Comparison of Isoflavone Contents and Antioxidant Effect in Cheonggukjang with Black Soybean Cultivars by Bacillus subtilis CSY191
- Author: Azizul Haque Md., Hwang Chung Eun, Lee Hee Yul, Ahn Min Ju, Sin Eui-Cheol, Nam Sang Hae, Joo Ok Soo, Kim Hyun Joon, Lee Shin-Woo, Kim Yun-Geun, Ko Keon Hee, Goo Young-Min, Cho Kye Man
- Publish: Korean Journal of Environmental Agriculture Volume 35, Issue1, p62~71, 31 March 2016
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ABSTRACT
BACKGROUND: Soybeans are the rich sources of isoflavones. To date, the changes of isoflavone contents in various black soybeans
cheonggukjang during fermentation byBacillus subtilis CSY191 has not been investigated.METHODS AND RESULTS: This study investigated the changes of total phenolic and isoflavone contents and antioxidant effects during
cheonggukjang fermentation made with four black soybean (BS) cultivars includingCheongja, Cheongja #3,Geomjeong #5, andIlpumgeomjeong with a potential probioticBacillus subtilis CSY191. The total phenolic contents, isoflavone-malonylglycoside and -aglycone contents, and antioxidant activity were increased incheonggukjang at 48 h fermentation, while the content of isoflavone-glycosides was decreased duringcheonggukjang fermentation. In particular, theCheongja #3 soybean fermented at 37℃ for 48 h displayed the highest antioxidant activities, compared to those of the other BS cultivars tested. Also, the highest levels of total phenolic, daidzein, glycitein, and genistein were present at concentrations of 17.28 mg/g, 283.7 g/g, 39.9 g/g, and 13.2 g/g at the end ofCheongja #3 soybean fermentation.CONCLUSION: The results from this study suggested that the enhanced antioxidant activity of
cheonggukjang of BS might be related to increased levels of total phenolic, isoflavon-aglycone, and malonyl-glycoside contents achieved during fermentation. Furthermore, fermentedCheongja #3 soybean showed the highest levels of enhanced antioxidant activities than the other BS cultivars.
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KEYWORD
Antioxidant , Bacillus subtilis CSY191 , Black soybean , Cheonggukjang , Isoflavone
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In general, soybeans [
Glycine max (L.) Merril] have been consumed as an important protein source to complement grain protein in Asian countries. However, they are also enriched with isoflavones, anthocyanins, saponins, lipids, and oligosaccharides (Kimet al. , 2011). The several soy-foods, such ascheonggukjang ,doenjang ,douche ,temphe , andtofu , are being prepared from soybeans through fermentation process.Among several soybean varieties, black soybean (BS) is increasingly sought after in food and medicinal industries because of their various beneficial effects (Lee
et al. , 2012). The beneficial properties of BS are due to the many phytochemicals present in the crop, including isoflavone, flavanol, flavan-3-ol, anthocyanin, and saponin (Leeet al. , 2012; Leeet al. , 2014). BS is very much popular in Korea and are used to prepare several different traditional fermented foods, includingmeju (soybean cake),cheonggukjang (soybean cook),kanjang (soybean sauce), anddoenjang (soybean paste). In particular,cheonggukjang is manufactured in a traditional way in homes using different types of processes, depending on the region: thus its physicochemical and functional properties vary due to differences in soybeans, microorganisms, and fermentation time (Namet al. , 2012).In raw soybeans, isoflavones are present in four chemical forms: malonylglycosides (70-80%), acetylglycosides (5%), glycosides (25%), and aglycones (2%) (Lee
et al. , 2011). Isoflavones conjugated glycoside are converted to aglycones under acidic or alkaline conditions or by the action of -glycosidase. Importantly, the aglycone forms show greater potential for absorption in the intestine than the glycoside forms (da Silvaet al. , 2011). Thus, incorporation of β-glycosidase has attempted to increase the content of isoflavone- aglycones incheonggukjang in several studies (Yanget al. , 2006; Choet al. , 2011). Moreover, some studies reports that total phenolic and isoflavone-aglycone contents increased, depending on whether antioxidant activities increased aftercheonggukjang fermentation (Choet al. , 2009; Huet al. , 2010). Recently, we reported that the total phenolic and isoflavone- aglycone contents were enhanced duringcheonggukjang fermentation made with two wild varieties of black soybeans (Hwanget al. , 2013) and brown soybean (Shinet al. , 2014) using a potential probioticBacillus subtilis CSY191 (Choet al. , 2009; Choet al. , 2011).In this study, the antioxidant activities in
cheonggukjang made with four BS cultivars includingCheongja ,Cheongja #3,Geomjeong #5, andIlpumgeomjeong by the potential probioticB. subtilis CSY191 were investigated. Moreover, the possibility of antioxidant enhancing effect duringcheonggukjang fermentation made with BS cultivars that may be related to the total phenolic contents and isoflavone compositions of this product were also investigated.> Black soybeans, microorganism, and chemicals
Four BS cultivars, including
Cheongja ,Cheongja #3,Geomjeong #5, andIlpumgeomjeong , were harvested in 2012 and were procured from the National Institute of Crop Science (NICS) of the Rural Development Administration (RDA) in Miryang, Korea. The collected BS samples were packaged in labeled vacuum pouches to prevent their degradation. The potential probioticBacillus subtilis CSY191 previously isolated from Korean traditional soybean paste (doenjang ), were used as the starter organism (Choet al. , 2011). The twelve standard isoflavones were purchased as described previously (Hwanget al. , 2014). Glacial acetic acid, Folin-Cicalteu phenol reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS), potassium persulfate, ferric chloride, sodium acetate, 2,4,6-tripyridyl-s-triazine (TPTZ), were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). HPLC-grade H2O, methanol, and acetonitrile were purchased from Fisher Scientific Korea ltd. (Gangnam-gu, Seoul, Korea). All other reagents were of analytical grade.Approximately 1,000 g of BS from each cultivar were separately washed and soaked with 2.5 volume tap water at 25±2℃ for 12 h, and steamed for 15 min at 121±1℃. The steamed HBS were left to stand for 1 h at 40±2℃ to cool down. After that, cooked BS were inoculated with 5.0% (w/w) strain CSY191 (7.65 log cfu/mL) and fermented for 48 h at 37±2℃ in an incubator and sampled at 0 and 48 h. The method was adapted as previously described (Hwang
et al. , 2013).> Viable cell number, pH, and β-glucosidase activity
One gram of
Cheonggukjang was mixed with 9 mL of 0.85% NaCl solution and the diluted suspension (0.1 portions) was spread on a TSA plate. The plate was incubated at 37±1℃ for 48 h and after which colony counts were carried out. A 10 g portion of thecheonggukjang of BS samples was dissolved in 90 mL of distilled water at room temperature for 12 h and was filtered through Whatman No. 4 filter paper (Whatman International, Ltd., Maidstone, England). The residue pH was measured by pH meter (MP 200, UK). Moreover, the β-glucosidase activity of theCheonggukjang crude extract was measured as previously described (Hwanget al. , 2013).> Isoflavone extraction and analysis
The four BS
Cheonggukjang samples isoflavone were extracted and analyzed by HPLC according to previously described method (Hwanget al. , 2013).> Total phenolic contents (TPCs)
A method based on gallic acid equivalents (GAE) was used to quantify the TPCs of 50% methanol extract of four BS
Cheonggukjang samples according to Choet al. (2009).> Diphenyl picrylhydrazyl (DPPH) radical scavenging activity
The 50% methanol extracts of BS
Cheonggukjang (0.2 mL) were prepared and mixed with 0.8 mL of 1.5×10-4 mM DPPH methanolic solution. The mixture was vortexed vigorously and allowed to stand for 30 min at room temperature in the dark. The absorbance of the mixture at 517 nm was determined using a spectrophotometer. The scavenging activity was expressed as a percentage using the following formula: DPPH radical scavenging activity (%) = (1-absorbance of sample/absorbance of control)×100. The DPPH radical scavenging activity ofCheonggukjang extracts was carried out according to Kimet al. (2013) that was adapted from Choet al. (2011).> ABTS radical scavenging activity
ABTS+ was dissolved in methanol to a finial concentration of 7 mM. This radical cation was produced by reacting the ABTS+ stock solution with 2.45 mM potassium persulfate (final concentration) and by leaving the mixture for 12-16 h until the reaction was complete and the absorbance was stable. The ABTS+ stock solution was diluted in ethanol to an absorbance of 0.7±0.02 at 734 nm. After adding 0.9 ml of the diluted ABTS+ solution to 0.1 mL of the sample and mixing them, the absorbance was taken 3 min later. The ABTS radical scavenging activity (%) of four cultivars of
Cheonggukjang extracts was expressed as a percentage using the following formula: ABTS radical scavenging activity (%) = (1-absorbance of sample / absorbance of control)×100 (Kimet al. , 2014).> Ferric reducing/antioxidant power assays
1.5 mL of working ferric reducing/antioxidant power (FRAP) reagent pre-warmed to 37℃ was mixed with 50 μL of the test samples and standards. After vortexing, the mixture absorbance was read at 593 nm against a reagent blank. The assay was conducted at 37℃ for 15 min. The FRAP assay was conducted according to Choi
et al. (2012).Data were expressed as the means SD (standard deviation) of three replicates. The results were subjected to analysis of variance followed by the Tukey's multiple range tests at
p <0.05 using the SAS program (Version 9, USA).> Viable cell numbers, pH, and β-glucosidase activity during black soybean fermentation
The viable cell numbers, pH, and β-glycosidase activities in each
cheonggukjang made with four BS cultivars were increased during fermentation (Table 1). As the results, the bacterial cell numbers in fermentedcheonggukjang made with BS ofCheongja ,Cheongja #3,Geomjeong #5, andIlpumgeomjeong cultivar were increased to 2.43-, 2.38-, 2.35-, and 2.35-fold, respectively. In addition, the β-glucosidase activities incheonggukjang made with BS ofCheongja ,Cheongja #3,Geomjeong #5, andIlpumgeomjeong cultivars were increased to 4.8-, 5.2-, 5.26-, and 4.82-fold by the end of fermentation (48 h), respectively. In fact, the pH of fermentedcheonggukjang made with BS from all four cultivars was raised approximately to 8.39 to 8.51. The viable cell numbers and β-glycosidase activities were markedly increased duringcheonggukjang fermentation in previous studies (Yanget al. , 2006; Choet al. , 2011; Hwanget al. , 2013; Shinet al. , 2014), which is a very good agreement with the current study.> Changes of total phenolic and isoflavone contents during BS fermentation
The change in the TPCs in
cheonggukjang made with BS ofCheongja ,Cheongja #3,Geomjeong #5, andIlpumgeomjeong cultivars is shown in Fig. 1. The TPCs in each fermentedcheonggukjang made with BS from all cultivars were increased than that of the unfermented BS. In particular, the TPCs in fermentedcheonggukjang made with BS were increased to 1.53-, 1.3-, 1.43-, and 1.31-folds by the end of fermentation (48 h) for theCheongja ,Cheongja #3,Geomjeong #5, andIlpumgeomjeong cultivars, respectively (Fig. 1). Phenolics are usually found in conjugated forms through hydroxyl groups with sugars and glycosides in plant materials (Juan & Chou, 2010). Catalyzing the release of the total phenolic contents from the BS during fermentation may thus lead to an increase in the content of those compounds, as shown in Fig. 1. Our previous study reported that the TPCs in fermentedcheonggukjang made with brown soybean (Galmi ) and wild BS cultivars (Seoritae andSeomoktae ) were increased in amount with potential probioticBacillus subtilis CSY191 by the end of fermentation (Hwanget al. , 2013; Shinet al. , 2014). These results suggested thatBacillus subtilis CSY191 is a strong potential candidate strain for the biotransformation of soybean biopolymers into beneficial phenolics duringcheonggukjang fermentation made with wild or breeding soybean seeds. Meanwhile, some studies reported that the total phenolic content increased during soybean fermentation in foods, such ascheonggukjang andnatto (Choet al. , 2009; Choet al. , 2011; Shonet al. , 2007).In the case of
chenoggukjang made with BS ofCheongja cultivar, the isoflavone-malonylglycoside and-aglycone contents increased throughout fermentation to approximately 1.24- and 1.85-fold relative to their starting amounts (23.1% and 6.8%, respectively), but the isoflavone-glycoside contents decreased from 70.1 to 58.7% at the end of fermentation. Similarly, the levels of isoflavone-malonylglycoside and -aglycone in thechenoggukjang made with BS ofCheongja #3,Geomjeong #5, andIlpumgeomjeong cultivars increased throughout fermentation to approximately 1.47- and 2.52-fold, 1.02- and 2.48-fold, and 1.27- and 1.72-fold relative to their starting amounts (20.4 and 7.5%, 32.9 and 8.8%, and 28.0 and 5.1%), but the isoflavone-glycoside contents decreased from 72.1 to 51.0%, 62.3 to 54.6%, and 66.9 to 55.5%, respectively, at the end of the fermentation (Fig. 2). In particular, daidzin of the glycoside type decreased from 432.9 μg/g to 258.7 μg/g and the corresponding daidzein of the aglycone type increased from 96.5 μg/g to 283.7 μg/g inchenoggukjang made with BS ofCheongja #3 cultivar at the end of fermentation (Table 2). In fact, the total amount of isoflavone was higher in unfermented and fermented (chenoggukjang ) BS ofCheongja #3 cultivar than those of the other BS cultivars examined. However, it is apparently shown that the isoflavone-glycosides decreased, while the isoflavone-aglycones increased duringcheonggukjang fermentation made with all the four BS cultivars (Fig. 3A-H). These results suggested that the glycoside type isoflavone content daidzin, glycitin, and genistin concentrations were decreased inchenoggukjang made with all BS cultivars, while the aglycones type isoflavone content daidzein, glycitein, and genistein concentrations were increased at the end of fermentation. In addition, the malonylglycosides concentrations were also increased in the fermentation of BSchenoggukjang than that of the unfermented BS. The content and composition of these isoflavones vary in soybean foods depending on the soybean varieties and processing techniques used, such as fermentation. It has been reported that the isoflavone levels in soybean-containing foods, such astofu ,douchi , andcheonggukjang , decrease depending on the processing conditions (Yanget al. , 2006; Choet al. , 20011; Cowardet al. , 1998; Prabhakaranet al. , 2006). Janget al. (2006) reported that the total isoflavone content in raw soybeans was 2.87 μg/g, which decreased by approximately 50% during cooking prior tocheonggukjang fermentation. In a related study, Yanget al. (2006) reported that the total isoflavone content decreased from 1,055 μg/g (0 h) to 870 μg/g (36 h) duringcheonggukjang fermentation byB. subtilis . Meanwhile, Choet al. (2011) reported that the total isoflavone contents incheonggukjang fermentation decreased approximately 64% from an initial 2923.21 μg/g to 1051.59 μg/g after 60 h of fermentation. Hwanget al. (2013) reported that the total isoflavone content incheonggukjang made with wild soybeans was decreased by 13.15% and 8.3% for theSeoritae andSeomoktae cultivars usingBacillus subtilis CSY191. In this study, the total isoflavone content decreased by approximately 6.6, 9.84, 0.7, and 15.1% after fermentation processing in BS ofCheongja ,Cheongja #3,Geomjeong #5, andIlpumgeomjeong cultivars at the end of fermentation (48 h), respectively (Table 2).In general, most isoflavones in soybean are present in glycoside form, and they are converted into aglycones during fermentation by microbial β-glycosidase activity (Velioglu
et al. , 1998; Yanget al. , 2006; Choet al. , 2011). It is important to note that the levels of isoflavone-aglycones and the β-glycosidase activity increased and isoflavone-glycosides decreased duringcheonggukjang fermentation by the potential probioticB. subtilis CS90 (Choet al. , 2011; Hwanget al. , 2013). In this study, we found that the starter potential probioticsB. subtilis CSY191 had the effect of increasing the β-glycosidase activity, and the algycone contents increased at the end of fermentation. In contrast, Yanget al. (2006) reported that the addition ofB. subtilis had no effect on β-glycosidase activity, and the aglycone contents did not increase duringcheonggukjang fermentation.> Change of antioxidant activities during Black Soybean fermentation
To examine the hydrogen donating activity, the DPPH radical scavenging activity of the unfermented black soybean (UFBS) and fermented black soybean (FBS) were tested. As the result, the DPPH radical scavenging activities of
chenoggukjang made with different BS cultivars were increased about to 1.4-, 1.3-, 1.3-, and 1.25-folds at the end of fermentation, respectively (Fig. 4). This result suggested that the hydrogen donating activities of BS were increased afterchenoggukjang fermentation. Importantly, thechenoggukjang made with BS ofCheongja #3 cultivars has shown the greater DPPH radical scavenging activity (78.3%) than those of thechenoggukjang made with other BS cultivars in this study.Moreover, to determine the hydrogen-donating antioxidants and chain-breaking antioxidants, the ABTS radical scavenging ability of
chenoggukjang made with UFBS and FBS were tested. The levels of ABTS radical scavenging activity in chenoggukjang ofCheongja ,Cheongja #3,Geomjeong #5, andIlpumgeomjeong was increased about to 1.35-, 1.25-, 1.27-, and 1.22- folds at 48 h of fermentation (Fig. 5). However, like DPPH radical scavenging activity, thechenoggukjang ofCheongja #3 has shown the greater ABTS radical scavenging activity (80.64%) than those of the otherchenoggukjang examined in this study.The FRAP assay directly measure the total antioxidant power of plant extracts. Thus, we further determine the antioxidant activity of the unfermented and FBSs. In fact, the chenoggukjang of
Cheongja ,Cheongja #3,Geomjeong #5, andIlpumgeomjeong , the values resulting from the FRAP assay of the fermented soybeans increased about to 1.52-, 1.21-, 1.25-, and 1.2-fold at 48 h fermentation, respectively (Fig. 6). Importantly, like DPPH and ABTS radical scavenging activity, the FRAP assay value (1.42) ofchenoggukjang made with BS ofCheongja #3 cultivar was greater than those of the otherchenoggukjang examined.Several studies revealed that phenolic compounds were responsible for the antioxidant activity in fruits, vegetables, and grains (Velioglu
et al. , 1998; Kwaket al. , 2005). Pratt (1980) reported that the combined isoflavones and phenolic acids account for nearly all thein vitro antioxidant activity of soybean and soy product. In fact, the TPCs were measured as an overall indicator of the contents of these molecules with antioxidant properties (Slavinet al. , 2009). It was reported that methanol extract ofcheonggukjang exhibited radical-scavenging activity of 69-87% and total phenolic contents of 0.13-0.27 mg/g (Shonet al. , 2007). Interestingly, fermentation enhances the TPC as well as antioxidant activity of the BS extract (Juanet al. , 2010). However, isoflavones have direct free radical quenching ability, with daidzein and genistein being particularly effective (Shonet al. , 2007; Choet al. , 2009; Choet al. , 2011). Moreover, Kimet al. (2008) reported that thecheonggukjang extract and its constituents, genistein and daidzein, exhibited significant antioxidant activityin vitro . In the present study, higherin vitro antioxidant activities were found shown in FBS incheonggukjang than the UFBS of the four cultivars tested. This result indicates that enhanced antioxidant effects ofcheonggukjang made with BS were caused by the increased amount of total polyphenol content and aglycone isoflavone than that of the unfermented BS. In addition, the increased malonyl glucoside concentration during fermentation may also contribute to enhance the antioxidant activity of FBS (cheonggukjang ) compared to the UFBS. In our previous study, the radical scavenging activity was increased from 53.6% to 93.9% according to the total phenolic and isoflavone-aglycone (daidzein) contents duringcheonggukjang fermentation with potential probioticB. subtilis CS90 (Choet al. , 2011). Recently, we found that the stronger antioxidant activity of cheonggukjnag made with two wild cultivars of BS as well as brown soybeans might be related to the markedly higher TPCs and isoflavone-aglycones and -malonylgycosides achieved during fermentation (Hwanget al. , 2013; Shinet al. , 2014), which is consistent with the current study and with Kwaket al. (2007).In conclusions, this study first documented the changes in the TPCs and in the contents of isoflavones during
cheonggukjang fermentation made with BS cultivars with a potential probioticB. subtilis CSY191. In the case of four BS cultivars, including,Cheongja ,Cheongja #3,Geomjeong #5, andIlpumgeomjeong , the TPCs and isoflavone-aglycone contents were markedly increased, while the isoflavone-glycosides were decreased according to the β-glycosidase activities. Importantly, the TPCs, total isoflavone contents, and antioxidant activities were higher incheonggukjang made with BS ofCheongja #3 cultivar than those of thecheonggukjang made withCheongja ,Geomjeong #5,Ilpumgeomjeong cultivars at 48 h of fermentation. Therefore, it is supposed that high antioxidant activity ofcheonggukjang made with BS might be related to the higher TPCs and isoflavone-aglycone contents achieved during fermentation withB. subtilis CSY191. Moreover,cheonggukjang extract made with hybrid BS seeds supposed to be used for the commercial production of functional foods in near future.-
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2. Cho K. M., Hong S. Y., Math R. K., Lee J. H., Kambiranda D. M., Kim J. M., Islam S. M. A., Yun M. G., Cho J. J., Lim W. J., Yun H. D. (2009) Biotransformation of phenolics (isoflavones, flavanols and phenolic acids) during the fermentation of cheonggukjang by Bacillus pumilus HY1. [Food Chemistry] Vol.114 P.413-419
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23. Shin E. C., Lee J. H., Hwang C. E., Lee B. W., Kim H. T., Ko J. M., Baek I. Y., Shin J. H., Nam S. H., Seo W. T., Cho K. M. (2014) Enhancement of total phenolic and isoflavone-aglycone contents and antioxidant activities during cheonggukjang fermentation of brown soybeans by the potential probiotic Bacillus subtilis CSY191. [Food Science and Biotechnology] Vol.23 P.531-538
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[Table 1.] Change of viable cell, pH, and β-glucosidase activity during four black soybean cultivars with cheonggukjang fermentation by B. subtilis CSY191
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[Fig. 1.] Change of total phenolic contents during cheonggukjang fermentation with four black soybean cultivars by B. subtilis CSY191. UFBS, unfermented black soybeans; and FBS, fermented black soybeans at 37℃ for 48 h. All values are means of determinations in three independent experiments. Means with different lowercase letters (a, b, c, d, and e) indicate significant differences of fermentation times by Tukey’s multiple range test (p< 0.05).
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[Fig. 2.] Change of isoflavone β-glucoside, -malonyl- β-glucoside, and -aglycones and total isoflavones contents during cheonggukjang fermentation with four black soybean cultivars by B. subtilis CSY191. UFBS, unfermented black soybeans; and FBS, fermented black soybeans at 37℃ for 48 h.
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[Fig. 3.] Typical HPLC chromatograms of isoflavones. HPLC chromatogram of isoflavone extract in cheonggukjang made with black soybean of (A) Cheongja cultivar according to fermentation period (0 h), (B) Cheongja cultivar according to fermentation period (48 h), (C) Cheongja#3 cultivar according to fermentation period (0 h), (D) Cheongja#3 cultivar according to fermentation period (48 h), (E) Geomjeong#5 cultivar according to fermentation period (0 h), (F) Geomjeong#5 cultivar according to fermentation period (48 h), (G) Ilpumgeomjeong cultivar according to fermentation period (0 h), and (H) Ilpumgeomjeong cultivar according to fermentation period (48 h). 1, diadzin; 2, glycitin; 3, genistin; 4, malonyldaidzin; 5, malonyl glycitin; 6, malonyl genistin; 7, daidzein; 8, glycitein; and 9, genistein.
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[Table 2.] Distributions of isoflavone contents in cheonggukjang made of four black soybean cultivars by the B. subtilis CSY191
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[Fig. 4.] Change of diphenyl picrylhydrazyl (DPPH) radical scavenging activity during cheonggukjang fermentation with four black soybean cultivars by B. subtilis CSY191. UFBS, unfermented black soybeans; and FBS, fermented black soybeans at 37℃ for 48 h. All values are means of determinations in three independent experiments. Means with different lowercase letters (a, b, c, and d) indicate significant differences of fermentation times by Tukey’s multiple range test (p<0.05).
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[Fig. 5.] Change of ABTS radical scavenging activity during cheonggukjang fermentation with black soybean cultivars by B. subtilis CSY191. UFBS, unfermented black soybeans; and FBS, fermented black soybeans at 37℃ for 48 h. All values are means of determinations in three independent experiments. Means with different lowercase letters (a, b, c, and d) indicate significant differences of fermentation times by Tukey’s multiple range test (p< 0.05).
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[Fig. 6.] Change of ferric reducing/antioxidant power value during cheonggukjang fermentation with black soybean cultivars by B. subtilis CSY191. UFBS, unfermented black soybeans; and FBS, fermented black soybeans at 37℃ for 48 h. All values are means of determinations in three independent experiments. Means with different lowercase letters (a, b, c, and d) indicate significant differences of fermentation times by Tukey’s multiple range test (p< 0.05).
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