Microbial Levan Biosynthesis from Palmyra Palm Sap Using Bacillus Subtilis and Its Characterization

Abstract

Objective: Evaluate the suitability of palmyra (Borassus flabellifer L.) sap as a fermentation substrate for microbial levan production using Bacillus subtilis. Methodology: Pasteurized palmyra sap was first analyzed for pH and then used as a substrate for fermentation under optimized conditions (pH 6–7, 30 ℃, 72 h). Following fermentation, levan was extracted and quantified. Structural and physicochemical properties of the obtained levan were characterized using FTIR to confirm fructofuranosyl linkages, XRD to determine crystallinity, TEM and FESEM for morphological visualization, NMR for structural elucidation, HRMS for molecular confirmation, and DSC for thermal behaviour. Rheological analysis was conducted to assess flow properties and structural recovery. Antioxidant activity was measured using DPPH and FRAP assays. Results and conclusions: Fermentation yielded 4.35 ± 0.43 g/L of levan. FTIR confirmed β-(2→6) fructofuranosyl linkages, and XRD showed an amorphous pattern with a broad peak at 2θ = 21°. TEM and FESEM revealed compact, carbon-rich structures typical of dense polysaccharides. NMR confirmed characteristic fructan proton and carbon shifts supporting β-(2→6) linkages and HRMS verified mass fragments consistent with levan. DSC indicated thermal stability with a glass transition at 54.46 ℃ and decomposition onset near 320 ℃. The levan showed shear-thinning behaviour with recovery on cooling. Antioxidant activity was notable, with 69% DPPH scavenging and 468 ± 0.37 μg AAE/g FRAP value. Overall, palmyra sap demonstrated strong potential as a natural substrate for producing structurally stable and bioactive microbial levan suitable for functional and biotechnological applications.