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 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.     

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     

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’.     

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.     

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.     

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.     

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.     

SOLUBILITY OF HOP α- AND β-ACIDS IN LIQUID CARBON DIOXIDE*: ADDENDUM: SOLUBILITY OF PESTICIDES IN LIQUID CARBON DIOXIDE

A procedure is described for determining the solubility of hop α- and β-acids in liquid carbon dioxide. Results have shown that the optimum temperature range for the extraction of hops with liquid carbon dioxide is +5 to +10°C. A number of pesticides used by hop growers are appreciably soluble in liquid carbon dioxide.     

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.     

Phenolic diterpene and α‐tocopherol contents in leaf extracts of 60 Salvia species

BACKGROUND: Carnosic acid and carnosol, the two major phenolic diterpenes present in rosemary and sage extracts, have received attention in food science and biomedicine because of their potent antioxidant properties. In plants, these compounds have been identified as being present in some species of the family Lamiaceae, but there is still little information about their distribution within the plant kingdom. The aim of the present study was to gain a better understanding of the occurrence of these compounds within leaf extracts of the genus Salvia and also to examine to what extent leaf senescence may influence their accumulation in relation to that of α‐tocopherol (vitamin E). RESULTS: Methanolic leaf extracts of 60 species of the genus Salvia were tested for the presence of carnosic acid and carnosol. These two diterpenes were detected in 48 and 27 species respectively. In contrast, α‐tocopherol was present in all species examined. Leaf senescence in Salvia officinalis resulted in α‐tocopherol increases by up to 5.5‐fold, while carnosic acid and carnosol increased by up to 18 and 290% respectively. Isorosmanol, an oxidation product of carnosic acid, increased by up to 2.1‐fold in senescing leaves. CONCLUSION: It was found that, while α‐tocopherol is ubiquitous in the genus Salvia, phenolic diterpenes are present in several but not all species. It was also shown that leaf senescence may increase the yield of both phenolic diterpenes and α‐tocopherol in sage extracts. Copyright © 2008 Society of Chemical Industry     

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     

FATTY ACIDS IN ISOMERIZED HOP EXTRACTS AND GUSHING

A procedure is described for the analysis of fatty acids (C₁₂-C₁₈ individually and C₁₈₊ as a group) in hops, hop extracts and isomerized hop extracts, and analytical results are quoted for twenty-one different samples of hops or hop products. Isomerized extracts varied widely in their content of fatty acids and isomerization and processing of hops appeared to eliminate some fatty acids selectively so that isomerized extracts were enriched in palmitic acid, linoleic acid and linolenic acid. Some hop extracts had a surprisingly high content of lauric acid. The analytical results are discussed in relation to gushing.     

TRACE VOLATILE CONSTITUENTS OF BEER. HOP SEED OIL

The fatty acid profile of hop seed oil has been examined and shown to be very similar to the fatty acid profile of the lipid fraction of both hops from which the seeds have been removed and seedless hops. The fates of the hop lipids and in particular the fatty acid fraction, both in conventional hopping procedures and in the preparation of an isomerised extract are discussed with reference to their possible effect on beer flavour.     

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     

In vitro inhibitory effects of cranberry bean (Phaseolus vulgaris L.) extracts on aldose reductase, α‐glucosidase and α‐amylase

The in vitro inhibitory activities of different seed extracts prepared from cranberry bean mutant SA‐05 and its wild‐type variety Hwachia against aldose reductase, α‐glucosidase and α‐amylase were examined. The results indicated that the polyphenolics‐rich extracts obtained using 800 g kg^?1 methanol and 500 g kg^?1 ethanol demonstrated inhibitory activities against aldose reductase (IC₅₀ of 0.36–0.46 mg mL^?1) and α‐glucosidase (IC₅₀ of 1.32–1.94 mg mL^?1). The 500 g kg^?1 ethanol extracts also showed α‐amylase inhibitory activities (IC₅₀ of 70.11–80.22 μg mL^?1). Subsequent extracts, prepared further with NaCl and H₂O from precipitates of 800 g kg^?1 methanol or 500 g kg^?1 ethanol extracts, exhibited potent α‐amylase inhibitory activities (IC₅₀ of 17.68–38.68 μg mL^?1). A combination of 500 g kg^?1 ethanol extraction plus a subsequent H₂O extraction produced highest polyphenolics and α‐amylase inhibitors. The SA‐05 α‐amylase inhibitor extracts showed greater inhibitory activities than that of Hwachia. Thus, cranberry bean mutant SA‐05 is an advantageous choice for producing anti‐hyperglycaemic compounds.     

CHEMISTRY OF HOP CONSTITUENTS. PART XXXIII REACTIONS OF β-ACIDS

Reactions of β-acids in wort and in mineral acids and alkali have been investigated. Under acidic conditions colupulone lost isopentenyl side chains forming two isomeric butenols. Under alkaline conditions two after-bitter compounds were formed, the structures of which were elucidated. They were also found in an isomerized hop extract.     

HOP PRODUCTS

Hop products have established an important and permanent position in the brewing raw materials market. Hop pellets and hop extract offer significant advantages to traditional brewers. However, the ultimate hop product is the isomerised hop extract, which can now be produced without the involvement of organic solvents. Still to be resolved and hence controlled is the contribution of the hop oil fraction to beer flavour.     

TOTAL AND INDIVIDUAL BARLEY (1–3), (1–4)-β-D-GLUCANASE ACTIVITIES IN SOME GREEN AND KILNED MALTS

Total β-glucanase activity was measured in extracts of malts prepared from three winter and three spring cultivars of barley. Samples were taken at intervals during modification and, after 116 hr, from malt kilned to 65°C. Good malting varieties showed highest levels of total β-glucanase activity, with a high correlation (r = 0.926) with HWE. Angora had the highest activity in the intermediate stages of malting, least loss of activity after heating extracts to 48°C for 10 mins and least loss of activity on kilning. Separation of isozymes by HPLC⁹ confirmed the greater heat stability of isozyme II, but, unlike previous studies on Australian cultivars, we found considerable activity of isozyme I after kilning, even up to 85°C. However, total β-glucanase activity was destroyed by heating extracts of all varieties to 60°C. Angora showed the highest proportion of total activity as isozyme II after kilining. Cultivar differences suggested some scope for breeding varieties with increased total activity by combining high activities of each isozyme. The high correlation of total activity with HWE suggests β-glucanase activity as a rapid test of malting quality.