ENZYMIC BREAKDOWN OF ENDOSPERM PROTEINS OF SORGHUM AT DIFFERENT MALTING TEMPERATURES

Enzymic breakdown of endosperm proteins of sorghum was more effective at 20°C than at 25°C and 30°C, as regards total protein solubilization, α-amino nitrogen and peptide production. Although the embryos (axes and scutella), of the three temperature treatments contained similar quantities of protein, it appeared that less proteins, in terms of amino acids and peptides, were transferred to the roots during malting at 30°C than at 25°C and 20°C. During mashing, higher levels of peptides but lower levels of α-amino nitrogen and total soluble nitrogen were released in an infusion mash at 65°C than in a decantation mash where enzymically active wort was decanted and used to mash gelatinized sorghum starch at 65°C. Although more of the maltose-producing enzyme—β—amylase was found in sorghum malts made at 25°C and 30°C than at 20°C, it would seem that, for sorghum, malting temperature of 20°C to 25°C were optimal as regards protein breakdown during malting. The protein breakdown produced when sorghum is malted at 20°C is comparable to that found in barley malt and should support similar levels of adjuncts and yeast growth during brewing.     

Effect of Mashing Temperatures and Endo-β-Glucanase on β-Glucan Content of Malt Worts

Similar basal levels of β-D-glucans were released into worts produced at 45°C from enzymically active or inactivated flours of milled malts. In contrast, significantly higher levels of β-D-glucans were found in worts derived from either enzymically active or inactivated malt flours mashed at 65°C. In general, mashing temperature may play a more important role in releasing β-D-glucans during mashing than enzymes described as β-glucan-releasing. In this context, the physical release of β-D-glucan during mashing should be separated from the enzymic release and degradation of β-D-glucan which occur during malting.     

THE VISCOSITY OF WORTS IN RELATION TO THEIR CONTENT OF β-GLUCAN

A procedure is given for assessing that proportion of wort viscosity which is attributable to β-glucan. Worts obtained from unkilned samples of malt which have been processed for 54 or 72 h show enhanced viscosity. This is principally due to β-glucan although the contribution of other constituents, absent from the wort of fully modified malt, is of significance. Barley variety is shown to have a pronounced effect on wort viscosity. Insoluble β-glucan is brought into solution in mashes at 65° C. The β-glucan isolated from malt which has been inactivated using aqueous ethanol prior to extraction at 65° C, is of higher specific viscosity than that isolated from control worts prepared at the same temperature. The introduction of a rest by mashing initially at 40° C results in the production of wort of lower viscosity, a decrease in the β-glucan content of the wort and a reduction in the specific viscosity of the β-glucan. There is no apparent relationship between the endo-β-glucanase content of the malts and either the viscosity of derived worts or the degree of breakdown of β-glucan which occurred during malting and mashing. Abrasion of barley, which is a factor assisting the distribution of enzymes during malting, acts to reduce wort viscosity.     

ROLE OF ALPHA-GLUCOSIDASE IN THE FERMENTABLE SUGAR COMPOSITION OF SORGHUM MALT MASHES*

The cause of the high glucose to maltose ratio in sorghum malt worts was studied. Mashing temperature and pH strongly affected both the amount of glucose and the proportion of glucose relative to total fermentable sugars. The relative proportion of glucose was higher when mashing was performed. at pH 4.0, close to the pH optimum for sorghum alpha-glucosidase, than at the natural pH of the mash (pH 6.0–5.5). Mashing according to the EBC procedure using an enzymic malt extract with pre-cooked malt insoluble solids producing a wort containing maltose and glucose in an approximately 4:1 ratio, whereas mashing with a malt extract without pre-cooking the malt insoluble solids resulted in a wort with approximately equal amounts of maltose and glucose. Both treatments gave the same quantity of total fermentable sugars and amount of wort extract. Sorghum alpha-glucosidase was confirmed to be highly insoluble in water. All or virtually all activity was associated with the insoluble solids. Hence, it appears that the high amount of glucose formed when sorghum malt is mashed conventionally is due to alpha-glucosidase activity. Pre-cooking the malt insoluble solids inactivates the alpha-glucosidase, preventing the hydrolysis of maltose to glucose.     

Free α-amino nitrogen production in sorghum beer mashing

Free α-amino nitrogen (FAN) is an essential nutrient for yeast growth during fermentation. Under normal conditions of sorghum beer mashing, 60°C at pH 4.0, production of FAN by proteolysis accounts for approximately 30% of wort FAN, the remaining 70% being preformed in the malt and adjunct. The quality of the FAN in sorghum beer worts is good as it does not contain a high percentage of proline. Optimum conditions for FAN production during mashing are 51°C and pH 4.6. Wort FAN was increased proportionally by raising the ratio of sorghum malt to adjunct and conversely decreased by raising the ratio of adjunct to malt. FAN was also increased by the addition to the mash of a microbial proteolytic enzyme. Wort FAN is directly proportional to malt FAN.     

FAILURE OF MERCURIC CHLORIDE TO SELECTIVELY INHIBIT β-AMYLASE IN SORGHUM MALT

Mercuric chloride has been reported to be a suitable reagent for the determination of α-amylase activity in sorghum malt, based on its ability to selectively inhibit β-amylase. In this re-investigation, the α- and β-amylase activities of eight sorghum malts were determined after treatment of malt extracts with various concentrations of mercuric chloride. At a malt: mercuric chloride ratio of 8.3 × 10³: 1, incomplete inhibition of β-amylase activity, as measured by the Betamyl assay, occurred in all extracts. However, this concentration resulted in significant inhibition of α-amylase activity in all extracts, as measured by both the Ceralpha assay and the Phadebas assay. In addition, α-amylase activity was found to be significantly inhibited at malt: mercuric chloride ratios as low as 1.0 × 10⁵: 1, when measured by the AmyloZyme assay. These findings do not support the original report that a malt: mercuric chloride ratio of 4.0 × 10³: 1 will selectively inhibit β-amylase in sorghum malt. Furthermore, in this context it should be emphasised that the original report was based upon inhibition studies conducted on β-amylase derived from barley, not sorghum malt .     

EXTRUDED SORGHUM AS A BREWING RAW MATERIAL

Small scale mashes (50 g total grist) with grists containing up to 50% by weight of extruded whole sorghum produced worts of high extract yield and low viscosity. Increasing the proportion of extruded sorghum in the grist resulted in decreasing wort filtration volume, total nitrogen and free amino nitrogen content. The wort filtration behaviour of mashes containing sorghum extruded at 175°C was superior to that of mashes containing sorghum extruded at 165°C or 185°C. The results from such small scale mashing experiments suggested that extruded sorghum compared favourably to extruded barley and extruded wheat as a brewing adjunct. Worts and beers were produced on a pilot brewery scale (100 1) from grists comprising 70% malt + 30% extruded sorghum and 100% malt under isothermal infusion mashing conditions. Mashes containing sorghum extruded at 175°C showed comparable wort filtration behaviour to that of the all malt control mash whereas mashes containing sorghum extruded at 165°C or 185°C showed poor wort filtration behaviour. Worts produced from grists containing extruded sorghum fermented more quickly than the control wort and attained lower values of final gravity. The resulting beers were filtered without difficulty. Beers produced from grists containing extruded sorghum contained lower levels of total nitrogen and free amino nitrogen compared to the control beer consistent with extruded sorghum contributing little or no nitrogenous material to the wort and beer. Beers brewed from grists containing extruded sorghum were of sound flavour and showed reasonable foam stability behaviour.     

DEVELOPMENT OF A MASHING PROFILE FOR THE USE OF MICROBIAL ENZYMES IN BREWING WITH RAW SORGHUM (80%) AND MALTED BARLEY OR SORGHUM MALT (20%)

Different time and temperature programmes were used to evaluate the production of hot water extract (HWE) and free amino nitrogen (FAN) from mashes containing raw sorghum and either malted sorghum or malted barley in the presence of microbial enzymes. Two malted varieties of sorghum (SK 5912 and Zaria) were used. The former gave higher HWE but lower FAN than the latter. Sorghum malts were unable to provide enzyme activity for starch extraction and exogenous enzymes were always needed. Seventeen commercially available enzyme preparations were assessed. A double-mash process was developed. Inclusion of calcium ions (200 ppm) was beneficial but adjustment of mash pH had little effect. Raw sorghum was gelatinised at 100°C for 30–40 min in the presence of a heat-stable α-amylase followed by mixing with a malt mash (started at B0°C) to give a temperature of 65°C with a total mash time of 167 min (127 min from mixing the mashes). The inclusion of a single commercial enzyme preparation (containing both proteolytic and amylolytic activities) was sufficient to achieve satisfactory HWE and FAN. Addition of different activities or combinations of activities gave no significant advantages. To obtain levels of FAN of 100–140 mg/l however excessive amounts of enzymes were required.     

Quality characteristics of triticale malts and worts

Worts from triticale malts, in particular well modified malts, separated poorly from mashes. Worts prepared at 70° C had high viscosities (10–27 cSt) indicating that problems would occur during filtration in brewing. The viscosities of triticale worts were higher than those of worts from barley malts. In addition, worts from well modified malts were generally turbid. Proteinaceous material (partly degraded prolamins) was the primary cause of this turbidity. Although the degree of malt modification did influence the rate of wort separation, it had little effect on wort viscosity. High viscosity was caused by pentosans dissolved from the triticale malt during mashing. Oxidative gelation was not observed with these pentosans. Grains and malts were fractionated, and the high molecular weight fractions were analysed for their sugar and acyl components. All were rich in arabinose and xylose. There was a rough inverse correlation between the solubility of the poly saccharide fractions and the levels of substitution with acetyl and feruloyl residues. The poor wort separation from triticale malt grists appeared to be related to the particle size distributions, which were narrow. The sedimentation values of the grist ‘fines’ were high.     

Malt characteristics of sorghum vulgare varieties from ghana

Two local varieties, a white type and a red type, of Sorghum vulgare cultivated in Ghana were malted, using a micro-malting method, and their malt characteristics studied and compared to that of a commercial barley malt. The optimal germination time, at 30°C, to produce a good malt of high diastatic power and extract from local varieties of these varieties was 4-5 days. The local varieties were also found to have high diastatic activities of between 55 and 68% of the commercial barley malt. Hot-water extracts of the well malted sorghum varieties were also found to have higher and sustainable amounts of free amino nitrogen than the commercial malt. The hot-water extracts of sorghum malts were lower than the commercial barley malt, yielding about 66-77% of the barley malt, but contained a higher glucose to malt ratio. In terms of varietal superiority, the white sorghum yielded higher malt extracts than the red type.     

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 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.     

Quality Assessment of a Sorghum Variety Malted Commercially under Tropical Conditions and Controlled Tropical Temperatures in the Laboratory

A sorghum variety was supplied as commercial malt and as an unmalted cereal by a maltster. The commercial sample had been malted in a tropical country. Sub‐samples of the unmalted cereal were malted in the laboratory under controlled germination temperatures of 28°C and 30°C. Laboratory and commercially malted sorghum were studied for their brewing qualities. The α‐amylase development in sorghum malt was enhanced when germination was carried out at the higher temperature of 30°C rather than at 28°C. Hot water extract (HWE) was more variable. With infusion mashing, results showed a significant difference for germination time (3–6 days), but no significant difference relating to germination temperature. With the decantation mashing method the reverse was observed. The low numerical values of HWE obtained from sorghum malt in the infusion mashing process confirmed that this process is not suitable to produce optimal extract development from malted sorghum. The 28°C germinated sorghum released more FAN products into the worts than the 30°C malt, using both the infusion and decantation methods. With regard to the parameters tested, commercially malted sorghum gave lower analytical values than laboratory malted sorghum. It was also observed that variations in malting temperatures and mashing processes can cause unexpected variations in the analyses of sorghum malt. These findings suggest that careful process control is required during the malting and mashing of sorghum.     

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.     

Influence of malted barley and exogenous enzymes on the glucose/maltose balance of worts with sorghum or barley as an adjunct

The sugar profile of wort from laboratory malted barley, malted sorghum, unmalted barley and unmalted sorghum grains mashed with commercial enzyme preparations were studied. Similar levels of glucose to maltose (1:7) were observed in wort of malted barley and malted sorghum. Mashing barley or sorghum grains with commercial enzymes changed the glucose to maltose ratio in both worts, with a greater change in wort from sorghum grains. Although hydrolysis with commercial enzymes released more glucose from maltose in sorghum wort, the same treatment retained more maltose in barley wort. Adding malted barley to sorghum grains mashed with commercial enzymes restored the glucose to maltose ratio in sorghum mash. Fermentation of wort produced from all barley malt (ABM) mash and commercial enzyme/barley malt/sorghum adjunct (CEBMSA) mash of similar wort gravity was also studied. ABM and CEBMSA worts exhibited similar glucose to maltose ratios and similar amino acid spectra. However, ABM released more individual amino acids and five times more proline than wort from commercial enzyme/barley malt/sorghum adjunct. ABM produced 27% more glucose and 7% more maltose than CEBMSA. After fermentation, ABM mash produced 9.45% ABV whilst commercial enzyme/barley malt/sorghum adjunct mash produced 9.06% ABV. Restoration of the glucose/maltose ratio in the CEBMSA mash produced wort with a sugar balance required for high gravity brewing. © 2020 The Institute of Brewing & Distilling     

COMPARATIVE STUDIES OF KILNED AND FREEZE-DRIED MALTS

The effect of the drying process on malt qualities has been investigated. Green malt samples were dried in a pilot plant kiln, in a brewery kiln or in a pilot plant sublimator (freeze dryer). Some increase of α-amylase activity as dextrinizing power in kilned malts was observed, but at the same time the β-amylase and total diastatic activity decreased as compared with freeze-drying. Modification, amylolytic activity as a whole and the yield of extract were significantly better in the freeze-dried malts, which gave slightly poorer results with regard to colour, turbidity, pH and viscosity of worts.     

Effect of malting time,mashing temperature and added commercial enzymes on extract recovery from a Nigerian malted yellow sorghum variety

Preliminary microbiological studies carried out on sorghum grains showed that the major microorganisms found were mainly bacteria and that aflatoxin‐producing fungi were not found. The effect of added commercial enzyme preparations and different infusion mashing temperatures on extract yield, from sorghum malted at 30 °C, was studied. The infusion mashing method (65 °C) developed for mashing well‐modified barley malt produces poor extract yields with sorghum malt. The extract yield from the sorghum malt in this study was very low with infusion mashing at 65 °C, without the addition of commercial enzyme preparations. A higher extract yield was obtained from the sorghum malt, without the commercial enzyme addition, when using infusion mashing at 85 °C. Both infusion mashing temperatures (65 and 85 °C) showed an improved extract yield over the control malt when commercial enzyme preparations were used during mashing of the sorghum malt. The added enzyme preparations resulted in a higher extract yield from the germinated sorghum when infusion mashing was performed at 65 °C over mashing at 85 °C. The use of individual commercial enzymes (α‐amylase, β‐glucanase, protease, xylanase, saccharifying enzyme and combinations of some hydrolytic enzyme) increased extract yields, when complemented with the enzymes that had developed in the sorghum malt. Copyright © 2016 The Institute of Brewing & Distilling     

β-GLUCAN AND β-GLUCAN SOLUBILASE IN MALTING AND MASHING

During malting the water-insoluble β-glucan of barley is diminished whilst water-soluble gum is little decreased. The amount of β-glucan surviving into malt depends on variety but barleys rich in glucan give malts with high β-glucan levels. The β-glucan content of barley depends on variety and growth site. β-Glucan solubilase survives mashing and catalyses the release of hemicellulose into solution. There is no correlation between the β-glucan content of malt and the amount released into wort. However, barley adjuncts containing high levels of β-glucan give worts rich in β-glucan. β-Glucan dissolution in mashing is dependent on time, temperature, grist particle size and liquor: grist ratio. Use of adjuncts derived from barley contribute relatively more β-glucan in wort, coinciding with reduced rates of wort separation, but these can be increased by using a β-glucanase produced by growing the fungus Trichoderma viride on spent grains.     

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.     

Effect of unmalted oats (Avena sativa L.) on the quality of high-gravity mashes and worts without or with exogenous enzyme addition

Barley malt is the preferred brewing material these days because of its high extract content and high enzyme activities. However, when substituting malted barley with oats to create a unique beer flavor and aroma, endogenous malt enzymes become the limiting factor. Therefore, the objectives of this study were to evaluate the effect of 10–40 % unmalted oats on the quality of high-gravity mashes/worts and to investigate the limitations of endogenous malt enzymes as well as the benefits of the application of industrial enzymes. The enzyme mix Ondea^® Pro was found to be particularly suitable for mashing with unmalted oats and was therefore used in the present rheological tests and laboratory-scale mashing trials. In order to gain detailed information about the biochemical processes occurring during mashing, the quality of mashes was comprehensively analyzed after each mash rest using standard methods described by Mitteleuropäische Brautechnische Analysenkommission and Lab-on-a-Chip capillary electrophoresis. Mashing with up to 40 % oats resulted in increased mash consistencies, color/pH (20 °C) values, β-glucan concentrations, wort viscosities 12.0 %, and filtration times as well as decreased FAN and extract contents. The application of Ondea^® Pro enormously increased the color of worts despite lower pH values but considerably improved the quality and processability of 30 or 40 % oat-containing mashes/worts. However, the substitution of up to 20 % barley malt with unmalted oats can easily be realized without the addition of exogenous enzymes.