Effect of composition, casein genetic variants and glycosylation degree on bovine milk whipping properties.

This study investigated the individual and combined effects of ĸ-Casein (ĸ-CN; AA, AB, BB), ß-Casein (ß-CN; A1A1, A1A2, A2A2) and high and low ratios of glycosylated ĸ-CN to total ĸ-CN, referred to as the glycosylation degree (GD), on bovine cream whipping properties. The genetic variants of individual cows were identified using reversed-phase high-performance liquid chromatography (RP-HPLC) and verified through liquid chromatography-mass spectrometry (LC-MS). A previously discovered relationship between days-in-milk and GD was validated and used to obtain high and low GD milk. Whipped creams were created through the mechanical agitation of fat standardised cream from milk of different ĸ-CN, ß-CN, and GD combinations, and whipping properties (the ability to whip, overrun, whipping time and firmness) were evaluated. No significant correlation was measured in whipping properties for cream samples from milks with different ĸ-CN and ß-CN genetic variants. However, 80 % of samples exhibiting good whipping properties (i.e., the production of a stiffened peak) were from milk with low GD suggesting a correlation between whipping properties and levels of glycosylation. Moreover, cream separated from skim milk of larger casein micelle size showed superior whipping properties with shorter whipping times (<5 min), and higher firmness and overrun. Milk fat globule (MFG) size, on the other hand, did not affect whipping properties. Results indicate that the GD of κ-CN and casein micelle size may play a role in MFG adsorption at the protein and air interface of air bubbles formed during whipping; hence, they govern the dynamics of fat network formation and influencing whipping properties.

Structure of biomimetic casein micelles: Critical tests of the hydrophobic colloid and multivalent-binding models using recombinant deuterated and phosphorylated ß-casein.

Milk contains high concentrations of amyloidogenic casein proteins and is supersaturated with respect to crystalline calcium phosphates such as apatite. Nevertheless, the mammary gland normally remains unmineralized and free of amyloid. Unlike κ-casein, ß- and αS-caseins are highly effective mineral chaperones that prevent ectopic and pathological calcification of the mammary gland. Milk invariably contains a mixture of two to five different caseins that act on each other as molecular chaperones. Instead of forming amyloid fibrils, several thousand caseins and hundreds of nanoclusters of amorphous calcium phosphate combine to form fuzzy complexes called casein micelles. To understand the biological functions of the casein micelle its structure needs to be understood better than at present. The location in micelles of the highly amyloidogenic κ-casein is disputed. In traditional hydrophobic colloid models, it, alone, forms a stabilizing surface coat that also determines the average size of the micelles. In the recent multivalent-binding model, κ-casein is present throughout the micelle, in intimate contact with the other caseins. To discriminate between these models, a range of biomimetic micelles was prepared using a fixed concentration of the mineral chaperone ß-casein and nanoclusters of calcium phosphate, with variable concentrations of κ-casein. A biomimetic micelle was also prepared using a highly deuterated and in vivo phosphorylated recombinant ß-casein with calcium phosphate and unlabelled κ-casein. Neutron and X-ray scattering experiments revealed that κ-casein is distributed throughout the micelle, in quantitative agreement with the multivalent-binding model but contrary to the hydrophobic colloid models.

The role of glycosylation in amyloid fibril formation of bovine κ-casein.

In order to explore the functions of glycosylation of κ-Casein (κ-CN) in bovine milk, unglycosylated (UG) and twice glycosylated (2G) forms of κ-CN B were purified by selective precipitation followed by anion exchange chromatography from κ-CN BB milk and tested for their amyloid fibril formation and morphology, oligomerisation states and protein structure. The diameter of self-assembled κ-CN B aggregates of both glyco-form were shown for the first time to be in the same 26.0-28.7 nm range for a 1 mg mL-1 solution. The presence of two bound glycans in the protein structure of 2G κ-CN B led to a greater increase in the maximum amyloid fibril formation rate with increasing protein concentration and a difference in both length (82.0 ± 29.9 vs 50.3 ± 13.7 nm) and width (8.6 ± 2.1 vs 13.9 ± 2.5 nm) for fibril morphology compared to UG κ-CN B. The present results suggest that amyloid fibril formation proceeds at a slow but steady rate via the self-assembly of dissociated, monomeric κ-CN B proteins at concentrations of 0.22-0.44 mg mL-1. However amyloid fibril formation proceeds more rapidly via the assembly of either aggregated κ-CN present in a micelle-like form or dissociated monomeric κ-CN, packed into reorganised formational structures above the critical micellar concentration to form fibrils of differing width. The degree of glycosylation has no effect on the polarity of the adjacent environment, nor non-covalent and disulphide interactions between protein molecules when in the native form. Yet glycosylation can influence protein folding patterns of κ-CN B leading to a reduced tryptophan intrinsic fluorescence intensity for 2G compared to UG κ-CN B. These results demonstrate that glycosylation plays an important role in the modulation of aggregation states of κ-CN and contributes to a better understanding of the role of glycosylation in the formation of amyloid fibrils from intrinsically disordered proteins.

Effects of Raw and Pasteurized Camel Milk on Metabolic Responses in Pigs Fed a High-Fat Diet.

Evidence suggests that camel milk (CM) can have insulin-like actions, although the mode of action is not understood. Using the pig as a monogastric model, this pilot experiment examined the effects of CM consumption on metabolic responses to an in vitro glucose tolerance test (IVGTT). Twenty female Large White × Landrace pigs were individually housed for 6 wks and randomly allocated to one of the following four diets (fed ad libitum; n = 5): control (Con); high fat (HF; ~16% fat); raw CM (the HF diet plus 500 mL CM/ day); or pasteurized CM (PCM). Blood samples were collected on two occasions (weeks 2 and 5). At week 6, the pigs were fitted with an ear vein cannula and the following day an in vitro glucose tolerance test (IVGTT) was conducted (0.3 g/kg BW glucose). Plasma fatty acids and cholesterol concentrations were greater in the pigs fed the HF diet and greatest in those fed CM, while there was no effect of diet on insulin concentrations. The pigs fed CM tended to have a reduced peak insulin (p = 0.058) and an increased glucose nadir (p = 0.009) in response to the IVGTT. These preliminary results tend to support the hypothesis that feeding CM can improve glycemic control in pigs.

Investigating casein gel structure during gastric digestion using ultra-small and small-angle neutron scattering.

This study aimed to understand the structural devolution of 10% w/w rennet-induced (RG) and transglutaminase-induced acid (TG) gels in H2O and D2O under in vitro gastric conditions with and without pepsin. The real-time devolution of structure at a nano- (e.g. colloidal calcium phosphate (CCP) and micelle) and micro- (gel network) level was determined using ultra-small (USANS) and small-angle neutron scattering (SANS) with electron microscopy. Results demonstrate that gel firmness or elasticity determines disintegration behaviour during simulated mastication and consequently the particle size entering the stomach. Shear of mixing in the stomach, pH, and enzyme activity will also affect the digestion process. Our results suggest that shear of mixing primarily results in erosion at the particle surface and governs gel disintegration behaviour during the early stages of digestion. Pepsin diffusivity, and hence action, occur more readily in the latter stages of gastric digestion via access to the particle interior. This occurs via the progressively larger pores of the looser gel network and channels created within the larger, less dense casein micelles of the RG gels. Gel firmness and brittleness were greater in the D2O samples compared to H2O, facilitating gel disintegration. Despite the higher strength and elasticity of RG compared to TG, the protein network strands of the RG gels become more compact when exposed to the acidic gastric environment with comparatively larger pores observed through SEM imaging. This led to a higher degree of digestibility in RG gels compared to TG gels. This is the first study to examine casein gel structure during simulated gastric digestion using scattering and highlights the benefits of neutron scattering to monitor structural changes during digestion at multiple length scales.

Nanoparticle Tracking Analysis of ß-Casein Nanocarriers.

Nanoparticle tracking analysis is an excellent tool for the characterization of mono- and polydisperse nanoparticle systems within the 10-2000 nm size range. The suitability of this technique relies on its ability to track all particles in solution simultaneously based on their Brownian motion giving an accurate size distribution. The tracked rate of particle movement is related to the particle's hydrodynamic radius using the Stokes-Einstein equation for determining the size distribution. Here we describe the characterization ß-casein nanocarriers encapsulating a model hydrophobic compound, 8-anilino-1-naphthalenesulfonic acid, and the natural bioactive curcumin using the Malvern NanoSight NS300. Utilizing both normal light scattering and fluorescent modes of the NS300 enabled the differentiation of particles that had encapsulated the two fluorescent molecules and provided an accurate size distribution of the nanocarriers.

Sequence characteristics responsible for protein-protein interactions in the intrinsically disordered regions of caseins, amelogenins, and small heat-shock proteins.

Milk caseins and dental amelogenins are intrinsically disordered proteins (IDPs) that associate with themselves and others. Paradoxically, they are also described as hydrophobic proteins, which is difficult to reconcile with a solvent-exposed conformation. We attempt to resolve this paradox. We show that caseins and amelogenins are not hydrophobic proteins but they are more hydrophobic than most IDPs. Remarkably, uncharged residues from different regions of these mature proteins have a nearly constant average hydropathy but these regions exhibit different charged residue frequencies. A novel sequence analysis method was developed to identify hydrophobic and order-promoting regions that would favor conformational collapse. We found that such regions were uncommon; most hydrophobic and order-promoting residues were adjacent to hydrophilic or disorder-promoting residues. A further reason why caseins and amelogenins do not collapse is their high proportion of disorder-promoting proline residues. We conclude that in these proteins the hydrophobic effect is not large enough to cause conformational collapse but it can contribute, along with polar interactions, to protein-protein interactions. This behaviour is similar to the interaction of the disordered N-terminal region of small heat-shock proteins with either themselves during oligomer formation or other, unfolding, proteins during chaperone action.

Immobilization of tobacco etch virus (TEV) protease on a high surface area protein nanofibril scaffold.

Tobacco etch virus (TEV) protease is widely used for the removal of poly-histidine affinity tags from proteins. In solution, it is a one-time use enzyme for tag cleavage that has low stability, and is therefore a good candidate for immobilization. Amyloid fibrils can act as a versatile nanoscaffold by providing a large surface area for biomolecule immobilization. Immobilization of TEV protease to amyloid fibrils grown from the surface of a small glass bead, using physisorption, successfully immobilized active TEV protease. The bead retained activity over several uses and successfully cleaved a poly-histidine tag from several his-tagged proteins. This is first time that TEV protease has been immobilized to insulin amyloid fibrils, or any protein based support. Such functionalized surface assembled amyloid fibrils show promise as a novel nanosupport for the creation of functional bionanomaterials, for example, active surface coatings for the production of fine chemicals, chemical detoxification, or biosensing. Insulin amyloid fibrils provide a new nanosupport for the immobilization of TEV protease, which could allow for the reuse of the enzyme, saving on production costs for recombinantly expressed poly-histidine tagged proteins. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1506-1512, 2018.

Coaggregation of κ-Casein and ß-Lactoglobulin Produces Morphologically Distinct Amyloid Fibrils.

The unfolding, misfolding, and aggregation of proteins lead to a variety of structural species. One form is the amyloid fibril, a highly aligned, stable, nanofibrillar structure composed of ß-sheets running perpendicular to the fibril axis. ß-Lactoglobulin (ß-Lg) and κ-casein (κ-CN) are two milk proteins that not only individually form amyloid fibrillar aggregates, but can also coaggregate under environmental stress conditions such as elevated temperature. The aggregation between ß-Lg and κ-CN is proposed to proceed via disulfide bond formation leading to amorphous aggregates, although the exact mechanism is not known. Herein, using a range of biophysical techniques, it is shown that ß-Lg and κ-CN coaggregate to form morphologically distinct co-amyloid fibrillar structures, a phenomenon previously limited to protein isoforms from different species or different peptide sequences from an individual protein. A new mechanism of aggregation is proposed whereby ß-Lg and κ-CN not only form disulfide-linked aggregates, but also amyloid fibrillar coaggregates. The coaggregation of two structurally unrelated proteins into cofibrils suggests that the mechanism can be a generic feature of protein aggregation as long as the prerequisites for sequence similarity are met.

Characterisation of minimalist co-assembled fluorenylmethyloxycarbonyl self-assembling peptide systems for presentation of multiple bioactive peptides.

UNLABELLED: The nanofibrillar structures that underpin self-assembling peptide (SAP) hydrogels offer great potential for the development of finely tuned cellular microenvironments suitable for tissue engineering. However, biofunctionalisation without disruption of the assembly remains a key issue. SAPS present the peptide sequence within their structure, and studies to date have typically focused on including a single biological motif, resulting in chemically and biologically homogenous scaffolds. This limits the utility of these systems, as they cannot effectively mimic the complexity of the multicomponent extracellular matrix (ECM). In this work, we demonstrate the first successful co-assembly of two biologically active SAPs to form a coassembled scaffold of distinct two-component nanofibrils, and demonstrate that this approach is more bioactive than either of the individual systems alone. Here, we use two bioinspired SAPs from two key ECM proteins: Fmoc-FRGDF containing the RGD sequence from fibronectin and Fmoc-DIKVAV containing the IKVAV sequence from laminin. Our results demonstrate that these SAPs are able to co-assemble to form stable hybrid nanofibres containing dual epitopes. Comparison of the co-assembled SAP system to the individual SAP hydrogels and to a mixed system (composed of the two hydrogels mixed together post-assembly) demonstrates its superior stable, transparent, shear-thinning hydrogels at biological pH, ideal characteristics for tissue engineering applications. Importantly, we show that only the coassembled hydrogel is able to induce in vitro multinucleate myotube formation with C2C12 cells. This work illustrates the importance of tissue engineering scaffold functionalisation and the need to develop increasingly advanced multicomponent systems for effective ECM mimicry. STATEMENT OF SIGNIFICANCE: Successful control of stem cell fate in tissue engineering applications requires the use of sophisticated scaffolds that deliver biological signals to guide growth and differentiation. The complexity of such processes necessitates the presentation of multiple signals in order to effectively mimic the native extracellular matrix (ECM). Here, we establish the use of two biofunctional, minimalist self-assembling peptides (SAPs) to construct the first co-assembled SAP scaffold. Our work characterises this construct, demonstrating that the physical, chemical, and biological properties of the peptides are maintained during the co-assembly process. Importantly, the coassembled system demonstrates superior biological performance relative to the individual SAPs, highlighting the importance of complex ECM mimicry. This work has important implications for future tissue engineering studies.

Immobilization of organophosphate hydrolase on an amyloid fibril nanoscaffold: towards bioremediation and chemical detoxification.

Organophosphate hydrolase has potential as a bioremediation and chemical detoxification enzyme, but the problems of reusability and stability need to be addressed to use this enzyme on an industrial scale. Immobilizing the enzyme to a nanoscaffold may help to solve these problems. Amyloid fibrils generated from insulin and crystallin provided a novel nanoscaffold for the immobilization of organophosphate hydrolase, using glutaraldehyde as the crosslinking reagent. Electrophoretic, centrifugation, and temperature stability experiments, together with transmission electron microscopy were undertaken to verify that crosslinking had successfully occurred. The resulting fibrils remained active towards the substrate paraoxon and when immobilized to the insulin amyloid fibrils, the enzyme exhibited a significant (∼ 300%) increase in the relative temperature stability at 40, 45, and 50°C (as measured by comparing the initial enzyme activity to the activity remaining after heating), compared to free enzyme. This confirms that amyloid fibrils could provide a new type of nanoscaffold for enzyme immobilization.