Journal of the Science of Food and Agriculture J Sci Food Agric 79 :648–652 (1999)

Reduction in flatulence factors in mung beans(Vigna radiata) using low-dose c-irradiationJP Machaiah,* MD Pednekar and Paul ThomasFood Technology Divis ion, Bhabha Atomic Res earch Centre, Fiply , Trombay,Mumbai-400 085, India

Abstract : Mungbeans (Vigna radiata), control and c-irradiated at insect disinfestation dose levels

(0.25 and 0.75 kGy) were germinated (0–6 days) and the qualitative and quantitative changes in soluble

carbohydrates were studied in detail. The key ýatulence-producing raffinose family oligosaccharides

in mungbeans were degraded in the irradiated samples at the onset of the germination (0–2 days)

compared to the control where it occurred much later (> 4 days). However, the reducing sugars,

mainly glucose, fructose and galactose, which are metabolised easily, were enhanced in the irradiated

samples. At low dose (0.25 kGy), irradiation had no eþ ect on germination and sprout length, indicating

that irradiated beans are suitable for use as sprouted beans. These observations clearly indicate that

c-irradiation at insect disinfestation dose levels improved the digestibility and nutritional quality of

mung beans by reducing the content of oligosaccharides responsible for intestinal gas production.

1999 Society of Chemical Industry(

Keywords: mung beans ; oligosaccharides ; c-irradiation; germination

INTRODUCTION

Legumes, stored as dry seeds, are a good source ofprotein, carbohydrates, vitamins and minerals. Ger-mination enhances the digestibility of legumes.Sprouted legumes form an important food item inmany Asian countries including India, consumedeither alone or in combination with other prep-arations like noodles, salads etc. Legumes, however,contain ýatulence-causing factors resulting in theformation of intestinal gas and discomfort1 whichimpedes their full nutritional utilisation. Flatulenceis the result of action of intestinal ýora on the raff-inose family oligosaccharides comprising mainlyverbascose, stachyose and raffinose. They containa(1-6) galactosido-ýucose and a(1-6) galactosido-galactose bonds which are indigestible to mammalianenzymes.2

Reduction in ýatulence in germinated legumes dueto breakdown of these oligosaccharides has beenobserved.3 Considerable reduction in gas productioncould also be achieved by aqueous or alcoholicextraction of legumes, altering pH, temperature,moisture levels and treatment with a-D-galactosidase(EC 3.2.1.22) from plant or microbial sources. Theseprocesses, however, downgrade their acceptability insensory evaluation.4

Gamma-irradiation treatment at low doses of 0.25–0.75kGy has been recommended for disinfestation oflegumes5 infested with all life stages of storage insectpests. Earlier studies from our laboratory haveshown that irradiation treatment of mungbeans at 2.5

to 10kGy eþectively reduced the ýatulence-producing oligosaccharides.3 The doses used here,however, were much greater than those required fordisinfestation of legumes. The present study wastherefore undertaken to evaluate if the recommendeddoses of radiation for disinfestation of legumes couldalso upgrade the nutritional quality of mungbeans byreducing the ýatulence factors. The qualitative andquantitative changes in the extractable carbohydratesin mung beans, irradiated at 0.25kGy to 0.75kGydose levels during germination were investigated.

MATERIALS AND METHODS

Mungbeans (Vigna radiata) were purchased from thelocal market and one single batch was used through-out the studies. All the standard carbohydrates,except verbascose, used in these studies wereobtained from Sigma Chemical Co USA. Verbascosewas a kind gift from Dr E Cristafaro, Nestle� Pro-ducts Technical Assistance Ltd (La Tour-De-Peilz,Switzerland). All the reagents used were made fromanalytical grade chemicals and the solvents used wereall redistilled prior to the experiment.

Irradiation

Mungbeans (100g lots) packed in polyethylene bags,were irradiated to doses of 0.25 and 0.75kGy gammarays at 25¡C in a Cobalt 60 Gamma-cell 220 (AECL,Canada) at a dose rate of 21.6Gy min~1. The over-dose ratio was 1 : 1.25. The irradiated samples were

* Corres pondence to : Food Technology Divis ion,JP Machaiah,

Bhabha Atomic Res earch Centre, Fiply, Trombay, Mumbai-400

085, India.

(Received 2 April 1997 ; revis ed vers ion received 24 December

1997; accepted 6 Augus t 1998)

( 1999 Society of Chemical Industry. J Sci Food Agric 0022–5142/99/$17.50 648

Irradiation of mung beans to reduce ýatulence factors

stored for 2 weeks before the commencement of theexperiments.

Germination

Control and irradiated mungbeans (0.25 and0.75kGy) in 2g lots were surface sterilised with 1%Na-hypochlorite solution for 15min, rinsed andimbibed in distilled water for 16hrs at 25¡C and ger-minated in petri dishes for 2, 4 and 6 days on ülterpapers moistened with distilled water containing5lg ml~1 of penicillin and streptomycin to inhibitmould growth. The seeds just after imbibition wereconsidered as germinated for 0 day. Height of theseedlings was measured in all the samples on the 4thand 6th day of germination.

Extraction of soluble carbohydrates

Dry seeds and samples germinated as above (bothendosperms and shoots included) were ground in amortar and transferred into a round bottomed ýaskand defatted by reýuxing over a boiling water bathwith (1 : 2v/v), 10ml g~1 for 3h,hexane : CHCl3with two changes.6 Thereafter the solvent layerswere discarded and the residue was reýuxed for 2hwith 80% aqueous ethanol (10ml g~1) and the super-natant was decanted and the reýuxing was continuedfurther for 1h with fresh 80% ethanol. The pooledextracts representing the total soluble carbohydrates(both reducing and non-reducing) were evaporatedunder vacuum and the residue was ültered and thevolume made to 10ml g~1 with distilled water.

Estimation of carbohydrates

Total soluble carbohydrates and reducing sugarswere estimated colorimetrically by the phenol sul-phuric acid7 and Somiyogi’s methods,8 respectively,using glucose as the standard. The total oligosac-charides which are non-reducing were estimated bythe thiobarbituric acid method9 using sucrose as thestandard.

Separation of carbohydrates

Carbohydrate extracts (5ml) were puriüed furtherby deionisation by passing through Dowex 50 H`and Amberlite IR-45 resins. For complete removalof chloride they were treated with Dowex 1-x-8-

and evaporated to dryness under vacuum.HCO3The residue was redissolved in a small volume ofdistilled water (300ll). Aliquots of these samples(15–20ll) containing approximately 300lg of totalcarbohydrates were subjected to paper chromatog-raphy for 40h at room temperature using

(6 : 4 : 3v/v) as then : butanol : pyridine : H2Osolvent system. Sugar spots were detected by alkalinesilver nitrate staining techniques.10 For quantitationthe corresponding sugar spots from the unstainedchromatograms were cut and eluted with distilledwater and estimated by the phenol sulphuric acidmethod.

The sugars were also analysed by HPLC (LKB-Pharmacia LCC-2252) on micro Bondapack carbo-hydrate column using (75 : 25v/v)acetonitrile : H2Oas the eluting solvent. The ýow rate was 1ml min~1and the sensitivity of the RI detector was adjusted toX/64. An aliquot (20ll) containing approximately250–300lg of total carbohydrates was used for injec-tion. Initially the chart speed was maintained at10mm min~1 and after 11.4min of the run it wasdecreased to 2mm min~1 as the oligosaccharides hada longer retention time. The sugars were identiüedby comparing their values and retention timesRfwith that of authentic standards.

RESULTS AND DISCUSSION

Germination

The preliminary stage in the germination processinvolves the breakdown of seed reserves followed bytheir utilisation in diþerent synthetic processes.Though no visible diþerences were noticeable in theearly germination between the control and 0.25kGytreated mung seedlings, the reduction in sproutlength of the 0.75kGy treated sample was quite sig-niücant (Table 1); the sprout length was reduced by24.5 and 30.4% on the 4th and 6th day of germi-nation, respectively. The diþerence increased withgermination time, which suggests some of the meta-bolic processes in the later stages of germination arealtered in the mung beans irradiated at higher doses.

Inhibition of seed germination and elongation ofshoots and roots in irradiated cereal grains andlegumes11 and in half-embryos removed from seedsof irradiated grapefruits, oranges and lemons12h14have been suggested as tests for detection of irradia-tion treatment in these commodities. The elongationof roots and shoots, particularly roots, was inhibitedin husked rice15 and wheat16 irradiated with dosesexceeding 0.3kGy. Our ündings that sprout (shoot)length of germinating mungbean seeds was notaþected by irradiation at 0.25kGy but was inhibitedat 0.75kGy corroborates the above reports. Lan et

Table 1. Effect of irradiation on the s prout lengths of germinating

mungbeans

Germination Sprout length in cm

(days )

Control 0 .25 kGy 0.75 kGy

4 4.2^ 0.8 4.6^ 0.6 3.2^ 0.36 5.4^ 1.0 4.9^ 1.1 3.8^ 0.9

In the germinating control and irradiated s amples the length of

the s prout was meas ured us ing a thread and the actual length in

cm was calculated on a graduated ruler. The values given here

repres ent the mean^SD of 12 s prouts each from twoindependent experiments in each s ample. The difference

between control and 0.25 kGy was not s ignificant while at

0.75 kGy it was s ignificant (P\ 0.001)

J Sci Food Agric 79 :648–652 (1999) 649

JP Machaiah, MD Pednekar, P Thomas

al17 found that irradiation at doses of 0.1 to 1kGyhad no eþect on germination and root length whendetermined 3 days after germination of green beans.

Carbohydrate composition

Germination generally enhances the nutritive valueof legumes ; the raffinose family oligosaccharideshave been shown to be degraded by the activation ofhydrolases like a(1,6)-galactosidase3 leading toreduction in ýatulence. The total soluble carbo-hydrates both in control and irradiated samplesincreased during germination. They constitutedabout 35 to 40mg g~1 of the seeds on dry weightbasis and increased to nearly two-fold on the 4th dayof germination and then decreased signiücantly onthe 6th day (Fig 1). This pattern was comparablewith the irradiated samples also.

Similarly, the total reducing sugars, which were aslow as 0.3mg g~1 in the dry seeds (Fig 1), rosesharply to about 6mg g~1 on the 6th day in thecontrol. In the irradiated samples the reducingsugars’ proüle was comparable to that of controluntil the 4th day ; however, on the 6th day a drasticreduction was noticed (Fig 1).

The total oligosaccharides varied between 1–2mg g~1 in the dry control beans. Following inhibi-tion, the oligosaccharides contents of the irradiatedsamples (0.25 and 0.75kGy) decreased by 20–25%compared to the control. Their concentrationincreased by 30–50% during germination in thecontrol while this increase was signiücant in the irra-diated samples (Fig 1).

Although these observations indicated radiationinduced decrease in oligosaccharide content irrespec-tive of germination time, it does not indicate whichof the oligosaccharides was most susceptible. Hence

Figure 1. Content of s oluble carbohydrates in germinating

mungbeans . The contents of total s oluble carbohydrates ,

reducing s ugars and oligos accharides were es timated by

colorimetric methods des cribed in the text, the values are

expres s ed as mggÉ1 of total dry weight and repres ent the means

of four s eparate extractions analys ed in duplicate.

the soluble carbohydrates were analysed further bypaper chromatography and HPLC.

Paper chromatography and HPLC conürmed thepresence of 9 sugars of which 8 could be identiüed asxylose, fructose, glucose, galactose, sucrose, raff-inose, stachyose and verbascose. The key ýatulencefactors, namely stachyose and verbascose, comprisednearly 50–60% of the total soluble carbohydrates asrevealed by quantitative assays ; they had very poorreducing capacity and low refractive index, so muchso they appeared as weak spots in paper and smallpeaks on HPLC proüles. In the control samplesthese oligosaccharides decreased gradually on germi-nation, but in the irradiated samples the reduction ingermination was drastic and occurred much earlierand was noticeable on the 0 day itself (Fig 2). On thesecond day of germination there was a reduction of68 and 83% in the 0.25kGy and 0.75kGy samples,respectively, while it decreased by only 10% in thecontrol samples. On the 6th day of germinationabout 90% reduction was observed in all thesamples. These results conclusively indicate that thedegradation of oligosaccharides occurred in a dosedependent manner and was higher at 0.75kGy com-pared to 0.25kGy samples, although no correlationcan be drawn between this reduction and thedecreased sprout length (Table 1) at higher doses,Earlier studies3 have indicated that there is no diþer-ence in the activity proüle of a-galactosidase in thecontrol and irradiated (10kGy) samples up to 80h of

Figure 2. The degradation of oligos accharides in germinating

mungbeans . The main oligos accharides , verbas cos e, s tachyos e

and raffinos e, contents were added in control and irradiated

s amples on different days of germination. The% decreas e ineach s ample was calculated bas ed on their total concentration

pres ent in the res pective dry s eeds . The values repres ent the

mean of quantitation data from four independent

chromatographic runs , repres enting four independently

germinated and extracted s amples .

650 J Sci Food Agric 79 :648–652 (1999)

Irradiation of mung beans to reduce ýatulence factors

germination. It was also observed3 that irradiation ofaqueous solution of raffinose (1–10kGy) resulted information of simple sugars in a dose dependentmanner. It is quite likely that the enhanced degrada-tion of oligosaccharides in our studies is also a radi-ation related process where the susceptibility ofsubstrate to the enzyme action is enhanced; thisneeds to be studied separately.

The total reducing sugars which were present invery low amounts in the dry seeds increased manyfold during germination (Fig 1). The proüle wascomparable between the control and irradiatedsamples until the 4th day of germination. On the 6thday, however, a sharp decrease was observed in theirradiated samples. The quantitative results present-ed a diþerent picture. The fold increase in reducingsugars was much more in the irradiated sample(F1)than the control and this increase continued as thegermination progressed. The reducing sugars,mainly glucose, fructose, galactose and xylose, whichconstituted about 20–30% of total soluble carbo-hydrates, decreased initially in the control and thenincreased steeply (2-fold) on the 4th day. Theincrease was much greater (5–7-fold) in the irradi-ated samples (Fig 3); glucose content was signiü-cantly enhanced, particularly at higher dose(0.75kGy).

It may be presumed that the observed rapidincrease in the total reducing sugars in the extractsfrom the control samples could be due to reducingcomponents other than the reducing sugars quanti-

Figure 3. Comparative profile of increas e in reducing s ugars .

The values depicted here repres ent the total concentration of the

main reducing s ugars (glucos e, fructos e and galactos e). The fold

increas e was calculated taking the corres ponding value in the

res pective dry s amples (control, 0.25 kGy and 0.75 kGy) as 1. The

values are obtained as the means of data from four different

chromatographic runs , repres enting four independently

proces s ed s amples .

tated by paper chromatography. The maximumobserved increase was due to rise in glucose whichcomprised 52 and 64% of the total reducing sugars inthe control and irradiated (0.75Gy) samples, respec-tively. The increase in the irradiated samples may beattributed to increased depolymerisation of starch,18rendering it more susceptible to enzymatic degrada-tion. Galactose, mainly arising as a degradationproduct of oligisaccharides, was also higher the irra-diated samples.

Earlier reports on cooked unirradiated and high-dose irradiated (5.0kGy) mungbeans observed nosigniücant diþerence in their sensory acceptabilitythough marginal diþerences in the scores of ýavourand colour were noticed.19 Recent studies(unpublished results) from our laboratory have alsoindicated that at 0.25 and 0.75kGy dose levels, thesensory characteristics of either sprouted or cookedmungbeans were not altered and were readily accept-able.

Compared to other methods4 available forreduction of ýatulence, low-dose irradiation ofmungbeans would make them more acceptable bothnutritionally as well as in sensory attributes com-bined with an enhanced shelf life by insect disin-fection. Mungbeans contain high protein (25%) andcompared to other legumes their starch has highcapacity to make lysine and methionine available.20Thus low-dose radiation treatment for disinfestationcould optimise utilisation of these beans as an effi-cient substitute for protein food of animal origin,which is high cost and whose availability is becominglimited; this could go a long way in combating theprotein calorie malnutrition prevalent in many partsof the globe. Our results also indicate that irradiationat 0.25kGy had no eþect on germination and sproutlength and such treated beans are suitable for use assprouted beans.

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