EVALUATION OF BARLEY AND MALT QUALITY USING NEAR-INFRARED REFLECTANCE TECHNIQUES

A scanning near-infrared reflectance spectrophotometer was calibrated for the prediction of barley aleurone colour and malt moisture. The malt moisture was predicted on malt ground for the determination of malt extract (coarse grind) making the method suitable for moisture correction in malt extract estimation. Calibrations for the prediction of malt extract and endosperm modification from barley and malt were also attempted. A correlation (r= 0.851 n = 135) was found between malt hot water extract and the percentage of the endosperm estimated as being modified by microscopy following staining with Calcofluor. Probably because of this influence of modification on malt extract, the use of near-infrared reflectance to predict malt extract was most successful at predicting the malt extract values obtained following micro-malting in the absence of the additives, gibberellic acid and potassium bromate.     

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).     

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.     

SEVERAL NEW FACTORS INFLUENCING THE MEASUREMENT OF β-GLUCAN CONTENT USING CALCOFLUOR FLOW-INJECTION ANALYSIS METHOD

Factors such as temperature, calcofluor concentration, sample injection volume, void volume of the mixing tube and other factors have been reported to influence the calcofiuor flow-injection analysis (FIA) of β-glucan. In addition to these factors, we revealed the following factors and elucidated their effects on the FIA of β-glucans: the inside diameter and arrangement of the mixing tube, calibration method using peak area or peak height of the edition patterns, and the quality of the calcofluor reagent. An apparently lower value was obtained when (1) the mixing tube had a smaller inside diameter, (2) the mixing tube was looped in a ring of smaller diameter, or (3) the mixing tube was wound as double coils in a figure eight instead of a loop. Furthermore, it was indicated that the peak area calibration produced higher measures values than the peak height calibration. The reagent also affected the results; a calcofluor reagent from Sigma Chemical Co. gave a higher value than one from Polyscience Inc. It was concluded that these troublesome phenomena were derived from the difference between the test sample and the standard solution of β-glucan, that is, the molecular weight distribution of β-glucans and/or the sugar and ethanol in the sample solutions. Based on these findings, it is suggested that international standardization of the FIA method for β-glucan be made.     

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.     

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.     

A PBS2 homologue from Debaryomyces hansenii shows a differential effect on calcofluor and polymyxin B sensitivity in Saccharomyces cerevisiae

The PBS2 gene encodes a MAP kinase kinase that plays a pivotal role in osmosensing signal-transduction pathway in the yeast Saccharomyces cerevisiae. Mutation in the PBS2 gene has a pleotropic effect. Besides being osmosensitive, pbs2 mutants show altered sensitivity to polymyxin B and calcofluor. Recent studies revealed that Pbs2p plays a different role in osmoadaptation and calcofluor sensitivity. We have isolated a gene homologous to PBS2 from the highly salt-tolerant yeast Debaryomyces hansenii by phenotypic complementation. DNA sequencing of the clone revealed that the gene encoded a protein of 683 amino acid residues. Like Pbs2p, this protein also has a proline-rich motif. Further characterization revealed that this gene could complement polymyxin B sensitivity but did not affect calcofluor sensitivity. Thus, it appeared that Pbs2p also has an independent role in these two physiological processes. The GenBank Accession No. of this sequence is AF371315.