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.     

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.     

ADDENDUM: SHORT-TERM STORAGE OF PELLETS MADE FROM SIX GROWTHS OF WYE TARGET HOPS

Pellets made from powdered Wye Target hops lost both α-acids and essential oil appreciably less rapidly than did cone hops during storage at ambient temperature for up to 2 years. Pelleting Wye Target hops is more effective than cold storage of cone hops in minimising losses of α-acids and maintaining brewing value. There are differences in the rate of loss of α-acids from pellets obtained when hops from a single growth of Wye Target hops were processed by four different processors. Cold storage of pellets from two processors resulted in a loss of just 5% of their initial α-acids after 27 months.     

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.     

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.     

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.     

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。     

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.     

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.     

THE AROMA OF HOPS I. ORIGIN AND IDENTIFICATION OF HOP AROMA SUBSTANCES

Gas chromatography of the head space was used to follow the evolution of hop aroma under different conditions of storage. Aroma from fresh hops contains mainly myrcene, which disappears as the hops get older. Meanwhile new, more volatile substances are formed, especially by degradation of the well-known α- and β-acids; when hops are kept in closed storage the aroma therefore soon takes a composite character. When the formed volatiles are allowed to escape, the aroma of hops becomes poorer with age, to disappear almost completely in the end. Twenty-five compounds have been identified in the more volatile fraction of hop extracts.     

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 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.     

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.     

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.     

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).     

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.     

HOP RESINS AND BEER FLAVOUR V. THE SIGNIFICANCE OF OXIDIZED HOP RESINS IN BREWING

Oxidation products of α- and β-acids have been used to flavour beers. Ion-exchange chromatographic examinations of extracts of these beers are described. Examination of old hops has revealed the presence of certain oxidation products of hop resins.     

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.     

DETERIORATION OF PELLETED HOP POWDERS DURING LONG-TERM STORAGE

During prolonged storage there is a reduction in the bittering potential of commercial samples of pellets made from either powdered Northern Brewer or Styrian Golding hops. These losses are particularly pronounced for Northern Brewer pellets stored at ambient temperature in ruptured packs. Hop pellets can also lose substantial quantities of essential oil during prolonged storage at ambient temperature and usually develop marked off-aromas. Such pellets are clearly unsuitable for dry hopping beer or for late additions to the copper. Beers brewed using pellets which have been stored warm usually have shorter shelf-lives than similar beers brewed using pellets which have been stored chilled. Changes in brewing performance can be minimized by storing hop pellets in sound packs at temperatures below 12 °C. Experience on the commercial scale has shown that whole hops stored at ambient temperature appear to lose up to 21% of their initial bittering value over a period of 18 months.     

THE STORAGE OF PELLETED POWDERS MADE FROM WYE NORTHDOWN HOPS

Pelleted hop powder from seeded Wye Northdown hops of the 1976 crop shows small losses in lead conductance value (LCV) when stored in commercial packs for ca. 12 months after processing. The losses for hop pellets stored at ambient temperature are slightly more rapid than for those stored at 0 to 4°C. Losses in LCV of pellets during storage in the cold or at ambient temperature are substantially less than those of seeded Wye Northdown cone hops stored in pockets under comparable conditions. The hop pellets showed no significant loss in oil content or bittering potential over the storage period. Taste panels were unable to detect differences in flavour when comparisons were made of beers bittered with pellets stored in the cold and at ambient temperature.