Examining Molecular Biodiversity Using Aniline Blue to Detect β‐1,3‐Glucans

β-1,3-Glucans are widely used in food and pharmaceuticals, such that testing for them is crucial to their appropriate application. Using the aniline blue method is a convenient way to test for β-1,3-glucans. However, the conditions used to detect them are quite varied and need to be standardized for all investigators. Here, we established a protocol that optimizes key parameters, including NaOH concentration (0.6 M), depolymerization temperature (40°C), reaction time (10 min), and dye-binding temperature (30°C) using curdlan as a reference. We found that the fluorescence intensity of aniline blue binding was negatively correlated with branching in β-1,3-glucans and positively correlated with molecular weight. A correction factor CF = -0.471 + 0.163·ln(a) × (1 - 0.550b), with a for molecular weight and b for branching degree, was developed to correct for structural diversity. This enabled accurate quantification among structurally different β-1,3-glucans. Overall, our results will greatly improve the accuracy and precision to detect β-1,3-glucans and provide a reliable method for their analysis.