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

摘要

We investigated the applicability of cryo-electron tomographyas a method to quantify changes in the majorconstituents of casein micelles (i.e., casein proteins,putative colloidal calcium phosphate nanoclusters, andserum-filled voids and chanNE_Ls) in response to theirenvironment. Skim milk diluted 20-fold in milk serumwas used for this study. Tomograms were generated formultiple casein micelles at 2 different pH values (6.7and 6.0) and pixel intensity thresholds were identifiedfor each constituent. The volume of each constituentwas determined using these thresholds and expressed asa fraction of micelle volume. At the given dilution, a significantdecrease in the volume fractions of casein proteins(~37%) and putative colloidal calcium phosphatenanoclusters (~67%) was observed with the reductionof pH from 6.7 to 6.0. Assessment of casein micellefraction obtained by ultracentrifugation of correspondingskim milk samples produced comparable results.When using such an approach, the imaging conditions,denoising methods, and thresholding approaches usedcan all affect the precision of the measurements, butthe overall trends in constituent volumes are able to betracked. The primary advantage of using cryo-electrontomography is that analysis can be done at the levelof individual micelles, within a 3-dimensional morphologicalcontext. This workflow paves the way for highthroughputexploration of milk micelles and how theirenvironment shapes their composition and structure.