Food-derived protein hydrolysates frequently contain both collagen-associated hydroxyproline (Hyp) and branched-chain amino acids (BCAAs), but the biological consequences of their combination remain unclear. This study tested whether Hyp changes the effects of leucine (Leu), isoleucine (Ile), and valine (Val) on lifespan and healthspan-related phenotypes in Caenorhabditis elegans and whether these treatment effects are accompanied by coordinated microbial and metabolic remodeling. Amino acid concentrations that did not strongly disrupt bacterial food clearance were selected for lifespan, stress-resistance, motility, muscle-integrity, biochemical, microbiome, metabolomic, and transcriptomic analyses. Hyp+BCAA combinations improved lifespan- and healthspan-related phenotypes in a BCAA- and dose-dependent manner. The strongest combined treatments enhanced resistance to thermal and oxidative stress, preserved age-associated motility and muscle structure, reduced oxidative and lipofuscin-associated markers, and increased ATP-related readouts. Multi-omics profiling showed that these direct treatment effects were accompanied by distinct C. elegans-associated microbiota signatures, altered NAD precursor-related metabolites including nicotinic acid ribonucleoside (NaR), and a Leu+Hyp transcriptional response enriched for energy metabolism, proteostasis, and extracellular-matrix remodeling. Integrated microbiome-metabolome analysis identified a positive association between Rhodococcus abundance and NaR levels. These findings demonstrate that, under the tested culture conditions, Hyp+BCAA exposure improves longevity-related phenotypes and identifies microbial composition, NAD precursor metabolism, and host energy homeostasis as candidate, testable mediators.
