Selective enrichment of milk phospholipids via cryo-crystallization: application to various dairy matrices and elucidation of sphingomyelin crystallization mechanisms.

Milk sphingomyelin (SM) is a bioactive phospholipid involved in infant neurodevelopment and gut health; however, its proportion in dairy by-products is around 20% of total phospholipids, compared to over 30% in human milk, limiting its application in infant formula products. The molecular mechanisms underlying the selective enrichment of SM during cryo-crystallization were elucidated. Cryo-crystallization increased total phospholipid content by 2.12-fold, while the SM proportion increased from 27% to 41%. Lipidomics revealed preferential enrichment of very-long-chain fatty acid-containing phospholipids. Structural analyses showed enhanced molecular ordering and intermolecular hydrogen bonding. Differential scanning calorimetry identified ~70% ethanol as a thermodynamic window favoring SM crystallization over glycerophospholipids. For raw buttermilk, low initial phospholipid content limited direct cryo-crystallization, requiring ethanol pre-extraction optimized at 100% ethanol, a 1:20 solvent-to-material ratio, 55 °C, and 90 min; phospholipid recovery decreased at 70 °C. The strategy was applicable to multiple dairy matrices for producing SM-enriched phospholipids.