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     

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.     

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’.     

ADDITION OF HOP EXTRACTS TO BEER USING LIQUID CARBON DIOXIDE*

A method is described for imparting hop character to beer using extract prepared by treating hops with liquid carbon dioxide. The long contact times required for conventional dry-hopping techniques are avoided, and the use of liquid carbon dioxide as a medium for dispersal of the extract obviates problems due to residual organic solvent or emulsion stabilizers. Beers in which hop character was introduced in this way were found to be similar in flavour to control beers treated with portions of the same hop extract dissolved in ethanol.     

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 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.     

HOP RESIN ANALYSIS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY ON MICROPARTICULATE SILICA

A high-performance liquid chromatographic separation of hop resins on a commercially available microparticulate silica has been developed. The chromatography involves a new mechanism in hop resin separation. A solvent containing di-n-butylammonium acetate is used. The method gives good separation of α-acid, iso-α-acid, β-acid and humulinic acid, and has been used for the quantitative analysis of α-acid and iso-α-acid in hop extracts and isomerised hop extracts.     

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 STABILITY OF HOP OILS IN LIQUID CARBON DIOXIDE EXTRACTS OF HOPS

Portions of extracts obtained by treating three varieties of hops with liquid carbon dioxide were stored in the cold and at ambient temperature for 18 months. During this period the composition of nop oils remained stable, except for the formation of two terpene methyl sulphides in extracts from all three varieties. Ales were ‘dry-hopped’ using portions of the stored extracts and, despite formation of the two sulphur compounds, the resulting hop character was satisfactory in all cases.     

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.     

USE OF EXTRACTS PREPARED WITH LIQUID CARBON DIOXIDE AS A SUBSTITUTE FOR DRY HOPS

Liquid carbon dioxide hop extracts were prepared for use as a substitute for dry hops. Many of these extracts were oil rich fractions containing up to 14% (w/w) hop oil. Extracts of Styrian Golding, Wye Target and Wye Northdown hops were used to treat ales prior to completion of beer processing and subsequent packaging. Emulsion/suspension systems were developed as carriers to facilitate addition of these extracts to beers before the chilling process. A loss of hop character from beer was found to occur during fining and filtration. Beers, treated with the extracts, exhibited natural though varietally distinct dry hop character. Production scale trials using these extracts have been successfully carried out to dry hop a brewery conditioned draught bitter and to match a traditionally dry hopped premium pale ale. The quantities of extract used indicated that economies in hop usage can be achieved when substituting liquid carbon dioxide hop extracts for dry hops.     

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.     

PREPARATION OF OIL RICH HOP EXTRACTS AND THEIR ADDITION TO BEER ON THE PILOT-SCALE USING LIQUID CARBON DIOXIDE*

The introduction of carbon dioxide hop extracts dissolved in liquid carbon dioxide to green beer during transfer from fermentation vessel to cold conditioning tank results in a sound dry hop flavour in the finished product. A system has been developed for injecting a solution of extract into a beer main. Extracts which are rich in hop oils are particularly suitable for imparting hop character to beer and a liquid-liquid countercurrent procedure is described for producing extract fractions containing enhanced levels of essential oil.     

PRODUCTION-SCALE APPARATUS FOR THE INJECTION OF HOP EXTRACTS INTO BEER USING LIQUID CARBON DIOXIDE

Extracts of hops made with liquid carbon dioxide can be substituted for dry hops to introduce the flavour of hop oils into beer. Apparatus has been devised so that such extracts can be redissolved in liquid carbon dioxide and then metered into beer. Successful trials have been carried out on the commercial scale.     

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.     

PILOT PLANT EXTRACTION OF HOPS WITH LIQUID CARBON DIOXIDE AND THE USE OF THESE EXTRACTS IN PILOT AND PRODUCTION SCALE BREWINGa

Several varieties of hop have been satisfactorily extracted using liquid carbon dioxide in a plant operating on the 1 kg scale. The extracts have been used in small scale (0·6 and 16 hl) and production scale (700–800 hl) brewing trials where both ales and lagers were produced. All beers were of sound quality and could not be distinguished organoleptically from controls brewed with either hops or commercial hop extracts.     

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     

Isomerization of hop extract α‐acids

An isomerization process of the α‐acids contained in hop extract (with magnesium oxide, potassium hydroxide and magnesium peroxide as catalysts at ambient temperature) was carried out. The influence of two factors (the amount of applied catalyst and the isomerization time) was studied. Ultra‐high performance liquid chromatography was used for the evaluation of the isomerization process. The best results were obtained with magnesium oxide. In this case, the influence of the operating variables on the isomerization process and optimal process parameters were determined using statistical methods. The isomerization method described above could be carried out with high efficiency without heating and could be easily adopted and applied on an industrial scale. Copyright © 2016 The Institute of Brewing & Distilling     

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.