Water-extractable arabinoxylan (WEAX) may cause major problems during clarification processes in a brewery owing to its ability to form gel networks. However, high WEAX contents can also enhance the nutritional quality of the final product as they play an important role in the human diet. Therefore, precise quantification of WEAX is required. Current methods are very time- and resource-consuming as well as limited in the number of samples and in some cases provide low accuracy. Thus, a reproducible high-throughput method for the quantification of WEAX optimized for beer was developed, reaching recovery rates (RRs) of almost 100%. The assay is based on Douglas's colorimetric method. Hydrolysis was conducted using glacial acetic acid to induce the formation of red color complexes resulting from the interaction between pentose degradation products and phloroglucinol. The method was successfully transferred to a multi-mode microplate reader to minimize the loss of color intensity over time and to obtain a high throughput. By using 96-well plates, up to 40% of the previous analysis time could be saved, and a larger number of samples could be analyzed in one batch. The collected data determined xylose as an optimal calibration standard due to high accuracy and reproducibility. The respective AX control standards showed RR within the range of 95-105% without exception. To validate and show the ruggedness of the modified method, WEAX concentration in seven commercial German beers (e.g., lager, pilsner, wheat beer, non-alcoholic beer) was quantified. Interfering hexose sugars that lead to measurement errors when analyzing samples with high amounts of fermentable sugars (e.g., non-alcoholic beer produced by limited fermentation) were eliminated by Saccharomyces diastaticus fermentation. Further investigations were carried out by means of LC-MS in order to obtain additional information about the reddish product in the hydrolyzed samples. In this context, C16H12O6 could be identified as one of numerous condensation products, contributing to the coloring. The collected data showed the impact of diverse factors on the measured AX concentration and helped optimize the experimental procedure for a high sample throughput with precise and highly reproducible results. The proposed quantification method should be primarily used in completely fermented finished beer to emphasize the time aspect. Wort samples and non-alcoholic beer produced by limited fermentation can be also analyzed, but only after fermentation with S. diastaticus.
Keywords: WEAX; acid hydrolysis; arabinoxylan; high throughput; phloroglucinol; quantification; recovery rate; xylose.