Digestion of Barley Starch Granules by the Combined Action of α- and β-amylases Purified from Barley and Barley Malt

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1 Agricultural and Biological Chemistry ISSN: (Print) (Online) Journal homepage: Digestion of Barley Starch Granules by the Combined Action of α- and β-amylases Purified from Barley and Barley Malt Iwao Maeda Shigeo Kiribuchi & Michinori Nakamura To cite this article: Iwao Maeda Shigeo Kiribuchi & Michinori Nakamura (1978) Digestion of Barley Starch Granules by the Combined Action of α- and β-amylases Purified from Barley and Barley Malt Agricultural and Biological Chemistry 42: DOI: / To link to this article: Published online: 09 Sep Submit your article to this journal Article views: 180 View related articles Citing articles: 1 View citing articles Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 02 December 2017 At: 12:58

2 Agric. BioI. Chern. 42 (2) 259~ Digestion of Barley Starch Granules by the Combined Action of a- and p-amylases Purified from Barley and Barley Malt Iwao MAEDA* Shigeo KIRIBUCHI** and Michinori NAKAMURA Department of Agricultural Chemistry The University of Tokyo Bunkyo-ku Tokyo 113 Received June a-amylase (EC ) and ti-amylase (EC ) were isolated and purified each to a state free from contaminating enzymes from the barley malt and ungerminated barley grains respectively. Starch granules isolated from the same sources served as substrates to study the mode of action of amylases on the digestion of starch granules in vitro.!'i-amylase alone had a very small activity on starch granules and formed maltose as a sole digestion product. a-amylase played a major role in the digestion of starch granules and the combined action of a- and ti-amylases was more effective than the action of a-amylase alone but was less effective on the same enzyme activity basis than the crude extract from barley malt which was thought to represent in vitro the enzyme system for the digestion of starch granules in the germinating barley grains. The possible roles to be played by a debranching enzyme a-glucosidase and/or glucosyl- or glucanotransferases are discussed. During their studies on the mechanism and mode of digestion of starch granules in germinating barley endosperms Kiribuchi and Nakamura!2) investigated the time-dependent changes of starch-degrading enzyme activities and carbohydrate contents. They found that the most rapid phase of starch digestion (4 to 8 days after the imbibition) coincided with the appearance of the maximum activity of u- and a-amylases. As there was no measurable decrease in the starch content in the first day of germination when there still was no u amylase although /I-amylase activity was already present at the level about half as much as the maximum value they postulated that u amylase might be mainly responsible for the digestion of starch granules during the germination. In a preliminary experiment it was shown (and was ascertained in this paper) that the crude extract from the barley malt (after 6 days of germination) could digest the barley starch granules in vitro at a reasonable rate and that * Present address: Hayashi Gakuen Women's Junior College Konan-shi 483. ** Present address: Department of Food Science Otsuma Women's University Sanbancho Chiyoda-ku Tokyo 102. the mode of digestion of starch granules by the crude extract as observed under a scanning electron microscope was very much alike if not identical to that occurring in vivo in the germinating barley endosperms. Therefore it was thought that the crude malt extract contained all the enzymes necessary for the digestion of raw starch granules in vivo. As stated above among the starch-degrading enzymes found in the crude extract the most conspicuous were u- and a-amylases from the point of view of their absolute activities and the time courses of fluctuation of their activities during the course of germination. In order to understand the mechanism and mode of digestion of starch granules in the germinating barley endosperms it seemed necessary to purify the starch-degrading enzymes and study the mechanism and mode of digestion of starch granules by these purified enzymes both alone and in combination. As a first step to this aim u- and p-amylases were purified extensively and the action of these two enzymes each alone and in combination on the starch granules in vitro was studied. The crude extract from malted barley served as a control representing the action of starchdegrading enzymes in vivo.

3 260 I. MAEDA S. KlRIBUCHI and M. NAKAMURA MATERIALS AND METHODS Reagents. Soluble starch (for the assay of amylase activity) was obtained from Wako Pure Chemicals & Co. and used after treatment with sodium borohydride. B ) p-amylase limit dextrin (P-LD) was prepared from waxy rice starch as described previously1) and after removal of the resulting maltose by dialysis and concentration in vacuo P-LD was precipitated from the solution by ethanol. Other chemicals were commercial products of the best quality. Preparation 0/ barley starch granules. Barley grains and malted barley (variety "New Golden") were kind gifts from the Sapporo Brewery Co. Tokyo. Starch granules were prepared from both sources essentially by the method of Banks et al. 4 ) as described by Taniguchi et au) Determination 0/ amylase activity 1) a-amylase. a-amylase activity was assayed by the International Association for Cereal Chemistry method as described by Perten 6 ) and modified by Kiribuchi and Nakamura1i so that the initial absorbance (E) at 575 nm of the iodine color reaction of the substrate P-LD was approximately 0.8. Within the range where a plot of log E against the reaction time in min gives a linear relation a-amylase activity is calculated using the following equation: units of a-amylase activity =300:< (log E1 -log E2) I (tz -t1) where E1 and Ez are the absorbance at 575 nm at time t 1 and 1 2 respectively and the factor 300 is used arbitrarily for convenience. 2) p-amylase. p-amylase activity was assayed essentially by the method of Bernfeld 7l at 30 C One unit is defined as the amount of enzyme that liberates one I'mole of maltose in one min under the defined conditions. Protein was determined by the method of Lowry et al. 8 ) and the specific activity of enzyme was given as units of enzyme activity per mg of protein. Purification of amylases 1) a.-amylase. Finely powdered barley malt (500 g) was extracted with 5 volumes of 2 % calcium acetate solution at 30 C for 2 hr with occasional stirring. The mixture was centrifuged at x g for 30 min and the supernatant solution was treated at 70 C for 15 min. The solution was cooled immediately and precipitates formed were centrifuged off at x g for 30 min (heat-treated crude extract). To the heat-treated crude extract cooled to O C were added maize starch granules which had been heated previously at a temperature a little below the gelatinization temperature in 20 % ammonium sulfate solution. The amount of starch to be added was determined by a preliminary small scale experiment. The mixture was stirred for 30 min to adsorb the a-amylase on the starch granules. The starch granules were collected on a Buchner funnel and washed with 2 % calcium acetate solution. a-amylase was then eluted with 0.1 M Trismaleate buffer ph 7.0 containing 0.5 % calcium acetate at 45 C for 1 hr and precipitated with 60% saturated ammonium sulfate. Further purification was carried out essentially by the method of Mitchel!.") The enzyme was subjected twice to Bio-gel P-100 column chromatography (2.6 x 60 cm) with 1 mm acetate buffer ph 5.5 containing 10 mm calcium chloride. u Amylase-active fractions which coincided with the 280 nm-absorbing peak were combined. The specific activity of this solution was units per mg of protein; it was approximately 57 times of purification over the heat-treated crude extract (190 units per mg of protein). No degradation product from pull ulan and no maltopentaose and glucose formation from maltotriose1 ) were detected by paper chromatography with this purified enzyme; therefore the purified enzyme was free from debranching enzyme (R-enzyme) and disproportionating enzyme (D-enzyme) activities. 2) p-amylase. p-amylase was purified essentially by the method of Visuri and Nummi. 11 ) Finely powdered ungerminated barley grains (1 kg) was extracted with 2.5 liters of water containing 2.5 ml of 2-mercaptoethanol and 3.5 g of dis odium EDTA per liter at 30 C for 2 hr. The crude extract obtained by centrifugation of the mixture at 7200 x g for 30 min was purified by ammonium sulfate fractionation (20 ~ 40 % saturation) phosphate fractionation and DEAE-cellulose (Whatman DE--52) (4.8 x 13 cm) and Sephadex G-100 (2.6/90 cm) column chromatographies. Active fractions were pooled and the enzyme was precipitated by the addition of 30 g of ammonium sulfate per 100 ml. The enzyme was kept as a paste in a refrigerator. Specific activity of the purified enzyme was 218 units per mg of protein. Digestion of starch granules. To 1.0 g of starch granules isolated from either lulgerminated barley grains or germinated barley malt were added 2.5 ml of 0.5 M acetate buffer ph 4.8 and 17.5 ml of 0.2 % calcium acetate solution containing amylase and the mixture was shaken at 90 rpm at 30 C. a-amylase at units and p-amylase at 1200 units were used either singly or in combination. The malt extract was prepared by extracting 20 g of finely powdered barley malt with 40 ml of 0.2 % calcium acetate solution at room temperature for 2 hr with occasional stirring. After centrifugation the supernatant solution was dialyzed against several changes of 0.2 % calcium acetate solution. The dialyzed malt extract contained approximately lulits of a-amylase and 1200 units of

4 Digestion of Barley Starch Granules by a- and /i-amylases!i-amylase and these amounts were added to the digestion mixture. At intervals 2 ml portions were withdrawn and after mild centrifugation the supernatant solutions were assayed for solubilized total sugars by the anthrone method12) and the percentage of digestion of starch granules was calculated from these determinations. Starch granules obtained after centrifugation of the reaction mixtures were washed with water three times dehydrated in ethanol dried under reduced pressure and served for scanning electron microscopic observations. 261 Gs I. Paper chromatography. Degradation products of starch by enzymes were separated and detected by paper chromatography. A definite amount of the solution was spotted with a micropipette on a sheet of Toyo filter paper No. 50 (20 x 20 cm) and subjected to chromatography by the double ascending method at 65 ~ 75 e. The solvent system used was 65 % n-propanol in water. Mter the chromatography the paper was treated with glucoamylase to intensify the color due to maltooligosaccharides13) and then the color was developed by immersion of the paper in the alkaline acetone-silver nitrate reagent. 14) Scanning electron microscopy. Scanning electron microscopic observations of starch granules isolated from barley grains or from enzymic digestion mixtures were carried out as described by Kiribuchi and Nakamura ) 30 Standard Standard Incubation ti me (min) FIG. I. Digestion Products of Soluble Starch by Purified Barley Malt a-amylase (I). Each reaction mixture containing 0.5 ml of I %soluble starch in 0.05 M acetate buffer ph 4.8 and 0.1 ml of 0.2 % calcium acetate solution containing 193 units of a-amylase was incubated at 30 0 e for the times specified under the chromatogram. '. RESULTS Digestion of soluble starch by a-amylase To test tubes each containing 0.5 ml of 1 % soluble starch in 0.05 M acetate buffer ph 4.8 were added 0.1 ml of a-amylase (193 units) in 0.2 %calcium acetate solution and the reaction mixtures were incubated at 30 e. At intervals the reaction was terminated by heating the tube in a boiling water bath for 1 min and 5u1 portions were spotted on a filter paper. The chromatogram showing the time course of the digestion of soluble starch by a-amylase is shown in Fig. 1. At the early stage of digestion intense spots corresponding to maltohexaose (G a) maltoheptaose (G7) and maltooctaose (Ga) appeared. With the progress of the reaction G a disappeared and then G 7 decreased while maltopentaose (G6) and smaller oligosaccharides increased. After 30 min G a was the most intense spot. 1 '. <. I Standard In cubation tim e (min). 30 Standard FIG. 2. Digestion Products of Soluble Starch by Purified Barley Malt a-amylase (2). The experimental conditions are the same as in Fig. I except the amount of a-amylase added to the reaction mixture is 1930 units (i. e. ten times as much as that in Fig. I).

5 J. MAEDA S. KIRmuc HI and M. NAKAMURA 262 Figure 2 shows the chromatogram of the digestion products of soluble starch when the amount of a-amylase added (1930 units) was ten times as much as in Fig. 1 under otherwise identical conditions. As early as 1 min after starting the reaction the spot corresponding to G 8 was absent G 7 and G 8 were intense and G 1 through G~ were already discernible. After 5 min G 7 disappeared and with the progress of the reaction G 6 also disappeared. During the course of the reaction G 4 appeared temporarily but disappeared after 30 min. At the last stage of the reaction maltose was the most abundant and along with glucose and maltotriose constituted the major portion of the digestion products of soluble starch by a-amylase. These results are in accord with the report of Hanes17) who showed that barley malt aamylase digested starch first into dextrins of mean degree of polymerization of 6 to 8; subsequent formation of glucose and maltose represented the second stage of digestion when there was a sufficient amount of a-amylase as compared to the substrate starch. Digestion products by fj-amylase To 0.5 ml of 1 % soluble starch or 2 %P-LD was added 5 pi (50 units) of purified fj-amylase 0 and the reaction mixture was incubated at 30 e for 60 min. The reaction was terminated by heating in a boiling water bath for 1 min and 5 pi portions were spotted on a filter paper. The chromatogram is shown in Fig. 3. Soluble starch gave only maltose and no product was formed from p-ld. These results show that the purified p-amylase is free from contaminating a-amylase and maltase activities. Action of a-amylase and/or fj-amylase on starch granules from ungerminated barley grains Figure 4 shows the time course of the Gs 10 o ---II Incubation time (hours) Time Course of Digestion of Starch Granules Isolated from Ungerminated Barley Grains. FIG. - Standard A B c D Standard 3. Digestion Products of Soluble Starch and II-Limit Dextrin by Purified Barley II-Amylase. FIG. The reaction mixture contained 0.5 ml of 1 % soluble starch (A and B) or 2 % fj-limit dextrin (C and D ) and 5 J.'I of water (A and C) or fj-amylase solution (50 units) (B and D ). The reaction mixtures were incubated at 30 C for 60 min. Each reaction mixture contained 1.0 g. of starch granules 2.5 ml of 0.05 M acetate buffer ph 4.8 and 17.5 ml of 0.2 % calcium acetate solution containing amylases. The reaction mixtures were shaken at 90 rpm at 30 C. Percentage digestion was calculated from the amount of solubilized total sugars determined by the anthrone method in the supernatant of the centrifuged reaction mixture. Amylases add~ were: cross 1200 units of fj-amylase; circle units of a-amylase; triangle units of a-amylase and 1200 units of fj-amylase; square the dialyzed malt extract (containing units of a-amylase and 1200 units of fj-amylase).

6 Digestion of Barley Starch Granules by digestion of barley starch granules by the actions of a- and B-amylases. Digestion of starch granules by the dialyzed malt extract was also included in the figure for the purpose of comparison. Purified B-amylase alone showed a very slight if any action on barley starch granules; the percentage of digestion obtained after 3 days was only 0.8 %. a-amylase on the other hand digested starch granules very well; the percentage of digestion after 3 days reached 42.7 %. B-Amylase although having almost no activity on starch granules showed a synergistic action on a-amylase in the digestion of starch granules; the percentage of digestion obtained by the combined action of both enzymes was 52.3 % after 3 days. However the combined action of a- and B-amylases was far inferior to the action of the dialyzed malt extract (85.9 %) having the same enzyme activities with respect to a- and B-amylases. Figure 5 shows the chromatogram of the solubilized digestion products from starch granules after 24 hr of reaction. It is apparent G G G} (1- and p-amylases 263 from the chromatogram that maltose is produced from starch granules by the action of B-amylase alone. a-amylase with or without B-amylase produced along with glucose maltose and maltotriose a series of oligosaccharides corresponding to G 5 and higher members. These oligo saccharides may represent mostly a- or ab-limit dextrins which are branched oligosaccharides. In contrast however the digestion products by the malt extract gives very little of these branched oligosaccharides and glucose maltose and mal totriose constitute the main digestion products. The absence of branched oligo saccharides in the digestion products indicates that the malt extract contains besides a- and B-amylases an enzyme probably a debranching enzyme which converts branched oligo saccharides into linear saccharides which are now susceptible to the further action of a- and B-amylases. Action of a-amylase and/or B-amylase on starch granules from barley malt Results of experiments similar to those given in the preceding section but using starch granules isolated from barley malt in place of starch granules prepared from ungerminated barley grains are shown in Fig. 6. The rate of G. 80 G; 70 c: ~ 40.'" G '" C 30 I 20.".' -'....0' - A"''''' "' Jf.o' / "/ / / / / 'ti' 'A' pl({ I" s I I J:{ jv 0 10 A B -"'" C D s '" FIG. 5. Solubilized Digestion Products of Starch Granules Isolated from Ungerminated Barley Grains. The reaction mixtures were the same as in Fig. 4; A B C and D in this figure stand for cross circle triangle and square in Fig. 4. The reaction was terminated after 24 hr and the supernatant of the centrifuged reaction mixture was subjected to paper chromatography. - o K 48 ~ 72 lncubation time (hours) FIG. 6. Time Course of Digestion of Starch Granules Isolated from Barley Malt. The reaction mixtures were the same as in Fig. 4 except starch granules isolated from barley malt were used in place of starch granules isolated from ungerminated barley grains.

7 264 I. MAEDA S. KIRIBUCHI and M. NAKAMURA digestion by a-amylase alone or in combination with p-amylase is greater with starch granules from germinated barley malt than with those from ungerminated barley grains (compare Figs. 4 and 6). The difference between the rate of digestion by the malt extract and that by purified enzymes was smaller with starch granules from germinated barley malt than with those from ungerminated barley grains; there was still an apparent difference between the rates by malt extract and by purified u- and p-amylases. However there was no essential difference between the chromatograms of the solubilized digestion products of starch granules prepared from either ungerminated barley (Fig. 5) or germinated barley malt (not shown). As the digestion proceeds the number of pits increases and the size of pits becomes larger. Some of the pits deepen vertically toward the center of the granule and the interior of the granule is digested preferentially keepingtbe approximate appearance of the granule intact. Some of the attacked granules were cracked at the equatorial region into halves probably during the preparation of the samples supporting the view that the interior of the barley starch granule is more susceptible to amylase digestion than the outer surface region of the granule (Figs. 9 and 10). The mode of digestion of starch granules by the dialyzed malt extract is very much alike to that by purified u- and p-amylases (Figs. 11 and 12). Double and/or triple pits as are shown in Observations with a scanning electron microscope Observations under a scanning electron microscope of starch granules prepared from ungerminated and germinated barley grains were reported previously.1516) Starch granules from ungerminated barley grains are consisted of two groups of granules; one is smaller-sized granules (av. diam. 5 ft) of almost spherical shape and the other is larger-sized granules (av. diam" 25 ft) of ellipsoidal or lense shape (Fig. 7). Some of the larger-sized granules bear a cluster of shallow pits reminescent of a chrysanthemum flower on the surface of the granule; what cause is responsible to this type of pits is not known at present. When starch granules are acted upon in vitro by a-amylase with or without {J-amylase there appear at an early stage several shallow pits at random on the surface of starch granules (Fig. 8) Fig. 12 may indicate the presence of the layered structure in starch granules each layer forming an obstacle to the action of amylases. Selec. tivity in the amylase digestion of starch granules has been reported as early as in 1961 by Leach and Schoch. 1S ) No essential difference was observed in the mode of digestion of starch granules by purified enzymes in vitro and by starch-degrading enzymes in vivo during the course of germination. DISCUSSION According to Walker and Hope 19 ) and Dunn20) p-amylases from sweet potato and barley did not act on maize starch granules. In this paper however the purified p-amylase from barley grains was shown to be able to attack barley starch granules although to a very small extent forming maltose as the sole degradation product. The purified p-amylase used in this study is free from contaminating FIGS. 7~12. Scanning Electron Micrographs of Barley Starch Granules Obtained under Various Conditions. See the text for explanation. FIG. 7. Starch granules prepared from ungerminated barley grains. FIG. 8. Starch granules treated with a-amylase for 24 hr (22 % digested). FIG. 9. Starch granules treated with a- and /i-amylases for 72 hr (52 % digested). FIG. 10. The same as in Fig. 9 but showing another microscopic field. The interior region of the granule was digested preferentially and the granule was cracked at the equatorial region into halves. FIG. II. Starch granules treated with the dialyzed malt extract for 72 hr (86 % digested). FIG. 12. The same as in Fig. 11 but showing another microscopic field. Double and/or triple pits indicate the presence of tbe layered structure in starch granules forming an obstacle to the amylase action.

8 Digestion of Barley Starch Granules by u- and p-amylases 265

9 266 I. MAEDA S. KIRIBUCHI and M. NAKAMURA a-amylase activity as is evident from the chromatograms shown in Figs. 3 and 5. Whether the difference in the source and intactness of starch granules or the difference in the assay conditions is responsible to these different results is not known at present. Also the significance of this finding in the digestion of starch granules in germinating barley endosperms is not known because the rate of digestion of starch granules by the purified p-amylase is very small indeed. Following the report of Reichert21) who showed that a-amylase could digest raw starch granules Kneen et at.22) Walker and Hope/g) Leach and Schoch'8) Clary et al."3) and Sandstedt and Ueda 24 ) studied the digestion the dialyzed crude extract from germinating barley malt which may represent the starchdegrading enzyme system in vivo. Addition of purified p-amylase (in an amount to give the same f3-amylase activity as in the malt extract) to the purified a-amylase increased the rate of digestion to a moderate extent although still much slower than that obtained with the malt extract. As the purified f3-amylase itself could hardly attack starch granules the accelerating effect of B-amylase on a-amylase is synergistic rather than additive. B-Amylase is thought to spare the a-amylase activity by digesting the dextrins solubilized by the action of a-amylase on starch granules which are to be digested further by the a-amylase itself in the absence of raw starch granules by various kinds of of added f3-amylase. Thus in the presence of amylases and their specificities. These studies added B-amylase a-amylase can devote its however employed various combinations of amylases and starches of different origins and made little contribution to the understanding of the digestion of starch granules in the germinating plant seeds where for instance barley starch granules are digested by the barley enzymes present in the end os perms of germinating barley seeds. Therefore these studies can serve only as auxiliary aids to this study the ai11l of which is to study the mode of digestion of starch granules by the degradative enzymes of the same plant source during the activity to the digestion of starch granules rather than to the digestion of solubilized dextrins its own digestion products. The slower rate of digestion obtained by a combination of purified 1.- and B-amylases than that obtained by the malt extract suggests that either there may be another enzyme which is able to digest starch granules directly or there may be another auxiliary enzyme or enzymes besides f3-amylase. The chromatogram of Fig. 5 shows that in the malt extract system glucose is produced in a much larger amount course of germination. From his model experiments on the degradation of starch granules in higher plants < Dunn ZO ) claims that (1) a-amylase is the sole enzyme that can attack starch granules and (2) other starch-degrading enzymes (such as f3-amylase limit dextrinase and a-glucosidase) play auxiliary roles in degrading dextrins solubilized by the action of a-amylase on starch granules. The results of this study support the view of Dunn with minor reservations. < It was shown < in this paper that purified;a-'amylase could effectively digest starch gramdes isolated from barley grains. However when compared on the same a-amylase activity basis the rate of «digestion' of starch granules by the purified <;1-amylase was much slower than the rate by and there is very little of branched dextrins. These results indicate that a debranching enzyme (R-enzyme) and at least one of glucoseforming enzymes (e.g. maltase a-glucosidase D-enzyme) may be operative in the malt extract system although the participation of an enzyme that is able to attack starch granules directly can not be excluded. The fact that starch granules isolated from germinated barley malt are more susceptible to amylase digestion than those from ungerminated barley grains is supported by the finding reported previously2) that the former < starch contained a larger proportion of short linear fractions than the latter starch. This suggests thilt a debranching enzyme may "'\.. ~-". " participate in the digestion of starch granules directly converting some of the branched

10 Digestion of Barley Starch Granules by u- and /i-amylases 267 fraction into the linear fraction although the digestion products are still attached to the granule and not solubilized. However there is as yet no evidence for the direct participation of a debranching enzyme on the digestion of starch granules. The possible role of a debranching enzyme in the digestion of starch granules is now under investigation. REFERENCES 1) S. Kiribuchi and M. Nakamura Nippon Nogeikagaku Kaishi (1973). 2) S. Kiribuchi and M. Nakamura ibid (1973). 3) D. H. Strumeyer Anal. Biochem (1967). 4) W. Banks C. T. Greenwood and D. D. Muir Starke (1973). 5) H. Taniguchi I. Maeda and M. Nakamura Agric. BioI. Chem. 42 in press (1978). 6) H. Perten Cereal Chem (1966). 7) P. Bernfeld "Methods in Enzymology" Vol. 1 ed. by S.P. Colowick and N.O. Kaplan Academic Press Inc. New York 1955 p ) O. H. Lowry N. J. Rosebrough A. L. Farr and R. J. Randall J. BioI. Chem (1951). 9) E. D. Mitchell Phytochemistry (1972). 10) E. Y. C. Lee J. J. Marshall and W. J. Whelan. Arch. Biochem. Biophys (1971). 11) K. Visuri and M. Nummi Eur. J. Biochem (1972). 12) W. E. Trevelyan and J. S. Harrison Biochem. J (1952). 13) K. Kainuma and D. French FEBS Lett (1969). 14) W. E. Trevelyan O. D. Procter and J. S. Harrison Nature (1950). 15) S. Kiribuchi and M. Nakamura Denpun Kagaku (1973). 16) S. Kiribuchi and M. Nakamura ibid (1973). 17) C. S. Hanes New Phyto/ (1973). 18) H. W. Leach and T. J. Schoch Cereal Chem (1961). 19) G. J. Walker and P. M. Hope Biochem. J (1963). 20) G. Dunn Phytochemistry (1974). 21) E. T. Reichert Publ. Carnegie lnst. No. 173 Part 1 (1913). 22) E. Kneen O. C. Beckord and R. M. Sandstedt. Cereal Chem (1941). 23) J. J. Clary G. E. Mitchell and C. O. Little J. Nutrition (1965). 24) R. M. Sandstedt and S. Veda Denpllil Kogyo Gakkaishi (1969).