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’.     

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.     

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.     

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.     

The Significance of Iso‐α‐Acids for Beer Quality Cambridge Prize Paper

The iso‐α‐acids and their chemically‐modified variants play a disproportionately large role in the final quality of beer. Here, fundamental aspects of two of these quality issues — foam and bitterness — are discussed. A common feature of both issues is the dependence on the hydrophobic character of the hop compounds on both bitterness potency and ability to stabilise foams. Thus the isocohumulones appear significantly less bitter than the other, more hydrophobic hop compounds. Also apparent were the differences in bitterness between the cis‐ and the trans‐isomers, with the former being the more potent. Also described are the differences in the partitioning of the cis‐ and trans‐iso‐α‐acids into beer foam. The trans‐isomers are enriched in foam relative to their cis‐counterparts and may account for the observed enrichment of cis‐isomers in the final beer relative to the common ratios observed upstream.     

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.     

The effect of hopping regime,cultivar and β‐glucosidase activity on monoterpene alcohol concentrations in wort and beer

Previous studies show that the complexity of hop aroma in beer can be partly attributed to the hydrolysis of glycosidically bound monoterpene alcohols extracted from hops during the brewing process to release volatile aglycones. However, fundamental studies that examine the extraction of glycosides during brewing and their subsequent hydrolysis by yeast have not been performed. Furthermore, extraction of other hop‐derived compounds into beer shows a strong dependency on the hop cultivar being used and the point at which it is added. This study focused on the extent of glycoside extraction owing to hopping regime and cultivar, and their hydrolysis by yeast β‐glucosidase activity. Glycoside concentrations of wort made with three different hopping regimes and three cultivars were measured by the difference in volatile aglycone concentrations between samples treated with purified β‐glucosidase and untreated samples. Aglycone concentrations were measured by solid‐phase microextraction gas chromatography–mass spectrometry. Additionally, β‐glucosidase activities for 80 different yeast strains and their effect on aglycone concentration in wort were determined. Results showed that yeast have a wide range of abilities to hydrolyse glycosides with a maximum hydrolysis occurring after 3 days of fermentation regardless of yeast activity. Although it was shown that yeast are capable of glycoside hydrolysis, glycoside concentrations in wort are low and make small contributions to hop aroma. These results help explain the extent to which different brewing yeasts and hopping regimes contribute to hoppy beer aroma through the hydrolysis of non‐volatile hop‐derived compounds. Copyright © 2017 The Institute of Brewing & Distilling     

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 .     

THE APPLICATION OF SALT FORMATION IN THE CHEMISTRY AND TECHNOLOGY OF HOP RESINS

In natural product chemistry the satisfactory isolation of chemical compounds of scientific and/or commercial significance normally follows a pattern of fractionation which involves the utilization of a range of physical and chemical properties of the compounds present in the mixture. Vapour pressure and solubility are the physical properties traditionally made use of in the initial stages of fractionation. Chemical properties normally enable somewhat more specific fractionation to be achieved. One chemical property that is widely used is the ability of a large number of compounds to form salts. In the case of the hop resins it is this specific chemical property which has played the most significant role in their isolation, characterization and commercial utilization. The historical development of this aspect of hop chemistry has been reviewed.     

Alterations of the Profiles of Iso‐α‐Acids During Beer Ageing,Marked Instability of Trans‐Iso‐α‐Acids and Implications for Beer Bitterness Consistency in Relation to Tetrahydroiso‐α‐Acids

Age‐induced decomposition of iso‐α‐acids, the main bittering principles of beer, determines the consistency of the beer bitter taste. In this study, the profiles of iso‐α‐acids in selected high‐quality top‐fermented and lager beers were monitored by quantitative high‐performance liquid chromatography at various time intervals during ageing. The degradation of the iso‐α‐acids as a function of time is represented by the ratio, in percentage, of the sum of the concentrations of trans‐isocohumulone and trans‐isohumulone to the sum of the concentrations of cis‐isocohumulone and cis‐isohumulone. This parameter is relevant with respect to the evaluation of bitterness deterioration in aged beers. Trans‐iso‐α‐acids having a shelf half‐life of less than one year proved to be significantly less stable than cis‐iso‐α‐acids, but it appears feasible to counteract degradation if a suitable beer matrix is available. The fate of the trans‐iso‐α‐acids in particular adversely affects beer bitterness consistency. In addition to using hop products containing low amounts of trans‐iso‐α‐acids, brewers may profit of the remarkable stability of tetrahydroiso‐α‐acids, even on prolonged storage, for the production of consistently bitter beers.     

β-GLUCANASE: THE SUCCESSFUL APPLICATION OF GENETIC ENGINEERING

The now well established principles of genetic engineering are described in relation to the solution of problems associated with β-glucans in beer. The application of these techniques has enabled the isolation of a Bacillus subtilis endo-1, 3–1, 4-β-D-glucanase gene which expresses a biologically active enzyme in yeast.^15,16 Although this enzyme is capable of hydrolysing beer β-glucans during fermentation, thereby enhancing beer filtration, insufficient β-glucanase is produced in yeast to enable successful commercial implementation. The requirements for the efficient production of β-glucanase in genetically manipulated brewing yeast are described.     

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     

IONIZATION BEHAVIOUR OF HOP COMPOUNDS AND HOP-DERIVED COMPOUNDS

The ionization behaviour of colupulone, (?)-humulone, trans-isohumulone and trans-humulinic acid was investigated using a number of techniques. When a potentiometric technique was employed, precise estimates of pKa could not be obtained for any of the compounds, since a drift in the observed pKa values occurred during the titrations. Approximate, or equilibrium pKa values for the most acidic function of each of the compounds were: colupulone, 6.1; (?)-humulone, 5.0; trans-isohumulone, 3.1; trans-humulinic acid, 2.7. Evidence from spectroscopic measurements (UV, ¹H-NMR, ¹³C-NMR), conductiometric experiments and measurements of the pH dependency of solvent partition behaviour and aqueous solubility of the compounds is consistent with the possibility that, in aqueous solution, compounds such as trans-isohumulone are present as covalent hydrates. It is likely that reversible hydration occurs at one or more carbonyl groups. As a consequence of this behaviour, no single pKa value is sufficient to describe the behaviour of any one hop compound or hop-derived compound. The potential significance of this phenomenon is discussed.     

Isolation of individual hop iso-α-acids stereoisomers by β-cyclodextrin

Individual iso-α-acids that are responsible for the bitter taste of beer need to be isolated because these acids are required as reference standards in quantitative analysis and when studying the parameters which effect the quality of beer. However, these pure compounds are very expensive, due to inefficient isolation methods. In this study a new isolation method has been developed, in order to reduce the isolation cost. β-Cyclodextrin has been used for the isolation of trans- and cis-iso-α-acids. The separation from the mixture of stereoisomers was achieved by complexation, using ethanol:water (1:2, v/v) as a solvent at a temperature of 50 °C for 30 min. The molar ratio of iso-α-acids sample to β-cyclodextrin for complexation was 1:1. Precipitation time varied between 9 h and 2 days, depending on the iso-α-acid. Release of the guest from the cyclodextrin complex was successfully accomplished by elution with methanol.     

QUANTITATIVE ANALYSIS OF HOPS AND HOP EXTRACTS BY HIGH PRESSURE ION EXCHANGE CHROMATOGRAPHY

A new analysis for hop acids in hops and hop extracts is described. It is based on recent developments in high pressure liquid chromatography using pellicular anion exchange column filling material. Quantitative evaluation of α-acids, β-acids and oxidation products in hops and hop extracts is carried out by standard addition of pure humulone. The α-acids are completely separated from other hop substances before quantitation. The results of α-acids determinations must therefore be more accurate than was formerly possible and they are compared with conductometric titration results which are systematically higher. This is to be expected as it becomes more and more obvious that paper strip and conductometric analysis are not selective enough and determine fractions as “α-acids” which are in fact oxidation products of the hop acids.     

The Contribution of Geraniol Metabolism to the Citrus Flavour of Beer: Synergy of Geraniol and β‐Citronellol Under Coexistence with Excess Linalool

The behaviour of hop‐derived monoterpene alcohols during fermentation by lager yeast was previously investigated in this laboratory. It was suggested that the concentration of geraniol and β‐citronellol in the finished beer increased depending on the initial concentration of geraniol in the wort. In addition, an additive effect among linalool, geraniol and β‐citronellol was found and 5 ug/L of geraniol and β‐citronellol was enough for this effect. In this paper, conditions regarding the enrichment of the initial concentration of geraniol in the wort were investigated. From the screening of various hop cultivars, Citra hop was selected as a geraniol‐rich cultivar. In addition, it was observed that coriander seed, which can be used in beer production as a flavourant, contained not only linalool but also geraniol at high levels. The use of Citra hop or coriander seed was effective for enriching the concentration of geraniol and β‐citronellol in the finished beers. In the Citra beer and in the coriander beer, the content of linalool was excess in comparison with the content of geraniol and β‐citronellol. Therefore, the synergy of geraniol and β‐citronellol, under coexistence of excess linalool, was examined. It was found that the flavour impression of excess linalool became more fruity and citrus by coexistence with geraniol and β‐citronellol and that the coexistence of all three monoterpene alcohols was effective for this synergy. The flavour characteristics of the Citra and coriander beer and the importance of geraniol metabolism for a citrus flavour in beer are discussed.     

CAMBRIDGE PRIZE LECTURE. STUDIES ON THE SENSITIVITY OF LACTIC ACID BACTERIA TO HOP BITTER ACIDS

Hop bitter acids act as mobile-carrier ionophores. They inhibit the growth of beer-spoilage bacteria by dissipating the transmembrane pH gradient. Their activity is pH dependent. Low pH favours antibacterial activity but high pH reduces it. Resistance to hop bitter acids is a stable character, associated only with beer-spoilage lactic acid bacteria. Hop-resistant organisms can maintain a larger transmembrane pH gradient and ATP pool than can hop-sensitive organisms. Prior exposure of bacteria to trans-isohumulone does not influence the degree of resistance to hop bitter acids. However, in some strains, exposure to trans-isohumulone does induce the ability to spoil beer. The chemistry of these compounds is more complex than previously thought. In aqueous solutions, such as beer, hop acids bind to metal ions and may be covalently hydrated.     

The Effects of Hop‐α‐Acids and Proline‐Specific Endoprotease (PSEP) Treatments on the Foam Quality of Beer

Influences on foam stability and cling were compared by brewing trials investigating beer hopping rate, hopping type and modification of beer protein composition by the inclusion of a proline specific protease (PSEP). The comparison of the NIBEM, Rudin and lacing foam assessment methods with the level of hopping demonstrated the superiority of hydrogenated hop α‐acids with respect to foam stability and particularly lacing. In addition, the NIBEM and Rudin foam analysis tests appear to respond relatively similarly with respect to hopping rate and hop type, with the NIBEM being somewhat more responsive in terms of foam stability measurments. The PSEP trials suggested that protein composition may only have a subtle effect on foam stability. Although more specific to haze active proteins, PSEP treatment in the small and pilot scale trials generally, but not always, resulted in a minor reduction in foam stability. This effect was not observed in 20 hL pilot and industrial scale beer productions. It was verified that both NIBEM and Rudin were positively influenced by increased levels of foam positive proteins. Although both foam tests were responsive to hopping rate and type, it is suggested that the Rudin foam test is somewhat biased towards foam positive proteins, particularly albuminous foam positive proteins (LTP1 and protein Z4), while in comparison the NIBEM foam test appears somewhat biased towards hordein foam positive proteins.     

COMPARISON OF TIME-INTENSITY WITH CATEGORY SCALING OF BITTERNESS OF ISO-α-ACIDS IN MODEL SYSTEMS AND IN BEER*

A 17-point non-numerical category scale indicated a direct, linear relationship between average perceived bitterness and concentration of iso-α-acids in water and in a commercial lager. Time-intensity (T-I) tracings yielded additional information: the rate of increase in bitterness after taking the sample into the mouth; the rate of decrease after reaching maximum intensity; the events associated with swallowing; and total duration, which ranged from 13 to 42 s across judges. The T-I curves revealed a burst of bitterness intensity immediately after swallowing, which was proportional to the concentration of iso-α-acids for water solutions, but not for beer. Addition of 2.6% ethyl alcohol to the lager enhanced bitterness, particularly at low levels of added iso-α-acids, whereas addition of 2.0% glucose reduced bitterness in the control as well as in beer with added iso-α-acids. In water, 20 and 30 ppm of iso-α-acids were more bitter and had a longer duration than in beer. Among judges there were marked differences in the patterns of the T-I tracings, but there was excellent reproducibility within judges across replications.     

SEPARATION AND QUANTIFICATION OF ALL MAIN HOP ACIDS IN DIFFERENT HOP CULTIVARS BY MICROEMULSION ELECTROKINETIC CHROMATOGRAPHY

Microemulsion electrokinetic chromatography has been applied to the analysis of hop acids in extracts of different hop cultivars. With this new analytical method, the 6 main hop bitter acids are completely separated within 10 minutes. Quantitative analyses of extracts from Saaz, Nugget and Wye Target cultivars have been compared to those obtained with reversed-phase high-performance liquid chromatography. The cohumulone and colupulone ratios are in good agreement. Microemulsion electrokinetic chromatography however is much faster and provides, in addition, data for the adhumulone and adlupulone ratios.