Assessing constituent volumes and morphology of bovine casein micelles using cryo-electron tomography.

We investigated the applicability of cryo-electron tomography as a method to quantify changes in the major constituents of casein micelles (i.e., casein proteins, putative colloidal calcium phosphate nanoclusters, and serum-filled voids and channels) in response to their environment. Skim milk diluted 20-fold in milk serum was used for this study. Tomograms were generated for multiple casein micelles at 2 different pH values (6.7 and 6.0) and pixel intensity thresholds were identified for each constituent. The volume of each constituent was determined using these thresholds and expressed as a fraction of micelle volume. At the given dilution, a significant decrease in the volume fractions of casein proteins (∼37%) and putative colloidal calcium phosphate nanoclusters (∼67%) was observed with the reduction of pH from 6.7 to 6.0. Assessment of casein micelle fraction obtained by ultracentrifugation of corresponding skim milk samples produced comparable results. When using such an approach, the imaging conditions, denoising methods, and thresholding approaches used can all affect the precision of the measurements, but the overall trends in constituent volumes are able to be tracked. The primary advantage of using cryo-electron tomography is that analysis can be done at the level of individual micelles, within a 3-dimensional morphological context. This workflow paves the way for high-throughput exploration of milk micelles and how their environment shapes their composition and structure.

Morphology of complexes formed between ß-lactoglobulin nanofibrils and pectins is influenced by the pH and structural characteristics of the pectins.

For the optimal use of ß-lactoglobulin nanofibrils as a raw material in biological composites an in-depth knowledge of their interactions with other constituents is necessary. To understand the effect of electrostatic interactions on the morphology of resulting complexes, ß-lactoglobulin nanofibrils were allowed to interact with pectins in which the amount of available negative charge was controlled by selecting their degree of methylesterification. In this study, citrus pectins having different degrees of methylesterification (∼48, 67, 86, and 97%) were selected and interacted with nanofibrils at pH 2 and pH 3, where they possess a net positive charge. Electrostatic complexes formed between ß-lactoglobulin nanofibrils and all pectin types, except for the sample having a degree of methylesterification of 97%. The morphology of these complexes, however, differed significantly with the degree of methylesterification of the pectin, its concentration, and the pH of the medium, revealing that distinct desired biological architectures can be attained relatively easily through manipulating the electrostatic interactions. Interestingly, the pectin with a degree of methylesterification of 86% was found to crosslink the ß-lactoglobulin nanofibrils into ordered 'nanotapes'.

ß-Lactoglobulin nanofibrils can be assembled into nanotapes via site-specific interactions with pectin.

Controlling the self-assembly of individual supramolecular entities, such as amyloid fibrils, into hierarchical architectures enables the 'bottom-up' fabrication of useful bionanomaterials. Here, we present the hierarchical assembly of ß-lactoglobulin nanofibrils into the form of 'nanotapes' in the presence of a specific pectin with a high degree of methylesterification. The nanotapes produced were highly ordered, and had an average width of 180 nm at pH 3. Increasing the ionic strength or the pH of the medium led to the disassembly of nanotapes, indicating that electrostatic interactions stabilised the nanotape architecture. Small-angle X-ray scattering experiments conducted on the nanotapes showed that adequate space is available between adjacent nanofibrils to accommodate pectin molecules. To locate the interaction sites on the pectin molecule, it was subjected to endopolygalacturonase digestion, and the resulting products were analysed using capillary electrophoresis and size-exclusion chromatography for their charge and molecular weight, respectively. Results suggested that the functional pectin molecules carry short (<10 residues) enzyme-susceptible blocks of negatively charged, non-methylesterified galacturonic acid residues in the middle of their homogalacturonan backbones (and possibly near their ends), that specifically bind to sites on the nanofibrils. Blocking the interaction sites on the nanofibril surface using small oligomers of non-methylesterified galacturonic acid residues similar in size to the interaction sites of the pectin molecule decreased the nanotape formation, indicating that site-specific electrostatic interactions are vital for the cross-linking of nanofibrils. We propose a structural model for the pectin-cross-linked ß-lactoglobulin nanotapes, the elements of which will inform the future design of bionanomaterials.

Formation of ß-lactoglobulin nanofibrils by microwave heating gives a peptide composition different from conventional heating.

A novel procedure involving microwave heating (MH) at 80 °C can be used to induce self-assembly of ß-lactoglobulin (ß-lg) into amyloid-like nanofibrils at low pH. We examined the self-assembly induced by MH, and evaluated structural and compositional differences between MH fibrils and those formed by conventional heating (CH). MH significantly accelerated the self-assembly of ß-lg, resulting in fully developed fibrils in ≤2 h. However, longer MH caused irreversible disintegration of fibrils. An increase in the fibril yield was observed during the storage of the 2 h MH sample, which gave a yield similar to that of 16 h CH sample. Fourier transform infrared (FTIR) and circular dichroism (CD) spectra suggested that the fibrils formed by the two methods do not show significant differences in their secondary structure components. However, they exhibited differences in surface hydrophobicity, and mass spectrometry showed that the MH fibrils contained larger peptides than CH fibrils, including intact ß-lg monomers, providing evidence for a different composition between the MH and CH fibrils, in spite of no observed differences in their morphology. We suggest MH initially accelerates the self-assembly of ß-lg due to its nonthermal effects on unfolding, nucleation, and subsequent stacking of ß-sheets, rather than promoting partial hydrolysis. Thus, MH fibrils are composed of larger peptides, and the observed higher surface hydrophobicity for the MH fibrils was attributed to the parts of the larger peptides extending out of the core structure of the fibrils.