Amyloid-like aggregation of recombinant ß-lactoglobulin at pH 3.5 and 7.0: Is disulfide bond removal the key to fibrillation?

Functional nanofibrils from globular proteins are usually formed by heating for several hours at pH 2.0, which induces acidic hydrolysis and consecutive self-association. The functional properties of these micro-metre-long anisotropic structures are promising for biodegradable biomaterials and food applications, but their stability at pH > 2.0 is low. The results presented here show that modified ß-lactoglobulin can also form nanofibrils by heating at neutral pH without prior acidic hydrolysis; the key is removing covalent disulfide bonds via precision fermentation. The aggregation behaviour of various recombinant ß-lactoglobulin variants was systemically studied at pH 3.5 and 7.0. The suppression of intra- and intermolecular disulfide bonds by eliminating one to three out of the five cysteines makes the non-covalent interactions more prevalent and allow for structural rearrangement. This stimulated the linear growth of worm-like aggregates. Full elimination of all five cysteines led to the transformation of worm-like aggregates into actual fibril structures (several hundreds of nanometres long) at pH 7.0. This understanding of the role of cysteine in protein-protein interactions will help to identify proteins and protein modifications to form functional aggregates at neutral pH.

Evaluation of oxygen partial pressure, temperature and stripping of antioxidants for accelerated shelf-life testing of oil blends using 1H NMR.

Lipid oxidation compromises the shelf-life of lipid-containing foods, leading to the generation of unpleasant off-flavours. Monitoring lipid oxidation under normal shelf-life conditions can be time-consuming (i.e. weeks or months) and therefore accelerated shelf-life conditions are often applied. However, little is known on their impact on the lipid oxidation mechanisms. In this study, different oxygen partial pressures (PO2; 10 and 21%), temperatures (20, 30 and 40 °C), and the removal of antioxidants through stripping of the oil were tested to accelerate lipid oxidation. Increasing the incubation temperature of stripped oil blends from 30 to 40 °C reduced the onset of lipid oxidation from 4 to 2 weeks, whereas the PO2 had no impact. Surprisingly, at room temperature, an increase in PO2 resulted in a longer onset time (10 weeks under 10% oxygen, 15 weeks under 21% oxygen). We hypothesize that this is due to a shift in (initiation) mechanism. In non-stripped oil, an increase in PO2 from 10 to 21% decreased the onset time from 16 to 10 weeks (40 °C). Temperature elevations and stripping led to a shift towards more trans-trans diene hydroperoxides, as compared to the cis-trans conformation. Additionally, oil stripping led to an increase in oxidized PUFAs with three or more double bonds in which the hydroperoxide group is located between the double bond pattern, instead of on the edge of it. Lastly, it was shown that small additions of LC-PUFAs (0, 0.3, 0.6, 1.2 and 2.3%, w/w) accelerate lipid oxidation, even in relatively stable stripped oils. In conclusion, increased PO2 and slightly elevated temperatures hold fair potential for accelerated shelf-life testing of non-stripped oils with a limited impact on the lipid oxidation mechanisms, whereas stripping significantly changes propagation mechanisms.