Food-Derived Compounds Extend the Shelf Life of Frozen Human Milk

Abstract

Breastmilk is known to provide optimal nutrition for infant growth and development. A cross-sectional analysis of nationally representative US data from 2016 to 2021 revealed that >90% of lactating mothers reported using breast pumps to express milk. We conducted a survey of n = 1049 lactating or recently lactating individuals from a US nationally representative population to explore breastmilk storage practices among this group. The data revealed that 83% of respondents store breastmilk in their homes, with 68% using freezers to do so for >1 month. The lowest available temperature in most household freezers is -20 °C, a temperature that is inadequate to maintain human milk's emulsified structure, leading to separation, degradation of fats, loss of key vitamins, and changes in palatability. We developed a first-of-its-kind high-throughput screening platform to identify food-derived compounds and combinations of compounds that, when added to human breastmilk, preserve fat content, retain antioxidant capacity, and reduce production of rancid-associated free fatty acids during extended freezer storage. Our screening identified pectin (0.5% w/v) and ascorbic acid (100 μg/mL) as optimal preservation agents. Compared to untreated controls, this formulation reduced glycerol production by approximately 60% and maintained antioxidant capacity after 6 months of storage at -20 °C. Lysozyme and protease activity were maintained at >75% of the levels in fresh breastmilk. This formulation represents a lead for the development of safe and affordable frozen breastmilk shelf-life extenders for at-home use to increase the longevity of stored breastmilk.

Keywords: breast pump; breastfeeding; freezer storage; high-throughput screening; household storage; human milk; lipolysis; milk preservation; milk storage.

Figure 2

Effects of pectin and ascorbic acid on antioxidant capacity, lipolysis, and glycerol accumulation in frozen human milk. (a) Antioxidant capacity and lipolysis in milk treated with 100 μg/mL ascorbic acid and/or 0.5% w/v pectin. No treatment (–) or the designated treatment (+) is indicated under the x-axis. Values are normalized to untreated fresh milk (antioxidant capacity) or untreated frozen milk (lipolysis). Data show means ± SDs of three independent replicates. Treatments significantly influenced antioxidant capacity (p = 0.0003) and lipolysis (p < 0.0001). Gray bars: untreated samples; colored bars: treated samples (blue: antioxidant capacity; orange: lipolysis). (b) Donor variation (n = 14) in antioxidant capacity (A₅₇₀ nm, blue) and lipolysis (BODIPY-FA RFU, orange) with and without the combined 100 μg/mL ascorbic acid and 0.5% w/v pectin treatment. Individual donor values are shown to illustrate inter-individual variability. Data show individual donor measurements. Statistical analysis revealed a significant effect of treatment on the measured outcomes (p < 0.0001). Gray: untreated; colored: treated samples. (c) Glycerol accumulation during −20 °C storage of human milk with and without the combined 100 μg/mL ascorbic acid and 0.5% w/v pectin treatment. Dashed lines indicate positive (100 μM glycerol) and negative (vehicle) controls. Data show means ± SDs of 14 biological replicates. Treatment across groups had a significant effect, with p < 0.0001, indicating that the intervention reduced glycerol accumulation compared to controls.

Figure 1

High-throughput screening of food-derived compounds for inhibition of lipolysis in frozen human milk. (a) Food-derived compounds screened for breastmilk lipolysis inhibition. Orange dots indicate compounds reducing lipolysis below the threshold (dashed line); gray dots show inactive compounds. Data represent the initial HTS readouts. (b) Validation of selected hit compounds from panel (a). The heatmap displays lipolysis inhibition in relative fluorescent units (RFU) at different compound concentrations. Compound concentrations are expressed as dilutions (Log₂(0) to Log₂(−7)) of the screening library stock solutions. Ne5Ac: N-Acetylneuraminic acid; 5-AVA: 5-Aminovaleric acid. Significant differences were observed across treatments and time points (p < 0.0001), indicating that these compounds influence milk lipolysis dynamics during freezer storage. Data represent the median of three independent replicates.

Figure 3

Changes in breastmilk quality during storage and the protective effects of ascorbic acid and pectin treatment. (a) Model depicting changes in human milk quality over storage time. At 1 week, degradation begins, leading to a decline in both nutritional value and structural integrity. Between 8 and 12 weeks, rancidification accelerates, marked by an increase in FFAs and other breakdown products, which further reduce nutritional quality and structural integrity. Rancidity continues to rise beyond 12 weeks. (b) Schematic model illustrating biochemical and structural changes that occur in human milk during storage. Untreated milk, left: Storage compromises MFG structure, enabling lipase-mediated fat hydrolysis and degradation. Lipolysis of triglycerides and lipid oxidation produces secondary and tertiary products that cause rancidity. Treated milk, right (model proposed in the current work): Pectin preserves MFG structural integrity, limiting lipase access to MFGs. Ascorbic acid provides protection from oxidation. Together, these two mechanisms suppress rancidification.