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.     

ON THE ANALYSIS OF HOP BITTER ACID

A High Performance Liquid Chromatography (HPLC) method for automated routine analysis of the hop bitter acids is described. It allows the simultaneous determination of α-acids, β-acids, cohumulone and colupulone. This analysis based on two chromatographic runs takes less than 1/2 h. With this new method as reference, the defects of existing conductometric titration techniques were studied and a new improved conductometric method based on toluene-buffer extraction was developed. The new methods are applicable to all forms of hop products containing α- and β-acids. Analysis for α-acids with ether-acid aqueous methanol extraction gives results which are too high because of several basic defects.     

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.     

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.     

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.     

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.     

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.     

DIRECT ANALYSIS OF INDIVIDUAL ANALOGUES OF ALPHA-ACIDS,BETA-ACIDS AND 4-DESOXYHUMULONES IN HOPS USING GAS-LIQUID CHROMATOGRAPHY AND NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

The techniques of gas-liquid chromatography and nuclear magnetic resonance allow simultaneous analysis of the individual α-acids and β-acids and the corresponding 4-desoxyhumulones. The rates of appearance in the growing hop-cone of humulone, lupulone, 4-desoxyhumulone and their co-homologues and ad-isomers are reported. The results indicate a relatively enhanced initial formation of the 4-desoxy compounds and β-acids. The α-acids reach their maximum concentration later than the desoxyhumulones and β-acids but eventually become the predominant group. It appears that formation of the α-acids is associated with a decline in the relative proportion of both β-acids and 4-desoxyhumulones. For each class of compounds, the co-homologues increase their relative concentrations during the growth of the hop; adhumulone and adlupulone remain in almost constant proportions during this period.     

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.     

酒花制品中异α-酸浓度的测定方法

采用分光光度法探索测定异α 酸、四氢异α 酸异构体、六氢异α 酸异构体等酒花制品中异α 酸的浓度 ,利用该方法对已知标准样品中异α 酸浓度进行检测 ,其重复性与精密度的结果表明 ,该方法的变异系数≤ 1 %,回收率在 99 8%～ 10 0 5 %之间 ;方法操作简单 ,可作为啤酒企业衡量评价异α 酸、四氢异α 酸异构体、六氢异α 酸异构体等酒花制品中异α 酸浓度的检测方法     

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.     

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 impact of different hop compounds on the growth of selected beer spoilage bacteria in beer

Beer spoiling lactic acid bacteria are a major reason for quality complaints in breweries around the world. Spoilage by a variety of these bacteria can result in haze, sediment, slime, off-flavours and acidity. As these bacteria occur frequently in the brewing environment, using certain hop products that inhibit the growth of these spoilers could be a solution to prevent problems. To investigate the impact of seven different hop compounds (α-acids, iso-α-acids, tetrahydro-iso-α-acids, rho-iso-α-acids, xanthohumol, iso-xanthohumol and humulinones) on the growth of six major beer spoilage bacteria (Lactobacillus brevis. L. backi, L. coryniformis, L. lindneri, L. buchneri, Pediococcus damnosous), two concentrations (10 and 25 mg/L) of each hop substance were added to unhopped beer. The potential growth of the spoilage bacteria was investigated over 56 consecutive days. A comparison of the results shows a strong inhibition of growth of all spoilage bacteria at 25 mg/L of tetrahydro-iso-α-acids closely followed by α-acids as the second most inhibitory substance. The results showed a high resistance of L. brevis to all hop compounds as well as an inhibition of L. coryniformis and L. buchneri at low concentrations of most hop components. In comparison with the control sample, L. lindneri showed increased growth in the presence of some hop compounds (rho-iso-α-acids, xanthohumol, iso-xanthohumol, humulinones). © 2020 The Authors. Journal of the Institute of Brewing published by John Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling     

Potential impact of medium characteristics on the isomerisation of hop α-acids in wort and buffer model systems

Many brewing variables, e.g., pH, boiling time, temperature, and the presence of metal catalysts, are essential in view of the isomerisation of hop α-acids into iso-α-acids. In this work, a comparative study on the impact of several selected factors on α-acids isomerisation was performed in both buffer model solutions and wort, in order to find out enhanced boiling conditions for higher isomerisation yields.     

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.     

Analysis of hop acids by capillary electrophoresis

Micellar electrokinetic chromatography (MEKC) was used to separate components of hop extracts. The separation of a sample of iso-α-acids by MEKC was better and faster than by an established HPLC method, giving <0.8% RSD on migration times and 5–10% RSD on peak areas. MEKC was also successfully used to separate the oxidation products of the α- and β-acids and thus to monitor the stability of hop products containing them. Furthermore, MEKC distinguished among samples of reduced iso-α-acids (rho-, tetrahydro- and hexahydro- derivatives).     

BICYCLIC ISOMERISATION PRODUCTS OF HUMULONE AND COHUMULONE IN BEER

Separation of five- and six-membered ring hop acids can be obtained selectively by High Performance Liquid Chromatography (HPLC) on anthracene-bonded silica gel. Photo-diode array detection combined with high resolution HPLC of the six-membered compounds of beer reveals the presence of I and II in Fig. 1. These compounds are formed by reaction of the respective α-acids, humulone (III) and cohumulone (IV), in brewing conditions.^1,2 Their concentration in beer is a few μgl^?1.     

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     

酒花制品有效成分稳定性研究

以α-酸、β-酸、总黄酮、黄腐酚含量为指标,比较了真空包装的压缩啤酒花、颗粒啤酒花、啤酒花浸膏在20℃、4℃、-20℃避光贮藏120d的成分变化.结果表明,随着温度的降低,压缩酒花、颗粒酒花、酒花浸膏中有效成分变化幅度逐渐变小,其中酒花浸膏中有效成分变化幅度最小,在20℃、4℃和-20℃贮藏条件下酒花浸膏中α-酸变化幅度分别为13.82％、7.39％、3.38％,β-酸17.13％、11.18％、6.80％,总黄酮4.48％、2.19％、1.56％,黄腐酚10.09％、5.68％、4.21％,以-20℃贮藏条件下酒花浸膏变化幅度最小.     

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.