Native disulphide-linked dimers facilitate amyloid fibril formation by bovine milk αS2-casein.

Bovine milk αS2-casein, an intrinsically disordered protein, readily forms amyloid fibrils in vitro and is implicated in the formation of amyloid fibril deposits in mammary tissue. Its two cysteine residues participate in the formation of either intra- or intermolecular disulphide bonds, generating monomer and dimer species. X-ray solution scattering measurements indicated that both forms of the protein adopt large, spherical oligomers at 20 °C. Upon incubation at 37 °C, the disulphide-linked dimer showed a significantly greater propensity to form amyloid fibrils than its monomeric counterpart. Thioflavin T fluorescence, circular dichroism and infrared spectra were consistent with one or both of the dimer isomers (in a parallel or antiparallel arrangement) being predisposed toward an ordered, amyloid-like structure. Limited proteolysis experiments indicated that the region from Ala81 to Lys113 is incorporated into the fibril core, implying that this region, which is predicted by several algorithms to be amyloidogenic, initiates fibril formation of αS2-casein. The partial conservation of the cysteine motif and the frequent occurrence of disulphide-linked dimers in mammalian milks despite the associated risk of mammary amyloidosis, suggest that the dimeric conformation of αS2-casein is a functional, yet amyloidogenic, structure.

Influence of Microbial Transglutaminase on Physicochemical and Cross-Linking Characteristics of Individual Caseins.

The effects of microbial transglutaminase (MTGase) cross-linking on the physicochemical characteristics of individual caseins were investigated. MTGase was used to modify three major individual caseins, namely, κ-casein (κ-CN), αS-casein (αS-CN) and ß-casein (ß-CN). The SDS-PAGE analysis revealed that MTGase-induced cross-linking occurred during the reaction and that some components with high molecular weights (>130 kDa) were formed from the individual proteins κ-CN, αS-CN and ß-CN. Scanning electron microscopy (SEM) and particle size analysis respectively demonstrated that the κ-CN, αS-CN and ß-CN particle diameters and protein microstructures were larger and polymerized after MTGase cross-linking. The polymerized κ-CN (~749.9 nm) was smaller than that of ß-CN (~7909.3 nm) and αS-CN (~7909.3 nm). The enzyme kinetics results showed KM values of 3.04 × 10-6, 2.37 × 10-4 and 8.90 × 10-3 M for κ-CN, αS-CN and ß-CN, respectively, and, furthermore, kcat values of 5.17 × 10-4, 1.92 × 10-3 and 4.76 × 10-2 1/s, for κ-CN, αS-CN and ß-CN, respectively. Our results revealed that the cross-linking of ß-CN catalyzed by MTGase was faster than that of αS-CN or κ-CN. Overall, the polymers that formed in the individual caseins in the presence of MTGase presented a higher molecular weight and larger particles.

Progressive ultrastructural changes in the casein matrix during the ripening of inadequately acidified feta cheese.

This study investigated the ultrastructural changes underlying the undesired softening of insufficiently acidified feta cheese during cold storage. Experimental feta cheeses with a range of pH values before brining were manufactured by allowing the cheese blocks to ferment overnight at 3 temperatures (35, 20, and 3°C), which resulted in pH values of 4.80, 4.88, and 5.17, respectively. Cheese blocks were stored in pH-adjusted whey brine solutions for up to 120 d, at which point significant decreases in the cheese firmness were confirmed with compression and shear tests. Samples for transmission electron microscopy were taken during the make procedure, after overnight fermentation, and after 7 and 90 d of cold storage. Increasing the initial pH from 4.80 to 5.17 resulted in a fundamentally different ultrastructure at d 90, with the protein matrix as the continuous phase having markedly decreased density compared with the typically open porous and discontinuous protein matrix of high density in the low-pH control feta cheese. Ultrastructural changes were progressive, and the first signs were evident after only 20 h (the overnight fermentation), when fine, proteinaceous material dissociated from the edges of the casein strands into the serum phase. By d 7, the serum phase was completely filled with the loosely aggregated casein closely surrounding the spheroidal fat globules. A further breakdown of the protein matrix was observed after 90 d, with the complete loss of open porous network structure. Image analysis quantitatively confirmed the progressive and significant decrease in density of the protein matrix. In summary, this is the first study to provide a comprehensive and in-depth view of the progressive and most likely irreversible ultrastructural changes that lead to this textural defect.

Changes in chemical and ultrastructural composition of ameroid constrictors following in vitro expansion.

OBJECTIVE: To (1) characterise the chemical and ultra-structural composition of ameroid constrictors, at a native state and during in vitro expansion and (2) determine the presence of irritant compounds at the surface or within the bulk of the constrictor. METHODS: Twelve sterile, commercially packaged ameroid constrictors (3 repeats of 3.5 mm, 5 mm, 6 mm and 7 mm internal diameter) were analysed by time-of-flight secondary ion mass spectrometry, Raman spectroscopy, attenuated total reflectance Fourier transform infrared spectroscopy and scanning electron microscopy. RESULTS: Ameroid constrictors have a composition commensurate with casein with little-to-no intra- or inter- constrictor variation. Microscopic analysis indicated that the topographical features of the constrictor surfaces were consistent between all constrictors. Following in vitro expansion there was a reproducible decrease in Ca+ ion content, little-to-no variation in secondary protein structure and morphological changes including the presence of surface aggregates present only at the inner surface of the ameroid constrictor. The potential irritant polydimethylsiloxane was found on the constrictor surface. A trace quantity of an ion fragment assigned as formaldehyde was detected; however, the extremely low level is thought highly unlikely to play a role as an inflammatory trigger clinically. DISCUSSION: There is a high degree of inter- and intra-constrictor homogeneity from different batches, and reproducible ultrastructural changes following in vitro expansion. Variations occur in both the surface chemistry and topography of the device during closure, which can potentially affect the biomaterial-host interface. Ameroid constrictor closure mechanism is likely involving calcium-mediated inter-protein interactions rather than the imbibition of water only.

Study of interactions between anionic exopolysaccharides produced by newly isolated probiotic bacteria and sodium caseinate.

The present study aims to evaluate the interactions between four exopolysaccharides (EPS) produced by probiotic bacteria and sodium caseinate (Cas) in order to simulate their behavior in dairy products. Complexation between the produced EPS samples and Cas was investigated as a function of polysaccharide to protein ratio. The highest turbidity and average size of complexes were formed at an EPS/Cas ratio of 3 (corresponding to 1 g/L of EPS and 0.33 g/L of Cas) as a result of the combination of individual complexes to form aggregates. Zeta potential measurements and Cas surface hydrophobicity results suggested that complex formation occurred essentially through electrostatic attractions with a possible contribution of hydrophobic interaction for EPS-GM which was produced by Bacillus tequilensis-GM. Afterwards, the effect of pH on the complexation between biopolymers was studied when EPS and Cas concentrations were maintained constant at 1 and 0.33 g/L, respectively. pH was adjusted to 3.0 and 3.5, respectively. Results showed that the highest amount and sizes of EPS/Cas complexes were formed at pH 3.5 and that EPS-GM enabled to obtain the biggest and highest amount of aggregates. Therefore, the obtained results support the fact that the simultaneous presence of EPS and Cas in dairy products results in complexes formation via electrostatic interactions depending on EPS/Cas ratio and pH of the medium.

High internal phase emulsion (HIPE)-templated biopolymeric oleofilms containing an ultra-high concentration of edible liquid oil.

We report, for the first time, the fabrication of oleofilms (containing more than 97 wt% edible liquid oil) using high internal phase emulsions (with oil volume fraction φoil = 0.82) as templates. Advanced microscopy studies revealed an interesting microstructure of these films where jammed oil droplets were embedded in a dried matrix of biopolymeric complexes.

Shear Bond Strength and Remineralisation Effect of a Casein Phosphopeptide-Amorphous Calcium Phosphate-Modified Glass Ionomer Cement on Artificial "Caries-Affected" Dentine.

This study investigated the effect of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP)-modified glass ionomer cement (GIC) on shear bond strength (SBS) and remineralisation of artificial "caries-affected" dentine. Human dentine slices were demineralised and allocated to three groups: group 1, conventional GIC; group 2, CPP-ACP-modified GIC; and group 3, resin-modified GIC. The SBS was measured using a universal testing machine (n = 16 per group). Remaining samples (n = 8 per group) were subjected to pH-cycling for 28 days. After pH-cycling, lesion depth and micro-mechanical properties at the sample-bonding interface were investigated using micro-computed tomography (micro-CT) and nano-indentation, respectively. The SBS for groups 1 to 3 were 4.6 ± 1.5 MPa, 4.2 ± 1.1 MPa, and 5.9 ± 1.9 MPa, respectively (p = 0.007; group 1, group 2 < group 3). Lesion depths determined by micro-CT for groups 1 to 3 were 186 ± 8 µm, 149 ± 14 µm, and 178 ± 8 µm, respectively (p < 0.001; group 2 < group 1, group 3). The mean (±SD, standard deviation) nano-hardness values for groups 1 to 3 were 0.85 ± 0.22 GPa, 1.14 ± 0.21 GPa, and 0.81 ± 0.09 GPa, respectively (p = 0.003; group 1, group 3 < group 2). The mean (±SD) elastic moduli for groups 1 to 3 were 1.70 ± 0.33 GPa, 2.35 ± 0.44 GPa, and 1.59 ± 0.13 GPa, respectively (p < 0.001; group 1, group 3 < group 2). The results suggest that the incorporation of CPP-ACP into GIC does not adversely affect the adhesion to artificial caries-affected dentine. Furthermore, CPP-ACP-modified GIC is superior to conventional GIC in promoting dentine remineralisation.

Nanoscale observation of the natural structure of milk-fat globules and casein micelles in the liquid condition using a scanning electron assisted dielectric microscopy.

Recently, aqueous nanoparticles have been used in drug-delivery systems for new type medicines. In particular, milk-casein micelles have been used as drug nanocarriers for targeting cancer cells. Therefore, nanostructure observation of particles and micelles in their native liquid condition is indispensable for analysing their function and mechanisms. However, traditional optical and scanning electron microscopy have difficulty observing the nanostructures of aqueous micelles. Recently, we developed a novel imaging technique called scanning electron-assisted dielectric microscopy (SE-ADM) that enables observation of various biological specimens in water with very little radiation damage and high-contrast imaging without staining or fixation at an 8-nm spatial resolution. In this study, for the first time, we show that the SE-ADM system is capable of high-resolution observation of whole-milk specimens in their natural state. Moreover, we successfully observe the casein micelles and milk-fat globules in an intact liquid condition. Our SE-ADM system can be applied to various biological particles and micelles in a native liquid state.

Solubilization of genistein by caseinate micellar system.

This study investigates the aggregation behavior of caseinate and the solubilization of genistein in aqueous caseinate solution. The critical aggregation concentration (CAC) of caseinate was obtained from the fluorescence intensity of 8-anilino-1-naphthalenesulfonic acid (ANS), which was enhanced by ANS-protein interactions and the hydrophobicity of caseinate. The increasing solubility of genistein in caseinate was confirmed by HPLC measurements; above and below the CAC, the genistein/caseinate molar ratio is 1:1 and 10:1, respectively. The latter ratio indicates that more caseinate molecules surround genistein below the CAC. However, the solubility of genistein in caseinate is unaffected by calcium ions. Atomic force microscopy (AFM) shows that casein sub-micelles are similarly structured in the presence and absence of genistein. In AFM phase images, the caseinate sub-micelle is brightened in the presence of genistein, implying that the particle becomes more rigid, probably because genistein attaches to the surface or to the narrow part of the sub-micelle. The diameter of sub-micelle aggregates is two times that of caseinate alone (24 nm versus 12 nm). These results were confirmed by cryo-TEM observations.

Properties of an αs-casein-rich casein fraction: influence of dialysis on surface properties, miscibility, and micelle formation.

In this study, the surface tension, miscibility, and particle size distribution of a solution containing an αs-casein (CN)-rich CN fraction (54 wt % αs-CN, 32 wt % ß-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.

Microwave initiated synthesis of polyacrylamide grafted casein (CAS-g-PAM)--its application as a flocculant.

Modification of the milk protein 'casein' has been carried out through microwave initiated graft copolymerization of acrylamide. The synthesis was optimized in terms of microwave irradiation time and monomer (acrylamide) concentration. The synthesized graft copolymers have been characterized by intrinsic viscosity measurement, elemental analysis, FTIR spectroscopy, scanning electron microscopy (SEM) and number average molecular weight determination through osmometry; taking the starting material (casein) as reference. Further, flocculation efficacy of various grades of the grafted product were studied and compared to that of the starting material 'casein' by Jar test and settling test procedure, in 1% coal-fine suspension.

Amyloid fibrils from readily available sources: milk casein and lens crystallin proteins.

Amyloid fibrils are a highly ordered and robust aggregated form of protein structure in which the protein components are arranged in long fibrillar arrays comprised of ß-sheet. Because of these properties, along with their biocompatibility, amyloid fibrils have attracted much research attention as bionanomaterials, for example as template structures (in some cases following modification) that can be used as biosensors, encapsulators, and biomimetic materials. To use amyloid fibrils for such a range of applications will require them to be obtained relatively easily in large quantities. In this chapter, we describe methods for isolating crystallin and casein proteins from readily available sources that contain abundant protein, i.e., the eye lens and milk, respectively, and the subsequent conversion of these proteins into amyloid fibrils.

Analysis of casein biopolymers adsorption to lignocellulosic biomass as a potential cellulase stabilizer.

Although lignocellulosic materials have a good potential to substitute current feedstocks used for ethanol production, conversion of these materials to fermentable sugars is still not economical through enzymatic hydrolysis. High cost of cellulase has prompted research to explore techniques that can prevent from enzyme deactivation. Colloidal proteins of casein can form monolayers on hydrophobic surfaces that alleviate the de-activation of protein of interest. Scanning electron microscope (SEM), fourier transform infrared spectroscopy (FT-IR), capillary electrophoresis (CE), and Kjeldahl and BSA protein assays were used to investigate the unknown mechanism of action of induced cellulase activity during hydrolysis of casein-treated biomass. Adsorption of casein to biomass was observed with all of the analytical techniques used and varied depending on the pretreatment techniques of biomass. FT-IR analysis of amides I and II suggested that the substructure of protein from casein or skim milk were deformed at the time of contact with biomass. With no additive, the majority of one of the cellulase mono-component, 97.1 ± 1.1, was adsorbed to CS within 24 h, this adsorption was irreversible and increased by 2% after 72 h. However, biomass treatment with skim-milk and casein reduced the adsorption to 32.9% ± 6.0 and 82.8% ± 6.0, respectively.

Microencapsulation of conjugated linolenic acid-rich pomegranate seed oil by an emulsion method.

Controlled release of food ingredients and their protection from oxidation are the key functionality provided by microencapsulation. In the present study, pomegranate seed oil, rich in conjugated linolenic acid, was microencapsulated. As encapsulating agent, sodium alginate or trehalose was used. Calcium caseinate was used as the emulsifier. Performances of the two encapsulants were compared in respect of the rate of release of core material from the microcapsules and stability of microcapsules against harsh conditions. Microencapsulation was carried out by preparation of an emulsion containing calcium caseinate as the emulsion stabilizer and a water-soluble carbohydrate (either sodium alginate or trehalose) as the encapsulant. An oil-in-water emulsion was prepared with pomegranate seed oil as the inner core material. The emulsion was thereby freeze-dried and the dried product pulverized. External morphology of the microcapsules was studied under scanning electron microscope. Micrographs showed that both types of microcapsules had uneven surface morphology. Release rate of the microcapsules was studied using UV-spectrophotometer. Trehalose-based microcapsules showed higher release rate. On subjecting the microcapsules at 110 °C for specific time periods, it was observed that sodium alginate microcapsules retained their original properties. Hence, we can say that sodium alginate microcapsules are more heat resistant than trehalose microcapsules.

Structural and thermal investigations of biomimetically grown casein-soy hybrid protein fibers.

A hybrid protein fiber from different protein sources such as casein and soybean using wet-spinning technique was prepared. The casein/soybean hybrid fibers were synthesized at different weight ratios such as 100/0 (casein), 75/25, 50/50, 25/75, and 0/100 (soy) and characterized. Electron microscopic analysis confirmed the growth of pure and hybrid fibers and shows an increased surface roughness as the soy concentration increases in the hybrid fibers. Infrared spectra did not exhibit any significant changes in the functional groups between pure and hybrid fibers. X-ray diffraction pattern indicates slight increase in the diffraction peak values of hybrid fibers compared with the neat fibers. Thermal analyses show a moderate increase in the thermal stability of hybrid fibers when compared with the pure fibers. These results implicitly indicate that the casein and soy proteins are homogeneous in the hybrid fiber form. It has been demonstrated that the hybrid fiber with ≥50 wt.% casein content exhibits better morphology and increased thermal stability, which has scope for application in technical and medical industries.

A new cryo-STEM method for imaging food colloids in the scanning electron microscope.

A new cryo-scanning transmission electron microscopy (cryo-STEM) technique for imaging casein micelles in a field emission scanning electron microscope is presented. Thin films of micellar casein suspensions on lacey carbon grids were prepared using a modified sample holder developed by Gatan UK. Bright and dark field images were obtained at -135°C showing casein micelles in their frozen hydrated state and in the size range 30-500 nm. Results were compared favorably with published images of casein micelles obtained with conventional cryo-transmission electron microscopy, suggesting that cryo-STEM is a useful alternative technique for visualizing food colloids close to their native state.

The dissociated form of kappa-casein is the precursor to its amyloid fibril formation.

Bovine milk kappa-casein forms a self-associating oligomeric micelle-like species, in equilibrium with dissociated forms. In its native form, intra- and inter-molecular disulfide bonds lead to the formation of multimeric species ranging from monomers to decamers. When incubated under conditions of physiological pH and temperature, both reduced and non-reduced kappa-casein form highly structured beta-sheet amyloid fibrils. We investigated whether the precursor to kappa-casein fibril formation is a dissociated state of the protein or its oligomeric micelle-like form. We show that reduced kappa-casein is capable of forming fibrils well below its critical micelle concentration, i.e. at concentrations where only dissociated forms of the protein are present. Moreover, by regulating the degree of disulfide linkages, we were able to investigate how oligomerization of kappa-casein influences its propensity for fibril formation under conditions of physiological pH and temperature. Thus, using fractions containing different proportions of multimeric species, we demonstrate that the propensity of the disulfide-linked multimers to form fibrils is inversely related to their size, with monomeric kappa-casein being the most aggregation prone. We conclude that dissociated forms of kappa-casein are the amyloidogenic precursors to fibril formation rather than oligomeric micelle-like species. The results highlight the role of oligomerization and natural binding partners in preventing amyloid fibril formation by disease-related proteins in vivo.

Microstructural changes in casein supramolecules during acidification of skim milk.

Pasteurized skim milk was acidified using different levels of glucono-delta-lactone at 10, 20, 30, and 40 degrees C to give slow, medium, and fast rates of acidification. Milk coagulation was monitored by measuring turbidity and curd firmness, and microstructural changes during acidification were observed on glutaraldehyde-fixed, agar-solidified milk samples using transmission electron microscopy. Rate of acidification had little influence on changes observed during acidification, except at 10 degrees C. At 40 degrees C, the casein supramolecules were spherical throughout acidification, whereas at lower temperatures they became progressively more ragged in appearance. All of the milks gelled at the same pH (pH 4.8), as measured by curd firmness, whereas increases in turbidity, assumed to be brought about by an increase in number of light-scattering particles, were observed to start at about pH 5.2 to 5.4. As the milk was acidified, aggregates of loosely entangled proteins were observed, presumably originating from proteins that had dissociated from the casein supramolecules. These aggregates were often as large as the casein supramolecules, particularly as the pH of the milk approached the isoelectric point of the caseins. Larger aggregates were observed at 40 degrees C than at the lower temperatures, suggesting the involvement of hydrophobic interactions between the proteins. A 3-phase model for acid-induced gelation of milk is proposed in which the first phase involves temperature-dependent dissociation of proteins from the casein supramolecules, with more dissociation occurring as temperature is decreased. Dissociation continues as milk pH is lowered, with the released proteins forming into loosely entangled aggregates, some as large as the casein supramolecules. The second phase of acid gelation of milk occurs between pH 5.3 and pH 4.9 and involves a reassociation of proteins with loosely entangled protein aggregates forming into more-compact colloidal particles or combining with any remaining casein supramolecules. The third and final phase involves rapid aggregation of the colloidal casein supramolecules into a gel network at about pH 4.8. Different gel structures were formed based on temperature of acidification, with a coarse-stranded gel network formed at 40 degrees C and a fine-stranded gel network at 10 degrees C.

Stretched extracellular matrix proteins turn fouling and are functionally rescued by the chaperones albumin and casein.

While evidence is mounting that cells exploit protein unfolding for mechanochemical signal conversion (mechanotransduction), what mechanisms are in place to deal with the unwanted consequences of exposing hydrophobic residues upon force-induced protein unfolding? Here, we show that mechanical chaperones exist that can transiently bind to hydrophobic residues that are freshly exposed by mechanical force. The stretch-upregulated binding of albumin or casein to fibronectin fibers is reversible and does not inhibit fiber contraction once the tension is released.

Kinetics of fibril formation of bovine kappa-casein indicate a conformational rearrangement as a critical step in the process.

S-carboxymethylated (SCM) kappa-casein forms in vitro fibrils that display several characteristics of amyloid fibrils, although the protein is unrelated to amyloid diseases. In order to get insight into the processes that prevent the formation of amyloid fibrils made of kappa-caseins in milk, we have characterized in detail the reaction and the roles of its possible effectors: glycosylation and other caseins. Given that native kappa-casein occurs as a heterogeneous mixture of carbohydrate-free and carbohydrate-containing chains, kinetics of fibril formation were performed on purified glycosylated and unglycosylated SCM kappa-caseins using the fluorescent dye thioflavin T in conjunction with transmission electron microscopy and Fourier transform infrared spectroscopy for morphological and structural analyses. Both unglycosylated and glycosylated SCM kappa-caseins have the ability to fibrillate. Kinetic data indicate that the fibril formation rate increases with SCM kappa-casein concentration but reaches a plateau at high concentrations, for both the unglycosylated and glycosylated forms. Therefore, a conformational rearrangement is the rate-limiting step in fibril growth of SCM kappa-casein. Transmission electron microscopy images indicate the presence of 10- to 12-nm spherical particles prior to the appearance of amyloid structure. Fourier transform infrared spectroscopy spectra reveal a conformational change within these micellar aggregates during the fibrillation. Fibrils are helical ribbons with a pitch of about 120-130 nm and a width of 10-12 nm. Taken together, these findings suggest a model of aggregation during which the SCM kappa-casein monomer is in rapid equilibrium with a micellar aggregate that subsequently undergoes a conformational rearrangement into a more organized species. These micelles assemble and this leads to the growing of amyloid fibrils. Addition of alpha(s1)-and beta-caseins decreases the growth rate of fibrils. Their main effect was on the elongation rate, which became close to that of the limiting conformation change, leading to the appearance of a lag phase at the beginning of the kinetics.