Bioactive fructans from Bacillus subtilis and the multifunctional role of L3 with cytotoxic, anticoagulant, and antimicrobial activities.

This study examined how culture conditions affect the molecular weight (MW), structure, and function of four Bacillus subtilis fructans (L1-L4). The polysaccharides (170-34 kDa) were characterized using TLC, FTIR, and NMR, revealing clear MW-dependent bioactivities. The highest-MW fructan, L1 (170 kDa), showed broad antimicrobial effects and strong prebiotic activity with minimal cytotoxicity. L2 (61 kDa) demonstrated exceptional anticoagulant efficacy, extending clotting time to 187 min, surpassing heparin. The low-MW L3 (34 kDa), enriched in extended β-(2 → 6) linkages, exhibited the strongest anticancer effect against HCT-116 cells (IC₅₀ = 44.7 μg), inducing apoptosis via P53 and Bax upregulation and Bcl-2 suppression. L3 also retained antimicrobial and antioxidant activity (21% DPPH scavenging) alongside prebiotic benefits. All fructans exceeded Hemoclar in fibrinolytic potential. Overall, the results establish a clear MW-function relationship and highlight L3 as a promising multifunctional candidate for biomedical use.