P-11 - An Improved Calcofluor Assay for Beta-glucan Determination

Beta-glucan is a direct measure of malt quality and modification for maltsters and of practical importance to brewers because of its significant contribution to wort viscosity.  As such, beta-glucan determination is one of the key assays of malt quality determination. One of the ASBC-approved methods for determining beta-glucan content relies on the binding of the fluorescent dye Calcofluor to beta-glucan and detection of the complex via fluorimeter. However, studies in oats suggest that the binding of calcofluor to beta-glucan can be improved by the addition of NaCl to the dye solution; specifically, Na⁺ ions dampen the negative surface charges of beta-glucan molecules, allowing increased calcofluor binding and improved resolution. Previous reporting utilized extracted and purified oat beta-glucan, and it’s unclear if a complex matrix such as malt wort produces the same effects. This experiment compares the ASBC method for beta-glucan determination using a segmented flow analysis system to a novel method which includes 200 mM NaCl in the dye solution.  Beta-glucan standards were prepared in the approximate range of 60 – 600 ppm and analyzed using both methods. The traditional method revealed a second-order response curve (R² > 0.998) exhibiting a diminishing response with increasing beta-glucan. In the published protocol, the curve is approximated as linear below 300 ppm, limiting the dynamic range of the assay to this range. Conversely, the novel method demonstrated a linear response over the entire 60-600 ppm range (R² > 0.998).

Analysis of wort samples revealed discrepancies in values obtained by the traditional method and the novel method. Subsequent analysis of wort samples via an enzymatic digestion method of beta-glucan determination yielded results supporting those of the novel method. Results were also compared to the Congo Red method of beta-glucan determination. Results suggest the accuracy, resolution, and dynamic range of the ASBC method Wort-18B can be improved by the addition of salt to the dye solution, and further investigation is merited.