International Journal of Food Science and Technology 2009, 44, 799–806799 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,adiphenyl-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, which is prepared by fermenting soybean with fungus, such as Rhizopus oligosporus, Rhizopus oryzae, or other beneficial bacteria. The high protein content (up to 40% of the dry mass) makes tempeh as a good substitute of meat products for vegetarians Researches reported that tempeh contained plenty of ergosterol (provitamin D2), vitamin B12 and minerals (Truesdell et al.,1987; Wiesel et al.,1997; Eklund-Jonsson et al., 2006; Feng et al., 2007). Besides its nutrient properties, tempeh has many beneficial effects, such as lowering serum cholesterol level, inhibiting development of tumour, improving diarrhoea symptoms and iron-deficient anaemia, reducing hypertension as well as lipid oxidation (Gyo¨rgy et 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 and soybean. For instance, phenolic compounds such as isoflavones in soybean have been considered to be the major sources of antioxidant (Vattem & Shetty, 2002; Randhir et al., 2004; Vattem et al., 2004; Nakajima *Correspondent: Fax: 886 4 26320027; e-mail:
[email protected] et al., 2005); however, isoflavones in tempeh may not be the key compounds in tempeh exerting antioxidant activity. Ikehata et al. (1968) found that isoflavones isolated from tempeh did not show antioxidant effect in soybean oil. Esaki et al. (1996) reported that 3-hydroxyanthranilic acid (HAA) was the principal antioxidant responsible for the protective effect on the auto-oxidative activity of soybean oil; even Gyo¨rgy et al. (1964) suggested 6,7,4¢-trihydroxyisoflavone was the most important antioxidant in tempeh. The difference in the antioxidants between tempeh and soybean may be mainly because of the process of microbial fermentation. For example, Sheih et al. (2000) reported that only one-thirds of the antioxidative capacity in tempeh was contributed by isoflavones; the other two-thirds were contributed by the peptides derived from proteolyzing soy proteins during microbial fermentation. Many researches have demonstrated that microbial fermented soybean products obtained higher antioxidant capacities than unfermented soybean (Santiago et al., 1992; Esaki et al., 1994, 1999; Berghofer et al., 1998; Lin et al., 2006). An in vivo study showed that Bacillus subtilis or Aspergillus oryzae fermented red bean was more effective than the unfermented red bean in raising the antioxidant levels in the liver tissue (Chou et al., 2008). Esaki et al. (1996) prepared tempeh by fermenting cooked soybean with R. oligosporus for 1, 2 doi:10.1111/j.1365-2621.2009.01907.x 2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology Murakami et al. In this experiment. 1. Starter preparation Superoxide anion-scavenging effect Rhizopus oligosporus (BCRC 31750) was inoculated on potato-dextrose agar (PDA. a-tocopherol (a-Toc). the antioxidant activities in different extract solutions (hexane. 5 and 10 days) on the antioxidant activity of tempeh fermented with R. The DPPH radical scavenging effect of different extracts was determined in terms of hydrogen donating or radical scavenging ability with the stable DPPH radical.3 mL) was mixed with 120 lm phenazine methosulphate solution (300 lL). and the supernatant was concentrated and hexane was removed by vacuum concentrating. Superoxide radicals were generated in PMS-NADP system by oxidation of NADH and assayed by the reduction of NBT. oligosporus.-T. Taiwan. Decreased absorbance of the reaction mixture indicated increased superoxide anion-scavenging activity. The mixture was kept at room temperature in the dark for 20 min. After cooling down to room temperature. 2% dextrose and 2% bacto-agar) slant (5 mL) and incubated at 25 C for 3 days. 95% ethanol and water) from tempeh were also compared. nitroblue tetrazolium (NBT). USA) and BCA protein assay kit from Pierce (Rockford. containing 1% potato. 5 and 10 days respectively. 2. and Atest was the absorbance of the extracts and standards. (1984) found that the major isoflavones in tempeh were genistein (5. where Acont was the absorbance of control reaction.5 cm (W) · 4. where Acont was the Preparation of tempeh Soybean (Glycine hispida Maxim Kaohsiung No. Chang et al. tempeh was dried by lyophilisation and ground into powder.2. potassium ferricyanide.7.5 with lactic acid and then cooked in boiling water for 30 min. The soybean was soaked. Preparation of tempeh extracts Tempeh powder (400 g) was mixed with 1 L of hexane and then stirred at 4 C for 8 h. MO. produced by digesting genistein and daidzein. the pH adjusted to about 3. Materials and methods a. and then the residues were lyophilised. dihydronicotinamide adenine dinucleotide (NADH). The reaction mixture was kept at room temperature in the dark for 14 min. IL.4¢trihydroxyisoflavone) and daidzein (7. and found that the methanol extract of 2day fermented tempeh had the strongest inhibition on lipid peroxidation. The capacity of scavenging DPPH was calculated using the following equation: DPPH-scavenging effect (%) = [(Acont ) Atest) ⁄ Acont] · 100. crude extract (0. de-hulled. The objective of this study was to evaluate the effect of fermentation time (2. with b-glucosidase during R. The spore suspension (approximately 109–1010 spores mL)1) was prepared by washing the PDA slant with 1 mL of sterile water. Journal compilation 2009 Institute of Food Science and Technology .a-Diphenyl-b-pricrylhydrazyl (DPPH). petroleum ether. (St Louis. Gallic acid was used as a standard. Kaoshiung.800 Antioxidant activities of tempehs C.5cm (H) stainless steel container covered with aluminium foil and incubated at 25 C for 0. 3. Subsequently. a. 6-diyl] bisbenzenesulfonic acid] (ferrozine) were purchased from Sigma Chemicals Co. The percentage inhibition of superoxide anion generation was calculated using the following formula: Super oxide anion-scavenging activity (%) = [(Acont ) Atest) ⁄ Acont] · 100. Taiwan. Measurement of superoxide anion-scavenging effect of tempeh crude extracts was based on the method described by Robak & Gryglewski (1988).4¢-[3-(2-pyridinyl)1. ether.a-Diphenyl-b-pricryl-hydrazyl radical scavenging effect Materials Rhizopus oligosporus (BCRC 31750) was purchased from Bioresource Collection and Research Center (BCRC). Shinchu.5 mL). trichloroacetic acid and [4. ether. 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. All other chemicals were reagent grade or purer. 4. USA). oligospous fermentation. These literatures support that the difference in the antioxidants between tempeh and soybean may be as a result of microbial activities. Trolox was used as a standard. The mixture was centrifuged at 3000 · g for 5 min. Meanwhile. then the absorbance was measured at 517 nm. The lyophilised residues were further extracted orderly with petroleum ether. 936 lm NADH (300 lL) and 300 lm NBT (300 lL). FeCl2. Food Industry Research and Development Institution.4-triazine-5. 95% ethanol and deionised water using the same procedures as the hexane extraction. and the absorbance was measured at 560 nm.4¢-dihydroxyisoflavone). spore suspension per 400 g soybean) into cooked soybean (1 kg) in a 28 cm (L) · 20. and 4 days. respectively. solid state fermentation of the cooked soybean was performed by inoculating the spore suspension (100 mL of International Journal of Food Science and Technology 2009 2009 The Authors. (1998) with modification. butylated hydroxytoluene. FeCl3. phenazine methosulfate (PMS). According to the method of Yamaguchi et al. 10) was obtained from Kaoshiung District Agriculture Improvement Station. The supernatant (500 lL) was mixed with equal volume of TBA solution (1%) and incubated in a water bath at 80 C for 30 min.59 ± 0.Antioxidant activities of tempehs C.16 ± 0.5 mm copper sulphate (25 lL) and crude extracts (125 lL) at 37 C for 24 h. Generally. microbial fermentation increased the DPPH radical scavenging activities for all extracts. The percentage inhibition of lipid peroxidation was calculated using the following equation: Inhibition of lipid peroxidation (%) = [(Acont ) Atest) ⁄ Acont] · 100.05 were regarded significant. In Hoppe et al. Chang et al. the DPPH radical scavenging activities from fermented tempeh were superior to those from the unfermented. 2009 The Authors. antioxidant components of tempeh were extracted with hexane ⁄ ethanol (2:1. DPPH-radical scavenging activity Figure 1 shows the dose–response curves of DPPH radical scavenging activities of the extracts from tempeh. tempeh fermented for 0 and 10 days had the highest DPPH radical scavenging activity. 2000). After incubating at room temperature for 14 min. (1984) believed that a synergistic effect of tocopherols (present in the soybean) and amino acid (liberated during the fermentation process) was responsible International Journal of Food Science and Technology 2009 801 . both water and 95% ethanol were excellent solvents to extract antioxidants from tempeh but hexane was an inadequate one (Table 1). After centrifugation at 9168 g for 10 min. Ishikawa et al. Vitamin C was used as a standard. it performed as good as 10-day fermented sample when high concentration (120 mg mL)1) was used. Aliquots of trichloroacetic acid (150 lL. petroleum ether. and then the serum samples were stored at )20 C and analysed within 2 months. Sheih et al.67 ± 0. 0.1%) were added to the mixture. At the highest concentration (120 mg mL)1). (1997). Among the solvents used. It was noted that the amount of substances distributed in different solvents was not affected significantly by the duration of microbial fermentation (data not shown). 1. at a dosage of £60 mg mL)1. 10%).2 m. is considered as an indicator of lipid peroxidation. The DPPH radical scavenging effects of all the crude extracts from tempeh fermented at different days were dose-dependent. Only little amount of substance (<0. The mixture was incubated at 50 C for 20 min. and Atest was the absorbance of the extracts. 95% ethanol and water.08. distilled water (600 lL) and freshly prepared FeCl3 solution (120 lL. 2.79 respectively. One-way analysis of variance (anova) followed by Duncan’s multiple-range test were performed in the experiments. absorbance of control reaction. and Atest was the absorbance of the extracts and standards. pH 6. the absorbance of the supernatant was measured at 532 nm. Results and discussion Extraction yields Reducing power The reducing powers of extracts were determined according to Yen & Chen (1995). For the water extracts.022 and 11.6) and 1% potassium ferricyanide [K3Fe(CN)6]. The blood samples of human adult males were centrifuged at 825 g for 10 min to collect the serum.-T. Decreased absorbance of the reaction mixture indicated increased superoxide anion-scavenging activity. Inhibition of lipid peroxidation The level of thiobarbituric acid reactive substances (TBARS).5%) was recovered by extracting with petroleum ether. Murakami et al.63 ± 0. mainly malonyldialdehyde (MDA). The protein concentration was measured by the BCA assay (Pierce) and adjusted to 1 mg mL)1 before tests. the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. where Acont was the absorbance of control reaction.58.25 ± 0. After incubation of the serum (1000 lL). As compared with the half-scavenging concentration (IC50) value of DPPH radical scavenging effects. MDA can react with TBA when heated under acidic condition to produce a pink colour chromogen having an absorption maximum at 532 nm. 2. Journal compilation 2009 Institute of Food Science and Technology The extraction yields (%) of tempeh extracted with different solvents.78. ether. Statistical analysis Data are presented as mean ± standard deviation (SD). hexane extraction resulted in the highest extraction yield followed by water. Even 0-day fermented sample showed low DPPH radical scavenging activity when lower amount (£60 mg mL)1) was applied in the reaction system. 0. In ether extracts and 95% ethanol extracts. Different extracts (150 lL) were mixed with equal volume of phosphate buffer (0. hexane extracts from tempeh fermented at 1 and 10 days and petroleum ether extracts of tempeh fermented for10 days exhibited the highest DPPH radical scavenging activity. vol ⁄ vol) and 5-(d-tocopheroxy)-dtocopherol was identified as the active compound. the mixture (750 lL) was added to equal volume of 30% TCA solution and centrifuged at 3300 g for 10 min (Liao & Yin. P-values <0. were 16. in the order of hexane. (1984) reported that isoflavones were the major compounds responsible for the antioxidant activity in tempeh. (2000) stated that two-thirds of the antioxidant capacities in tempeh were derived from the oligopeptides by proteolysis during fermentation. a IC50 are given as mean ± SD (mg mL)1. 1 day fermented.68dw 6.07 18. n = 3). (a) Hexane extract (b) ether extract (c) petroleum ether extract (d) 95% ethanol extract (e) water extract.36ez 2.36gy 1.8 0.7 1. 4.33 71.05).9 2.09cw Ratio 1.6 77.2 1. Trolox.59ew 0. a Table 1 The half-scavenging concentration (IC50) of a.0 0. Each value represents mean ± SD (n = 3).83 ± ± ± ± ± 4.73ex 1.04fy 0.84 28.a-Diphenyl-b-pricryl-hydrazyl radical.54 21.0 1.06 24.58 27. Chang et al. c–g Values in the same column with different superscripts show significant difference (P < 0.95fy 1.27gy 5.09dz 3.7 1.08fy 3.05dx 5. w–z Values in the same row with different superscripts show significant difference (P < 0.a-diphenyl-b-pricryl-hydrazyl (DPPH) radical of tempeh extracts and relative DPPHscavenging effects of tempeh extractsb Hexane Petroleum ether Fermentation time (day) IC50 (mg mL)1) 0 1 2 5 10 80.44cx Ether 95% Ethanol Ratio IC50 (mg mL)1) 1. b Data were obtained by divided extraction yield with IC50 value and compared with that of 0-day fermented extract which was assigned as 1.77cy Ratio IC50 (mg mL)1) 1.11 13.82ex 2.59cx Ratio IC50 (mg mL)1) 1. 5 days fermented.3 Trolox was used as the assay standard with the IC50 of 0.29ez 3. .0 1.36fx 2.2 1.64 38.21cw 0. International Journal of Food Science and Technology 2009 2009 The Authors. Figure 1 Scavenging effects of the tempeh extract on a.8 0.3 4.0.52dx 1.06 44.05).63fw 0.8 3.61 29. Journal compilation 2009 Institute of Food Science and Technology .-T.07317 (mg mL)1).8 38. 10 days fermented.7 2.42dx 2.59dw 1.74 20. ).08 39.89 ± ± ± ± ± 3.87 52.11 45.01 49.31 ± ± ± ± ± 1.802 Antioxidant activities of tempehs C. unfermented.70 ± ± ± ± ± 1. h.9 39.0 1. . 2 days fermented.70 ± ± ± ± ± 1.41dx Water Ratio IC50 (mg mL)1) 1.2 1.9 2.1 1.63ex 0.8 1. r.76 9.15 47.3 2.43 72.31 21.64 26.0 1.9 45. all hexane extracts of tempeh did not show significant inhibitory effect on lipid peroxidation. in which data were obtained by dividing extraction yield with IC50 value and compared with that of 0-day fermented extract which was assigned as 1. Inhibition of lipid peroxidation All hexane extracts of tempeh did not show significant inhibitory effect on lipid peroxidation (data not shown).Antioxidant activities of tempehs C. free amino acids and peptides were responsible for the antioxidant activity of tempeh. during R.. The inhibition effect of 95% ethanol Figure 2 Inhibition of lipid oxidation of the tempeh extract. Watanabe et al. and they also found that isoflavone aglycones. Sparringa & Owens (1999) also stated that approximately eight times increase in the amount of amino acids and peptides in tempeh was found after 3 days fermentation. 2006). 5 days fermented. 1984). ether. 95% ethanol and water. 2 days fermented. In this study. we expect that hexane extract should exhibit strong inhibition on lipid peroxidation because tocopherol should be present mainly in hexane extract. however. Chang et al. (a) 95% Ethanol extract (b) water extract. Many researches demonstrated that the DPPH radical scavenging activities of soybean could be enhanced through microbial fermentation (Santiago et al. of that of unfermented soybean. 1998. and also the hexanes extracts obtained higher IC50 value of DPPH radical scavenging effects than the water extracts and 95% ethanolic extracts. 1 day fermented. 2). such as genistein and daidzein. . 95% ethanol tempeh extracts inhibited lipid peroxidation and the inhibitory effect was increased with the increase in concentrations of the extract (Fig. both ether extracts and petroleum ether extracts were excluded because of their low yields. (1997). the antioxidant components were extracted with hexane followed by petroleum ether. As mentioned above.-T. Our results somehow agreed with the judgments of Murakami et al. Each value represents mean ± SD (n = 3). On the other hand.and 3. h. ). 10 days fermented. According Hoppe et al. Even though hexane had the highest extraction yield. the optimum fermentation time to obtain the extracts with the highest DPPH radical scavenging activities was 10 days. and this might be as a result of the b-glucosidase activity on isoflavones. for the antioxidant effect of tempeh. it showed the highest amount needed to reach the IC50. and water soluble and ethanol soluble antioxidants increased up to 4. the 5-day fermented tempeh showed the highest DPPH radical scavenging activities. 2006). but it was not a matter of time dependant. Fermentation time did significantly affect the DPPH radical scavenging activities for all extracts. Antioxidants with the DPPH radicals scavenging activity were dramatically increased through microbial fermentation during tempeh preparation. Water extract of 10-day fermented tempeh had the highest relative DPPH-scavenging effects.. (1984) and Sheih et al.7-fold. For the rest of assessments.. 2009 The Authors.. r. superoxide anion-scavenging activity and reducing power.. unfermented. we expected that isoflavones-derived compounds and oligopeptides were produced during microbial fermentation and provided the antioxidant activities for our tempeh samples. (2007) modified the fermentation condition for preparing tempeh with an additional 5-h anaerobic fermentation after the end of aerobic fermentation. 1992. respectively. oligospous fermentation (Murakami et al. (2000).0 (Lin et al. . tocopherol or tocopherol-like compounds were not concerned as the key active compound in our samples.3. Journal compilation 2009 Institute of Food Science and Technology International Journal of Food Science and Technology 2009 803 . including inhibition of lipid peroxidation. Berghofer et al. High concentrations of genistein and daidzein were detected in 2-day fermented tempeh. Lin et al. Except for 95% ethanol extract. Therefore. as 95% ethanol extracts and water extracts of tempeh obtained greater DPPH radical scavenging activities among these extracts and their main compounds were expected to be isoflavones and peptides respectively. Table 1 also shows the relative DPPH-scavenging effects of tempeh by counting extraction yield and IC50 value together. For the 95% ethanol extracts. HAA. 10 days fermented.57 ± 1. It had been reported that tempeh oil was much stable against lipid oxidation than unfermented soybean oil when they were extracted with hexane ⁄ ethanol in a ratio of 1:2 (Gyo¨rgy et al.19cw 1. extract from tempeh fermented for 10 days was superior to those fermented for other times.02 (mg mL)1).23dx 5.66dw 10. Chang et al. 4. But in the deionised water extracts. Each value represents mean ± SD (n = 3).97 ± 1.24 ± 1.66 ± 0.19 ± 2.05). 2 days fermented. Journal compilation 2009 Institute of Food Science and Technology . from methanol extract of the tempeh with the capacity in preventing the autooxidation of soybean oil. w–x Values in the same row with different superscripts show significant difference (P < 0.55bw <0. a Values are given as mean ± SD (n = 3).. (1996) isolated an antioxidant. and they believed that isoflavones in soybean protein played the major role.69 9. (a) 95% Ethanol extract (b) water extract.75 ± 2.22 ± 5. 1984). (1996) compared tempeh with soybean on their activities of anti-lipid peroxidation by measuring oxygen absorption of soybean oil. Murakami et al.-T. Esaki et al. Fermentataion time (day) 0 1 2 5 10 Superoxide anion radical IC50 (mg mL)1) Inhibition of lipid peroxidation IC50 (mg mL)1) 95% Ethanol 95% Ethanol >120 >120 3. ). 1 day fermented. oligosporous. and also 2-day fermented tempeh obtained higher antioxidant activity than 4-day fermented tempeh. Both daidzein and genistein are soluble in either 95% ethanol or water system. Kerry & Abbey (1998) found that genistein inhibited copper-induced low density lipoprotein (LDL) oxidation and reduced the concentration of malondialdehyde in plasma effectively. All 95% ethanol extracts had lower IC50 values than the water extracts. gallic acid.13 ± 2.91 ± 1.804 Antioxidant activities of tempehs C.52 ± 0. and found that methanol extract of tempeh showed much higher antioxidant activity than that of soybean. Esaki et al.15bw <0.94 <0. Researches showed that tempeh was very stable to rancidity development. HAA was not found in soybean.75 ± 0. and Tikkanen et al.29 ± 0.05).04c cw 9. the activity of tempeh fermented for 5 days was the highest. Sekizaki et al. Our results indicated that antioxidant compounds of tempeh Figure 3 Scavenging effects of the tempeh extract on superoxide anion. 1964..39cw 5. b–d Values in the same column with different superscripts show significant difference (P < 0. International Journal of Food Science and Technology 2009 2009 The Authors.94 Water Table 2 The half-scavenging concentration (IC50) of superoxide anion radical and inhibition of lipid peroxidation of tempeh extractsa 10. h. unfermented.72dw 11. .44b 8. 5 days fermented. .26dx 15.66 ± 1.18c Gallic acid was used as the assay standard with the IC50 of <0. r. Tempeh fermented for 5 days seemed to be the optimum based on the concentration required for 50% inhibition towards lipid peroxidation (Table 2). but was produced during tempeh preparation with R. (1998) also reported that daidzein and genistein could increase the lag time for LDL oxidation and showed antioxidant activities in plasma.94 Water >120 >120 5. (1993) conducted an animal study showing that soybean protein could reduce the concentrations of lipid peroxidants in rats.36bx 48. Berghofer. Y. dissolved in ethanol contributed the anti-lipid peroxidation activity dramatically and we suggested HAA might play an important role as it is soluble in ethanol ⁄ water system (Esaki et al. Bioscience. Mundigler.. the superoxide anion-scavenging activity was increased with the fermentation time. Table 2 also shows that 10-day fermented and 2-day fermented tempehs had the lowest IC50 value of superoxide anion-scavenging activity for the 95% ethanol extracts and water extracts respectively. h. N. the ethanol extracts showed higher reducing powers than the water extracts.. soybean. J.-T. Effects of caminobutyric acid-enriched tempeh-like fermented soybean (GABA-tempeh) on the blood pressure of spontaneously hypertensive rats. Ethanol extracts of tempeh exhibited higher superoxide anion-scavenging activity than water extract. All extracts exhibited high DPPH radical scavenging activities indicating that tempeh contained diverse antioxidant compounds. oligospous fermentation. while the water extract from tempeh fermented for 2-day obtained the highest activity among the water extracts. Their financial support is greatly appreciated. 1 day fermented. The fermented tempehs were superior to unfermented one (soybean).. . (a) 95% Ethanol extract (b) water extract. r. Furuya. Sentall. 2 days fermented. H. Figure 4 Reducing powers of the tempeh extract. K. Each value represents mean ± SD (n = 3). Superoxide anion-scavenging activity According to our data. Chang et al. vitamin C. unfermented. 5 days fermented.. Grzeskowiad. (1998). Endo. W.B. & Fujimoto. & Walcak. E. Antioxidative properties of faba-. 95% ethanol seemed to be the most applicable solvent to extract antioxidants from tempeh. Acknowledgment This research was partially supported by National Science Council. the hexane extracts was unavailable for testing their superoxide anion-scavenging activity or reducing power. Fermentation time affected the antioxidant activities of tempeh and fermentation for 10 days seemed to be applicable based on the results of DPPH radical scavenging activity. As a result of the limitation of sample solubility. Journal compilation 2009 Institute of Food Science and Technology International Journal of Food Science and Technology 2009 805 . Taiwan (NSC94-2313-B-126-009).Antioxidant activities of tempehs C. The reducing power of 95% ethanol extract Aoki. (2003). 1806–1808. 4. 10 days fermented. . In water extracts. exhibited dose–response relationship and the activity of 5-day fermented tempeh showed the highest activity. 68. B.and 2009 The Authors. Conclusion Tempeh made from soybean obtained great antioxidant activity because of R. ). Among the different solvents applied in this study.. Y. 3). Reducing power References Figure 4 shows the reducing powers for the 95% ethanol extracts and water extracts and the results showed that microbial fermentation increased the reducing powers in both 95% ethanol and water extracts. Biotechnology and Biochemistry.. water extract of unfermented soybean showed little activity in low concentration (<5 mg mL)1) and showed no additional superoxide anion-scavenging activity when the concentration increased further (Fig. the highest activity was obtained from 10-day fermented tempeh while unfermented soybean exhibited little reducing power. Generally. For the 95% ethanol extract.1996). Randhir. M. Proceedings of the National Academy of Science. (2005). Vattem. M. 203. Murata. Nancy. P. A. (2004). Vihma. (2006). H... 62. & Yamane. M.A.Y.C. S. H. Sandberg. (2007). Esaki.. & Osawa. M. Nature. Process Biochemistry. C. R-69 from Indonesia. 23. M.W. 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