Effect of the differences in protein structure on digestibility

Food is a complex combination of chemically diverse structures, influenced by composition andprocessing. The nutritional quality of food cannot be solely based on individual contents in nutrientsas food structure will strongly modulate the bioaccessibility of nutrients upon digestion. In previousstudies, the most common approach for measuring the extent of proteolysis and nutrient uptake wasto compare the initial macroscopic features of ingested food to final concentrations in thebloodstream. However, there is limited literature to the support underlying mechanisms of thedigestion of food in the stomach and enzyme diffusion in solid food substrates. In the present work,the influence of casein protein gel microstructure on enzymatic digestion was investigated for thefuture development of personalised foods. Casein gels with an identical composition, but differing bythe coagulation mode, were characterized and submitted to simulated gastric digestion. The changesin gel structure over time were studied using confocal microscopy and small angle x-ray scattering(SAXS). The molecular interactions that occur as a gel network is formed, and subsequently brokendown under digestive conditions will be discussed

Immobilising biomolecules on amyloid fibrils for biotechnology applications

Amyloid fibrils are an insoluble, highly ordered, fibrous protein structure, which have increasingly been recognised as having bionanotechnology applications. Their ability to selfassemble allows a bottom-up approach to material design. Their nanometre dimensions affords them a high surface-to-volume ratio and their proteinaceous building blocks from which they are assembled allow for decoration with biomolecules and chemicals through amino acid residues. Amyloid fibrils are therefore a potential nanoscaffold for immobilisation of biomolecules. Immobilisation offers a solution to the problems associated with the use of enzymes in in vitro applications, by increasing their stability, reusability, and in some cases, enhancing catalytic activity. Nanosupports offer a high surface-to-volume ratio compared to classical planar 2-D supports, potentially affording them dramatic increases in immobilisation capacity. To investigate the potential of amyloid fibrils as a novel nanoscaffold, organophosphate hydrolase (OPH), cytochrome P450BM3 (P450BM3), green fluorescent protein (GFP), tobacco etch virus protease (TEV), and glucose oxidase (GOD) were immobilised in solution to the model amyloid fibril forming protein, bovine insulin. Covalently immobilised OPH was found to have a ~300 % increase in relative thermostability at 40 and 50 °C. P450BM3 was not successfully immobilised in its active state, most likely due to unfolding of the enzyme on the amyloid fibril surface. Covalently immobilised GFP retained full fluorescence and acted as a fluorescent protein tag. TEV was shown to have a physical interaction with the nanoscaffold and retain activity. GOD was immobilised and retained activity. Although not all proteins retained activity, a range of different protein structures were successfully immobilised onto the insulin amyloid fibril nanoscaffold. Attachment to the crystallin amyloid fibril nanoscaffold remains a work in progress due to the complexities associated with post-translational modifications of these fibrils. Crystallin amyloid fibrils were assembled on a surface for the first time. Their surface assembled structure was found to resemble spherulites, not previously seen before with crystallin amyloid fibrils. Bovine insulin amyloid fibrils were assembled on the surface of glass beads to increase the available surface area for biomolecule immobilisation. The surface assembled bovine insulin nanoscaffold was first functionalised with GOD, demonstrating that the nanoscaffold provides more surface area for biomolecule immobilisation, although in this case the increase was limited due to high non-specific binding of GOD to the unmodified glass surface. GFP was successfully employed as a fluorescent protein tag to assess the degree of nanoscaffold coverage, confirming the nanoscaffold affords the glass bead a greater surface area. Moreover, a reusable immobilised TEV protease-bead system was developed that was able to sequentially cleave the poly-histidine tags of three different proteins. In conclusion, bovine insulin amyloid fibrils have been shown to be a versatile nanoscaffold for the immobilisation of a range of biomolecules. The surface characteristics of the nanoscaffold allows for both covalent and physical immobilisation of biomolecules. Thus, amyloid fibrils have exciting potential in the creation of novel bionanotechnologies

Effect of the differences in protein structure on digestibility

Food is a complex combination of chemically diverse structures, influenced by composition and processing. The nutritional quality of food cannot be solely based on individual contents in nutrients as food structure will strongly modulate the bioaccessibility of nutrients upon digestion. In previous studies, the most common approach for measuring the extent of proteolysis and nutrient uptake was to compare the initial macroscopic features of ingested food to final concentrations in the bloodstream. However, there is limited literature to the support underlying mechanisms of the digestion of food in the stomach and enzyme diffusion in solid food substrates. In the present work, the influence of casein protein gel microstructure on enzymatic digestion was investigated for the future development of personalised foods. Casein gels with an identical composition, but differing by the coagulation mode, were characterized and submitted to simulated gastric digestion. The changes in gel structure over time were studied using confocal microscopy and small angle x-ray scattering (SAXS). The molecular interactions that occur as a gel network is formed, and subsequently broken down under digestive conditions will be discussed

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

Coaggregation of κ-Casein and β-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 β-sheets running perpendicular to the fibril axis. β-Lactoglobulin (β-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 β-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 β-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 β-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

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

Tailoring the structure of casein micelles through a multifactorial approach to manipulate rennet coagulation properties

International audienceThe properties of casein micelles are known to be affected by modifications to the environment, such as variations in pH or the addition of salts, yet the scientific literature typically considers the effects of one factor at a time, while in industrial processes, several modifications are performed simultaneously. The aim of this study was to assess the impact of multifactorial environmental modifications on the colloidal, structural and rennet coagulation properties of casein micelles in a simplified model system. A key finding was that dense regions (~20 nm in size) could be released from the casein micelle. The addition of NaCl and CaCl2 had opposing effects, i.e. enhancing or limiting this micellar disruption, respectively. A decrease in pH had the strongest impact on the mineral balance, causing the colloidal CaP to solubilize and the micelle to swell. The rennet clotting time was impacted by variations in pH and NaCl content. Interestingly, a consideration of all three levels of casein micelle structure and their interactions was needed to explain variations in the firmness of rennet gels. This study illustrates the complex interplay of factors affecting micellar structure and improves our understanding of how micelles can be manipulated to control their properties