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.

Oat and barley bran are high in dietary fiber (β-glucans), minerals, and antioxidants, have high activity of enzymes, but possess also antinutrients. This study aimed to investigate the influence of high-intensity ultrasound on enzyme and antioxidant activities, phytic acid content, as well as functional and rheological properties of oat and barley bran. Ultrasonic treatment was performed at 24 kHz on a 15% bran water suspension, at three specific energies (87, 217.5, and 348 kJ/kg), with or without pulsation (5 sec every 10 sec). Bran was assessed for β-glucanase and phytase activity, phytic acid and total phenolic content, antioxidant activity, hydration, and rheological properties. β-glucanase from oat bran was inactivated up to 82% and from barley bran up to 55%, in dependence of ultrasound specific energy and pulsation. In both bran types, phytase activity increased by 40-44% after treatments with 87 kJ/kg but decreased by 89-94% at 348 kJ/kg. Phytic acid was reduced on average in oat bran by 17% and in barley bran by 39%. Depending on the energy and pulsation, the ultrasonication of both bran types reduced total phenolic content (27-55%), antioxidant activity (by 28-48%), complex viscosity (62-71%) and maximum stress tolerated by the sample (46-68%). In contrast, water swelling (42-48%) and water retention capacity (44–59%) increased for both bran types. Hence, high-intensity ultrasound is a useful technique in reducing antinutrients, while altering the enzyme activity and functional properties of the bran. These results could help wider application of bran in food production.