Effect of Transglutaminase Post-Treatment on the Stability and Swelling Behavior of Casein Micro-Particles.

Casein microparticles are produced by flocculation of casein micelles due to volume exclusion of pectin and subsequent stabilization by film drying. Transglutaminase post-treatment alters their stability, swelling behavior, and internal structure. Untreated particles sediment due to their size and disintegrate completely after the addition of sodium dodecyl sulfate. The fact that transglutaminase-treated microparticles only sediment at comparable rates under these conditions shows that their structural integrity is not lost due to the detergent. Transglutaminase-treated particles reach an equilibrium final size after swelling instead of decomposing completely. By choosing long treatment times, swelling can also be completely suppressed as experiments at pH 11 show. In addition, deswelling effects also occur within the swelling curves, which enhance with increasing transglutaminase treatment time and are ascribed to the elastic network of cross-linked caseins. We propose a structural model for transglutaminase-treated microparticles consisting of a core of uncross-linked and a shell of cross-linked caseins. A dynamic model describes all swelling curves by considering both casein fractions in parallel. The characteristic correlation length of the internal structure of swollen casein microparticles is pH-independent and decreases with increasing transglutaminase treatment time, as observed also for the equilibrium swelling value of uncross-linked caseins.

Virus particle assembly into crystalline domains enabled by the coffee ring effect.

Tobacco mosaic virus particles can be rapidly assembled into 3D-domains by capillary flow-driven alignment at the triple contact-line of an evaporating droplet. Virus particles of ∼150 Šdiameter can be resolved within individual domains at the outer rim of the "coffee-ring" type residue by atomic force microscopy. The crystalline domains can also be probed by X-ray microdiffraction techniques. Both techniques reveal that the rod-like virus particles are oriented parallel to the rim. We further demonstrate the feasibility of collection of hk0 reflection intensities in GISAXS geometry and show it allows calculating a low-resolution electron density projection along the rod axis.

Stabilizing interactions of casein microparticles after a thermal post-treatment.

Casein microparticles from milk are important carrier materials for bioactive substances with stability and swelling properties that can be influenced by heat treatment. Microparticles produced by depletion flocculation and film drying remain stable in acidic media but swell and disintegrate under slightly alkaline conditions. Heat treatment after formation can stabilize the microparticles via a disulfide bridge network and newly formed hydrophobic contacts. Temperatures >60 °C are required so that denatured whey protein initiate formation of disulfide bridges via thiol exchange reactions. The particles then swell in a two-step process and exhibit an overshooting effect. If formation of disulphide bridges is prevented during heat treatment by adding N-methylmaleimide, overshooting swelling disappears and microparticles continue to expand instead. The analysis with parallel system dynamics models is based on the swelling of uncross-linked caseins, which is limited by the expansion capacity of cross-linked caseins.

Regenerated Fibers from Rennet-Treated Casein Micelles during Acidification.

Micellar casein fibers of defined size and internal structure can be produced by the extrusion of cold-renneted casein micelles into a warm, calcium-rich coagulation bath. Calcium phosphate contacts within the casein matrix are important for fiber stability and production but become less important under acidic pH conditions. We demonstrate this with swelling experiments in media with pH < 2, which we adjust with citric acid of different molarities. In contrast to the simple swelling of dried casein fibers in water, a two-phase process takes place in citric acid similar to swelling in 1 N HCl. However, instead of a second deswelling step, we observe in citric acid that the fiber swells further. The observation is explained by a pH-dependent transition from a rennet casein gel to an acidified rennet gel. This can be simulated with a kinetic model that couples two second-order rate equations via a time-varying ratio. The final swelling values decrease with increasing proton concentration via a scaling relation, which is also confirmed by swelling in other acids (HCl or acetic acid) in this pH range. We attribute this to a decrease in the molecular weights of the aggregated casein structures within the strands of the gel network.

Physical Characteristics of Egg Yolk Granules and Effect on Their Functionality.

Eggs are among the most nutritious foods in the world, a versatile ingredient in many food applications due to their functional attributes such as foaming, emulsifying, and coloring agents. Many studies have been reported on egg yolk fractionation and characterization in the last decade because of its nutritional and health benefits, especially egg yolk granules. This has led to the development of new food products and packaging materials. However, the influence of their physical characteristics during processing significantly impacts the functionality of yolk granules. In this overview, the egg yolk, the granule fraction's separation, fractionation, components, and molecular protein structure are first presented. Secondly, recent studies on egg yolk granules published over the past decade are discussed. Furthermore, the application of the granules in different industries and current specific scientific challenges are discussed. Finally, it simplifies the changes in the physical characteristics of the granules during different treatment methods and the impact on the functionalities of the resulting products in the food (emulsifiers, edible films), pharmaceutical, and health (encapsulation systems and biosensors) sectors.

Nearly Reversible Expansion and Shrinkage of Casein Microparticles Triggered by Extreme pH Changes.

Solvent flows in the fL/s range across the total surface of a casein microparticle cause its expansion and shrinkage. Microparticles prepared from the milk protein casein have a porous and flexible inner structure with water-filled channels and cavities. Solvent uptake occurs in two phases and results in disintegration if de-swelling is not triggered by acidification. So far, nothing is known about the reversibility of the swelling/de-swelling steps. We performed pH jump experiments between pH 11 and pH 1 on a single micro-particle and analyzed the swelling-induced size changes with system dynamics modeling. Both the swelling steps and the subsequent de-swelling process proceed reversibly and at an unchanged rate over a sequence of at least three pH exchange cycles. We observed that the duration of the first swelling step increased during the sequence, while the second step became shorter. Both of the time intervals are negatively correlated, while a statistical evaluation of only one swelling cycle for an ensemble of microparticles with different stabilities did not reveal any significant correlation between the two parameters. Our results indicate that the pH-induced swelling/shrinkage of casein microparticles is, to a large extent, reversible and only slightly influenced by the acid-induced decomposition of colloidal calcium phosphate.

Citrate effect on the swelling behaviour and stability of casein microparticles.

Casein microparticles obtained from casein micelles by volume exclusion of added pectin and subsequent film drying remain stable in the acidic and neutral pH range, but swell strongly in the basic range. Calcium significantly impacts on the stability and water-binding behavior of phosphorylated caseins and the aggregates and gels formed from them. For a future effective and controlled use as a carrier for bioactive substances, e.g. via the gastrointestinal tract, we therefore investigated how the addition of the calcium chelating agent citrate affects the swelling and stability of the microparticles. Citrate concentrations of 2 mM and above cause a stronger swelling of the microparticles at pH 8, while above 4 mM the second characteristic swelling step starts earlier and thus can also be investigated within the observation time of 120 min. All swelling kinetics can be simulated using seven parameters of a dynamic model, which reproduces the individual swelling steps via volume inflows and outflows into a reservoir. While the rate coefficient for swelling step 1 increases linearly with citrate concentration, no such dependence could be found for swelling step 2. The more citrate is used, the faster the microparticles decompose in turbidity experiments after the addition of sodium dodecyl sulfate, which can be related to a weakening of the hydrophobic interactions.

In situ muGISAXS: I. Experimental setup for submicron study of protein nucleation and growth.

In this study, we used microbeam grazing-incidence small-angle x-ray scattering (muGISAXS) to investigate in situ protein nucleation and crystal growth assisted by a protein nanotemplate, and introduced certain innovations to improve the method. Our aim was to understand the protein nanotemplate method in detail, as this method has been shown to be capable of accelerating and increasing crystal size and quality, as well as inducing crystallization of proteins that are not crystallizable by classical methods. The nanotemplate experimental setup was used for drops containing growing protein crystals at different stages of nucleation and growth. Two model proteins, lysozyme and thaumatin, were used under unique flow conditions to differentially probe protein crystal nucleation and growth.

In situ muGISAXS: II. Thaumatin crystal growth kinetic.

The formation of thaumatin crystals by Langmuir-Blodgett (LB) film nanotemplates was studied by the hanging-drop technique in a flow-through cell by synchrotron radiation micrograzing-incidence small-angle x-ray scattering. The kinetics of crystallization was measured directly on the interface of the LB film crystallization nanotemplate. The evolution of the micrograzing-incidence small-angle x-ray scattering patterns suggests that the increase in intensity in the Yoneda region is due to protein incorporation into the LB film. The intensity variation suggests several steps, which were modeled by system dynamics based on first-order differential equations. The kinetic data can be described by two processes that take place on the LB film, a first, fast, process, attributed to the crystal growth and its detachment from the LB film, and a second, slower process, attributed to an unordered association and conversion of protein on the LB film.

Influence of ß-lactoglobulin and calcium chloride on the molecular structure and interactions of casein micelles.

Targeted processing of casein micelles (CM) requires a basic understanding of their molecular structure as well as their interactions with each other and with other components. In this study, angle- and concentration-dependent static and dynamic light scattering is applied to investigate changes in the molecular weight, size, and intermolecular interactions of CM after the addition of ß-lactoglobulin (ß-Lg) and calcium chloride. Addition of a surplus of ß-Lg impairs the colloidal stability of CM. In the presence of 0.5wt% ß-Lg and natural calcium chloride concentrations (10mM), the molecular weight of CM is reduced and the radius of gyration is increased. Both changes can be explained by the release of αS2-casein and κ-casein, which were determined in higher concentration free in solution by High performance liquid chromatography. In contrast, the structure of casein micelles is not altered by the presence of ß-Lg at elevated calcium chloride concentrations. The repulsive forces between the CM show no significant dependence on ß-Lg for all calcium chloride concentrations tested.

The Combined Effect of High Hydrostatic Pressure and Calcium Salts on the Stability, Solubility and Gel Formation of ß-Lactoglobulin.

Stability, aggregation and gelation of ß-Lactoglobulin are affected by high pressure and salts of the Hofmeister series. Little is known about their combined effects on structure formation processes of ß-Lactoglobulin, mainly because many salts of the series are not suitable for use in food. Here, we investigate the effect of calcium salts on the strength of pressure-induced gels, inspired by the fact that high pressure and salts change the water structure in a similar way. We find that the larger the applied pressures, the higher the strength of the gels. In addition to pressure, there is a significant influence by the type of anions and the amount of added calcium salts. Gel strength increases in the order CaCl2 < Ca (NO3)2 < CaI2. This trend correlates with the position of the salts in the Hofmeister series. The results are explained by analogy with the thermal aggregate formation by taking reaction rates for unfolding and aggregation, as well as specific/non-specific salts effect into consideration.

Application of confocal Raman microscopy to investigate casein micro-particles in blend casein/pectin films.

Pectin triggers formation of casein micro-particles during solution casting. Confocal Raman microscopy revealed their composition and spatial dimension in resulting films. Peaks in the Raman spectra corresponded to those found in films prepared by either casein or pectin. This suggested that no conformational changes occurred after mixing. Raman images revealed incompatibility of both polymers because particles consisted of casein only and the surrounding matrix of pectin. Deformation of micro-particles into an oblate shape took place during film formation. In dried films, an empty space between casein and pectin was found in lateral dimension. In contrast, casein micro-particles overlapped with the pectin matrix in the vertical dimension.

Simulation of the shape and size of casein micelles in a film state.

Size fractionated casein micelles (CMs) form homogeneous films in which they are densely packed. The lateral size of CMs in films can be well resolved by surface-sensitive methods, but the estimation of their heights is still a challenge. We show that height information can be obtained from scattering patterns of GISAXS experiments on highly ordered casein films. We use an elastic scattering approach within the distorted wave Born approximation (DWBA) to simulate for the first time the two-dimensional intensity distribution of a GISAXS experiment of the CM near their critical angle. The model which fits the GISAXS data best considers an ellipsoidal form factor for the CM and an arrangement on a hexagonal lattice. Our results indicate that during film formation the spherical solution structure of CMs becomes compressed in the direction perpendicular to the film surface. In the film state, the micelles assume an oblate ellipsoidal shape with an aspect ratio of 1.9. Hence, their surface and contact area to the surrounding increases. As a result, the density of κ-casein on the micellar surface decreases, which could influence the functional properties of coatings and films.

Structure and stabilizing interactions of casein micelles probed by high-pressure light scattering and FTIR.

Caseins form heterogeneous micelles composed of three types of disordered protein chains (α, ß, κ), which include protein-bound calcium phosphate particles. We probe the stability limits of the micelle by applying hydrostatic pressure. The resulting changes of the size distribution and the average molecular weight are recorded in situ with static and dynamic light scattering. Pressure induces irreversible dissociation of the micelles into monomers above a critical value depending on their size. The critical pressure increases with temperature, pH, and calcium concentration due to the interplay of hydrophobic and electrostatic interactions. The pressure transition curves are biphasic, reflecting the equilibrium of two micelle states with different stability, average size, entropy, and calcium bound. The fast process of pressure dissociation is used to probe the slow equilibrium of the two micelle states under various conditions. Binding and release of ß-casein from the micelle is suggested as the molecular mechanism of stabilization associated with the two states. In situ FTIR spectroscopy covering the P-O stretching region indicates that bound calcium phosphate particles are released from serine phosphate residues at pressures above 100 MPa. The resulting imbalance of charge triggers the complete decomposition of the micelle.

Structural ordering of casein micelles on silicon nitride micro-sieves during filtration.

The paper reports on the structure and formation of casein micelle deposits on silicon nitride micro-sieves during the frontal filtration. The most frequent radius of the fractionated casein micelles we use is R=60 nm as detected by static light scattering (SLS) and atomic force microscopy (AFM). We estimate the size and size distribution of the casein micelles which pass through the micro-sieve during the filtration process. A sharpening of the size distribution at the beginning of the filtration process (t=40s) is followed by a broadening and a shift of the most frequent radii towards smaller sizes at later times (t=840 s). The size distribution of the micelles deposited on the micro-sieve during filtration is bimodal and consists of the largest and smallest micelles. At larger filtration times, we observe a shift of both deposited size classes towards smaller sizes. The atomic force micrographs of the reference sample reveal a tendency of the casein micelles to order in a hexagonal lattice when deposited on the micro-sieves by solution casting. The deposition of two size classes can be explained by a formation of a mixed hexagonal lattice with large micelles building up the basis lattice and smaller sizes filling octahedral and tetrahedral holes of the lattice. The accompanied compression with increasing thickness of the casein layer could result from preferential deposition of smaller sizes in the course of the filtration.

Characterization of the boundary zone of a cast protein drop: fibroin beta-sheet and nanofibril formation.

We have studied a cast fibroin drop with grazing incidence small-angle X-ray scattering, imaging, and spectroscopy techniques. Optical microscopy shows that the dried drop forms a boundary zone. Grazing-incidence small-angle X-ray scattering performed with a synchrotron radiation microbeam in the boundary zone suggests the formation of nanometer-sized domains with one-dimensional paracrystalline order. Atomic force microscopy and scanning electron microscopy support a nanofibrillar morphology. MicroRaman spectroscopy shows the formation of beta-sheet secondary structure in the boundary zone, which is attributed to a shearing effect due to the retraction of the drop boundary upon evaporation.

Structural changes of casein micelles in a calcium gradient film.

Calcium gradients are prepared by sequentially filling a micropipette with casein solutions of varying calcium concentration and spreading them on glass slides. The casein film is formed by a solution casting process, which results in a macroscopically rough surface. Microbeam grazing incidence small-angle X-ray scattering (microGISAXS) is used to investigate the lateral size distribution of three main components in casein films: casein micelles, casein mini-micelles, and micellar calcium phosphate. At length scales within the beam size the film surface is flat and detection of size distribution in a macroscopic casein gradient becomes accessible. The model used to analyze the data is based on a set of three log-normal distributed particle sizes. Increasing calcium concentration causes a decrease in casein micelle diameter while the size of casein mini-micelles increases and micellar calcium phosphate particles remain unchanged.

Size distribution of pressure-decomposed casein micelles studied by dynamic light scattering and AFM.

Reversible and irreversible states of pressure-dissociated casein micelles were studied by in situ light scattering techniques and ex situ atomic force microscopy. AFM experiments performed at ambient pressure reveal heterogeneities across the micelle, suggesting a sub-structure on a 20 nm scale. At pressures between 50 and 250 MPa, the native micelles disintegrate into small fragments on the scale of the observed sub-structure. At pressures above 300 MPa the micelles fully decompose into their monomeric constituents. After pressure release two discrete populations of casein aggregates are observed, depending on the applied initial pressure: Between 160 and 240 MPa stable micelles with diameters near 100 nm without detectable sub-structures are formed. Casein micelles exposed to pressures above 280 MPa re-associate at ambient pressure yielding mini-micelles with diameters near 25 nm. The implications concerning structural models are discussed.