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When 29 malts representing a wide variation in free amino nitrogen of wort were mashed for 3 h at 45°C, the increases in TCA-soluble amino nitrogen varied from 47 to 116 μmoles per gramme of malt. The carboxypeptidase activities of the malts showed only small positive correlations with the increase of amino nitrogen during mashing. Inactivation of the different malt carboxypeptidases by 70 to 90% with diisopropylfluorophosphate decreased the liberation of amino nitrogen by only 5 to 25%. Moreover, addition of purified malt carboxypeptidase I to the mashing suspensions had no effect on the liberation of amino nitrogen. It is therefore evident that the carboxypeptidases do not have a major rate-limiting role in the liberation of amino acids in mashing, although it has earlier been shown that they are essential enzymes in this process. The proteinase activities of the malts, determined using two methods, correlated strongly with the liberation of amino nitrogen in mashing. Addition of crude malt proteinase to the mash increased, and addition of purified barley proteinase inhibitor decreased the measured proteinase activity and the liberation of amino acids roughly to the same extent. These results indicate that the most important rate-limiting enzyme activity in the liberation of amino acids in mashing is the proteinase activity of the malt.

The properties and enzymic activity of endoglucanases (EC 3.2.1.4) of the fungus Trichoderma reesei were studied by means of immunological methods and by using polyglycosidic substrates. Endoglucanases exist in the culture liquid as a series of immunologically related components. The most active endoglucanase component has an Mr of 43 000 and pI value of 4.0. The most abundant components have a value of pI about 5.0, an Mr of 56 000-67 000 and specific activity only one-fifth of that of the pI-4.0 component. During purification and storage the endoglucanases are spontaneously modified; the relative proportion of components having greater Mr values, more alkaline pI values and lower specific activities is increased. The hexose content of the endoglucanase components is 2-7%. Endoglucanases hydrolyse soluble β-1,4 glycans. The enzymes described here differ from endoglucanase preparations described previously in not showing activity towards insoluble substrates. The role of endoglucanases in wood hydrolysis is consequently limited to the stage where wood constituents are already in soluble form.

Insoluble xylan was prepared from ground birch (Betula pubescens) pulp by alkali extraction and precipitation with ethanol. The only sugar detected after acid hydrolysis of the preparation was xylose. The insoluble xylan was used as substrate in a nephelometric assay to determine the xylanase (EC 3.2.1.8, 1,4-beta-D-xylan xylanohydrolase and EC 3.2.1.37, 1,4-beta-D-xylan xylohydrolase) activities of Aspergillus and Trichoderma enzymes. The nephelometric method is reliable in evaluating xylanase hydrolysis of insoluble xylan.

The search for renewable raw materials to replace fossile fuel has directed research towards the utilization of various cellulosic materials. Cellulosic plant materials consist of three main components: cellulose, hemicellulose, and lignin. In order to develop an economically feasible process, it is necessary to utilize all these materials. Cellulose and hemicellulose can be decomposed either by enzymatic or acid hydrolysis to yield sugars. In the case of both hardwoods and annual plants the main part of the hemicellulose consists of pentosans yielding xylose on hydrolysis (Table 1). The hydrolysate thus contains mainly glucose and xylose which must be fermented to ethanol in order to obtain a combustible motor fuel. Glucose can easily be fermented by conventional yeasts or by some bacteria, e.g., Zymo-monas, but xylose presents a more difficult problem. Recently, there has been much interest in this field and the results are promising although no commercial process is available as of yet.

Les nouveaux developpements de la biotechnologie et leurs applications en malterie brasserie : utilisation et developpement de nouvelles varietes d'orge de brasserie, nouvelles techniques de maltage et de fermentation

The initial steps of D-xylose catabolism in Fusarium oxysporum have been studied. The presence of the oxidoreductase pathway for D-xylose catabolism was demonstrated. The enzymes involved, D-xylose reductase and xylitol dehydrogenase, were found to be inducible and relatively specific for D-xylose and xylitol. D-xylose isomerase was not detected.

A 1,4-beta-D-glucan cellobiohydrolase (EC 3.2.1.91) was purified from the culture liquid of Trichoderma reesei by using biospecific sorption on amorphous cellulose and immunoaffinity chromatography. A single protein band in polyacrylamide-gel electrophoresis and one arc in immunoelectrophoresis corresponded to the enzyme activity. The Mr was 65 000. The pI was 4.2-3.6. The purified enzyme contained about 10% hexose. The enzyme differs from previously described cellobiohydrolases in being more effective in the hydrolysis of cellulose.

Pectinatus cerevisiophilus strains were subjected to mild hydrochloric acid extraction. Electron microscopic examination revealed that the bacterial cells still retained their original form. The extract contained vesicles, derived mostly from the outer membranes of the bacteria. The extracted material from ten isolates was studied by SDS polyacrylamide gel electrophoresis and a number of different protein patterns were found. These allowed two main groups and four subgroups to be distinguished among the isolates. The molecular weights of the major proteins varied between 13 and 62 kilodaltons. The mild hydrochloric acid extractable protein patterns provide a useful tool for typing different strains.

A method for the analysis of zearalenone, T-2 toxin, neosolaniol and HT-2 toxin from the grains of barley, wheat and oats has been developed. Toxins are extracted with ethyl acetate, purified on a kieselgel TLC-plate and analysed on a HPTLC-plate. The limits of detection are 0.2 mg/kg for zearalenone and T-2 toxin and 5 mg/kg for neosolaniol and HT-2 toxin. For more accurate estimation the purified toxins are analysed as their trimethysilyl derivatives by gas chromatography, in which the detection limit for all toxins in 50 μg/kg and the accuracy ±10%–30%. The percentage recovery in both methods is 80%.

Steeping without any aeration or bubbling the steep water with nitrogen or carbon dioxide causes retardation of initial germination. However, no elongation of the germination period is needed to obtain malt of normal quality. Thus the malt yield is significantly increased, by 1% when using nitrogen and by 2-2½% when using carbon dioxide. The best results were obtained using carbon dioxide with a steeping procedure involving dry-steeping. The use of a dry-steeping period results in a more even germination.

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.

Barley contains a proteolytic enzyme which hydrolyses the synthetic substrate, benzoyl-arginine-p-nitroanilide. This enzyme has been purified by chromatography on DEAE-cellulose and gel filtration. During purification the activity of the enzyme increased 3–10 fold, an effect which is possibly due to removal of an unstable dissociable inhibitor. The enzyme is inhibited by SH-reagents only to the extent of 50–70%. Thus the thiol group does not seem to be at the active centre of the enzyme but somewhere sufficiently close to it to interfere sterically with the formation of the enzyme-substrate complex.

(1964). Modification of Barley after Embryonic Destruction by Freezing. Proceedings. Annual meeting - American Society of Brewing Chemists: Vol. 22, Proceedings of the Annual Meeting 1964, pp. 86-96.

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.

A carboxypeptidase has been purified from germinated barley. The preparations are homogeneous in ultracentrifugal analysis. In disc electrophoresis traces of impurities can be detected at high concentration. The purified enzyme liberates carboxyl-terminal amino acid residues from a wide range of carbobenzoxy (Z)-dipeptides. Peptides containing proline are not attacked. It does not possess any endopeptidase, aminopeptidase, or dipeptidase activity. The enzyme has a pH optimum at pH 5.2, is inhibited by di-isopropylphosphofluoridate and p-chloromercuribenzoate, and has a molecular weight of about 90,000.

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.

Sterile excised barley embryos were allowed to germinate in a medium containing U-14C- and 15N-l-leucine. After periods of 3 and 5 days, the whole embryos were subjected to acid hydrolysis and the resulting amino acids were analysed for isotopic labelling. While there was no significant spread of the 14C labelling to other amino acids, the appearance of 15N in all the amino acids showed that extensive transamination had occurred.

When using a mixture of two different malts, the wort nitrogen content remains in some cases lower than the value calculated according to the proportions of each malt. A similar effect can be obtained by the use of a mixture of malt and barley, and is especially seen in the amino acid content of the wort; this fall is caused by proteolytic inhibitors present in barley. The presence of both large and small molecular inhibitors has been demonstrated. A mixture of equal parts of barley and malt extracts gives a 50% inhibition of the endopeptidase activity. Owing to these inhibitory effects, the use of barley together with overmodified malt is advantageous in mashing.

2,4-Dichlorophenoxyacetic acid (2,4-D) reduces the growth of rootlets in malting to a minimum without seriously affecting the quality of the resulting malt as judged from analytical values. Gibberellin could advantageously be used together with 2,4-D, Small amounts of gibberellin did not affect the rootlet growth-retardation caused by 2,4-D. The extract yield was high in these makings. 2t4-D had no harmful effects on fermentation, but high concentrations seemed to impair the taste of the final beer. However, the concentration originally present in wort decreased by 50% during fermentation* Part of the 2,4-D was evidently metabolized by yeast. The results indicate that the combined use of gibberellin and some root growth restricting compound might lead to appreciable advantages.

The influence of moisture content and temperature during storage of grain on the formation of Fusarium metabolites was studied. Naturally and artificially contaminated barley grain samples were stored at 15%, 25%, and 30% moisture contents, and at temperatures of + 5°C + 25°C, and + 30°C. Time of storage varied between one week and six months and the occurrence of Fusarium species and metabolites was analysed. The only Fusarium metabolite detected was zearalenone. The extent of Fusarium contamination decreased during storage whilst the concentration of zearalenone increased. To avoid the danger of mycotoxicoses, grains must be dryed immediately after harvest and then stored at a low temperature.

The application of control engineering to biotechnology is a complex field requiring deep knowledge of both fundamental biological processes and highly developed technology. The possibilities of monitoring the course of a microbiological process by measuring changes in the culture liquid are limited. By modelling we may be able to solve the problem of controlling events in the cell. This and other aspects of the interactions between biotechnology and control engineering are discussed.

A comparative study has been made of the β-amylases of barley, wheat, rye, oats and sweet-potato by means of exclusion chromatography and immunochemical analysis. The reactivity of barley malt and wheat β-amylase was compared with different anti-barley and anti-wheat sera. In exclusion chromatography on Sephadex G100, barley β-amylase yielded four, and both wheat and rye, two active components, whereas oat and sweet-potato had only one active component. During the storage of barley, wheat and rye β-amylases the large-molecule components were split into smaller ones; no changes occurred in oat and sweet-potato β-amylases. On analysis against a specific barley β-amylase antiserum, wheat and rye β-amylase gave a reaction which indicated that they were immunologically partly identical with barley β-amylase, and identical with each other. This serum induced no reaction in β-amylases of sweet-potato and oats. The rye β-amylase precipitation line did not display enzymic activity after reaction with this antiserum. Analyses with different antisera of barley and wheat confirmed the partial immunological identity of barley malt and wheat β-amylase. With some barley antisera, partial inhibition of wheat β-amylase activity was observed. A similar phenomenon was apparent when barley malt β-amylase was precipitated with some wheat antisera.

Publisher Summary Cellulolytic enzymes do not hydrolyze efficiently well-ordered areas of cellulose, but attack only amorphous regions. This chapter presents a method in which a more refined amorphous substrate has been prepared by ball milling. This substrate is completely dissolved by cellulases and can be used to measure the initial velocity of the reaction. Initial hydrolysis is followed by means of a nephelometer and the slopes of nephelometric plots are used to calculate the cellulase activity. The initial reaction rate can also be estimated with a photometer where the activity is measured as the decrease in absorbance of a suspension of amorphous cellulose. A good correlation has been found between the initial rate of reaction and the amount of sugar released at given times.

Barley grain contains about 10% insoluble reserve proteins. When the grain germinates the reserve proteins are hydrolysed to amino acids and transported to the growing tissues of the seedling. In the resting grain most of the reserve proteins are 'packed' into the non-living storage tissue, the starchy endosperm. During germination the internal pH of the starchy endosperm is about 5, and it contains high activities of proteinases (secreted by the living aleurone cells) and carboxypeptidases, all with pH optima between 4 and 6. As a whole the starchy endosperm of a germinating grain resembles a giant secondary lysosome. Adjacent to the starchy endosperm is a specialized absorptive and processing tissue, the scutellum. This organ contains very high activities of the 'acid carboxypeptidases' and also two 'alkaline peptidase': a leucine aminopeptidase and a dipeptidase, both pH optima at 8 to 10. The high peptidase activities in the scutellum suggest that the hydrolysis products of the reserve proteins are absorbed from the starchy endosperm as a mixture of amino acids and small peptides, which are hydrolysed to amino acids in the scutellum before transport to the growing seedling tissues.

A new reference serum (No. 2) for barley and malt proteins has been released by the Barley Proteins Sub-Committee of the European Brewery Convention. Samples can be obtained from the Secretariat. The new antiserum, which provides better resolution than the first antiserum released in 1967, has been prepared by the immunization of a horse with a mixture of barley and malt proteins. As a result, it also reacts with malt proteins, such as α-amylase, which the first antiserum did not.