THE VISCOSITY OF WORTS IN RELATION TO THEIR CONTENT OF β-GLUCAN

A procedure is given for assessing that proportion of wort viscosity which is attributable to β-glucan. Worts obtained from unkilned samples of malt which have been processed for 54 or 72 h show enhanced viscosity. This is principally due to β-glucan although the contribution of other constituents, absent from the wort of fully modified malt, is of significance. Barley variety is shown to have a pronounced effect on wort viscosity. Insoluble β-glucan is brought into solution in mashes at 65° C. The β-glucan isolated from malt which has been inactivated using aqueous ethanol prior to extraction at 65° C, is of higher specific viscosity than that isolated from control worts prepared at the same temperature. The introduction of a rest by mashing initially at 40° C results in the production of wort of lower viscosity, a decrease in the β-glucan content of the wort and a reduction in the specific viscosity of the β-glucan. There is no apparent relationship between the endo-β-glucanase content of the malts and either the viscosity of derived worts or the degree of breakdown of β-glucan which occurred during malting and mashing. Abrasion of barley, which is a factor assisting the distribution of enzymes during malting, acts to reduce wort viscosity.     

SOLUBILIZATION OF β-GLUCAN DURING MASHING

β-Glucan can be solubilized during mashing by an enzyme or enzymes differing from, and more heat-stable than, the endo-barley-β-glucanase which reduces the viscosity of solutions of barley β-glucan.     

Effects of arabinoxylan solubilization on wort viscosity and filtration when mashing with grist containing wheat and wheat malt

Arabinoxylans are partially water-extractable, high-molecular-weight polymers that contribute to the problems of viscosity and membrane filterability during beer brewing. These problems are more pronounced when wheat and wheat malt are used as adjuncts due to their higher arabinoxylan contents and higher molecular weights. This paper aimed at investigating the effects of mashing temperature, time, grist size and liquor:grist ratio on the solubilization of arabinoxylans. Results indicated that increasing the mashing temperature generally increased the amount of arabinoxylans released into the wort. When greater proportions of wheat or wheat malt were used as adjunct, higher arabinoxylan contents in the final wort were observed. The more finely ground the grist, the more were arabinoxylans released into the wort. When more diluted mashes were used, more efficient solubilization of arabinoxylans was observed. The effects of arabinoxylan content and β-glucan content on the wort viscosity were also examined using a General Linear Model (GLM). There was a good correlation (R² = 0.98) between wort viscosity and its arabinoxylan and β-glucan contents.     

EVALUATION OF MALTING QUALITY IN A BARLEY BREEDING PROGRAMME USE OF α-AMYLASE AND β-GLUCAN LEVLES IN MALT AS PRESELECTION TOOLS

Analysis according to the EBC protocol, immunological determination of a α-amylase and estimation of malt β-glucan using the Calcofluor-FIA method, were used to screen 327 barley breeding lines for malting quality. The results obtained with the α-amylase and β-glucan methods are highly correlated to the important malt quality paramters: extract yield and β-glucan content in the wort. It is recommended that either of the two methods, which are simple to perform are used as prescreening tools in breeding programmes for malting barley.     

Changes in β-glucan and other carbohydrate components of barley during malting

Changes in total (1→3), (1→4)-β-glucan content were followed during the micro-malting of nine varieties of barley with a wide range of malting qualities. These changes were related to estimates of endosperm modification based upon staining with Calcofluor. β-Glucan content declined from an average of 3.54% in the barley to 0.75% in the malt. Pentosan and total starch (including starch-derived oligosaccharides) levels showed comparatively little change during malting. β-Glucan composition of the barley was a poor indicator of malting performance. However, the β-glucan, starch and xylose contents of the malt all showed significant correlations with malt extract. Estimation of malt β-glucan content gave the best indication of malt quality. Direct determination of β-glucan may be of more value in assessing malt quality than indirect techniques based upon assessing modification of stained grains.     

THE INFLUENCE OF β-GLUCANASE ON EFFICIENCY OF WORT SEPARATION

Treatment of mixed grists of malt and either barley flakes or barley flour with β-glucanase during infusion mashing at 65°C improves both the rate of run-off of wort and the total yield of extract. The total amount of non-starchy polysaccharide in solution is increased but the high molecular weight β-glucan fraction is decreased by the addition of β-glucanase. An improvement in wort separation is associated with a lowering of wort viscosity but it is concluded that the major factor influencing wort separation is the structure of the mash bed and the presence of inert material to which protein, glucan and pentosans can bind. A micro-mashing unit is described which simulates the problems of wort separation encountered with certain grists in deep infusion mash tuns.     

β-GLUCAN AND β-GLUCANASE IN BREWING

The development of endo β-glucanase during the micro-malting of barley, with and without the addition of gibberellic acid, was compared. Results indicated that the stimulative effect of gibberellic acid on the enzyme system was of only marginal practical importance. From the assessment of the enzyme activity in a number of commercial malts it would appear that the germination time used for some malts is too short to take full advantage of the critical phase of very rapid enzyme development. The viscosity-reducing power of β-glucanase was demonstrated in miniature brewery mashing experiments and details of the progressive degradation of β-glucan by the enzyme system were analysed by gel filtration methods. The β-glucan content of a number of varieties of barley was established.     

THE DEVELOPMENT OF β-GLUCAN SOLUBILASE DURING BARLEY GERMINATION

β-Glucan solubilase in either germinating barley or in endosperm slices treated with gibberellic acid is synthesized before endo-β-glucanase, α-amylase and protease. In common with these enzymes, β-glucan solubilase is synthesized much sooner in endosperm slices than in whole grain. Gibberellic acid stimulates β-glucan solubilase synthesis in endosperm slices and most of the activity is rapidly released into the surounding medium, irrespective of whether the hormone is present. Inhibitors of RNA and protein synthesis block the formation of β-glucan solubilase. Unlike β-glucanase, α-amylase and protease, β-glucan solubilase is present in significant quantity in untreated barley where it is concentrated in the embryo-containing half of the grain. The only β-glucan solubilase activity in barley is due to an acidic carboxypeptidase. Malt contains a small amount of a second solubilizing enzyme which appears to be an endo-β1, 3-glucanase.     

Comparison of the impact on the performance of small‐scale mashing with different proportions of unmalted barley,Ondea Pro®, malt and rice

The impact of using different combinations of unmalted barley, Ondea Pro® and barley malt in conjunction with a 35% rice adjunct on mashing performance was examined in a series of small scale mashing trials. The objective was to identify the potential optimal levels and boundaries for the mashing combinations of barley, Ondea Pro®, malt and 35% rice (BOMR) that might apply in commercial brewing. Barley and malt samples used for the trials were selected from a range of Australian commercial barley and malt samples following evaluation by small‐scale mashing. This investigation builds on previous studies in order to adapt the technology to brewing styles common in Asia, where the use of high levels of rice adjunct is common. Mashing with the rice adjunct, combined with differing proportions of barley, Ondea Pro® and malt, resulted in higher extract levels than were observed for reference mashing, using either 100% malt reference or 100% barley reference and Ondea Pro® enzymes. Synergistic mashing effects between barley, Ondea Pro® and malt were observed for mash quality and efficiency parameters, particularly wort fermentability. The optimum levels of barley in the grist (with the relative level of Ondea Pro®) were assessed to be in the range 45–55% when paired with 10–20% malt and 35% rice. When the proportion of malt was reduced below 10% of the grist, substantial reductions in wort quality were observed for wort quality parameters including extract, lautering, fermentability, free amino nitrogen and haze. Extension of this new approach to brewing with rice adjuncts will benefit from further research into barley varietal selection in order to better meet brewer's quality requirements for the finished beer. Copyright © 2016 The Institute of Brewing & Distilling     

THE RELATIONSHIP BETWEEN TOTAL β-GLUCAN OF MALT AND MALT QUALITY

The total β-glucan content of barley and malts has been determined using a direct enzyme degradation method incorporating measures to ensure inactivation of any contaminating amyloglucosidase. Results for barleys range between 2·7 and 4·4% w/w and indicate genetic variation in the β-glucan content. Malts produced by both a laboratory micromalting procedure and commercially have been analysed for total β-glucan, extract and 70°C mash viscosity at different stages of germination and in the end product. A very good correlation has been found between total β-glucan and fine-concentrated extract difference values showing that in a fully modified malt having a negligible extract difference value, all the β-glucan material is degraded. The extract difference value had been demonstrated earlier to be closely linked with brewhouse extract yield. The total β-glucan of malt, therefore, is directly associated with achievable extract in the brewhouse and is the most important biochemical factor determining the extractability of a malt.     

EFFECTS OF CULTIVAR AND ENVIRONMENT ON β-GLUCAN CONTENT AND MALTING QUALITY OF TURKISH BARLEYS

Eight barley cultivars grown under the same agronomic conditions and samples of Tokak cultivar grown at six different sites of Turkey were used in this study. There were significant differences among the barley cultivars and growing locations in terms of β-glucan content (p<0.05). Among malt quality criteria tested for the 8 barley cultivars; friability, viscosity, Kolbach index and extract difference showed significant correlations (p<0.05) with the total β-glucan content. Similar correlations were also observed between the malt quality criteria (Kolbach index and extract difference) and β-glucan contents for the Tokak samples grown at different sites.     

INHIBITOR OF FLUORESCENCE REACTIONS BETWEEN CALCOFLUOR AND β-(1,3)(1,4)-D-GLUCAN IN BEER AND WORT

An inhibitor of calcofluor fluorescence reaction in beer and wort was shown by analyzing beers and worts containing no β-glucan. The inhibitory activity was enhanced against the fluorescence reaction which is intensified by linkage of calcofluor with β-glucan compared to the fluorescence of calcofluor itself. The investigation indicated that the inhibitor was derived from malt and is produced during the germination process. The inhibitory activity in beer and wort changed with the degree of malt modification and the ratio of malt in the grist. Japanese beers had a wide range of inhibitory activities against the calcofluor fluorescence, lowering the apparent β-glucan contents by 12 to 20%. The inhibitor in beer and wort appeared to be an acidic compound with a molecular weight of less than 1000 daltons.     

THE EBC METHODS FOR DETERMINATION OF HIGH MOLECULAR WEIGHT β-GLUCAN IN BARLEY,MALT, WORT AND BEER

Two new methods for the determination of β-glucan in barley, malt, wort and beer are accepted as EBC Standard Methods for β-glucan by the Analysis Committee of the European Brewery Convention. One method (The fluorimetric method) is based upon the specific interaction of the fluorochrome Calcofluor towards high molecular weight β-glucan. The method is suitable where a high number of determinations have to be done routinely. The other method (The enzymatic method) utilizes a specific degradation of the β-glucan by purified enzymes. This method is more laborious, but it can be carried out using apparatus available in an analytical laboratory. The methods, which are described in detail, have been tested in a EBC Collaborative Test with 14 participating laboratories. Barley, malt and beer samples at 6 levels of concentration were tested for β-glucan. The collaborative test resulted in acceptable statistical values for repeatability and reproductibility for both methods. Comparison of the results from the two methods, showed that they are highly correlated and that the actual values of β-glucan content are comparable.     

DETERMINATION OF TOTAL β-GLUCAN IN MALT

An enzymic method for the estimation of total β-glucan in barley has been modified to make it suitable for determination of the small amounts of β-glucan present in malt. Interference from the high levels of reducing sugars in malt has been eliminated by reducing the free sugars in the sample with sodium borohydride rather than extracting them using 80% (v/v) ethanol. The reduction procedure also inactivates endogenous carbohydrate hydrolases in the sample. Because it is no longer necessary to extract the samples with ethanol and centrifuge repeatedly, the modified method is also advantageous in the analysis of barley β-glucan. Errors associated with extraction are eliminated and the speed of analysis of large batches is greatly increased.     

Carbohydrate content and structure during malting and brewing: a mass balance study

A holistic view of the fate of barley starch, arabinoxylan and β-glucan throughout malting and brewing is largely missing. Here, an industrial scale malting trial and pilot brewing trial were performed, and the concentration and structural characteristics of carbohydrates were analysed at 28 key points in the process. The barley starch content decreased during malting from 75.0% to 69.7%. During mashing, malt starch was converted to fermentable sugars (75.3%), dextrin (22.8%) or was retained in spent grains (1.8%). Arabinoxylan was partially hydrolysed during malting. Despite mashing-in at 45°C, no further solubilisation of arabinoxylan was observed during mashing. However, the average degree of polymerisation of the soluble arabinoxylan fraction decreased slightly. During fermentation, the arabinoxylan content decreased to 2.5 g/L. The amount of barley β-glucan decreased gradually in time during malting. Of the solubilised β-glucan, 31% was retained in the spent grains during wort filtration, slightly lowering the β-glucan content in the wort. The β-glucan content remained at 0.5 g/L during fermentation. Sucrose was hydrolysed during mashing, probably by barley invertases. From the total amount of malt used, 41.0% was converted to fermentable sugars. This mashing yield could have been improved by the full hydrolysis to fermentable sugars of the present β-glucan (to 41.1%), the remaining starch in spent grains (to 42.0%) and dextrin in wort (to 50.3%). These results provide more insight into the carbohydrate conversions during malting and brewing and can act as a baseline measurement for future work. © 2020 The Institute of Brewing & Distilling     

THE ACTION OF ENDO-β-1,3-GLUCANASES ON BARLEY AND MALT β-GLUCANS

The β-glucan extracted from ungerminated barley with water at 40 °C has a much lower specific viscosity than the corresponding material isolated from a wort prepared at 65 °C from a two-day germinated barley malt. Both glucans are similar in that they are polymers of β-D-glucose, with approximately 74% of the linkages in the β-1,4 configuration and 26% in the β-1,3 configuration. However, the two glucans are not hydrolysed to the same extent either by a partially purified bacterial endo-β-1,3-glucanase or by a homogeneous endo-β-1,3-glucanase from malted barley. The malt glucan is readily hydrolysed, causing a rapid decrease in specific viscosity but with no measurable increase in reducing power, whereas barley glucan undergoes only limited hydrolysis under similar conditions. Thus, different β-glucan preparations from barley or malt may be identical in the proportion of β-1,3 to β-1,4-linkages but the overall arrangement of linkages, and hence susceptibility to enzyme attack, differs according to the source and the method of extraction of the glucan. The molecular weights of both β-glucan preparations and the products of their enzyme hydrolysis have been determined by agarose gel permeation chromatography. A simple model which illustrates the underlying structural relationships of the β-glucans from barley and malt is suggested.     

Effects of Mashing Parameters on Mash β‐Glucan,FAN and Soluble Extract Levels

To gain further technological knowledge of mashing, pilot scale mashing trials were carried out varying mashing programme (upward/isothermal mashing), milling procedure, grist:liquor ratio, time of mash stands, and grist modification level (well and poorly modified malt). During mashing β‐glucan, free amino nitrogen (FAN) and extract contents were analysed as key indicators for cytolysis, proteolysis, and amylolysis, respectively. The malt modification was of major impact for the β‐glucan release in contrast to a variation of milling procedure and of grist:liquor ratio. Extended stands lead to increased final values only for poorly modified malt. Similarly, FAN release was predetermined by malt modification while variation of milling and of grist:liquor ratio was not relevant in contrast to stand extension. None of the variations applied influenced extract yield as long as gelatinization temperature was reached. Greatest gains occurred around 57°C. In conclusion, wort quality is critically determined by malt modification. Mashing with well modified malt in combination with short stands should result in a mash of low β‐glucan and sufficient FAN level without losing extract yield. However, for poorly modified malt the variation of mashing parameters has an impact on the key indicators in which cytolysis plays the dominating role.     

Release of phenolic flavour precursors during wort production: Influence of process parameters and grist composition on ferulic acid release during brewing

In this study, the effects of mashing variables such as mashing-in temperature, time and pH, mash thickness, grist coarseness and composition, and stirring regime on the release of ferulic acid were examined. Ferulic acid is a precursor for the formation of flavour-active volatile phenols and a potent natural antioxidant in beer. Given one barley malt variety, the multitude of choice in setting various process parameters and adding brewery adjuncts during brewhouse operations can give rise to worts with widely varying ferulic acid levels. A clear difference in temperature- and pH-dependence between the release of the water-extracted and the enzymatically hydrolyzed fraction was found. The T,t-dependencies of arabinoxylan-degrading enzyme activities were correlated with ferulic acid release during mashing. Results from laboratory-scale mashing experiments were validated with those from a pilot-scale (5 h) wort production process. Enhancing the enzymatic release of phenolic flavour precursors from bound forms during mashing can greatly enhance the phenolic aroma potential of wort. Optimising this precursor release during mashing may be a means for controlling final volatile phenol levels in beer.     

AN ENZYMIC METHOD FOR THE MEASUREMENT OF TOTAL AND WATER-SOLUBLE β-GLUCAN IN BARLEY

Crude cellulase preparations from Trichoderma reesei, freed of amyloglucosidase by heating, will completely hydrolyse barley β-glucan to glucose. Apparent non-quantitative recovery of glucose is due to its adsorption by the high levels of protein present. Methods are given for the measurement of total and water-soluble β-glucan. Dehusking does not lower the amount of β-glucan which is measured.     

FIELD FUNGI AND β-GLUCAN SOLUBILASE IN BARLEY KERNELS*

Significant amounts of β-glucan solubilase activity have been found in barleys harvested from a number of test sites. Enzyme activity appeared to be related to the climatic conditions at crop maturity, indicating that β-glucan solubilase was generated, possibly, by microflora on the barley grain. Species of the most common field fungi genera, Alternaria, Cladosporium, Epicoccum and Helminthosporium and two bacterial cultures were isolated from barley kernels and incubated on autoclaved barley for solubilase examinations. All the fungal isolates studied showed abilities to reduce the viscosity of carboxymethyl cellulose and to solubilise barley β-glucan. The molecular size distribution of the solubilised β-glucan products resembled that obtained for products formed by a partially purified preparation of solubilase from barley. It has been concluded, therefore, that the common field fungi associated with the hull and seed cot of barley may be the source of β-glucan solubilase.