Diurnal variation of microbial colony counts in frozen human milk: An experimental study

Freezing human milk for long periods may reduce its antibacterial properties. We assessed antimicrobial effects of human milk against Escherichia coli after 6 months of freezing and to explore daily variations in both fresh and frozen milk to optimize storage protocols.

A study analyzed 60 breast milk samples from 30 healthy mothers with infants aged 15-30 days, conducted from February to May 2025. The human milk expressed during the daytime and at nighttime was tested for antimicrobial effects against E coli. Fresh samples were analyzed within 24 h, and frozen samples were stored at -80°C for 6 months. Bacterial growth was assessed by diluting milk with E coli, and statistics were calculated using colony counts on MacConkey agar.

Fresh human milk had a median E coli count of 6.5, much lower than 225 in frozen samples stored for 6 months (P < 0.001), showing a 97.1% drop in antimicrobial quality. Daytime frozen samples had lower microbial counts (median 95) than nighttime samples (median 120, P = 0.006). Higher nighttime counts appeared in frozen samples from primiparous women, cesarean deliveries, and male infants. Maternal age correlated positively with daytime frozen milk microbial counts (r = 0.421, P = 0.021), whereas gestational age correlated negatively with counts in both daytime (r = -0.583, P < 0.001) and nighttime samples (r = -0.562, P = 0.001).

Frozen human milk samples substantially lose antibacterial properties after 6 months, with variations influenced by time of day. Samples from primiparous, breastfeeding, male sex infants and those who had cesarean deliveries show diurnal variations following the cryopreservation process.