FLUOROMETRIC DETERMINATION OF CYSTEINE IN BEER BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH PRECOLUMN DERIVATISATION

A high-performance liquid chromatographic procedure (HPLC) is described for the fluorometric determination of cysteine (CySH) in beer. Reduced CySH in beer is reacted with a fluorescent reagent, N-(9-acridinyl) maleimide (NAM), at pH 8·8 for 15 h to form a stable fluorescent derivative. The resulting reaction mixture is analysed by reversed-phase gradient elution HPLC. Under the optimised conditions for chromatography, ca 0·1 mg of CySH per litre of beer can be determined. Total CySH is determined by electrolytic reduction at a Hg pool electrode prior to the HPLC analysis. The levels of reduced and total CySH in 28 brands of commercial lager beers ranged from 0·2 to 0·9 mg/litre, and from 2·1 to 13·5 mg/litre, respectively.     

PURIFICATION AND PROPERTIES OF THE MAJOR ANTIGENIC BEER PROTEIN OF BARLEY ORIGIN

The major antigenic beer macromolecule, Antigen 1, has been isolated from commercial lager beer by succesive use of ultrafiltration, alcohol precipitation, anion exchange, cation exchange and gel filtration chromatography. Amino acid analyses showed a composition identical with that of a major barley albumin, Protein Z, except for a 16% lower content of lysine. Like Protein Z, Antigen 1 showed a molecular weight (MW) near 40,000 in gel filtration and SDS-gel electrophoresis. Unlike Protein Z, Antigen 1 contained 2·5% carbohydrate and was more acidic. When properly standardised, the amount of Antigen 1 in beer could be determined immunoelectrophoretically. Contents ranged from 22 mg/litre in a pale ale (7·7°P; 36% brewers adjunct) to 170mg/litre in a stout (18·8°P). The results suggest that Antigen 1 may account for more than 10% of the total non-dialysable proteinaceous material in beer, and that about 25% of the Protein Z present in the brewing materials may be recovered with an almost unmodified protein structure as Antigen 1 of beer. Apparently, Antigen 1 was not affected by stabilisation of the beer with insoluble PVP or with papaya proteinases.     

PHENOLIC ACIDS IN BEERS AND WORTS

The total contents of phenolic acids measured by high-performance liquid-chromatography were 5–8 mg/litre in beers brewed in Ireland whereas 16–40 mg/litre were present in four other beers. In all beers the predominant phenolic acids were vanillic, p-coumaric and ferulic acids. Free phenolic acids were extracted from Emma barley grains and malt in very small amounts (15–28 mg/kg) but larger quantities (191 mg/kg) were released on mashing the malt. Little change occurred in the contents of phenolic acids on processing a lager wort through to the finished beer. Treatment with excess Polyclar AT removed astringent flavour and phenolic acids from an experimental ale but this flavour loss could not be accounted for by the adsorption of phenolic acids. The flavour threshold for a nine-component phenolic acid mixture in lager was between 50 mg/litre and 100 mg/litre.     

Estimation of the fungal toxins,zearalenone and aflatoxin,contaminating opaque maize beer in Zambia

The concentration of zearalenone and aflatoxin was estimated in Zambian homebrewed and commercial opaque maize beer. Levels of aflatoxin were negligible. Zearalenone was present up to 4.6 mg/litre with a mean concentration of 0.92 mg/litre. Zearalenone was detected in the maize and maize malt used in beer preparation and it was found to dissolve preferentially in the liquid fraction of the beer. The concentration was related to the district of maize growth.     

DETERMINATION OF SILICONE ANTIFOAM IN BEER

An atomic absorption method is described for the determination of polydimethylsiloxane residues in beer, in the concentration range 0.02–0.5 mg/litre. Actual levels found were 0.02–0.15 mg/litre from fermentations treated with 2–3 mg/litre silicones.     

Effect of Adenosine-Nucleotides and Their Derivatives on the Denaturation of Myofibrillar Proteins in vitro during Frozen Storage at −20°C

To investigate the effect of adenosine-nucleotides and their derivatives on the denaturation of myofibrillar proteins, 235 nmoles/mL adenosine-5′-diphosphate (ADP), adenosine-5′-monophosphate (AMP), inosine-5′-monophosphate (IMP), inosine (HxR), or hypoxantine (Hx), was added to 3 mg/mL actomyosin (AM) solution suspended in 0.10M KC1 solution and stored at ?20°C for 12 wk. The AM was extracted from milktish dorsal muscle. Protein denaturation was evaluated by measuring solubility, Ca-ATPase and Mg(EGTA)-ATPase activity of AM, by analyzing changes in electrophoretic profiles and transmission electron microscopy. Inosine and hypoxanthine accelerated protein denaturation compared to control samples. Infrared spectrum analyses indicated that negatively charged groups of these nucleotides interacted with amino or imino groups on AM after addition. ADP, AMP, and IMP had a protective effect on denaturation of AM during frozen storage at ?20°C.     

A NEW COLOURIMETRIC ASSAY FOR FLAVANOIDS IN PILSNER BEERS

A new colourimetric assay (based upon the reaction of their A-rings with the chromogen p-dimethylaminocinna-maldehyde) has been developed for flavanoids in beer. The flavanoid content for 19 different Belgian Pilsner beers by this method varied between 4·8 and 39·5 mg/litre with a mean of 26·3 mg/litre. These results were compared with the analytical data for total polyphenolics (EBC, Singleton), anthocyanogens (Jerumanis), and flavanoids (vanillin procedure). Good correlations existed between the results by the new method and the analytical data for total polyphenolics (EBC) and anthocyanogens (Jerumanis). Analysis by the new assay requires only one reagent and it is clear from the analytical data that the method is highly reproducible and sensitive     

QUANTITATIVE ANALYSIS OF DIACETYL,PENTANEDIONE AND THEIR PRECURSORS DURING BEER FERMENTATION BY AN ACCURATE GC/MS METHOD

A GC/MS method previously described for diacetyl was developed for the quantification of 2,3-pentanedione, and the derivatization procedure was modified for the determination of α-acetohydroxy acid. The reaction of 2,3-pentanedione with 4,5-dichloro-1,2-diaminobenzene produced 6,7-dichloro-2-methyl-3-ethylquinoxaline (DCMEQ), which was extracted with toluene and determined by gas chromatography using a mass selective detector. The formation of DCMEQ was linearly correlated with the 2,3-pentanedione concentration. The method was very simple and sensitive. The detection limit of the 2,3-pentanedione derivative and diacetyl derivative was 0.0007 mg/litre and 0.0002 mg/litre of the toluene extract respectively, and the determination limit was 0.001 mg/litre and 0.0007 mg/litre, respectively. Cautious sample treatment led to a low (10%) and controlled conversion of α-acetohydroxy acids to vicinal diketones. This reproducible percentage of conversion made it possible to determine precisely free vicinal diketones and α-acetohydroxy acids . The method was applied to the determination of precursors and vicinal diketones concentrations during beer fermentation .     

DETERMINATION OF ANIONS IN BEER BY ION CHROMATOGRAPHY

A method relying on ion chromatography, with suppressed ion detection, for the determination of anions in beer, has been collaboratively tested by members of the American Society of Brewing Chemists, the European Brewery Convention and the Brewery Convention of Japan. Precision values obtained for the determination of chloride, sulphate and phosphate in beer were judged to be acceptable. Repeatability (r₉₈) and reproducibility (R₉₈) values for chloride were 5.7, 12.6, 12.5 and 15.0, 38.4, 36.8 respectively at corresponding mean levels of 68.7, 218.6 and 322.5 mg/litre. r₉₈ and R₉₈ values for sulphate were 7.5, 6.2, 7.6 and 44.8, 54.0, 46.5 respectively at corresponding mean levels of 101.4, 205.1 and 122.6 mg/litre. r₉₈ and R₉₉ values for phosphate were 14.1, 11.9, 24.9 and 78.7, 53.8, 84.0 at corresponding mean levels of 411.5, 224.1 and 397.5 mg/litre. Whilst the r₉₈ value for nitrate was acceptable, the value for R₉₈ was unsatisfactory. The ion chromatographic method for determining chloride, sulphate and phosphate in beer is recommended for use and inclusion in Analytica -EBC as an International Method.     

ESTIMATION OF ALGINATE,CARRAGEENAN AND FURCELLARAN IN BEER

Quantitative and semi-quantitative methods are given for estimating alginate, carrageenan and furcellaran at low concentrations in beer. A number of commercial beers and experimental beers known to have been brewed with either copper finings or auxiliary finings containing these acidic polysaccharides as active ingredients were analysed for alginate, carrageenan and furcellaran residues. The results show that alginate and carrageenan do not occur in finished beers at levels greater than 1 mg/litre. Using a serological technique which is more sensitive for furcellaran, it was found that levels of furcellaran in most finished beers were below 0.5 mg/litre, though a few beers contained this polysaccharide at about 0.5 mg/litre.     

GAS CHROMATOGRAPHIC DETERMINATION OF TRIHYDROXYOCTADECENOIC ACIDS IN BEER

A rapid and quantitative method for measuring the content of trihydroxyoctadecenoic acids in beer is described. The acids are extracted from degassed beer with ethyl acetate and methylated with diazomethane. After washing, the methylated compounds are silylated and analysed by gas chromatography. The coefficient of variation of the method is 3·2%. The amounts of total trihydroxyoctadecenoic acids in commercial beer samples varied from 4 to 12 mg/litre. The effects of these acids on beer flavour and head retention are discussed.     

The analysis of 5′-mononucleotides in infant formulae by HPLC

A method is described for the determination of four 5′-mononucleotides (cytidine 5′-monophosphate, uridine 5′-monophosphate, adenosine 5′-monophosphate and guanosine 5′-monophosphate) in infant formulae. Nucleosides which may be formed during processing can also be analysed simultaneously. This method is based on deproteinisation of samples and direct analysis by ion-pair HPLC using two Nucleosil 120-C₁₈ columns in series, followed by diode-array detection. This method gives good recoveries of 5′-mononucleotides from spiked infant formula products. However, some chromatographic interferences were observed when analysing hypoallergenic infant formulae containing hydrolysed proteins which made peak quantification difficult. To overcome this problem a strong anion-exchange solid-phase extraction (SPE) column was used. Four SPE columns from different suppliers were evaluated, but the best recoveries of all four 5′-mononucleotides and highest reproducibility of results were obtained with Bakerbond® quaternary amine columns. Nucleosides, which may occur in very low concentrations in hypoallergenic products, are not retained on the SPE columns and so cannot be analysed by this technique.     

THE CONTRIBUTION OF DEXTRINS TO BEER SENSORY PROPERTIES PART I. MOUTHFEEL

The effect of dextrins on the mouthfeel of beer was assessed using instrumental and sensory techniques. By measuring the viscosity of beer with a rheogoniometer, it was shown that beer is a newtonian fluid, i.e., shear stress increased linearly with shear rate. A capillary viscometer and a rheogoniometer gave viscosity measurements that did not differ significantly for six beers ranging in viscosity from 1.1 to 2.6 centipoise (cP). In carbonated beer, the amount of dextrins needed to produce an increase in viscosity detectable by judges was 52 g/litre, which raised the viscosity by 0.31 cP as measured by capillary viscometry. Since beer usually contains between 10 and 50 g/litre of dextrins, it is concluded that dextrins are not the sole determinant of beer viscosity.     

Development and application of a liquid chromatographic method for analysis of nucleotides and nucleosides in milk and infant formulas

A method for the simultaneous determination in milk of the 5′-mononucleotides adenosine 5′-monophosphate, cytidine 5′-monophosphate, guanosine 5′-monophosphate, inosine 5′-monophosphate and uridine 5′-monophosphate and their corresponding nucleosides is described. Following deproteinisation, the sample extract was analysed by reversed-phase liquid chromatography, whereby chromatographic separation was achieved using a polymer grafted silica C₁₈ column, gradient elution with a simple binary mobile phase and UV detection by photodiode array. Performance parameters included recoveries of 95.5–105.2% and precision evaluated as 3.42–6.38% relative standard deviation. The described technique has been applied to the analysis of bovine and human milk, a range of commercial bovine milk-based infant and follow-on formulas, a seasonal study of skim milk powders and an assessment of alkaline phosphatase influence on nucleotide retention.     

PROCESSES FOR PRODUCING BEER WITH A LOW TENDENCY TO FORM HAZE

It was confirmed that wort from malt resteeped in a solution of formaldehyde (1000 mg./litre), had a low level of anthocyanogens. It was shown that beer brewed from this malt had a lesser tendency to form haze than beer brewed from a malt resteeped in water. Malt yielding wort with usefully reduced levels of anthocyanogens could be prepared by adding formaldehyde (500–1000 mg./litre), to the final steep, in an otherwise conventional malting sequence. The rapid rate of haze formation that occurred in beers to which formalin had been added was shown to be a useful, quick guide to their stability under different storage conditions. When hydrogen peroxide (100 mg./litre) mashing liquor was added to mashes it reduced the anthocyanogen levels of the wort. The beers prepared from treated mashes were remarkably slow to form haze. The effect was greatest when hydrogen peroxide was added in small increments throughout the mashing period. The other alterations in wort characteristics resulting from this process, including marginal increases in colour and decreases in fermentability, were small. In most trials the treatment did not significantly alter the flavour of the beer. Charcoal (Norit, N.K.), added to the mash (500 mg./kg. grist), or to the copper (500 mg./kg. grist), reduced the anthocyanogen contents of the worts; the final beers had greatly enhanced shelf-lives. Charcoal was most effective when spread over the surface of the mash at the start of sparging.     

GAS CHROMATOGRAPHIC DETERMINATION OF TYROSOL AND TRYPTOPHOL IN WINES AND BEERS

A gas chromatographic method for the determination of tyrosol and tryptophol in wines and beers has been developed. The aromatic fusel alcohols are extracted with ether or ethyl acetate from a sample made alkaline with Na₂CO₃ and saturated with NaCl. In beer determinations the extraction is performed with ethyl acetate in order to avoid complications caused by emulsion. Most of the solvent is removed by distillation and the rest is cautiously allowed to evaporate. The gas chromatographic determination of tyrosol and tryptophol is performed in a 0·5-m. Apiezon M-DEGS column at 190°C. According to the infra-red spectra, both components are eluted without being decomposed at this temperature. In the grape wines investigated, tyrosol was found in amounts of 10–40 mg. per litre, in two Finnish berry wines 10–15 mg. per litre and in two different types of pale lager beers 5–10 mg. per litre. The same samples contained about 1–4 mg. tryptophol per litre. The two white grape wines and one red berry wine were exceptional, with only 0·2–0·3 mg. tryptophol per litre.     

A CHEAP,DIRECT METHOD FOR THE ESTIMATION OF DISSOLVED AIR IN BEER

A plastic medical syringe is used to collect a beer sample and to apply vacuum to de-gas it. The beer is then replaced with sodium hydroxide solution and alkali-insoluble gas is displaced into a calibrated plastic capillary tube. Dissolved-air contents in the range 2 ml/litre to 50 ml/litre have been estimated with a repeatability adequate for many process investigations.     

Hydrolytic Stability at Intermediate pHs of the Common Purine Nucleotides in Food, Inosine-5'-monophosphate, Guanosine-5'-monophosphate and Adenosine-5'-monophosphate

When the purine nucleotides, inosine-5′-monophosphate (IMP), guanosine-5′-monophosphate (GMP) and adenosine-5′-monophosphate (AMP) were subjected to canning temperatures (121°C) at pHs between 3 and 8, extensive phosphate bond hydrolysis to the corresponding nucleoside occurred. The half life for hydrolysis at pH 5 was 63, 41, and 51 min for IMP, GMP, and AMP, respectively. Rates of hydrolysis were even greater at pH 3 because both hydrolysis of the phosphate bond and the glycosidic bond occurred simultaneously. An Arrhenius plot of data collected at different temperatures illustrated that the nucleotides were very stable at room temperature. Half lives for IMP, GMP, and AMP were estimated to be 36, 19, and 40 yr, respectively, at 23°C and a pH of 5.     

DETERMINATION OF VICINAL DIKETONES IN BEER BY GAS CHROMATOGRAPHY (HEADSPACE TECHNIQUE)—COLLABORATIVE TRIAL

Two methods for the determination of vicinal diketones in beer have been collaboratively tested by the Analysis Committee of the Institute of Brewing and are recommended for use. Both methods employ gas chromatography and are essentially the same, except that one relies on the use of a packed column and the other on a capillary column. For diacetyl it was judged that repeatability (r₉₅) values were independent of concentration over the range 0.04 to 0.19 mg/litre. Over this range, r₉₅ values for diacetyl of 0.028, 0.020 and 0.026 mg/litre were obtained for capillary, packed columns and combined results respectively. Values for reproducibility (R₉₅) were judged to be dependent on the mean level (m). R₉₅ values were 0.032 + 0.68 m, 0.01 + 0.47 m and 0.005 + 0.67 m were obtained for capillary, packed columns and combined results respectively. For both methods the r₉₅ and R₉₅ values for 2,3-pentanedione were judged to be independent of concentration over the range 0.02 to 0.07 mg/litre. For capillary columns, packed columns and combined results respectively, r₉₅ values were 0.009, 0.009, 0.010 mg/litre and R₉₅ values were 0.037, 0.042 and 0.038 mg/litre.