva , Concentración de Iones de Hidrógeno , Leche/enzimología , Proteínas de la Leche/química , Desnaturalización Proteica , Proteína de Suero de LecheRESUMEN
Tests were made to determine whether surface plasmon resonance (SPR) could be used as a technique to study the dissociation properties of bovine casein micelles or of sodium caseinate and the interactions between these protein particles and different polysaccharides. Surfaces of bound micelles or caseinate were made, and the changes in refractive index of these layers were used to define changes in the structures of the chemisorbed material. The technique appears to have some potential for studying details of the dissociation of casein micelles and of the binding of different polysaccharides to caseins.
Asunto(s)
Caseínas/química , Polisacáridos/química , Resonancia por Plasmón de Superficie/métodos , Animales , Bovinos , Cinética , Micelas , Unión ProteicaRESUMEN
The acid-induced interactions between different protein particles in milk (casein micelles and serum protein/kappa-casein complexes) were studied in a series of different mixtures of heated and unheated proteins using diffusing wave spectroscopy (DWS) and small deformation rheology. The measurements were made as functions of pH during acidification by addition of glucono-delta-lactone (GDL). Heat treatment (85 degrees C, 10 min) affected the composition of the serum and the reactivity of casein micellar surface based on the pH at which the casein micelles aggregated during acidification. It was observed that the gel points as defined by DWS and rheology did not always coincide. The experiments showed that all systems containing heated serum proteins gelled at a higher pH than those containing unheated serum proteins. For systems containing heated micelles, an intermediate network can be formed between heat-induced aggregates of serum proteins and kappa-casein formed at the surfaces of the micelles and dispersed as soluble complexes in the serum. This can explain the observation that DWS measurements detected aggregation of casein micelles at an earlier stage than did rheology. For systems containing unheated micelles and soluble complexes from heated milk, the results appear to be explained only by a direct interaction between soluble serum protein complexes and the casein micelles themselves, once the pH has decreased to below about 5.5. Comparison of the different systems studied gives a more complete description of the possible mechanism of interaction of the different protein materials during the acid-induced coagulation of milk-based systems.
Asunto(s)
Proteínas Sanguíneas/química , Caseínas/química , Geles/química , Calor , Leche/química , Animales , Concentración de Iones de Hidrógeno , Micelas , Reología , Análisis EspectralRESUMEN
The technique of forward-scattering diffusing wave spectroscopy has been used to study the rennet-induced gelation of skim milk. The results allow the comparison of a colloidal suspension at a realistic concentration (Phi approximately 10%) compared with well-established measurements made on highly-diluted milk samples. It is shown that the partially renneted casein micelles do not begin to approach one another until the extent of breakdown of kappa-casein has reached about 70%; above this point, they interact increasingly strongly with the extent of proteolysis. This interaction initially restricts the diffusive motion of the particles rather than causing true aggregation. Only after more extensive removal of the protective kappa-casein does true aggregation occur, with the appearance of a space-filling gel (defined by rheology as having a value of tandelta<1). The results show in greater detail than hitherto the progress of interactions between the particles in a system where the steric stabilization is progressively destroyed, and suggest that the renneting of milk at its normal concentration cannot be described simply by reactions between freely-diffusing particles.
Asunto(s)
Leche/química , Análisis Espectral/métodos , Animales , Caseínas/análisis , Cinética , Fragmentos de Péptidos/análisisRESUMEN
The effect of the pH of heating (6.3-7.3) on the composition of sera in reconstituted skimmed milks was investigated. A combination of SDS-PAGE analysis and size exclusion chromatography (SEC) combined with an original approach to the analysis of the SEC profiles was performed. The composition of the sera varied greatly when the pH of heating was adjusted below and above the natural pH of milk. The formation, composition, and concentration of heat-induced soluble complexes depended on the combination of the effect of adjusting the pH of the milk and the heat treatment. Two types of mechanism for the formation of soluble aggregates appeared to exist, depending on the pH of the milk. The first type results from the formation of WP/kappa-casein aggregates at the surface of the micelle, and these were detached partially into the serum in larger amount as the pH increased up to 6.7, where it reaches a maximum. The second type of complexes, whose amount increased as the pH of heating increased from 6.7 to 7.3, may be formed between caseins (kappa- but also perhaps some alpha(s)-casein) and aggregated WP resulting in complexes that are smaller in size and with a higher kappa-casein/whey protein ratio than the first type.
Asunto(s)
Calor , Leche/química , Animales , Caseínas/análisis , Fenómenos Químicos , Química Física , Electroforesis en Gel de Poliacrilamida , Grasas/análisis , Concentración de Iones de Hidrógeno , Lactalbúmina/análisis , Lactoglobulinas/análisis , Tamaño de la PartículaRESUMEN
Milk gels induced by partial proteolysis of the kappa-casein followed by acidification were studied, and their gelation behavior was compared to that of milk gels induced by simultaneous acidification and renneting, using dynamic rheology. There were generally two stages (at pH values below and above 5.0) in the gelation of the milk whose kappa-casein had been partially proteolyzed and acidified. The onset of gelation was at higher pH as the degree of kappa-casein proteolysis increased. The development of G' immediately after the onset of gelation was faster in the milk gels induced by simultaneous acidification and renneting, because of the continuing kappa-casein proteolysis. Preheat treatment caused the onset of gelation to occur at higher pH than for unheated milk. However, the maximum tan delta during gelation always occurred at the same pH (for a given concentration of acidulant), and its value and position were independent of the extent of renneting and whether the milks had been heat treated. The results are discussed in terms of the interactions between casein micelles occurring during gelation.
Asunto(s)
Caseínas/química , Caseínas/metabolismo , Leche/química , Péptido Hidrolasas/metabolismo , Animales , Quimosina/metabolismo , Geles , Calor , Concentración de Iones de Hidrógeno , Micelas , ReologíaRESUMEN
The acid-induced aggregation of casein micelles from milk, in the presence of different whey protein preparations from heated and unheated milk, has been studied using diffusing wave spectroscopy (DWS). In particular, the study focused on the turbidity (or l*) parameter obtainable from DWS, which can give information on the interactions between particles in aggregating systems. The experiments provided evidence that the presence of small, soluble, whey protein/kappa-casein aggregates derived from heated milk gave rise to interactions with both heated and unheated casein micelles over a pH range of 5.6 down to 5.2. Comparison of heated and unheated milks, together with milks whose sera had been exchanged, showed that direct interactions were indeed occurring, even between untreated casein micelles and soluble whey protein complexes. Comparison of the behavior of the whey protein aggregates in emulsion preparations where they could not interact with the large particles confirmed that the effect was specific to the presence of casein micelles and could not arise simply from the aggregation of the whey proteins themselves.
Asunto(s)
Caseínas/química , Micelas , Proteínas de la Leche/química , Dimerización , Emulsiones , Calor , Concentración de Iones de Hidrógeno , Unión Proteica , Desnaturalización Proteica , Suero , Análisis Espectral , Proteína de Suero de LecheRESUMEN
The effects of heat at temperatures in the range of 80-90 degrees C on mixtures of reconstituted skim milk powder (RSMP) and sodium caseinate have been determined. In the absence of caseinate, the action of heat on RSMP produces soluble complexes of whey proteins and kappa-casein, as well as complexes of whey protein with the casein micelles. When sodium caseinate was added to RSMP at levels of 0.5 and 1.0%, the denaturation of the whey protein and the production of the soluble complexes in the serum were hardly affected, either in rate or in amount. However, during the heating, the caseinate disappeared from the serum. Further studies on model mixtures of the different components showed that it was probable that the bulk of the caseinate associated with the casein micelles during heating, probably by binding inside the surface layer of kappa-casein, because no increase in the diameters of the casein micelles could be observed.
Asunto(s)
Caseínas/administración & dosificación , Calor , Leche/química , Animales , Caseínas/química , Fenómenos Químicos , Química Física , Cromatografía Líquida de Alta Presión , Micelas , Proteínas de la Leche/química , Tamaño de la Partícula , Proteína de Suero de LecheRESUMEN
Transmission diffusing wave spectroscopy has been used to study and compare three milk gelling systems (acid gelation of heated and unheated milks and rennet coagulation of unheated milk). In all cases, DWS was able to demonstrate the point of gelation as indicated by a rapid increase in particle size, as well as the small decreases in casein micelle radius attributed to the collapse or removal of the hairy kappa-casein layer. More importantly, the photon transport mean free path (l(*)) was measured. This parameter is unique to transmission DWS and can potentially give information about developing microstructures and the mechanical properties between different types of gels. The values of l(*) changed during the gelation processes, and these changes were manifested earlier than any change in particle aggregation or rheology of the systems. All three different gelling systems showed different changes in l(*) with time, showing the development of different interactions as the acidification or renneting reactions proceeded. Although a full analysis of the l(*) parameter and its changes cannot be made, it is concluded that they can provide important information on the pre-gelation states of aggregating systems.
Asunto(s)
Quimosina/química , Geles/química , Leche/química , Análisis Espectral/métodos , Animales , Difusión , Gluconatos/química , Concentración de Iones de Hidrógeno , Lactonas , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Heat-induced (90 degrees C, 10 min, pH 6.7) intermolecular disulfide bond formation in 1:1 mixtures of beta-lactoglobulin B (beta-Lg) and kappa-casein A (kappa-CN) was studied by enzymatic digestion with trypsin or glu-C, reverse-phase HPLC, and MALDI-TOF-MS. Observed masses were compared to theoretically calculated masses of disulfide-bonded peptide dimers and trimers, and the number of different masses matching peptide combinations involving each bond was used as a measure of confidence of identification. The beta-Lg cysteine residues 121 or 119 were involved in bonds with both cysteines of kappa-CN and all cysteines of beta-Lg. This agrees with the supposed initiatory role of beta-C121 in heat-induced SH/SS interchange. The largest numbers of matches corresponded to bonds linking beta-C119/C121 with kappa-C11 or with beta-C66. Multiple matches were recorded for beta-C119/C121 bonding with beta-C119/C121, with beta-C160, or with kappa-C88. However, beta-C106 was observed only in bonds with beta-C119/C121 and did not appear to bond to kappa-CN, suggesting it remains buried in the core of the protein.
Asunto(s)
Caseínas/química , Disulfuros/química , Calor , Lactoglobulinas/química , Cromatografía Líquida de Alta Presión , Concentración de Iones de Hidrógeno , Soluciones , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The physical aggregation of commercial whey protein isolate (WPI) and purified beta-lactoglobulin was studied by ultrasound spectroscopy. Protein samples were dialyzed to achieve constant ionic strength backgrounds of 0.01 and 0.1 NaCl, and gelation was induced in situ at constant temperatures (from 50 to 75 degrees C) or with a temperature ramp from 20 to 85 degrees C. Changes in the ultrasonic properties were shown in the early stages of heating, at temperatures below those reported for protein denaturation. During heating, the relative ultrasound velocity (defined as the difference between sample velocity and reference velocity) decreased continuously with temperature, indicating a rearrangement of the hydration layer of the protein and an increase in compressibility of the protein shell. At temperatures <50 degrees C the ultrasonic attenuation decreased, and <65 degrees C both velocity and attenuation differentials showed increasing values. A sharp decrease in the relative velocity and an increase in the attenuation at 70 degrees C were indications of "classical" protein denaturation and the formation of a gel network. Values of attenuation were significantly different between samples prepared with 0.01 and 0.1 M NaCl, although no difference was shown in the overall ultrasonic behavior. WPI and beta-lactoglobulin showed similar ultrasonic properties during heating, but some differences were noted in the values of attenuation of WPI solutions, which may relate to a less homogeneous distribution of aggregates caused by the presence of alpha-lactalbumin and other minor proteins in WPI.
Asunto(s)
Calor , Lactoglobulinas/química , Proteínas de la Leche/química , Ultrasonido , Fenómenos Químicos , Química Física , Geles , Concentración Osmolar , Desnaturalización Proteica , Análisis Espectral , Temperatura , Proteína de Suero de LecheRESUMEN
Intermolecular disulfide bond formation in pure beta-lactoglobulin (beta-Lg) B and in its 1:1 mixture with alpha-lactalbumin (alpha-La), heated at 85 degrees C for 10 min in solutions of low and high (100 mM NaCl) ionic strength and pH 6.0, was studied by reverse-phase HPLC and MALDI-TOF mass spectrometry. Disulfide bonding between beta-Lg monomers was more extensive than reported in the literature for a temperature of 68.5 degrees C, including formation of trimers connected by two of the three adjacent cysteines, C106/C119/C121. The participation of the different thiol groups in disulfide bonds appeared to depend on their location in the native structure, with surface-located cysteines more involved than internally located ones. This also applied to alpha-La-beta-Lg interactions, where the predominant participants were the surface-located alphaC111, alphaC120, alphaC61, and alphaC6. The least active participant was alphaC28, suggesting that it becomes sterically inaccessible during unfolding of the protein. High ionic strength apparently promoted disulfide bonding. The order of cysteine participation at the high ionic strength was similar to that at low ionic strength, with fewer native-location bonds observed and a lower activity of some groups, such as beta-C106/C119/C121 and alphaC61.
Asunto(s)
Disulfuros/química , Calor , Lactalbúmina/química , Lactoglobulinas/química , Cromatografía Líquida de Alta Presión , Cisteína/química , Concentración de Iones de Hidrógeno , Concentración Osmolar , Pliegue de Proteína , Cloruro de Sodio , Soluciones , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Gel formation was monitored by low amplitude rheometry during acidification at 40 degrees C with 1.5% glucono-delta-lactone in combined milk systems containing soluble and/or micelle-bound heat-induced (95 degrees C/10 min) aggregates of denatured whey proteins and kappa-casein and in heated dairy mixes with varying micellar casein/whey protein ratio (CN/WP). Both soluble and micelle-bound aggregates increased gelation pH and gel strength. Micelle-bound aggregates seemed to modify the micelle surface so that micelles were destabilized at a pH of 5.1 (instead of 4.7), while soluble aggregates precipitated at their calculated pI of approximately 5.3, and initiated an early gelation by interacting with the micelles. Decreasing the CN/WP ratio produced larger aggregates with higher whey protein: kappa-casein ratio, which gave more elastic gels. The specific effects of the micellar and soluble aggregates on gel strength are discussed with respect to their relative proportions in the heated milk.
Asunto(s)
Geles/química , Calor , Micelas , Proteínas de la Leche/química , Leche/química , Animales , Caseínas/análisis , Caseínas/química , Elasticidad , Concentración de Iones de Hidrógeno , Proteínas de la Leche/análisis , Solubilidad , Viscosidad , Proteína de Suero de LecheRESUMEN
The formation of heat-induced aggregates of kappa-casein and denatured whey proteins was investigated in milk-based dairy mixtures containing casein micelles and serum proteins in different ratios. Both soluble and micelle-bound aggregates were isolated from the mixtures heated at 95 degrees C for 10 min, using size exclusion chromatography. Quantitative analysis of the protein composition of the aggregates by reverse phase high-performance liquid chromatography strongly suggested that primary aggregates of beta-lactoglobulin and alpha-lactalbumin in a 3 to 1 ratio were involved as well as kappa-casein, and alpha(s2)-casein in micellar aggregates. The results gave evidence that heat-induced dissociation of micellar kappa-casein was implicated in the formation of the soluble aggregates and indicated that a significant amount of kappa-casein was left unreacted after heating. The average size of the aggregates was 3.5-5.5 million Da, depending on the available kappa-casein or the casein:whey protein ratio in the mixtures. The size and density of these aggregates relative to those of casein micelles were discussed.