LIPOLYTIC AND OXIDATIVE CHANGES OF BARLEY LIPIDS DURING MALTING AND MASHING

Lipolytic and oxidative changes of barley lipids were studied during malting and mashing. The amount of lipid decreased by 23% during malting and changes in the composition of lipid classes were minor. On the other hand, during mashing the amount of free fatty acids (FFA) increased which indicated, that lipid hydrolysis had occurred. The same phenomenon was seen when malt flour was soaked in water at 23°C. The triglyceride (TG) and polar lipid (PL) contents were reduced and the proportion of FFA in total lipids was increased. Following similar soaking of barley flour, TG and PL were reduced but the accumulation of FFA and especially linoleic acid (LA) was slight. The results were consistent with the data on lipoxygenase activity (LOX) during malting. During steeping LOX decreased and was 15–20% of the activity of raw barley at the beginning of germination. The activity remained low during germination but rose sharply in the middle of kilning only to decrease again to a very low level at the end of kilning (5%). This in combination with the fact that the proportion of FFA remained high in the soaked malt samples suggests that the oxidation by LOX is negligible in the malt samples. However, the data suggest that mashing of barley, but not that of malt, includes the potential for the formation of highly polar lipid oxidation products.     

CHANGES IN ACTIVITY OF SORGHUM LIPASE DURING MALTING AND MASHING

Lipase activity was monitored during malting and mashing of sorghum grains. All three sorghum varieties contained detectable lipase activity in the ungerminated form. Lipase activity changed only slightly during steeping for 24 hours but increased several fold in the course of germination. Between 24% and 60% of the lipase activity of the green malt was retained after kilning at 48°C but no activity was detected in the wort after mashing at 65°C. About 68% of the lipase activity of 72 hours old malt was detected in the plumule, while 29% and 3% were detected in the endosperm and radicle, respectively. Optimum activity was observed at pH 7.0.     

CHANGES IN THE CONTENTS OP BARLEY PROTEOLYTIC INHIBITORS DURING MALTING AND MASHING

Barley contains three types of large-molecular proteolytic inhibitors, viz., inhibitors active against malt endopeptidases, a specific trypsin inhibitor, and a group of inhibitors of microbial proteinases and chymotrypsin. The inhibitors of malt endopeptidases disappear at an early stage of germination, just before the endopeptidase activity begins to increase. The total amount of these inhibitors is so small, however, that the great increase in endopeptidase activity occurring during germination cannot be due to the breakdown of an “equivalent” amount of inhibitors. The contents of the other two types of inhibitor in barley are much higher; they decrease slowly during germination, but high activities are still present in malt. The inhibitors of microbial proteinases are progressively inactivated during mashing. The trypsin inhibitor retains full activity through mashing, but is completely inactivated during boiling and hopping.     

I. CONDITIONS OF MASHING AND CARBOHYDRATE COMPOSITION OF THE WORT

Experimental mashings of ungerminated barley and 5–10% of malt with addition of the debranching enzyme pullulanase have been carried out. Worts with high attenuation are obtained in good yield. Of the fermentable sugars, there is less glucose, and more maltose and maltotriose than normally observed. The dextrin pattern is different from, but not necessarily inferior to, that traditionally seen. The worts resulting from the action of pullulanase are deprived of the dextrins with 8–14 glucose units, whereas the amounts of dextrins with 4–6 glucose units are close to those normally observed. The pullulanase preparations used are accompanied by proteolytic activity. It is suggested that debranching enzymes such as pullulanase offer an alternative choice of carbohydrases to be used in brewing from unmalted cereals.     

QUANTITATIVE CHANGES IN THE CONCENTRATIONS OF THE MAJOR COMPONENTS OF FOUR GENOTYPES OF BARLEY GRAINS DURING MALTING AND MASHING

Four genotypes of barley, including good and poor malting varieties, were sampled as grain, green malt, kilned malt and spent grains. Each of these samples were analysed for total protein, aggregated protein, total and soluble β-glucan, starch and husk contents. Protein sub-units were separated using sodium dodecyl sulphate polyacrylamide electrophoresis. Activities of β-glucanase, endopeptidase and α-amylase were measured and starch from each sample was purified and separated into large and small granules, and analysed for total protein and sub-unit protein content. Results calculated as % of dry weight and as a proportion of the weight of dry grain showed quantitatively the changes which occurred in the components of the grain during malting and mashing. Comparisons of the composition of the genotypes at the various stages showed that the best malting variety studied, Ark Royal, was better because of moderate superiority in several characters rather than a fundamental difference in a single attribute and supports the thesis that to further improve malting quality plant breeders should select for several characters which are independently inherited.     

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.     

SOME RELATIONSHIPS BETWEEN THE PROTEIN NITROGEN OF BARLEY AND THE PRODUCTION OF AMYLOLYTIC ENZYMES DURING MALTING

Barleys studied (Chariot and Delibes) contained different levels of extractable β-amylase enzymes. The potential levels of β-amylase enzymes of the two varieties studied were similar at 1.4 to 1.7% total nitrogen. Higher values of potential β-amylase enzyme were observed in the Delibes barley of higher total nitrogen of 1.9%. The higher level of β-amylase found in the barleys with the highest total nitrogen was not reflected in the protein banding patterns as revealed by SDS-PAGE protein fractionation. Extraction of barley proteins was largely influenced by the different extractants used. The alcohol soluble proteins, M_r 97 kDa (D-hordeins), were only extracted when mercaptoethanol was included in the extracting solution. Although barleys with the highest nitrogen (1.9%) had the highest apparent potential to develop β-amylase enzymes, the better modified low nitrogen barleys produced higher levels of β-amylase and α-amylase when malted. Dehusking revealed that the high nitrogen barleys contained more steely grains. In contrast, the low nitrogen barleys contained more mealy grains. Steely grains contained more nitrogen than mealy grains and had the greater potential to develop β-amylase. Notwithstanding, the results of this study suggested that the proteins of the lower nitrogen barleys (1.4–1.7%) were capable of producing higher levels of β-amylase and α-amylase than the higher nitrogen barleys (1.9%) over comparable periods of malting. The high apparent β-amylase potential of the barley was linked to high nitrogen levels and associated high levels of steeliness, whilst the corresponding high β-amylase levels of malt were linked to the efficiency of endosperm modification of the malted grain.     

THE EFFECTS OF MASHING TEMPERATURE AND MASH THICKNESS ON WORT CARBOHYDRATE COMPOSITION

Temperature and mash thickness are shown to affect both mash performance and enzyme activity. Alpha amylase was found to be considerably more resistant to heat inactivation than was beta amylase. This difference was reflected by changes in wort fermentability that were manifest at temperatures below those which affected levels of extract. Increasing the mashing temperature from 65°C to 80°C had only a slight effect on extract but reduced wort fermentability from over 70% to less than 30%. At 85°C and over, when temperature had a significant effect on alpha amylase, as well as on beta-amylase, extract was lost and starch was present in the wort. Diluting the mash with liquor had a similar effect to that of increasing temperature on both the amylolytic enzymes and on the mash performance. Thin mashes contained more starch and fewer fermentable sugars than did thick mashes at the same temperature. These changes can be related to the stability of the amylolytic enzymes.     

HEAT SENSITIVITY,OPTIMUM pH AND CHANGES IN ACTIVITY OF SORGHUM PEROXIDASE DURING MALTING AND MASHING

Peroxidase activity was demonstrated in dry and germinating sorghum seeds. The specific activity increased about 14-fold during malting for a 96-hour period. On the average about 41% of peroxidase activity was located in the endosperm, and the remaining 56% in the acrospire and rootlet of sorghum malt. The crude enzyme extract retained 77%, 17.5% and 7.6% of activity after heating at 60°, 70° and 80°C, respectively. More than 50% of the peroxidase activity in the finished malt survived mashing at 65°C. Optimum activity was recorded at pH 5.5 which falls within the observed pH range of sorghum worts. The level of residual peroxidase activity in the wort differed with sorghum species.     

THE DEGRADATION OF β-GLUCAN DURING MALTING AND MASHING: THE ROLE OF β-GLUCANASE

Significant β-glucanolysis takes place during mashing and is catalysed by a β-glucanase which is specific to mixed-linkage β-glucans. The enzyme develops during the germination of barley, but is rapidly and extensively destroyed in kilning. Partially-purified preparations of β-glucanase are protected from denaturation by heat if their solutions are adjusted to pH 4 or if bovine serum albumin is added. However the most effective stabiliser of the enzyme is reduced glutathione. Oligosaccharides containing three and four glucosyl units are produced by the action of β-glucanase and they are further converted during malting and mashing by a different enzyme(s) to disaccharides and glucose.     

THE FATE OF AMINO ACIDS DURING MASHING AND HOP BOILING

In an infusion mash system the concentration of all α amino acids in sweet wort reaches a maximum value when approximately one-third of the total volume extracted has been run from the mashing vessel. The relative concentrations of each α amino acid in wort vary throughout the mash tun extraction owing to differences in rate of extraction of the amino acids and to the quantities produced by peptidase activity in the mash tun. The relative proportions of proline, alanine, glutamine and asparagine in the total α amino nitrogen content of wort decrease appreciably as the mashing process proceeds. Copper boiling results in the partial destruction of methionine, glutamic acid, lysine, arginine and valine present in sweet wort together with an increase in the concentration of ammonium ion in the wort.     

THE FATE OF HUMULONE DURING WORT BOILING AND COOLING

Variations in the nature of the grist, mashing temperature, gravity and content of anthocyanogens of wort influenced the yield of break obtained after boiling with humulone, but not the bitterness of the resulting worts. Only small amounts of resin were found on the break and although increased break formation resulted in increased adsorption of resin this did not lead to measurable differences in the bitterness of wort. The bitterness of the cooled wort was independent of the degree of aeration and speed of cooling.     

THE PROTEIN RESERVES OF THE BARLEY GRAIN AND THEIR DEGRADATION DURING MALTING AND BREWING

The hordeins of barley are the main components of the grain protein, comprising B (sulphur rich), C (sulphur poor) and D (high relative molecular mass) species. A gel fraction can be isolated that consists of the D and part of the B hordein. Other important components of the reserve protein are β-amylase, protein Z and enzyme inhibitors as well as endosperm cell wall protein and protein associated with the starch granules. Degradation of protein is mediated by peptidase enzymes, most important of which are the endopeptidases and the exopeptidases; the latter having specificity for the carboxy- or amino-terminal region of a polypeptide chain. A very high activity of carboxypeptidase develops in the germinating barley grain. Five carboxypeptidases have been identified in malt and three fully characterized. Several endopeptidases have been identified, which have optimum activity close to pH 5.0 (that of the starchy endosperm) and studies with active site inhibitors suggest that sulphydryl dependent species are the most important. However, only limited use has been made of hordein as a substrate in the measurement of peptidase activity in malt. Lack of a complete understanding on the peptidases and their regulation in the germinating barley is a major limiting factor in the knowledge on the mobilization of the protein reserves during malting and mashing. Both de novo synthesis and activation of the peptidases have been demonstrated and the hormones gibberellic acid and abscisic acid appear to have a regulatory role. A controlled modification of the protein reserve is required so that there is an appropriate supply of amino acids for the brewers yeast but retention of proteins important in the desirable foaming component of beer. It is also important that proteins which can interfere in the final stages of brewing, e.g. those with chill haze forming potential, are broken down. An early hydrolysis is required of cell wall proteins and those proteins intimately associated with the starch granules.     

EFFECT OF MALTING TEMPERATURE AND TIME ON ENZYME DEVELOPMENT AND SORGHUM WORT PROPERTIES

The effect of malting temperature and time on enzyme development and wort properties of an improved Nigerian sorghum cultivar (Ex-Kwara) were investigated. Malting was carried out at two temperature regimes, 20°C and 25°C for eight days. Parameters evaluated included α- and β;-amylase development, hot water extract (HWE), soluble extract, fermentability, fermentable extract, viscosity, filtration rate, reducing sugars, α;-amino nitrogen and total soluble nitrogen (TSN). For virtually all the parameters studied, germination at 25°C produced higher values on the 4th day after which temperature appeared to have little influence. α;-Amylase development continued throughout the germination period while β;-amylase peaked on the 6th day. Optimal values of total soluble nitrogen (TSN) were recorded at both 25°C and 20°C at the 6th and 8th day of germination respectively .     

THE INFLUENCE OF ASSIMILABLE NITROGEN COMPOUNDS IN WORT ON THE ABILITY OF YEAST TO REDUCE DIMETHYL SULPHOXIDE

Dimethyl sulphoxide (DMSO) reductase activity increased at the end of the growth cycle of Saccharomyces cerevisiae NCYC 240 on complex medium. On defined medium, DMSO reductase and thioredoxin reductase activities and thioredoxin were all elevated in cultures limiting in nitrogen, but not in nitrogen-sufficient cultures. In nitrogen limiting cultures it was also shown that the reduction of DMSO (measured as disappearance of [¹⁴C] from the medium) was much increased compared with conditions of nitrogen excess. This applied whether ammonium or glutamate was the limiting nitrogen source. When methionine was used as the limiting nitrogen source, DMSO reduction was higher still. Methionine-limited cells accumulated [¹⁷C] methionine much more rapidly than did cells from methionine-sufficient cultures, and [¹⁴C] methionine sulphoxide could be identified in the cells after 1 min incubation with labelled methionine. It is concluded that DMSO reductase activity is controlled by nitrogen catabolite repression and may be involved in methionine uptake by yeast. The amino nitrogen content of wort will in consequence have an important effect on levels of dimethylsulphide (DMS) produced during fermentation.     

THE USE OF RAPID SCREENING TESTS TO COMPARE CHANGES DURING MALTING IN SORGHUM

Eight sorghum cultivars were malted and samples were taken after steeping and after each of 5 days germination. In genotypes with low levels of grain nitrogen, up to 35% of the nitrogen solubilisation during the malting process occurred in the steeping phase. After 5 days germination, extracts ranged from very low to moderate levels and correlated highly with diastase and half-grain mash results. In addition, genotypes producing higher extracts showed greater reductions in milling energy between 2 and 5 days germination. During malting, there was considerable genotype X day interaction for several quality characters, suggesting variation in rates of development as well as final quantities.     

DETERMINATION OF FERMENTABLE SUGARS AND NITROGENOUS COMPOUNDS IN WORT BY NEAR- AND MID-INFRARED SPECTROSCOPY

Near-infrared (NIR) and mid-infrared (MIR) spectroscopy techniques were evaluated for the analysis of brewery worts. Good calibrations were obtained for maltose (SEP_NIR 2.6 g/l, SEP_MIR 2.0 g/l), glucose SEP_NIR 0.6 g/l, SEP_MIR 0.4 g/l) and the sum of fermentable sugars (SEP_NIR 2.8 g/l, SEP_MIR 2.6 g/l) with both methods. Satisfactory calibration was obtained for maltotriose (SEP_NIR 1.4 g/l, SEP_MIR 1.5 g/l), nitrogen (SEP_NIR 122 mg/l, SEP_MIR 92 mg/l) and free alpha-amino nitrogen (SEP_NIR 29 mg/l, SEP_MIR 23 mg/l). MIR spectroscopy gave slightly better results, but sample handling was more difficult than in the NIR region. The NIR measurement is simple and rapid and could be used for on-line process control during mashing. No sample preparation other than filtration is needed.     

SYSTEM OF WORT ANALYSIS. IV: THE ESTIMATION OF THE TRUE PROTEIN NITROGEN OF WORTS AND BEERS

A method is given in which wort or beer is heated with sufficient sodium nitrite and acetic acid to deaminate the nitrogen compounds present. On cooling a precipitate forms which, the experiments suggest, represents the true protein* present, since it appears to correspond to the albumin and globulin of barley extracts and to include the nitrogen compounds of wort which are coagulable by boiling. It is possible that the amount of this fraction bears a relation to non-biological haze, while a subsequent paper suggests an important effect on fermentation. The results indicate that boiling conditions play an important part in controlling the amount, as also may the amount of tannin in hops and fermentation conditions.     

THE LOSSES OF BITTER SUBSTANCES DURING FERMENTATION

Almost all of the bitter substances which are lost during pilot-scale brewery fermentations using yeast N.C.Y.C. 240 can be recovered unchanged. The largest losses of bitter substances are associated with the dirty head formed during the first 18 hours of a fermentation. Hop utilization can be improved by folding this dirty head back into the fermenting wort. This process is most effective when carried out after the ethanol concentration of the wort has reached about 1%, but before the bitter substances have had the opportunity to undergo extensive oxidative degradation. Losses of bitter substances are lower when 25% of a malt grist is replaced by wheat flour. There is no consistent relationship between losses of bitter substances and either the assimilable or total nitrogen of wort. With increase in wort O.G. there is increasing loss of bitter substances, such losses being principally associated with the dirty head.     

THE CHEMISTRY OF NITROSAMINE FORMATION: RELEVANCE TO MALTING AND BREWING

This paper reviews the chemistry of nitrosamine formation with special reference to malting and brewing. Whilst the main emphasis is on the formation of nitrosodimethylamine (NDMA), the chemistry is also related to the formation of other nitrosamines and certain other nitrocompounds. It seems likely that in malting and brewing the only important source of the nitroso group is the mixture of oxides of nitrogen (NO_x) in the hot air in contact with malt during kilning. Under normal commercial conditions there appears to be little if any formation of nitrosamines at other stages of malting or brewing. It is suggested that much of the nitrosation is due to NO_x which exists temporarily in the form of nitrogen trioxide (N₂O₃) and nitrogen tetroxide (N₂O₄) as it dissolves in water in the moist malt or in malt lipids. The dissolved NO_x subsequently reaches equilibrium with nitrite and nitrate ions formed from it. The origin of the dimethylamine portion of the NDMA is still not clear. The most likely sources are free DMA, gramine and hordenine, all of which are produced in germinating barley. On balance it seems that hordenine is the most important source of NDMA in the malt at the end of kilning. The reasons for the low yield of nitrosamines from the precursors and NO_x present during kilning are discussed and related to the formation of other nitroso- and nitro-compounds. The occurrence of competing reactions which reduce nitrosamine formation is also described.