α-AMYLASE IN DEVELOPING BARLEY KERNELS — A REAPPRAISAL1

The outer layers that can be dissected readily from immature barley kernels were identified by light microscopy. α-Amylase was located in the outer pericarp of developing Bonanza and Himalaya barley. Only low pl α-amylase (α-amylase 1) was detected in the pericarps but only one of the two major low pl components of α-amylase from germinated Himalaya was found in the pericarp tissue. These enzymes appeared to hydrolyze the small starch granules (2–3 μm) present in the outer pericarp of developing barley kernels. The even smaller starch granules (0.25–2μm) present in the inner pericarp were hydrolyzed at a later stage of kernel development.     

Effects of Germination of Wheat,Oats, and Pearl Millet on Alpha-Amylase Activity and Starch Degradation

Hard red winter wheat, oats, and pearl millet were germinated at 15°C for periods up to 14 days. Endosperm areas in kernels fractured with a razor blade and starches isolated from the malted flours were examined with a scanning electron microscope to determine the morphology of starch granules and the nature of α-amylolytic attack on granules. Free sugars content, damaged starch content, and α-amylase activity of the flours were determined to establish relationships between physical changes in the cereal grains and formation of α-amylase during germination. Starch granules in the three cereals were degraded during germination by α-amylase accompanied by production of free sugars and increases in the damaged starch of flours milled from the germinated grains. The cementing material embedding starch granules in endosperms of wheat and oats decreased and disappeared during germination; these changes were not obvious in germinated millet endosperm. Amorphous-appearing material seemed to cover starch granules in the endosperm of wheat and oats during later stages of germination, but were not observed in the isolated starch granules. Although wheat starch granules had the highest α-amylase activity, this enzymic degradation was less than expected. Enzymic attack was evidenced as channels or pits on the surfaces of wheat and millet starch granules. Compound oat starch granules were relatively resistant to enzymic attack and no evidence of erosion of the granule surface was observed; small granula were released from the compound granules during germination. The most extensive degradation was obtained with millet starch, appearing as discrete holes leading to the interior of the granule. Concentric shells were visible in the interior of wheat and millet starch granules, but were not observed in oat starch granules.     

TISSUE DISTRIBUTION OF α-AMYLASE AND PHOSPHORYLASE IN DEVELOPING BARLEY GRAIN

Approximately half the total α-amylase and phosphorylase activity detectable in crude homogenates of the tissues of developing barley grain was localized in the pericarp. This tissue is particularly active in the early stages of grain development. It is suggested that this activity may influence the starch type and content of the mature barley grain.     

In vitro Degradation of Starch Granules by α-Amylase Isomers from Mature Triticale

Starch granules and α-amylases (green, α-I, and germination, α-II) were isolated from a triticale line with a high frequency of kernel shrivelling. The isolated starch granules were treated with the α-amylase ismomers for different periods of time. Degradation of the starch granules was followed by analyses of reducing sugar produced, and by scanning electron microscopy. About similar degrees of starch degradation were observed after treatment with α-I- and α-II-amylases. After a few hours, the degrading effects of the enzymes were manifested as holes or craters on the surface of starch granules and as erosion and dilation of the equatorial groove. These features were also characteristic of starch granules present in shrivelled kernels. Amylase treatments for a longer time caused more profound degradation, leading to collapse and fragmentation of the starch granules. The lack of such effects in shrivelled kernels means that amylases are not the sole factor in the formation of kernel cavities associated with shrivelling. The occurrence of mitotically aberrant and degenerating cells, already existing at early stages of endosperm development, and disparity between the processes that determine kernel size and kernel filling potential are also of relevance in this connection.     

THE IN VIVO AND IN VITRO DEGRADATION OF BARLEY AND MALT STARCH GRANULES

The susceptibilities to amylolytic hydrolysis of the two different types of starch granule in barley and malt have been investigated. The large and small granules from both germinated and ungerminated grain were subjected to the sole action of malt α-amylase under conditions which otherwise simulated those of a conventional infusion mash. Large starch granules from barley are hydrolysed at a slower rate than those from malt. The faster conversion of the latter is attributed to prior modification of the starch granule structure during germination. The small granules from barley are extremely resistant to attack by α-amylase and even pre-cooking does not appreciably increase their susceptibility to amylase attack. Although the small starch granules from malt are less susceptible than the corresponding large granule fraction, they can be hydrolysed to a greater extent than can the small granules from barley. The increased susceptibility of small granules from malt is probably due to the partial removal of their protecting surface layer of protein. Although during malting the small granules of barley are hydrolysed more rapidly than the large granules, the situation is reversed during mashing. Very little loss of extract can be attributed to the enzymic resistance of small starch granules in all-malt mashes. If raw barley is used in the grist, substantial amounts of small starch granules may remain in the mash.     

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.     

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.     

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.     

Screening of α-Amylase Suitable for Evaluating the Degree of Starch Retrogradation

To estimate the degrees of starch retrogradation in the complex foods, an enzymatic method using α-amylase from Bacillus subtilis was proposed in the previous report (Tsuge, H. et al.: Starch/Stärke 42 (1990), 213–216). However, actual digestibility of the enzyme for the native starch granules was not checked at that time. A comparative study to see the digestibility of native starch granules was carried out using four different α-amylase preparations and digestion of retrograded wheat starch was tested by two α-amylase preparations. Pancreas α-amylase preparation digested some native starch granules to a great extent, while Aspergillus oryzae enzyme did not digest native starch granules virtually. In conclusion, α-amylase preparation from A. oryzae was an ideal enzyme as the tool to distinguish between raw and gelatinized starches. It was justified for the use of A. oryzae enzyme as well as B. subtilis α-amylase to evaluate the retrograded starch contents in the complex foods.     

A GEL FILTRATION STUDY ON THE ACTION OF BARLEY α-AMYLASE ISOENZYMES ON GRANULAR STARCH

Small starch granules are hydrolysed better than large granules by barley α-amylase isoenzymes. The major isoenzyme in barley is less active against both types of granules than the minor isoenzyme. Molecular weight distribution analysis of the hydrolysates shows that the action pattern of the two enzymes is almost identical. Though minor differences in the intermediate products are obtained, the major product from both starches is dextrins with a degree of polymerisation of 6–8.     

Some Properties and Degradability of Isolated Starch Granules

Starch granules of 11 starchy feedstuffs were isolated. An isolation procedure is presented. The procedure did not damage the starch granules, as could be proven by scanning electron microscopy. The starch granules were nearly completely defatted by the procedure and only about 0.5% of the original protein remained in the starch, except for rice starch. The content of apparent and real amylose was measured in the isolated starch granules. Degradability of raw materials and isolated starch granules was determined with α-amylase and rumen fluid. Starch of tapioca and rice was relatively easy to degrade by α-amylase and rumen fluid, while potato starch was relatively difficult to degrade by α-amylase. Starches containing a low amount of amylose were found to degrade faster than starches containing a high amount of amylose.     

玉米微孔淀粉制备及其显微结构研究

以玉米淀粉为原料,研究比较酶法及酸法水解处理玉米淀粉制备微孔淀粉工艺,并借助于 扫描电子显微镜(SEM)、差式扫描量热计(DSC)、动态热机械分析(DMA)等分析手段对产品显微 结构及热相变过程进行分析。     

Comparison of Size Characterization of Barley Starch Granules Determined by Electron and Optical Microscopy,Low Angle Laser Light Scattering and Gravitational Field-Flow Fractionation

Several methods were used for the characterization of starch granules isolated from barley kernels. A procedure based on a combination of alkaline digestion, toluene treatment and filtration over sieves with pore diameters of 70 and 40 μm was used for isolation and purification of starch granules from kernels. The released starch granules were characterized by various methods: scanning electron microscopy (SEM), image analysis of optical microscopy data (IAOM), low angle laser light scattering (LALLS), and gravitational field-flow fractionation (GFFF). All methods showed the bimodal size distribution of the isolated starch granules, however, they differed in the ratio of large and small starch granules. LALLS and GFFF were also used for determination of the ratio of large and small starch granules (ratio A/B) isolated from two malting barley cultivars Kompakt and Akcent. Both techniques determined the higher ratio A/B for the cultivar Akcent. SEM was also used to examine the extent of digestion. The micrographs indicate that a significant proportion mainly of small granules are still embedded into residues of endosperm and a more extensive digestion must be performed to release all starch granules from barley kernels.     

INHIBITION BY HORDEIN OF STARCH DEGRADATION

Large and small starch granules prepared from Proctor barley contain high levels of firmly bound protein. Experiments with α-amylase under simulated mashing conditions suggest that this protein limits the rate of starch breakdown during mashing. Gel electrophoresis shows hordein to be a principal component. Treatment with cysteine or malt endopeptidase changes the nature of the associated protein.     

The Rapid Enzymatic Determination of Starch Damage in Flours from Sound and Rain Damaged Wheat

A starch damage analytical method for use on wheat flours has been developed which is rapid, simple in operation, analytically precise, and is independent of any α-amylase present in flour, as when milled from rain damaged wheat. The new method is based on the use of an analytical grade fungal α-amylase enzyme, which is shown to be free of contaminating amyloglucosidases, and α-glucosidases which produce glucose rather than maltose. The level of analytical α-amylase per starch damage determination was obtained by reference to thin layer chromatography of the hydrolysates so as to obtain maltose as the principal reducing sugar. This ensured that adequate but not excess analytical α-amylase was present, as the undamaged starch granules are some what susceptible to α-amylase.     

青稞β-葡聚糖对淀粉体外消化性的影响

对比青稞全粉和普通小麦粉体外消化性,研究青稞β-葡聚糖质量浓度和分子质量对青稞淀粉体外消化性的影响,从青稞β-葡聚糖流变学特性及其与α-淀粉酶的相互作用方面,探究青稞降血糖的可能机理。结果表明,青稞全粉中淀粉消化率明显低于小麦粉;随青稞β-葡聚糖质量浓度和分子质量的增大,其溶液黏弹性增大,对α-淀粉酶活性的抑制效果越明显,延缓淀粉体外消化效果越显著。青稞β-葡聚糖形成的高黏性环境是青稞全粉低淀粉消化率的潜在机理。     

AMYLOLYSIS OF LARGE STARCH GRANULES FROM BARLEYS IN RELATION TO THEIR GELATINISATION TEMPERATURES

Large starch granules, prepared from a range of barley samples, show considerable variation in their susceptibility to attack by α-amylase at 65°C. Susceptibility is not correlated with their protein content, amylose/amylopectin ratio or gelatinisation temperature as measured by loss of birefringence. It does correlate with gelatinisation as measured by swelling of the granules or their staining with Congo Red. When granular expansion is taken as the measure of gelatinisation, the more resistant starches show gelatinisation curves displaced to higher temperatures.     

THE UPTAKE OF GIBBERELLIC ACID BY ABRADED BARLEY

The view that abrasion of barley allows gibberellic acid to enter the grain at the distal end by rupturing the pericarp is re-examined. Cumulative evidence is presented that once gibberellic acid has access to the pericarp surface, it can enter the grain and α-amylase is released. Hence, the abrading process is effective simply by removal of husk.     

Labeling of Starch Granules by Bombardment with Tritium Atoms

Starch granules were labeled by exposure to tritium atoms produced by thermal dissociation of tritium gas at a tungsten filament. Activity was shown by α-amylase etching experiments to be confined to the surface of the granule. Dextrins and low molecular weight compounds could be partially resolved on Sephadex G-75. The dextrin produced during tritiation had an elution volume corresponding to dextrin of 20–35 glucose units and was hydrolyzeable by α-amylase. Labeled starch was hydrolyzed first with ß-amylase and the limit dextrin hydrolyzed further with α-amylase. A comparison of the specific activities of the maltoses produced by the two enzymes demonstrated that labeling was more heavily concentrated in outer brunches than in inner branches. Amylose obtained from labeled potato starch was purified by crystallization and on DEAE-Sephadex columns. The ion-exchange purified potato amylose was hydrolyzed with ß-amylase with 50% of the activity retained in maltose. Tritium was found to be concentrated at the nonreducing ends of the amylose molecules. These results suggest that starch molecules are organized in granules with nonreducing ends oriented to the surface.     

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.