This study aims to investigate the production of natural flavor compounds through the utilization of Bacillus subtilis-fermented soybean meal extract and evaluate their biological potential. The experiment involved a comprehensive in vitro investigation to assess the capabilities and effects of the produced flavor compounds. The resulting flavor compounds were subjected to various in vitro tests to assess their properties, including cytotoxicity, antioxidant activity, anticancer potential, antiviral activity, and antimicrobial activity. To enhance the fermentation process, soybean meal extract was fortified with a combination of L-Lysine and L-Threonine. Gas chromatography-mass spectrometry (GC/MS) analysis was conducted on the fermented soybean meal using two strains of Bacillus subtilis, namely NRCH123 and NRCZ144. This analysis revealed the presence of various volatile compounds in all extracts, including Butylated hydroxytoluene. The fermented soybean extract with bacillus subtilis NRCZ144 (B2) fortified with a combination of 2.5% (w/w) L-Lysine and 2.5% w/w L-threonine (SLT2) exhibited a rich profile of flavor compounds, with Eucalyptol being identified as the predominant compound. The antioxidant activity of the SLT₂ extract was found to be 72.04% at a concentration of 100 μg/mL, indicating significant antioxidant potential. Furthermore, when tested against the human liver cancer cell line HepG2, the extract demonstrated anticancer activity with an IC50 value of 2.26 μg/mL. The extract exhibited potent cytotoxicity, with an IC50 value of 1.02 μg/mL. Importantly, the SLT₂ extract displayed strong antibacterial and antifungal activity, even at very low concentrations. The extract's antimicrobial properties indicate its potential for inhibiting the growth of bacteria and fungi.

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