Egg white and wheat gluten protein amyloid (like) fibrillation under food processing relevant conditions

Proteins are essential components of the human diet. In addition, they have important properties in food production and products (e.g. gelling and foaming properties, visco-elasticity, etc.). In a food context, proteins are often categorized as either of animal or plant origin. Egg white (EW) and wheat gluten (WG) proteins are relevant examples of each of the two main groups, respectively. EW is highly nutritious and recognized for its excellent foaming and gelling properties. WG is the most important cereal protein consumed and an essential structural component of many baked products. The functionality of proteins is intrinsically related to their structure. However, developing protein structures with specific functionality is still a major challenge. Amyloid fibrils (AFs) are protein structures composed of highly ordered β-sheet structures which hold potential to deliver specific functionality in food products. Unfortunately, EW and WG protein fibrillation has mainly been studied at conditions which are not relevant for food processing (e.g. very acidic pH or extended incubation times). Against the above background, the AIM of this doctoral dissertation was to study EW and WG protein fibrillation under food system relevant processing conditions by providing answers to the following research questions: what is the impact of drying on EW and WG protein fibrillation, to what extent, hydrothermal treatments promote EW and WG protein fibrillation, what is the potential of enzymatic hydrolysis to enhance WG protein fibrillation, and to what extent does adding limited amounts of chaotropic or enzymatic agents contribute to the extraction of protein fibrils from heated EW and WG. In a FIRST PART, the impact of heating conditions resembling slow cooking (78 °C, 22 h) or boiling on EW protein fibrillation was studied. Both types of conditions promoted the formation of aqueous soluble ovalbumin (OVA; the main protein in EW) protein fibrils, albeit to a larger extent under the former conditions. Sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) containing media (either in combination or separately) were not suitable for extracting protein fibrils from gels obtained by boiling EW for 15 min. An enzyme-assisted extraction with proteinase K of protein fibrils from boiled EW was developed. Proteinase treatment (37 °C, 48 h, 150 rpm) on boiled EW solubilized mainly amorphous aggregates. Following centrifugation, a dense network of worm-like protein structures was solubilized from the pellets with 0.01 M HCl (room temperature, 1 h, 150 rpm) as observed with transmission electron microscopy (TEM). Fourier transform infrared spectroscopy (FTIR), X-ray diffraction analysis and green birefringence upon staining with Congo red confirmed the presence of AFs in such pellets. Approximately 1.5–3.0% of the protein in EW assembled into AFs during boiling. In addition, treating heated OVA (78 °C, 22 h) with trypsin (37 °C, 48 h, 150 rpm) allowed to demonstrate that it contained AFs. The developed enzyme-assisted extraction method and the analytical techniques mentioned above were used to further investigate AF formation under other food relevant processing conditions. EW powders are commonly used in the food industry. In a SECOND PART, the influence of drying modes and hydrothermal treatment conditions on protein fibrillation in solutions of dried EW was investigated. Drying influenced EW protein fibrillation and spray-drying promoted this process to a larger extent than freeze-drying. Nonetheless, specific dry heating conditions [i.e. storage for one week either at 50 °C and 50% relative humidity (RH) (EWSD 50°C/50%) or at 60 °C and 80% RH (EWSD 60°C/80%)] had limited impact on spray-dried EW protein (EWSD) fibrillation. A response surface design allowed determining optimal fibrillation conditions for solutions of freeze-dried OVA and EW (OVAFD and EWFD). These were found to be 2.0% (wprotein/v), pH 7.0, 23 h, 76 °C and 0.5% (wprotein/v), pH 7.0, 24 h, 85 °C, respectively. Under such conditions, OVAFD solutions contained a higher level of β-sheet structures and larger worm-like aggregates than EWFD solutions. A higher extent of fibrillation was found in EWSD 60°C/80% dispersions heated [0.5% (wprotein/v), pH 7.0, 24 h, 85 °C] than in those of EWSD 50°C/50%, illustrating the impact of different dry heating conditions. In a THIRD PART, the fibrillation of WG protein under heating conditions that were successful to induce EW protein fibrillation was studied. Unheated and heated WG samples were treated with proteinase K and trypsin (37 °C, 48 h, 150 rpm) to solubilize the non-fibrillated proteins, while protein fibrils were extracted with 0.05 M sodium phosphate buffer (pH 7.0) from the undissolved fraction obtained by the same enzymatic treatment. Unheated WG dispersions already contain some protein fibrils presumably as a result of the industrial drying process during their isolation. Heating caused the level of protein fibrils to increase. Heated WG dispersions (78 °C, 22 h) induced the formation of straight fibrils (ca. 700 nm in length), whereas boiling WG for at least 15 min resulted in longer (ca. 1 to 2 μm) and straight fibrils. The latter showed the typical green birefringence of AFs when stained with Congo red and its X-ray fiber diffraction pattern showed the typical reflection (4.7 Å) for inter β-strand spacing. Two weaker additional reflections were observed at 7.8 Å and 12.1 Å. The latter most probably indicated the distance between packed b-sheets (i.e. 10 Å). These results combined with those of FTIR validated the identification of β-rich amyloid-like fibrils (ALFs) in dispersions of boiled WG. Approximately 0.1% to 0.5% of WG proteins assembled into ALFs during boiling. In a FOURTH PART, the formation of ALFs from wheat gluten peptides (WGPs) was investigated under different hydrothermal conditions. Firstly, tryptic WGP [degrees of hydrolysis 2.0% (DH 2) or 6.0% (DH 6)] fibrillation was optimized under hydrothermal conditions using a response surface design. DH 6 WGPs had a higher propensity to fibrillate than did DH 2 WGPs. Heating DH 6 WGPs at 2.0% (w/v) for 38 h at 85 °C and pH 7.0 resulted in optimal fibrillation. Secondly, trypsin, chymotrypsin, thermolysin, papain and proteinase K were individually used to produce different DH 6 WGP samples. After enzyme inactivation and subsequent heating at optimal fibrillation conditions, chymotrypsin and proteinase K DH 6 WGPs produced small worm-like fibrils whereas fibrils prepared from trypsin DH 6 WGPs were long (ca. 500 nm to 1.3 μm) and straight. The average peptide size and surface hydrophobicity of the peptides were key for fibrillation, as heat-induced fibrillation occurred to a larger extent in tryptic WGPs containing an average larger peptide size with increased hydrophobicity than in other enzyme-prepared peptides. Thirdly, tryptic peptides from the WG components gliadin and glutenin fractions separately formed mainly worm-like protein structures. As the morphology and the size of these fibrils did not resemble those of tryptic WGPs protein fibrils, it is believed that both WG protein fractions jointly contribute to fibrillation. In conclusion, this dissertation demonstrates that food relevant processing conditions induced the formation of ALFs or even AFs in EW and WG proteins, suggesting their presence in the human diet. In addition, the enzyme-assisted extraction protocol developed in this doctoral research can be of value to study AFs in complex food matrices. Based on previous reports, it is hoped that ALFs or even AFs prepared under food relevant processing conditions have specific techno-functional properties that can be beneficial for the quality of food products

Jaar van publicatie:2021

Toegankelijkheid:Open