Multi-omics reveals the microbial succession-driven metabolic regulation during sequential fermentation of fresh Spirulina.

Fermented Spirulina has superior sensorial and bioactive properties, but the impact of microbial succession on metabolic regulations of these attributes remains unclear. This study investigates metabolic regulations via integrated multi-omics analyses that link microbial succession with coordinated shifts in volatile and non-volatile metabolites. Sequential fermentation enhanced the sensory score of Spirulina from 40.09 to 89.29 points and improved its in vitro hypoglycemic and hypolipidemic activities as depicted by higher α-glucosidase inhibition (90.2%), pancreatic lipase inhibition (36.5%), and sodium taurocholate binding (40.2%). Fermentation kinetics suggests coordinated metabolic transition, characterized by high microbial viability (>8 log CFU/mL), enhanced organic acid production (lactic acid up to 2318.8 mg/L), and increased levels of TCA (tricarboxylic acid) intermediates (citrate, malate, succinate). Volatile metabolite profiling showed 50-fold increase in total volatile organic compounds, accompanied by a shift from aldehyde-dominating grassy notes to alcohol/ester dominating floral/fruity notes. Integrated 16S/18S sequencing suggests a structured microbial succession from cyanobacteria dominance (>98%) in unfermented sample to Kluyveromyces marxianus dominance in first-stage fermentation, to Lactiplantibacillus plantarum and indigenous Proteobacteria dominance during second-stage fermentation. Metabolomics revealed an extensive metabolic reprogramming (>700 significantly altered metabolites), indicating transition from primary (amino acids, lipids) to secondary (esters, alcohols, organic acids) metabolites. Integrated correlation and pathway analyses suggest division of labor where K. marxianus dominant fermentation was associated with the generation of aroma precursors and TCA flux, while L. plantarum dominant stage potentially contributed to proteolysis, acidification, and bioactive compound generation. These findings provide a mechanistic rationale for the targeted valorization of microalgal biomass into palatable functional foods.