Food Hydrocolloids VolA no.5 pp.365-377 , 1991

Studies on mung bean starch: granule stability

e.G.Oates

Department of Biochemistry, National University of Singapore , 10, Kent RidgeCrescent, Singapore 0511

Abstract . Physicochemical measurements confirm that mung bean starch exhibits restricted swellingand high granular stability. The effect of a non-specific protease upon the fractionation and type ofprodu ct obtained after hydrolysis with amylases was investigated. Successful fractionation wasachievable only after treatment with the protease. Limited debranching and reduced l3-amylaseactivity was shown; such phenomena are typical of cross-bonded starch. Further study usingproteolytic enzymes suggests that the amylopectin fraction of mung bean starch is stabilized by thepresence of peptide cross-links. Electrophoresis indicated the presence of two major polypeptides oflow molecular weight associated with the amylopectin fraction.

Introduction

The major commercial application of mung bean starch is in the production ofmung bean noodles. In such a product the high rate and extent of retrogradation,of this starch can be fully utilized . Further, this starch gives a brabender curvewhich is characteristic of restricted swelling starches. Amylographs with nopasting peak but rather a constant increase in viscosity during cooking at 95°Cand constant cold paste viscosity when agitated at 50°C for 1 h characterize thisstarch. Related to the restricted nature of this starch , further interestingproperties include: low solubility, resistance to a-amylase attack, and highgelation temperature (1) which ensures the inclusion of this starch in a widerange of products. The high level of granular stability exhibited by this starch isin common with other legume starches. These starches possess a high degree ofcrystallinity. Evidence in support of a highly crystalline structure has beenprovided by a number of authors; Hoover and Sosulski (2) have correlated thedegree of crystallinity of legume starch granules with their resistance tohydrolysis by a-amylase, whilst Biliaderis et al. (3) propose that the restrictednature of legume starches is due to the more rigid structure of their amylopectinchains. Tolmasquim et al. (4) have proposed that these chains may well bestabilized by the inclusion of a number of natural cross-links, but unfortunatelythese authors do not give any indication of the nature of these stabilizingbridges. Natural starch phosphodiester bonds have been suggested as possiblecross-links, but the low phosphorus levels of 0.010-0.016% commonly encoun­tered in legume starches would preclude any significant contribution of suchcross-links to granule stability. In support of Tolmasquim et al. (4) and Hooverand Sosulski (5) have also suggested that covalent bonds may be present withinthe starch granule.

Lipids associated with starch granules can restrict the adsorption of water andrelease of soluble material by these starch granules. The lipid content of legumestarches of 0.12-0.20% would be too low to result in the extent of restrictionexhibited by these starches .

© Oxford University Press 365

C.G.Oates

The purpose of this work was to investigate the possible existence of peptidecrosslinks and to determine their influence on the stability of mung bean starchgranules.

Methods

Materials

Sweet potato f3-amylase (EC 3.2.1.2), pullulanase obtained from Enterobacteraerogenes (EC 3.2.1.41) and bromelain, sp. act. 2900 units/g protein (EC3.4.22.4) were purchased from Sigma Chemical Company, USA. Isoamylasefrom Pseudomonas amyloderamosa (EC 3.2.1.68) was purchased from Hayash­ibar Biochemical Laboratories Inc ., Japan. All other chemical were at leastanalytical grade and were also purchased from Sigma Chemical Company, USA.

A Tris buffer was prepared as follows: Tris (0.055 mol/drrr' , pH 6.8), sodiumdodecylsulphate (2.3%), 2-mercaptoethanol (5.0%) and glycerol (10%) madeup with distilled water.

Starch isolation and preparation

Starch was isolated from Thai mung beans by the procedure of Schoch andMayweld (1) with minor modifications. Mung beans , purchased from the localmarket, were washed free of any extraneous matter and soaked in sodiumsulphite for 20 h. Pre-soaked beans were ground in a blender with a minimumquantity of water, slurried with distilled water (200 ml/lOO g) and the slurryfinally passed through muslin cloth. The procedure was repeated three times toensure maximum extraction of the starch. The filtrates were combined andallowed to settle, the water and brown residue decanted off , and the starchpurified with six successive sedimentations in 0.1 mol/drrr' NaC!. Purificationwas achieved by suspending the starch in 0.1 mol/dnr' NaCl/toluene (0.2%) ,shaking for several hours , and washing with distilled water until all traces oftoluene had been removed. Finally the starch was ethanol-washed and dried at40°C in a convection oven.

Suspensions (10%) of starch in phosphate buffer (pH 5.2) were incubated at45°C for 48 h. To some of the suspensions was added 1.2 units bromelain/gstarch. Samples were again dried after washing with ethanol and diethyl ether.

Starch swelling power and solubility

Swelling power and solubility determinations were carried out for the tempera­ture range 70-90°C by the procedure of Ring (6). Starch granule size wasmeasured using a microscope fitted with a calibrated eye piece . The longestdimension of 100 granules was taken as an estimate of the granular size.

Starch fractionation

Mung bean starch was fractionate according to the method of Takeda andHizukuri (7). It was found that this starch had to be re-dissolved three times in

366

Granule stability of mung bean starch

dimethyl sulphoxide (DMSO), with heating, before satisfactory solubility inwater could be achieved.

Samples of amylose were incubated with bromelain (1.2 units/g amylose) at45°C for 48 h. The amylose was subsequently re-crystallized three times in hotsaturated I-butanol before finally washing with ethanol and diethyl ether priorto drying under vacuum.

Gel permeation studies

Starch granules were gelatinized following suspension in 25% DMSO andheating at 90°C overnight in a convection oven. After gelatinization the solutionswere diluted to give a final DMSO concentration of 10%. The solutions weremade 0.25 mol/drrr' with respect to KOH and separated using sephacryl S500 in a100 X 25 mm column, with an eluting phosphate buffer (pH 8) flow rate of 8­9 ml/h. Total sugar was determined by the method of Dubois et al. (8).

Enzymatic studies were performed using crystalline suspensions of sweetpotato !3-amylase, isoamylase and pullulanase. Samples of amylopectin weredissolved in phosphate buffer (pH 5.2); some of these samples, prior toenzymatic hydrolysis, had been incubated with bromelain (0.1 %) at pH 5.2 for48 h at 45°C. Prior to hydrolysis with !3-amylase and/or pullulanse or isoamylase,all samples were heated at 100°C for 15 min and analysed for reducing sugar.Hydrolysis was carried out as described by Hood and Mercier (9). Gelpermeation chromatography on sephacryl S200 was carried out in a 100 X

25 mm column at a flow-rate of 8-9 ml/h with a phosphate buffer (pH 5.2) as theeluant. Total sugar was determined by the method of Dubois et al. (8) andreducing sugar by the neocuproine method (10).

Electrophoresis

Starch granules were washed by re-suspension and centrifugation in the Trisbuffer three times. The granules were washed a further two times with distilledwater and dried under vacuum. Protein extraction was carried out by a modifiedGoldner and Boyer (11) method. Starch granules (10 mg), amylopectin,amylose, or 30 mg bromelain-pretreated amylopectin were mixed with the Trisbuffer (0.1 ml) and heated in a boiling water bath for 2 min. To the gelledsolution, after cooling on ice, 0.2 ml of Tris buffer + bromophenol blue wasadded. Finally, the preparation was centrifuged and 20 or 30 ILl used forelectrophoresis.

Electrophoresis was carried out on 1.5 mm-thick vertical slab gels accordingto Laemmli (12). Denaturing 12% SDS-polyacrylamide gels were used, alongwith 4.0% stacking gel. Silver staining of the gels was carried out according toWray et al. (13).

Scanning electron microscopy

Starch samples were sprinkled on double-sided tape attached to a stub. Sampleswere coated with a 20 nm carbon-gold layer and examined with a Philips SEM515 scanning electron microscope at 10 kV.

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Results

Swelling power and solubility

Mung bean starch showed a limited two-stage swelling and solubility pattern(Figure 1) typical of legume starches and cross-bonded starches. Followingreaction with bromelain, a non-specific protease, both swelling and solubilitywere significantly increased. Over a similar range of temperatures the meangranule size was greater for those granules previously pre-treated withbromelain. Both granule populations exhibited the same mean size prior toheating in water (Table I).

Effect of bromelain on starch fractionation

Concentrations of bromelain in the range used in this study when incubated withamylopectin have no significant effect on the amount of reducing sugar detectedafter incubation, as compared with the material incubated without the protease(Table II). Higher concentrations (10 x recommended level), when incubatedwith amylopectin, can result in a slight increase in the reducing value (0.009%).

...~/

'",-,-

/'

30

...Gl~oQ.

01C

20

10

15

9080

Te mperature (C)

O~-----r------~----~O70 100

Fig. 1. Swelling and solubility profile for mung bean starch. Bromelain-treated starch: (.) swelling,(A) solubility; non-treated starch: (0) swelling, (L',) solubility.

368

Granule stability of mung bean starch

Table I. Percentage increase in the size of mung bean starch granules following heating

Temperature(DC)

Native708090

Granule size (% native size)

Bromelain-treated

100137.5187.5237.5

Untreated

100100137.5175

Table II. Effect of pre-incubation with different concentrations of bromelain on the percentageincrease in reducing value recorded in amylopectin solutions

Bromelain concentration(units/g amylopectin)

0.010.11.0

Increase in reducing value(%)

0.0050.0050.009

The surfaces of bromelain-treated granules show no signs of amylolytic attackwhen viewed by SEM (Figure 2).

The elution profile of gelatinized mung bean starch separated on sephacrylS500 exhibited both a major peak at Yo and a minor peak centred at an elutionvolume of 300 ml (Figure 3). In contrast, two well-defined peaks were obtained,one at Vo and the second centred at an elution volume of 300 ml, if this starchwas digested with bromelain following gelatinization. This would suggest thatthe amount of amylose leached out of the starch granule is increased if thegelatinized granule is further incubated with bromelain. Amylose fractionatedby repeatedly precipitating with I-butanol was not pure as the elution profile ofthis material indicated the presence of a high-molecular-weight componentwhich could not be removed despite repeated re-crystallization 12 times with 1­butanol (Figure 4). However, apparently pure amylose fractions, free fromcontamination by high-molecular-weight material, were obtained followinghydrolysis with bromelain and subsequent re-crystallization with l-butanol(Figure 4). The peak maxima at 320 ml of amylose before and after digestionwith bromelain were the same.

Amylopectin analysis

Amylopectin extracted from mung bean starch was found to be relativelyresistant to l3-amylase (Table III); only 30% hydrolysis of the amylopectin wasachieved following incubation with l3-amylase. This constituted only 60% of theamount of hydrolysis after pre-digestion with bromelain. Further, morecomplete hydrolysis by the combined action of pullulanase and l3-amylase wasshown only by bromelain-treated samples. The non-treated material was only59.5% hydrolysed.

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C.G.Oates

a

b

""'..., ;..f"'r\ (.

'.~.t ..1 " •• .: " =1It~ ~

. ' . -=-~_..-=/.,~~ " ~.~ .-~ .~ .

Fig. 2. Scanning electron micrographs of mung bean starch granules. Bar = 10 urn. (a) Nativegranules . (b) Granules pre-treated with bromelain.

The elution profile of pullulanase de-branched amylopectin (Figure 5) andisoamylase debranched amylopectin (not shown) show a broad peak centred atan elution volume of 352 ml and a much smaller peak immediately after Vo(Figure 5). The elution profile of amylopectin after de-branching for bothenzymes used and following bromelain pre-treatment exhibited the double peaktypically associated with de-branched amylopectin (14) . Peak maxima wereevident at 312 and 352 ml; the small peak immediately after Vo was very muchreduced or not evident.

Electrophoresis studies

Both amylopectin and starch granules contained major polypeptide componentswith mol. wts of 55 000 and 50 000, as shown by the electrophoretic separation

370

Granule stability of mung bean starch

400300

VOLUME (mU

--- -- - - "">, "-""'--- \.\

-,'r ....

200

ELUTIONV o

...I-Z:J

>a:l(a:I-IIIa: I

l(I

w II

D IJ

0 I

Fig. 3. Effect of bromelain pre-treatment on the gel permeation chromatograph y elution profile ofgelatin ized mun g bean starch. Bromel ain-tr eated (-) and non-treated (-_.) .

of low concentrations of the protein extract. High concentrations of thesesamples revealed the presence of an additional three minor polypeptides of45 000, 42 000 and 27 500. Bromelain pre-treated amylopectin and amyloseapplied even at a high concentration exhibited either reduced intensity of theprotein bands or, as in the case of amylose, no bands at all (Figures 6 and 7).

Discussion

Mung bean starch showed a limited two-stage swelling pattern which isindicative of strong bonding by two sets of forces within the ' granule (15). Suchrestricted swelling patterns are also common for cross-bonded starches (16). It isinteresting that the swelling and solubility profiles are increased after incubationwith bromelain. This phenomena is difficult to explain as it is generally acceptedthat molecules with a molecul ar weight greater than 1000 will not readilypenetrate into the starch granule (mol. wt of bromelain: 19-20000). The result sthough do suggest some form of structural change; SEM photomicrography ofthe granules before and after bromelain treatment do not indicate any grosschang e in the structure, so that it would seem likely that changes are at the level

371

e.G.Oates

....Z:::l

>a:«a:....ma:4

co

/'

/ '/

//

( //

1\ /1\ /I I I1\ /, . r ::I .... _/IIIIIIIJ

\\

\\\\\\\\\

50~ELUTION

e!50

VOLUME ml

4!50t;

Fig. 4. Effect of bromelain pre-t reatment on the gel permeation chrom atog raph y elution profile offractionated amylose . Bromelain -treated (--) and non-treated (---) .

Table III . Extent of hydrol ysis by 13-amylase and 13-amylase with pulluJanase *

Sampl e

StarchStarch with bromelain

* Values are mean % ± SD

13-Amylase

30.0 ± 4.8553.6 ± 2.92

13-Amylase with pullulanase

59.5 ± 3.32102.0 ± 6.77

of the fine structure. Further work is being undertaken to investigate thephen omena of bromelain action on whole granules .

Incubation of amylopectin with bromelain in the concentration range 0.12-12units/g did not result in any significant degradation of the amylopectin structure .Further, sta rch granules incubated with bromelain for an extended period (48 h,50°C) do not show any indication of amylolytic activity when observed by SEM .No significant shift in peak maxima as indicated by the elution profiles (Figure 4)would also suggest that this enzyme has no amylolytic action on the major chainlength populations of amylose or amylopectin, though increased amounts of low­molecular-weight material are liberated after hydrol ysis with the protease. Theresults previously presented would suggest that this is reflective of the fact that

372

Granule stability of mung bean starch

...

,\

\\,\\\

\

\\\\

/-,/ \

/ \

I \

I "1/ \

~/ \/~

II

II

I/

II

/I

II

h

(At

I II II \

I "I \I \

I 'I "I -,

./ "_______ I

co

~

2:J

>II:CI:II:~

IIII:CI:

120 '10

240

ELUTION VOLUME EmU

360

Fig. 5. Effect of bromelain pre-treatment on the gel permeation chromatography profile ofpullulanase de-branched amylopectin. Bromelain-treated (--) and non-treated (---).

LANE

1 2 3 4 5 6 7 MWT

16000

66000

97400

3470045000

-- t--- -- t---

I--

-1--11f--

Fig. 6. Diagram of 50S-PAGE separation of polypeptides extracted from starch granules andfractions of amylose and amylopectin. Lane 1: Starch granules (30 ul}; 2: starch granules (20 ul); 3:amylopectin (30 u.l}; 4: amylopectin (20 ul); 5: amylose (30 ul); 6: amylose (20 ul}; 7: molecularweight standards.

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C.G.Oates

29K

45K

7·4 1<i16K

Fig. 7. SDS-PAGE separation of polypeptides extracted from starch granules and fractions ofamylose and amylopectin. Lane 1: molecular weight standards; 2 and 3: starch granules (30 u.l); 4and 5: amylopectin (30 ul); 6: amylose (30 u.l); 7: bromelain pre-treated amylopectin (30 fl.l); 8:molecular weight standards.

the low-molecular-weight material is more easily diffusible after proteaseactivity as opposed to any major degradation of the polymers. Bromelain is aglycoprotein and, as such, care must be taken to correct for the reducing activityinherent in the enzyme; this may well account for the slight increase in reducingvalue observed following the use of the high levels of this enzyme.

Following gelatinization, a limited amount of amylose is leached from thestarch granules; the appearance of a second peak after pre-incubation withbromelain would suggest that greater quantities of amylose are leached from thegranule after this treatment. Unfortunately, the relative amount of amylosewhich is released is not known, though it does not seem possible for all of theamylose to be leached from the granule; further study is in progress tounderstand the nature of the amylose remaining in the granule after leaching.The increased tendency for amylose leaching after bromelain digestion could bethe consequence of the breakdown of intra-chain peptide links. The elutionprofiles for treated and non-treated amylopectin have been shown to be similar(Oates, in preparation); it is suggested that this result is due to the disparitybetween the cut-off point of S-500 and the molecular size of amylopectin. It isinteresting that even after the multiple purification procedure recommended byTakeda et at. (17) for the isolation of homogeneous samples of rice amylose, it isnot possible to obtain a pure amylose fraction from mung bean starch; however,it is possible to achieve an homogeneous sample following pre-incubation withbromelain and subsequent re-crystallization. Analysis of the fine structure ofmung bean and other legume starches has not been carried out usinghomogeneous amylose fractions; previously reported results have all indicatedthe presence of a small amount of contaminating amylopectin (18,19). This workis suggestive of the fact that pre-incubation with bromelain could well be an

374

Granule stability of mung bean starch

efficient method for obtaining pure fractions of mung bean starch for structuralanalysis (e.G.Oates, in press).

The nature of the high-molecular-weight fraction remaining after de­branching is unknown, though Hood and Mercier (9) suggest that this fractionmay be composed of undebranched amylopectin. A similar fraction present afterde-branching either chemically cross-linked cassava starch (9) or other legumestarches (20) has been found. De-branching of amylopectin with pullulanase orisoamylase results in the liberation of only one major population of polysacchar­ide chains; further work using a calibrated column (e.G. Oates, in press) wouldsuggest that this material has an average chain length of 14. Followingincubation with bromelain and subsequent hydrolysis with pullulanase a secondpopulation of polysaccharide chains is liberated with an average chain length of42. It is therefore apparent that in the non-bromelain-incubated material onlyspecific branches are cleaved by the action of de-branching enzymes; thesebranches are relatively short and correspond to the material of Dp 11-16, whichis regarded as the outer branches of amylopectin by a number of authors(9,21 ,22) . This is suggestive of the fact that the outer 1-6 glucosidic bonds ofmung bean amylopectin are preferentially hydrolysed by the action of the de­branching enzymes. As a consequence of pre-hydrol?,sis with bromelain andsubsequent exposure to de-branching enzymes the fraction eluting immediatelyafter Vo is lost and a population of larger chain length material liberated. Thismay indicate that bromelain is able to penetrate into the amylopectin structure.Such accessibility of amylopectin to bromelain as opposed to the amylase couldwell be reflective of either the smaller mol. wt of the protease [bromelain: mol.wt 19000-20000 (23); pullulanase: mol. wt 90000-100 000 (24); ~-amylase:

mol. wt 197 000 (25); isoamylase: mol. wt 90000 (26)] or that its mode of actionis such that a gradual loosening of the amylopectin molecule takes place as themore peripheral polypeptide cross-links are progressively hydrolysed.

Results reported by Hood and Mercier (9) imply that the presence ofmodifying groups attached to amylopectin chains physically inhibit the action ofstarch hydrolysing enzymes-though this work suggests also that, as aconsequence of structural restraints , amylase activity is inhibited in mung beanstarch. The mechanism resulting in resistance to the action of amylases bychemically modified starch and mung bean starch would appear to be different.It is unlikely that bromelain will remove amino acid substitution from thecarbohydrate moiety but will rather cleave .polypeptide bonds randomly alongthe polypeptide chain. The increased amylase activity therefore seems to takeplace despite the presence of amino acid 'stubs'. If the existence of suchsubstitutions does not inhibit amylase activity, it is possible that the 1-6glycosidic bonds are protected by a general 'tightening' of the amylopectinstructure.

Several different molecular weight proteins have been extracted from thegranules of starch obtained from cereal crops; the number of proteins detectedin a granule (between 3 and 25) is dependent upon the variety of the cereal fromwhich the starch was originally isolated. The mol. wts of these proteins havebeen determined (11); such analysis has revealed that certain proteins are

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e.G.Oates

common to most cereal starches. A protein with a mol. wt of 68000 has beenfound in all the cereal starches examined, whilst a second protein of mol. wt60 000 is common to 81% of the starches. Analysis of the granular proteincomponent of mung bean starch suggests the presence of a number of proteinswith different molecular weights ranging from 27 500 to 55 000. The rigoroustreatment of the starch granules prior to extraction guarantees that the detectedprotein has not originated from the surface of the granule (11). The proteinsisolated from starch granules, it has been implied, relate to starch synthases (27)or glucotransferases (28). The mol. wts of the proteins present in mung beanstarch granules would suggest that these proteins are too small to be either ofthese enzymes (starch synthases and glucotransferases have mol. wts >70 (00).It is of further interest that the protein component isolated from the granules ofmung bean starch is also present in amylopectin but not in amylose fractions.This may indicate that amylopectin alone is stabilized by polypeptide cross-linkswhilst amylose remains free to move in and out of the granule. The inabilitysuccessfully to obtain pure samples of amylopectin and amylose by fractionationof mung bean starch could well be the result of a tightly compacted amylopectinstructure as opposed to any cross-linking between the two fractions.

This work would therefore suggest the possible existence of peptide cross­links within the amylopectin fraction of mung bean starch, which are responsiblefor maintaining the high level of granular integrity observed for this material.

Acknowledgements

The author would like to thank for technical assistance Miss Lee Woan Peng,Miss S.L.Sim and Miss B.l.Sim, and gratefully acknowledges the support of theNational University of Singapore.

References

1. Schoch,T.J. and Mayweld,E.C. (1968) Cereal Chern., 45, 564-573.2. Hoover,R. and Sosulski,F. (1985) Starch/Starke, 37, 181-191.3. Biliaderis,C.G., Maurice,J.J. and Vose,J.R. (1980) J. Food su., 45, 1669-1674.4. Tolmasquim,E., Correa,A.M.N. and Tolmasquim,S.T. (1972) Cereal Chern., 48,132-135.5. Hoover,R. and Sosulski,F. (1985) Starch/Starke, 37, 397-404.6. Ring,S.G. (1985) Starch/Starke, 37, 80-82.7. Takeda,Y. and Hizukuri,S. (1986) Carbohydr. Res., 148,299-308.8. Doubois,M., Gilles,K.A., Hamilton,J.K., Rebers,P.A. and Smith,F. (1956) Anal. Chern., 28,

350-354.9. Hood,L.F. and Mercier,C. (1978) Carbohydr. Res., 61, 53-66.

10. Dygert,S., Li,L.M., Florida,D. and Thoma,J.A. (1965) Anal. Biochern., 13,367-374.11. Goldner,W.R. and Boyer,C.D. (1989) Starch/Starke, 41, 250-254.12. Laemmli,U.K. (1970) Nature, 227, 680-685.13. Wray,W., Boulikas,T., Wray,V.P. and Hancock,R. (1981) Anal. Biochern., 118, 197-203.14. Hizukuri,S. (1985) Carbohydr. Res., 141, 295-306.15. Leach,H.W., McCowen,L.D. and Schoch,T.J. (1959) Cereal Chern., 36, 534-544.16. Hoover,R. and Sosulski,F. (1986) Starch/Starke, 38, 149-155.17. Takeda,Y., Tokunaga,N., Takeda,C. and Hizukuri,S. (1986) Starch/Starke, 38, 345-350.18. Biliaderis,C.G., Grant,D.R. and Vose,J.R. (1981) Cereal Chern., 58, 496-502.19. Banks,W. and Greenwood,C.T. (1967) Starch/Starke, 19, 197-206.20. Biliaderis,C.G. (1982) J. Agric. Food Chern., 30, 925-930.21. Takeda,Y., Shitaozono,T. and Hizukuri,S. (1988) Starch/Starke, 40, 51-54.

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Granule stability of mung bean starch

22. Enevoldsen,B.S. and Juliano,B.O. (1988) Cereal Chem., 65, 424-427.23. Feinstein,G. and Whitaker,J.R. (1964) Biochemistry, 3, 1050-1054.24. Norman,B.E. (1983) J. lap. Soc. Starch Sci., 30, 200-204.25. Spradlin.J, and Thoma,J.A. (1970) l. Bioi. Chem., 245,117-127.26. Yokobayashi,K., Misaki,A. and Harada ,T, (1970) Biochim. Biophys. Acta 212, 458-469.27. Baxter,E.D. and Duffus,eM. (1971) Phytochemistry, 10,2641-2647.28. Preis.L, McDonald,F.D., Singh,B.K., Robinson,N. and McNamara,K. (1985) In Hill,R.D. and

Munck.L. (eds), New Approaches to Research on Cereal Carbohydrates. Elsevier, Amsterdam.

Received on May 10, 1990; accepted on September 11, 1990

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