The Significance of Iso‐α‐Acids for Beer Quality Cambridge Prize Paper

The iso‐α‐acids and their chemically‐modified variants play a disproportionately large role in the final quality of beer. Here, fundamental aspects of two of these quality issues — foam and bitterness — are discussed. A common feature of both issues is the dependence on the hydrophobic character of the hop compounds on both bitterness potency and ability to stabilise foams. Thus the isocohumulones appear significantly less bitter than the other, more hydrophobic hop compounds. Also apparent were the differences in bitterness between the cis‐ and the trans‐isomers, with the former being the more potent. Also described are the differences in the partitioning of the cis‐ and trans‐iso‐α‐acids into beer foam. The trans‐isomers are enriched in foam relative to their cis‐counterparts and may account for the observed enrichment of cis‐isomers in the final beer relative to the common ratios observed upstream.     

Liquid Chromatography‐Diode Array and Electrospray High‐Accuracy Mass Spectrometry of Iso‐α‐Acids in DCHA‐Iso Standard and Beer

Excellent liquid chromatographic (LC) separation of cis/trans stereoisomers of iso‐α‐acids has been achieved with reversed‐phase sorbent XTerra MS C18. An isocratic alkaline mobile phase, consisting of a mixture of 5 mmol l^?1 ammonium acetate (pH 8)‐acetonitrile‐methanol, 62:21:17 (v/v/v), was used. In the DCHA‐Iso international calibration standard trans‐isoposthumulone was identified combining photo diode array (PDA) spectra and electrospray high‐accuracy mass spectrometric (MS) data. Moreover, the molecular mass of two degradation products resulting from the in‐solution storage of the DCHA‐Iso standard was determined. The presence of trans and cis isomers of isoposthumulone, isocohumulone, isoadhumulone and iso‐n‐humulone in beer samples was confirmed. The trans isomers of iso‐α‐acids showed characteristic and reproducibly slightly different ultraviolet absorbance spectra with respect to cis isomers.     

Relationships of Sensory Bitterness in Lager Beers to Iso‐α‐Acid Contents

Iso‐α‐acids and their chemically modified variants play a large role in evoking the bitter sensory attributes of lager character, but individual consumers may vary in their perception of bitterness. Sixteen lagers were scored in rank‐rating for bitterness by 14 trained assessors and the concentrations of the six bitter components in these beers were determined by high performance liquid chromatography. Relationships between bitterness intensity and the bitter components were modelled well using partial least square regression with a correlation value of 0.92. When 8 assessors carried out time‐intensity scoring of bitterness, profiles for single products were very different. However, single assessor profiles for multiple products showed qualitative similarities but quantitative differences. That individual assessors perceived bitter characters differently in relation to time has implications for new product development.     

Enhanced Quantitative Extraction and HPLC Determination of Hop and Beer Bitter Acids

A pleasant and consistent bitterness is an essential flavour attribute of beer. Hop‐derived iso‐α‐acids are largely responsible for beer bitterness and accurate determination of these primary flavour compounds is very important in relation to quality control. The most widely used way to determine beer bitterness is based on spectrophotometry, measuring the absorbance of an iso‐octane extract of acidified beer. However, this approach is far from specific as it measures all of the extracted compounds, including non‐bittering principles. For that particular reason, High Performance Liquid Chromatography (HPLC) is increasingly applied for the quantitative determination of hop‐derived iso‐α‐acids and, if present, reduced iso‐α‐acids. However, to obtain accurate data on beer bitterness profiles, both quantitative sample preparation and state‐of‐the‐art HPLC are essential. In this paper, several extraction procedures based on solid phase extraction (SPE) and liquid‐liquid extraction (LLE), respectively, were evaluated and an optimised extraction methodology using H₃PO₄ for sample acidification prior to extraction is presented. The proposed extraction/HPLC methodology allows for the quantitative recovery and analysis of hop‐derived beer bitterness.     

Influence of the Hopping Technology on the Storage‐induced Appearance of Staling Aldehydes in Beer

In this paper, the involvement of iso‐α‐acids in the appearance of stale flavour in beer during storage was examined. Flavour instability upon storage, by far the most important quality problem of beer, may be caused by a multitude of reactions, in particular, the degradation of trans‐iso‐α‐acids is pivotal. In order to gain improved understanding of the beer ageing process, the behaviour of the stereoisomers of the bitter acids was studied for the first time in pilot beers at particularly high and low proportions of the trans‐iso‐α‐acids concentration relative to the cis‐iso‐α‐acids concentration. Therefore, pure trans‐ respectively cis‐isomers were required and obtained on a pilot scale by the separation of trans‐iso‐α‐acids as β‐cyclodextrin inclusion complexes from a commercial isomerised hop extract, permitting the quantitative dosage to beer in mg/L amounts. The relationship between the storage‐induced degradation of these iso‐α‐acids in authentic beer samples and the increase in staling aldehydes, especially 2‐methylpropanal, 2‐methylbutanal and 3‐methylbutanal, is the subject of further investigations. A remarkable increase in the relative concentration of cis‐iso‐α‐acids was observed in the beers bittered with purified trans‐iso‐α‐acids, which may be ascribed to partial conversion, i.e., reverse isomerisation, of trans‐iso‐α‐acids via α‐acids into cis‐iso‐α‐acids. In spite of the trans‐specific degradation observed during ageing, the decomposition of these bitter acids leading to volatile carbonyl compounds is of minor importance. Aldehyde formation as a function of forced ageing was irrespective of the mode of bittering, emphasising that malt quality and the brewing process itself are probably the most important factors regarding the flavour instability of beer.     

Degradation of Iso‐α‐Acids During Wort Boiling

A detailed study on the degradation of iso‐α‐acids was conducted. Because of the complexity of the wort matrix and interfering interactions during real wort boiling, the investigation of degradation kinetics was performed in an aqueous solution. Degradation was investigated as a function of time (0–90 min), temperature (80–110°C), pH value (4–7), original gravity (10–18°P) and ion content of the water (0–500 ppm Ca²⁺ and Mg²⁺). After 90 min of boiling, over 20% of the dosed iso‐α‐acids could no longer be detected. A strong dependence of degradation could be shown due to high temperature, low pH, high original gravity and a high Mg²⁺ content. The cis:trans ratio and co‐iso‐α‐acid content did not change significantly. Losses of isohumulones could be lowered by reducing the temperature and original gravity, as well as by heightening the pH value. High amounts of Ca²⁺ and Mg²⁺ salts also led to an increase in degradation products. Solutions to decrease degradation and thereby possible improvements in sensory bitter quality are discussed.     

Relationship of iso‐α‐acid content and endogenous antioxidative potential during storage of lager beer

One of the critical issues regarding the quality of beer is the change in its chemical composition that occurs during storage. Decomposition of iso‐α‐acids results in an undesirable decrease in bitterness as well as a deterioration in the sensory profile of the beer. These changes are caused by the susceptibility of iso‐α‐acids to degradation owing to the influence of reactive oxygen species and light. The aim of this study was to investigate the influence of storage conditions (temperature, light) on the degradation of iso‐α‐acids during aging, with the main focus on monitoring the relationship between the turnover of iso‐α‐acids, the sulphur dioxide content and the antioxidative potential of stored beer as measured by electron spin resonance spectroscopy. In agreement with previous investigations, a significant decrease in the content of bitter compounds (up to 18 % relative to the original level, depending on storage conditions) was observed. A significant decrease in the antioxidant potential of beer was recorded simultaneously and the data confirmed a strong correlation between these parameters. The decline in beer bitterness could become a marker for estimating oxidative damage during storage. Copyright © 2014 The Institute of Brewing & Distilling     

An improved HPLC method for single‐run analysis of the spectrum of hop bittering compounds usually encountered in beers

A single‐run reverse phase‐high performance liquid chromatography method for the quantification of humulinones, α‐acids, iso‐α‐acids and reduced iso‐α‐acids (where present) in commercial beer samples is presented. The method utilizes a binary solvent system consisting of (A) 1% v/v acetic acid and (B) 0.1% v/v orthophosphoric acid in acetonitrile. Separation was achieved on a Purospher® star RP‐18 column (250 × 46 mm, 3 µm) with a flow rate of 0.5 mL/min. The compounds of interest eluted within 32 min. The method was fully validated according to International Conference on Harmonization guidelines and subsequently applied to monitor degradation of hop acids in a storage trial where four lager beers were aged at 28 and 38 °C for 70 and 60 days, respectively. Results confirmed the widely reported degradation through storage of trans‐iso‐α‐acids whilst demonstrating that the HLPC method was sufficiently sensitive to monitor and model this degradation. One beer exhibited a significantly lower (P < 0.05) rate of trans‐iso‐α‐acid degradation than the other conventionally hopped beers in the study, which might have been linked to its higher pH (4.71 vs 4.36). The relative stability of reduced iso‐α‐acids during ageing was also confirmed.     

Inhibition of Aldose Reductase Activity by Extracts from Hops

The inhibitory effect of beer on aldose reductase was investigated. Components present in beer strongly inhibited aldose reductase. Inhibition activity was observed primarily in the methanol‐soluble fraction of the beer and active components were identified to be of hop origin. Further, a component was identified as iso‐α‐acids, which are well known as main bitter components of beer. The inhibition rate of iso‐α‐acids was similar to quercetin, which is known to be a strong inhibitor of aldose reductase activity.     

一种检测啤酒老化的新参数

通过HPLC，研究了在啤酒贮存过程中，酒花苦味物质的变化。观察到异律草酮浓度在此过程中变化显著不同。反式异律草酮的浓度在啤酒贮存过程中逐渐减少。而顺式异律草酮的浓度没有变化。根据色谱峰面积的大小。得到反式-顺式异棒草酮的比例，该比例对于新鲜啤酒，虽然啤酒的种类不同，但总是相差不大。但对于陈酒来说，它是逐渐减少的，其减少的程度与啤酒的种类有关。同时证实了反式-顺式异律草酮的比例与啤酒陈腐味强度有相关性。反式-顺式异律草酮的比例反映了啤酒变质过程中的氧化程度。     

Sensory and Analytical Characterisation of Reduced,Isomerised Hop Extracts and Their Influence and Use in Beer

The use of reduced, isomerised hop extracts became very popular during the last decade in terms of achieving both better foam and light stability. Today the most common products are those with tetra‐hydro‐iso‐α‐acids (THIA) or rho‐iso‐α‐acids (RHIA). There is still not clarity concerning the taste properties of these products in comparison with iso‐α‐acid Bitter Units (BU). The experiments conducted showed that the bitter conversion factors determined in previous work (1.6 for THIA and 0.7 for RHIA) represented only perceived bitterness when presented in tap water. In the test beers a bitter conversion factor of 1.0–1.1 for THIA was determined and the factor of 0.7 for RHIA in beer was confirmed. Photometric methods led to considerable deviations when used for the determination of THIA and RHIA, whereas the HPLC method, in combination with either Solid Phase Extraction (SPE) or Liquid Liquid Extraction (LLE), led to very good results. RHIA was found to have a foam stabilising effect and it was also shown that the foam stabilising effect of THIA in unhopped beer was higher than in hopped beer. The quality of bitterness decreased with an increase of THIA and RHIA. A dependency on the alcohol content in regard to the bitter intensity was ascertained.     

Content of Individual Phenolic Acids in Worts and Beers and their Possible Contribution to the Antiradical Activity of Beer

Phenolic acids are widely distributed in foods and raw materials. They are easily absorbed by humans due to their simplicity. Once they enter the blood plasma, they act as antioxidants. Beer can be a rich source of phenolic acids in the diet. The aim of this study was to determine the concentrations of phenolic acids in two experimental worts and beers as well as in nine market beers (using HPLC‐UV). An examination of the total antiradical activities of phenolic acids with in vitro model systems (using ABTS and DPPH free radicals), at the concentrations comparable to those detected in beers, was performed. Only low fractions of the main phenolic acids present in barley malt (ferulic, vanillic and p‐coumaric acid) were detected in the experimental worts. Moreover, the concentrations of phenolic acids significantly decreased until the last steps of beer production. The main beer phenolic acids (vanillic and ferulic acid) exerted a lower share of total antiradical activity against both free radicals (calculated as the sum of the individual activities of all acids detected in beer) than the minor phenolic acids (caffeic, chlorogenic, o‐coumaric, sinapic or syringic acid). The synergies, between individual phenolic acids in pairs, were also studied with in vitro model solutions using free radicals. The total antiradical activity of the compounds studied in pairs, was at the most as high as the sum of the antiradical activity of the individual phenolic acids, but in most cases it was considerably lower (i.e. no synergy was detected).     

A critical review of methodologies used in determination of relative bio‐availability ratio of RRR‐α‐tocopheryl acetate and all‐rac‐α‐tocopheryl acetate

Bio‐availability of different α‐tocopherol forms in livestock animals is measured by the increase in plasma or tissue concentrations of α‐tocopherol after oral administration. It is generally accepted that RRR‐α‐tocopheryl acetate (natural source vitamin E derived from vegetable oil) has a higher bio‐availability compared to all‐rac‐α‐tocopheryl acetate (synthetic vitamin E, i.e. α‐tocopherol produced by chemical synthesis). However, different bio‐availability ratios have been reported in the literature. The major reason for conflicting results in literature studies was the inability to separate the proportion of α‐tocopherol originating from test materials, from the proportion of α‐tocopherol originating from basal dietary ingredients and pre‐feeding. This causes significant variability. For bio‐availability determination, a baseline or control treatment is essential. The estimation of bio‐availability without correction for basal vitamin E status will lead to incorrect interpretation of the results. When using proper methodologies, it is possible to correct for the impact of α‐tocopherol intake from basal ingredients and α‐tocopherol originating from pre‐feeding, therefore yielding results reflecting the true relative bio‐availability of different α‐tocopherol substances. When reviewing literature data a critical evaluation of the method used in determination of relative bio‐availability is recommended. Copyright © 2010 Society of Chemical Industry     

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     

ANALYTICAL AND BREWING SIGNIFICANCE OF A TRICYCLIC OXIDATION PRODUCT OF HUMULONE (TRICYCLODEHYDROISOHUMULONE)

Tricyclodehydroisohumulone (TCD) is an oxidation product of humulone formed during storage of hops. It is about 70% as bitter as isohumulone, and considerably more stable, both chemically and in the brewing process. When used to bitter beer by addition to the copper the overall utilization of TCD is around 75% and the quality of bitterness is excellent. TCD is a normal constituent of beer brewed from old hops, and accounts for a part of the non iso-α-acid bitterness of such beers. TCD mimics iso-α-acids in some standard analytical procedures for bitterness determination, and interferes in others; the analytical significance is discussed.     

Characterization of the Supermolecular Structure of Polydatin/6‐O‐α‐Maltosyl‐β‐cyclodextrin Inclusion Complex

Polydatin is the main bioactive ingredient in many medicinal plants, such as Hu‐zhang (Polygonum cuspidatum), with many bioactivities. However, its poor aqueous solubility restricts its application in functional food. In this work, 6‐O‐α‐Maltosyl‐β‐cyclodextrin (Malt‐β‐CD), a new kind of β‐CD derivative was used to enhance the aqueous solubility and stability of polydatin by forming the inclusion complex. The phase solubility study showed that polydatin and Malt‐β‐CD could form the complex with the stoichiometric ratio of 1:1. The supermolecular structure of the polydatin/Malt‐β‐CD complex was characterized by ultraviolet–visible spectroscopy (UV), Fourier transform infrared spectroscopy (FT‐IR), X‐ray diffractometry (XRD), thermogravimetric/differential scanning calorimetry (TG/DSC), and proton nuclear magnetic resonance (¹H‐NMR) spectroscopy. The changes of the characteristic spectral and thermal properties of polydatin suggested that polydatin could entrap inside the cavity of Malt‐β‐CD. Furthermore, to reasonably understand the complexation mode, the supermolecular structure of polydatin/Malt‐β‐CD inclusion complex was postulated by a molecular docking method based on Autodock 4.2.3. It was clearly observed that the ring B of polydatin oriented toward the narrow rim of Malt‐β‐CD with ring A and glucosyl group practically exposed to the wide rim by hydrogen bonding, which was in a good agreement with the spectral data.     

Isolation of individual hop iso-α-acids stereoisomers by β-cyclodextrin

Individual iso-α-acids that are responsible for the bitter taste of beer need to be isolated because these acids are required as reference standards in quantitative analysis and when studying the parameters which effect the quality of beer. However, these pure compounds are very expensive, due to inefficient isolation methods. In this study a new isolation method has been developed, in order to reduce the isolation cost. β-Cyclodextrin has been used for the isolation of trans- and cis-iso-α-acids. The separation from the mixture of stereoisomers was achieved by complexation, using ethanol:water (1:2, v/v) as a solvent at a temperature of 50 °C for 30 min. The molar ratio of iso-α-acids sample to β-cyclodextrin for complexation was 1:1. Precipitation time varied between 9 h and 2 days, depending on the iso-α-acid. Release of the guest from the cyclodextrin complex was successfully accomplished by elution with methanol.     

Assessment of changes in hop resins and polyphenols during long‐term storage

Changes in the content and composition of hop secondary metabolites during storage are reflected in beer quality and in the economics of beer production. A 12‐month storage experiment with T90 pellets of four hop varieties showed different dynamics of hop aging in relation to both storage conditions and hop variety. Negligible effects on the α‐ and β‐acids were detected during storage without air access at +2°C. Storage at +20°C resulted in a final loss of 20–25% α‐acids, but the content of β‐acids did not change significantly. Large decreases in α‐acids (64–88%) and in β‐acids (51–83%) were found in hops stored with access to air at +20°C. The rate of decline accelerated markedly after 6 months of storage. In terms of hop resin changes, Premiant and Sládek were the most and the least stable varieties, respectively. After 12 months, the content of the total polyphenols and flavonoids decreased by 30–40% and by 20–30%, respectively, irrespective of storage conditions. The rate of decline accelerated strongly after 6 months. The DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) antiradical potential decrease was significant only in hops stored under aerobic conditions. The depletion was 9–25% after 1 year; Saaz was the most stable variety. Copyright © 2012 The Institute of Brewing & Distilling     

Inhibitory potential of phenylpropanoid glycosides from Ligustrum purpurascens Kudingcha against α‐glucosidase and α‐amylase in vitro

The leaves of Ligustrum purpurascens are used in a Chinese traditional tea called small‐leaved kudingcha, which is rich in phenylpropanoid glycosides (PPGs) and has many beneficial properties. Two critical exoacting glycoside hydrolase enzymes (glucosidases) involved in carbohydrate digestion are α‐glucosidase and α‐amylase. We investigated the properties of PPGs from L. purpurascens for inhibiting α‐amylase and α‐glucosidase activity in vitro and found IC₅₀ values of 1.02 and 0.73 mg mL^?1, respectively. The patterns of inhibiting both α‐amylase and α‐glucosidase were mixed‐inhibition type. Multispectroscopy and molecular docking studies indicated that the interaction between PPGs and α‐amylase and α‐glucosidase altered the conformation of enzymes, with binding at the site close to the active site of enzymes resulting in changed enzyme activity. Our studies may help in the further health use of small‐leaved kudingcha.