Abstract PumpKin Baby is developing a method to extend the shelf life of breastmilk using food-derived bioactive ingredients that preserve the structural integrity of breastmilk and retain its vital fats, vitamins, proteins, and taste during freezer storage and subsequent thawing. Human milk is the gold standard for infant nutrition, containing a complex blend of key nutrients and components that are essential to meeting life’s critical early milestones. The World Health Organization recommends exclusive breastfeeding for the first six months of an infant’s life to deliver an ideal source of nutrition, immune instruction, pathogen defense, and microbiome development. To facilitate continued breastfeeding in light of challenges with return to work, nursing mothers often rely on freezer storage of expressed breastmilk, and those with lactation difficulties seek donor breastmilk, which is also stored frozen. However, the temperature of most household freezers (-20 °C) does not preserve the complex emulsified structure of human breastmilk, resulting in fat breakdown and the generation of rancid flavor compounds during storage and thawing processes. In addition to loss of fat/caloric content and rancidity, the nutritional value of freeze-thawed breastmilk is significantly degraded compared to fresh breastmilk, particularly for proteins, carbohydrates, antioxidant capacity, and bactericidal capacity. With ~74% of breastfeeding mother freezing their milk for over three months, and ~42% of parents finding frozen milk more difficult to feed than fresh breastmilk, these challenges contribute to suboptimal infant feeding regimes (e.g., discontinuation of breastfeeding) and infant undernutrition. The only established method to prevent milk spoilage is pasteurization, which alters breastmilk's emulsified structure and decreases its nutritional content and is impractical for caregivers to perform at home. Addressing these gaps, PumpKin’s affordable technology would be the first to offer an easy to use, at- home solution for breastmilk preservation. PumpKin’s breastmilk-preserving formulations have been developed through rapid, high-throughput screening of 2,750+ food-derived and/or generally recognized as safe (GRAS) compounds and have already been shown to inhibit >60% of fat breakdown over six months. Building on these promising findings, the following Phase I Specific Aims are proposed: 1) Develop GRAS-based formulation(s) demonstrated to significantly preserve fat stability in frozen breastmilk; 2) Investigate benefits of formulations on the preservation of other high-value, storage-sensitive biological components in human milk (i.e., maternal cell viability and milk fat globule membranes; and 3) Assess performance under realistic home storage conditions and determine the minimum formulation dose needed for human breastmilk preservation. These aims will position PumpKin to further develop this technology in Phase II, which will involve exploring additional...