THE INFLUENCE OF ACCIDENTAL ABRASION OF BARLEY ON THE RESPONSE TO GIBBERELLIC ACID

Studies of short grown malt (3 days growth) indicate that gibberellic acid applied to non-abraded barley has little or no effect on its modification. Although exogenous gibberellic acid may enter the grain through the micropyle, or the region of the pericarp ruptured by the emerging chit, it is apparent that after 3 days such gibberellic acid does not significantly improve modification. Mild damage occurs during the ordinary threshing and dressing of barley, giving rise to abrasive lesions of the pericarp-testa in about 25% of the grains. It seems that most, if not all, of the increased modification observed when gibberellic acid is applied to germinating barley is due to the hormone gaining direct access to aleurone tissue through these lesions.     

EFFECTS OF BROMATE AND GIBBERELLIC ACID ON MALTING BARLEY

Barley was micromalted with or without gibberellic acid and with or without additions of potassium bromate. Bromate initially depressed and subsequently enhanced levels of soluble nitrogen. This can be explained by a combination of reduced proteolysis, reduced respiratory losses, reduced rootlet growth and a decline with time in the inhibitory effects of the bromate. It was most useful when applied at steep-out. Bromate added alone at steep-out enhanced malt extract. However when added with gibberellic acid (GA₃) it did not increase the extract above that obtained with GA₃ alone. This contrasts with industrial experience, and suggests a weakness in current micromalting techniques.     

MODIFICATION OF ABRADED AND NORMAL BARLEY GRAINS

Microscopic examination of the distal (non embryo) areas of the endosperms of abraded and control malts confirms earlier work that accelerated modification occurs when the corresponding areas of the original barley grains are effectively abraded.     

ULTRASTRUCTURE OF THE CELL WALLS OF THE TRANSPORT PATHWAY FOR GIBBERELLIC ACID IN BARLEY ALEURONE LAYER

Transmission and scanning electron microscopic studies of the scutellum-aleurone junction of barley suggest that, in the absence of plasmodesmata connections, the transport of gibberellic acid from the scutellum to the aleurone layer is effected by apoplastic transport across 3–4 μm thick cell walls. Transport of gibberellic acid along the aleurone could be apoplastic as well as symplastic because large numbers of plasmodesmata are present in the 4 μm thick cell walls, through which the hormone could pass from cell to cell. Structurally, the scutellum is separated from the starchy endosperm by a 30 μm thick layer of compressed cell wall material across which transfer of either gibberellic acid to the aleurone or small quantities of non-specific β-glucanase enzymes (E1) from the scutellum to the starchy endosperm would be restricted. No such barrier is present between the aleurone layer and the starchy endosperm, where cell wall thickness is about 4 μm, and the release of endosperm-degrading enzymes into the starchy endosperm is not restricted .     

THE DETERMINATION AND UTILIZATION OF GIBBERELLIC ACID IN GERMINATING BARLEY

A barley endosperm bioassay, utilizing the induction of acid phosphatase in barley endosperm seed halves, has been developed and applied to study both the development of endogenous gibberellin in grain germinating under malting conditions and the utilization of externally applied gibberellic acid. The assay is affected by barley age and variety and these observations may have practical implications. After 24 h germination the level of endogenous gibberellin-like material present in the grain was approximately 1·4 ng/corn. The biological activity of the total gibberellin-like material extracted from grain treated with gibberellic acid fell during germination although significant quantities of applied gibberellic acid had entered the grain during the first 24 h germination.     

COMBINED ACIDULATION AND GIBBERELLIC ACID TREATMENT IN THE ACCELERATED MALTING OF ABRADED BARLEY*

Pilot-scale malting trials show that when slight acidulation of the steeping water is combined with a high level of gibberellic acid, abraded barley produces a full extract in only 72 h. Acidulation also inhibits growth of the rootlets, curbing malting loss, in addition to encouraging higher levels of total soluble nitrogen.     

INCREASED ENDOSPERM MODIFICATION OF ABRADED BARLEY GRAIN AFTER GIBBERELLIC ACID TREATMENT*

Physiological and structural observations on barley grains revealed that the pericarp is impermeable to exogenous gibberellic acid. The use of a mechanical hand-mill abraded the pericarp at the non-embryo (distal) end of the grain without damaging the embryo or the overlying husk. When grown in gibberellic acid, the α-amylase content of these abraded grains was significantly higher than that of the controls. Malts prepared from abraded grains were visually identical with control malts prepared with gibberellic acid, but they gave higher hot water extracts. Grain abrasion permitted exogenous gibberellic acid to reach a larger number of aleurone cells, thus increasing the extent of endosperm modification during growth.     

CHANGES IN THE CATIONIC ISOENZYMES OF PEROXIDASE DURING THE MALTING OF BARLEY II: THE EFFECT OF GIBBERELLIC AND ABSCISIC ACIDS

During germination, alpna-amylase activity in barley (variety: Chariot) increased 29-fold, while peroxidase activity increased 4.2-fold. Increasing concentrations of GA₃ increased the rate of development of alpha-amylase activity while the rate of increase of peroxidase activity remained unchanged, but there was an overall increase in the amount of peroxidase synthesized. ABA caused a concentration-dependent suppression of activity and a decrease in the rate of expression of both enzymes. Three different types of response of individual peroxidase isoenzymes occurred during germination. Six isoenzymes increased in activity during malting, four decreased in activity and nine showed a rise at the beginning of germination followed by a significant fall. The majority of the isoenzymes were positively GA₃-sensitive, ie increased in activity or were synthesized earlier in the presence of GA₃, and negatively ABA-sensitive. Only four isoenzymes were negatively GA₃-sensitive, and only three were positively ABA-sensitive. Detailed data for the synthesis during germination of the six major peroxidase isoenzymes that survive into the finished malt is presented .     

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.     

DIFFERING SUSCEPTIBILITIES OF BARLEY SAMPLES TO ABRASION

Abrasion trials on different varieties of barley from the 1971 harvest show that susceptibility to abrasion at the distal (non-embryo) end of the grain is not correlated with malting quality as judged by conventional criteria. In general, barleys of small corn size (less than 29 g per 1000 corn weight) are difficult to abrade because their pointed distal ends are covered by relatively greater quantities of husk material than are barleys of larger size. Effective abrasion is related to scarification of the pericarp layer at the distal end of the grain rather than to general loss of husk material.     

PATTERNS OF MODIFICATION IN MALTING BARLEY

The modified regions of the starch endosperms of malted grains were fragile and, in thin sections, the cell walls in these regions did not stain readily with Congo Red or Trypan Blue, although scanning electron microscopy demonstrated some cell-wall material remained. Initially the enzymes causing modification came from the scutellum, but later more came from the aleurone layer. Patterns of modification in different grains differed significantly, but usually resembled those recorded previously⁶ except that often modification had advanced further by the nucellar sheaf cells. In grains treated with gibberellic acid modification advanced faster, particularly beneath the aleurone layer, after two days germination. In tumbled or commercially abraded grains, malted with gibberellic acid, modification was even more rapid, but in more than 99% of the grains the same patterns of modification occurred. Two-way modification was a rare event. Rapid, general sub-aleurone modification occurred in grains cut at the apex and dosed with gibberellic acid. Decorticated grains modified exceptionally quickly and, when treated with gibberellic acid, massive subaleurone modification occurred. The cell walls of the tissues that resisted modification fluoresced strongly in ultra-violet light, in contrast to those of the starchy endosperm.     

IMPROVED METHOD FOR DETERMINING THE DEGREE OF ABRASION OF BARLEY

Current methods for assessing whether barley has been abraded are slow and liable to misinterpretation. An improved method is described which is rapid, simple to interpret, and shows the positions of the abrasive lesions of the pericarp-testa of each grain.     

THE EFFECT OF ABRASION ON DORMANT BARLEY

The primary purpose of barley abrasion is to facilitate the passage of gibberellic acid to the aleurone tissue, particularly at the distal end of the grain. Abrasion has now been shown to alleviate dormancy as a result of the damage suffered by the proximal areas of the barley grain during the abrasion treatment. This effect alone should make the abrasion process useful to the malting industry whether or not subsequent treatment with gibberellic acid is desired, particularly when multi-hit rather than single-hit abrasion machines are used.     

THE UPTAKE AND DISTRIBUTION OF BROMATE IN GERMINATING BARLEY

Bromate applied to steeped barley enters the grain rapidly, within 4 h of application; it is reduced to bromide as germination proceeds but persists in the endosperm in measurable quantities until kilning. Bromate has not been detected in kilned malt. Examination of lateral sections of grain shows that bromate concentration is highest at the embryo end, lowest in the middle and has an intermediate concentration at the distal end. These results, obtained with selected undamaged grains of both Proctor barley and Nackta (a naked variety), suggest that bromate can enter the grain at the distal end and over the grain surface as well as through the embryo.     

THE PERMEABILITY OF THE SURFACE LAYERS OF CEREAL GRAINS,AND IMPLICATIONS FOR TESTS OF ABRASION IN BARLEY

The husk of barley, and the pericarps of naked, husked and ‘stripped’ barleys, of wheat and rye are more or less permeable to aqueous solutions of salts, or Eosin. The pericarps of stripped barley grains conduct aqueous solutions so that, for example, they conduct solutions of Eosin, or [¹⁴C]-gibberellic acid from the apex to the base of the grain where it accumulates in the embryo region. On the other hand the husk and pericarp are not so readily permeated by aqueous Trypan Blue. The testa/nucellar cuticle, together with the pigment strand, limits the inward penetration of salts and dyes. Gibberellic acid, and apparently water also, traversed the testae of some, but not all, decorticated barley grains, as demonstrated by the consequent modification of the starchy endosperms. However in most instances gibberellic acid solution does not traverse the surface layers of stripped grains and induce modification in their endosperms.     

COMPARATIVE MALTING PERFORMANCE OF OLD AND NEW BARLEY VARIETIES

A comparison was made of the malting behaviour of eight barley varieties representing a range from ancient to modern types. Plants were grown under controlled conditions in a glasshouse with two different nitrogen treatments and the grain obtained was subjected to a factorial micro-malting analysis. Under the conditions of this experiment there was little evidence of an improvement in malting ability between Plumage Archer and Ark Royal and there is limited scope for further increases in potential hot water extract. It would therefore appear that emphasis should now be given to the production of varieties with good agronomic characteristics which maintain this potential and modify rapidly without the need for abrasion and exogenous GA₃.     

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.     

TRANSPORT OF [14C] GIBBERELLIC ACID IN THE BARLEY EMBRYO

Radioactive gibberellic acid has been used to obtain direct evidence that, during germination of barley, transport occurs along vascular strands of the scutellum towards the dorsal (non-furrowed) surface of the grain. Preferential stimulation of the dorsally-placed aleurone cells therefore accounts for the normal asymmetric pattern of enzymic modification that occurs during malting. In the scutellum, gibberellic acid is transported at a rate of about 2.5 mm per hour. It is suggested that rapid development of the vascular system in the scutellum may be an important feature of barleys that malt rapidly.     

ASSESSING MALTING QUALITY BY THE AUTOLYSIS OF BARLEY

There has been prolonged speculation over the past thirty years on the relationship between the properties of aqueous extracts of barley and malting quality. On reviewing the literature on this subject, the concensus of opinion is that the total amount of gum or β-glucan in the barley does not correlate with malting quality. The cytolytic potential of both barley and malt, on the other hand, appears the more important parameter. Present evidence is that measurements made on aqueous extracts, either by viscosity or β-glucan determination, are more indicative of barley cytolytic activity than they are a true measure of gum content. Low β-glucan values in some barleys may therefore be artefacts of enzymic hydrolysis.     

CHARACTERISTICS OF WATER UPTAKE OF AUSTRALIAN POLISHED BARLEY IN SHOCHU-MAKING

Characteristics of water uptake during steeping of 70% polished Australian barley, used as material for shochu making, were studied on 6 grain samples. There were some similarities in water uptake curves in spite of the differences in varieties and regions of harvest. Polished barley abruptly absorbed water at the same time when in contact with water. After 5 h the water uptake content reached 52 to 56, 54 to 59, and 58 to 63% grain wet weight at 15, 20, and 30°C respectively. The relationship between water uptake and steeping time may be described by the equation Y=aX^b, where Y is the water uptake (%), X is the time (min), and a and b are coefficients¹. From the data obtained with Schooner (South), the water uptake curve in steeping at 15°C was described as Y=5.50X^0.40 (r²=0.993). Furthermore a log-log plot of water uptake (%) against integrated steeping temperature (1ST), which was presented by the product of temperature and time, showed a very clear linear relationship, and could be represented by Y=2.047(T · t)^0.382 (r²=0.987). The coefficient values a and b determined the relationship of water uptake and 1ST on 6 samples. The values of 5 samples, excluding Stirling (West) were close (a=2.05 to 2.33 and b= 0.37 to 0.38), and no differences were apparent amongst these varieties and regions. Stirling (West) with a=1.81 and b=0.40 were similar to the Japanese barley cultivar Nishinochikara (a=1.99, b=0.39). The Schooner (South) equation could generally be applied to control water uptake during steeping on 70% polished Australian barley supplied to our factory. The water uptake values from the steeping experiments were between 35.1 and 36.7% when the objective value was set at 35% .