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 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.     

PRODUCTION OF α-AMYLASES IN GERMINATING WHOLE AND INCUBATING DE-EMBRYONATED BARLEY KERNELS*

α-Amylases produced in germinated barley and incubated de-embryonated barley kernels (cv. Bonanza), in the absence and presence of gibberellic acid (GA₃), were analyzed qualitatively by polyacrylamide gel isoelectric focusing (PAG-IEF) and quantitatively by chromatofocusing. Identical patterns of α-amylase components were obtained for both germinated barley and incubated de-embryonated barley kernels at each germination/incubation stage, in the absence or presence of GA₃. Total α-amylase increased rapidly in the germinating whole seed whereas in the incubating de-embryonated grain the α-amylase activity increase was much slower. Addition of exogenous GA₃ did not induce production of higher levels of α-amylase in either the germinating whole or incubating de-embryonated barley kernel. Quantitative chromatofocusing analysis revealed that the proportion of α-amylase III to α-amylase II activity decreased linearly with germination time in the whole grain but remained constant in the incubating de-embryonated grain in the absence or presence of GA₃. The major proportion of α-amylase activity in the germinating whole grain and incubating de-embryonated grain was synthesized in the form of α-amylase II components. However, α-amylase I represented a larger proportion of the total α-amylase activity produced in the incubating de-embryonated grain, as compared to the germinating whole seed in the absence or presence of GA₃. These results suggest that embryo excision differentially affects production of α-amylase II as compared to α-amylase I.     

RELATIONSHIPS BETWEEN β-AMYLASE POLYMORPHISMS IN DEVELOPING,MATURE AND GERMINATING GRAINS OF BARLEY

A β-amylase polymorphism (electrophoretic forms Sd1 and Sd2) in barley malt is shown to be closely associated with the proportion of free to total (free plus latent) β-amylase, but not with the level of total β-amylase in the mature grains of 46 cultivars. All of the cultivars with Sd1 malts have a proportion of free β-amylase less than 50% (usually from 30% to 40%) of total whereas Sd2 types have free to total β-amylase (F/T) ratios greater than 50% (usually between 62% and 78%). These polymorphisms are also correlated with forms of β-amylase in the developing grain, although, in the latter, Sd1 cultivars can be divided into two types, Sd^d and Sd^e which cannot be distinguished either in malts or on the basis of F/T ratio. Unusual F/T ratios of an intermediate type (approximately 50%) and a very low type (under 30%) also occurred in these experiments. These may result from environmental effects or may be new genetic types.     

PLANT HORMONES IN FUNGI AND BACTERIA FROM MALTING BARLEY

40 Fungi and 16 strains of bacteria, isolated from the grains of three cultivars of matting-grade barley (Kymppi, Pokko and Kustaa, of 1990 harvest), were screened for the production of the plant hormones gibberellic acid (GA₃, abscisic acid (ABA) and indole-3-acetic acid (IAA). Four fungal strains were found capable of GA₃ production and four of ABA production. IAA production was common among both fungi (58% of strains active) and bacteria (88% of strains active). To get an estimate of the physiological significance of the presence of plant hormone producing microbes, the plant hormone production per microbial unit in the liquid growth media of the cultured organisms was weighed against the microbial counts and the endogenous hormone concentrations of barley grains. It was concluded that bacterial IAA production could be of significance in imbibed grains. This presupposes, however, that the conditions be ideal for the propagation of the active species and also, for the production of IAA by those same species and lastly, that similar production occurs in vivo as well as in vitro. Microbial GA3 and ABA production, on the other hand, were estimated to occur in negligent amounts.     

THE DETERMINATION OF GERMINATED GRAINS IN BARLEY

A method for determining germinated grains in barley by detecting the presence of α-amylase activity in individual corns is recommended.     

THE DISTRIBUTION OF WATER IN GERMINATING BARLEY

During germination, the embryo of barley absorbs water initially from the film surrounding the corn and later from the endosperm. If growth is stimulated during steeping, absorption of water by the embryo modifies the pattern of moisture distribution and this is usually associated with reduced extractability in the final malt.     

PURIFICATION AND PROPERTIES OF THE MAJOR ANTIGENIC BEER PROTEIN OF BARLEY ORIGIN

The major antigenic beer macromolecule, Antigen 1, has been isolated from commercial lager beer by succesive use of ultrafiltration, alcohol precipitation, anion exchange, cation exchange and gel filtration chromatography. Amino acid analyses showed a composition identical with that of a major barley albumin, Protein Z, except for a 16% lower content of lysine. Like Protein Z, Antigen 1 showed a molecular weight (MW) near 40,000 in gel filtration and SDS-gel electrophoresis. Unlike Protein Z, Antigen 1 contained 2·5% carbohydrate and was more acidic. When properly standardised, the amount of Antigen 1 in beer could be determined immunoelectrophoretically. Contents ranged from 22 mg/litre in a pale ale (7·7°P; 36% brewers adjunct) to 170mg/litre in a stout (18·8°P). The results suggest that Antigen 1 may account for more than 10% of the total non-dialysable proteinaceous material in beer, and that about 25% of the Protein Z present in the brewing materials may be recovered with an almost unmodified protein structure as Antigen 1 of beer. Apparently, Antigen 1 was not affected by stabilisation of the beer with insoluble PVP or with papaya proteinases.     

ISOELECTRICFOCUSING AND IMMUNOCHEMICAL ANALYSIS OF GERMINATED BARLEY α-AMYLASES AFTER FREEZE-DRYING AND KILNING

Isoelectricfocusing analysis showed that germinated barley contained three groups of α-amylase—α-amylase I a minor component and major components α-amylases II and III. During kilning there was a significant conversion of α-amylase III to α-amylase II. These two enzymes were shown to have immunochemical identity.     

ON-LINE MEASUREMENT OF THE MICROBIAL IMPACTS ON THE GERMINATION OF BARLEY DURING MALTING

During germination, the metabolic activity of sterile barley was more pronounced than the activity of barley contaminated by its endogenous microbial population. CO₂ and heat production profiles showed higher peak values when microbial activity was eliminated. Moreover, the maxima in the profiles appeared sooner than with barley in the presence of its natural microflora. More intensive growth of the acrospire was observed with sterile barley and a malt was obtained with a higher and more homogeneous modification. Reverse effects were noticed when sterile barley was inoculated with an Erwinia sp. strain and germination was evaluated. The results provide clear evidence for the strong interaction between microbial and plant metabolism, even for non dormant and non water sensitive barley. The ratio of CO₂ production to heat production levels was a useful tool for on-line monitoring of microbial activity during germination. In this way, the effects of the microbial activity can be taken into account in the development of a more effective control of the malting process. All experiments were performed using a specially designed solid-state reactor.     

GENETIC AND ENVIRONMENTAL VARIATION OF GUM CONTENT IN BARLEY

The influence of genetic vs environmental factors on the gum content of the barley grain has been studied by growing 4 varieties of barley, at 4 locations in the South of Spain, with 6 sowing dates at each location. The genetic factors proved to be the more important, accounting for 89% of the variation over locations and 71·5% of the variation among locations and sowing dates. Of the environmental factors studied, the sowing date was the most important. The variety × sowing date interaction was also statistically significant. Furthermore, the later the sowing date the higher the gum content of the grain. There was no variety × location interaction and, therefore, the varieties ranked in the same order at each location. It was also demonstrated that there was a different pattern of response for the varieties when grown in environments which were able to induce high or low gum content, suggesting that low gum content-low response varieties should be grown if the grain is to be used as an adjunct in brewing.     

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 ULTRASTRUCTURE OF GERMINATING SORGHUM AND MILLET GRAINS

The parenchyma tissues of ungerminated and germinated sorghum and millet grains contain abundant fat and protein deposits, which undergo various metabolic changes during germination and seedling growth. The presence of these and other organelles which are characteristic of the mature plant cell was confirmed by a combination of light and electron microscopy. During the early period of the germination process in the millet scutellum, evidence of starch synthesis was apparent, suggesting that fat to starch conversion possibly occurred in the cells of this tissue. In the endosperm of 72 h germinated sorghum and millet grains, starch degradation occurred although such degradation was more extensive in sorghum than in millet.     

THIOLS AND DISULPHIDES IN QUIESCENT AND GERMINATING BARLEY GRAINS,BOTH DORMANT AND MATURE

Reduced and oxidised glutathione, cysteine, cystine and traces (too small to quantify) of γ-glutamyl-cysteine and cysteinylglycine were detected, in about the same amounts, in extracts of the embryos of dormant and mature barleys. During micromalting the levels of the thiols and disulphides altered in various ways, but altered in the same ways in dormant and mature samples of grain until germination began. An early decline in the glutathione content of embryos was mirrored by a rise in the amount in degermed grains. Histochemical tests and analyses of isolated tissues showed that in the quiescent grains thiols were concentrated in the embryo (particularly the scutellum) and in the aleurone layer and isolated embryos released thiols into an incubation medium. We conclude that the endogenous thiols and disulphides are not obviously involved in the regulation of dormancy.     

THE ROLE OF PROLINE IN THE AMINO ACID METABOLISM OF GERMINATING BARLEY

The changes in nitrogen composition in germinating barley have been studied under malting conditions. It has been shown that most amino acids are produced in the endosperm, by peptidase activity, in the correct concentrations required for protein synthesis in the embryo. During growth the total concentrations of free and combined glutamic acid, glycine and ammonia decrease with the concurrent increases in the total concentration of free and combined proline, aspartic acid and lysine. No evidence has been found for the presence of an end product inhibition of peptidase activity in the endosperm. Thus in malting the growth of the embryo is shown to serve a useful purpose in the removal of amino acids from the endosperm.     

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.     

THE STRUCTURE OF THE PERICARP AND TESTA OF BARLEY

Detailed microscopic study of the covering layers of the mature barley grain shows that beneath the husk the pericarp consists of highly compressed cell material bound by a thin outer cuticle. The testa is a double cell layer, also bound by an outer cuticle. An innermost cuticular membrane separates the testa from the remains of the nucellus. This membrane is of nucellar origin and not part of the testa. The outer cuticle of the testa is incomplete in the ventral furrow and in the micropylar area of the grain. The absence of the testa cuticle and the relatively uncompressed nature of the pericarp at the micropyle may facilitate uptake of solutes by the embyro.     

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.     

VARIETAL IDENTIFICATION OF BARLEY AND MALT*

A vertical polyacrylamide — SDS electrophoretic technique, including whole protein extraction and staining steps, was improved with a view to developing it for routine laboratory use with single barley kernels. The pattern consisted of 4 zones: A (albumins-globulins). B and C (hordeins) and D (possibly glutelins) displaying unequal varietal polymorphisms (1, 13, 13 and 4 types respectively). 28% of the barley samples (77 varieties), including most cultivars grown in France, could be unambiguously identified from qualitative differences only, in the B, C and D zones. Adding three other characteristics (hairs and furrow hairiness, peroxidase, zymogram, esterase zymogram), as many as 78% of the varieties could be identified, the other 22% consisting of very closely related barleys. After slight modification of protein extraction conditions, the same methods could be used with malt, based on the same electrophoretic types. A graphic tablet connected to a microcomputer was used for automatic acquisitions of records and comparisons of electrophoretic data.     

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% .