ABSTRACT / PROJECT SUMMARY Currently recommended strategies for primary maintenance of bone mass with age are limited to consuming a diet rich in vitamins and minerals and performing weight bearing activity. Without more specific and effective ways to maintain bone mass with age, older adults will develop osteoporosis and elevated fracture risk necessitating prescription drugs, with side effects and restrictive costs. Hence, there is an unmet need for safe and effective dietary interventions for the metabolic processes underlying bone loss. Our long term goal is to develop safe and effective prebiotic/probiotic combination supplements for the dietary management of the metabolic processes underlying osteopenia and osteoporosis. The objective of this project is to test the efficacy of an probiotic/prebiotic combination (“synbiotic”) on the skeleton in a clinical trial that is designed to provide mechanistic insights into the action of the synbiotic. Our central hypothesis is that a novel plant-derived medical food synbiotic (“SBD111”) will improve dual energy x-ray absorptiometry (DXA) derived lumbar spine bone mineral density (BMD), quantitative computed tomography (QCT) derived bone strength — aka Biomechanical Computed Tomography analysis, or BCT— and volumetric BMD (vBMD), markers of bone turnover, inflammation, and gut microbiome function in older women (>60 years) compared to placebo. Guided by strong preliminary data this hypothesis will be tested by performing an 18-month randomized, double-blinded, placebo- controlled clinical trial in 220 older women. Our specific aims are: Aim 1a and 1b To determine the effect of 18 months of daily intake of SBD111 on the primary outcome of lumbar spine DXA-BMD and secondary outcomes (BCT-derived vertebral compressive bone strength, vBMD, and markers of bone turnover) in women. Aim 2a and 2b To determine the effect of 18 months of daily intake of SBD111 on markers of inflammation and gut microbiome function in women. We will employ whole metagenome and meta-transcriptome shotgun sequencing to dissect changes in gut microbiome function [defined as changes in glycosyl hydrolase, menaquinone 7, short chain fatty acid (SCFA), and microbial diversity], which can mediate the effect of SBD111 on bone turnover and inflammation. We will also consider intake of total dietary fiber, prebiotic fiber, and probiotic foods that could modify the effect of SBD111 on bone, inflammation, and gut microbiome. If successful, this proposed trial will lead to developing potentially safe, inexpensive health promoting strategies for the dietary management of the metabolic processes underlying osteopenia and osteoporosis. 1

PROJECT NARRATIVE and Relevance to Public Health Our proposed study is an 18-month randomized, double-blinded, placebo-controlled clinical trial in 220 postmenopausal women aged 60 years and above. Our objective is to test the efficacy of a probiotic/prebiotic combination (“synbiotic”) on the skeleton in a clinical trial that is designed to provide mechanistic insights into the action of the symbiotic supplement. Understanding these mechanisms might lead to the development of medical food supplements to aid in the dietary management of aging associated bone loss. 1