Novel approaches to the automated assay of β-glucanase and lichenase activity.
Mangan, D., Liadova, A., Ivory, R. & McCleary, B. V. (2016). Carbohydrate Research, 435, 162-172.
We report herein the development of a novel assay procedure for the measurement of β-glucanase and lichenase (EC 188.8.131.52) 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 184.108.40.206). 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.
Soluble, dye-labeled polysaccharides for the assay of endohydrolases.
McCleary, B. V. (1988). Methods in Enzymology, 160, 74-86.
A range of methods has been developed for the assay of polysaccharide endohydrolases, and these include viscosimetric and nephelometric methods and procedures based on the measurement of increase in reducing sugar equivalents and on the rate of release of soluble, dye-labeled fragments on hydrolysis of chromogenic polysaccharide substrates. Assays based on the use of chromogenic (dye-labeled) substrates have several advantages over more conventional assays including specificity and simplicity in use. However, as dyeing generally reduces the solubility of the polysaccharide, most commercially available dye-labeled substrates are insoluble and have the inherent disadvantages of heterogeneity in the assay tube and the difficulties associated with dispensing a solid substrate routinely with accuracy. This chapter describes methods for the preparation of soluble dye-labeled substrates for the assay β-D-mannanase, endo-1,4-β-D-glucanase , endo-1,3(4)-β-o-glucanase, and α-amylase.
New developments in the measurement of α-amylase, endo-protease, β-glucanase and β-xylanase.
McCleary, B. V. & Monaghan, D. (2000). “Proceedings of the Second European Symposium on Enzymes in Grain Processing”, (M. Tenkanen, Ed.), VTT Information Service, pp. 31-38.
Over the past 8 years, we have been actively involved in the development of simple and reliable assay procedures, for the measurement of enzymes of interest to the cereals and related industries. In some instances, different procedures have been developed for the measurement of the same enzyme activity (e.g. α-amylase) in a range of different materials (e.g. malt, cereal grains and fungal preparations). The reasons for different procedures may depend on several factors, such as the need for sensitivity, ease of use, robustness of the substrate mixture, or the possibility for automation. In this presentation, we will present information on our most up-to-date procedures for the measurement of α-amylase, endo-protease, β-glucanase and β-xylanase, with special reference to the use of particular assay formats in particular applications.
Measurement of dietary fibre components: the importance of enzyme purity, activity and specificity.
McCleary, B. V. (2001), “Advanced Dietary Fibre Technology”, (B. V. McCleary and L. Prosky, Eds.), Blackwell Science, Oxford, U.K., pp. 89-105.
Interest in dietary fibre is undergoing a dramatic revival, thanks in part to the introduction of new carbohydrates as dietary fibre components. Much emphasis is being placed on determining how much fibre is present in a food. Linking a particular amount of fibre to a specific health benefit is now an important area of research. The term 'dietary fibre' first appeared in 1953, and referred to hemicelluloses, celluloses and lignin (Theandere/tf/. 1995). Trowell (1974) recommended this term as a replacement for the no longer acceptable term 'crude fibre'. Burkitt (1995) has likened the interest in dietary fibre to the growth of a river from its first trickle to a mighty torrent He observes that dietary fibre 'was first viewed as merely the less digestible constituent of food which exerts a laxative action by irritating the gut', thus acquiring the designation 'roughage' - a term later replaced by 'crude fibre' and ultimately by 'dietary fibre'. Various definitions of dietary fibre have appeared over the years, partly due to the various concepts used in deriving the term (i.e. origin of material, resistance to digestion, fermentation in the colon, etc.), and partly to the difficulties associated with its measurement and labelling (Mongeau et al. 1999). The principal components of dietary fibre, as traditionally understood, are non-starch polysaccharides (which in plant fibre are principally hemicelluloses and celluloses), and the non-carbohydrate phenolic components, cutin, suberin and waxes, with which they are associated in nature. In 1976, the definition of dietary fibre was modified to include gums and some pectic substances, based on the resistance to digestion of these components in the upper intestinal tract. For the purposes of labelling, Englyst et al. (1987) proposed that dietary fibre be defined as 'non-starch polysaccharides (NSP) in the diet that are not digested by the endogenous secretions of the human digestive tract'. Methods were concurrently developed to specifically measure NSP (Englyst et al. 1994).
Measurement of malt beta-glucanase.
McCleary, B. V. (1986). Proceedings of the 19th Convention of the Institute of Brewing (Aust. and N.Z. section), 181-187.
A Procedure has been developed for the assay of malt β-glucanase [a(1→3)(1→4)-β-D-glucanase] which employs as substrate, barley β-glucan dyed with Remazolbrilliant Blue and chemically modified with carboxymethyl groups to increase solubility. The described assay procedure together with a modified extraction format allows analysis of up to ten malt samples in less than 80 min. Also, the procedure is specific for enzymes active on barley β-glucan, is accurate and reliable, and can be readily applied to the analysis of β-glucanase in malt, green malt and wort.
A soluble chromogenic substrate for the assay of (1→3)(1→4)-β-D-glucanase (lichenase).
McCleary, B. V. (1986). Carbohydrate Polymers, 6(4), 307-318.
A simple procedure for the assay of (1→3)(1→4)-β-D-glucanase (lichenase) has been developed. This assay employs as substrate barley (1→3)(1→4)-β-D-glucan dyed with Remazolbrilliant Blue R and chemically modified with carboxymethyl groups to increase solubility. Preparation of this substrate required the development of an improved procedure for the extraction and purification of barley β-glucan. Assays based on the use of the described chromogenic substrate at pH 6•5 are sensitive and specific for enzymes active on barley β-glucan.
Problems caused by barley beta-glucans in the brewing industry.
McCleary, B. V. (1986). Chemistry in Australia, 53, 306-308.
Brewing, the oldest application of bio-technology is now a mix of trade art and modern science. This article describes new applications of enzyme chemistry to trouble-shooting in beer production.
Assay of malt β-glucanase using azo-barley glucan: an improved precipitant.
McCleary, B. V. & Shameer, I. (1987). Journal of the Institute of Brewing, 93(2), 87-90.
A procedure recently described for the assay of malt β-glucanase, which employs a dye-labelled and chemically-modified barley β-glucan substrate, has been improved by changing the precipitant solution used to terminate the reaction. The new precipitant solution contains 0•4% (w/v) zinc acetate and 4% (w/v) sodium acetate dissolved in 80% (v/v) aqueous methyl cellosolve. With this precipitant the procedure can be directly applied to the assay of cellulase activity, and with minor modification, to the assay of lichenase activity.
The influence of germination conditions on beta-glucan, dietary fibre and phytate during the germination of oats and barley.
Hübner, F., O’Neil, T., Cashman, K. D. & Arendt, E. K. (2010). European Food Research and Technology, 231(1), 27-35.
This study aimed to quantify the changes caused by varying germination conditions on the contents of some bioactive compounds in barley and oats. Samples of the two grains were germinated at temperatures between 10 and 20°C for a period of 2–6 days, using a two-dimensional central composite design. The germination temperature had only minor effect in comparison with the germination time. Slight changes in the mineral content of the malts were observed, mainly caused by steeping. Phytate has been seen as an anti-nutritional compound, as it complexes minerals and lowers their bioavailability. The phytate content in barley malts was considerably lower than in the native kernels. Variations in the germination conditions did not have a significant effect on phytate content. In oats, degradation of phytate was significantly enhanced by prolonging the germination period. It was possible to retain the amounts of soluble dietary fibre, when short germination periods were applied. However, long germination periods caused an extensive breakdown of soluble dietary fibre, especially beta-glucan. The content of insoluble fibre, however, was increased by applying long germination periods for oat malts.
Malting of barley with combinations of Lactobacillus plantarum, Aspergillus niger, Trichoderma reesei, Rhizopus oligosporus and Geotrichum candidum to enhance malt quality.
Hattingh, M., Alexander, A., Meijering, I., van Reenen, C. A. & Dicks, L. M. T. (2014). International Journal of Food Microbiology, 173, 36-40.
Good quality malt is characterised by the presence of high levels of fermentable sugars, amino acids and vitamins. To reach the starch-rich endosperm of the kernel, β-glucan- and arabinoxylan-rich cell walls have to be degraded. β-Glucanase is synthesized in vast quantities by the aleurone layer and scutellum during germination. Secretion of hydrolytic enzymes is often stimulated by addition of the plant hormone gibberellic acid (GA3) during germination. We have shown an enhanced β-glucanase and α-amylase activity in malt when germinating barley was inoculated with a combination of Lactobacillus plantarum B.S1.6 and spores of Aspergillus niger MH1, Rhizopus oligosporus MH2 or Trichoderma reesei MH3, and L. plantarum B.S1.6 combined with cell-free culture supernatants from each of these fungi. Highest malt β-glucanase activity (414 Units/kg malt) was recorded with a combination of L. plantarum B.S1.6 and spores of A. niger MH1. Highest α-amylase activities were recorded with a combination of L. plantarum B.S1.6 and spores of R. oligosporus MH2 (373 Ceralpha Units/g malt). Highest FAN levels were recorded when L. plantarum was inoculated in combination with spores of either R. oligosporus MH2 or T. reesei MH3 (259 and 260 ppm, respectively). This is the first study showing that cell-free culture supernatants of Aspergillus, Rhizopus and Trichoderma have a stimulating effect on β-glucanase and α-amylase production during malting. A combination of L. plantarum B.S1.6, and spores of A. niger MH1 and R. oligosporus MH2 may be used as starter cultures to enhance malt quality.
Detection, localization, and variability of endogenous β-glucanase in wheat kernels.
Vatandoust, A., Ragaee, S., Wood, P. J., Tosh, S. M. & Seetharaman, K. (2012). Cereal Chemistry, 89(1), 59-64.
Clinical studies with isolates of β-glucan have shown that the health benefits are regulated not only by the polysaccharide concentration but also by the molecular weight and concentration in solution, because these health benefits are controlled, inter alia, by viscosity in the gut. The degradation of β-glucan in baked products is likely caused by baking ingredients or processes, or by endogenous enzymes in wheat flour. The objectives of the present study were to quantify β-glucanase in wheat kernels and to determine factors that influence the levels of this enzyme. A modified protocol to quantify β-glucanase was developed and then confirmed through high-performance size-exclusion chromatography (HPSEC) with Calcofluor detection. Under this protocol, it was shown that the concentration of β-glucanase activity was the highest in the bran fraction of the kernel in ungerminated wheats, whereas it was distributed throughout the entire kernel following germination. Furthermore, investigation on different wheat cultivars planted in the same and different locations showed that genotype, environment, and agronomic practice all can have an effect on β-glucanase activity level in wheat kernels.
Effect of extraction conditions on yield, composition, and viscosity stability of barley β-glucan gum.
Burkus, Z. & Temelli, F. (1998). Cereal Chemistry, 75(6), 805-809.
Cereal β-glucan can function as a thickener, but endogenous β-glucanase enzymes of the grain cleave β-glucan, reducing its viscosity. Although different extraction techniques have been developed, the viscosity stability of β-glucan gum has not been reported. The objective of this study was to investigate the effect of extraction treatments on the yield, purity, and viscosity stability of barley β-glucan (BBG) gum. A regular barley cultivar, Condor, and a waxy cultivar blend were extracted at pH 7–10 and 55°C for 0.5 hr. Four extraction conditions were evaluated: 1) extraction at high pH with no additional heat treatment; 2) boiling of extract; 3) prior refluxing of flour with 70% ethanol; and 4) treatment of extract with thermostable α-amylase for purification. Viscosity of extracts was monitored for ≥24 hr at 25°C. The highest β-glucan purities were achieved with a boiled Condor extract at pH 7 (81.3% db, 4.1% yield) and with refluxed waxy barley extracted at pH 8 and treated with α-amylase and (79.3% db, 5.1% yield). Gums extracted without subsequent heat treatment or prior refluxing of flour had high protein (>17%) and starch (>24%) impurities, respectively. The viscosity of gums obtained without heating was unstable. Prior refluxing treatment was not sufficient to stabilize final extracts. Boiling extracts resulted in stable but low viscosity. Reflux followed by purification treatment produced the highest stable viscosity for 0.5% solutions of both Condor (64 mPa sec-1, pH 7) and waxy (48.8 mPa sec-1, pH 8) extracts. Stable BBG gum with high viscosity can be obtained using thermal treatments in combination with high pH. The potential use of such gums as thickeners in food systems needs to be assessed.
Influence of germination time and temperature on the properties of rye malt and rye malt based worts.
Hübner, F., Schehl, B. D., Gebruers, K., Courtin, C. M., Delcour, J. A. & Arendt, E. K. (2010). Journal of Cereal Science, 52(1), 72-79.
The effects of germination time and temperature on the quality of rye malt and worts derived thereof were investigated using Response Surface Methodology. Amylolytic and proteolytic enzyme activities were increased by long germination periods, while β-glucanase activity was not influenced. Total and Soluble Nitrogen content were also not significantly affected by the variations in germination conditions. Free Amino Nitrogen (FAN) was found in higher amounts in worts prepared from rye malts with long germination times. Extract contents were higher in rye malt than in the control barley malt and could be increased by a favourable germination regime, while no such impact on wort fermentability was found. High wort viscosities could be significantly reduced by a long germination period at low temperatures, but were still unacceptably high. The same conditions favoured the development of endoxylanase activity. Arabinoxylan (AX) accumulated during the germination process and their extractability increased. The results suggest that longer germination periods resulted in an increased number of AX molecules with lower molecular mass. Optimal rye malt qualities within the limits of this study were found for a germination time of 144 h at 10°C, which resulted in an acceptable FAN content and the lowest measured viscosity.
Spectroscopic and chemical fingerprints in malted barley.
Tarr, A., Diepeveen, D. & Appels, R. (2012). Journal of Cereal Science, 56(2), 268-275.
A unique “Matrix” of malted barley samples was produced to validate spectroscopic procedures for monitoring the malting process. Three critical factors that were examined in controlling the rate of germination were moisture content, temperature and germination time. Of interest to the malting industry, the analysis indicates the potential to identify new germplasm that, under optimized malting conditions, would produce suitably modified malt in three days of germination. It is also clear that the control of both moisture and temperature is essential for undertaking malting studies. The study suggests that Raman and FTIR could usefully complement NIR spectroscopy for monitoring grain during the malting process. For whole grain NIR measurements, the differences between test grain and control grain at optimal wavelengths of 1280 nm and 2224 nm were found to be valuable parameters for tracking progress during the malting process. The study showed the whole grain NIR most likely assessed changing properties of the periphery of the grain. This research suggested that specific calibration models using NIR for predicting malt quality attributes such as diastatic activity on whole malt are misleading and difficult to interpret because they are highly correlated with other carbohydrate/protein-related attributes of the malt.
Diastatic enzymes malting process optimisation of African finger millet for biotechnological uses.
Kolawole, A. N. & Kolawole, A. O. (2015). African Journal of Biochemistry Research, 9(6), 81-88.
This study sought to understand the importance of variation of steeping and germination conditions (temperature, pH and salts) on the quality of African finger millet malt in terms of diastic power (α-amylase and β-amylase), endo- (1,3) (1,4)-β-D-glucanase, β-glycan content and protein profile. The results show that the physiological responses of African finger millet malted seeds are correlated to pH (acidity and alkalinity) but inversely correlated to temperature stress. The effect of the stresses on the activity of α-amylase, β-amylase and endo-(1,3)(1,4)-β-D-glucanase as well as β-glycan content was significantly different in magnitude except for the β-amylase activities obtained after acidic and alkaline treatment at 40°C which are not statistically different. Alkaline pH and heat stress at 30°C were the dominant factors for malting optimization from the result of diastic power indices. α-amylase activity is a better predictor of diastic power. The grains subjected to the steeping and germination process carried out in Tris-HCl buffer solution (25 mM, pH 9) containing 100 mM NaCl at 30°C during 96 h showed higher α-amylase and β-amylase activity. This shows that for a salt–alkali-heat mix stress, a reciprocal enhancement among salt stress, alkali and heat stress was a characteristic feature with no significant change in the hordein protein expression. The influential effect of the stress conditions indicate that alkaline pH steeping and 30°C malting is the most effective condition for producing malted African millet flour with a promising potential of distinct malting quality metrics.
Impact of hydrothermal and mechanical processing on dissolution kinetics and rheology of oat β-glucan.
Grundy, M. M. L., Quint, J., Rieder, A., Ballance, S., Dreiss, C. A., Butterworth, P. J. & Ellis, P. R. (2017). Carbohydrate Polymers, 166, 387-397.
Oat mixed-linkage β-glucan has been shown to lower fasting blood cholesterol concentrations due notably to an increase in digesta viscosity in the proximal gut. To exert its action, the polysaccharide has to be released from the food matrix and hydrated. The dissolution kinetics of β-glucan from three oat materials, varying in their structure, composition and degree of processing, was investigated by incubating the oats at 37°C over multiple time points (up to 72 h). The samples were analysed for β-glucan content, weight-average molecular weight and rheological behaviour. Regardless of the materials studied and the processing applied, the solubilisation of β-glucan was not complete. Mechanical and hydrothermal processing led to differences in the viscosity flow curves of the recovered solutions, with the presence of particulates having a marked effect. This study revealed that the structure and processing methods applied to oat materials resulted in varied and complex rheological properties, especially when particulates are present.
Variation in barley (1→ 3, 1→ 4)-β-glucan endohydrolases reveals novel allozymes with increased thermostability.
Lauer, J. C., Cu, S., Burton, R. A. & Eglinton, J. K. (2017). Theoretical and Applied Genetics, 130(5), 1053-1063.
Rapid and reliable degradation of (1 → 3, 1 → 4)-β-glucan to produce low viscosity wort is an essential requirement for malting barley. The (1 → 3, 1 → 4)-β-glucan endohyrolases are responsible for the primary hydrolysis of cell wall β-glucan. The variation in β-glucanase genes HvGlb1 and HvGlb2 that encode EI and EII, respectively, were examined in elite and exotic germplasm. Six EI and 14 EII allozymes were identified, and significant variation was found in β-glucanase from Hordeum vulgare ssp. spontaneum (wild barley), the progenitor of modern cultivated barley. Allozymes were examined using prediction methods; the change in Gibbs free energy of the identified amino acid substitutions to predict changes in enzyme stability and homology modelling to examine the structure of the novel allozymes using the existing solved EII structure. Two EI and four EII allozymes in wild barley accessions were predicted to have improved barley β-glucanase thermostability. One novel EII candidate was identified in existing backcross lines with contrasting HvGlb2 alleles from wild barley and cv Flagship. The contrasting alleles in selected near isogenic lines were examined in β-glucanase thermostability analyses. The EII from wild barley exhibited a significant increase in β-glucanase thermostability conferred by the novel HvGlb2 allele. Increased β-glucanase thermostability is heritable and candidates identified in wild barley could improve malting and brewing quality in new varieties.
Accumulation and degradation of two functional constituents, GABA and β-glucan, and their varietal differences in germinated barley grains.
Kihara, M., Okada, Y., Iimure, T. & Ito, K. (2007). Breeding Science, 57(2), 85-89.
The changes in the contents of two functional constituents, γ-aminobutyric acid (GABA) and (1→3)(1→4)-β-D-glucans (β-glucan), during germination were investigated in two malting barley varieties in order to determine the optimal conditions for accumulation and degradation of these constituents in dried barley grains after germination treatment. A time-course analysis during germination treatment revealed that the content of GABA remarkably increased in the 48 hour imbibition process and decreased in the subsequent germination process. On the other hand, β-glucan, which is known to be decomposed during malting, did not undergo substantial degradation after the 48 hour imbibition process. These findings thus indicated that it is possible to produce germinated barley grains containing these functional constituents at high levels by optimal germination treatment. A total of 43 barley varieties were surveyed for the GABA and β-glucan contents in germinated grains. Our results also indicated that there were varietal differences in the content of each ingredient. A waxy starch variety in which the contents of both β-glucan and GABA were higher than those in the other barley varieties tested, could become a promising source of the two functional constituents.