Abstract
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
Highlights
It is always required to determine total carbohydrates in aqueous solution for polysaccharide application development in most cases [1,2]
UV spectra of levan solutions in the range of 200 to 400 nm were scanned and the derivative spectra were transformed by UVWin software (Figures 2 and 3)
Where dA/dλ is the difference between the firstorder derivative absorbance at 306 nm (A’306 nm) and the first-order derivative absorbance at 337 nm (A’337 nm); A is the absorbance at 320 nm (A 320 nm); and C is the concentration of sugar
Summary
It is always required to determine total carbohydrates in aqueous solution for polysaccharide application development (with a good reproducibility) in most cases [1,2]. Diversity and heterogeneity of polysaccharides have led to the development of numerous chromatographic and spectroscopic methods for measuring carbohydrates [3,4]. These methods require sophisticated skills and advanced equipment. Derivative ultraviolet-visible absorption spectrophotometry (DS) method has been widely used to enhance the signal and decrease the interference of impurities with closely adjacent peaks in complex mixtures for determining drugs, food and other compounds [7]. DS, found few applications in determining polysaccharides due to its poor absorbance in the UV region
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