TOTAL AND INDIVIDUAL BARLEY (1–3), (1–4)-β-D-GLUCANASE ACTIVITIES IN SOME GREEN AND KILNED MALTS

Total β-glucanase activity was measured in extracts of malts prepared from three winter and three spring cultivars of barley. Samples were taken at intervals during modification and, after 116 hr, from malt kilned to 65°C. Good malting varieties showed highest levels of total β-glucanase activity, with a high correlation (r = 0.926) with HWE. Angora had the highest activity in the intermediate stages of malting, least loss of activity after heating extracts to 48°C for 10 mins and least loss of activity on kilning. Separation of isozymes by HPLC⁹ confirmed the greater heat stability of isozyme II, but, unlike previous studies on Australian cultivars, we found considerable activity of isozyme I after kilning, even up to 85°C. However, total β-glucanase activity was destroyed by heating extracts of all varieties to 60°C. Angora showed the highest proportion of total activity as isozyme II after kilining. Cultivar differences suggested some scope for breeding varieties with increased total activity by combining high activities of each isozyme. The high correlation of total activity with HWE suggests β-glucanase activity as a rapid test of malting quality.     

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.     

STUDIES ONα-GLUCOSIDASE IN BARLEY AND MALT*

A specific and sensitive method was used to determine α-glucosidase activity in barley and malt. Reliable results were obtained only after extracts of barley and malt had been dialyzed extensively to remove low molecular weight carbohydrates that interfered with the enzyme assay, α-Glucosidase was present in immature kernels of Bonanza and Ellice barley shortly after anthesis but enzyme levels fell rapidly as the kernels matured. A high proportion of the activity was present in pericarp tissue. Enzyme activity increased rapidly in Bonanza and Klages barley during initial stages of germination and fell only slightly during kilning. A high proportion of enzyme activity was present in the scutellum of 4-day germinated barley with lesser amounts in the aleurone and endosperm.     

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 .     

A Simulation Model for Malt Enzyme Activities in Kilning

The drying and survival of enzyme activities during the kilning of malt were modelled. A set of experiments at the micro‐malting scale was carried out for model identification and validation. The dynamic models predict the effects of the kilning programme, i.e. the temperature profile on grain moisture, activities of β‐glucanase, α‐amylase and limit‐dextrinase, and diastatic power during kilning. The process behaviour was analysed by simulations. The predictions match the malting experience well. The models increase the general understanding of enzyme inactivation and can be used in planning the kilning programme.     

Malting Performance of Normal Huskless and Acid‐Dehusked Barley Samples

Sulphuric acid dehusked barley had a higher germinative energy and lower microbial infection than normal huskless (naked) barley, suggesting that the pericarp layer harboured microbial infection which may have limited the germination rate. Dehusking the normal huskless barley using sulphuric acid resulted in lower microbial infection, and increased germinative energy. The normal huskless barley sample had a higher β‐glucan content than the acid‐dehusked barley and had slower β‐glucan breakdown during malting. This resulted in the release of seven times more β‐glucan during mashing, and the production of wort of higher viscosity. The normal huskless barley sample had a higher total nitrogen content than the acid‐dehusked barley but both samples produced similar levels of amylolytic (α‐ and β‐amylase) activity over the same malting period. No direct correlation was found between barley total nitrogen level and the amylolytic activity of the malt. When barley loses its husk at harvest, the embryo is exposed and may be damaged. This may result in uneven germination which can reduce malting performance and hence malt quality.     

A MICROBIOLOGICAL EVALUATION OF BARLEY MALT PRODUCTION

The development of the microflora of barley malt was examined by direct and dilution plating. At all stages of the malting process mesophilic bacteria predominated. Viable counts of bacteria on green malt were 85–600 times greater than on the original barley, but fell to less than one-half of the original level with kilning. Corresponding increases in yeast and, especially, mould counts during malting were smaller. The yeast-like mould Geotrichum candidum was prominent in green malt. Although counts of yeasts and most moulds were considerably reduced by kilning, Mucor spp. proliferated during kilning.     

Control of Superoxide Dismutase Activity during Malting Using Plackett‐Burman and Box‐Behnken Experimental Design and Its Effect on Reducing Power of Wort

The Plackett‐Burman multifactorial design was employed to screen the important malting parameters for superoxide dismutase (SOD) in final malt of Ganpi‐3. The eight factors screened for SOD were steeping temperature, steeping time, peroxide hydrogen concentration in steeping water, germination temperature, germination time, withering temperature, drying temperature and kilning temperature. Variance analysis showed that steeping time, germination temperature and kilning temperature were significant for SOD activity. Box‐Behnken experimental design was further used to optimize the levels of the above three factors. By response surface methodology and canonical analysis, the optimal malting factors for higher SOD activity in final malt were: steeping time 42.2 h, germination temperature 16.9°C and kilning temperature 82.9°C. Under these conditions, the model predicted a SOD activity of 2234 U/g of dry weight malt. Verification of the optimization showed that a SOD activity of 2220 U/g was observed under optimal conditions. It showed that the experimental data could be reliably predicted by the polynomial model. Besides Ganpi‐3, three other barley varieties including Ganpi‐4, Ken‐2 and Hamelin were malted under optimal and common conditions under laboratory conditions. To some extent, SOD activities were higher in malts from the optimal malting process than those from the common malting process. Especially, SOD activities in Ganpi‐3 and Hamelin increased by 18.8% and 15.3%, respectively. Furthermore, twenty‐nine samples of malts, including eleven imported malts and eighteen domestic malts, were used. Relationships between SOD activity in malt and the reducing power of wort were examined. There was significant correlation between SOD activity and the reducing power of wort (R² = 0.8069).     

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.     

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.     

Malt Performance is More Related to Inhomogeneity of Protein and β‐Glucan Breakdown than to Standard Malt Analyses

Nitrogen analyses of the grains of samples of commercial malts indicate that β‐glucan breakdown and the uniformity of malt modification are influenced by uniformity of distribution of grain protein. It is proposed that for normal malting barley, variations in malt modification are related to the different percentages of grains which contain high levels and different types of proteins which resist enzymic modification during malting. This kind of inhomogeneity of malt modification can cause brewhouse problems but cannot be detected with precision by standard malt analyses.     

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.     

Effects of Pulses of Higher Temperature on the Development of Enzyme Activity During Malting

The increase of temperature at the beginning, in the middle and at the end of malting has been evaluated in terms of quality parameters (malting losses, index of acrospire development, friability, HWE, viscosity, SNR) and enzyme (β‐glucanase and α‐amylase) development, in a good quality malting barley (Otis) and a higher protein‐higher β‐glucan content barley used for feed (Extra). A shift from 15 to 20°C at the beginning of malting was shown to increase acrospire development, friability, HWE and SNR and to reduce viscosity, without significantly affecting malting losses. This effect was related to higher β‐glucanase and α‐amylase activities within each variety. However, the same enzyme activities were not directly related to a better malting quality when the two genotypes were compared. This confirms previous indications that diversity in malting performance between genotypes cannot simply be traced back to differences in enzyme activities; but, indeed, it suggests that, for a defined barley lot, changes in the levels of enzyme activities following different malting procedures may have a direct effect on malt quality.     

Changes in β-glucan and other carbohydrate components of barley during malting

Changes in total (1→3), (1→4)-β-glucan content were followed during the micro-malting of nine varieties of barley with a wide range of malting qualities. These changes were related to estimates of endosperm modification based upon staining with Calcofluor. β-Glucan content declined from an average of 3.54% in the barley to 0.75% in the malt. Pentosan and total starch (including starch-derived oligosaccharides) levels showed comparatively little change during malting. β-Glucan composition of the barley was a poor indicator of malting performance. However, the β-glucan, starch and xylose contents of the malt all showed significant correlations with malt extract. Estimation of malt β-glucan content gave the best indication of malt quality. Direct determination of β-glucan may be of more value in assessing malt quality than indirect techniques based upon assessing modification of stained grains.     

The Evolution of Dextrins During the Mashing and Fermentation of All‐malt Whisky Production

The production of malt whisky involves the mashing of barley malt, followed by the fermentation of the resulting wort without further treatment. While this process has many parallels to the production of an all‐malt beer, one of the main differentiating steps during substrate preparation is the inclusion of a boiling step for the wort in the production of beer. Other than the destructive action of the boiling process on microorganisms, the boiling also destroys all malt enzyme activity. Since a typical whisky wash is not boiled it carries through a certain proportion of microbial activity associated with the malt, but more importantly it retains some enzyme activity that has been activated during the malting and mashing processes. The changes in sugars and dextrins during both mashing and fermentation of the resulting wash were investigated. Evidence of the continuous amylolytic activity during an unboiled, all‐malt wash fermentation is shown; while no ongoing amylolytic activity could be deduced during the fermentation of a boiled all‐malt wort. Furthermore, the data suggests that the amylolytic activity during mashing and fermentation are different with regards to α‐amylase action linked to its multiple‐attack action pattern as a function of substrate conformation, temperature, and effectiveness of potential hydrolytic events.     

‘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.     

THE DEVELOPMENT OF β-D-GLUCANASES DURING THE GERMINATION OF BARLEY AND THE EFFECT OF KILNING ON INDIVIDUAL ISOENZYMES†

Two endo-1,3;1,4-β-D-glucanase isoenzymes developed in response to gibberellic acid, during the germination of barley. Two endo-1, 3-β-D-glucanases, one present in ungerminated, steeped grain, also developed but did not appear to be markedly stimulated by the hormone. A comparison of crude and partially purified malt extracts highlighted the errors that are involved in the specific determination of endo-1, 3;1, 4-β-glucanase activity in crude extracts. The development and effect of kilning on individual malt isoenzymes was demonstrated by carboxymethylcellulose (CM-cellulose) chromatography profiles. Kilning and dry-milling of germinated barley caused losses of 80–90% in the specific endo-1,3;1,4-glucanase activity. The effect was less pronounced if wet-milling was substituted for dry-milling. Extraction studies and CM-cellulose chromatography profiles indicated that both endo-1,3;1, 4-β-glucanase isoenzymes were heat labile and were particularly susceptible to oxidation. In contrast, endo-1,3-β-glucanase activity and cellobiase activity in malt extracts were less affected by the kilning process or extraction procedures. Preliminary results suggested that one of the endo-1,3-β-glucanase isoenzymes was more sensitive to kilning.     

Malting of Sorghum: Further Studies on Factors influencing α‐Amylase Activity

The effects of steep regime, nature of alkaline steeping agent, and kilning condition on α‐amylase development were studied for four Nigerian sorghum cultivars. Malt α‐amylase activity was highly significantly (p<.001) influenced by all the four factors as well as their various assortments of interaction. Generally malts from the Local Red (LR) variety produced the highest a‐amylase values, followed by those of SK 5912, Local White and KSV 8 in the above sequence. The presence/absence of air‐rest processes in steep regimes was a significant factor (p<.001) influencing malt α‐amylase response to final warm steeping as well as to the other factors under study. Similarly, the nature of the steeping agent was a very significant determinant of malt α‐amylase response to kilning condition and regime of steeping. Of significant interest was the observation that Ca (OH)₂ steeps enhanced malt α‐amylase activity at the higher temperature of kilning. The significantly lower α‐amylase values given under similar conditions by the other alkaline liquors suggest a possible increase in malt thermostability due to steeping in Ca (OH)₂. Additionally, the fact that the extent of enhancement of malt α‐amylase activity by Ca (OH)₂, at 50°C Kiln temperature, was regime‐dependent, suggests that the latter was an important modulator of sorghum germination physiology.     

Effects of Grain and Malt â‐Glucan on Distilling Quality in a Population of Hull‐less Barley

A population of barley lines, derived by mutation in the hull‐less variety, Penthouse, was included in a replicated trial, along with Penthouse and the hulled malting cultivar, Optic. Samples were assessed for a range of grain quality traits, then malted, with germination for either 4 or 5 days, prior to kilning. Most lines had grain β‐glucan contents lower than that of Penthouse, but there was no significant correlation between grain and malt β‐glucan content. Malt β‐glucan levels were indicative of differences in cell wall breakdown between 4 and 5 days germination, but negative associations with distilling parameters Extract and Alcohol Yield, were not statistically significant. It was concluded that the lines differed in the rate and extent of cell wall breakdown and that grain shape may influence modification in distal parts of the grain. However, a malting regime, optimised to suit Optic may be less suited to discriminating between hull‐less lines of reasonable quality.     

The Influence of Barley Storage on Respiration and Glucose‐6‐Phosphate Dehydrogenase During Malting

Malt is produced by the controlled, but limited germination of barley. To produce good quality malt, the barley employed must be able to germinate rapidly and synchronously. Dormancy is a seed characteristic that can interfere with the rapid and uniform germination of barley, thereby reducing the resultant malt quality. Various studies have shown that post harvest storage can be used to remove dormancy and enhance barley germination characteristics and malt quality. Because of its complexity, the fundamental basis of dormancy induction, maintenance and termination remain unknown. Glucose‐6‐phosphate dehydrogenase (G6PDH) is the rate limiting enzyme of the pentose phosphate pathway and has been associated with dormancy decay and increased seed vigor of a variety of different seeds. The aim of this study was to determine if changes in barley germination vigour were associated with respiration and/or G6PDH changes during malting. Commercially grown barley (cv. Gairdner) was obtained from various states of Australia and stored at room temperature for up to 7 months. At 1, 3 and 7 months, samples were taken and stored at ?18°C. The germinative energy (GE) and germinative index (GI) of these samples were measured. Samples were micro‐malted and the α‐amylase activity, respiration rate, and G6PDH activity of the germinating grains were measured at various stages of malting. It was found that storage of barley for up to seven months significantly improved the germination characteristics and increased the α‐amylase activity during malting. However, these improvements were not associated with concomitant changes in respiration rate or G6PDH activity during malting.