this is due to the presence of a large number of human pathogens in food , especially in seeds . the food and drug administration ( fda ) has approved the use of ionizing radiation to control microbial pathogens in seeds . however , the higher doses of radiation could have adverse effects on sensorial and nutritional quality . soybean and its processed products have been acclaimed as health foods due to their high content of protein and essential amino acids , omega-3 fatty acids , fat - soluble vitamins , polysaccharides , and insoluble fibers . besides these constituents , soybeans also contain isoflavones that are of wide interest due to their beneficial effects on humans , such as prevention of cancer , cardiovascular diseases , osteoporosis , and menopausal symptoms . gamma irradiation causes oxidative stress and affects biomolecules by causing conformational changes , oxidation , rupture of covalent bonds and formation of free radicals . the hydroxyl ( ho ) and superoxide anion ( o2 ) radicals that are generated by radiation could modify the molecular properties of the proteins and lipids causing oxidative modifications of the proteins and lipid peroxidation ( lp ) , . chemical changes of the proteins that are caused by -irradiation are fragmentation , cross - linking , aggregation and oxidation caused by oxygen radicals which are generated by water radiolysis . radiation processing by -radiation at dose of 1 kgy has been recommended for quarantine treatment of legumes including soybeans , whereas exposure to higher doses ( up to 5 kgy ) resulted in improvement in quality such as reduction in cooking time and improvement in texture without production of off - flavor . degradation of isoflavones , the major phenolic constituents in soybean seeds , has been shown to occur in soy and soy products during food processing and storage , resulting in changes in their bioactivities . however , studies of the effect of irradiation on the phenolic content and the impact of these changes on the antioxidant properties of soybeans were poor and insufficient . there are some reports showing that stress caused by low doses of -irradiation could induce antioxidant activities in plant material including soybean . our previous studies concerning antioxidant activities in stressed seeds indicated that low doses of herbicides could induce antioxidant activity in seeds of beans , lettuce and pea . therefore , the aim of our study was to investigate the effects of stress caused by medium dose of -irradiation ( 110 kgy ) on phenolic content , lipid and protein oxidation and free radical scavenging ability of soybean . the results obtained by investigating the influence of medium doses of -irradiation ( 110 kgy ) on some antioxidant properties of soybean seed are presented in figures 15 . use of -irradiation dose up to 10 kgy did not provoke significant changes in lp ( figure 1 ) . as shown in figure 1 , the above mentioned doses of -irradiation caused slight increase in lp . the maximum increase in lp was observed with a 2 kgy dose ( 11.0 % when compared to the unirradiated control ) . other authors also established that doses up to 10 kgy caused insignificant changes in total lipids , fatty acid composition , peroxide value and trans fatty acid content . medium doses of -irradiation caused insignificant changes in soluble protein content ( figure 2a ) , without practical significance . data obtained by other authors also showed that gamma irradiation , using a doses up to 10 kgy , did not induce significant loss in water soluble components such as minerals , nitrogenous constituents , sugars and proteins . quantity of carbonyl - groups in oxidatively modified proteins significantly increased only at dose of 6 kgy ( 5.3 % ) . decrease in quantity of carbonyl groups was observed after other doses were applied , but it was significantly decreased only at dose of 10 kgy ( 19.3 % ) , ( figure 2b ) . introduction of carbonyl groups into amino acid residues of proteins was a hall - mark for oxidative modification . presented results implicated that protein oxidation intensity was more sensitive parameter of oxidative stress than the lipid peroxidation in irradiated soybean seeds , which was positively correlated with high protein content in soybean seeds . decline in protein oxidation by dose of 10 kgy could be explain by the high and significant increase of dpph radical scavenger capacity ( figure 5 . ) . this observation implicate that dpph radical scavengers , induced by dose of 10 kgy , act as protective agents against protein oxidation . our results concerning total phenol and tannin contents ( figure 3 . ) and total antioxidant activity ( figure 4 . ) demonstrated high values of all parameters at the dose of 10 kgy , what could also provoke the decrease in protein oxidation . other authors also established that some phenolic compounds can act as antioxidants by retarding protein oxidation reaction or by binding to the proteins . the antiradical mechanism in phenol - protein aggregates may be due to ability of phenolic compounds to transfer oxidative damage from one phenolic site to other , protecting proteins from oxidation . significant increase in total phenolic and tannin contents was caused by applied doses of -irradiation ( figure 3a and 3b ) . the maximum increase of these parameters was observed at dose of 1 kgy ( 10.0 % for total phenols and 21.6 % for tannins ) . even at the highest dose of 10 kgy , increase in total phenolic ( 7.6 % ) and tannin ( 11.0 % ) contents was observed . in plant tissues many phenolic compounds are potential antioxidants : flavonoids , tannins and lignin precursors may act as ros ( reactive oxygen species ) scavenging compounds . observed increase in total phenolic and tannin contents was beneficial for antioxidant properties of soybean seeds due to polymerisation of phenolic constituents and also cross - linking and fragmentation , which were the key reactions controlling the properties of macromolecules such as proteins . total antioxidant activity determined by frap method was significantly increased at doses of 1 kgy ( 17.0 % ) ; the increase was insignificant at 4 kgy ( 2.2 % ) and 10 kgy ( 6.7 % ) ( figure 4 ) . the irradiation induced increase in total antioxidant activity at dose of 1 and 10 kgy could be the result of high total phenol and tannin accumulation in soybean by these doses ( figure 3a , b ) . it is well known that among ascorbate , phenols are the most active ferric reducing agents . dpph radical scavenger capacity ( dpph rsc ) was increased corresponding to dose of -irradiation ( up to 49.2 % ) . polynomial relationship between dpph rsc and -irradiation dose ( y = 41.01 + 1.18x  0.0429x ; r = 0.94366 ) is presented in figure 5b . it was also reported that -irradiation doses up to 5 kgy enhanced dpph rsc of soybean butanolic extracts . higher levels of dpph antiradical activity were in correlation with no enzymatic antioxidants , especially plant polyphenols   what was also in agreement with results given in figures 3a and 3b . our results indicated that medium doses of -irradiation ( 110 kgy ) led to an antioxidant response in seed tissue of soybean genotype ana . antioxidant response was manifested in the increase in total phenolic and tannin contents , also in the increase of dpph scavenger ability at all applied doses . reduced protein oxidation intensity , was noticed when higher doses of -irradiation were applied . furthermore the lp increased only slightly which could be the consequence of the appropriate antioxidant response . therefore our results led to conclusion that -irradiation increased antioxidant capacity and protein stability of soybean seeds ( genotype ana ) , which is of great importance for health , nutritive and processing characteristics of soybean seeds . soybean seeds [ glycine max ( l. ) merr . ] of genotype ana were obtained from the institute of field and vegetable crops in novi sad . soybean seeds were irradiated with following doses of -irradiation : 1 , 2 , 4 , 6 , 8 and 10 kgy . for that purpose seed irradiation was performed in the laboratory for radiation chemistry and physics  gama  at the institute of nuclear sciences in vina , belgrade . irradiated and nonirradiated soybean seeds ( 100 seeds ) plant material ( 1 g ) was extracted with 25 ml 70 % aqueous ethanol ( 0.1 m hcl ) under 30 min sonication in an ultrasonic bath at ambient temperature . the extracts were rapidly vacuum - filtered through a sintered glass funnel and kept refrigerated . this extract was used for total phenolic and tannin contents , dpph radical scavenger capacity and total antioxidant power determinations . for lipid peroxidation , protein oxidation intensity and soluble protein content , one gram of plant material was extracted with 50 ml 0.1 m k2hpo4 at ph 7.0 after 30 min sonication in an ultrasonic bath at ambient temperature . after 10 minutes centrifugation at 4 c and 15 000 g , the aliquots of the supernatant were used for above mentioned determinations . lipid peroxidation intensity was determined by the tbars ( thiobarbituric acid reactive substances ) method . crushed sample ( 0.30 g ) was homogenized in 1.25 ml trichloroacetic acid ( tca ) ( 0.1 % ) sodium dodecyl sulfate salt ( sds ) ( 1 % ) . the homogenate was centrifuged at 12,000 g for 5 min . to 300 l aliquot of the supernatant the mixture was heated at 95 c for 30 min and then quickly cooled in an ice - bath . it was determined as amount of 2,4-dinitrophenylhydrazone ( dnph ) formed upon reaction with dnph . after cells disruption and centrifugation , samples ( > 1.5 mg protein ) were treated with 10 mm dnph in 2 m hcl at room temperature for 60 min . proteins were precipitated by addition of trichloroacetic acid up to final concentration 10 % , centrifuged at 4000 g for 10 min at 4 c , and washed three times with 1 ml solution ethanol : ethyl acetate ( 1:1 ) . the final pellets were dissolved in 6 m guanidine hydrochloride in 5 % ( v / v ) phosphoric acid . aliquots ( 0.1 ml ) of aqueous acetone extracts were transferred into the test tubes and their volumes adjusted up to 0.5 ml with distilled water . after addition of 0.25 ml folin - ciocalteu reagent and 1.25 ml 20 % aqueous sodium carbonate solution , tubes were vortexed and absorbance of blue colored mixtures recorded after 40 min at 725 nm against a blank containing 0.1 ml of extraction solvent . the amount of total phenols was calculated as a catechin equivalent from the calibration curve of catechin standard solutions ( covering the concentration range between 0.1 and 1.0 mg ml ) , and expressed as g catechin kg dry plant material . total tannin content was determined by folin - ciocalteu procedure as above , after removal of tannins by their adsorption on insoluble matrix ( polyvinylpolypyrrolidone , pvpp ) . insoluble , cross - linked pvpp ( sigma , germany ; 100 mg ) was weighed into test tubes and 1.0 ml aqueous acetone extracts added . after 15 min at 4 c , tubes were vortexed and centrifuged for 10 min at 4350 g. aliquots of supernatant ( 0.2 ml ) were transferred into test tubes and nonabsorbed phenolics determined as described . calculated values were subtracted from total phenolic contents and total tannin contents expressed as g catechin kg dry plant material . total antioxidant capacity was estimated according to the frap ( ferric reducing antioxidant power ) assay . frap value was calculated using formula : where asample is the change in absorbance of the sample and astandard is the change in absorbance of the standard ( 100 mol / dm fe ) after 4 min incubation at 593 nm . the percent inhibition of the dpph radical ( dpph rsc ) by the samples was calculated using the formula : where ac is absorbance of the control and ax is absorbance of the sample after 30 min of incubation . differences between means were evaluated for significance using tukey - test ( p < 0.05 ) . soybean seeds [ glycine max ( l. ) merr . ] of genotype ana were obtained from the institute of field and vegetable crops in novi sad . soybean seeds were irradiated with following doses of -irradiation : 1 , 2 , 4 , 6 , 8 and 10 kgy . for that purpose seed irradiation was performed in the laboratory for radiation chemistry and physics  gama  at the institute of nuclear sciences in vina , belgrade . irradiated and nonirradiated soybean seeds ( 100 seeds ) plant material ( 1 g ) was extracted with 25 ml 70 % aqueous ethanol ( 0.1 m hcl ) under 30 min sonication in an ultrasonic bath at ambient temperature . the extracts were rapidly vacuum - filtered through a sintered glass funnel and kept refrigerated . this extract was used for total phenolic and tannin contents , dpph radical scavenger capacity and total antioxidant power determinations . for lipid peroxidation , protein oxidation intensity and soluble protein content , one gram of plant material was extracted with 50 ml 0.1 m k2hpo4 at ph 7.0 after 30 min sonication in an ultrasonic bath at ambient temperature . after 10 minutes centrifugation at 4 c and 15 000 g , the aliquots of the supernatant were used for above mentioned determinations . lipid peroxidation intensity was determined by the tbars ( thiobarbituric acid reactive substances ) method . crushed sample ( 0.30 g ) was homogenized in 1.25 ml trichloroacetic acid ( tca ) ( 0.1 % ) sodium dodecyl sulfate salt ( sds ) ( 1 % ) . the homogenate was centrifuged at 12,000 g for 5 min . to 300 l aliquot of the supernatant the mixture was heated at 95 c for 30 min and then quickly cooled in an ice - bath . it was determined as amount of 2,4-dinitrophenylhydrazone ( dnph ) formed upon reaction with dnph . after cells disruption and centrifugation , samples ( > 1.5 mg protein ) were treated with 10 mm dnph in 2 m hcl at room temperature for 60 min . proteins were precipitated by addition of trichloroacetic acid up to final concentration 10 % , centrifuged at 4000 g for 10 min at 4 c , and washed three times with 1 ml solution ethanol : ethyl acetate ( 1:1 ) . the final pellets were dissolved in 6 m guanidine hydrochloride in 5 % ( v / v ) phosphoric acid . aliquots ( 0.1 ml ) of aqueous acetone extracts were transferred into the test tubes and their volumes adjusted up to 0.5 ml with distilled water . after addition of 0.25 ml folin - ciocalteu reagent and 1.25 ml 20 % aqueous sodium carbonate solution , tubes were vortexed and absorbance of blue colored mixtures recorded after 40 min at 725 nm against a blank containing 0.1 ml of extraction solvent . the amount of total phenols was calculated as a catechin equivalent from the calibration curve of catechin standard solutions ( covering the concentration range between 0.1 and 1.0 mg ml ) , and expressed as g catechin kg dry plant material . total tannin content was determined by folin - ciocalteu procedure as above , after removal of tannins by their adsorption on insoluble matrix ( polyvinylpolypyrrolidone , pvpp ) . insoluble , cross - linked pvpp ( sigma , germany ; 100 mg ) was weighed into test tubes and 1.0 ml aqueous acetone extracts added . after 15 min at 4 c , tubes were vortexed and centrifuged for 10 min at 4350 g. aliquots of supernatant ( 0.2 ml ) were transferred into test tubes and nonabsorbed phenolics determined as described . calculated values were subtracted from total phenolic contents and total tannin contents expressed as g catechin kg dry plant material . total antioxidant capacity was estimated according to the frap ( ferric reducing antioxidant power ) assay . frap value was calculated using formula : where asample is the change in absorbance of the sample and astandard is the change in absorbance of the standard ( 100 mol / dm fe ) after 4 min incubation at 593 nm . the percent inhibition of the dpph radical ( dpph rsc ) by the samples was calculated using the formula : where ac is absorbance of the control and ax is absorbance of the sample after 30 min of incubation . differences between means were evaluated for significance using tukey - test ( p < 0.05 ) .
the effect of medium doses of -irradiation ( 110 kgy ) on total phenolic and tannin contents , lipid peroxidation , protein oxidation intensity and soluble protein content of soybean seeds , ( genotype ana ) , was investigated . 
 screening for antioxidant ability was performed using frap and dpph methods . 
 total phenolic and tannin contents and dpph scavenger activity were increased , while protein oxidation intensity was decreased by applied doses of -irradiation . 
 gamma irradiation provoked insignificant changes in lipid peroxidation and soluble protein content , while protein oxidation intensity was significantly decreased when dose of 10 kgy was applied . 
 presented results implicated that increased antioxidant capacity and protein stability of soybean seeds were increased after application of -irradiation .