Alterations of the Profiles of Iso‐α‐Acids During Beer Ageing,Marked Instability of Trans‐Iso‐α‐Acids and Implications for Beer Bitterness Consistency in Relation to Tetrahydroiso‐α‐Acids

Age‐induced decomposition of iso‐α‐acids, the main bittering principles of beer, determines the consistency of the beer bitter taste. In this study, the profiles of iso‐α‐acids in selected high‐quality top‐fermented and lager beers were monitored by quantitative high‐performance liquid chromatography at various time intervals during ageing. The degradation of the iso‐α‐acids as a function of time is represented by the ratio, in percentage, of the sum of the concentrations of trans‐isocohumulone and trans‐isohumulone to the sum of the concentrations of cis‐isocohumulone and cis‐isohumulone. This parameter is relevant with respect to the evaluation of bitterness deterioration in aged beers. Trans‐iso‐α‐acids having a shelf half‐life of less than one year proved to be significantly less stable than cis‐iso‐α‐acids, but it appears feasible to counteract degradation if a suitable beer matrix is available. The fate of the trans‐iso‐α‐acids in particular adversely affects beer bitterness consistency. In addition to using hop products containing low amounts of trans‐iso‐α‐acids, brewers may profit of the remarkable stability of tetrahydroiso‐α‐acids, even on prolonged storage, for the production of consistently bitter beers.     

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.     

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.     

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.     

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.     

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.     

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     

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.     

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     

Interactions between polypeptides derived from barley and other beer components in model foam systems

The impact of a range of variables on the foaming properties of hordein‐ and albumin‐derived fractions from barley has been investigated. When foamed using nitrogen, the hordein‐derived polypeptides produce the most stable foams. However, when carbon dioxide was the foaming agent, the previously reported observations were repeated, namely that proteolysis weakens the foaming capabilities of albumin, whereas partial hydrolysis enhances the stability of foams derived from hordein. At lower levels of bitterness (iso‐α‐acid) addition there is a reduction in foam stability of albumin‐derived polypeptides, although hordein‐derived polypeptides can withstand this destabilising effect. Higher bitterness levels in both instances lead to a stabilisation of foam. It is likely that this involves ionic interactions between the acid anion (pK of iso‐α‐acids is ca 3.1) and divalent cations, because higher pHs (in the range 3.8–4.6) and the addition of zinc lead to an enhancement of foam stability, particularly of the albumin‐derived fractions. The foaming of the latter, however, is significantly impaired by the presence of ethanol, whereas hordein‐based foams are enhanced by increasing concentrations of ethanol. Copyright © 2003 Society of Chemical Industry     

Humulus lupulus – a story that begs to be told. A review

The hop cones of the female plant of the common hop species Humulus lupulus L. are grown almost exclusively for the brewing industry. Only the cones of the female plants are able to secrete the fine yellow resinous powder (i.e. lupulin glands). It is in these lupulin glands that the main brewing principles of hops, the resins and essential oils, are synthesized and accumulated. Hops are of interest to the brewer since they impart the typical bitter taste and aroma to beer and are responsible for the perceived hop character. In addition to the comfortable bitterness and the refreshing hoppy aroma delivered by hops, the hop acids also contribute to the overall microbial stability of beer. Another benefit of the hop resins is that they help enhance and stabilize beer foam and promote foam lacing. In an attempt to understand these contributions, the very complex nature of the chemical composition of hops is reviewed. First, a general overview of the hop chemistry and nomenclature is presented. Then, the different hop resins found in the lupulin glands of the hop cones are discussed in detail. The major hop bitter acids (α‐ and β‐acids) and the latest findings on the absolute configuration of the cis and trans iso‐α‐acids are discussed. Special attention is given to the hard resins; the known δ‐resin is reviewed and the ε‐resin is introduced. Recent data on the bittering potential and the antimicrobial properties of both hard resin fractions are disclosed. Attention is also given to the numerous essential oil constituents as well as their contributions to beer aroma. In addition to the aroma contribution of the well‐known essential oil compounds, a number of recently identified sulfur compounds and their impact on beer aroma are reviewed. The hop polyphenols and their potential health benefits are also addressed. Subsequently, the importance of hops in brewing is examined and the contributions of hops to beer quality are explained. Finally, the beer and hop market of the last century, as well as the new trends in brewing, are discussed in detail. Hop research is an ever growing field of central importance to the brewing industry, even in areas that are not traditionally associated with hops and brewing. This article attempts to give a general overview of the different areas of hop research while assessing the latest advances in hop science and their impact on brewing. Copyright © 2014 The Institute of Brewing & Distilling     

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.     

The foaming properties of proteins isolated from barley

Albumin and hordein protein fractions have been isolated from barley by extraction with salt and aqueous ethanol respectively. Both produce stable foams as assessed by a shaking procedure, and in each case foaming was enhanced by denaturation of the proteins. Foam power was increased rather more for albumin than for hordein, especially by heat, and this was accompanied by an increase in hydrophobic character as assessed by the fluorochrome 1‐anilino‐8‐napthalenesulphonic acid. Iso‐α‐acids and reduced iso‐α‐acids increased the foam stability of both albumin and hordein, with there being a proportionately greater increase for the undenatured proteins. Albumin is resistant to hydrolysis by the cysteine proteinases ficin and papain but is hydrolysed by the serine proteinase trypsin. In the latter instance, hydrolysis is accompanied by a loss of foam stability. By contrast, hordein is resistant to digestion by trypsin but is susceptible to the action of ficin and papain. In these instances, limited proteolysis leads to a substantial enhancement of foam stability from hordein, though prolonged proteolysis is detrimental. Proteinase A from yeast hydrolyses both protein classes and lowers their ability to give stable foams. It is concluded that polypeptides derived from both albumin and hordein could make a contribution to the foam potential of beer. © 2002 Society of Chemical Industry     

The Effects of Hop‐α‐Acids and Proline‐Specific Endoprotease (PSEP) Treatments on the Foam Quality of Beer

Influences on foam stability and cling were compared by brewing trials investigating beer hopping rate, hopping type and modification of beer protein composition by the inclusion of a proline specific protease (PSEP). The comparison of the NIBEM, Rudin and lacing foam assessment methods with the level of hopping demonstrated the superiority of hydrogenated hop α‐acids with respect to foam stability and particularly lacing. In addition, the NIBEM and Rudin foam analysis tests appear to respond relatively similarly with respect to hopping rate and hop type, with the NIBEM being somewhat more responsive in terms of foam stability measurments. The PSEP trials suggested that protein composition may only have a subtle effect on foam stability. Although more specific to haze active proteins, PSEP treatment in the small and pilot scale trials generally, but not always, resulted in a minor reduction in foam stability. This effect was not observed in 20 hL pilot and industrial scale beer productions. It was verified that both NIBEM and Rudin were positively influenced by increased levels of foam positive proteins. Although both foam tests were responsive to hopping rate and type, it is suggested that the Rudin foam test is somewhat biased towards foam positive proteins, particularly albuminous foam positive proteins (LTP1 and protein Z4), while in comparison the NIBEM foam test appears somewhat biased towards hordein foam positive proteins.     

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 effect of hopping regime,cultivar and β‐glucosidase activity on monoterpene alcohol concentrations in wort and beer

Previous studies show that the complexity of hop aroma in beer can be partly attributed to the hydrolysis of glycosidically bound monoterpene alcohols extracted from hops during the brewing process to release volatile aglycones. However, fundamental studies that examine the extraction of glycosides during brewing and their subsequent hydrolysis by yeast have not been performed. Furthermore, extraction of other hop‐derived compounds into beer shows a strong dependency on the hop cultivar being used and the point at which it is added. This study focused on the extent of glycoside extraction owing to hopping regime and cultivar, and their hydrolysis by yeast β‐glucosidase activity. Glycoside concentrations of wort made with three different hopping regimes and three cultivars were measured by the difference in volatile aglycone concentrations between samples treated with purified β‐glucosidase and untreated samples. Aglycone concentrations were measured by solid‐phase microextraction gas chromatography–mass spectrometry. Additionally, β‐glucosidase activities for 80 different yeast strains and their effect on aglycone concentration in wort were determined. Results showed that yeast have a wide range of abilities to hydrolyse glycosides with a maximum hydrolysis occurring after 3 days of fermentation regardless of yeast activity. Although it was shown that yeast are capable of glycoside hydrolysis, glycoside concentrations in wort are low and make small contributions to hop aroma. These results help explain the extent to which different brewing yeasts and hopping regimes contribute to hoppy beer aroma through the hydrolysis of non‐volatile hop‐derived compounds. Copyright © 2017 The Institute of Brewing & Distilling     

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.     

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     

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