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.     

ISO-α-ACIDS CONTENT OF ISOMERIZED HOP EXTRACTS

Two chromatographic procedures for the analysis of iso-α-acids in isomerized extracts have been tested. Although one of the methods—a variant of the Hansen, Hetzel & Miller procedure—performed better than the other, it still falls short of the demands of commercial transactions. The methods available for the assay of iso-α-acids are reviewed.     

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.     

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.     

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     

ABEO-ISO-α-ACIDS IN ENGLISH BEERS

Polyphenols interfere in thin layer chromatographic estimations of abeo-iso-α-acids. A paper chromatographic technique for separating polyphenols from abeo-iso-α-acids has been developed. English beers examined contained less than 6 ppm of abeo-iso-α-acids.     

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.     

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 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.     

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.     

Purification and physicochemical characterization of ovine β‐lactoglobulin and α‐lactalbumin

Ovine whey proteins were fractionated and studied by using different analytical techniques. Anion‐exchange chromatography and reversed‐phase high‐performance liquid chromatography (HPLC) showed the presence of two fractions of β‐lactoglobulin but only one of α‐lactalbumin. Gel permeation and sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis allowed the calculation of the apparent molecular mass of each component, while HPLC coupled to electrospray ionisation‐mass spectrometry (ESI‐MS) technique, giving the exact molecular masses, demonstrated the presence of two variants A and B of ovine β‐lactoglobulin. Amino acid compositions of the two variants of β‐lactoglobulin differed only in their His and Tyr contents. Circular dichroism spectroscopy profiles showed pH conformation changes of each component. The thermograms of the different whey protein components showed a higher heat resistance of β‐lactoglobulin A compared to β‐lactoglobulin B at pH 2, and indicated high instability of ovine α‐lactalbumin at this pH.     

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.     

Heat-induced aggregation of β-lactoglobulin A and B with α-lactalbumin

β-Lactoglobulin A and β-lactoglobulin B were heated at 75°C in the absence and presence of α-lactalbumin, and the aggregation products were characterized by size exclusion chromatography in combination with multi-angle laser light scattering and electrophoretic techniques. α-Lactalbumin did not form aggregates when heated alone, but in admixture with β-lactoglobulin it was incorporated into both the disulphide-bonded and the hydrophobically associated aggregates as well as forming α-lactalbumin dimers and other oligomers. The presence of α-lactalbumin diminished the proportion of smaller aggregates and increased the number of very large aggregates within both variant protein mixtures. In the presence of α-lactalbumin, β-lactoglobulin A was converted into a series of disulphide-bonded and the hydrophobically associated aggregates more slowly, but with a greater proportion of hydrophobically associated aggregates, than β-lactoglobulin B. These patterns are similar to that when β-lactoglobulin A or B are heated on their own. These and other results indicate that the mechanism of aggregation of α-lactalbumin/β-lactoglobulin mixtures is governed by β-lactoglobulin.     

The effects of α-amylase inhibitors on insect storage pests: Inhibition of α-amylase in vitro and effects on development in vivo

Protein α-amylase inhibitors were prepared from wheat and their effects tested against insect storage pests both in vitro against the insect α-amylases and in vivo in insect feeding trials. Inhibitor fraction A was found to inhibit porcine pancreatic α-amylase but not insect α-amylases, whereas fractions B, C and D (0.28) did not inhibit porcine pancreatic α-amylase but were strong inhibitors of digestive α-amylases from larvae of Tribolium confusum, a storage pest of wheat products, and Callosobruchus maculatus, a storage pest of legume seeds. Fraction D, which was a single polypeptide of M_r 13 000 was the most effective inhibitor in vitro. It would appear that the degree of inhibition by the wheat α-amylase inhibitor preparations can be correlated with the presence of the M_r 13 000 (0.28) polypeptide since the purer this polypeptide the stronger was the inhibition; fraction A which contained two polypeptides of M_r 60 000 and 58 000 caused no inhibition. The effects of fractions B and C on larval development were determined in insect feeding trials. With C. maculatus both fractions were toxic, their relative effectiveness being directly paralleled by their effectiveness observed in vitro. Only fraction C was tested against T. confusum in feeding trials. Despite this fraction being equally effective against both pests in vitro it had very little effect upon larval development of T. confusum in vivo, thus suggesting that this organism is able to detoxify the wheat α-amylase inhibitors. As far as the authors are aware, this is the first time that the effects of identified inhibitor fractions have been monitored both in vitro and in vivo. The results, in contrast to previous proposals, suggest that selecting wheat varieties for high α-amylase inhibitory activity may not be a very reliable criterion in selecting for insect resistance.     

Thermal modifications of structure and codenaturation of α‐lactalbumin and β‐lactoglobulin induce changes of solubility and susceptibility to proteases

Study of heat denaturation of major whey proteins (β‐lactoglobulin or α‐lactalbumin) either in separated purified forms, or in forms present in fresh industrial whey or in recomposed mixture respecting whey proportions, indicated significant differences in their denaturation depending on pH, temperature of heating, presence or absence of other co‐denaturation partner, and of existence of a previous thermal pretreatment (industrial whey). α‐Lactalbumin, usually resistant to tryptic hydrolysis, aggregated after heating at ⪈85°C. After its denaturation, α‐lactalbumin was susceptible to tryptic hydrolysis probably because of exposure of its previously hidden tryptic cleavage sites (Lys‐X and Arg‐X bonds). Heating over 85°C of β‐lactoglobulin increased its aggregation and exposure of its peptic cleavage sites. The co‐denaturation of α‐lactalbumin with β‐lactoglobulin increased their aggregation and resulted in complete exposure of β‐lactoglobulin peptic cleavage sites and partial unveiling of α‐lactalbumin tryptic cleavage sites. The exposure of α‐lactalbumin tryptic cleavage sites was slightly enhanced when the α‐lactalbumin/β‐lactoglobulin mixture was heated at pH 7.5. Co‐denaturation of fresh whey by heating at 95°C and pH 4.5 and above produced aggregates stabilized mostly by covalent disulfide bonds easily reduced by β‐mercaptoethanol. The aggregates stabilized by covalent bonds other than disulfide arose from a same thermal treatment but performed at pH 3.5. Thermal treatment of whey at pH 7.5 considerably enhanced tryptic and peptic hydrolysis of both major proteins.     

Free α-amino nitrogen production in sorghum beer mashing

Free α-amino nitrogen (FAN) is an essential nutrient for yeast growth during fermentation. Under normal conditions of sorghum beer mashing, 60°C at pH 4.0, production of FAN by proteolysis accounts for approximately 30% of wort FAN, the remaining 70% being preformed in the malt and adjunct. The quality of the FAN in sorghum beer worts is good as it does not contain a high percentage of proline. Optimum conditions for FAN production during mashing are 51°C and pH 4.6. Wort FAN was increased proportionally by raising the ratio of sorghum malt to adjunct and conversely decreased by raising the ratio of adjunct to malt. FAN was also increased by the addition to the mash of a microbial proteolytic enzyme. Wort FAN is directly proportional to malt FAN.     

ASSESSMENT OF BACILLUS LICHENIFORMIS α-AMYLASE AS A CANDIDATE ENZYME FOR GENETIC ENGINEERING OF MALTING BARLEY1

Bacillus licheniformis α-amylase, a thermostable starch-degrading enzyme, has been assessed as a candidate enzyme for the genetic transformation of malting barley. The temperature optimum, pH optimum and thermostability of B. licheniformis α-amylase were compared with those of barley α-amylase. The bacterial enzyme has a higher pH optimum (?9), a higher temperature optimum (?90°C) and much higher thermostability at elevated temperatures than the barley enzyme. The specific activity of the bacterial enzyme under conditions of pH and temperature relevant to the brewing process (pH 5.5, 65°C) is ?1.5-fold higher than that of the barley enzyme. Measurements of α-amylase activity during a micro-mash showed that the bacterial enzyme is at least as stable as the barley enzyme under these conditions, and that a level of expression for the bacterial enzyme corresponding to ?0.5% of total malt protein would approximately double the α-amylase activity in the mash. B. licheniformis α-amylase activity was rapidly eliminated by boiling following mashing as would occur during brewing. The combined results suggest that barley expressing the bacterial enzyme may be useful in the brewing process.     

α-GLUCOSIDASE ACTIVITY OF SORGHUM AND BARLEY MALTS

Sorghum malt α-glucosidase activity was highest at pH 3.75 while that of barley malt was highest at pH 4.6. At pH 5.4 employed in mashing sorghum malt α-glucosidase was more active than the corresponding enzyme of barley malt. α-Glucosidase was partly extracted in water but was readily extracted when L-cysteine was included in the extraction buffer, pH 8. Sorghum malt made at 30°C had higher α-glucosidase activities than the corresponding malts made at 20°C and 25°C. Nevertheless, the sorghum malts made at 20°C and 25°C produced worts which contained more glucose than worts of malt made at 30°C. Although barley malts contained more α-glucosidase activity than sorghum malts, the worts of barley had the lowest levels of glucose. The limitation to maltose production in sorghum worts, produced at 65°C, is due to inadequate gelatinization of starch and not to limitation to β-amylase and α-amylase activities. Gelatinization of the starch granules of sorghum malt in the decantation mashing procedure resulted in the production of sorghum worts which contained high levels of maltose, especially when sorghum malt was produced at 30°C. Although the β-amylase and α-amylase levels of barley malt was significantly higher than those of sorghum malted optimally at 30°C, sorghum worts contained higher levels of glucose and equivalent levels of maltose to those of barley malt. It would appear that the individual activities of α-glucosidase, α-amylase and β-amylase of sorghum malts or barley malts do not correlate with the sugar profile of the corresponding worts. In consequence, specifications for enzymes such as α-amylase and β-amylase in malt is best set at a range of values rather than as single values.