Effect of Fermentation Time on the Antioxidant Activity of Tempeh

Original article

Effect of fermentation time on the antioxidant activities of tempeh

prepared from fermented soybean using Rhizopus oligosporus

Chen-Tien Chang,1 Cheng-Kuang Hsu,2 Su-Tze Chou,1 Ya-Chen Chen,1 Feng-Sheng Huang1 & Yun-Chin Chung1*

1 Department of Food and Nutrition, Providence University, No. 200, Chungchi Rd, Shalu, Taichung 433, Taiwan, China

2 Department of Health and Nutrition Biotechnology, Asia University, No. 500, Lioufeng Road, Wufeng Shiang, Taichung 433, Taiwan, China

(Received 7 August 2008; Accepted in revised form 15 December 2008)

Summary The objective of this study was to evaluate the effect of fermentation time on the antioxidant activity of

tempeh, a fermented product from soybean. Rhizopus oligosporus was used to ferment soybean for 0, 1, 2, 5

and 10 days. Lyophilised tempeh powder was extracted with hexane followed by petroleum ether, ether, 95%

ethanol and water. Antioxidant activities of the extracts were evaluated with various models including a,a-diphenyl-b-pricryl-hydrazyl (DPPH) and superoxide-scavenging activities, reducing power and inhibitory

activity towards lipid peroxidation. The results revealed that tempeh showed greater antioxidant activities

than unfermented soybean. On behalf of a DPPH scavenger, water extracts were as good as 95% ethanol

extracts. In comparison of superoxide anion-scavenging activity, inhibition of lipid peroxidation and

reducing power, 95% ethanol extracts were superior to water extracts. Tempeh fermented with R. oligosporus

for 10 days exhibited the highest antioxidant activities than the others.

Keywords Antioxidant activities, extract, fermentation time, Rhizopus oligosporus, tempeh.

Introduction

Tempeh is a traditional Indonesian food product, whichis prepared by fermenting soybean with fungus, such asRhizopus oligosporus, Rhizopus oryzae, or other benefi-cial bacteria. The high protein content (up to 40% of thedry mass) makes tempeh as a good substitute of meatproducts for vegetarians Researches reported that tem-peh contained plenty of ergosterol (provitamin D2),vitamin B12 and minerals (Truesdell et al.,1987; Wieselet al.,1997; Eklund-Jonsson et al., 2006; Feng et al.,2007). Besides its nutrient properties, tempeh has manybeneficial effects, such as lowering serum cholesterollevel, inhibiting development of tumour, improvingdiarrhoea symptoms and iron-deficient anaemia, reduc-ing hypertension as well as lipid oxidation (Gyorgyet al.,1964,1974; Murakami et al.,1984; Aoki et al.,2003).Although tempeh is made from soybean, the active

compounds can be quite different between tempeh andsoybean. For instance, phenolic compounds such asisoflavones in soybean have been considered to be themajor sources of antioxidant (Vattem & Shetty, 2002;Randhir et al., 2004; Vattem et al., 2004; Nakajima

et al., 2005); however, isoflavones in tempeh may not bethe key compounds in tempeh exerting antioxidantactivity. Ikehata et al. (1968) found that isoflavonesisolated from tempeh did not show antioxidant effect insoybean oil. Esaki et al. (1996) reported that 3-hydroxy-anthranilic acid (HAA) was the principal antioxidantresponsible for the protective effect on the auto-oxida-tive activity of soybean oil; even Gyorgy et al. (1964)suggested 6,7,4¢-trihydroxyisoflavone was the mostimportant antioxidant in tempeh.The difference in the antioxidants between tempeh

and soybean may be mainly because of the process ofmicrobial fermentation. For example, Sheih et al. (2000)reported that only one-thirds of the antioxidativecapacity in tempeh was contributed by isoflavones; theother two-thirds were contributed by the peptidesderived from proteolyzing soy proteins during microbialfermentation. Many researches have demonstrated thatmicrobial fermented soybean products obtained higherantioxidant capacities than unfermented soybean (San-tiago et al., 1992; Esaki et al., 1994, 1999; Berghoferet al., 1998; Lin et al., 2006). An in vivo study showedthat Bacillus subtilis or Aspergillus oryzae fermented redbean was more effective than the unfermented red beanin raising the antioxidant levels in the liver tissue (Chouet al., 2008). Esaki et al. (1996) prepared tempeh byfermenting cooked soybean with R. oligosporus for 1, 2

*Correspondent: Fax: 886 4 26320027;

e-mail: [email protected]

International Journal of Food Science and Technology 2009, 44, 799–806 799

doi:10.1111/j.1365-2621.2009.01907.x

� 2009 The Authors. Journal compilation � 2009 Institute of Food Science and Technology

and 4 days, and found that the methanol extract of 2-day fermented tempeh had the strongest inhibition onlipid peroxidation. Murakami et al. (1984) found thatthe major isoflavones in tempeh were genistein (5,7,4¢-trihydroxyisoflavone) and daidzein (7,4¢-dihydroxyisof-lavone), produced by digesting genistein and daidzein,respectively, with b-glucosidase during R. oligospousfermentation. These literatures support that the differ-ence in the antioxidants between tempeh and soybeanmay be as a result of microbial activities.The objective of this study was to evaluate the effect

of fermentation time (2, 3, 4, 5 and 10 days) on theantioxidant activity of tempeh fermented withR. oligosporus. Meanwhile, the antioxidant activities indifferent extract solutions (hexane, petroleum ether,ether, 95% ethanol and water) from tempeh were alsocompared.

Materials and methods

Materials

Rhizopus oligosporus (BCRC 31750) was purchasedfrom Bioresource Collection and Research Center(BCRC), Food Industry Research and DevelopmentInstitution, Shinchu, Taiwan. a,a-Diphenyl-b-pricryl-hydrazyl (DPPH), a-tocopherol (a-Toc), FeCl2, FeCl3,potassium ferricyanide, butylated hydroxytoluene,nitroblue tetrazolium (NBT), phenazine methosulfate(PMS), dihydronicotinamide adenine dinucleotide(NADH), trichloroacetic acid and [4,4¢-[3-(2-pyridinyl)-1,2,4-triazine-5, 6-diyl] bisbenzenesulfonic acid] (ferro-zine) were purchased from Sigma Chemicals Co. (StLouis, MO, USA) and BCA protein assay kit fromPierce (Rockford, IL, USA). All other chemicals werereagent grade or purer.

Starter preparation

Rhizopus oligosporus (BCRC 31750) was inoculated onpotato-dextrose agar (PDA, containing 1% potato, 2%dextrose and 2% bacto-agar) slant (5 mL) and incu-bated at 25 �C for 3 days. The spore suspension(approximately 109–1010 spores mL)1) was preparedby washing the PDA slant with 1 mL of sterile water.

Preparation of tempeh

Soybean (Glycine hispida Maxim Kaohsiung No. 10)was obtained from Kaoshiung District AgricultureImprovement Station, Kaoshiung, Taiwan. The soybeanwas soaked, de-hulled, the pH adjusted to about 3.5with lactic acid and then cooked in boiling water for30 min. After cooling down to room temperature, solidstate fermentation of the cooked soybean was per-formed by inoculating the spore suspension (100 mL of

spore suspension per 400 g soybean) into cookedsoybean (1 kg) in a 28 cm (L) · 20.5 cm (W) · 4.5cm(H) stainless steel container covered with aluminium foiland incubated at 25 �C for 0, 1, 2, 5 and 10 daysrespectively. Subsequently, tempeh was dried by lyo-philisation and ground into powder.

Preparation of tempeh extracts

Tempeh powder (400 g) was mixed with 1 L of hexaneand then stirred at 4 �C for 8 h. The mixture wascentrifuged at 3000 · g for 5 min, and the supernatantwas concentrated and hexane was removed by vacuumconcentrating, and then the residues were lyophilised.The lyophilised residues were further extracted orderlywith petroleum ether, ether, 95% ethanol and deionisedwater using the same procedures as the hexaneextraction.

a,a-Diphenyl-b-pricryl-hydrazyl radical scavenging effect

The DPPH radical scavenging effect of different extractswas determined in terms of hydrogen donating orradical scavenging ability with the stable DPPH radical.According to the method of Yamaguchi et al. (1998)with modification, an aliquot of crude extract (100 lL)was added to its original solvent (400 lL) and 250 lm

solution of DPPH in 95% ethanol (0.5 mL). Themixture was kept at room temperature in the dark for20 min; then the absorbance was measured at 517 nm.Trolox was used as a standard. The capacity ofscavenging DPPH was calculated using the followingequation: DPPH-scavenging effect (%) = [(Acont ) A-

test) ⁄ Acont] · 100, where Acont was the absorbance ofcontrol reaction, and Atest was the absorbance of theextracts and standards.

Superoxide anion-scavenging effect

Measurement of superoxide anion-scavenging effect oftempeh crude extracts was based on the methoddescribed by Robak & Gryglewski (1988). Superoxideradicals were generated in PMS-NADP system byoxidation of NADH and assayed by the reduction ofNBT. In this experiment, crude extract (0.3 mL) wasmixed with 120 lm phenazine methosulphate solution(300 lL), 936 lm NADH (300 lL) and 300 lm NBT(300 lL). The reaction mixture was kept at roomtemperature in the dark for 14 min, and the absorbancewas measured at 560 nm. Gallic acid was used as astandard. Decreased absorbance of the reaction mixtureindicated increased superoxide anion-scavengingactivity. The percentage inhibition of superoxide aniongeneration was calculated using the following formula:Super oxide anion-scavenging activity (%) =[(Acont ) Atest) ⁄Acont] · 100, where Acont was the

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International Journal of Food Science and Technology 2009 � 2009 The Authors. Journal compilation � 2009 Institute of Food Science and Technology

absorbance of control reaction, and Atest was theabsorbance of the extracts and standards.

Reducing power

The reducing powers of extracts were determinedaccording to Yen & Chen (1995). Different extracts(150 lL) were mixed with equal volume of phosphatebuffer (0.2 m, pH 6.6) and 1% potassium ferricyanide[K3Fe(CN)6]. The mixture was incubated at 50 �C for20 min. Aliquots of trichloroacetic acid (150 lL,10%), distilled water (600 lL) and freshly preparedFeCl3 solution (120 lL, 0.1%) were added to themixture. After incubating at room temperature for14 min, the absorbance was measured at 700 nm.Increased absorbance of the reaction mixture indicatedincreased reducing power. Vitamin C was used as astandard.

Inhibition of lipid peroxidation

The level of thiobarbituric acid reactive substances(TBARS), mainly malonyldialdehyde (MDA), is con-sidered as an indicator of lipid peroxidation. MDAcan react with TBA when heated under acidiccondition to produce a pink colour chromogen havingan absorption maximum at 532 nm. The bloodsamples of human adult males were centrifuged at825 g for 10 min to collect the serum, and then theserum samples were stored at )20 �C and analysedwithin 2 months. The protein concentration was mea-sured by the BCA assay (Pierce) and adjusted to1 mg mL)1 before tests. After incubation of the serum(1000 lL), 2.5 mm copper sulphate (25 lL) and crudeextracts (125 lL) at 37 �C for 24 h, the mixture(750 lL) was added to equal volume of 30% TCAsolution and centrifuged at 3300 g for 10 min (Liao &Yin, 2000). The supernatant (500 lL) was mixed withequal volume of TBA solution (1%) and incubated ina water bath at 80 �C for 30 min. After centrifugationat 9168 g for 10 min, the absorbance of the superna-tant was measured at 532 nm. Decreased absorbanceof the reaction mixture indicated increased superoxideanion-scavenging activity. The percentage inhibition oflipid peroxidation was calculated using the followingequation: Inhibition of lipid peroxidation (%) =[(Acont ) Atest) ⁄Acont] · 100, where Acont was theabsorbance of control reaction, and Atest was theabsorbance of the extracts.

Statistical analysis

Data are presented as mean ± standard deviation (SD).One-way analysis of variance (anova) followed byDuncan’s multiple-range test were performed in theexperiments. P-values <0.05 were regarded significant.

Results and discussion

Extraction yields

The extraction yields (%) of tempeh extracted withdifferent solvents, in the order of hexane, petroleumether, ether, 95% ethanol and water, were 16.63 ± 0.78,0.25 ± 0.08, 1.59 ± 0.58, 2.16 ± 0.022 and11.67 ± 0.79 respectively. Among the solvents used,hexane extraction resulted in the highest extraction yieldfollowed by water. Only little amount of substance(<0.5%) was recovered by extracting with petroleumether. It was noted that the amount of substancesdistributed in different solvents was not affected signif-icantly by the duration of microbial fermentation (datanot shown).

DPPH-radical scavenging activity

Figure 1 shows the dose–response curves of DPPHradical scavenging activities of the extracts from tempeh.The DPPH radical scavenging effects of all the crudeextracts from tempeh fermented at different days weredose-dependent. Generally, microbial fermentation in-creased the DPPH radical scavenging activities for allextracts, at a dosage of £60 mg mL)1. At the highestconcentration (120 mg mL)1), hexane extracts fromtempeh fermented at 1 and 10 days and petroleum etherextracts of tempeh fermented for10 days exhibited thehighest DPPH radical scavenging activity. In etherextracts and 95% ethanol extracts, the DPPH radicalscavenging activities from fermented tempeh were supe-rior to those from the unfermented. For the waterextracts, tempeh fermented for 0 and 10 days had thehighest DPPH radical scavenging activity. Even 0-dayfermented sample showed low DPPH radical scavengingactivity when lower amount (£60 mg mL)1) was appliedin the reaction system, it performed as good as 10-dayfermented sample when high concentration(120 mg mL)1) was used.As compared with the half-scavenging concentration

(IC50) value of DPPH radical scavenging effects, bothwater and 95% ethanol were excellent solvents to extractantioxidants from tempeh but hexane was an inadequateone (Table 1). Murakami et al. (1984) reported thatisoflavones were the major compounds responsible forthe antioxidant activity in tempeh. Sheih et al. (2000)stated that two-thirds of the antioxidant capacities intempeh were derived from the oligopeptides by prote-olysis during fermentation. In Hoppe et al. (1997),antioxidant components of tempeh were extracted withhexane ⁄ ethanol (2:1, vol ⁄vol) and 5-(d-tocopheroxy)-d-tocopherol was identified as the active compound.Ishikawa et al. (1984) believed that a synergistic effect oftocopherols (present in the soybean) and amino acid(liberatedduring the fermentationprocess)was responsible

Antioxidant activities of tempehs C.-T. Chang et al. 801

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Figure 1 Scavenging effects of the tempeh

extract on a,a-Diphenyl-b-pricryl-hydrazylradical. Each value represents mean ± SD

(n = 3). (a) Hexane extract (b) ether extract

(c) petroleum ether extract (d) 95% ethanol

extract (e) water extract. , unfermented; h, 1

day fermented; r, 2 days fermented; ),

5 days fermented; , 10 days fermented; 4,

Trolox.

Table 1 The half-scavenging concentration (IC50) of a,a-diphenyl-b-pricryl-hydrazyl (DPPH) radical of tempeh extractsa and relative DPPH-

scavenging effects of tempeh extractsb

Fermentation

time (day)

Hexane Petroleum ether Ether 95% Ethanol Water

IC50 (mg mL)1) Ratio IC50 (mg mL)1) Ratio IC50 (mg mL)1) Ratio IC50 (mg mL)1) Ratio IC50 (mg mL)1) Ratio

0 80.87 ± 3.08fy 1.0 39.15 ± 1.59ew 1.0 77.08 ± 1.36gy 1.0 45.64 ± 4.36fx 1.0 38.74 ± 1.63fw 1.0

1 52.43 ± 3.09dz 1.7 47.64 ± 0.95fy 0.8 39.58 ± 1.63ex 1.1 38.31 ± 2.82ex 1.3 20.01 ± 0.68dw 1.8

2 72.33 ± 3.36ez 1.2 26.61 ± 1.05dx 0.8 27.06 ± 0.42dx 1.9 21.11 ± 2.59dw 2.8 49.84 ± 6.27gy 0.7

5 71.11 ± 2.29ez 1.2 29.54 ± 5.52dx 1.9 44.06 ± 2.04fy 2.2 13.07 ± 1.21cw 3.7 28.76 ± 5.73ex 1.3

10 45.89 ± 3.77cy 1.9 21.70 ± 1.44cx 2.6 24.31 ± 0.59cx 1.9 18.83 ± 0.41dx 2.8 9.70 ± 1.09cw 4.3

Trolox was used as the assay standard with the IC50 of 0.07317 (mg mL)1).aIC50 are given as mean ± SD (mg mL)1, n = 3).bData were obtained by divided extraction yield with IC50 value and compared with that of 0-day fermented extract which was assigned as 1.0.c–gValues in the same column with different superscripts show significant difference (P < 0.05).w–zValues in the same row with different superscripts show significant difference (P < 0.05).



for the antioxidant effect of tempeh. In this study, theantioxidant components were extracted with hexanefollowed by petroleum ether, ether, 95% ethanol andwater. According Hoppe et al. (1997), we expect thathexane extract should exhibit strong inhibition on lipidperoxidation because tocopherol should be presentmainly in hexane extract; however, all hexane extractsof tempeh did not show significant inhibitory effect onlipid peroxidation, and also the hexanes extractsobtained higher IC50 value of DPPH radical scavengingeffects than the water extracts and 95% ethanolicextracts. As mentioned above, tocopherol or tocoph-erol-like compounds were not concerned as the keyactive compound in our samples. Our results somehowagreed with the judgments of Murakami et al. (1984)and Sheih et al. (2000), as 95% ethanol extracts andwater extracts of tempeh obtained greater DPPH radicalscavenging activities among these extracts and theirmain compounds were expected to be isoflavones andpeptides respectively. Watanabe et al. (2007) modifiedthe fermentation condition for preparing tempeh withan additional 5-h anaerobic fermentation after the endof aerobic fermentation, and they also found thatisoflavone aglycones, free amino acids and peptideswere responsible for the antioxidant activity of tempeh.Fermentation time did significantly affect the DPPH

radical scavenging activities for all extracts, but it wasnot a matter of time dependant. Except for 95% ethanolextract, the optimum fermentation time to obtain theextracts with the highest DPPH radical scavengingactivities was 10 days. For the 95% ethanol extracts,the 5-day fermented tempeh showed the highest DPPHradical scavenging activities. Table 1 also shows therelative DPPH-scavenging effects of tempeh by countingextraction yield and IC50 value together, in which datawere obtained by dividing extraction yield with IC50

value and compared with that of 0-day fermentedextract which was assigned as 1.0 (Lin et al., 2006).

Water extract of 10-day fermented tempeh had thehighest relative DPPH-scavenging effects. Antioxidantswith the DPPH radicals scavenging activity weredramatically increased through microbial fermentationduring tempeh preparation, and water soluble andethanol soluble antioxidants increased up to 4.3- and3.7-fold, respectively, of that of unfermented soybean.Even though hexane had the highest extraction yield, itshowed the highest amount needed to reach the IC50.Many researches demonstrated that the DPPH radicalscavenging activities of soybean could be enhancedthrough microbial fermentation (Santiago et al., 1992;Berghofer et al., 1998; Lin et al., 2006). High concen-trations of genistein and daidzein were detected in 2-dayfermented tempeh, and this might be as a result of theb-glucosidase activity on isoflavones, such as genisteinand daidzein, during R. oligospous fermentation(Murakami et al., 1984). Sparringa & Owens (1999)also stated that approximately eight times increase in theamount of amino acids and peptides in tempeh wasfound after 3 days fermentation. Therefore, we expectedthat isoflavones-derived compounds and oligopeptideswere produced during microbial fermentation andprovided the antioxidant activities for our tempehsamples.For the rest of assessments, including inhibition of

lipid peroxidation, superoxide anion-scavenging activityand reducing power, both ether extracts and petroleumether extracts were excluded because of their low yields.


All hexane extracts of tempeh did not show significantinhibitory effect on lipid peroxidation (data not shown).On the other hand, 95% ethanol tempeh extractsinhibited lipid peroxidation and the inhibitory effectwas increased with the increase in concentrations of theextract (Fig. 2). The inhibition effect of 95% ethanol

Figure 2 Inhibition of lipid oxidation of the

tempeh extract. Each value represents

mean ± SD (n = 3). (a) 95% Ethanol

extract (b) water extract. , unfermented; h,

1 day fermented; r, 2 days fermented; ),

5 days fermented; , 10 days fermented.



extract from tempeh fermented for 10 days was superiorto those fermented for other times. But in the deionisedwater extracts, the activity of tempeh fermented for5 days was the highest. Tempeh fermented for 5 daysseemed to be the optimum based on the concentrationrequired for 50% inhibition towards lipid peroxidation(Table 2). All 95% ethanol extracts had lower IC50

values than the water extracts.Researches showed that tempeh was very stable to

rancidity development. Sekizaki et al. (1993) conductedan animal study showing that soybean protein couldreduce the concentrations of lipid peroxidants in rats,and they believed that isoflavones in soybean proteinplayed the major role. Kerry & Abbey (1998) found thatgenistein inhibited copper-induced low density lipopro-tein (LDL) oxidation and reduced the concentration ofmalondialdehyde in plasma effectively, and Tikkanenet al. (1998) also reported that daidzein and genisteincould increase the lag time for LDL oxidation and

showed antioxidant activities in plasma. Both daidzeinand genistein are soluble in either 95% ethanol or watersystem. Esaki et al. (1996) compared tempeh withsoybean on their activities of anti-lipid peroxidation bymeasuring oxygen absorption of soybean oil, and foundthat methanol extract of tempeh showed much higherantioxidant activity than that of soybean, and also 2-dayfermented tempeh obtained higher antioxidant activitythan 4-day fermented tempeh. Esaki et al. (1996)isolated an antioxidant, HAA, from methanol extractof the tempeh with the capacity in preventing the auto-oxidation of soybean oil. HAA was not found insoybean, but was produced during tempeh preparationwith R. oligosporous.It had been reported that tempeh oil was much stable

against lipid oxidation than unfermented soybean oilwhen they were extracted with hexane ⁄ ethanol in a ratioof 1:2 (Gyorgy et al., 1964; Murakami et al., 1984). Ourresults indicated that antioxidant compounds of tempeh

Table 2 The half-scavenging concentration

(IC50) of superoxide anion radical and

inhibition of lipid peroxidation of tempeh

extractsaFermentataion

time (day)

Superoxide anion radical

IC50 (mg mL)1)


IC50 (mg mL)1)

95% Ethanol Water 95% Ethanol Water

0 >120 >120 9.66 ± 1.69cw 10.52 ± 0.72dw

1 >120 >120 9.97 ± 1.39cw 11.24 ± 1.66dw

2 3.75 ± 0.19cw 5.29 ± 0.36bx 5.19 ± 2.55bw 10.57 ± 1.23dx

5 1.66 ± 0.15bw 48.13 ± 2.26dx <0.94 5.75 ± 2.44b

10 <0.94 15.22 ± 5.04c <0.94 8.91 ± 1.18c

Gallic acid was used as the assay standard with the IC50 of <0.02 (mg mL)1).aValues are given as mean ± SD (n = 3).b–dValues in the same column with different superscripts show significant difference (P < 0.05).w–xValues in the same row with different superscripts show significant difference (P < 0.05).

Figure 3 Scavenging effects of the tempeh

extract on superoxide anion. Each value

represents mean ± SD (n = 3). (a) 95%

Ethanol extract (b) water extract. ,

unfermented; h, 1 day fermented; r, 2 days

fermented; ), 5 days fermented; , 10 days

fermented; 4, gallic acid.



dissolved in ethanol contributed the anti-lipid peroxi-dation activity dramatically and we suggested HAAmight play an important role as it is soluble inethanol ⁄water system (Esaki et al.,1996).

Superoxide anion-scavenging activity

According to our data, water extract of unfermentedsoybean showed little activity in low concentration(<5 mg mL)1) and showed no additional superoxideanion-scavenging activity when the concentrationincreased further (Fig. 3). The fermented tempehswere superior to unfermented one (soybean). For the95% ethanol extract, the superoxide anion-scavengingactivity was increased with the fermentation time;while the water extract from tempeh fermented for2-day obtained the highest activity among the waterextracts. Table 2 also shows that 10-day fermentedand 2-day fermented tempehs had the lowest IC50

value of superoxide anion-scavenging activity for the95% ethanol extracts and water extracts respectively.Ethanol extracts of tempeh exhibited higher superox-ide anion-scavenging activity than water extract. As aresult of the limitation of sample solubility, the hexaneextracts was unavailable for testing their superoxideanion-scavenging activity or reducing power.

Reducing power

Figure 4 shows the reducing powers for the 95%ethanol extracts and water extracts and the resultsshowed that microbial fermentation increased thereducing powers in both 95% ethanol and waterextracts. The reducing power of 95% ethanol extract

exhibited dose–response relationship and the activity of5-day fermented tempeh showed the highest activity. Inwater extracts, the highest activity was obtained from10-day fermented tempeh while unfermented soybeanexhibited little reducing power. Generally, the ethanolextracts showed higher reducing powers than the waterextracts.

Conclusion

Tempeh made from soybean obtained great antioxidantactivity because of R. oligospous fermentation. Fermen-tation time affected the antioxidant activities of tempehand fermentation for 10 days seemed to be applicablebased on the results of DPPH radical scavenging activity.Among the different solvents applied in this study, 95%ethanol seemed to be the most applicable solvent toextract antioxidants from tempeh. All extracts exhibitedhigh DPPH radical scavenging activities indicating thattempeh contained diverse antioxidant compounds.

Acknowledgment

This research was partially supported by NationalScience Council, Taiwan (NSC94-2313-B-126-009).Their financial support is greatly appreciated.

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Effect of Fermentation Time on the Antioxidant Activity of Tempeh