ANALYSIS OF HOPS AND HOP PRODUCTS FOR α-ACIDS OR ISO-α-ACIDS

Two methods based on the resolution of mixtures of hop compounds by chromatography on Sephadex columns have been adopted by E.B.C. and A.S.B.C. as ‘International Methods’.     

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.     

ESTIMATION OF α-, β- AND ISO-α-ACIDS IN HOPS,HOP PRODUCTS AND BEER USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Methods are described in which high performance liquid chromatography (HPLC) is used to estimate α-, β- and iso-α-acids in hops, hop products and beer. The chromatography relies on an isocratic elution of components from a polythene ‘cartridge’ column, and the method is calibrated with the pure substances as primary standards. Using such a column over 1000 analyses have been carried out without any significant loss in resolution or precision. The procedures are sufficiently rapid for use in commercial transactions and for quality control purposes. For hops and hop extracts coefficients of variation (%) of 2·5 and 0·8 were obtained respectively for α-acids. Values of 0·9 and 0·3 were obtained for iso-α-acids in isomerised extracts and beers respectively. For some isomerised extracts it has been observed that peaks in addition to those given by the iso-α-acids are present on the chromatogram. The current method recommended by the EBC over estimates the iso-α-acid content since these other constituents are included in the analysis.     

ENZYMIC DEGRADATION OF α-ACIDS IN HOPS

Evidence is presented for the biological oxidation of α-acids in hop cones by an enzyme having molecular weight 17,000 daltons, K_m 6–0 and Mn as the active metal ion. The enzyme is located in bracts and bracteoles and not in lupulin glands. The presence of an endogenous inhibitor was demonstrated. The specific activity of protein in hop cones increased with maturity and this was probably associated with release of α-acids from lupulin glands.     

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     

SOLUBILITY OF HOP α- AND β-ACIDS IN LIQUID CARBON DIOXIDE*: ADDENDUM: SOLUBILITY OF PESTICIDES IN LIQUID CARBON DIOXIDE

A procedure is described for determining the solubility of hop α- and β-acids in liquid carbon dioxide. Results have shown that the optimum temperature range for the extraction of hops with liquid carbon dioxide is +5 to +10°C. A number of pesticides used by hop growers are appreciably soluble in liquid carbon dioxide.     

AUTOMATED REPORTING ON THE QUALITY OF HOPS AND HOP PRODUCTS

The quality of a hop variety or a hop product can readily be assessed by a fully automated sequence of selective extraction, fractionation and quantitative analysis. To illustrate the elegance of the method, nine hop varieties and three hop extracts were compared with respect to the content of important marker compounds in the hop oils and of the hop acids. Supercritical fluid extraction at different densities of carbon dioxide was applied to extract selectively, the hop oils and the hop acids, respectively. The hop oils were further fractionated into an apolar and a polar fraction by solid phase extraction and consecutive elution with n-hexane and ethyl acetate. Separation and identification were achieved by capillary gas chromatography coupled to mass spectrometry. Myrcene, β;-caryophyllene, α;-humulene and β;-farnesene in the apolar fraction, linalool, undecan-2-one, tridecan-2-one and humuladienone in the polar fraction were selected for quantitative evaluation of the respective hop oils. Sulphur-containing compounds were revealed by capillary gas chromatography using sulphur-selective atomic emission detection. Complete separation and quantification of all hop α;-acids and β;-acids was effected by microemulsion electrokinetic chromatography coupled to diode array detection .     

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.     

ABEO-ISO-α-ACIDS IN ENGLISH BEERS

Polyphenols interfere in thin layer chromatographic estimations of abeo-iso-α-acids. A paper chromatographic technique for separating polyphenols from abeo-iso-α-acids has been developed. English beers examined contained less than 6 ppm of abeo-iso-α-acids.     

ISO-α-ACIDS CONTENT OF ISOMERIZED HOP EXTRACTS

Two chromatographic procedures for the analysis of iso-α-acids in isomerized extracts have been tested. Although one of the methods—a variant of the Hansen, Hetzel & Miller procedure—performed better than the other, it still falls short of the demands of commercial transactions. The methods available for the assay of iso-α-acids are reviewed.     

QUANTITATIVE DETERMINATION OF HOP BITTER SUBSTANCES AND THEIR DERIVATIVES IN HOP EXTRACTS BY THIN LAYER CHROMATOGRAPHY

A method is described which allows the separation of hop bitter components and derivatives present in hop extracts by thin-layer chromatography on a mixture of silica gel and cellulose. This method can be used to evaluate the purity of hop extracts, isomerized hop extracts and complementary base extracts.     

SIMULTANEOUS DETERMINATION OF α, β AND ISO-α ACIDS IN HOPS AND HOP PRODUCTS

A method for the simultaneous analysis of α, β and iso-α acid in hops, hop extracts and isomerised hop extracts is described. It is based on the use of reversed phase high performance liquid chromatography and quantitative evaluation of the hop compounds is carried out with a computing integrator. The isomerisation reaction can be examined in detail, particularly in connection with the production of hop derived haze forming compounds in isomerised hop extracts used for post fermentation bittering.     

ANALYTICAL EVALUATION OF α-ACIDS IN HOP EXTRACTS AND IN HOPS*

The results of new investigations on hop extract analysis are presented. The main findings are:

• 1 The specific extinction (257) of α-acids at 276 nm in iso-octane used at present in CCD and paper strip analyses is 3·5% too high: analytical results have therefore been too low. The new extinction value to be used is 248.
• 2 The result of paper strip analysis depends very much on the amount of α-acids placed on the strip. The three-point paper strip method can only partly correct this. In general, figures with paper strip analysis have a tendency to be too high.
• 3 Conductometric titration of α-acids produces a figure about 2 to 10% too low. This can be partly corrected by adding 20% dimethylsulphoxide or dimethylformamide to the titration medium.
• 4 Conductometric analysis of hop extracts with removal of “iso-α-acids like material” before titration is possible by a very simple procedure (pH 7 buffer extraction).

New photometric and conductometric procedures based on these results give figures which are in agreement for something which must be practically true α-acids.     

EFFECT OF REDUCTION IN HOP OIL CONTENT ON RATE OF DETERIORATION OF ALPHA-ACID IN HOPS

Hops in which the essential oil content has been reduced by steam distillation under vacuum lose α-acid on storage more slowly than do untreated hops.     

THE DETERMINATION OF BARLEY α-AMYLASE ACTIVITY

Starch-iodine colour (S.I.C.) units of α-amylase activity are re-defined. Two improved, alternative methods of calculating results are given, either (a) by a revised graphical procedure used in conjunction with a standard graph, or (b) by a graphical procedure using points calculated from the experimental results with an empirical equation using a programmed electronic calculator. Semiautomation of the measurement of starch-iodine colours usefully reduces the work load involved in this enzyme assay. Enzyme activities may be expressed in S.I.C. units, or as a rate of increase in reducing power.     

THE HOP MARKET—PRESENT AND PROSPECTIVE

The world production of hops currently exceeds the demand by brewers. Factors contributing to this situation are reviewed together with the effect that this has produced on price levels.     

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.     

CHROMATOGRAPHIC STUDY OF THE INFLUENCE OF LINOLEIC ACID AND ISO-α-ACIDS IN THE FORMATION OF CARBONYLS IN BEER

The influence of the concentration of linoleic acid, iso-α-acids and metabisulphite in the formation of volatile carbonyls in beer has been studied. The ageing of the beer is effected by means of a 1.5 hour reflux at pH=2 and the carbonyl compounds are determined by high pressure (HPLC) liquid chromatography of the corresponding 2.4 dinitrophenylhydrazones (DNPS). It has been shown that carbonyls with more than 5 carbon atoms mainly come from the autoxidation of linoleic acid. The inhibiting action of metabisulphite has also been confirmed     

QUANTITATIVE DETERMINATION OF BITTER SUBSTANCES IN HOPS BY THIN LAYER CHROMATOGRAPHY

A method is described which allows the separation of hop bitter components and derivatives present in hops by thin-layer chromatography on a mixture of silica gel and cellulose. This method can be used to evaluate ageing and the quality of the hop.     

THE DEGRADATION OF β-GLUCAN DURING MALTING AND MASHING: THE ROLE OF β-GLUCANASE

Significant β-glucanolysis takes place during mashing and is catalysed by a β-glucanase which is specific to mixed-linkage β-glucans. The enzyme develops during the germination of barley, but is rapidly and extensively destroyed in kilning. Partially-purified preparations of β-glucanase are protected from denaturation by heat if their solutions are adjusted to pH 4 or if bovine serum albumin is added. However the most effective stabiliser of the enzyme is reduced glutathione. Oligosaccharides containing three and four glucosyl units are produced by the action of β-glucanase and they are further converted during malting and mashing by a different enzyme(s) to disaccharides and glucose.