Determination of Levan from Bacillus  licheniformis    by Ultraviolet Spectrophotometry

Purpose: To establish a first-order derivative ultraviolet spectrophotometric (FODUS) method with good reproducibility for the determination of levan, a D-fructofuranosyl polymer with a β-(2-6) backbone and β-(2-1) branching.
Methods: Levan was isolated from fermentation broth by alcohol precipitation and ultrafiltration. Factors influencing the determination of the ultraviolet (UV) spectrophotometric method was compared with a single-factor analysis. The UV spectra of levan reaction solutions in the absorbance range of 200 - 400 nm were obtained. An orthogonal experimental design was applied to optimize the sulfuric acid hydrolysis conditions in the spectrophotometric determination. FODUS method was validated by analyzing its linearity, reproducibility, stability and recovery.
Results: Factors influencing absorbance for the determination were confirmed and two regression equations were established. UV absorbance at 320 nm of sulfuric acid-hydrolyzed sample was stable for 5 h. The FODUS method developed had a good reproducibility (RSD = 2.1 %, n = 5), linearity (ranging from 1.6 μg/mL to 12.8 μg/mL), R² = 0.9996) and recovery (95.90 %, RSD = 1.7 %, n = 3).
Conclusion: The developed FODUS method is convenient, efficient and robust for the determination of microbial levan. The method provides a valuable approach for the determination of polysaccharides.

Keywords
Derivative spectrophotometry; Bacillus licheniformis; Levan; Total carbohydrate determination