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 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     

ABEO-ISO-α-ACIDS

During boiling, substantial amounts of hop α-acids are transformed into oxidation products containing the same cyclopentane nucleus as the iso-α-acids: these oxidation products, which have been called abeo-iso-α-acids I, II and III, are separable by countercurrent distribution. Lager beers contained between 88 and 160 mg. of abeo-iso-α-acids per litre. Although the abeo-iso-α-acids are almost devoid of bitterness, they have strong foam-producing properties.     

啤酒花浸膏的异构化工艺研究

α-酸是啤酒花软树脂中的一个重要组分,在啤酒酿造过程中会生成苦味更强、水溶性更好的异α-酸,是啤酒苦味的主要来源,也是提供啤酒防腐能力的主要成分。以正交试验为基础,直接以啤酒花浸膏为原料,不分离浸膏中的啤酒花油和β-酸等软树脂类,考察了溶剂、反应温度、反应体系的pH值、催化剂镁盐的使用量、以及反应时间等多个因素对异构化反应的影响。试验结果表明,尽管啤酒花浸膏的黏度较高,但无需添加溶剂稀释即可完成反应;在保证浸膏中α-酸完全异构,而啤酒花风味和β-酸基本不发生变化的情况下,镁盐与α-酸的摩尔比为2．0：1、反应温度为100℃、反应时间为60min、pH值为11．0作为异构化反应的最佳条件,并在此条件下进行了放大验证,浸膏中的异α-酸收率超过96％。     

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     

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.     

ANALYTICAL EVALUATION OF α-ACIDS IN HOP EXTRACTS AND IN HOPS*

The results of new investigations on hop extract analysis are presented. The main findings are:

• 1 The specific extinction (257) of α-acids at 276 nm in iso-octane used at present in CCD and paper strip analyses is 3·5% too high: analytical results have therefore been too low. The new extinction value to be used is 248.
• 2 The result of paper strip analysis depends very much on the amount of α-acids placed on the strip. The three-point paper strip method can only partly correct this. In general, figures with paper strip analysis have a tendency to be too high.
• 3 Conductometric titration of α-acids produces a figure about 2 to 10% too low. This can be partly corrected by adding 20% dimethylsulphoxide or dimethylformamide to the titration medium.
• 4 Conductometric analysis of hop extracts with removal of “iso-α-acids like material” before titration is possible by a very simple procedure (pH 7 buffer extraction).

New photometric and conductometric procedures based on these results give figures which are in agreement for something which must be practically true α-acids.     

Hop bitter acids inhibit carbohydrate metabolism,enhance biogenic amine metabolism and alter L-malic acid,glutamic acid and arginine metabolism of Lactobacillus brevis 49

Beer contains only limited amounts of readily fermentable carbohydrates and amino acids. Beer spoilage lactic acid bacteria have to come up with metabolic strategies in order to deal with selective nutrient content. The research was performed to investigate the influence of iso-α-acids on the metabolism of organic acids, biogenic amines (BAs), off-flavour compounds, carbohydrates and amino acids of Lactobacillus brevis 49. Only glyoxylic acid and ethyl formate in de man, rogosa, sharpe broth was consumed, and multiple organic acids, BAs and off-flavour compounds were produced. By supplementing a series of concentrations of hop iso-α-acids, consumption of L-malic acid, glutamic acid and arginine and generation of BAs were found benefit for bacteria to develop hop-resistance. In the metabolism of carbohydrates, glucose was preferred over maltose and maltriose, and hops significantly inhibited the utilisation of carbohydrates. The results provide comprehensive information of metabolites of L. brevis 49 under various concentrations of hop stress.     

HUMULINIC ACID IN ISOMERIZED HOP EXTRACTS

Humulinic acid, which is not bitter, behaves similarly to iso-α-acids in some analytical estimations of beer bitterness. The humulinic acid content of a number of isomerized hop extracts was estimated using thin layer chromatography.     

OXIDATION PRODUCTS OF ISOHUMULONES

A complex mixture of oxidation products is formed from iso-α-acids during wort boiling. These substances are already present in hops and persist to beer. Seven major constituents of such a mixture, obtained from pure humulone, were isolated by countercurrent distribution and their structures were elucidated, mainly by mass spectroscopy. The bitterness of the new beer substances was evaluated against trans isohumulone as a reference standard. These oxidation products of iso-α-acids are not extracted with iso-octane and they therefore escape the usual beer bitterness assay.     

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.     

Structure determination and sensory evaluation of novel bitter compounds formed from β-acids of hop (Humulus lupulus L.) upon wort boiling

In order to screen for the bitter compounds generated from hop-derived precursors during the wort boiling process, an ethanolic hop extract was fractionated; the fractions obtained were thermally treated under model wort boiling conditions and, then, sensorially evaluated for their bitterness. Besides the isomerisation of the α-acids into the intensely bitter iso-α-acids, the bitterness of the fraction containing the β-acids was also found to be enhanced after wort boiling. To gain first insights into the β-acid-derived bitter compounds, the β-acid colupulone was isolated, thermally treated under wort boiling conditions and, then, investigated for bitter tasting degradation products by means of a taste dilution analysis (TDA). Besides the cohulupone, five previously unreported bitter-tasting colupulone degradation products, all of which exhibited a lingering, β-acid-like bitter taste with low recognition thresholds between 37.9 and 90.3 μmol/l, were isolated and their structures determined as two tricyclocolupone epimers, two dehydrotricyclocolupone epimers, and nortricyclocolupone, respectively, by means of LC–TOF–MS and 1D/2D-NMR spectroscopy.     

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.     

EFFECT OF STORAGE ON HOP EXTRACTS

Representative commercial hop extracts suffered no appreciable loss either of α- or β-acids or of bittering value when kept for 2 to 21 months in closed containers. Though there was loss of α- and β-acids on prolonged exposure of the extracts to air, it did not lead to a corresponding loss of bittering value. Of a range of commercial isomerized extracts, most proved satsifactorily stable on storage. However, when large losses of iso-α-acids did occur they were accompanied by corresponding reductions in bittering value. With reference to bittering compounds, there appeared to be no significant differences in the storage stability of beers bittered using hops or extracts of various types.     

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。     

100 YEARS OF HOP CHEMISTRY AND ITS RELEVANCE TO BREWING

Chemical research has shown that the α-acids are the main quality factors of hops and that the bitter taste of hopped beer derives from the iso-α-acids. This has provided the basis for improvements in hop breeding, conditioning, extraction, use, analysis etc. Structural chemical development of hop constituents was completed about ten years ago. At the present moment the accent of chemical research is on analytical aspects, mainly using High Performance Liquid Chromatography. From the standpoint of the brewery the situation of chemical research is not completely satisfactory, since a clear understanding of the more subtle chemical quality factors of hops and their impact on beer quality is still lacking. Hops are much more important for beer than is generally estimated, even in brewery circles.     

HPLC法检测麦汁及啤酒中的异α-酸

建立了HPLC法测定麦汁和啤酒的异α-酸，在25分钟内同时分离检测3种异α-酸（异合律草酮、异棒草酮和异加律草酮）、2种α-酸（合律草酮、律草酮＋加律草酮）和2种β-酸（合蛇麻酮、蛇麻酮＋加蛇麻酮），方法快速、简单，重复性好。为进一步控制和研究啤酒苦味提供了数据支持。     

采用高效液相色谱法鉴别啤酒中异构化α-酸的种类

采用高效液相色谱法(HPLC)分析异构化α-酸,给出同一色谱条件下异α-酸、二氢、四氢和六氢异α-酸的HPLC谱图和各组分的紫外光谱。用该方法分析未知啤酒样品,将其与标准品进行对照,从而鉴别出样品中异构化α-酸的种类。     

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 .     

LACTIC ACID PRODUCTION IN SWEET WORTS

The production of D-(-)-lactic acid in sweet wort was due to growth of Lactobacillus delbrueckii whereas L-(+)-lactic acid was produced by Bacillus coagulans. Incubation of sweet worts at 45°C favoured the production of D-(-)-lactic acid whereas L-(+)-lactic acid was produced in greatest amounts at 55°C. Low concentrations of hop iso-α-acids (4–12 ppm) inhibited the growth of both B. coagulans and L. delbrueckii at 55 °C.