OBJECTIVES/SPECIFIC AIMS: Aim 1: To compare frozen MOM to fresh MOM over time as an agent to inoculate DHM and measure the enrichment of commensal microbes and their beneficial bioactive components similar to MOM. Hypothesis: Frozen or fresh MOM inoculated in DHM will produce similar microbial content to MOM over time allowing for the production of beneficial bacterial compounds that may contribute to host immune response. Aim 2: To determine the effect of MOM storage (fresh vs frozen) on the expansion of bioactive components from live microbiota in DHM. Hypothesis: Both fresh and frozen MOM will produce similar results when inoculated into DHM to restore the microbial content (including their bioactive components) similar to each MOM sample. Aim 3: To compare the microbiome found in a mother’s MOM to the microbiome in her infant’s stool. Hypothesis: The mother/infant pair will share a common microbiome between the mother’s MOM and her infant’s stool. METHODS/STUDY POPULATION: Subjects will include 12 pump-dependent mothers of infants born < 34 weeks gestation admitted to the University of Florida Health Shands Hospital, Neonatal Intensive Care Unit (NICU). Inclusion criteria consists of mothers expressing over 100 ml of MOM per day, producing at least 45 ml of MOM at an expression session, at least 18 years of age, and speak English. Mothers are excluded if they have taken antibiotics within 3 days of sample collection, are HIV+, or delivered an infant who has a chromosomal abnormality or is severely ill. An expressed MOM sample will be collected and divided into two fractions: (A) fresh and (B) frozen at -20C for 24 h. The fresh fraction (A) will be processed immediately while the frozen fraction (B) will be processed after 24 h. Each MOM will be inoculated in DHM at dilutions of 10% and 30% and incubated at different time points: 0 h (T0), 2 h (T2), 4 h (T4) at 37°C. At each time point, total viable cell counts as well as microbiome analysis through 16S ribosomal shotgun sequencing will be performed and compared for differences. Bacteria isolated from each MOM will be saved, identified through 16S ribosomal sequencing and grown in culture for future studies. Fecal samples from each corresponding infant will be collected within 48 h after collection of the MOM sample. Stools will be homogenized and subjected to DNA extraction to perform 16S ribosomal shotgun sequencing. Microbiome analysis will be conducted, compared between fecal samples as well as with the microbiome of the MOM. RESULTS/ANTICIPATED RESULTS: Study is ongoing. We anticipate similar results with fresh or frozen MOM to that of a previous pilot study, where enriched microbiota similar to MOM was found when fresh MOM was inoculated and incubated in DHM. The microbiome analysis of the infant fecal samples may illustrate the influence that the microbiome of the MOM may have on the development of the infants’ gastrointestinal microbiota. DISCUSSION/SIGNIFICANCE OF IMPACT: The purpose of this study is to provide evidence on the ability, timing, and efficacy of inoculating DHM with fresh and frozen MOM. Study results will inform future studies to support the implementation of an inoculation procedural protocol to be used in clinical practice and human milk banking. The description of the MOM microbiome, as well as the gastrointestinal microbiome, will expand scientific knowledge on the role breast milk has on the origins of health and disease.