Changes in bioactive proteins and serum proteome of human milk under different frozen storage

•Human milk serum proteins stored at −60°C has less changes compared to that of −18°C.•The migration of MFGM proteins to serum is much more at −18°C storage than −60°C.•Ice-induced denaturation may result in reduction of protein bioactivity.•Fast cooling, ultra-low constant temperature are recommended for human milk storage. This study aimed to investigate changes in macronutrients, total bacterial count, and serum proteome of human milk (HM) under different frozen storage (−18°C and −60°C, 60 d and 180 d) by using IBT Labeling proteomics techniques and ELISA kit. The results indicated that total protein concentrations and total aerobic bacterial counts were significantly decreased at −18°C, while no difference at −60°C. A total of 1617 proteins were identified and quantified, and 173 proteins were significantly different. The −18°C storage had much higher influence on HM serum protein profiles than that of −60°C. Increased milk fat globule membrane (MFGM) proteins at −18°C are highly related to the damage of MFGM and transfer of MFGM proteins. The reduction of bioactive proteins is probably related to the ice-induced denaturation. In conclusion, fast cooling and ultra-low constant temperature are more suitable for the cryopreservation of human milk.