Fractionation and characterisation of hard milk fat crystals using atomic force microscopy

The hard milk fat (HMF) fraction of milk fat was isolated via dry, thermal fractionation, followed by a solvent washing process. The resulting HMF crystals were visibly free of entrapped liquid fat, and subsequently characterised by thermal analysis, X-ray diffraction, and electron microscopy. The HMF crystals were found to be mostly β′ and β′₂ crystalline structures, with a lamellar thickness of 42.7–44.1 Å. Additionally, crystal size was determined to be ≥1 μm in length and 0.4–1 μm in width. Atomic force microscopy (AFM) was used to further characterise the HMF crystals. AFM enabled 3D mapping and visualisation of crystal layering, as well as simple determination of layer thickness (∼4.2 ± 0.8 nm); a value in close agreement with the results obtained via X-ray analysis. The AFM characterisation approach provides a simple method of characterising HMF crystals, without suffering the limitations of other widely used techniques.