Enhanced Production, Characterization, Functional Evaluation, and In-Vitro Cytotoxicity on HepG2 Liver Cancer Cells of <em>Bacillus subtilis</em>-Derived Oligofructans

Oligofructans are fructose-based molecules composed of oligosaccharides and fructans linked by -glycosidic bonds. With the growing market demand for functional foods and bioactive ingredients, oligofructans have attracted considerable interest in their health-promoting properties. This study enhanced the production of oligofructans powder based on Bacillus subtilis TISTR 001 fermentation and analyzed the physicochemical properties of viscosity at concentrations of 10 %, 30 %, and 50 % (w/v), as well as stability at different pH levels (3, 5, 7, 9, and 11) and temperatures (25, 75, 100, and 125 °C) over time. Structural characterization was investigated using advanced analytical techniques (FTIR, NMR, and MALDI-TOF-MS). Based on the results, oligofructans production was successful, achieving a maximum concentration of 75.86 g/L under the optimized conditions at pH 6.5, 32.5 °C, and 250 rpm for 48 h. The oligofructans solution had low viscosity (10 cP at 50 % (w/v)) with Newtonian flow behavior and maintained excellent stability under neutral pH and moderate temperature conditions. The structural analysis confirmed the presence of characteristic fructose-based glycosidic linkages. Based on the results of the functional property assessment, there were dual bioactivity functions, with antioxidant activity having an IC₅₀ value of 7.11 mg/mL and the cytotoxicity studies indicating significant inhibitory effects on HepG2 liver cancer cells at concentrations ≥100 mg/mL. These findings supported the potential of oligofructans as multifunctional ingredients for application in the food, nutraceutical, and pharmaceutical industries. Further research will focus on process optimization for industrial-scale production and stability testing in various food matrices, along with investigating the bioactive and therapeutic potential, particularly for liver health and cancer prevention.