Probiotic-Assisted Semi-Solid-State Fermentation of Amaranth Flour Using Response Surface Methodology for Enriched Bioactives

Semi-solid‑state fermentation (S-SSF) of black amaranth flour, supplemented with okara (a by-product from soy beverage production), offers a sustainable route to generate value‑added bioactives from underutilized crops. A three-factor, three-level central composite design was employed in this study for predicting the effects of fermentation time (40 - 80 h), okara concentration (3.30 - 11.70 %) and temperature (17 - 34°C) on total titratable acidity, soluble protein index, total polyphenols content, flavonoids content, antioxidant activity (ABTS), and antifungal activities against Penicillium expansum MIUG M11 strain and Aspergillus niger MIUG M5 strains, of the fermented products. Twenty experimental runs, including six center‑point replicates, were performed with Lactiplantibacillus pentosus MIUG BL24 probiotic strain as the starter culture. For instance, titratable acidity, soluble protein index and antifungal activity increased with longer fermentation time and higher okara supplementation, whereas flavonoid and antioxidant activities showed antagonistic interactions between time and temperature due to phenolics biotransformation. The desirability function approach yielded an overall desirability of 0.785 at 48 h, 29.54°C and 9.63 % (w/v) okara. Under these conditions, the model predicted titratable acidity of 9.90 mL NaOH 0.1N, soluble protein index of 0.74 mg/g DW, TPC of 3.52 mg GAE/g DW, TFC of 1.91 mg CE/g DW, antioxidant activity (ABTS scavenging assay) of 40.93 %, and antifungal activities of 70.20 % (against P. expansum) and 16.66% (against A. niger). Validation experiments at 48 h, 30°C and 9.60% okara confirmed the model’s accuracy, with all measured responses falling within the 95 % confidence limits. These results demonstrate that combining nutrient enrichment from okara with controlled S-SSF conditions enhanced the functional properties of amaranth‑based fermented products.