Structural impact of cholesterol depletion and phytosterol enrichment in milk fat globule membranes

Cholesterol was depleted from the milk fat globule membrane (MFGM) by up to 94% using β-cyclodextrin and partially replaced by β-sitosterol to determine if this phytosterol can form liquid-ordered domains. Substitution by β-sitosterol cream produced larger fat globules compared to cholesterol-depleted and native globules. Globule integrity was not affected as assessed by confocal laser scanning microscopy and ζ-potential measurements. Sterol substitution decreased the total phospholipid content with sphingomyelin content unchanged. Xanthine oxidase activity, used as a proxy for membrane disruption, rose after cholesterol removal and decreased after β-sitosterol substitution, indicating partial recovery of outer MFGM bilayer integrity. Confocal Raman microscopy revealed sterol-dependent changes in lipid chain organization within the MFGM. Cholesterol depletion reduced lipid alkyl chain packing and increased membrane heterogeneity, whereas β-sitosterol substitution partially restored chain packing but promoted a more disordered, compositionally heterogeneous membrane state, suggesting that β-sitosterol may not fully reproduce cholesterol-dependent membrane organization under the physicochemical conditions of the experimental model.