Rethinking Casein Micelle Structure Using Electron Microscopy

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Journal of Dairy Science



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Numerous models for the structure of casein micelles have been proposed in the past. Based on the chemical and physical properties of micelles, these models fall into two general categories, models of internal structure and models of submicelles. Electron microscopy has been used to provide evidence for these models, although the available techniques imposed limits. With the development of newer electron microscopes, the structure of the bovine casein micelle has been further examined. Even at high resolution, scanning electron microscopy does not produce satisfactory images of casein micelles because the application of metal coatings (2 to 10 nm thickness) obscures the proteins from view, and the resultant images of casein micelles resemble a lumpy sphere. Transmission electron microscopy has been used with varying results. Freeze-fracture replica methods coat the fine protein structure with metal, as occurs with scanning electron microscopy. Negative staining that includes air-drying introduces artifacts into the sample during drying. Plastic embedding can yield fairly representative images, but not without dimensional compression and some chemical modification. These chemical modifications occur predominantly from 1) chemical fixation, causing crosslinking of protein and thus shifting of their spatial resolution; 2) ethanol, causing coagulation of the proteins during dehydration; and 3) protein loss during infiltration with plastic. The inability to avoid these problems in the past has limited the ability to provide an accurate image of casein micelles. In this study, a cryopreparation technique has been applied to examine single casein micelles while retaining the maximum in situ structure. To avoid problems of metal coating and chemical modifications, casein micelles were absorbed to parlodion-coated grids, immersed in uranyl oxalate counterstain, quick frozen, and freeze-dried. This method yields images of whole, spherical casein micelles and shows the fine structural detail of the micellar proteins. Stereo images showed that the largest subaggregate particles within the casein micelle matrix were approximately 8 to 10 nm in size.

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