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 .     

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.     

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.     

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.     

PREPARATION,PURIFICATION AND SEPARATION BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF HUMULINIC ACIDS,DEHYDROHUMULINIC ACIDS,AND HULUPONES

Modification of published procedures are described for the preparation of humulinic acids, dehydrohumulinic acids and hulupone, using iso-α-acids as starting material. These procedures provide facile routes for preparation of these compounds in higher yields than was hitherto reported, and also in sufficient purity for their use as standards in quantitative High Performance Liquid Chromatography. An alternative and more feasible route to hulupones, using β-acids as starting material, is also outlined.     

THE BITTERNESS OF HOP-DERIVED MATERIALS IN BEER

The bitterness of trans- and cis-isohumulones obtained both from beer and by alkaline isomerization of α-acids has been compared with the bitterness of hop resins and other materials extractable from beer. Trans-isohumulones and cis-isohumulones, obtained either from beer or by alkaline isomerization of α-acids, and hulupones have been shown to be the most bitter materials. Hulupones were only slightly less bitter than trans-and cis-isohumulones, which showed little difference in bitterness value when prepared directly, although the collective tasting results show a bias for a greater bittering associated with the cis-component. The hulupones, like the parent β-acid, possessed a distinctive aromatic flavour. α-Acids and other unidentified materials obtained from beer brewed with hops of a normal age have been found to possess a much lower level of bitterness. The observation that aged hops impart normal levels of tastable bitterness to beer has been confirmed at significant brewery level (60 brl.) using grists consisting solely of six year old hops. Approximately 35% of this bitterness is provided by isohumulones and the greater part of the remainder arises from materials soluble in low-boiling paraffinic hydrocarbon solvents. A known β-acid oxidation material has been isolated from fresh and old hops and from beers brewed with either fresh or old hops.     

PREPARATION OF HOP EXTRACTS RICH IN PARTICULAR CONSTITUENTS*

When a column containing powdered hops is extracted with liquid carbon dioxide, chromatographic separation of hop components occurs. They are extracted in the order essential oils, β-acids, α-acids; and the separation is enhanced when finely milled hops are extracted. Early fractions (～0·5 hour) contain a high proportion of the available essential oils when hops are extracted at ?20°C and such extracts are suitable as a replacement for dry hops. Fractions can be obtained from extractions at ～7°C which are rich in α-acids and contain low levels of β-acids. Small amounts of fats and waxes are normally present in fractions collected towards the end of a run when seeded hops are extracted.     

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.     

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     

KINETICS OF HOP STORAGE

After an initial ‘delay period’ the storage losses of α- and β-acids follow a first-order rate equation and not a zero-order equation as has been suggested by others.     

STABILITY OF RESINS IN PELLETISED HOPS

The α-acids content and Lead Conductance Value of hop pellets, manufactured from seedless Pride of Ringwood hops grown in Australia and stored at 5°C, 20°C or 30°C, were monitored over a twelve month storage period. No decrease in α-acids content occurred during storage at 5°C, whereas at 20°C the decrease (ca. 7%) was approximately one-third that experienced for baled hops of the same variety. At both 5°C and 20°C the Lead Conductance Value decreased at approximately one-third the rate of that in baled hops. During twelve months storage at 30°C the α-acids content of the hop pellets decreased by approximately 40% and the Lead Conductance Value by 30%. The hop pellets stored at 20°C and 30°C developed rancid odours during the twelve months storage period.     

老化的颗粒酒花对啤酒发酵抗氧化能力的影响

分析老化后酒花的主要成分,研究发现在酒花老化过程中α酸和β酸发生氧化降解,前期生成异α酸,随着老化的进行,继而产生葎草灵酮(humulinone)和希鲁酮(hulupones)等衍生氧化物。酒花中的总多酚含量逐渐减少,具有抗氧化性的酚酸也发生氧化聚合,使用液相色谱检测几乎无响应峰。并测定总多酚、DPPH清除率、TRAP值、TBA值等抗氧化指标来分析老化的酒花对麦汁和啤酒的抗氧化能力影响,实验发现老化的酒花对麦汁和啤酒的抗氧化能力影响较大,尤其是风味稳定性系数SI值,波动最为明显。     

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.     

PROPOSALS FOR THE EFFICIENT AND ECONOMICAL USE OF HOPS IN BREWING

The hop lupulin glands, which contain the essential oils and resin acids, can be separated effectively from the bulky and worthless bracts and residues in a mechanical process which avoids solvent contamination. The separation is accomplished in an air stream through a centrifugal sifter after the lupulin has been rubbed off in a disc pin mill. The separated lupulin can be broken open by grinding in a colloid mill and used to obtain a dry hop aroma in beers by an ‘instant’ process. The lupulin can be used to bitter beers either directly in the copper or in a pre-boiling process. It can also be used in an aqueous extraction process to yield, nearly quantatively, pure and separate extracts of the α- and β-acids. The α-acids can then be isomerized almost quantitatively and the β-acids can be oxidized to give a 50% yield of hulupones. The hop residues, when boiled with wort, give an approximately 50% utilization of the remaining α-acids. These isohumulones can then be enhanced by those obtained from the extracted α-acids and the bitterness can be supplemented by the hulupones obtained from the β-acids. Overall, this process is calculated to give a bitterness utilization equivalent to 87% calculated on the original α-acids.     

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.     

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.     

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.     

ON THE SUNSTRUCK FLAVOUR OF BEER

It has been confirmed that sunstruck flavour is due to the formation of 3-methyl-2-butene-1-thiol formed by photolysis of iso-α-acids in the presence of sulphur-containing amino acids.     

ORGANOLEPTIC TRIALS WITH HOP BITTER SUBSTANCES

Pure hops-derived substances were added to unhopped beer. Illumination only produced a sun-struck flavour with cis-isohumulone, trans-isohumulone and racemic trans-isohumulone. It seems that 3-methyl-butenyl-mercaptan is not or not alone responsible for sun-struck flavour. Tests trials show that trans-isohumulone, cis-isohumulone, racemic trans-isohumulone, trans-₁-isohumulone and cis-₁-isohumulone do not show significant differences in bitter taste and bittering power. Fresh solutions of these bitter acids are not as bitter as solutions which have been kept for a few weeks.     

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.