Identifying Key Bioactive Components in Postbiotic Preparations: From Candidate Discovery to Functional Validation

Postbiotic preparations contain inanimate microbial cells and a diverse mixture of cell-wall structures, proteins, polysaccharides, lipids, metabolites, and vesicle-associated materials. Although many of these components have been proposed as key bioactives, most are identified through compositional profiling, enrichment in active fractions, or testing of purified molecules. Such findings demonstrate biological activity but do not necessarily show that a candidate contributes to the effect of the original preparation. This review examines the experimental approaches used to narrow candidate lists and evaluate functional contribution, including phenotype-guided comparison, activity-guided fractionation, selective depletion, multi-omics profiling, structural characterization, dose-response testing, mechanistic intervention, and reconstitution. A structured narrative search of Web of Science Core Collection, PubMed, and Scopus through 30 March 2026, supplemented by citation tracking, identified the relevant literature; 15 representative primary studies that examined defined candidates and provided evidence beyond compositional detection were selected and appraised across seven preparation-level attribution domains. An appraisal of representative studies shows that current evidence largely supports the activity of individual candidates, whereas preparation-level attribution remains uncommon. Stronger evidence requires quantification of the candidate in the source material, selective removal with appropriate controls, and restoration at a preparation-relevant dose. Structural heterogeneity, processing history, molecular state, dose, and experimental context must also be considered. This evidence-based approach can support bioactive-component validation, batch consistency, and the design of future preclinical and human studies.