antioxidant and free radical scavenging activity of methanol extract of chungkukjang

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Journal of Food Composition and Analysis 20 (2007) 113–118

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Original Article

Antioxidant and free radical scavenging activity of methanolextract of chungkukjang

Mi-Yae Shona, Jun Leeb, Je-Hun Choid, Sun-Young Choia, Sang-Hae Namc, Kwon-Il Seod,Sang-Won Leee, Nak-Ju Sunga, Seok-Kyu Parkd,�

aDepartment of Food and Nutrition, Gyeongsang National University, Gaza-dong, Jinju city, Republic of KoreabCollege of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea

cDepartment of Food Science, Jinju National University, Chilam-dong, Jinju city, Republic of KoreadDepartment of Food and Nutrition, Sunchon National University, Magok-dong, Suncheon city, Republic of KoreaeDepartment of Microbiological Engineering, Jinju National University, Chilam-dong, Jinju city, Republic of Korea

Received 26 August 2004; received in revised form 12 June 2006; accepted 25 August 2006

Abstract

An assessment of chungkukjang (traditional fermented soyfood) fermented at 42 1C for 72 h with six sample groups of Korean black

soybean (Glycine max) was carried out. Total phenol and isoflavone contents of large black soybean (LBS) group extracts were higher

than that of small black soybean (SBS) group extracts. Genistein contents were higher than daidzein in all groups. In total anthocyanin

pigment, LBS groups were similar to SBS groups. LBS group extracts exhibited good inhibition rate of about 70–90% in antioxidant and

some scavenging activities of free radicals as well as hydrogen peroxide. Bacillus megaterium SMY-212 was a suitable fermenting strain to

promote the antioxidant and free-radical scavenging activities in cooked black soybean.

r 2006 Elsevier Inc. All rights reserved.

Keywords: Black soybean chungkukjang; Glycine max; Daidzein; Genistein; Phenol; Anthocyanin; Antioxidant activity

1. Introduction

Dietary intake of soybean has been linked to a decreasedrisk of osteoporosis, cardiovascular disease and cancer,including breast, colon and prostate cancers (Adlercreutz,1995; Ruiz-Larrea et al., 1997; Stoll, 1997). Many studiesreport that certain components of soybean, especiallyisoflavone, phytic acid, anthocyanin pigment, saponinand unsaturated fatty acids, protect against oxidativestress (Djuric et al., 2001).

Isoflavones occur in many legumes and are derivedmainly from soybeans and various soybean-based foodproducts in the human diet. Isoflavones present in soybean,genistein and daidzein, have been reported to have anti-carcinogenic, antiatherogenic, antifungal and antioxidant

e front matter r 2006 Elsevier Inc. All rights reserved.

a.2006.08.003

ing author. Department of Food and Nutrition, Sunchon

rsity, Magok-dong, Suncheon city, Korea.

1 750 3652.

ess: [email protected] (S.-K. Park).

properties (Coward et al., 1993; Barnes, 1995; Naim et al.,1974; Wiseman et al., 2000; Anthony et al., 1997; Anthonyet al., 1998; Tikkanen et al., 1998). These polyphenols havethe potential to scavenge free radicals such as superoxideand nitric oxide (Parthasarathy and Santanam, 1994; Hogget al., 1993; Patel and Darley-Usmar, 1999). Isoflavoneshave direct free radical quenching ability, with genisteinand daidzein being particularly effective (Ruiz-Larrea et al,1997; Stoll, 1997; Arora et al., 1998). Soy isoflavones havebeen shown to exhibit antioxidant effects both in vitro andin vivo. Isoflavones may also result in decreased oxidativedamage in cells via indirect mechanisms, such as inductionof antioxidant-scavenging enzymes (Cai and Wei, 1996).Anthocyanin pigments are widely distributed in the

human diet through seeds and crops such as fruits andvegetables, which means that many anthocyanin pigmentsare ingested in a plant-based diet. Black soybean (Glycine

max) also contains anthocyanin derived from soybeanskin. It contains flavonoid and non-flavonoid molecules,including anthocyanins. Anthocyanins are polyphenolic

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ARTICLE IN PRESSM.-Y. Shon et al. / Journal of Food Composition and Analysis 20 (2007) 113–118114

compounds that have been shown to have anti-inflamma-tory (Wang et al., 1999) and antidiabetic (Cignarella et al.,1996) effects, with a daily intake in human evaluated to beas much as 180–215mg/per/day. The results of antioxida-tive activity of anthocyanin pigments indicate that as thenumber of hydroxyl substituents on the B-ring is increased,higher activity is achieved with the glycosides, while in thecase of the aglycons increased hydroxyl substituentsproduces weaker activity. It has been revealed that activeoxygen agents such as OHd and Od2� are thought to causeoxidative damage, and much attention has been focused onactive oxygen scavenging agents such as natural phenolicslike flavonoid and anthocyanin pigments in preventing celldamage. In general, the flavylium cation form of antho-cyanin is stable in the acidic condition, but the structurechanges in neutral and alkaline conditions and breaksdown (Brouillard, 1988). When anthocyanins scavengeactive oxygen or lipid hydroperoxide radicals, the structurealso would be broken, and reaction products showantioxidative activity may scavenge the radicals. Theresults obtained from the in vitro assay systems areexpected to play a role in reducing oxidative damage incells. Some anthocyanins are also expected to be useful forinhibition of low-density lipoprotein (LDL) oxidation andfor reducing coronary heart disease and atherosclerosis(Frankel et al., 1995).

Fermented foods are an important part of the diet ofpeople in certain areas of the world. Furthermore doenjang

and chungkukjang (fermented soy paste) are among themost important fermented foods and traditional foods inKorea. There is as yet no evidence that isoflavone andanthocyanin in black soybean chungkukjang for signifi-cantly affecting the antioxidant status of the organism. Inparticular, the beneficial effects of black soybean chung-

kukjang with isoflavone and anthocyanin pigment have notbeen investigated, and have largely been overlooked at thebiological and biochemical levels. However, we think thatanthocyanin and isoflavone are responsible for the anti-oxidant properties of black soybean chungkukjang. In thepresent study, we have investigated the antioxidant activityand free radical scavenging activity of the methanolextracts of black soybean chungkukjang.

2. Materials and methods

2.1. Materials

2,2-Diphenyl-picrylhydrazyl (DPPH), linoleic acid, poly-oxyethylene sorbitan monopalmitate (tween 40), sodiumpotassium tartarate, 2-thiobarbituric acid (TBA), 2-de-oxyribose, ethylenediaminetetraacetic acid (EDTA), potas-sium phosphate, potassium ferricyanide, b-carotene, lino-leic acid, ferric chloride and L-ascorbic acid were allpurchased from Sigma Chemical Co. (St. Louis, MO).Hydrogen peroxide (H2O2, 30%, v/v) was obtained fromAldrich Chemical Co. (Milwaukee, WI). All other solvents

and chemicals used were of analytical grade and purchasedfrom Sigma-Aldrich Co. (St Louis, MO).

2.2. Preparation of chungkukjang

Korean traditional chungkukjang was prepared usingtwo different sized kernels as follows: with large blacksoybeans (LBSs) using large kernels (35 g as weight of 100kernels); and with small black soybeans (SMSs; 10 g as theweight of 100 kernels). Raw black soybean (100 g) forfermentation was soaked in 300mL water at about 15 1Cfor 3 h. After draining, the soybean was cooked at apressure of 1.0 atm for 20min and then crushed to increasefermentation rate. When cooled to 45 1C, the crushedsoybean was inoculated with culture broth (1%, v/w) fromisolated Bacillus megaterium SMY-212, which containspotent enzyme activities (protease: 124.8U/mL, a-amylase:78.2U/mL, glucoamylase: 13.9U/mL), and B. subtilis

ATCC 6633. The inoculated soybean was incubated intriplicate at 42 1C for 72 h.

2.3. Preparation of methanol extract

Freeze-dried chungkukjang (30 g) was ground andextracted in 300mL methanol (75%, v/v) for 12 h, threetimes. The methanol extract was concentrated by using avacuum evaporator (EYELA, Japan). The extract waslyophilized and stored at 20 1C. Three separate samples ofchungkukjang were extracted (n ¼ 3), and all the experi-mental analyses of the methanol extract of each samplewere carried out in triplicate.

2.4. Determination of antioxidant components

The contents of total phenols were analyzed by readingsamples on a UV/Vis spectrophotometer at 760 nmaccording to the method of Folin and Denis (1915). Eachextract was mixed with 5mL of Folin–Denis phenolreagent, 10mL of Na2CO3 and diluted by a factor of 100with distilled water. The total phenol content of eachsolvent extract was estimated by comparison with astandard curve generated from analysis of caffeic acid.The contents of total anthocyanins were analyzed by theHPLC method. The crushed chungkukjang was mixed with1% HCl and extracted anthocyanins at 4 1C for 24 h. Theextracted anthocyanins were filtered with 0.45 mm mem-brane filter and adapted Sep-pak C18 cartridge (Waters,USA). After through the water layer, adhered anthocyaninpigments were dissolved with methanol. Methanol solutionwas analyzed by HPLC (Kim et al., 1997).

2.5. Determination of total daidzein and genistein contents

In order to verify the presence of total daidzein andgenistein contents in methanol extract, we employed anHPLC method. The crushed chungkukjang (2 g) was mixedwith 10mL of acetonitrile and 2mL of 0.1 N HCl and then

ARTICLE IN PRESSM.-Y. Shon et al. / Journal of Food Composition and Analysis 20 (2007) 113–118 115

was stirred for 2 h. Mixing solution was filtered withWhatman No. 42, was concentrated, transferred to vials,and then dissolved in 10mL of methanol. Methanolsolution was filtered with 0.45 mm membrane filter andanalyzed using HPLC (Kitagawa et al., 1984).

2.6. DPPH radical-scavenging activity

The scavenging activity of methanol extracts on DPPHradicals was measured according to the method of Shimadaet al. (1992). Ethanolic solutions of DPPH (10�4) andmethanol extracts solutions were mixed so that the finalmass ratios were extracts: DPPH ¼ 5.5: 1 and referencecompound: DPPH ¼ 0.5: 1. The samples were incubatedfor 15min in the dark at 30 1C, and the decrease inabsorbance at 517 nm was measured against ethanol usinga spectrophotometer. Ethanol was used to zero thespectrophotometer. A blank sample containing the sameamount of ethanol and DPPH was prepared and measureddaily, stored in a flask covered and kept in the dark at 4 1Cbetween the measurements.

2.7. Hydroxyl radical-scavenging activity

Hydroxyl radical scavenging activity of extracts wasmeasured according to the method of Halliwell et al.(1987). One milliliter of the final reaction solution consistedof aliquots (500 mL) of various concentrations of theextract, 1mM FeCl3, 1mM EDTA, 20mM H2O2, 1mM

L-ascorbic acid, and 30mM deoxyribose in potassiumphosphate buffer (pH 7.4). The reaction mixture wasincubated for 1 h at 37 1C, and further heated in a boilingwater bath for 15min after addition of 1mL of 2.8% (w/v)trichloroacetic acid and 1mL of 1% (w/w) 2-thiobarbituricacid. The color development was measured of 532 nmagainst a blank containing phosphate buffer.

2.8. Antioxidant assay using b-carotene-linoleate model

system

The antioxidant activity of extracts was evaluated usingthe b-carotene linoleate model system (Miller, 1971). Asolution of b-carotene was prepared by dissolving 2mg ofb-carotene in 10mL of chloroform. Two millilitres of thissolution were pipetted into a 100mL round-bottom flask.After the chloroform was removed under vacuum, 40mg ofpurified linoleic acid, 400mg of tween 40 emulsifier, and100mL of aerated distilled water were added to the flaskwith vigorous shaking. Aliquots (4.8mL) of this emulsionwere transferred into different test tubes containingdifferent concentration of the extract. BHA was used forcomparative purposes. As soon as the emulsion was addedto each tube, the zero time absorbance was measured at470 nm using a spectrophotometer (CE2021, CECIL,England). The tubes were placed at 50 1C in a water bath.Measurement of absorbance was continued until the colorof b-carotene disappeared for the blank, devoid of

b-carotene, were prepared for back ground subtraction.Antioxidant activity (AA) was calculated using the follow-ing equation:

AA ¼b�carotene content after 2 h of assay

initial b�carotene content� 100.

2.9. Reducing power activity

The reducing power of extracts was determined by themethod of Yen and Duh (1993). Different concentrationsof extracts were mixed with 2.5mL of phosphate buffer(200mM, pH 6.6) and 2.5mL of 1% potassium ferricya-nide. The mixtures were incubated for 20min at 50 1C.After incubation, 2.5mL of 10% trichloroacetic acid wereadded to the mixtures, followed by centrifugation at650� g for 10min. The upper layer (5mL) was mixed with5mL of distilled water and 1mL of 0.1% ferric chlorideand the absorbance of the resultant solution were measuredat 700 nm.

2.10. Hydrogen peroxide-scavenging activity

The extract was dissolved in 3.4mL of 0.1 M phosphatebuffer (pH 7.4) and mixed with 600 mL of 43mM solution ofhydrogen peroxide. The absorbance value (at 230 nm) ofthe reaction mixture was recorded at 10min intervalsbetween zero and 40min. For each concentration, aseparate blank sample was used for background subtrac-tion (Ruch et al., 1989).

3. Results and discussion

3.1. Isoflavone and antioxidant components

In total phenol contents, LBS+SMY 212 andSBS+SMY 212 gave about 0.27 and 0.17mg/g, respec-tively; thus chungkukjang fermented using B. subtilis startershowed lower levels than the others. In total anthocyanincontents, LBS groups were similar to SBS groups (Table 1).Changes in contents of two antioxidant compounds werenot observed between cooked soybean and chungkukjang.In total daidzein and genistein contents, LBS and SBSgroups gave in the range of 1.28–1.58 and 0.90–0.92mg/g,respectively. Genistein contents were higher than daidzeinin all groups (Table 2). In total isoflavone content,chungkukjang groups prepared with starter of B. subtilis

were lower than the other groups because of antibacterialactivity of black soybean and its fermented products (Shon,2001).

3.2. DPPH radical-scavenging activity

LBS and SBS group extracts showed DPPH radicalscavenging activity with 76–87% and 69–76% at concen-tration of 1000 mg, respectively (Table 3). But the effect wasrelatively lower as compared to that of BHT and ascorbic

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Table 1

Contents of total phenol and anthocyanin pigment of large (LBS) and

small (SBS) black soybean chungkukjang fermented at 42 1C for 72 h (dry

basis, mg/g)a

Samples Total anthocyanin Total phenol

LBS (cooked) 8.24 0.24

LBS+wild strainsb 8.32 0.26

LBS+B. subtilisc 7.79 0.19

LBS+SMY-212c 8.13 0.27

SBS (cooked) 8.79 0.13

SBS+wild strains 8.88 0.14

SBS+B. subtilis 8.62 0.08

SBS+SMY-212 8.98 0.17

aAll values are means of three independent determinations, n ¼ 3,

analysed in triplicate.bChungkukjang fermented with Bacillus spp. taken from cooked

soybean and originating from the environment.cChungkukjang fermented with pure culture of B. subtilis and SMY-212.

Table 2

Contents of total daidzein and genistein of large (LBS) and small (SBS)

chungkukjang fermented at 42 1C for 72 h (dry basis, mg/g)a

Samples Compounds

Daidzein Genistein Total

LBS (cooked) 0.09 0.14 0.23

LBS+wild strainsb 0.35 0.97 1.32

LBS+B. subtilisc 0.38 0.90 1.28

LBS+SMY-212c 0.40 1.18 1.58

SBS (cooked) 0.05 0.07 0.12

SBS+wild strains 0.30 0.60 0.90

SBS+B. subtilis 0.31 0.61 0.92

SBS+SMY-212 0.24 0.66 0.90




Table 3

Scavenging activities on DPPH radicals of methanol extract of large (LBS)

and small (SBS) soybean chungkukjang fermented at 42 1C for 72 h (%)a

Sample Concentration (mg/mL)

50 500 1000

L-Ascorbic acid 95 96 97

BHT 26 87 93

LBS (cooked) 10 23 42

LBS+wild strainsb 19 66 85

LBS+B. subtilisc 25 58 76

LBS+SMY-212c 21 65 87

SBS (cooked) 2 12 35

SBS+wild strains 27 64 70

SBS+B. subtilis 21 52 69

SBS+SMY-212 19 63 76




Table 4

Hydroxyl radical scavenging activities of methanol extract of large (LBS)

and small (SBS) soybean chungkukjang fermented at 42 1C for 72 h (%)a


50 500 1000

BHT 69a 87 95

L-Ascorbic acid 73 77 80

LBS (cooked) 12 25 47



LBS+SMY-212c 68 72 73

SBS (cooked) 24 32 39



SBS+SMY-212 59 61 65




M.-Y. Shon et al. / Journal of Food Composition and Analysis 20 (2007) 113–118116

acid. Of all samples tested, LBS+SMY 212 extract showedthe highest scavenging activity being 21–87% at50–1000 mg/mL, followed by the LBS, SBS+SMY 212extracts. Methanol extract of LBS+wild strain chungkuk-

jang naturally fermented with endospore and viable cells ofBacillus sp. originated from cooked soybean and the airdemonstrated higher scavenging activity as compared topure culture of B. subtilis. This is due to the fact thatgrowth of B. subtilis was strongly inhibited by anthocyaninof black soybean (Shon, 2001).

3.3. Hydroxyl radical-scavenging activity

LBS and SBS group extracts showed hydroxyl radicalscavenging activity with about 68–72% and 60–63% atconcentration of 500 mg/mL (Table 4). A concentrationdependent inhibition against hydroxyl radical induced

deoxyribose degradation was observed in the deoxyriboseassays. Furthermore, because LBS groups were high intheir phenol, anthocyanin and isoflavone aglycones, theirantioxidant compounds may well act as antioxidantsand scavenge hydroxyl radicals generated from theFenton reagent. However, even if the concentration ofextracts was changed, the inhibition curve was not largelyshifted, i.e.,extracts inhibited hydroxyl radicals generatingsystem, rather than scavenging hydroxyl radicals. There-fore, hydroxyl radicals scavenging activity was not due todirect scavenging but inhibition of hydroxyl radicalsgeneration by iron and copper chelating ion (Spenceret al., 1996).

ARTICLE IN PRESS

Table 5

Hydrogen peroxide scavenging activities of methanol extract (10 mg/mL)

of large (LBS) and small (SBS) black soybean chungkukjang fermented at

42 1C for 72 h (%)a

Sample Time (min)

0 10 20 30 40

BHT 92 87 85 84 83

LBS (cooked) 57 43 39 30 21

LBS+wild strainsb 88 85 76 73 70

LBS+B. subtilisc 93 84 82 77 71

LBS+SMY-212c 83 82 81 80 79

SBS (cooked) 43 35 21 20 16

SBS+wild strains 86 85 74 72 69

SBS+B. subtilis 82 81 74 71 65

SBS+SMY-212 84 83 80 73 68




Table 6

Reducing powers of methanol extract of large (LBS) and small (SBS)

black soybean Chungkukjang fermented at 42 1C for 72 h (OD at 700 nm)a


50 300 500 1000

L-Ascorbic acid 2.940a

LBS (cooked) 0.121 0.230 0.515

LBS+wild strainsb 0.519 0.735 1.057

LBS+B. subtilisc 0.412 0.625 0.942

LBS+SMY-212c 0.515 0.731 1.134

SBS (cooked) 0.091 0.134 0.321

SBS+wild strains 0.423 0.631 0.971

SBS+B. subtilis 0.421 0.624 0.845

SBS+SMY-212 0.513 0.750 1.152




Table 7

Antioxidant activities of methanol extract of large (LBS) and small (SBS)

black soybean chungkukjang fermented at 42 1C for 72 h in the b-carotene-linoleate system (%)a


1 5 10

L-Ascorbic acid 57a 64 85

BHT 39 56 98

LBS (cooked) 3 4 25



LBS+SMY-212c 47 55 82

SBS (cooked) 0 2 18



SBS+SMY-212 36 41 76




M.-Y. Shon et al. / Journal of Food Composition and Analysis 20 (2007) 113–118 117

3.4. Hydrogen peroxide-scavenging activity

Scavenging activity of hydrogen peroxide in LBS andSBS groups (10 mg) and BHT (10 mg) as reference com-pound was not remarkably different and shown to be 88,86 and 92% at initial time, respectively (Table 5). Thecomposition of hydrogen peroxide into water may occuraccording to the antioxidant compounds. Since antioxidantcompounds present in the extract are good electron donors,they may accelerate the conversion of H2O2 to H2O (Ruchet al., 1984).

3.5. Reducing power activity

At concentration of 500 mg/mL, LBS and SBS groupsshowed absorbance with about 0.62–0.74 and 0.62–0.75,respectively (Table 6). Thus, all groups exhibited reducingactivity. The reducing power might be due to hydrogen-donating ability (Shimada et al. (1992), and is generallyassociated with the presence of reductones (Duh, 1998).Thus, the free radical scavenging activity of chungkukjang

extracts may be mostly related to their genistein andanthocyanin pigments. Since phenolic compounds presentin the extract are good electron donors, they may acceleratethe conversion of H2O2 to H2O (Ruch et al., 1984). Theyshowed the reducing power as the good electron donors.

3.6. Antioxidant activity in b-carotene–linoleate system

LBS and SBS groups, BHT and ascorbic acid atconcentration of 10 mg/mL were found to give the higherantioxidant activity of 80%, 70%, 98% and 85%,respectively (Table 7). SBS groups except for SBS+SMY212 had greater activity at low concentration of extracts,while in contrast, LBS group was found to have greater

level at high concentration. Additionally, it seems thatantioxidant activity of all sample groups showed differencebetween fermented samples and cooked black soybean asraw material. The antioxidant activity of carotenoids isbased on the radical adducts of carotenoid with freeradicals from linoleic acid. The linoleic acid free radicalattacks the highly unsaturated b-carotene models. Thepresence of carotenoid shows not only a decrease of thefree radical concentration, but also the reduction of Fe3+

to Fe2+ by carotenoids. The presence of differentantioxidants can hinder the extent of b-carotene-bleachingby neutralizing the linoleate-free radical and other freeradicals formed in the system (Jayaprakasha et al., 2001).

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4. Conclusions

The methanol extracts of black soybean chungkukjang

showed antioxidant and free radical scavenging activities inall samples and then major antioxidative component seemsto be the natural phenolics like isoflavonoids andanthocyanin pigments. It is apparent that fermentationprocess of black soybean contributes to the increase ofbiological activities. In making chungkukjang, large blacksoybean was more effective substrate than small blacksoybean based on the conversion rate of isoflavone b-glycosides to aglycones and biological activities. Comparedwith wild type strains and B. subtilis, B. megaterium SMY-212 was a suitable fermenting strain to promote theantioxidant and free radical scavenging activities. Thepresent study suggests that black soybean chungkukjang

extracts are useful nutritional antioxidants and theirsupplementation decreases oxidative stress.

Acknowledgements

Technology Development Program supported this studyfor Agriculture and Forestry, Ministry of Agriculture andForestry, Republic of Korea.

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