Casein Micelles at Non-Ambient Pressure Studied by Neutron Scattering (original) (raw)
Related papers
kappa-Casein micelles: structure, interaction and gelling studied by small-angle neutron scattering
European Journal of Biochemistry, 1991
Small-angle neutron scattering (SANS) measurements on dilute and concentrated dispersions of K-casem micelles m a buffer at pH = 6 7 were made usmg the Dl l diffractometer m Grenoble Results indicate that the micelles have a dense core with a fluffy outer layer This outer layer appears to give rise to a steeply repulsive interaction on contact In fact, the hard-sphere model best fits the measured scattering intensities Adding chymosin to the dispersion mitiated a fractal flocculation of the micelles and consecutively a coalescence of the micelles This unexpected second process resembled that of spinodal demixing The dispersion phase thus separates into a water and a protein phase on a time scale of hours The observed phenomona contnbute to the understanding of the cheese-making process Fresh bovine milk contains 2 5% (by mass) casein which is associated in micelles The mam caseins components are found m a molar ratio of a sl a s2 (ft + T) K = 4 l 3 13 K-Casein plays a crucial role in the stabilization of the micelles The number of K-casem molecules/surface area is roughly constant although the micelle size vanes, mainly between \ 00 -250 nm m diameter Fairly detailed pictures of these micelles exist, but not all details are fully understood The basic model developed by Schmidt [1] was derived from electron microscopy studies It was suggested that cow milk casein micelles 'consisted of nearly spherical subunits with a diameter of 10 -15 nm' [1] Later Schmidt and Bucheim [2] made a more extensive electron microscopical study and reported size distributions for the different types of (sub)micelles between 5 -20 nm The picture was refined by Holt [3] and Walstra [4] who proposed a 'hairy' micelle model The hairs provide steric stabilization of the micelles and are identified as the K-casein The models of Schmidt, Holt and Walstra are essentially the same Differences are mainly topographical Their common feature is that the casein micelles show a highly regular conglomerate of highly uniform submicelles In neutron and Xray scattenng experiments such a conglomerate would give a very pronounced peak m the scattering intensities as a function of wave vector The small-angle neutron scattering (SANS) results of Stothart show only a very weak maximum in the structure factor Identifymg the position of this very broad peak to a correlation length seems justifiable, but it need not necessanly be the size of a submicelle It could well be the average size of condensed protein structures as is depicted by Griffin [7] The analysis of the small-angle X-ray data of Fessen et al is not correct since they add intensities instead of field strengths It thus seems that the picture of Griffin [7] is more realistic Enzyme Chymosin In all descnptions K-casem is situated at the surface of the micelle thus providing the 'mainly steric' stabilization The phenomenon of stenc stabilization is well understood (see e g Napper [9]) and plays an important role m other practical systems as ferro-fluids, black india mk, paint and the quality of motor oils The stabilization effect comes from the local increase m free energy where two particles meet The interpenetrating polymer layers cause a local increase in polymer segment density and thus a decrease in local entropy So quahtatively, the stabilizing effect of K-casem is understood and the influence of different conditions (e g pH, temperature and salt concentration) has been mvestigated by, for example, Schmidt and Koops [10] Several other studies have related the gelation of cow milk to the amount of K-casein on the surface of the micelles To our knowledge, there is to date no study about the strength and the range of the (mainly) steric barrier In view of the proposed flufïiness of the hairs and the probable presence of electncal charges, a so-called soft-sphere interaction is expected The aim of this paper was to study the interaction between K-casein 'hairs' usmg SANS In addition, the interaction precedmg and during the flocculation process when the hairs are cut by the enzyme chymosin was studied For this purpose submicelles of K-casem were prepared Such submicelles were formerly prepared and mvestigated by Vreeman et al Vreeman et al [12] suggested a model for the self-assembly of K-casein polymer chains based on placmg them on the surface of a sphere m such a way that each unit position is equivalent to all other positions Experimentally Vreeman et al found that the submicelles were practically monodisperse and contained on average 30 K-casein monomers Furthermore, Vreeman et al proposed a 'dense core' particle with the Cterrmnal part of the K-casem, usually called the glycomacropeptide part, danghng outwards Thurn et al came to essentially the same model m a SANS study at or below the cntical micelle concentration (which is about O 4% by mass at pH = 67) They, however, suggested that about 15
European Journal of Biochemistry
Small-angle neutron scattering (SANS) measurements on dilute and concentrated dispersions of K-casem micelles m a buffer at pH = 6 7 were made usmg the Dl l diffractometer m Grenoble Results indicate that the micelles have a dense core with a fluffy outer layer This outer layer appears to give rise to a steeply repulsive interaction on contact In fact, the hard-sphere model best fits the measured scattering intensities Adding chymosin to the dispersion mitiated a fractal flocculation of the micelles and consecutively a coalescence of the micelles This unexpected second process resembled that of spinodal demixing The dispersion phase thus separates into a water and a protein phase on a time scale of hours The observed phenomona contnbute to the understanding of the cheese-making process Fresh bovine milk contains 2 5% (by mass) casein which is associated in micelles The mam caseins components are found m a molar ratio of a sl a s2 (ft + T) K = 4 l 3 13 K-Casein plays a crucial role in the stabilization of the micelles The number of K-casem molecules/surface area is roughly constant although the micelle size vanes, mainly between \ 00 -250 nm m diameter Fairly detailed pictures of these micelles exist, but not all details are fully understood The basic model developed by Schmidt [1] was derived from electron microscopy studies It was suggested that cow milk casein micelles 'consisted of nearly spherical subunits with a diameter of 10 -15 nm' [1] Later Schmidt and Bucheim [2] made a more extensive electron microscopical study and reported size distributions for the different types of (sub)micelles between 5 -20 nm The picture was refined by Holt [3] and Walstra [4] who proposed a 'hairy' micelle model The hairs provide steric stabilization of the micelles and are identified as the K-casein The models of Schmidt, Holt and Walstra are essentially the same Differences are mainly topographical Their common feature is that the casein micelles show a highly regular conglomerate of highly uniform submicelles In neutron and Xray scattenng experiments such a conglomerate would give a very pronounced peak m the scattering intensities as a function of wave vector The small-angle neutron scattering (SANS) results of Stothart show only a very weak maximum in the structure factor Identifymg the position of this very broad peak to a correlation length seems justifiable, but it need not necessanly be the size of a submicelle It could well be the average size of condensed protein structures as is depicted by Griffin [7] The analysis of the small-angle X-ray data of Fessen et al is not correct since they add intensities instead of field strengths It thus seems that the picture of Griffin [7] is more realistic Enzyme Chymosin In all descnptions K-casem is situated at the surface of the micelle thus providing the 'mainly steric' stabilization The phenomenon of stenc stabilization is well understood (see e g Napper [9]) and plays an important role m other practical systems as ferro-fluids, black india mk, paint and the quality of motor oils The stabilization effect comes from the local increase m free energy where two particles meet The interpenetrating polymer layers cause a local increase in polymer segment density and thus a decrease in local entropy So quahtatively, the stabilizing effect of K-casem is understood and the influence of different conditions (e g pH, temperature and salt concentration) has been mvestigated by, for example, Schmidt and Koops [10] Several other studies have related the gelation of cow milk to the amount of K-casein on the surface of the micelles To our knowledge, there is to date no study about the strength and the range of the (mainly) steric barrier In view of the proposed flufïiness of the hairs and the probable presence of electncal charges, a so-called soft-sphere interaction is expected The aim of this paper was to study the interaction between K-casein 'hairs' usmg SANS In addition, the interaction precedmg and during the flocculation process when the hairs are cut by the enzyme chymosin was studied For this purpose submicelles of K-casem were prepared Such submicelles were formerly prepared and mvestigated by Vreeman et al Vreeman et al [12] suggested a model for the self-assembly of K-casein polymer chains based on placmg them on the surface of a sphere m such a way that each unit position is equivalent to all other positions Experimentally Vreeman et al found that the submicelles were practically monodisperse and contained on average 30 K-casein monomers Furthermore, Vreeman et al proposed a 'dense core' particle with the Cterrmnal part of the K-casem, usually called the glycomacropeptide part, danghng outwards Thurn et al came to essentially the same model m a SANS study at or below the cntical micelle concentration (which is about O 4% by mass at pH = 67) They, however, suggested that about 15
A novel application of neutron scattering on dairy products
Food Hydrocolloids, 2007
The successful application of spin echo small angle neutron scattering (SESANS) has been demonstrated for studying concentrated, turbid colloid suspensions with respect to their structure without dilution. The structure could be studied in a distance range between 50 nm and 3 mm. Data on the size distribution of casein micelles in non-diluted fat-free milk was obtained. This distribution is similar to earlier observations. The structure of casein gels resulting from processes aimed to simulate curdling (early stage of cheese making) and yoghurt production (acidification) was investigated and found to be not very different, the yoghurt gel being somewhat less dense. r
k -Casein micelles: structures, interaction, and gelling studied by small-angle neutron scattering
Small-angle neutron scattering (SANS) measurements on dilute and concentrated dispersions of K-casem micelles m a buffer at pH = 6 7 were made usmg the Dl l diffractometer m Grenoble Results indicate that the micelles have a dense core with a fluffy outer layer This outer layer appears to give rise to a steeply repulsive interaction on contact In fact, the hard-sphere model best fits the measured scattering intensities Adding chymosin to the dispersion mitiated a fractal flocculation of the micelles and consecutively a coalescence of the micelles This unexpected second process resembled that of spinodal demixing The dispersion phase thus separates into a water and a protein phase on a time scale of hours The observed phenomona contnbute to the understanding of the cheese-making process Fresh bovine milk contains 2 5% (by mass) casein which is associated in micelles The mam caseins components are found m a molar ratio of a sl a s2 (ft + T) K = 4 l 3 13 K-Casein plays a crucial role in the stabilization of the micelles The number of K-casem molecules/surface area is roughly constant although the micelle size vanes, mainly between \ 00 -250 nm m diameter Fairly detailed pictures of these micelles exist, but not all details are fully understood The basic model developed by Schmidt [1] was derived from electron microscopy studies It was suggested that cow milk casein micelles 'consisted of nearly spherical subunits with a diameter of 10 -15 nm' [1] Later Schmidt and Bucheim [2] made a more extensive electron microscopical study and reported size distributions for the different types of (sub)micelles between 5 -20 nm The picture was refined by Holt [3] and Walstra [4] who proposed a 'hairy' micelle model The hairs provide steric stabilization of the micelles and are identified as the K-casein The models of Schmidt, Holt and Walstra are essentially the same Differences are mainly topographical Their common feature is that the casein micelles show a highly regular conglomerate of highly uniform submicelles In neutron and Xray scattenng experiments such a conglomerate would give a very pronounced peak m the scattering intensities as a function of wave vector The small-angle neutron scattering (SANS) results of Stothart show only a very weak maximum in the structure factor Identifymg the position of this very broad peak to a correlation length seems justifiable, but it need not necessanly be the size of a submicelle It could well be the average size of condensed protein structures as is depicted by Griffin [7] The analysis of the small-angle X-ray data of Fessen et al is not correct since they add intensities instead of field strengths It thus seems that the picture of Griffin [7] is more realistic Enzyme Chymosin In all descnptions K-casem is situated at the surface of the micelle thus providing the 'mainly steric' stabilization The phenomenon of stenc stabilization is well understood (see e g Napper [9]) and plays an important role m other practical systems as ferro-fluids, black india mk, paint and the quality of motor oils The stabilization effect comes from the local increase m free energy where two particles meet The interpenetrating polymer layers cause a local increase in polymer segment density and thus a decrease in local entropy So quahtatively, the stabilizing effect of K-casem is understood and the influence of different conditions (e g pH, temperature and salt concentration) has been mvestigated by, for example, Schmidt and Koops [10] Several other studies have related the gelation of cow milk to the amount of K-casein on the surface of the micelles To our knowledge, there is to date no study about the strength and the range of the (mainly) steric barrier In view of the proposed flufïiness of the hairs and the probable presence of electncal charges, a so-called soft-sphere interaction is expected The aim of this paper was to study the interaction between K-casein 'hairs' usmg SANS In addition, the interaction precedmg and during the flocculation process when the hairs are cut by the enzyme chymosin was studied For this purpose submicelles of K-casem were prepared Such submicelles were formerly prepared and mvestigated by Vreeman et al Vreeman et al [12] suggested a model for the self-assembly of K-casein polymer chains based on placmg them on the surface of a sphere m such a way that each unit position is equivalent to all other positions Experimentally Vreeman et al found that the submicelles were practically monodisperse and contained on average 30 K-casein monomers Furthermore, Vreeman et al proposed a 'dense core' particle with the Cterrmnal part of the K-casem, usually called the glycomacropeptide part, danghng outwards Thurn et al came to essentially the same model m a SANS study at or below the cntical micelle concentration (which is about O 4% by mass at pH = 67) They, however, suggested that about 15
Structure of casein micelles studied by small-angle neutron scattering
European Biophysics Journal, 1996
The structure of casein micelles has been studied by small-angle neutron scattering and static light scattering. Alterations in structure upon variation of pH and scattering contrast, as well as after addition of chymosin, were investigated. The experimental data were analyzed by a model in which the casein micelle consists of spherical submicelles. This model gave good agreement with the data and gave an average micellar radius of about 100-120 nm and a submicellar radius of about 7 nm both with a polydispersity of about 40-50%. The contrast variation indicated that the scattering length density of the submicelles was largest at the center of the submicelles. The submicelles were found to be closely packed, the volume fraction varying slightly with pH. Upon addition of chymosin the submicellar structure remained unchanged within the experimental accuracy.
In the native bovine casein micelle the calcium sensitive caseins (α S1 -, α S2 -and β-casein) sequester amorphous calcium phosphate in nanometer-sized clusters, whereas the calcium-insensitive κ-casein limits the growth of the micelle. In this paper, we further investigate the self-association of κand β-casein, which are two of the key proteins that control the substructure of the milk casein micelle, using neutron and light scattering techniques and cryogenic transmission electron microscopy. Results demonstrate that κ-casein can, apart from the known self-assembly, form amyloid-like fibrils already at temperatures of 25°C when subject to agitation. This extended aggregation behavior of κ-casein is inhibited by β-casein, as reported by others. These findings have implications for the structure and stability of casein micelles. The neutron scattering data was used to gain information on the self-assembly structure of κcasein. β-Casein shows similar self-association behavior as κ-casein, but unlike κ-casein, the self-association exhibits temperature dependence within the studied temperatures (6 and 25°C). Here, we will discuss our extended study of the known self-assembly of casein in the context of the fibrillation of κ-casein.
Dynamics of casein micelles in skim milk during and after high pressure treatment
Food Chemistry, 2006
The effect of high hydrostatic pressure on turbidity of skim milk was measured in situ together with casein micelle size distribution. High pressure (HP) treatment reduced the turbidity of milk with a stronger pressure dependency between 50 and 300 MPa when the temperature was decreased from 20 to 5°C, while at 30°C (50-150 MPa) turbidity exceeded that of untreated milk. At 250 and 300 MPa turbidity decreased extremely. During pressurization of milk at 250 and 300 MPa, the turbidity initially decreased, but treatments longer than 10 min increased the turbidity progressively, indicating that re-association followed dissociation of casein micelles. Especially at 40°C and at 250 and 300 MPa, the turbidity increased beyond untreated milk. Dynamic light scattering was used to investigate casein micelle sizes in milk immediately after long time (up to 4 h) pressurization at 250 and 300 MPa and casein micelle size distributions were bimodal with micelle sizes markedly smaller and markedly larger than those of untreated milk. Pressure modified casein micelles present after treatment of milk at 250 and 300 MPa were concluded to be highly unstable, since the larger micelles induced by pressure showed marked changes toward smaller particle sizes in milk left at ambient pressure.
Substructure of bovine casein micelles by small-angle X-ray and neutron scattering
Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2003
The casein micelles of cow's milk are polydisperse, more-or-less spherical, protein particles of up to several hundred nanometer in size, containing about 7% by dry weight of calcium phosphate. Small-angle neutron scattering with contrast variation and small-angle X-ray scattering were used in critical tests of models of casein micelle substructure. An inflexion in the neutron scattering curve near Q 0/0.35 nm (1 was observed in heavy water which became a more pronounced subsidiary maximum at the match point of the protein. In water-rich buffers, where the contrast between protein and calcium phosphate is small, the inflexion was less apparent. The position of the inflexion and its variation in shape and relative importance with contrast matching are explained poorly, if at all, by the submicelle models of casein micelle substructure. However, the observations are explained by a model in which a relatively uniform protein matrix contains a disordered array of calcium phosphate ion clusters. A notable achievement of the model is the prediction of the position of the subsidiary maximum from independent measurements of the intrinsic viscosity of micelles, their mass fraction of calcium phosphate and the mass of the core of a calcium phosphate nanocluster. # (C. Holt).
Chemical Physics Letters, 2006
Small-angle neutron scattering (SANS) of solutions of glucose/xylose isomerase from Streptomyces rubiginosus was measured as a function of pressure. It is shown that the structure of the enzyme in solution as seen by SANS is practically the same as that in the crystal and does not change with pressure up to 150 MPa. This reflects the unusually high structural stability of this material, which makes it extremely interesting to use as a secondary standard for pressure-dependent SANS experiments. This lack of pressure dependence of the SANS data also indicates that any possible change in hydration of the protein induced by pressure is not visible in the SANS curves. An appropriate correction procedure must be used for the SANS data in order to account for the distortion of the intensity curve due to hard-sphere and electrostatic interactions. After this correction, the isomerase can be readily used as a secondary standard for SANS measurements. research papers J. Appl. Cryst. (2009). 42, 461-468 Ewa Banachowicz et al. Glucose isomerase conformation in solution 463
2. THE USE OF SMALL ANGLE NEUTRON SCATTERING FOR THE STUDY OF SOLUTIONS OF PROTEINS AND POLYMERS
2000
Aqueous solutions of polymers have excited some recent interest for their ability to solubilize otherwise poorly soluble species without the involvement of organic solvents. Such systems include cloud point extraction, micellar extraction, PEO-PPO co-polymer solutions, and aqueous biphasic systems (ABS). It is well known that the UV-Visible absorption spectra of a wide range of organic compounds show alterations in the position intensity and shape of the absorption bands in solvents of different polarity. Such effects have been used for many years to derive empirical scales (Linear Solvation free energy relationships LSERs) with which to relate the polarity of solvents to such diverse phenomena as chemical reactivity, quantitative structure activity relationships, and solvent extraction. In the latter case scales of solvent polarity have been established for a wide range of systems and the solvatochromic method has also recently been used for the characterization of micellar extraction systems. We report on the use of the wavelength shift of the absorption spectrum of Reichardt's carboxylated pyridinium N-phenoxide betaine dye in a PEG-2000/potassium triphosphate system. The behavior of this dye in both the monophasic region and in the separated phases from within the binodal curve will be discussed. The apparent polarity of the system as reported by the bathochromic shift of the dye will be discussed in the context of conventional solvents used in solvent extraction. The results will also be compared to similar determinations made in micellar and cloud point extraction systems.