PRODUCTION OF BEER USING IMMOBILIZED YEAST ENCODING α-ACETOLACTATE DECARBOXYLASE

Genetically modified brewer's yeast encoding α-acetolactate decarboxylase (α-ALDC) was tested in immobilized yeast bioreactors for main fermentation of beer. The α-ALDC enzyme produced by the transformant catalyzes the direct conversion of α-acetolactate to acetoin without formation of diacetyl. The long lagering period required for beer maturation in conventional brewing can thus be shortened or even omitted. Three different packed bed bioreactors were employed, with volumes of 1.6 dm³, 5 dm³ and 25 dm³. The 5 dm³ column had a slightly conical geometry in contrast to the others which had cylindrical shapes. Sintered glass beads were chosen as the carrier material on the basis of experiments with the parent strain. The brewing performance of the transformant compared well with that of the parent strain in the immobilized system. Fermentation, utilization of amino acids (including isoleucine, valine and leucine) and flavour formation were practically identical with both strains, the only difference being a marked decrease in the formation of diacetyl by the transformant. Small differences were, however, observed in the long-term biochemical stability. By using yeast encoding α-ALDC in the immobilized yeast system the total (primary and secondary) fermentation time could be reduced to approximately 2–6 days, compared with 3–6 weeks in a conventional batch process.     

DIFFERENCES IN UTILISATION OF THE ESSENTIAL OIL OF HOPS DURING THE PRODUCTION OF DRY-HOPPED AND LATE-HOPPED BEERS

Late-hopped and dry-hopped beers were prepared and their lipophilic constituents extracted using Amberlite XAD-2 resin. Examination of the volatile constituents by GC-MS confirmed that most of the hop oil added towards the end of wort boiling is lost by evaporation. Part of the material which survives boiling is chemically transformed by yeast during fermentation. Dry-hopped beer contained compounds more representative of the original hop oil than did the corresponding late-hopped beer. A liquid carbon dioxide extract of hops, rich in essential oil, has been fractionated by column chromatography on alumina-silica giving preparations which simulate either late-hop or dry-hop character.     

OCCURRENCE OF DIKETONES AND α-ACETOHYDROXYACIDS IN FERMENTATIONS

Diketone and α-acetohydroxyacid levels have been monitored during ale fermentations both in stirred fermentors and in a pilot brewery. It was found necessary to bring samples to neutral pH and protect them from air until assayed. Little diketone was formed during normal fermentations. Addition of sodium metabisulphite trapped α-acetohydroxyacids and high concentrations persisted in the beer. Lowering the wort pH decreased the peak concentrations of α-acetohydroxyacids without a corresponding increase in diketone concentrations. Alterations in the oxygen supply during preculture of the yeast or at pitching had little effect on diketone formation. α-Acetohydroxyacids have been detected in beer in keg from a continuous fermentation.     

SUPPRESSION OF α-ACETOLACTATE FORMATION IN BREWING WITH IMMOBILIZED YEAST

Immobilized yeast cells extensively produced the diacetyl precursor, α-acetolactate, during alcohol fermentation. The activity of acetohydroxy acid synthetase, which is responsible for the formation of α-acetolactate from pyruvic acid, was high in cell-free extracts of immobilized yeast cells compared with that of free yeast cells. It was suggested that the expression of AHA synthase of immobilized yeast cells was increased during growth in the carrier as compared with free yeast cells. When the initial immobilizing yeast cell concentration was changed from 1.0 × 10⁶ cells/ml to 1.0 × 10⁹ cells/ml, production of α-acetolactate was reduced from 0.94 mg/l to 0.30 mg/l. Furthermore, during continuous fermentation for 10 d, the concentration of α-acetolactate in beer was 0.30 mg/l.     

CONVERSION OF α-ACETOLACTATE AND REMOVAL OF DIACETYL A KINETIC STUDY

Brewers' yeast does not form diacetyl during fermentation, but α-acetolactate which spontaneously is converted to diacetyl. This non-enzymic reaction is mainly dependent upon pH, temperature and substrate concentration. The present kinetic studies demonstrate that temperature and pH have a dominating effect on the spontaneous conversion of α-acetolactate during primary fermentation. From the reported kinetic studies it is possible to predict the conversion rate of α-acetolactate present in the fermenting liquid, and to calculate the time necessary to convert a given amount of α-acetolactate to diacetyl. As long as yeast is present in the fermenting liquid, diacetyl can not be detected analytically due to the high diacetyl-reducing activity of the yeast cells. The specific activity is strain-dependent and decreases gradually during fermentation. The diacetyl-reducing activity of the yeast will, however, normally be high enough to compete with the diacetyl formed from α-acetolactate.     

α-GLUCOSIDASE, α-GLUCOSIDE PERMEASE,MALTOSE FERMENTATION AND LEAVENING ABILITY OF BAKER'S YEAST

α-Glucosidase and α-glucoside permease are synthesized simultaneously in two different strains of baker's yeast (Saccharomyces cerevisiae) when the cells are induced with maltose. α-Thioethyl D-glucopyranoside (α-TEG) inhibits the transport of maltose into the cells, and the transport of maltose and α-TEG appeared to be mediated by the same permease system. There is an obvious correlation between α-glucosidase, α-glucoside permease and maltose fermentation activities in the yeasts while no correlation between these and the leavening ability of the yeasts can be demonstrated. Apparently the glucose concentration in dough is high enough to inhibit the permease-mediated transport of maltose into the cells thus impairing leavening ability from the maltose fermenting system.     

ROLE OF WORT AERATION IN THE BREWING PROCESS PART 1: OXYGEN UPTAKE AND BIOSYNTHESIS OF LIPID BY THE FINAL YEAST

While yeast at the end of fermentation (final yeast) contained minimal amounts of lipid, no significant differences could be found in the lipid compositions of various strains of final yeast obtained from several practical brewing plants. Lipid content of the final yeast increased during storage in cold water until the 3rd or 4th day. External nutrients were found to be necessary for the final yeast to synthesize well-balanced lipid during pre-aeration. The final yeast required more than 30 mg of available oxygen/g of dry yeast synthesizing the sufficient amounts of lipid which are necessary for desirable fermentation.     

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     

PRODUCTION OF α-AMYLASES IN GERMINATING WHOLE AND INCUBATING DE-EMBRYONATED BARLEY KERNELS*

α-Amylases produced in germinated barley and incubated de-embryonated barley kernels (cv. Bonanza), in the absence and presence of gibberellic acid (GA₃), were analyzed qualitatively by polyacrylamide gel isoelectric focusing (PAG-IEF) and quantitatively by chromatofocusing. Identical patterns of α-amylase components were obtained for both germinated barley and incubated de-embryonated barley kernels at each germination/incubation stage, in the absence or presence of GA₃. Total α-amylase increased rapidly in the germinating whole seed whereas in the incubating de-embryonated grain the α-amylase activity increase was much slower. Addition of exogenous GA₃ did not induce production of higher levels of α-amylase in either the germinating whole or incubating de-embryonated barley kernel. Quantitative chromatofocusing analysis revealed that the proportion of α-amylase III to α-amylase II activity decreased linearly with germination time in the whole grain but remained constant in the incubating de-embryonated grain in the absence or presence of GA₃. The major proportion of α-amylase activity in the germinating whole grain and incubating de-embryonated grain was synthesized in the form of α-amylase II components. However, α-amylase I represented a larger proportion of the total α-amylase activity produced in the incubating de-embryonated grain, as compared to the germinating whole seed in the absence or presence of GA₃. These results suggest that embryo excision differentially affects production of α-amylase II as compared to α-amylase I.     

THE EFFECTS OF ETHANOL,HEXAN-1-OL,AND 2-PHENYLETHANOL ON CIDER YEAST GROWTH,VIABILITY, AND ENERGY STATUS; SYNERGISTIC INHIBITION

An ethanol-tolerant strain of Saccharomyces cerevisiae, used for cider manufacture, when grown aerobically in the presence of either 1.7 M ethanol (10%, v/v), 3.35 mM hexan-1-ol (0.05%), or 25 mM 2-phenylethanol (0.25%), showed decreased growth rates (about 60%, 60% and 75%) and diminished viability. The normal changes in medium pH (increased acidity over the first 24 h from pH 6.0 to 5.4 followed by a decreased acidity to pH 7.8 by 70 h) were altered in the presence of an added alcohol. “Acidification power” (glucose-driven pH change) was also markedly reduced by growth with the alcohols from a value of 1.77 for control cultures, to 0.5, 1.35 or 0.6 respectively after 48 h. Adenylate energy charge values were decreased from 0.8 to 0, 0.3 and 0.2 respectively after 115 h; control cultures without an added alcohol showed a final value of 0.4. Ethanol plus either hexan-l-ol or 2-phenylethanol gave much more pronounced decreases in viability, medium pH changes during growth, acidification power, and adenylate energy change values than predicted by summation of individual effects. It is concluded that synergy between ethanol and the higher alkanol or aryl-alcohol occurs to produce membrane-associated lesions with deleterious effects during cider fermentation.     

EVALUATION OF MALTING QUALITY IN A BARLEY BREEDING PROGRAMME USE OF α-AMYLASE AND β-GLUCAN LEVLES IN MALT AS PRESELECTION TOOLS

Analysis according to the EBC protocol, immunological determination of a α-amylase and estimation of malt β-glucan using the Calcofluor-FIA method, were used to screen 327 barley breeding lines for malting quality. The results obtained with the α-amylase and β-glucan methods are highly correlated to the important malt quality paramters: extract yield and β-glucan content in the wort. It is recommended that either of the two methods, which are simple to perform are used as prescreening tools in breeding programmes for malting barley.     

EVIDENCE FOR THE PRESENCE OF MALTASE AND α-GLUCOSIDASE ISOENZYMES IN BARLEY

An examination of the development of α-glucosidase and maltase activities (as measured by the hydrolysis of p-nitrophenyl α-D-glucoside and maltose respectively) indicated that two genotypes of Glacier barley had the same general pattern of enzyme development. However, the development of α-glucosidase activity followed a different course from that of maltase activity suggesting that separate enzyme proteins are involved in hydrolysing these substrates. Further evidence that separate enzyme proteins were responsible for hydrolysis of maltose and p-nitrophenyl α-glucoside was obtained by column chromatography of extracts of germinated barley which indicated the presence of two maltases and two α-glucosidases. The maltases and the α-glucosidases differed in molecular weight, pH of optimum activity and in thermostability. When isomaltose was used as a substrate the optimum pH and behaviour on gel chromatography were coincident with that of maltase activity but different from the α-glucosidases.     

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.     

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.     

MEASUREMENT OF (1 → 3)(1 → 4)-β-d-GLUCAN IN MALT,WORT AND BEER

A method developed for the quantification of (1 → 3)(1 → 4)-β-d -glucan in barley flour has been modified to allow its use in the measurement of this component in malt, wort, beer and spent grain. For malt samples, free d -glucose was first removed with aqueous ethanol. Quantification of the polymer in wort and beer samples involved precipitation of the β-glucan with ammonium sulphate followed by washing with aqueous ethanol to remove free d -glucose. Spent grain was lyophilised and milled and then analysed by the method developed for malt. In all cases, the β-glucan was depolymerised with lichenase and the resultant β-gluco-oligosaccharides hydrolysed to d -glucose with β-d -glucosidase. The released d -glucose was then specifically determined using glucose oxidase-peroxidase reagent.     

DETERMINATION OF α- AND β-AMYLASE IN MALT

The Analysis Committee of the European Brewery Convention carried out a collaborative trial on malts using the specific analysis methods for α- and β-amylase activities based on dyed substrates supplied by MegaZyme (Aust.) Pty. Ltd. The repeatability and reproducibility values for the methods were judged to be unsatisfactory and consequently the methods were not recommended for Analytica-EBC.     

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.     

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     

ENZYMIC QUANTIFICATION OF (1→3) (1→4)-β-D-GLUCAN IN BARLEY AND MALT

A simple and quantitative method for the determination of (1→3) (1→4)-β-D-glucan in barley flour and malt is described. The method allows direct analysis of β-glucan in flour and malt slurries. Mixed-linkage β-glucan is specifically depolymerized with a highly purified (1→3) (1→4)-β-D-glucanase (lichenase), from Bacillus subtilis, to tri-, tetra- and higher degree of polymerization (d.p.) oligosaccharides. These oligosaccharides are then specifically and quantitatively hydrolysed to glucose using purified β-D-glucosidase. The glucose is then specifically determined using glucose oxidase/peroxidase reagent. Since barley flours contain only low levels of glucose, and maltosaccharides do not interfere with the assay, removal of low d.p. sugars is not necessary. Blank values are determined for each sample allowing the direct measurement of β-glucan in maltsamples.α-Amylasedoes not interfere with the assay. The method issuitable for the routineanalysis of β-glucan in barley samples derived from breeding programs; 50 samples can be analysed by a single operator in a day. Evaluation of the technique on different days has indicated a mean standard error of 0–1 for barley flour samples containing 3–8 and 4–6% (w/w) β-glucan content.