Protein nanostructures in food - Should we be worried?

Nanotechnology promises to affect many aspects of our lives with its development being greeted with both excitement and fear. The debate concerning nanotechnology has echoed that of genetically engineered organisms and their introduction into the environment and the food chain. Nanotechnology offers many potential advantages in the processing and manufacture of foods: enhanced bioavailability, colour and flavour; novel food textures; new delivery mechanisms; and access to biosensors to enhance food safety. In fact, many of the foods we have been consuming for centuries already contain nanostructures, leading many to assume that they are safe. The extent to which novel nanostructures may afford new risks has not been adequately resolved, however, leading to concern within some consumer groups. In this article, we use proteins as a case study to explore our current understanding of nanostructures in foods and the extent to which novel nanostructures may introduce new properties. It is well recognised that some protein nanostructures are toxic and are associated with disease, so there is legitimate concern as to whether such species should be deliberately introduced into our foods. This review highlights current literature on protein nanostructures in food and possible risks associated with their use. We aim to provide a balanced assessment to inform future decision-making regarding the utilisation of nanostructures in food

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

Investigation of Age Gelation in UHT Milk

Milk samples with twelve combinations of κ- and β-casein (CN) and β-lactoglobulin (β-Lg) variants were obtained to investigate the effect of protein variant on the mechanism/s of age gelation in ultra-high temperature (UHT) skim milk. Only milk groups with κ-CN/β-CN/β-Lg combinations AB/A1A2/AB and AB/A2A2/AB suffered from the expected age gelation over nine months storage, although this could not be attributed to the milk protein genetic variants. Top-down proteomics revealed three general trends across the twelve milk groups: (1) the abundance of intact native proteins decreases over storage time; (2) lactosylated proteoforms appear immediately post-UHT treatment; and (3) protein degradation products accumulate over storage time. Of the 151 identified degradation products, 106 (70.2%) arose from β-CN, 33 (21.9%) from αs1-CN, 4 (2.7%) from β-Lg, 4 (2.7%) from α-La, 3 (2%) from κ-CN and 1 (0.7%) from αs2-CN. There was a positive correlation between milk viscosity and 47 short peptides and four intact proteoforms, while 20 longer polypeptides and 21 intact proteoforms were negatively correlated. Age gelation was associated with specific patterns of proteolytic degradation and also with the absence of the families Bacillaceae, Aerococcaceae, Planococcaceae, Staphylococcaceae and Enterobacteriaceae, present in all the non-gelling milk groups pre-UHT

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