Low gamma irradiation effects on protein profile, solubility, oxidation, scavenger ability and...

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Please note: Images will appear in color online but will be printed in black and white.ArticleTitle Low gamma irradiation effects on protein profile, solubility, oxidation, scavenger ability and

bioavailability of essential minerals in black and yellow Indian soybean (Glycine max L.) varietiesArticle Sub-Title

Article CopyRight Akadémiai Kiadó, Budapest, Hungary(This will be the copyright line in the final PDF)

Journal Name Journal of Radioanalytical and Nuclear Chemistry

Corresponding Author Family Name SachdevParticle

Given Name ArchanaSuffix

Division Division of Biochemistry

Organization Indian Agricultural Research Institute

Address 110012, New Delhi, India

Email [email protected]

Author Family Name KrishnanParticle

Given Name VedaSuffix




Email

Author Family Name SinghParticle

Given Name ArchanaSuffix




Email

Author Family Name ThimmegowdaParticle

Given Name VinuthaSuffix




Email

Author Family Name Singh

Particle

Given Name BhupinderSuffix

Division Centre for Environment Science and Climate Resilient Agriculture(CESCRA)



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Author Family Name DahujaParticle

Given Name AnilSuffix




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Author Family Name RaiParticle

Given Name Raj DeoSuffix




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Received 18 November 2014

Revised

Accepted

Abstract Effect of low doses of gamma irradiation (0.25, 0.5 and 1.0 kGy) on protein oxidation, profile, solubility,ROS scavenging and in vivo bioavailability of minerals in black (BS1) and yellow (BRAGG) soybeanvarieties were investigated. Increased oxidation, altered protein profile with decreased solubility wasobserved higher in BRAGG compared with BS1. The most significant ROS scavenging effect, antioxidantactivity, least phytate content and improved bioavailability was found at 0.5 kGy in BS1 than BRAGG dueto anthocyanins, and phenolics. Still 1.0 kGy is considered as toxicologically and microbiologically safebut it causes biochemical alterations and thus 0.5 kGy can be the optimum dose with enriched nutraceuticalproperties.

Keywords (separated by '-') Soybean - Gamma ray - Protein solubility - Oxidation - Free radical-scavenging - In vivo bioavailability

Footnote Information Electronic supplementary material The online version of this article (doi:10.1007/s10967-015-4193-3)contains supplementary material, which is available to authorized users.

UNCORRECTEDPROOF

12

3 Low gamma irradiation effects on protein profile, solubility,

4 oxidation, scavenger ability and bioavailability of essential

5 minerals in black and yellow Indian soybean (Glycine max L.)

6 varieties

7 Veda Krishnan1 • Archana Singh1 • Vinutha Thimmegowda1 • Bhupinder Singh2 •

8 Anil Dahuja1

• Raj Deo Rai1

• Archana Sachdev1

9 Received: 18 November 201410 � Akademiai Kiado, Budapest, Hungary 2015

11 Abstract Effect of low doses of gamma irradiation (0.25,

12 0.5 and 1.0 kGy) on protein oxidation, profile, solubility,

13 ROS scavenging and in vivo bioavailability of minerals in

14 black (BS1) and yellow (BRAGG) soybean varieties were

15 investigated. Increased oxidation, altered protein profile with

16 decreased solubility was observed higher in BRAGG com-

17 pared with BS1. The most significant ROS scavenging ef-

18 fect, antioxidant activity, least phytate content and improved

19 bioavailability was found at 0.5 kGy in BS1 than BRAGG

20 due to anthocyanins, and phenolics. Still 1.0 kGy is con-

21 sidered as toxicologically and microbiologically safe but it

22 causes biochemical alterations and thus 0.5 kGy can be the

23 optimum dose with enriched nutraceutical properties.24

25 Keywords Soybean � Gamma ray � Protein solubility �

26 Oxidation � Free radical-scavenging � In vivo

27 bioavailability

28 Abbreviations

29 FDA Food and drug administration

30 PUFA Poly unsaturated fatty acids

31 CVD Cardio vascular diseases

32 BCA Bicinchoninic acid assay

33DNPH Dinitro phenyl hydrazine

34TPTZ 2,4,6-Tripyridyl-S-triazine

35FRAP Ferric ion reducing power

36PEB Protein extraction buffer

37DPPH 1,1-Diphenyl-2-pycril-hydrazil

38SDS-PAGE Sodium dodecyl sulphate-poly acrylamide

gel electrophoresis

39DNP Dinitro phenyl hydrazine

40Tris Tris(hydroxymethyl)aminomethane

41RSC Radical scavenging capacity

42RT Room temperature

43

44

45Introduction

46Outbreaks of food borne diseases have been acknowledged

47as a worldwide problem by health professionals mainly

48associated with the consumption of poor quality food

49which is due to the presence of large number of human

50pathogens in food, especially in seeds. Most of the post-

51harvest procedures used to control insects and moulds in

52the seeds are chemical, biological, physical or a combi-

53nation of these techniques. Food irradiation is one of the

54most common process in which products are exposed to

55ionizing energy such as gamma rays, electron beams and

56X-rays for a specified time, which effectively inactivates

57the food borne pathogens [1]. Soybean is now a preferred

58food ingredient in the products on the grocery shelves all

59over the world due to its rich content in protein and poly

60unsaturated fatty acids (PUFAs), as well as now considered

61as a smarter curer for multiple health disorders like cardio

62vascular diseases (CVD), cancer, fatty liver disease and

63metabolic syndromes [2]. Radiation processing up to

A1 Electronic supplementary material The online version of thisA2 article (doi:10.1007/s10967-015-4193-3) contains supplementaryA3 material, which is available to authorized users.

A4 & Archana Sachdev

A5 [email protected]

A6 1 Division of Biochemistry, Indian Agricultural Research

A7 Institute, New Delhi 110012, India

A8 2 Centre for Environment Science and Climate Resilient

A9 Agriculture (CESCRA), Indian Agricultural Research

A10 Institute, New Delhi 110012, India

AQ1

123Journal : Large 10967 Dispatch : 14-5-2015 Pages : 9

Article No. : 4193h LE h TYPESET

MS Code : JRNC-D-14-01306 h CP h DISK4 4

J Radioanal Nucl Chem

DOI 10.1007/s10967-015-4193-3

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http://dx.doi.org/10.1007/s10967-015-4193-3

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64 10 kGy of gamma rays is considered as safe by FDA

65 without any toxicological or microbiological hazards for

66 legumes like soybean till date but previous studies have

67 showed alterations in quality parameters [3, 4]. Higher

68 doses were observed with increased oxidative stress, free

69 radical generation as well as alterations in the conforma-

70 tions of biomolecules. The hydroxyl and superoxide anion

71 radicals generated by radiation modifies the molecular

72 properties of proteins and lipids causing oxidation and

73 peroxidation [5], which in case of soybean products, en-

74 hance the off-flavor problems and hence there is a need to

75 analyze the effect of low doses on various biochemical

76 parameters as well.

77 Effect of lower doses were initially indicated with no

78 substantial changes on various physiochemical parameters

79 in soybean and were considered as safe [6–8]; whereas

80 recent studies showed exposure of even lower doses

81 (0.5–2 kGy) resulted with decreased protein content; as

82 well as increased protein oxidation and lipid peroxidation

83 which reduced the palatability in various food products [9].

84 Studies conducted on various Indian soybean genotypes

85 irradiated till 5 kGy revealed a varied difference in the

86 grassy flavor which might be due to the difference in their

87 inherent antioxidant potential contributed by their inherent

88 genetic makeup. Colored soybean varieties especially black

89 has been reported with increased antioxidant potential

90 compared to green and yellow counter parts. Mild doses of

91 gamma irradiation also has been reported with enhance-

92 ment in total antioxidant potential, total phenolic as well as

93 flavonoid content in black soybean varieties [10], however

94 very few studies have been carried out to see the correlate

95 the effect of low doses of radiation on various biochemical

96 parameters as well with respect to seed coat differences,

97 Hence, the main purpose of this investigation was to op-

98 timize the most effective dose of gamma irradiation with

99 least biochemical alterations correlating to its seed coat

100 color in soybean addressing parameters like protein solu-

101 bility, oxidation, total antioxidant activity, free radical

102 scavenging ability, phytate content, in vivo bioavailability

103 of essential minerals and enriching nutraceuticals (antho-

104 cyanins and phenolics).

105 Materials and methods

106 Seed material and gamma irradiation

107 Soybean seeds (Glycine max L.) of two different genotypes

108 BS1 (black) and BRAGG (yellow) were obtained from

109 pulse laboratory, IARI, India and were cleaned thoroughly

110 with sterile water. Seeds were divided into four groups, i.e.,

111 control (non-irradiated, 0 kGy), 0.25, 0.5 and 1.0 in trip-

112 licates. Ten gram seeds of uniform size were selected and

113packed in polyethylene high density bags (4 mm thickness)

114and were irradiated at different dose level of gamma irra-

115diation (0.25, 0.5 and 1.0 kGy) at room temperature

116(24 ± 2 �C). Gamma irradiation was performed using 60Co

117gamma radiation chamber (Model GC-5000, BRIT Mum-

118bai) facility at Nuclear Research Laboratory, IARI, New

119Delhi India. All biochemical analyses were carried out

120after 24 h of storage at 4 �C.

121Protein extraction and quantification

122Irradiated and non-irradiated defatted soybean meal was

123extracted with 0.03 M Tris-HCl, pH 8.0 buffers at room

124temperature to yield whole buffer extract as described by

125Iwabuchi and Yamauchi [11], then centrifuged at

12610,0009g for 30 min at 4 �C. The aliquots of supernatant

127were used for estimations. Extract of non-irradiated seeds

128were used as control. Soluble proteins were estimated using

129bicinchoninic acid assay (BCA) by John et al. [12].

130SDS-PAGE

131100 lg protein samples were separated by SDS-PAGE on

132the basis of their molecular mass using Laemmli and

133Eiserling’s method [13]. Total proteins were extracted in a

134modified tris buffer containing 1 % triton X-100, 2 % SDS

135and 15 % glycerol [14]. Protein samples were mixed with

1366X sample buffer pH 6.8 and denatured for 5 min at 958C.

137Electrophoresis was carried out using 12 % separating and

1384 % stacking gel. Medium ranged protein molecular weight

139marker (Fermentas, USA) was loaded in a separate well.

140The gels were stained with Coomassie Brilliant Blue made

141in 5:5:1 (Methanol: Water: Acetic acid) solution. Gels were

142de-stained and image analyses were done using gel

143documentation system (Alfa Inno Tech, USA).

144Protein solubility

145Protein solubility was expressed as the ratio of soluble

146protein (aqueous medium) to total protein.

147Protein oxidation

148Protein oxidation intensity was developed using the content

149of carbonyl groups in protein and derivatizing by using

150Dinitro Phenyl Hydrazine (DNPH) reagent [15]. Powdered

151seed tissue was ground in a chilled mortar (0.5 g fresh

152weight/ml) in protein extraction buffer (PEB) containing

153100 mM Tris-HCl, pH 8.0, 2 % (v/v) b-mercaptoethanol,

1545 mM EGTA, 10 mM EDTA and 10 mM NaF. The soluble

155protein extracts were mixed with two volumes of 10 mM

156DNPH in 2 M HCl at room temperature for 30 min with

157gentle agitation. A control sample was mixed with two





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158 volumes of 2 M HCl. Five volumes of ice-cold phenol

159 (Tris-buffered, pH 7.9) was added to each tube. After

160 vortexing for 1 min, the mixture was centrifuged for

161 10 min at 10,000 g. The upper phase was removed and

162 discarded leaving the interface intact, and the phenol phase

163 was re-extracted twice with ice-cold Tris-HCl buffer

164 (50 mM, pH 8.0). Five volumes of cold 0.1 M ammonium

165 acetate in methanol was added to the lower phase and in-

166 cubated at -20 �C overnight. The following day, the

167 mixture was centrifuged at 10,0009g for 20 min, and the

168 resulting pellets were washed three times with 1 ml 0.1 M

169 ammonium acetate in methanol and once with 1 ml cold

170 ethanol. To each pellet, buffer (containing 7 M urea, 2 M

171 thiourea, 65 mM DTT, 20 ll 0.1 % bromophenol, and

172 20 ll buffer, pH 4–7) was added. The samples were in-

173 cubated for 2 h at room temperature, sonicated at low

174 power, and incubated for 1 h at room temperature. Insol-

175 uble material was removed by centrifugation at

176 10,0009g for 20 min at 4 �C and the supernatant was

177 transferred to a fresh tube. For 1-DE analysis, SDS-PAGE

178 was performed with 12 % gels with a loading of 100 lg

179 protein samples in each lane. Resolved proteins were

180 electrophoretically transferred to Immobilon-P (PVDF,

181 Millipore) membranes using Invitrogen electro blotting

182 technique. The oxidized proteins were detected using anti-

183 DNP antibodies (Sigma Aldrich, USA).

184 Total antioxidant activity

185 The total antioxidant activity of irradiated and control

186 soybean phenolic extracts was measured by ferric ion re-

187 ducing power (FRAP) assay [16] with some modifications.

188 Briefly, freshly prepared FRAP reagent (10:1:1) acetate

189 buffer 300 mM pH 3.6, 10 mM 2,4,6-tripyridyl-S-triazine

190 (TPTZ) in 40 mM HCl and 20 mM FeCl3.6H2O was mixed

191 with the sample and allowed to stand for 5 min at RT

192 before measuring absorption at 593 nm. Aqueous solutions

193 of Fe2? (FeSO4�6H2O) in the concentration range of

194 150–1500 lmol/L were used for calibration of FRAP as-

195 say. FRAP values were expressed as millimoles of Fe2? per

196 100 g sample (mmol of Fe2?/100 g of grains FW) and was

197 calculated using the Eq. (1).

FRAP value ¼ DA sampleð Þ=DA standardð Þ ð1Þ

199199 where DA (sample) is the change in the absorbance of the

200 sample and DA (standard) is the change in absorbance of

201 the standard after 5 min incubation when read at 593 nm.

202 Free radical scavenging capacity

203 Free radical scavenging capacity was determined using 1,1-

204 diphenyl-2-pycril-hydrazil radical (DPPH). Reduction of

205 DPPH radical was determined by measuring the

206disappearance of DPPH at 515 nm. DPPH radical scav-

207enging capacity (RSC) is expressed by percentage com-

208pared to control [17]. The percent inhibition of the DPPH

209radical (DPPH RSC) by the samples was calculated using

210the Eq. (2).

DPPH RSC ¼Ac� Ax½ �

Ac� 100 ð2Þ

212212where Ac is the absorbance of the control and Ax is ab-

213sorbance of the sample after 30 min of incubation.

214Estimation of phytic acid

215Phytic acid (phytate) was measured by an assay procedure

216specific for the measurement of phosphorus released, based

217on the available phosphorus from phytic acid, myo-inositol

218(phosphate)n and monophosphate esters by phytase and

219alkaline phosphatase using the Phytic Acid/Total Phos-

220phorus Assay Kit (Megazyme, Ireland).

221Bioavailable Fe21, Zn21 and Ca21 determination

222using in vivo simulation model

223The bioavailability of Fe2?, Zn2? and Ca2? were deter-

224mined by the in vitro digestion method described by ku-

225mari et al. [18]. Control and irradiated samples (5 g) in

226triplicate were suspended in 30 ml distilled water and di-

227gested under simulated gastrointestinal conditions, subse-

228quently using alpha amylase solution, stomach medium

229consisting lipase and pepsin, and pancreatic solution con-

230sisting of pancreatin and bile. After digestion, the suspen-

231sion was centrifuged at 36009g for 15 min. The

232supernatant was decanted and the pellet was discarded. The

233supernatants were pooled and filtered through a 0.45 mm

234filter. A blank was included consisting of 30 ml distilled

235water digested and filtered as described above. Both filtered

236supernatants from sample and blank were analyzed for

237Fe2?, Zn2? and Ca2?. Samples were corrected for added

238reagents/water by subtracting Fe2?, Zn2? and Ca2? con-

239tents of the blank from that of supernatants from samples.

240Fe2?, Zn2? and Ca2? content were measured by using the

241Atomic Absorption Spectrophotometer. The amounts of

242Fe2?, Zn2? and Ca2? (in supernatant) were regarded as

243soluble minerals. Percentage of soluble mineral was cal-

244culated as bioavailability %.

Bioavailability%¼ Amount of Fe2þ; Zn2þ andCa2þ

ðsupernatantÞ �Amount of Fe2þ;

Zn2þ andCa2þ ðblankÞ=Amount of Fe2þ;

Zn2þ andCa2þ in undigested sample

� 100:

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246246247 Statistical analysis

248 All determinations were done in triplicate. Statistical ana-

249 lysis were done using SPSS (version 10) program. Mean

250 and standard error were descriptive measures of quantita-

251 tive data using the analysis of variance test (ANOVA) for

252 in dependent samples. P values at P\ 0.05 were consid-

253 ered significant.

254 Results and discussion

255 Effect of low doses of gamma irradiation on soybean

256 protein profile

257 The objective of this investigation was to evaluate the ef-

258 fect of different low doses of gamma radiation on soybean

259 protein patterns with respect to its seed coat color varia-

260 tions. The results showed a differential protein profile with

261 respect to intensity and number of bands at different (0.25,

262 0.5 and 1 kGy) doses of gamma irradiation in both BS1 and

263 BRAGG varieties (Fig. 1). Appearance of a signature

264 protein band of approximately 60 kD and an absence of

265 40 kD protein was observed in the yellow soybean variety

266 BRAGG at 0.25 kGy which might be due to the modifi-

267 cations of physiochemical properties of proteins like

268oxidation which led to condensation, polymerization or

269aggregation [19]. The reason by which BRAGG variety

270was more prone to alterations in protein profile might be

271due to decrease in antioxidant molecules like anthocyanins

272in its seed coat which prevents protein oxidation to an

273extent (Supplementary Fig. 6b). An increasing in 79 kD

274protein was observed with increased dose of gamma ra-

275diation in BS1, in contrast to BRAGG. This result was in

276agreement with the Afify et al. [20], where they showed,

27779 kD protein a-conglycinin expression increased during

278gamma irradiation, and the difference observed might be

279the genotypic differences in the inherent antioxidant po-

280tential (differences in seed coat pigmentation). A 34 kD

281protein agglutinin increased by low doses (0.25, 0.5,

2821 kGy) of gamma irradiation in both BS1 and BRAGG

283varieties. On the other hand, most of the remaining protein

284did not change significantly. Mehlo et al. [21] reported that

285albumin and globulin content of sorghum flour decreased

286significantly even with low doses of irradiation which helps

287the protein to aggregate and might decrease its solubility.

288Effect of low doses of gamma irradiation on protein

289solubility

290The results in Table 1 clearly showed that the total protein

291content was not significantly affected by irradiation, while the

292solubility of total protein fraction was decreased and reached

293to theminimumat 1.0 kGy in both the varieties. Percentage of

294solubility shifted from 64.2 % (0 kGy) to 56.79 % (1 kGy)

295and 69.65 % (0 kGy) to 63.02 % (1 kGy) respectively in BS1

296and BRAGG varieties. Byun et al. [22] reported that partial

297degradation of 7S globulin into 2S protein and aggregation of

29811S globulin into 15S components because of ionizing ra-

299diation in turn reduces the soluble nature of protein. The

300maximum decrease in protein solubility observed (11.5,

30126.2 %) in BS1 and BRAGG respectively at 1 kGy dose of

302gamma irradiation justifies the evidence that even low doses

303leads to cross-linking of protein chains and its aggregation

304which increased the hydrophobic interactions and reduced

305protein solubility (Table 2).

306Effect of gamma irradiation on protein oxidation

307Introduction of carbonyl groups into amino acid residues of

308protein is a hall-mark for oxidative modification and the

309oxidized proteins were detected by immuno blotting using

310anti-DNP antibodies. The specific carbonyl levels were

311obtained using densitometry (Fig. 2a, b). A significant

312(P\ 0.05) increase in the levels of an oxidized protein—a-

313conglycinin at 79 kD in BRAGG was observed with in-

314creased dose of radiation as compared to BS1 (Fig. 2). The

315low protein oxidation observed in that black seed coat

316variety, BS1 might be due to its inherent characteristics of

Fig. 1 Electrophoretic separation of total protein from irradiated and

control of BS1 variety (a) and BRAGG (b) in SDS-PAGE using 12 %

resolving gel

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317 possessing more antioxidants like anthocyanins in their

318 seed coats which act as a buffering molecule to combat the

319 oxidative stress induced protein oxidation compared to

320 yellow seed coat variety (BRAGG). The previous results

321 are in agreement with the observation (Supplementary

322 Fig. 6). Previous studies reported by Zhao et al. [23] have

323 showed the radio-protective effects of anthocyanins mainly

324 by quenching the free radicals generated by irradiation.

325 The decrease in oxidation noticed in BS1 might also be due

326 to the increase in total phenolic content observed after ir-

327 radiating with low doses of gamma rays (Supplementary

328 Fig. 6a). BS1 being a black seed coat variety is rich in

329 phenolics which attributes an anti-radical mechanism by its

330 ability to transfer oxidative damage from one phenolic site

331 to other, protecting proteins from oxidation.

332 The traditional immunochemistry technique was used to

333 detect the oxidized proteins and to validate the results.

334 Effect of gamma irradiation on antioxidant activity

335 and scavenging free radicals

336 Free radicals generated by protein oxidation are neutralized

337 by the antioxidant molecules inherently present in the

338genotype especially attributed by the pigment in their seed

339coat. The results obtained from the analysis of the effect of

340low doses (0.25, 0.5, 1 kGy) of gamma irradiation on total

341antioxidant activity determined by FRAP method is pre-

342sented in Fig. 3a. Antioxidant activity was significantly

Table 1 Effect of low doses of

gamma irradiation on protein

solubility in BS1 and BRAGG

soybean varieties

No Variety Dose (kGy) Total protein (mg/100 g) Soluble protein (mg/100 g) % Solubility

1 BS1 Control 0.458 ± 0.07a 0.294 ± 0.03a 64.20

0.25 0.448 ± 0.12b 0.287 ± 0.04b 64.06

0.5 0.427 ± 0.05b 0.272 ± 0.09b 63.70

1.0 0.412 ± 0.08c 0.234 ± 0.11c 56.79

2 BRAGG Control 0.468 ± 0.07a 0.326 ± 0.15a 69.65

0.25 0.459 ± 0.03b 0.288 ± 0.09b 62.70

0.5 0.451 ± 0.16b 0.265 ± 0.08b 58.07

1.0 0.449 ± 0.07c 0.231 ± 0.08c 51.40

Values are mean ± SE of three determinations. Different superscripts in the same column among BS 1 and

BRAGG with different letters are significantly (P\ 0.05) different

Table 2 Effect of radiation doses on in vivo bioavailability of Fe2?,

Zn2? and Ca2? in percentage

Samples Fe2? Zn2? Ca2? (%)

BS1

Control 8.25 ± 0.08 7.5 ± 0.04 8.2 ± 0.09

0.25 kGy 8.98 ± 0.02 8.2 ± 0.02 8.7 ± 0.04

0.5 kGy 9.2 ± 0.03 8.5 ± 0.04 9.5 ± 0.02

1.0 kGy 9.5 ± 0.03 8.9 ± 0.06 9.8 ± 0.03

BRAGG

Control 9.48 ± 0.05 8.6 ± 0.08 9.15 ± 0.06

0.25 kGy 9.76 ± 0.04 8.8 ± 0.06 9.62 ± 0.04

0.5 kGy 9.93 ± 0.03 9.2 ± 0.05 9.98 ± 0.06

1.0 kGy 10.12 ± 0.05 9.5 ± 0.04 10.25 ± 0.04

Fig. 2 a Immunoblot analysis using anti-DNP antibodies following

derivatization of soluble proteins with DNPH in BS1 and BRAGG

varieties. b Carbonyl levels in BRAGG variety. c Ratio was

calculated based on the morphometric assessment of oxidized bands

compared with control on Image J software. Carbonyl levels in BS1

variety. Error bars indicate SEM for 3 samples in each group





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343 high in non-irradiated BS1 (0.34 mmol of Fe2?/100 g) than

344 in BRAGG (0.22 mmol of Fe2?/100 g). A further increase

345 in the antioxidant activity by 0.32 mmol of Fe2?/100 g,

346 0.43 mmol of Fe2?/100 g at 0.25 and 0.5 kGy respectively

347 in BRAGG and 0.45 mmol of Fe2?/100 g and 0.55 mmol

348 of Fe2?/100 g at 0.25 and 0.5 kGy respectively in BS1

349 were observed. The increasing of total antioxidant activity

350 at 0.25 and 0.5 kGy could be the result of high phenolic

351 and anthocyanin accumulation (Supplementary Fig. 6). It is

352 also well known that anthocyanins being polyphenolic in

353 nature are one of the most active ferric reducing agents [24,

354 25] and hence the decline in the antioxidant activity at

355 1.0 kGy might be due to decrease in anthocyanins which

356 are predominantly accumulated in seed coats. This is in

357 agreement with Mohajer et al. [26] where total anthocyanin

358 content was observed to be inversely proportional to irra-

359 diation intensity.

360 The free radical scavenging increasing as (35.7, 38.5,

361 41.1 %) BRAGG and (40.7, 47.5, 55.2 %) in BS1 at 0,

362 0.25, 0.5 kGy respectively were observed (Fig. 3b).A

363 maximum free radical quenching ability at 0.5 kGy as 26.2,

364 13.2 % in BS1 and BRAGG respectively were observed;

365 which followed by a damp in antioxidant activity at

366 1.0 kGy. In contrary, an enhanced DPPH RSC was ob-

367 served in soybean butanolic extracts irradiated till 5 kGy

368[27]. DPPH RSC being a measure of non-enzymatic an-

369tioxidant activity, its well in correlation with the conclu-

370sion that antioxidant molecules like anthocyanins and

371phenols present in the seed coat of BS1 might probably get

372induced by low doses of radiation to combat the chemical

373oxidation of biomolecules occurred due to free radicals

374preferentially in a better way compared to yellow variety.

375Effect of low doses of gamma irradiation on Phytate

376content and bioavailability of essential minerals

377(Fe21, Zn21 and Ca21) using an in vivo simulation

378model

379The phytic acid in BRAGG and BS1 that irradiated by

3800.25, 0.5 and 1 kGy was decreased significantly (P\ 0.05)

381by 13.7, 25.3, 35.6 % and 35.5, 46.6, 54.1 % respectively.

382To correlate the decline in phytate content with mineral

383bioavailability we used an in vivo mimicking model. The

384observed Fe2? bioavailability increase by 8.98 %

385(0.25 kGy), further increased to 9.2 % (0.5 kGy) and to

3869.5 % (1.0 kGy) in BS1. A similar increasing trend in Fe2?

387bioavailability was observed in BRAGG with an initial of

3889.48 % (control), 9.76 % (0.25 kGy), 9.93 % (0.5 kGy)

389and a maximum of 10.12 % (1.0 kGy) doses. The im-

390proved bioavailability of Fe2? after irradiation might be

391possibly due to the significant reduction in phytate levels

392observed (Fig. 4) and is in agreement with the findings of

393El-Niley [28]. Similar findings were reported by Hassan

394et al. [29] in Maize, Sorghum and soybean [30, 31]. Certain

395tannins and other polyphenols which chelates the minerals

396may also reduce during irradiation process as a result of

397formation of polyphenol complexes with proteins and also

398cause gradual degradation of oligosaccharides [32, 33].

399Such reduction in polyphenols may facilitate Fe2? ab-

400sorption. The bioavailability % of Zn2? increased from

4017.5 % (control), to 8.98 % (0.25 kGy); further to 9.2 %

Fig. 3 Effect of c-irradiation on a total antioxidant activity deter-

mined by FRAP method in terms of mmol Fe2?/100 g b DPPH

Radical Scavenger Capacity (in terms of DPPH RSC %) of BS1 and

BRAGG soybean seeds

Fig. 4 Effect of low doses of c-irradiation on phytic acid content in

BS1 and BRAGG soybean seeds. Error bars indicate SEM for 3

samples in each group





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402 (0.5 kGy) and to 9.5 % (1.0 kGy) in BS1. A similar pattern

403 of increase in Zn2? bioavailability % was seen in BS1 and

404 BRAGG. An initial of 9.48 % bioavailability was observed

405 in the control seeds, which increased to 9.76 % after

406 0.25 kGy and further scaled up to 9.93 % (0.5 kGy) and a

407 maximum of 10.12 % (1.0 kGy). Further, the main in-

408 hibitory factor of Zn2? bioavailability is phytic acid which

409 might get partially degraded as a result of irradiation and

410 thus improved its bioavailability [34]. Among these

411divalent ions, in vitro and animal experiments have im-

412plicated Ca2? as a potentiating factor because it reacts with

413phytate, and further binding of Zn2? ions to these com-

414plexes causes precipitation. The percentage of bioavail-

415ability of Ca2? increased from 8.2 % (control) to 8.7 % in

4160.25 kGy dose, which further improved to 9.5 % (0.5 kGy)

417and 9.8 % (1.0 kGy) in BS1. BRAGG, following a similar

418trend showed an initial rise of 9.15 % in control and further

419rose to 9.62 % (0.25 kGy), 9.98 % (0.5 kGy) and 10.25 %

Fig. 5 A significant (P\ 0.05)

negative correlation of phytic

acid with in vivo bioavailability

of Fe2? (a), Zn2? (b) and Ca2?

(c) was observed





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420 (1.0 kGy). Previously assumed that fiber negatively affects

421 the Ca2? balance by physical entrapment or by cationic

422 binding, but it is more likely that the phytate associated

423 with fiber rich products probably affects Ca2? absorption,

424 which decrease due to irradiation. The low extractability of

425 divalent cations in raw seeds can be attributed to their

426 covalent association with phytic acid which constitutes

427 about 2.2 and 2.8 % of the total dry weight of the soybean

428 seeds. The gamma irradiation had a beneficial effect on the

429 bioavailability of Fe2?, Zn2? and Ca2? and followed a

430 consistent trend with increase in the irradiated doses. De-

431 crease in the levels of phytic acid by irradiation as reported

432 by previous workers [35] in maize and sorghum as well as

433 in the present study, may possibly release these metallic

434 ions in the free form and account for their increased

435 bioavailability. Studies done by Sattar et al. [36] is in line

436 with the present study and they also reported that the extent

437 of reduction in phytic acid increased linearly with increase

438 in radiation dose. This reduction might be due to chemical

439 degradation of phytate to the lower inositol phosphates or

440 inositols by the action of free radicals produced by ra-

441 diation [37]. Another possible mode of phytate loss which

442 in turn improved the mineral bioavailability could have

443 been through cleavage of the phytate ring itself. A sig-

444 nificant (P\ 0.05) negative correlation of the antinutrient,

445 phytic acid, with bioavailable Fe2?, Zn2? and Ca2? ob-

446 served in the present study strengthens our findings and

447 clearly underlines the role of phytic acid in lowering the

448 bioavailability of divalent cations (Fig. 5).

449 Conclusion

450 The present study indicated that even 1.0 kGy dose which

451 is considered as toxicologically and microbiologically safe

452 for for legume seeds can cause unwanted biochemical

453 changes affecting quality. An increasing trend in protein

454 oxidation was observed with increasing dose of irradiation,

455 which indirectly might be responsible for the decreased

456 solubility observed. The enrichment in the inherent an-

457 tioxidant potential, free radical scavenging activity and

458 improved bioavailability of essential minerals like Fe2?,

459 Zn2? and Ca2? were observed higher in black (BS1)

460 compared to yellow (BRAGG) varieties after radiation

461 Therefore, our results led to the conclusion that 0.5 kGy

462 can be used as an optimum dose with least biochemical

463 alterations and enriched nutraceutical attributes, improved

464 mineral bioavailability and protein stability.

465 Acknowledgments We thank Dr. S. K. Lal for providing the sam-466 ples. This study was supported by grant in aid for scientific research467 by Indian Agricultural Research Institute, New Delhi, India.

468

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