Abstract
Psychobiotics are emerging as key players in gut–brain axis research, yet the role of Fructophilic Lactic Acid Bacteria (FLAB) remains largely unexplored. To address this gap, we employed a comprehensive strategy that first simulated gastrointestinal pre-digestion of soybeans to mimic host conditions, then harnessed FLAB fermentation to reshape the soybean metabolite landscape and finally applied in silico prediction to assess the neuroactive potential of the resulting compounds. Six strains were initially screened for gastrointestinal resistance, with only Apilactobacillus kunkeei (BEE4, PL34) and Fructobacillus fructosus (FCS3, PL22) showing high tolerance. Selected strains showed varied but notable functional and pre-technological enzymatic activities. When inoculated into pre-digested soybean, they showed high adaptation, with efficient acidification and short chain fatty acids release. While in vitro digestion alone enriched the amino acid profile, FLAB fermentation further enhanced it, particularly increasing gamma-aminobutyric acid in PL34_DS. Although digestion reduced most phenolic compounds, fermentation not only restored but also diversified the metabolite pool, yielding higher levels of daidzein, genistein, dihydrokaempferol, biochanin A, sinapic acid, caftaric acid, and naringenin. Similarly, peptide abundance rose after digestion and was further boosted by fermentation, especially with A. kunkeei PL34 and F. fructosus PL22, which increased low-molecular-weight peptides and generated novel sequences. Importantly, PL34_DS consistently showed superior antioxidant activity and the strongest reduction of antinutritional factors, highlighting its functional potential. In silico analyses further revealed that several released phenolics (e.g., daidzein) and peptides have gastrointestinal absorption and blood–brain barrier permeability, targeting neurotransmitter receptors, cytokine networks, and cholinergic enzymes. Taken together, these findings indicate that FLAB-driven soybean fermentation can enrich neuroactive metabolite profiles with promising psychobiotic potential, providing a foundation for future in vivo studies aimed at mental health modulation.