Malt β-Glucanase/Lichenase Assay Kit (MBG4 Method)

Colourimetric assays were used to measure the activities of six key hydrolases endogenous to barley: β‐glucanase, xylanase, cellulase, α-amylase, beta‐amylase and limit dextrinase. The analysed barley malt samples were previously characterised by 27 conventional malt quality descriptors. Correlations between enzymatic activities and brewing parameters such as extract yield, fermentability, viscosity and filterability were investigated. A single extraction protocol for all six hydrolases was optimised and used for multi‐enzyme analysis using fully automatable assay formats. A regression analysis between malt parameters was undertaken to produce a relationship matrix linking enzyme activities and conventional malt quality descriptors. This regression analysis was used to inform a multi‐linear regression approach to create predictive models for extract yield, apparent attenuation limit, viscosity and filterability using the Small‐scale Wort rapId Filtration Test (SWIFT) and two different mashing protocols – Congress and a modified infusion mash at 65^oC (MIM 65^oC). It was observed that malt enzyme activities displayed significant correlations with the analysed brewing parameters. Both starch hydrolases and cell wall hydrolase activities together with modification parameters (i.e. Kolbach index) were found to be highly correlated with extract yield and apparent attenuation limit. Interestingly, it was observed that xylanase activity in malts was an important predictor for wort viscosity and filterability. It is envisaged that the automatable measurement of enzyme activity could find use in plant breeding progeny selection and for routine assessment of the functional brewing performance of malt batches. This analytical approach would also contribute to brewing process consistency, product quality and reduced processing times.

We report herein the development of a novel assay procedure for the measurement of β-glucanase and lichenase (EC 3.2.1.73) in crude enzyme extracts. Two assay formats based on a) a direct cleavage or b) an enzyme coupled substrate were initially investigated. The ‘direct cleavage’ substrate, namely 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-3¹-cellotriosyl-β-glucopyranoside (MBG4), was found to be the more generally applicable reagent. This substrate was fully characterised using a crude malt β-glucanase extract, a bacterial lichenase (Bacillus sp.) and a non-specific endo-1,3(4)-β-glucanase from Clostridium thermocellum (EC 3.2.1.6). Standard curves were derived that allow the assay absorbance response to be directly converted to β-glucanase/lichenase activity on barley β-glucan. The specificity of MBG4 was confirmed by analysing the action of competing glycosyl hydrolases that are typically found in malt on the substrate. Manual and automated assay formats were developed for the analysis of a) β-glucanase in malt flour and b) lichenase enzyme extracts and the repeatability of these assays was fully investigated.