Methods of mixing donor human milk during bottling results in fat differences between samples within a pool

The influence of milk-banking processes on nutrients in donor human milk (DHM) is largely unknown. Previous studies have measured nutrients between pools of DHM, but within-pool nutrient differences (between bottles from the same pool) have yet to be elucidated. The objective of this study was to gain a better understanding of the effect of different mixing characteristics on the distribution of fat, protein, IgA, and lysozyme in bottled, raw DHM. Pools of DHM were created in a laboratory setting according to published human milk-banking guidelines and assigned to a mixing treatment (mixing during bottling method, pooling container material, and refrigerated hold time). Four mixing protocols using glass pooling containers and a 1-h refrigerated hold time were tested: control (no mixing during bottling); manual-A (Man-A, hand swirl after pouring 3 bottles); manual-B (Man-B, hand swirl after pouring every bottle); and mechanical-G (Mech-G, continuous stirring with a magnet). As secondary objectives, we compared the effect of a glass and a plastic pooling container with mechanical mixing (mechanical-P, Mech-P), and compared refrigerated delays of 1 and 24 h before bottling with manual mixing (manual-A24, Man-A24). To control for differences in nutrient content, comparisons between treatments were made using absolute percent difference from the treatment-specific mean; and comparisons within a treatment were made using the ratio of fat content in a bottle to fat content in the first bottle of the same pool. We did not observe differences in nutrient distribution between Man-A, Man-B, and Mech-G in pools held for 1 h, but all were significantly different from the control for fat. There were no differences between glass or plastic pooling containers when mechanical mixing was used. Holding a pool in the refrigerator for 24 h before bottling created significantly greater fat distribution than holding a pool for 1 h. Outcomes were the result of controlled experiments. In summary, manual and mechanical mixing of 1,700-mL DHM pools produces similar fat and protein distributions when DHM is pooled and bottled after a 1-h hold time. When DHM is held for 24 h before bottling, more research is needed to determine the duration of initial mixing needed to reduce fat variability between bottles.