Prediction of potential malt extract and beer filterability using conventional and novel malt assays.
Cornaggia, C., Evans, D. E., Draga, A., Mangan, D. & McCleary, B. V. (2019). Journal of Institute of Brewing, In Press.
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°C (MIM 65°C). 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.
Colourimetric and fluorometric substrates for measurement of pullulanase activity.
McCleary, B. V., Mangan, D., McKie, V., Cornaggia, C., Ivory, R. & Rooney, E. (2014). Carbohydrate Research, 393, 60-69.
Specific and highly sensitive colourimetric and fluorometric substrate mixtures have been prepared for the measurement of pullulanase and limit-dextrinase activity and assays employing these substrates have been developed. These mixtures comprise thermostable α- and β-glucosidases and either 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-maltotriosyl (1-6) α-maltotrioside (BzCNPG3G3, 1) as a colourimetric substrate or 4,6-O-benzylidene-4-methylumbelliferyl-β-maltotriosyl (1-6) α-maltotrioside (BzMUG3G3, 2) as a fluorometric substrate. Hydrolysis of substrates 1 and 2 by exo-acting enzymes such as amyloglucosidase, β-amylase and α-glucosidase is prevented by the presence of the 4,6-O-benzylidene group on the non-reducing end D-glucosyl residue. The substrates are not hydrolysed by any α-amylases studied, (including those from Aspergillus niger and porcine pancreas) and are resistant to hydrolysis by Pseudomonas sp. isoamylase. On hydrolysis by pullulanase, the 2-chloro-4-nitrophenyl-β-maltotrioside (3) or 4-methylumbelliferyl-β-maltotrioside (4) liberated is immediately hydrolysed to D-glucose and 2-chloro-4-nitrophenol or 4-methylumbelliferone. The reaction is terminated by the addition of a weak alkaline solution leading to the formation of phenolate ions in solution whose concentration can be determined using either spectrophotometric or fluorometric analysis. The assay procedure is simple to use, specific, accurate, robust and readily adapted to automation.
Colourimetric and fluorimetric substrates for the assay of limit dextrinase.
Mangan, D., McCleary, B. V., Cornaggia, C., Ivory, R., Rooney, E. & McKie, V. (2015). Journal of Cereal Science, 62, 50-57.
The measurement of limit-dextrinase (LD) (EC 22.214.171.124) in grain samples such as barley, wheat or rice can be problematic for a number of reasons. The intrinsic LD activity in these samples is extremely low and they often contain a limit-dextrinase inhibitor and/or high levels of reducing sugars. LD also exhibits transglycosylation activity that can complicate the measurement of its hydrolytic activity. A minor modification to the industrial standard Limit-Dextrizyme tablet test is suggested here to overcome this transglycosylation issue.
In addition, two new substrates are described that can be adopted for use in an auto-analyser format. 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-63-α-D-maltotriosyl-maltotrioside (BzCNPG3G3, Hexachrom) is not susceptible to transglycosylation and serves amiably as a routine quantitative assay tool with the potential to run kinetic assays due to the low pKa (∼5.5) of the chromogenic moiety while 4,6-O-benzylidene-4-methylumbelliferyl-β-63-α-D-maltotriosyl-maltotrioside (BzMUG3G3, Hexafluor) was found to be susceptible to transglycosylation with LD. It is anticipated that Hexafluor may find extensive use in applications where high sensitivity is required such as high throughput screening studies.
Gluten-free sources of fermentable extract: effect of temperature and germination time on quality attributes of teff [Eragrostis tef (zucc.) trotter] malt and wort.
Di Ghionno, L., Marconi, O., Lee, E. G., Marconi, O., Rice, C. J., Sileoni, V. & Perretti, G. (2017). Journal of Agricultural and Food Chemistry, 65(23), 4777-4785.
This study was conducted to evaluate the behavior of a white teff variety called Witkop during malting by using different parameters (germination temperature and duration) and to identify the best malting program. Samples were evaluated for standard quality malt and wort attributes, pasting characteristics, β-glucan and arabinoxylan content, and sugar profile. It was concluded that malting teff at 24°C for 6 days produced acceptable malt in terms of quality attributes and sugar profile for brewing. The main attributes were 80.4% extract, 80.9% fermentability, 1.53 mPa s viscosity, 7.4 EBC-U color, 129 mg/L FAN, and 72.1 g/L of total fermentable sugars. Statistical analysis showed that pasting characteristics of teff malt were negatively correlated with some malt quality attributes, such as extract and fermentability. Witkop teff appeared to be a promising raw material for malting and brewing. However, the small grain size may lead to difficulties in handling malting process, and a bespoke brewhouse plant should be developed for the production at industrial scale.
Optimization of the production of an extracellular and thermostable amylolytic enzyme by Thermus thermophilus HB8 and basic characterization.
Akassou, M. & Groleau, D. (2017). Extremophiles, 1-14.
The objective of this study was to determine the potential of Thermus thermophilus HB8 for accumulating a high level of extracellular, thermostable amylolytic enzyme. Initial production tests indicated clearly that only very low levels of amylolytic activity could be detected, solely from cells after extraction using the mild, non-ionic detergent Triton X-100. A sequential optimization strategy, based on statistical designs, was used to enhance greatly the production of extracellular amylolytic activity to achieve industrially attractive enzyme titers. Focus was placed on the optimal level of initial biomass concentration, culture medium composition and temperature for maximizing extracellular amylolytic enzyme accumulation. Empirical models were then developed describing the effects of the experimental parameters and their interactions on extracellular amylolytic enzyme production. Following such efforts, extracellular amylolytic enzyme accumulation was increased more than 70-fold, with enzyme titers in the 76 U/mL range. The crude extracellular enzyme was thereafter partially characterized. The optimal temperature and pH values were found to be 80°C and 9.0, respectively. 100% of the initial enzyme activity could be recovered after incubation for 24 h at 80°C, therefore, proving the very high thermostability of the enzyme preparation.