ENZYMIC QUANTIFICATION OF (1→3) (1→4)-β-D-GLUCAN IN BARLEY AND MALT

A simple and quantitative method for the determination of (1→3) (1→4)-β-D-glucan in barley flour and malt is described. The method allows direct analysis of β-glucan in flour and malt slurries. Mixed-linkage β-glucan is specifically depolymerized with a highly purified (1→3) (1→4)-β-D-glucanase (lichenase), from Bacillus subtilis, to tri-, tetra- and higher degree of polymerization (d.p.) oligosaccharides. These oligosaccharides are then specifically and quantitatively hydrolysed to glucose using purified β-D-glucosidase. The glucose is then specifically determined using glucose oxidase/peroxidase reagent. Since barley flours contain only low levels of glucose, and maltosaccharides do not interfere with the assay, removal of low d.p. sugars is not necessary. Blank values are determined for each sample allowing the direct measurement of β-glucan in maltsamples.α-Amylasedoes not interfere with the assay. The method issuitable for the routineanalysis of β-glucan in barley samples derived from breeding programs; 50 samples can be analysed by a single operator in a day. Evaluation of the technique on different days has indicated a mean standard error of 0–1 for barley flour samples containing 3–8 and 4–6% (w/w) β-glucan content.     

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.     

DETERMINATION OF HIGH MOLECULAR WEIGHT β-GLUCAN IN WORT AND BEER USING A POST-COLUMN CALCOFLUOR FLOW-INJECTION-ANALYSIS (FIA) METHOD

To eliminate the influence of maltose, ethanol, low molecular weight β-glucan and an inhibitor of the calcofluor fluorescence reaction in wort and beer on the measured values of a calcofluor-FIA, a post-column calcofluor-FIA method has been developed using a short-size gel permeation chromatography column (6.0 × 50 mm). A column packed with polyhydroxymethacrylate gel (molecular weight exclusion limit, 100,000) was found to be the most appropriate for this system. This short column allowed rapid and specific measurement of high molecular weight β-glucan in a few minutes without the influence of the fluorescence inhibitor, maltose and ethanol which have molecular weights of less than 1000 daltons. Because the low molecular weight β-glucan responsible for the scatter caused by a slight difference in measuring conditions such as temperature, calcofluor concentrations, sample volumes, etc., was separated through the use of the column, the measured values by the post-column calcofluor-FIA method hardly fluctuated under different conditions. Though it has been recognized that dilution of a sample could affect a calcofluor FIA, the new system was not influenced. This also made it possible to measure the β-glucan content in dark-coloured samples (even over 100 EBC colour units). The measured values by the post-column FIA showed a high correlation (r² = 0.993) with those obtained by the enzymatic method (the McCleary method).     

KINETICS OF β-GLUCAN DEGRADATION IN WORT BY EXOGENOUS β-GLUCANASES TREATMENT

Three commercial β-glucanases, one bacterial (Cereflo 200L from Novo) and two fungal (Biobeta from Gist-Brocades and Filtrase from Biocon) have been studied with regard to the hydrolysis of β-glucan in sweet and hopped wort. At temperatures below 70°C these processes follow first order kinetics with rate constants being directly proportional to the enzyme concentrations. The rate constant for bacterial β-glucanase Cereflo 200L shows a negative dependence on temperature but positive with wort pH, whereas the reverse is the case for the two fungal β-glucanases. Within the ranges of pH and temperature tested the bacterial β-glucanase has 2–5 times the activity of the fungal ones. No evidence for synergic or competitive effects between bacterial and fungal β-glucanases have been found.     

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.     

SOME BARLEY GRAIN AND GREEN MALT PROPERTIES AND THEIR INFLUENCE ON MALT HOT-WATER EXTRACT: I. β-GLUCAN, β-GLUCAN SOLUBILASE AND ENDO-β-GLUCANASE

A study has been made of the variation between varieties in some properties of barley and malt and how this variation relates to malt hot water extract (HWE). The development of enzyme activity along the grain during germination was investigated. In this first paper we have examined β-glucan-related characters and found significant varietal variation in maximum enzyme activities and in the activities in different sections of grain during germination. Varietal variation was greater than environmental variation for each character. The fraction of β-glucan soluble in acid was the character most highly correlated with HWE.     

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.     

THE RELATIONSHIP OF DIMETHYL SULPHIDE LEVELS IN MALT,WORT AND BEER

The half-life for the conversion of malt dimethyl sulphide (DMS) precursor to free DMS has been determined at various temperatures and pH values. At pH 5·2 the half-life of the precursor in wort (S.G. 1·060) at its boiling point is 38 min, and is doubled for each 6°C fall in temperature. At pH 5·5 the half-life at the boiling point is 32·5 min. Knowing the stability of the precursor at the various temperatures in the brewing process, the extent of conversion to free DMS in wort at pitching can be predicted for malt of a given precursor content and for a given set of process conditions. The results of DMS analyses of samples taken during brewery trials are in reasonable agreement with the predicted values. This work involved infusion mashing only, but the same principles apply to decoction mashing. The fate of precursor and free DMS during fermentation and conditioning has been followed on a production scale. With some brews, where high levels of free DMS were present at pitching, much free DMS was lost during fermentation. Also, precursor DMS reappeared in the beer after a few days and there was some increase in the level of free DMS. The DMS precursor in green malt has been isolated by ion-exchange and gel-filtration chromatography. A preparation has been obtained which has 0·6 mol potential DMS per mol amino nitrogen. Thin layer chromatography showed that the preparation and its hydrolysis product had the same R_f values as S-methylmethionine and homoserine respectively.     

METHODS FOR THE DETERMINATION OF VOLATILE NITROSAMINES IN MALT,WORT AND BEER

Methods for the analysis of beer, wort and malt for NDMA at the one in 10⁹ level using the thermal energy analyzer have been evaluated. The beer and wort procedure using Preptube extraction has been shown to give excellent results. For malt, the preparation of an aqueous extract, followed by analysis by the beer procedure, also gives excellent results. Vacuum distillation from mineral oil has also been examined for the analysis of malt. Providing sufficient aqueous phase is initially present, this procedure yields results similar to those from aqueous extraction. The methods will also detect NDEA, although no NDEA has been observed in any of the beer, wort or malt samples examined. NDMA has been observed to be present in bound forms in malt and Preptubes and is released by the addition of water     

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.     

APPLICATION OF A RADIAL DIFFUSION ASSAY FOR THE MEASUREMENT OF β-GLUCANASE IN MALT

An assay for the routine determination of β-glucanase by a gel diffusion technique has been established. The method has many features which make it preferable to the viscometric procedure currently employed.     

DETERMINATION OF α- AND β-AMYLASE IN MALT

The Analysis Committee of the European Brewery Convention carried out a collaborative trial on malts using the specific analysis methods for α- and β-amylase activities based on dyed substrates supplied by MegaZyme (Aust.) Pty. Ltd. The repeatability and reproducibility values for the methods were judged to be unsatisfactory and consequently the methods were not recommended for Analytica-EBC.     

A ROLE FOR CARBOXYPEPTIDASE IN THE SOLUBILIZATION OF BARLEY β-GLUCAN

An enzyme is described which catalyzes the release of soluble β-glucan from insoluble barley endosperm cell walls. This enzyme increases in activity throughout malting. It has been partially purified and found to behave in the same way as an acidic carboxypeptidase on isoelectric focusing and in its sensitivity to inhibitors and activators and to heating. The importance of the β-glucan solubilizing enzyme in malting and mashing is discussed. An improved method for β-glucan determination is described.     

THE INSTITUTE OF BREWING ANALYSIS COMMITTEE THE DETERMINATION OF ENDO-β-GLUCANASE ACTIVITY IN MALT

Various substrates for the determination of malt endo-β-glucanase activity have been tested in conjunction with the β-glucanase procedure advocated by Bourne and Pierce. It is recommended that one universal standard substrate should be used and that each determination should be measured against a standard check malt. Furthermore, great care must be exercised in the dissolution of the substrate to obtain consistent results.     

SELECTIVE DETERMINATION OF β-GLUCAN OF DIFFERING MOLECULAR SIZE,USING THE CALCOFLUOR-FLUORIMETRIC FLOW-INJECTION-ANALYSIS (FIA) METHOD

When using the calcofluor-fluorimetric flow-injection-analysis (FIA) method to determine β-glucan in wort and beer, the actual range of β-glucan molecular weights effectively complexed by the calcofluor, and consequently the total amount detected, is dependent on the ionic strength of the eluent. In the base eluent of very low ionic strength (20 ppm of calcofluor in aqueous 0.1 M TRIS, pH = 11), only β-glucan molecules of molecular weight greater than approximately 200, 000 daltons are fully complexed by calcofluor, and consequently fully detected. As the molecular weight of β-glucan molecules decreases, β-glucan is only partially complexed by calcofluor, and hence partially detected. By increasing the ionic strength of the base eluent through adding successive amounts of NaCl up to a maximum of 1%, the molecular weight of β-glucan which can be detected by the method decreases down to about 65, 000 daltons, whereas the molecular weights below about 10,000 daltons are not significantly complexed nor detected. Different size ranges of β-glucan have been isolated and characterized by enzymatic degradation of high molecular weight standard β-glucan, followed by classical gel permeation chromatography (GPC), using Sephacryl S-400 HR as stationary phase, pure water as eluent, and dextrans as molecular weight markers.     

SEPARATION OF NITROGENOUS CONSTITUENTS OF MALT,WORT AND BEER USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

High performance liquid chromatography has been used to separate groups of soluble nitrogenous compounds in malt. Malts are extracted with water at 5°C and the nitrogenous components separated using a column packed with a material designed specifically for the analysis of proteins and peptides. The procedure is suitable for routine use and can be readily adapted to study changes in the development of nitrogenous compounds during the brewing process.     

PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ENDO-β1,3-GLUCANASE FROM GREEN MALT

An endo-β1,3-glucanase from a green malt extract was purified by DEAE- and CM-cellulose ion exchange chromatography followed by molecular sieve chromatography on BioGel P-100. A final enzyme preparation had two protein components on disc electrophoresis, one of which was in-active. The enzyme had a pH optimum of 5·0 for activity on laminarin and 5·8 on carboxymethyl pachyman. The activity was stable up to 60°C and was stimulated by NaCl. The isoelectric point of the enzyme was 9·8.     

COMPARATIVE STUDIES OF THE β-D-GLUCAN RELEASED INTO SORGHUM AND BARLEY WORTS

Worts prepared from two cultivars of Nigerian grown sorghum six day melts — LI87 end SK5912 had β-D-glucan levels off five to seven times more than that of proctor barley. In contrast to barley, malting of the sorghums results in the release off more β-D-glucan into wort. Apparently, this is due to increasing levels of β-glucan solubilase and (1→3)-β-glucanase during malting with no significant (1→3, 1→4)-β-glucanase activity.     

THE RELATIONSHIP BETWEEN β-GLUCAN SOLUBILASE,BARLEY AUTOLYSIS AND MALTING POTENTIAL

There is a correlation between the autolysis of barleys and their β-glucan solubilase activities. There is no correlation between autolysis and nitrogen content, β-glucan level, Milling Energy or Zeleny sedimentation value of the barley. Activities of endo-β-glucanase are inversely related to coarse-grind Hot Water Extract obtained from malts grown for 4 days. Whilst β-glucanase is not involved in the early stages of autolysis, β-glucan solubilase, present in large amounts in untreated barley, has a role both in extracting and degrading β-glucan. Barleys with low β-glucan content or high β-glucan solubilase modify more rapidly.     

Preparation and characterization of molecular weight standards of low polydispersity from oat and barley (1→3)(1→4)-β- -glucan

Purified (1→3)(1→4)-β- -glucans (β-glucans) from oat and barley with broad molecular weight (MW) distribution were separated into seven fractions using gradient precipitation with ammonium sulfate (NH₄)₂SO₄. The MW of each fraction decreased consecutively with the concentration of (NH₄)₂SO₄ at which it was precipitated. The MW distribution of each fraction was much narrower compared to the parent sample and is comparable to commercially available pullulan MW standards. To determine whether the fractionation process was separating sub-fractions of different structure, the original β-glucan sample and each fraction were hydrolyzed by a (1→3)(1→4)-D-β-glucan-4-glucanohydrolase (lichenase, E.C.3.2.1.73) and the liberated oligosaccharides were analyzed by high performance anion exchange chromatography. The analysis revealed no differences in oligosaccharide pattern (DP 2–9) derived from each fraction and the parent sample. In particular, the tri/tetra oligosaccharide ratio remained constant for all fractions, indicating no fractionation based on structural features had taken place. The effect of starting β-glucan concentration on the fractionation process was studied. The results showed that it was possible to achieve good separation at overlapping parameter c[η] lower than 3.5. Further increase in starting β-glucan concentration hindered clear separation of the fractions. Temperature also affected the fractionation efficiency. The higher the temperature, the lower the amount of (NH₄)₂SO₄ that was necessary to precipitate the samples of same MW. A Mark Houwink relationship was derived from the measured MW and intrinsic viscosity for fractions from oat and barley, respectively.