Related Experiment Video
Updated: May 10, 2026

Individualized Reconstitution of Human Milk Microbiota: A Feasible Approach in Real-World Settings
Published on: February 7, 2025
Effect of Holder pasteurization and frozen storage on macronutrients and energy content of breast milk
Nadia Raquel García-Lara1, Diana Escuder Vieco, Javier De la Cruz-Bértolo
1Department of Neonatology, 12 de Octubre Hospital, Madrid, Spain. nadiaraquelg@yahoo.es
Objectives:
The aim of this study was to explore the effect of Holder pasteurization and frozen storage at -20°C after pasteurization on fat, total nitrogen, lactose, and energy content of breast milk. Both procedures are routinely practiced in human milk banks.
Methods:
A total of 34 samples of frozen breast milk, donated by 28 women, were collected. Once thawed, an aliquot of each sample was analyzed before pasteurization; the remaining milk was pasteurized (Holder method) and split into 8 aliquots. One aliquot was analyzed after pasteurization and the remainder frozen at -20°C and analyzed 30, 60, 90, 120, and 180 days later. For every aliquot, fat, total nitrogen, lactose, and energy content were determined using the device human Milk Analyzer.
Results:
We observed a significant reduction in fat (3.5%; -0.17 (-0.29; -0.04) g/dL) and energy content (2.8%; -2.03 (-3.60; -0.46) g/dL) after pasteurization. A significant decrease over time was observed for fat, lactose and energy content. No significant changes were observed for nitrogen content. Mean differences between day 0 postpasteurization and day 180 were -0.13 (-0.21; -0.06) g/dL for fat, -0.08 (-0.13; -0.03) g/dL for lactose, and -1.55 (-2.38; -0.71) kcal/dL for energy content. The relative decreases were 2.8%, 1.7%, and 2.2%, respectively. Overall (postpasteurization + frozen storage), a 6.2% and 5% decrease were observed for fat and energy, respectively.
Conclusions:
Holder pasteurization decreased fat and energy content of human milk. Frozen storage at -20°C of pasteurized milk significantly reduced fat, lactose, and energy content of human milk.
Related Concept Videos
Microbes in Food Production
Development of Human Microbiota
Physical Methods for Controlling Microbial Growth: Temperature
Microbial Spoilage of Food

