IMPROVED EXTRACTION AND ASSAY OF β-AMYLASE BROM BARLEY AND MALT

β-Amylase was extracted from barley or malt using four physical techniques to break up grists which had been prepared using a Moulinex coffee grinder. Grinding with a Polytron homogeniser apparently completely disrupted all cells, as determined by transmission electron microscopy, and increased the efficiency of extraction of β-amylase from barley by more than 30%. The other treatments tested were without value . The β-amylase activity in extracts of barley or malt was assayed by measuring the production of reducing sugars from reduced soluble starch, using a PAHBAH reagent. α-Amylase, which interferes with the quantitation of β-amylase in extracts of malt, was not totally inactivated by the chelating buffer used for enzyme extraction or by several other chelating agents. α-Amylase activity was quantified specifically using Phadebas. Using purified α-amylase a calibration was developed which related activity, as determined using Phadebas, to reducing power units. Thus the α-amylase activity present in an extract containing β-amylase could be determined using Phadebas and the reducing power equivalent activity subtracted from the total “apparent” activity to give the actual β-amylase activity. α-Glucosidase and limit dextrinase activities are believed to be too low to have a significant effect on the apparent β-amylase . The soluble and bound β-amylase activities were measured in samples taken from micromalting barley (Alexis). Dry weight losses increased to over 10% after 8 days germination. Antibiotics, applied during steeping, were used to control microbes in one experiment. However, their use checked germination and reduced malting losses to 8.4% in 8 days germination. The soluble enzyme present in extracts from steeped barley and early stages of germination was activated (20–40%) by additions of the reducing agent DTT .     

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.     

OPTIMIZED McCLEARY METHOD FOR MEASUREMENT OF TOTAL β-AMYLASE IN BARLEY AND ITS APPLICABILITY

The McCleary method for determination of β-amylase in malt has been modified in order to allow determination of total β-amylase in barley as well as malt. A ruggedness test, performed on the modified method, demonstrated that the method is quite robust and highly reproducible. When the variables α-amylase, β-amylase and diastatic power were measured in 90 malt samples, only β-amylase was significantly correlated to diastatic power (r² = 0.85 and p < 0.0001). The same high correlation was found between total β-amylase in 20 barley samples and diastatic power in the corresponding malts. The validity of this relationship was tested by predicting diastatic power in malt from total β-amylase in barley. Predicted values correlated highly to measured values (r² = 0.95). In breeding material a positive relationship was found between total β-amylase in barley and protein content. This relationship must be considered when evaluating new barley lines.     

MALT DIASTATIC ACTIVITY. PART II. A MODIFIED EBC DIASTATIC POWER ASSAY FOR THE SELECTIVE ESTIMATION OF BET A-AMYLASE ACTIVITY,TIME AND TEMPERATURE DEPENDENCE OF THE RELEASE OF REDUCING SUGARS

Ammoniumcitrate, sodium EDTA and ammonium oxalate were tested as possible inhibitors of α-amylase in the EBC diastatic power assay. A contact time of 90 min at a concentration of 0·1 M ammonium oxalate was shown to inactivate α-amylase completely in the enzymic malt extract while only very slightly enhancing β-amylase activity. The selective release of reducing sugars by β-amylase in the diastatic power assay was evaluated as a function of time using a p-hydroxybenzoic acid hydrazide assay and compared with that of the diastatic power procedure with the same colourimetric technique. The diastatic power results of eight different malts are well correlated with those of the β-amylase activity (r² = 0·98). In a linear regression model the diastatic power results at 20°C of the same malts correlated better with results obtained at 55°C (r² = 0·77) than with those obtained at 60°C (r² = 0·63). For the results of β-amylase activities we found r² = 0·86 for the relation between the activities at 20°C and at 55°C and r² = 0·92 for the relation between the activities at 20°C and 60°C. Since the temperature conditions (20°C) of the EBC-diastatic power assay do not correspond to those of brewing practice it is suggested that the activity of β-amylase as well as the malt diastatic power be evaluated at 55°C.     

QUANTITATIVE DETERMINATION OF α-AMYLASE ENZYMES IN GERMINATED BARLEY AFTER SEPARATION BY ISOELECTRICFOCUSING1

Quantitative extraction of malt and germinated barley α-amylases from polyacrylamide gels after isoelectricfocusing was achieved using bovine serum albumin (2 mg/ml) in the extracting medium. Sharp bands of activity were obtained when extracts from polyacrylamide gels were re-focused on another gel. This technique demonstrated that α-amylase III was the major component in malt and germinated barley extracts. This enzyme was converted to α-amylase II when such extracts were heated at 70°C.     

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.     

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 .     

EFFECT OF pH,TEMPERATURE, AND CALCIUM IONS ON BARLEY MALT α-AMYLASE ISOENZYMES

In this study we have detected the α-amylase isoenzymes in the two barley varieties ‘Ingrid’ and ‘Pomo’. We further report on the effect of different pH and temperatures on the activities of the α-amylase I and II (following the nomenclature of MacGregor & Daussant¹⁸) in ‘Pomo’ malt, as well as the stabilising effect of Ca²⁺ under such different conditions. The importance of enzyme concentration and the inhibition of Hg²⁺ is also considered.     

ENHANCEMENT OF AMYLOLYTIC POTENTIAL OF SORGHUM MALTS BY ALKALINE STEEP TREATMENT

The effect of alkaline steep liquor on sorghum maltability was investigated using three improved Nigeria sorghum cultivara. Germination was for four days at 30°C after steeping under four different regimes. Grain germinability, root length and malting loss were significantly (P < 0.001) repressed by steeping in alkaline liquor for all the cultivars. However, the extent of this repression seemed in all cases significantly dependent on cultivar and steep regime plus their possible interactions. Similarly, the development of diastatic enzymes activities appeared to be highly significantly dependent on steep liquor, steep regime, cultivar, plus their possible pairwise and three way interactions. For ICSV400, highest diastatic power and α-amylase development were attained on steeping grains in alkaline liquor under a continuous steep regime incorporating final warm steep treatment. Conversely, exposure of KSV8 and SK5912 to a regime incorporating air-resting and final warm steep significantly enhanced diastatic power and α-amylase development. β-Amylase activity was in all cases enhanced by alkaline steeping. In fact, β-Amylolytic activity constituted over 70% of total diastatic activity in most alkaline steeped ICSV400 malts. However, SK5912 exhibited relatively low hot water extract in spite of the improved amylolytic activity.     

AN INTER-LABORATORY STUDY OF STARCH DEGRADING ENZYME ASSAYS

The reliability of specific colorimetric assays for the main starch degrading enzymes, α-amylase, β-amylase and limit dextrinase was investigated by conducting an inter-laboratory collaborative study. Three malt samples were assayed in duplicate in 14 laboratories. The repeatability (r) within laboratories was good for α-amylase and β-amylase assays. Between-laboratory reproducibility (R) was acceptable (CV_R 9.2–14%). Errors for limit dextrinase were higher. Repeatability was good at high enzyme concentrations (CV_r = 2%) but increased at low enzyme concentrations. Reproducibility was relatively poor (CV_R-27–33%).     

INFLUENCE OF MALTING CONDITIONS ON QUALITY OF FINGER MILLET MALT

Effect of processing conditions on dry matter loss and quality characteristics of malt from INDAF-6 variety of finger millet was studied. Increasing the temperature of malting from 15 to 35°C caused increased dry matter loss. Germination at 15–20°C gave malts of higher α-amylase activities than those germinated at 30–35°C. Germination at 20°C for 4–5 days gave malt with maximum enzyme activity and acceptable dry matter loss. Drying the malt to 12% moisture and kilning at 70°C for about 45 min resulted in malt of desired aroma, maximum retention of α-amylase and minimum loss of available lysine. The α-amylase in the malted flour caused the slurries to have low hot paste viscosity.     

STUDIES ON COMPARATIVE MALTING CHARACTERISTICS OF SOME TROPICAL CEREALS AND MILLETS

Malts prepared from some of the tropical cereals and millets were evaluated for their suitability in weaning food formulations. Pearl millet and finger millet malts exhibited high α-amylase activity within 2–3 days of germination, while maize, sorghum, wheat and triticale malts showed high enzyme activity after 4–5 days of germination. Malting drastically lowered the paste viscosity of cereal flours especially millet. Finger millet malt had desirable flavour and taste besides high amylase activity. Wheat and triticale malts also had acceptable flavour. Rice malt was bitter and pearl millet malt developed rancid odour and bitterness within a week after preparation.     

PURIFICATION OF BARLET MALT α-AMYLASE BY IMMUNOAFFINITY CHROMATOGRAPHY

Immunoaffinity chromatography was used to purify the high pl α-amylase (α-amylase II) in a one step procedure after fractionation of the whole barley malt extract on Sephadex G25. The immunoglobulin G (IgG) fraction of an immune serum specific for the malt α-amylase II was immobilized on Ultrogel. A mild desorption procedure was used, combining distilled water elution with an interrupted elution. The purification was achieved within half a day including kernel extraction. The quality of the purification was assayed by SDS polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and isoelectric focusing. For the second technique, an immune serum was used which was polyspecific for malt proteins including the high pl α-amylase (α-amylase II). The effect of this procedure on the specific activity of the enzyme and on its antigenicity was evaluated. The results underline the efficiency of the purification procedure and indicate that α-amylase II accounts for a few percent of the total soluble protein in malts. However, the α-amylase II fraction was not completely free from α-amylase I. The procedure resulted in a partial loss of the enzymatic activity but not of the antigenicity.     

DEVELOPMENT OF AMYLOLYTIC ACTIVITIES IN SORGHUM AND BARLEY MALT

A Nigeria-grown sorghum cultivar was observed to develop more Diastatic Power (DP) and β-amylolytic activity than a previously studied sorghum cultivar grown in Australia. Barley malt produced more Diastatic Power and β-amylolytic activity than the Nigeria-grown sorghum cultivar. In general, the kilned barley malt retained less α-amylolytic activity than kilned sorghum malt. The different diastatic potential of sorghum cultivars may reflect differences in genetic character or growth conditions.     

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.     

‘FREE’ AND ‘BOUND’ β-AMYLASES DURING MALTING OF BARLEY. CHARACTERIZATION BY TWO-DIMENSIONAL IMMUNOELECTROPHORESIS

Major qualitative and quantitative changes in the β-amylases and in other salt soluble barley proteins occurred during the first four days of germination. Two soluble forms of barley β-amylase, ‘free’ β-amylase and β-amylase aggregated with a non-active protein Z, were found in extracts from all stages. A third enzyme form appeared during malting. Immunoelectrophoretic characterization seemed to support the possibility that this enzyme form could be a product of ‘bound’ β-amylase solubilization. All soluble forms of β-amylase and of protein Z in malt were electrophoretically heterogeneous. Two different, immunochemically related forms of protein Z present after malting retained their immunoelectrophoretic properties during brewing and were found to be dominant antigens in beer.     

α-AMYLASE COMPONENTS IN EXCISED,INCUBATED BARLEY EMBRYOS*

Excised embryos from kernels of Betzes and Bonanza barley were incubated on sand for 3 days in the presence of CaCl₂ (10-3³M), CaCl₂ + gibberellic acid (10-⁴M), CaCl₂ + casein hydrolysate (0·2%) and a mixture of all three. α-Amylase components in these embryos were analysed quantitatively by chromato-focusing. The α-amylase I component predominated in all samples. Addition of gibberellic acid or casein to the incubation medium did not increase α-amylase synthesis but addition of casein did appear to lower the proportion of α-amylase I synthesised.     

THE BREWING POTENTIAL OF “ACHA” (DIGITARIA EXILIS) MALT COMPARED WITH PEARL MILLET (PENNISETUM TYPHOIDES) MALTS AND SORGHUM (SORGHUM BICOLOR) MALTS

The malting and brewing characteristics of millets (Pennisetum typhoides and Digitaria exilis) and sorghum (Sorghum bicolor) were compared. Diastase, α-amylase, amyloglucosidase and proteases increased with malting time and the increase was associated with the modification. Development of hydrolytic enzymes was significantly higher in pearl millet and Digitaria exilis (“acha”) than in sorghum at P ≥ 0.01. The major starch degrading enzyme in the three varieties of pearl millet (SE composite, SE.13 and SE 2124) was α-amylase. On the other hand, β-amylase was the major starch degrading enzyme in “acha” (Digitaria exilis) which is similar to the pattern in barley. Gibberellic acid had a stimulating effect on the diastatic activity of pearl millets, Digitaria exilis (“acha”) and sorghum (KSV-4), but inhibited the diastatic activities of sorghum (Farafara). Gibbereltic acid inhibited the proteolytic activities in all the pearl millet varieties, Digitaria exilis and sorghum varieties. Potassium bromate had little or no effect in the reduction of malting losses. Although “acha” (Digitaria exilis) had a high β-amylase content, a high malting loss makes it uneconomical to brew with “acha” mart. A blend of “acha” malt with pearl millet malt or sorghum malt (composite malt) will produce a malt of the same profile as barley malt and this will enhance the quality of sorghum and pearl millet malt during the mashing process. Wort quality of all the samples was suitable for brewing conventional beer.     

Biochemical changes in phosphorus compounds and in the activity of phytase and α-amylase in the rice (Oryza sativa) grain during germination

Changes in the phytic acid, inorganic phosphorus and ATP contents, and in the activity of phytase and α-amylase in rice (Oryza sativa L) grains were determined during 18 days of germination in a dark room. The effect of phytic acid on α-amylase activity was studied in vitro. Rice grains immersed in sterilised deionised water at 14^°C germinated on the fifth day. Phytase activity, detected in the ripening rice grains, increased linearly until the eighth day and reached a maximum on the tenth day. There was a marked decrease in phytate and an increase in inorganic phosphorus accompanying germination. There was a good inverse correlation between the levels of both phytase activity and inorganic phosphorus, and phytate breakdown. α-Amylase activity was detected on the fourth day and increased markedly from the 12th to the 16th day of germination. ATP level increased from the second to the fourth day and slightly decreased from the fourth to the eighth day; it increased rapidly again from the eighth to the 18th day of germination. α-Amylase activity was influenced by both pH and phytic acid concentration in the assay system. At 75 mM phytic acid, α-amylase activity was lowered by 23%, 93% and 52% at pH 4–0, 5–0 and 6–0 respectively. When the enzyme, phytate and Ca²⁺ were incubated together at pH 5–0, the inhibition of α-amylase by phytic acid was markedly decreased by addition of Ca²⁺. The chemical affinity of Ca²⁺ for phytic acid was higher in the reaction at pH 5–0 than in those at pH 4–0 and pH 6–0, and over 98% of Ca²⁺ in the reaction system was precipitated as Ca-phytate.     

PRODUCTION OF AMYLOLYTIC ENZYMES BY SEVERAL YEAST SPECIES

Several amylolytic yeast species, endomycopsis spp, schwanniomyces spp, pichia spp and saccharomyces spp have been compared for their ability to synthesise α-amylase (E.C. 3.2.1.1), glucoamylase (E.C. 3.2.1.3) and pullulanase (E.C. 3.2.1.9). Endomycopsis fibuligera strain 240 possessed the highest glucoamylase activity (208 nmoles glucose/min) and second highest α-amylase activity (128 nmoles maltose/min) and produced the largest amount of biomass. Schwanniomyces spp were the only yeast species studied which exhibited significant debranching activity. It was found that in endomycopsis spp, schwanniomyces spp and pichia spp, glucose, at a concentration above 3.0 × 10^?3 m , appeared to repress α-amylase activity. However, glucoamylase activity was not repressed at that glucose concentration.