ACTIVITIES OF VARIOUS PEPTIDASES IN BARLEY,GREEN MALT AND MALT

The three groups of malt peptidases (carboxypeptidases, “naphthylamidases” and (amino) peptidases acting on Leu-Tyr and Ala-Gly) are present in unmalted barley; the activities are low and of a similar order of magnitude. On germination the activities of the different carboxypeptidases increase from 10- to 20-fold; the “naphthylamidases” increase only 2-fold, and the peptidase activities increase from 3- to 6-fold. None of these enzymes is inactivated during kilning to any significant extent. There are considerable differences between the carboxypeptidase activities of malts derived from different varieties of barley and the activities are correlated with α-amylase activity.     

INFLUENCE OF CULTIVAR AND GERMINATION CONDITIONS ON PROTEOLYTIC ACTIVITIES IN SORGHUM MALT

The effects of cultivar and germination conditions on proteinase and carboxypeptidase activities in sorghum malt were investigated using enzyme assays based on kafirin, the sorghum prolamin, as substrate. When different sorghum cultivars grown under identical environmental conditions were malted under identical conditions, the proteinase and carboxypeptidase activities of the various malts both differed significantly. This indicates that the activities of these enzymes are cultivar related. Steeping sorghum grain resulted in no significant change in proteinase or carboxypeptidase activity. Proteinase activity increased only moderately with germination time, up to a maximum at 4 days. Neither germination temperature (24–32°C) nor moisture affected proteinase activity. In contrast, there was a large increase in carboxypeptidase activity with germination time up to 4 days and both germination temperature and moisture affected carboxypeptidase activity.     

PURIFICATION AND PROPERTIES OF MALT CARBOXYPEPTIDASES ATTACKING HORDEIN

Carboxypeptidases capable of releasing amino acids from barley hordein have been extracted and purified from malted barley. Three enzymes can be distinguished, with similar molecular weights but slightly different ionic characteristics and specificities. The malt carboxypeptidases preferentially attack peptides containing an aromatic amino acid, although other peptides, including those containing proline, are also attacked. The purified carboxypeptidase fractions reduce the viscosity of β-glucan extracted from endosperm cell walls. It is suggested that these enzymes may be instrumental in the initial solubilization of endosperm cell walls, possibly by the hydrolysis of the peptide component of these walls. The pH optima and heat stability data indicate that carboxypeptidases are crucial for the release of amino acids both from hordein, and from peptides derived from hordein during both malting and mashing.     

EXTRACTION AND ASSAY OF PROTEOLYTIC ACTIVITIES IN SORGHUM MALT

A method has been developed to assay the proteinase and carboxypeptidase activities in sorghum malt. The method specifically addresses two problems encountered with some other assays for malt proteolytic activity; that of enzyme inextractability and the use of alien substrates. It was found that as with barley malt proteolytic enzymes, a high proportion of sorghum malt proteolytic activity was inextractable with sodium chloride solution. Systematic investigation into the extraction of proteolytic enzymes from sorghum malt led to the development of a novel extractant viz. 0.6 M Lithium iodide LiI plus 3.33 mM dithiothreitol. This extractant appears to solubilize a significantly higher proportion of the malt proteolytic activities with a single extraction than previously used buffers. Assay is carried out Using purified Kafirin, the sorghum prolamin storage protein, as substrate. Proteinase is measured in terms of total nitrogen solubilized and carboxypeptidase as free α-amino nitrogen solubilized.     

Modification of the Methods for the Extraction of Carboxypeptidase and Proteinase Activities from Sorghum Malts

The effects of two extractants on the activities of carboxypeptidase and proteinase enzymes from three sorghum varieties germinated for 3 and 5 days were studied. In all three varieties, cysteine hydrochloride (Cyst. HCl) proved a better extractant than 2‐mercaptoethanol (2‐ME) as shown by the increase in carboxypeptidase and proteinase activities with 5‐day malts. A three‐way analysis of variance (ANOVA) on the effect of germination time, extractant and variety on the carboxypeptidase activities of the three malts showed that all the factors plus their possible four‐way interactions highly significantly (P < 0.001) affected the extractability/activity of the enzyme. However, while the three factors had a highly significant (P < 0.001) effect on the proteinase activity, one of their four‐way interactions, specifically, extractant/variety, had no significant effect. This suggests that, unlike the carboxypeptidase enzyme, the effectiveness of the extractants in enhancing the extractability/activity of the proteinase enzyme in all the sorghum malt samples was amongst other factors, not variety‐dependent.     

THE INFLUENCE OF MASHING CONDITIONS ON WORT AND BEER QUALITY

From a comparison of the performance of ale and lager malts mashed by constant temperature infusion, by temperature programmed infusion, and by decoction procedures it emerges that malt modification and the pattern of kilning are as important in determining the composition of wort as are the mashing conditions. Worts with appropriate levels of amino and total nitrogen and of fermentable sugars may be obtained by selecting lightly-dried well-modified malts and mashing with a constant temperature infusion rather than by using less modified malt in conjunction with more complex mashing programmes. Ale malts yield worts of lower pH which is reflected in a slight reduction in hop utilization. Head retention is improved by the use of undermodified malts but colloidal stability is improved when well-modified malts are used. In the case of well-modified malts a high final curing temperature is not a prerequisite for achieving good colloidal stability in beer.     

Impact of Dark Specialty Malts on Extract Composition and Wort Fermentation

Dark specialty malts are important ingredients for the production of several beer styles. These malts not only impart colour, flavour and antioxidative activity to wort and beer, they also affect the course of wort fermentations and the production of flavour‐active yeast metabolites. The application of considerable levels of dark malt was found to lower the attenuation, mainly as a result of lower levels of fermentable sugars and amino acids in dark wort samples. In fact, from the darkest caramel malts and from roasted malts, practically no fermentable material can be hydrolysed by pilsner malt enzymes during mashing. Compared to wort brewed with 50% pilsner malt and 50% dark caramel malt or roasted malt, wort brewed with 100% pilsner malt contained nearly twice as much fermentable sugars and amino acids. Reduced levels of yeast nutrients also lowered the fermentation rate, ranging from 1.7°P/day for the reference pilsner wort of 9 EBC to 1.1°P/day for the darkest wort (890 EBC units), brewed with 50% roasted malt. This additionally indicates that lower attenuation values for dark wort are partially due to the inhibitory effects of Maillard compounds on yeast metabolism. The application of dark caramel or roasted malts further led to elevated levels of the vicinal diketones diacetyl and 2,3‐pentanedione. Only large levels of roasted malt gave rise to two significant diacetyl peaks during fermentation. The level of ethyl acetate in beer was inversely related to colour, whereas the level of isoamyl acetate appeared to be affected by the use of roasted malt. With large levels of this malt type, negligible isoamyl acetate was generated during fermentation.     

Influence of malt browning degree on lipoxygenase activity

The development of off-flavour in pale beer is generally attributed to the oxidation of the intermediate products of lipid degradation catalysed by lipoxygenase during mashing. This paper deals with the effect of speciality malts, having different browning levels, on malt lipoxygenase. The results obtained showed that lipoxygenase activity, which was found to be present only in the pale malt, was affected by mashing temperatures above 63 °C and by the presence of Maillard reaction products in the speciality malts. In particular, the addition to pale malt of speciality malts, having high inhibitory capacity towards lipoxygenase but low colouring potential, was effective in reducing the off-flavour development in pale beer.     

INACTIVATION OF MALT PEPTIDASES DURING MASHING*

Malt contains at least eight different peptidases: three carboxypeptidases (pH optima 4·8, 5·2, 5·7), three aminopeptidases active on aminoacyl-β-naphthylamides (pH optima in hydrolysis of peptides at pH 5·8-6 5), and two (amino)peptidases acting on Leu-Tyr and Ala-Gly at higher pH (pH optima 8·6, 7·8). We have studied the progressive inactivation of these peptidases during mashing with a temperature programme from 45° to 70° C (pH 5·8 → 5·7). All peptidases were stable at 45° C. The five aminopeptidases were inactivated at different rates during a 30-min incubation at 55° C. The carboxypeptidases retained 70–100% of their activities at this temperature, and one of them had 40% residual activity after 60 min at 70° C. Liberation of amino acids continued at a considerable rate during the incubation at 70° C, probably catalysed by the heat-stable carboxypeptidase. Malt carboxypeptidases are therefore more heat-stable in mashing conditions than the aminopeptidases. This property, in combination with their high activities and suitable pH optima, makes them the most important enzymes in the production of free amino acids in mashing.     

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.     

麦芽中SOD活性与麦汁还原力的关系以及糖化温度对它们的影响

研究了11种进口麦芽和18种国产麦芽中超氧化物歧化酶(SOD)活性的差异以及与协定麦汁还原力之间的关系。以甘啤-3号麦芽为对象,采用7种恒温糖化工艺考察了糖化温度对2者的影响。结果表明,不同品种麦芽中,SOD的差异比较明显(1205.6～2126.0U/g),而且麦芽的SOD活性与协定麦汁的还原力之间存在显著的正相关性(相关系数r=0.898,P<0.01);SOD活性随着糖化温度的升高而逐渐降低,55℃恒温糖化60min后有53.22%的酶活残存;当糖化温度升高到65℃时,SOD的活性大幅度下降,30min后仍有25.45%的酶活残存;糖化温度为70℃和80℃时SOD下降到极低的活性;麦汁的还原力随着糖化温度的升高而升高,80℃恒温糖化100min后麦汁的还原力高达3.75mmol/mL。     

Behavior of mono-, di-, and trihydroxyoctadecenoic acids during mashing and methods of controlling their production

The behavior of mono-, di-, and trihydroxyoctadecenoic acids was investigated during laboratory-scale mashing under various conditions with a view to controlling their production. Using a malt in which the lipoxygenase activity was at only a trace level (less than 0.01 U/g) or starting the mashing at a higher temperature than that conventionally used (65 degrees C instead of 48 degrees C) significantly decreased the production of these hydroxy fatty acids. Lowering the pH of the mash to inhibit lipoxygenase activity and preventing O2 uptake by the mash using carbon dioxide were also effective in reducing the amounts of these acids produced during mashing. From the viewpoint of industrial-scale beer production, the prevention of O2 uptake by the mash was selected as an appropriate method for reducing oxidation during wort production without affecting the subsequent brewing process or the taste of the finished beer. After introducing oxidation prevention procedures, the content of trihydroxyoctadecenoic acids decreased by about 30% and the foam stability and taste were improved in commercial products brewed using less than 25% malts.     

VARIATION IN pH AND LACTATE LEVELS IN MALTS

Malt pH, defined as the pH of worts prepared by the Institute of Brewing Methods of Analysis, was examined in commercial malts from a single malting plant over four crop years, 1990 to 1993. Batch to batch variability was found, in addition to a distinct pattern within two of the years, with a gradual decrease in the winter months following commencement of malting of the new crop barley, followed by a subsequent increase in the late summer. Variability in pH and the longer term trend were not related to any measured processing variables or characteristics of the barley used for malting. Possible reasons for the differences in pH were examined and a negative relationship between malt pH and wort lactate content was demonstrated in a selection of malts from two crop years. A method is proposed for extraction and purification of lactate directly from malt and using this method it was shown that lactate in wort derives mainly from malt with only small amounts being produced during mashing.     

SMALL SCALE MASHING EXPERIMENTS WITH GRISTS CONTAINING HIGH PROPORTIONS OF RAW SORGHUM

Small scale mashes (50 g total grist) with grists containing high proportions of raw sorghum (50%–80% malt replacement) showed high values of extract recovery and produced worts of lower total nitrogen, free amino nitrogen, viscosity and colour but higher values of pH compared to worts produced from all malt mashes. Increasing the proportion of raw sorghum in the grist relative to malt resulted in a decline in extract recovery, wort total nitrogen, free amino nitrogen and an increase in wort pH. Addition of industrial enzyme preparations to mashes containing raw sorghum resulted in higher values of extract recovery (enzyme preparations containing α amylase and β glucanase), higher values of wort total nitrogen and free amino nitrogen (enzyme preparations containing a neutral proteinase) and decreased wort viscosity (enzyme preparations containing β glucanase or cellulases) compared to worts produced from untreated mashes. Worts and beers were produced on a pilot brewery scale from 50% malt and 50% polished (whole) sorghum (single decoction mashing regime) and 20% malt and 80% raw sorghum supplemented with an industrial enzyme preparation (double mashing regime). Mashes comprising 50% malt and 50% polished sorghum showed comparable wort filtration behaviour (lautering) to that of control mashes (70% malt and 30% maize grists) whereas wort produced from 20% malt and 80% raw sorghum filtered slowly. Worts produced from grists containing sorghum were of high fermentability and showed lower levels of total nitrogen and free amino nitrogen compared to control worts. Analysis of worts produced from small scale mashes containing raw sorghum and a pilot brewery scale mash comprising 20% malt and 80% raw sorghum demonstrated that the levels of total nitrogen and free amino nitrogen were higher than expected from the reduction in the malt content of the mash, consistent with the release of nitrogenous components (polypeptides, peptides and amino acids) derived from sorghum into the wort. Beers produced from 50% malt and 50% polished sorghum and 20% malt and 80% raw sorghum were filtered without difficulty and were of sound flavour. Beers produced from 50% malt and 50% polished sorghum contained lower levels of isobutanol, 2-methylbutanol, dimethylsulphide and higher levels of n propanol and diacetyl compared to control beers.     

Effects of mashing on total phenolic contents and antioxidant activities of malts and worts

Effects of mashing on total phenolic contents (TPC), radical‐scavenging activity, reducing power and metal chelating activity of malts and corresponding worts were clarified in this study. Results showed that there were significant variations in TPC and antioxidant activity across malts and worts. An 8.8% of increase in TPC was observed during the early stage of mashing, accompanied by an increase in antioxidant activity. However, 2.6% of decrease in TPC and inconsistent changes in antioxidant activity were found during the mashing at higher temperature. Overall, mashing resulted in 6.0% and >10.0% of increases in TPC and antioxidant activity, respectively. Moreover, Pearson correlation analysis revealed that there were good correlations (ranging from 0.622 to 0.735, P < 0.01) between TPC and antioxidant activity of malts and worts. Additionally, mashing resulted in more positive correlations between TPC and antioxidant activity, emphasising the key role of malt phenolic compounds for wort antioxidant activity.     

WORT-BOILING IN RELATION TO BEER QUALITY

Comparisons of beers from worts mashed with pale ale malts (nitrogen 1·3%) at 150°F. (65·5°C.) and boiled for 1/2, 1 and 2 hr. show that increase in boiling time leads to slightly increased hop-utilization, decreased head-retention, improved non-biological stability and slight differences in flavour. Findings were very similar when the mashing temperature was either 145°F. (63°C.) or 155°F. (68°C.) except that, in the latter case, no difference in flavour was detectable between beers from long and short boiled worts. Stirring can replace vigorous boiling to secure normal hop-utilization and shelf-life but simmered worts give beers of characteristic flavour probably as a result of enhanced contents of hop oil. Use of a lightly kilned malt of high nitrogen content (1·8%) produced a characteristically different flavour from that obtained with pale-ale malt and reduced the utilization of hop substances by provoking a greater loss of this material during fermentation.     

Current Developments in Malting and Brewing Trials with Sorghum in Nigeria: A Review

Initially, large‐scale lager beer brewing with sorghum malts proved highly intractable due to a number of biochemical problems including: high malting losses estimated at 10–30% as against 8–10% for barley; high gelatinisation temperatures which limited starch solubilisation/ hydrolysis by the amylolytic enzymes during mashing; low extract yield/low diastatic power (DP) due to inadequate hydrolytic enzyme activities especially β‐amylase; low free α‐amino nitrogen (FAN) due to inadequate proteolysis limiting yeast growth during fermentation; high wort viscosities/beer filtration problems due to low endo‐β‐1,3; 1–4‐glucanase activities on the endosperm cell walls causing the release of some β‐glucans. Strident research efforts using improved Nigerian sorghum malt varieties (SK5912, KSV8 and ICSV400) have reported some encouraging results. The knowledge of the biochemical integrity of the endo‐β‐glucanases of the sorghum malt is helping to elucidate their mode of activity in the depolymerisation of the β‐glucans. This is bound to ensure process efficiency in sorghum beer brewing, reduce beer production costs and ultimately, produce a Pilsner‐type of lager beer with 100% sorghum malt.     

Effects of nitrogen application on malt modification and dimethyl sulfide precursor production in two Japanese barley cultivars

BACKGROUND: Nitrogenous components have a great influence on both malt and beer qualities. Barley storage proteins are degraded during the germination process, in which amino acids and small peptides are released. Some of these compounds relate to dimethyl sulfide precursor production in the malting process. In this study, barley and malt qualities were investigated using two Japanese barley cultivars, Sukai Golden and Mikamo Golden, with several different nitrogen (N) treatments. RESULTS: Nitrogen top‐dressing treatments efficiently increased N and sulfur (S) concentrations in grains. A difference in malt modification was induced by these treatments without any change in protease activity in malts. S‐Methyl methionine (SMM) concentration in malt of Sukai Golden with low‐N treatment was 1.8–2.1 times higher than that with higher‐N treatments. Methionine concentration in malts was not significantly affected by N treatments of both cultivars, while grain S level was not consistent under any treatments. CONCLUSION: Results show that low‐N treatment increases SMM concentration in malts despite major S‐containing amino acids of malts being not highly affected by the difference in nutrient status of grains. Further investigations are necessary into aspects of both metabolic profiles in barley germination and SMM degradation in the kilning process. Copyright © 2008 Society of Chemical Industry     

ISOLATION AND PARTIAL CHARACTERIZATION OF THE DIMETHYL SULPHIDE PRECURSOR IN GREEN MALT,AND ITS EFFECT ON BEER DIMETHYL SULPHIDE LEVELS

The component from which dimethyl sulphide (DMS) is produced when green malt worts are heated has been purified and its effects on wort and beer DMS levels studied. The green malt precursor can be split into S-methylmethionine and an as-yet uncharacterized ninhydrin-positive component. When the purified precursor is added to worts derived from kilned malts, it is rapidly taken up by yeast, but there is no resulting increase in DMS production. DMS production during fermentation occurs with worts made from kilned malts but not with worts from green malt; however, this difference is not due to an inhibitor being present in green malt worts. The evidence suggests that malt kilning affects the nature as well as the amount of DMS precursor in malts.     

Influence of carboxypeptidases on free amino acid,peptide and methylpyrazine contents of under‐fermented cocoa beans

A study of the action of two carboxypeptidases on free amino acids, peptides and methylpyrazines in under‐fermented cocoa beans was carried out. Carboxypeptidase B from porcine pancreas and carboxypeptidase Y from baker's yeast were used separately for digestion. Hydrophobic free amino acids (alanine, valine, isoleucine, leucine, phenylalanine and tyrosine) were predominantly produced in samples digested with both carboxypeptidase B and Y. The peptide patterns of samples digested with both carboxypeptidases were similar to that of the control. The concentration of 2,3,5,6‐tetramethylpyrazine in samples with carboxypeptidase B addition was significantly higher than those of 2,5‐dimethyl‐ and 2,3,5‐trimethylpyrazine; the concentration of 2,3,5‐trimethylpyrazine was highest (1727.86 µg per 100 g) in the sample with carboxypeptidase B addition that had been incubated for 24 h. These findings indicate that carboxypeptidase B from porcine pancreas was more prominent in the formation of cocoa‐specific aroma. © 2002 Society of Chemical Industry