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.     

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.     

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.     

THE EFFECT OF STORAGE TEMPERATURE ON THE STABILITY OF THE ALPHA-ACIDS CONTENT OF BALED HOPS

The α-acids content of baled hops (Pride of Ringwood) stored at ?20°C, ?5°C, +5°C and +22°C was monitored over a twelve month storage period. The α-acids content of the hops was found to decrease at a linear rate, i.e., by zero order kinetics, at each storage temperature. The relationship between reaction rate and storage temperature was found to accord with the predictions of classical reaction kinetics, enabling a prediction of the stability of hops stored at various temperatures to be made.     

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 .     

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     

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.     

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 STORAGE OF WYE TARGET HOPS: ADDENDUM: STORAGE OF FIFTEEN GROWTHS OF WYE TARGET HOPS OVER 12 MONTHS

Wye Target hops lose both α-acids and bittering potential when stored at ambient temperature. Although there is some variation in the rate that different growths lose α-acids, such losses can be substantially reduced by storing Wye Target hops in the cold.     

GAS CHROMATOGRAPHY AND GAS CHROMATOGRAPHY-MASS SPECTROMETRY OF SOFT RESINS AND RELATED COMPOUNDS

The α-acids, β-acids, iso-α-acids, hulupones and humulinic acid have been separated by gas chromatography utilising the cool on-column injection technique on various bonded phase capillary columns. The α-acids were separated into cohumulone and humulone; the β-acids into colupulone and lupulone; and the hulupones into cohulupone and hulupone. Iso-α-acids were separated into four peaks; cis-isocohumulone, trans-isocohumulone, cis-isohumulone and trans-isohumulone. Flame ionization detection (FID) and chemical ionization mass spectrometry (CIMS) were used for chromatographic detection. Best results were achieved by employing a DB-1 fused silica capillary column with isooctane as the sample solvent .     

RAPID SPECTROPHOTOMETRIC METHOD FOR DETERMINATION OF THE BITTERING VALUE OF ISOMERIZED HOP EXTRACTS

A method is described which allows the rapid determination of the bittering value of isomerized hop extracts. The method is also applicable in the case of isomerized hop extracts containing impurities such as α-acids and β-acids.     

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.     

THE EFFECT OF TIME OF DRESSING ON THE FLOWERING BEHAVIOUR AND YIELD OF HOPS VARIETY LATE CLUSTER

The Late Cluster variety of hops when introduced to Kashmir (India) from USA gave two flowering flushes in a growing season, thus giving two harvests. By adjusting the date of dressing (root pruning) from December to May 15 the abnormal flowering behaviour of the plant could be altered from two flushes to only one. Plants dressed late in spring, April 15 to May 1, gave the highest cone yield which was expressed in only one harvest. This also produced the highest α-acids content.     

啤酒花活性成分与抗氧化活性的相关性

以3 个品种的萨斯特啤酒花为原料,比较了其石油醚、氯仿、乙酸乙酯、正丁醇和水相等不同极性溶剂提取物中α-酸、β-酸、总黄酮和总多酚的含量,采用?OH、1,1-二苯基-2-三硝基苯肼自由基、β-胡萝卜素-亚油酸3 种不同的抗氧化活性评价体系对各极性溶剂提取物的抗氧化活性进行了研究,并与其活性成分的含量进行了相关性分析。结果表明,啤酒花各极性部位均具有不同程度的抗氧化活性。α-酸、β-酸在石油醚相中含量较高,在3 个啤酒花样品中的含量范围分别为α-酸444.29～583.81 mg/g、β-酸131.83～293.19 mg/g,在氯仿相中含量相对较低,其余提取物中均未检出;相关性研究表明α-酸、β-酸是石油醚和氯仿相抗氧化活性的主要物质基础。中等极性和极性较大溶剂提取物中啤酒花总黄酮、总多酚含量较高,相关性研究也表明黄酮、多酚类物质是这类提取物抗氧化活性的主要物质基础;其中,3 个啤酒花样品中总黄酮、总多酚在乙酸乙酯相中含量最高,分别为20.43～24.13 mg/g和34.97～40.24 mg/g,正丁醇次之,分别为9.45～11.68 mg/g和12.12～19.14 mg/g。     

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.     

α-GLUCOSIDASE ACTIVITY OF SORGHUM AND BARLEY MALTS

Sorghum malt α-glucosidase activity was highest at pH 3.75 while that of barley malt was highest at pH 4.6. At pH 5.4 employed in mashing sorghum malt α-glucosidase was more active than the corresponding enzyme of barley malt. α-Glucosidase was partly extracted in water but was readily extracted when L-cysteine was included in the extraction buffer, pH 8. Sorghum malt made at 30°C had higher α-glucosidase activities than the corresponding malts made at 20°C and 25°C. Nevertheless, the sorghum malts made at 20°C and 25°C produced worts which contained more glucose than worts of malt made at 30°C. Although barley malts contained more α-glucosidase activity than sorghum malts, the worts of barley had the lowest levels of glucose. The limitation to maltose production in sorghum worts, produced at 65°C, is due to inadequate gelatinization of starch and not to limitation to β-amylase and α-amylase activities. Gelatinization of the starch granules of sorghum malt in the decantation mashing procedure resulted in the production of sorghum worts which contained high levels of maltose, especially when sorghum malt was produced at 30°C. Although the β-amylase and α-amylase levels of barley malt was significantly higher than those of sorghum malted optimally at 30°C, sorghum worts contained higher levels of glucose and equivalent levels of maltose to those of barley malt. It would appear that the individual activities of α-glucosidase, α-amylase and β-amylase of sorghum malts or barley malts do not correlate with the sugar profile of the corresponding worts. In consequence, specifications for enzymes such as α-amylase and β-amylase in malt is best set at a range of values rather than as single values.     

THE STORAGE OF YEOMAN HOPS AND PELLETISED POWDERS

Studies have shown that on extended storage of up to 90 weeks at ambient temperature pockets of Yeoman hops lose α-acids less rapidly than do the other varieties of English hops which are rich in resins. As expected the rate of loss of α-acids is further reduced when Yeoman hops are stored in the cold. Pelletised powders made from Yeoman hops show excellent storage characteristics when commercial packs are kept for 12 months either at ambient temperature or in the cold.     

COMPARISON OF TETTNANGER,SAAZ, HALLERTAU AND FUGGLE HOPS GROWN IN THE USA,AUSTRALIA AND EUROPE

Analyses of essential oil from 16 samples and α-acids and β-acids in 9 samples of Tettnanger hops grown in the USA showed that they were not the same as Tettnanger hops grown in the Tettnang district of Germany. The composition of the essential oils and ratios of α-acid to β-acid in samples grown in the USA were consistent with them being the English variety Fuggle. Samples of USA clones of Tettnanger and Hallertau mittelfrüher grown in Australia also appeared to be Fuggle but a commercial sample of Saaz grown in Yakima was similar to Saaz grown in England, at Wye. The varieties Saaz and Tettnanger were indistinguishable by chemical methods .     

Characterisation of lactose crystallised in acidified aqueous ethanol

Three types of crystalline lactose hydrates having melting points (MP) of 218°C, 195°C and 169°C were crystallised from 680 ml litre^?1 ethanol solution with a lactose to solvent ratio of 1:14-1:24 at three pH values, ～ 4, 2.5 and 1.3. The three types were characterised on the basis of their optical rotation, phase transition and X-ray powder diffraction pattern. The samples with high and low MP were α-lactose hydrates whereas that with the intermediate MP (195°C) was a hydrated lactose containing both α- and β-forms.     

HOP INDUSTRY COMMITTEE: ANALYSIS OF HOPS OF THE 1977 CROP

Analytical values for α-acid content of thirteen varieties of hops from the 1977 crop are presented in relation to the district of production. For all varieties the tolerance aim of ±0.5% was easily achieved, the whole crop being less variable than in any previous year since these records were kept. The results are acceptable for commercial purposes. α-Acid values for seedless hops from Hampshire and from the Seedless Hop Trials are included and comparisons with seeded hops are shown over a three year period.