The Development of Enzymes During Germination and Seedling Growth in Nigerian Sorghum

The levels of enzymes responsible for the enzymic modification of sorghum endosperm have been followed during germination and seedling growth. Sorghum β-glucanase was shown to be inactive towards barley β-glucan. Gibberellic acid does not appear to control the levels of α-amylase. In contrast to barley, the synthesis of this enzyme occurs in the embryo but it subsequently acts on the starch granules in the endosperm. Limit dextrinase, on the other hand appears to be present in the endosperm as a zymogen. Proteases were also examined during germination and seedling growth. Amino acid-releasing enzymes develop in the embryo and are absent from the endosperm, whereas endoproteases can be detected in the embryo and to a greater extent in the endosperm. Amylolytic attack on endosperm starch in sorghum is very extensive during the early stages of grain growth. The significance of these results to the malting properties of sorghum is discussed.     

The sorghum embryo in relation to the hydrolysis of the endosperm during germination and seedling growth

Time-course changes in the structural and physiological properties of sorghum grain embryo have been investigated in relation to the hydrolysis of the endosperm during germination and seedling growth. Histochemical analysis showed that the reserve food materials of the scutellum tissue were metabolised rapidly during germination and seedling growth. Light microscope analysis of structural changes showed that scutellar epithelial cell elongation was associated with endosperm reserve mobilisation. Electron microscope studies verified preliminary histochemical and light microscope findings and showed that extensive metabolism of subcellular storage materials occurred during early germination and seedling growth. Physiological evidence indicated that both sorghum embryo and endosperm were insensitive to the gibberellic acid, GA₃. Other hormones such as indole-3-acetic acid and kinetin also failed to induce α-amylase production. This suggests that the hormonal response of the aleurone of sorghum is different from that of barley aleurone. Dissection of sorghum showed that the whole body of the scutellum was capable of producing α-amylase.     

MORPHOLOGY OF STARCH GRANULES IN CEREAL GRAINS AND MALTS

During malting, amylases have limited action on large starch granules of barley endosperm but rapidly degrade the small granules. In contrast, the small starch granules of wheat endosperm are resistant to enzymic attack. High levels of exogenous gibberellic acid increase the production of α-amylase and encourage the appearance of radial channels in the partially-degraded large starch granules. These endo-corroded granules are mainly found in the proximal (embryo) half of the endosperm where levels of α-amylase are much higher than at the distal end. Degradation of malt starch can therefore result from enzymic attack both outside and inside the granules. Malting of barley reduces the population of small starch granules which are slower to gelatinize than large granules at the infusion mashing temperatures of 65° C. During germination of barley multiple starch granules are rapidly synthesized in single amyloplasts in the scutellum. The endosperm of high amylose barley is devoid of small starch granules and the average size of the large granules is reduced. Steeliness in sorghum is related to the close packing of the starch-protein matrix rather than to unequal distribution of protein. The significance of these results is discussed, particularly in relation to the morphology of starch granules, the nature of their outer covering, the distribution of amylopectin and amylose within the granule, and the site of enzymic attack.     

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 .     

THE MILLING ENERGY OF MALTED BARLEY AND ITS RELATIONSHIP WITH HOT WATER EXTRACT AND α-AMYLASE ACTIVITY

Hot water extract is dependant on endosperm structure and its breakdown during malting. These endosperm characteristics can be rapidly assessed on 5 g samples by measuring the milling energy of malted barley. α-amylase determinations on the resultant flour indicate that good malting barleys have either moderate or high levels of the enzyme. High levels of α-amylase will not confer good malting quality where endosperm modification has proceeded slowly.     

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.     

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.     

A REASSESSMENT OF THE PATTERN OF ENDOSPERM HYDROLYSIS (MODIFICATION) IN GERMINATED BARLEY

Microscopic and enzymic studies of germinated barley have confirmed that excised barley embryos can produce α-amylase because the peripheral areas of the scutellar tissue contain aleurone cells. In contrast, the aleurone-free tissue of the scutellum is incapable of producing significant quantities of α-amylase. The potential of excised embryos to develop α-amylase is not correlated with the in vivo elongation of the scutellar epithelial cells in the grain because these cells do not elongate in excised embryos. Detailed anatomical studies revealed that the highly insoluble Intermediate layer of cell wall material, which is located between the embryo and the starchy endosperm, is broken-down asymmetrically, thus confirming that enzymic modification of the endosperm is under aleurone rather than scutellar control, in germinated barley. Other studies which have sited the scutellum of barley as inducing symmetric break-down of the endosperm have not linked structural changes, in vivo, with fluorescent or non-fluorescent staining patterns. Some of these studies have failed to recognise the possibility that grains such as sorghum and rice may have a different pattern of endosperm break-down from that of barley.     

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.     

DIFFERENCES IN MALTING PERFORMANCE BETWEEN BARLEYS GROWN IN SPAIN AND SCOTLAND

Two barley genotypes were grown, in 2 seasons, at sites in both Scotland and Spain. The development of enzyme levels and endosperm modification were assayed, over the final 3 days of malting. Spanish grown samples demonstrated faster and more extensive synthesis of both α-amylase and β-glucanase, more rapid cell wall modification and a greater reduction in milling energy during malting than Scottish grown samples. Malt milling energy was strongly associated with cell wall breakdown, which was a limiting step in modification of Scottish, but not Spanish, grown samples. Extract levels were not related to α-amylase activity, but Kolbach index exhibited an association with extract at both sites.     

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 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 .     

IMPROVED MALTING PERFORMANCE OF FRESHLY HARVESTED BARLEY AFTER ABRASION

Abrasion improved the malting performance of dried, freshly-harvested barley and provided an alternative to storage. The maltability of undried, unstored barley was improved by abrasion, but the undried grain was more difficult to abrade than was dried grain. Unabraded barley required storage periods of at least 3 weeks before adequate extracts could be obtained. Although acidulation reduced the malting losses of dried grain, it only improved the yield of extract with barleys stored for at least 9 weeks before malting. The potential of dried freshly-harvested barley to produce α-amylase and endo-β-glucanase, as indicated by the response of endosperm slices to gibberellic acid, was initially low and increased gradually as the barley was stored. It is considered that the dominant factor in the accelerated malting of freshly-harvested barley is the improved distribution of enzymes in the endosperm which results from abrasion.     

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.     

‘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.     

RELATIONSHIP BETWEEN LEVELS OF GIBBERELLIC ACID AND THE PRODUCTION AND ACTION OF CARBOHYDRASES OF BARLEY

Different hydrolytic enzymes require different levels of gibberellic acid to induce their maximal production and release into the endosperm of barley. Barley-endo-β-glucanase requires a higher level of gibberellic acid to induce maximal production than does α-amylase. Although gibberellic acid also increases the level of barley endo-β-1,3 glucanase, this enzyme, unlike the barley-endo-β-glucanase, develops to significant levels when gibberellic acid is absent. In gibberellic acid-treated aleurone layers β-glucanases degrade the cell wall mainly to glucose. Xylose and cellobiose appear when the aleurone wall has undergone extensive enzymic hydrolysis. Laminaribiose and arabinose are found whether or not gibberellic acid is present in the medium. In addition to the degradation of the endosperm cell walls, β-glucanases may also play an important role in the release of enzymes from the aleurone into the endosperm during malting.     

THE CHROMATOGRAPHIC PROPERTIES OF BARLEY AND MALT β-AMYLASES

Barley β-amlyase occurs as a heterogeneous, polydisperse enzyme in thiol-free extracts of Conquest barley. During malting, the polydisperse enzyme is altered, resulting in the formation of four distinct enzyme components which increase in activity as germination progresses. Addition of thioglycerol to a thiol-free extract of barley, or initial extraction with thioglycerol, produces extracts containing two discrete β-amylase enzymes. β-amylase I is the major component of the extract; β-amylase II occurs as a minor component. Similarly, malt extracts containing thioglycerol have two β-amylase enzymes, β-amylase III and IV. Barley β-amylase II and malt β-amylase III have similar chromatographic properties on CM-cellulose but it is not known whether these enzymes are identical. During the early stages of germination, barley β-amylase I disappears and cannot be detected in extracts of 1-day malt; β-amylase III is the major β-amylase enzyme in this extract. Malt β-amylase IV cannot be detected in barley extracts. It develops during germination until it becomes the major β-amylase in malt extracts.     

Effect of Germination Moisture and Time on Pearl Millet Malt Quality — With Respect to Its Opaque and Lager Beer Brewing Potential

The effect of germination moisture and time on pearl millet malt quality was investigated. Two pearl millet varieties SDMV 89004 and 91018 were germinated at 25°C under three different watering regimes for 5 days. As with sorghum malting, diastatic power, beta‐amylase activity, free α‐amino nitrogen (FAN), hot water extract and malting loss all increased with level of watering. However, pearl millet malt had a much higher level of beta‐amylase and higher FAN than sorghum malt and a similar level of extract. Malting losses were similar or lower than with sorghum. Thus, it appears that pearl millet malt has perhaps even better potential than sorghum malt in lager beer brewing, at least as a barley malt extender, especially in areas where these grains are cultivated and barley cannot be economically cultivated. Also, its increased use in commercial opaque beer brewing, where sorghum malt is currently used, could be beneficial.     

THE DEVELOPMENT OF β-GLUCAN SOLUBILASE DURING BARLEY GERMINATION

β-Glucan solubilase in either germinating barley or in endosperm slices treated with gibberellic acid is synthesized before endo-β-glucanase, α-amylase and protease. In common with these enzymes, β-glucan solubilase is synthesized much sooner in endosperm slices than in whole grain. Gibberellic acid stimulates β-glucan solubilase synthesis in endosperm slices and most of the activity is rapidly released into the surounding medium, irrespective of whether the hormone is present. Inhibitors of RNA and protein synthesis block the formation of β-glucan solubilase. Unlike β-glucanase, α-amylase and protease, β-glucan solubilase is present in significant quantity in untreated barley where it is concentrated in the embryo-containing half of the grain. The only β-glucan solubilase activity in barley is due to an acidic carboxypeptidase. Malt contains a small amount of a second solubilizing enzyme which appears to be an endo-β1, 3-glucanase.     

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.