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.     

Measurement of (1 → 3),(1 → 4)-β-D-glucan in barley and oats: A streamlined enzymic procedure

A commercially available enzymic method for the quantitative measurement of (1 → 3),(1 → 4)-β-glucan has been simplified to allow analysis of up to 10 grain samples in 70 min or of 100–200 samples by a single operator in a day. These improvements have been achieved with no loss in accuracy or precision and with an increase in reliability. The glucose oxidase/peroxidase reagent has been significantly improved to ensure colour stability for periods of up to 1 h after development. Some problems experienced with the original method have been addressed and resolved, and further experiments to demonstrate the quantitative nature of the assay have been designed and performed.     

MEASUREMENT OF (1 → 3)(1 → 4)-β-d-GLUCAN IN MALT,WORT AND BEER

A method developed for the quantification of (1 → 3)(1 → 4)-β-d -glucan in barley flour has been modified to allow its use in the measurement of this component in malt, wort, beer and spent grain. For malt samples, free d -glucose was first removed with aqueous ethanol. Quantification of the polymer in wort and beer samples involved precipitation of the β-glucan with ammonium sulphate followed by washing with aqueous ethanol to remove free d -glucose. Spent grain was lyophilised and milled and then analysed by the method developed for malt. In all cases, the β-glucan was depolymerised with lichenase and the resultant β-gluco-oligosaccharides hydrolysed to d -glucose with β-d -glucosidase. The released d -glucose was then specifically determined using glucose oxidase-peroxidase reagent.     

Cryogelation of cereal β-glucans: structure and molecular size effects

The effects of molecular size and fine structure of mixed-linkage cereal (1→3), (1→4) β- -glucans (β-glucans) on their cryogelation behavior were investigated. Values of apparent molecular weight (M_w) for oat β-glucans ranged between 65 and 200×10³, whereas the respective values for both barley and wheat β-glucan preparations were about 200×10³. The fine structure of cereal β-glucans, as assessed by high-performance anion-exchange chromatography of the cellulosic oligomers released by the action of lichenase, revealed differences in the relative amounts of 3-O-β-cellobiosyl- -glucose (DP3) and 3-O-β-cellotriosyl- -glucose (DP4) units only among the different genera of cereals; the weight percent of DP3 units estimated as 67.1, 63.3, and 55.3–55.8% for wheat, barley, and oat β-glucans, respectively. Aqueous β-glucan solutions (1–3% w/v) were subjected to 12 freezing (−18 °C for 24 h) and thawing (5 °C for 24 h) cycles. The phenomenological appearance of the gelled materials obtained after this process as well as the yield of cryostructurates were influenced by the initial solution concentration, the number of freeze–thaw cycles, as well as by the molecular features of the β-glucans. Such effects were unraveled by studying the cryogelation process with differential scanning calorimetry (DSC), small strain dynamic rheometry, and large deformation mechanical measurements. For the cereal β-glucan cryogels the storage modulus, G′, increased and the tan δ decreased with decreasing polysaccharide molecular size and with increasing initial solution concentration, number of freeze–thaw cycles, and trisaccharide segments in the polymeric chains. The apparent melting enthalpy values (ΔH) of β-glucan cryostructurates, as determined from the DSC endothermic peaks, increased with decreasing molecular size and with increasing amount of cellotriose units, but they were independent of the number of freeze–thaw cycles. The DSC melting temperature of the gel network was found to increase with the molecular size and amount of DP3 units of β-glucans. Moreover, large deformation mechanical tests (compression mode) revealed an increase in strength of cereal β-glucan cryogels with increasing molecular size and decreasing trisaccharide units in the polysaccharide preparation.     

Effect of partially hydrolyzed oat β-glucan on the weight gain and lipid profile of mice

Oat β-glucan hydrolysates with different molecular weights were prepared and their physicochemical, hypocholesterolemic, and weight-reducing characteristics were evaluated. The enzymatic hydrolysis by cellulase caused a decrease in the molecular weight of oat β-glucan (1450–370 × 10³ g/mol), which also affected swelling power and bile acid/fat binding capacities. In addition, mice were fed high-fat diet supplemented with β-glucans with three different molecular weights (1450, 730, 370 × 10³ g/mol). The diets treated with β-glucans significantly reduced the body weight of the mice. However, the molecular weight of β-glucans did not appear to significantly affect the serum lipid profile.     

Optimised methodology for carboxymethylation of (1→3)‐β‐d‐glucan from Yeast (Saccharomyces cerevisiae) and promotion of mechanical activation

With a view to utilise yeast (1→3)‐β‐d ‐glucan as biological response modifiers with better water solubility, carboxymethylation was carried out by a two‐step alkalisation and etherification with monochloroacetic acid. Four technological parameters of carboxymethylation were investigated by orthogonal experiments for obtaining the maximum degree of substitution (DS), apparent viscosity (η) and solubility of carboxymethyl derivatives. In view of the orthogonal analysis, the optimal technological parameters were reaction temperature 50 °C, total reaction time 5 h, 3 mL of 50% sodium hydroxide as the second alkali dosage and 15 mL of 4 m chloroacetic acid. In addition, it was found that ball milling pretreatment for original (1→3)‐β‐d ‐glucan can be an advantage for carboxymethylation. By contrast, DS, η and solubility of carboxymethyl product increased 24%, 6% and 22%, respectively, suggesting the effect of ball milling pretreatment could not be neglected on improvement of DS, η and solubility for carboxymethyl products.     

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.     

“Nata Puree,” a Novel Food Material for Upgrading Vegetable Powders,Made by Bacterial Cellulose Gel Disintegration in the Presence of (1,3)(1,4)-β-Glucan

Pulverization is a potentially powerful solution for the resource management of surplus- and non-standard agricultural products, maintaining their nutritional values for long and ensuring their homogeneity, whereas their original textures could disappear to narrow the application ranges. Therefore, new technologies should be developed for reconstructing the powders to provide them with new physical characteristics. Herein, we developed a novel food material, nata puree (NP), by nata de coco (bacterial cellulose gel) disintegration with a water-soluble polysaccharide using a household blender. The process worked well with (1,3)(1,4)-β-glucan (BGL) as the polysaccharide, which could be substituted with barley extract. Lichenase treatment of the NP dramatically modified its physical properties, suggesting the importance of the BGL polymeric forms. NP exhibited distinct potato powder and starch binding activities, which would be attributed to its interactions with the cell wall components and a physical capture of powders by the NP network, respectively. NP supplementation into the potato paste improved its firmness and enabled its printable range shift for 3D food printing to a lower powder-concentration. NP also promoted the dispersion of powders in its suspension, and designed gelation could also be successfully performed by the laser irradiation of an NP suspension containing dispersed curdlan and turmeric powders. Therefore, NP could be applied as a powder modifier to a wide range of products in both conventional cooking, food manufacturing, and next generation processes such as 3D food printing.     

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.     

Structure and physicochemical properties of β-glucans and arabinoxylans isolated from hull-less barley

The molecular characteristics of β-glucans and arabinoxylans from selected hull-less barleys were examined following their sequential extraction with water (45 and 95 °C), Ba(OH)₂, Ba(OH)₂/H₂O, and NaOH which resulted in five distinct fractions designated as WE45, WE95, Ba(OH)₂, Ba(OH)₂/H₂O, and NaOH, respectively. Both water-extractable fractions consisted mainly of β-glucans but up to 20% of arabinoxylans were found in WE45 of normal and high amylose barley varieties. Analysis of the water-extractable β-glucans, indicated differences in molecular features (weight average molecular weight (M_w), β-(1→4) to β-(1→3) linkage ratios, and amounts of cellulosic regions along the β-glucan chain) between WE45 and WE95, and among β-glucans from different cultivars. The variation observed in the viscoelastic behaviour of WE45 and WE95 fractions confirmed differences in the molecular weight among water-extractable β-glucans derived from various barley cultivars.Differences in the structure of arabinoxylans from the various fractions and cultivars were also observed. Water-extractable arabinoxylans (WE45 and WE95 fractions) had significantly lower xylose to arabinose (Xylp/Araf) ratios (1.47 and 1.52) than those extracted with Ba(OH)₂ (1.70), implying a greater degree of branching in the more readily soluble fractions. Small varietal differences in molecular weight (M_w), root mean square radius (R_g), and polydispersity (M_w/M_n) among Ba(OH)₂ fractions correlated with the substitution pattern of the xylan backbone.     

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.     

Purification and physicochemical characterization of ovine β‐lactoglobulin and α‐lactalbumin

Ovine whey proteins were fractionated and studied by using different analytical techniques. Anion‐exchange chromatography and reversed‐phase high‐performance liquid chromatography (HPLC) showed the presence of two fractions of β‐lactoglobulin but only one of α‐lactalbumin. Gel permeation and sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis allowed the calculation of the apparent molecular mass of each component, while HPLC coupled to electrospray ionisation‐mass spectrometry (ESI‐MS) technique, giving the exact molecular masses, demonstrated the presence of two variants A and B of ovine β‐lactoglobulin. Amino acid compositions of the two variants of β‐lactoglobulin differed only in their His and Tyr contents. Circular dichroism spectroscopy profiles showed pH conformation changes of each component. The thermograms of the different whey protein components showed a higher heat resistance of β‐lactoglobulin A compared to β‐lactoglobulin B at pH 2, and indicated high instability of ovine α‐lactalbumin at this pH.     

The (1–3, 1–4)‐β‐Glucanases in Malting Barley: Enzyme Survival and Genetic and Environmental Effects

Eighteen barley genotypes used in Brazilian malting barley breeding programs were characterized in relation to (1–3, 1–4)‐β‐glucanase activity in green and kilned malt. They were tested to determine the loss of enzyme activity during kilning in the malting process and the environmental effects on enzyme activity were measured. The genotypes analyzed showed great variation regarding the enzyme activity in both kinds of malt, in a range from 531.94 to 934.31 U/kg in green malt, and from 187.02 to 518.40 U/kg in dry malt. The mean enzyme activity loss during kilning was close to 60%, very similar to the results obtained in other studies. The loss among genotypes varied from 8.04% to 71.54%. The enzyme activity varied significantly under the different environments tested, showing existence of environmental effects on the genotypes analyzed. Embrapa 127 was the genotype that exhibited the highest enzyme activity in finished malt although it had shown a low activity in green malt, reflecting a negligible loss of activity during kilning. The data indicate promising results to malting barley breeding due to the wide variability exhibited by genotypes as to enzyme activity and levels of isoenzyme with high thermostability.     

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.     

NON-IMPLICATION OF HOP α- AND β- ACIDS AS SOURCES OF SULPHUR COMPOUNDS IN BEER

Whereas hop oil terpenoids can give rise to organoleptically undesirable sulphur compounds in beer brewed using hops dressed on the bine with sulphur, the hop resin α- and β-acids and their transformation products appear incapable of reactions with sulphur under analogous conditions. The evidence indicates that the hop resins are not potential sources of sulphur compounds in beer     

MODIFIED FLUORIMETRIC FLOW-INJECTION-ANALYSIS (FIA) METHOD FOR THE DETERMINATION OF (1–3) (1–4)–B—D—GLUCAN

A simple system has been constructed for the quantitative determination of barley β-glucans. Measurements were made by the flow-injection-analysis (FIA) technique using the fluorescent dye Calcofluor as a specific reagent for (1·3) (1·4)–B—D—linkages. In this paper a single-line system is described involving only one pump samples of β-glucan being directly injected into the reagent stream. The reaction results in an increased intensity of fluorescence detected by a flow-through-cell fluorimeter and evaluated as peak heights or peak areas by a printer-plotter. The analysis is completed within 20s. The operational parameters were set during an optimization procedure.     

Structural characterization of barley β-glucan extracted using a novel fractionation technique

A barley β-glucan concentrate prepared according to a novel technology was further purified and subjected to detailed structural characterization by NMR spectroscopy. β-Glucan was hydrolysed with β-glucan-4-glucanohydrolase (lichenase). Fractions of hydrolysate were collected using an HPLC-fraction collector. Intact β-glucan and the major fractions collected were subjected to MALDI-TOF–MS and NMR analyses. The two major oligosaccharides produced by lichenase hydrolysis of purified barley β-glucan were identified as β-d-Glc p-(1 → 4)- β-d-Glc p-(1 → 3)-β-d-Glc p and β-d-Glc p-(1 → 4)-β-d-Glc p-(1 → 4)-β-d-Glc p-(1 → 3)-β-d-Glc p based on ¹³C and ¹H NMR data. Spectrums were similar to those documented for barley β-glucan in the literature.     

THE DETERMINATION OF α- AND β-ACIDS IN HOPS AND HOP PRODUCTS USING HPLC

A rapid reversed phase HPLC method for the analysis for α- and β-acids in hops and hop products is described and has been evaluated. The method uses citric acid in the eluent as a complexing agent to overcome the irreversible adsorption effects shown by some columns, thus allowing optimum eluent pH to be selected. The precision of the method for analysis of hop extract has been determined giving relative standard deviations of 1·0% and 2·1% for α- and β-acids respectively. General agreement with results obtained using a polarimetric α-acids analysis method for hop extracts and hops has been demonstrated.     

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.     

ESTIMATION OF α- AND β-ACIDS IN HOP EXTRACTS BY HPLC

A procedure relying on high performance liquid chromatography for the estimation of α-acids and β-acids in hop extracts has been collaboratively tested by members of a Sub-Committee of the Institute of Brewing Analysis Committee and is recommended for use. No significant differences were found between precision values obtained using peak height and peak area measurements. For α-acids, the mean repeatability (r₉₅) and reproducibility (R₉₅) values were 1-3 and 2-4% m/m respectively over the range 41–62% m/m. For β-acids they were 0-9 and 2-0% m/m respectively over the range 11 to 35% m/m. The precision values were judged to be independent of concentration.