Pasteurizing donor human milk inactivates bacteria that may be of concern to the preterm infant. However, current practice for Holder Pasteurization (62.5 degrees C for 30 min) is detrimental to the bioactivity of human milk. An experimental pasteurizer was used to determine the maximum temperature at which 90% of secretory IgA, lysozyme, and lactoferrin were retained and whether this temperature was capable of inactivating five common bacterial contaminants. The retention of these proteins was also compared using a commercially available bottle immersion or holding chamber system. After pasteurization at 62.5 degrees C for 30 min, the retention across all three systems was 72.3 +/- 3.6%, 21.8 +/- 3.3%, and 39.4 +/- 11.5% for sIgA, lactoferrin, and lysozyme, respectively (n = 22). The retention of all three proteins was at least 90% when human milk was pasteurized at 57 degrees C for 30 min, and this temperature was also effective at removing 99.9% of all inoculated bacterial species. In addition, human milk that was pasteurized in the experimental system had a significantly higher proportion of lysozyme compared with samples pasteurized in the bottle immersion system. These findings suggest that optimizing pasteurization temperature and improving pasteurizer design enhances the quality of pasteurized donor human milk.