Bovine β-lactoglobulin (β-Lg) self-assembles into long amyloid-like fibrils when heated at 80 °C, pH 2, and low ionic strength (<0.015 mM). Heating β-Lg under fibril-forming conditions shows a lag phase before fibrils start forming. We have investigated the structural characteristics of β-Lg during the lag phase and the composition of β-Lg fibrils after their separation using ultracentrifugation. During the lag phase, the circular dichroism spectra of heated β-Lg showed rapid unfolding, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of samples showed increasing hydrolysis of β-Lg. The SDS-PAGE profiles of fibrils separated by ultra centrifugation showed that after six hours, the fibrils consisted of a few preferentially accumulated peptides. Two-dimensional SDS-PAGE under reducing and nonreducing conditions showed the presence of disulfide-bonded fragments in the fibrils. The sequences in these peptide bands were characterized by in-gel digestion electrospray ionization (ESI)-MS/MS. The composition of solubilized fibrils was also characterized by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS/MS. Both MS analyses showed that peptides in fibrils were primarily from the N-terminal region, although there was some evidence of peptides from the C-terminal part of the molecule present in the higher molecular weight gel bands. We suggest that although the N-terminal region of β-Lg is almost certainly involved in the formation of the fibrils, other peptide fragments linked through disulfide bonds may also be present in the fibrils during self-assembly.
There has been growing consumer interest in sheep and goat milk products as alternatives to cow milk products. The physicochemical characteristics of milk vary not only between ruminant species, but also during different seasons; they determine the nutritional quality and processing properties of the milk. In this study, we characterized sheep and goat milks from New Zealand over the seasons for their composition (macronutrients, macro- and micro-minerals, fatty acids, and proteins) and physicochemical properties (e.g., ionic calcium, fat globule size, casein micelle size, viscosity, and melting behavior of milk fat). Heat-induced (95 °C for 5 min) protein interactions and changes in the physical properties of the milks were also investigated. The compositional and structural features of sheep and goat milks were identified and compared with those reported for cow milk. Seasonal variations in the milk characteristics were more pronounced for sheep milk than goat milk and were probably affected by the production systems. Sheep milk, particularly in the late season, had the largest heat-induced increases in casein micelle size and viscosity, probably arising from the greater casein–whey protein and casein–casein interactions during heat treatment. This study provides comprehensive information on the properties of sheep and goat milks and highlights the interaction effects between species, season, and processing.
β-Lactoglobulin (β-lg) forms fibrils when heated at 80 °C, pH 2, and low ionic strength (<0.015 mM). When formed at protein concentrations <3%, these fibrils are made up of peptides produced from the acid hydrolysis of the β-lg monomer. The present study investigated the effects of the polyhydroxy alcohols (polyols) glycerol and sorbitol (0-50% w/v) on β-lg self-assembly at pH 2. Glycerol and sorbitol stabilize native protein structure and modulate protein functionality by preferential exclusion. In our study, both polyols decreased the rate of β-lg self-assembly but had no effect on the morphology of fibrils. The mechanism of these effects was studied using circular dichroism spectroscopy and SDS-PAGE. Sorbitol inhibited self-assembly by stabilizing β-lg against unfolding and hydrolysis, resulting in fewer fibrillogenic species, whereas glycerol inhibited nucleation without inhibiting hydrolysis. Both polyols increased the viscosity of the solutions, but viscosity appeared to have little effect on fibril assembly, and we believe that self-assembly was not diffusion-limited under these conditions. This is in agreement with previous reports for other proteins assembling under different conditions. The phenomenon of peptide self-assembly can be decoupled from protein hydrolysis using glycerol.
In this study we investigated the effects of different levels of glucosylation and lactosylation on β-Lg self-assembly into nanofibrils at 80 °C and pH 2. Fibrils in heated samples were detected with the thioflavin T assay and transmission electron microscopy, while SDS-PAGE was used to investigate the composition of the heated solutions and fibrils. Glycation had different effects in the nucleation and growth phases. The effect of glycation on the nucleation phase depended on the degree of glycation but not the sugar type, whereas both the type of sugar and the degree of glycation affected the rate of fibril growth. Glycation by either sugar strongly inhibited self-assembly in the growth phase, and lactosylation produced a much stronger effect than glucosylation. We suggest that the varying glycation susceptibility of different lysine residues can explain these observations. The large, polar sugar residues on the glycated fibrillogenic peptides may inhibit fibril assembly by imposing steric restrictions and disrupting hydrophobic interactions.
The structural and physicochemical characteristics of oil bodies from hemp seeds were explored in this study. Oil bodies from several plant-based sources have been previously studied; however, this is the first time a characterisation of oil bodies from the seeds of industrial hemp is provided. The morphology of oil bodies in hemp seeds and after extraction was investigated using cryo-scanning electron microscopy (cryo-SEM), and the interfacial characteristics of isolated oil bodies were studied by confocal laser scanning microscopy (CLSM). Proteins associated with oil bodies were characterised using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The effect of pH and ionic strength on colloidal properties of the oil bodies was investigated. Oil bodies in hemp seeds appeared spherical and sporadically distributed in the cell, with diameters of 3 to 5 μm. CLSM images of isolated oil bodies revealed the uniform distribution of phospholipids and proteins at their interface. Polyunsaturated fatty acids were predominant in the lipid fraction and linoleic acid accounted for ≈61% of the total fatty acids. The SDS-PAGE analysis of washed and purified oil bodies revealed major bands at 15 kDa and 50–25 kDa, which could be linked to membrane-specific proteins of oil bodies or extraneous proteins. The colloidal stability of oil bodies in different pH environments indicated that the isoelectric point was between pH 4 and 4.5, where oil bodies experienced maximum aggregation. Changes in the ionic strength decreased the interfacial charge density of oil bodies (ζ-potential), but it did not affect their mean particle size. This suggested that the steric hindrance provided by membrane-specific proteins at the interface of the oil bodies could have prevented them from flocculation at low interfacial charge density. The results of this study provide new tertiary knowledge on the structure, composition, and colloidal properties of oil bodies extracted from hemp seeds, which could be used as natural emulsions or lipid-based delivery systems for food products.
A new iron–casein complex (ICC) has been developed for iron (Fe) fortification of dairy matrices. The objective was to assess the impact of ascorbic acid (AA) on its in vitro bioavailability in comparison with ferrous sulfate (FeSO4) and ferric pyrophosphate (FePP). A simulated digestion coupled with the Caco-2 cell culture model was used in parallel with solubility and dissociation tests. Under diluted acidic conditions, the ICC was as soluble as FeSO4, but only part of the iron was found to dissociate from the caseins, indicating that the ICC was an iron chelate. The Caco-2 cell results in milk showed that the addition of AA (2:1 molar ratio) enhanced iron uptake from the ICCs and FeSO4 to a similar level (p = 0.582; p = 0.852) and to a significantly higher level than that from FePP (p < 0.01). This translated into a relative in vitro bioavailability to FeSO4 of 36% for FePP and 114 and 104% for the two ICCs. Similar results were obtained from water. Increasing the AA to iron molar ratio (4:1 molar ratio) had no additional effect on the ICCs and FePP. However, ICC absorption remained similar to that from FeSO4 (p = 0.666; p = 0.113), and was still significantly higher than that from FePP (p < 0.003). Therefore, even though iron from ICC does not fully dissociate under gastric digestion, iron uptake suggested that ICCs are absorbed to a similar amount as FeSO4 in the presence of AA and thus provide an excellent source of iron.
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