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Characteristics and Antioxidant Effect of Garlic in the Fermentation of Cheonggukjang by Bacillus amyloliquefaciens MJ1-4
Characteristics and Antioxidant Effect of Garlic in the Fermentation of Cheonggukjang by Bacillus amyloliquefaciens MJ1-4
Journal of Microbiology and Biotechnology. 2014. Jul, 24(7): 959-968
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : October 18, 2013
  • Accepted : March 29, 2014
  • Published : July 30, 2014
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About the Authors
Jeong Hwan Kim
Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 660-701, Republic of Korea
Chung Eun Hwang
Department of Food Science, Gyeongnam National University of Science and Technology, Jinju 660-758, Republic of Korea
Chang Kwon Lee
Mong-Go Foods Co., Ltd., Changwon 641-465, Republic of Korea
Jin Hwan Lee
National Institute of Chemical Safety, Ministry of Environment, Daejeon 305-343, Republic of Korea
Gyoung Min Kim
Namhae Garlic Research Institute, Namhae 668-812, Republic of Korea
Seong Hoon Jeong
Namhae Garlic Research Institute, Namhae 668-812, Republic of Korea
Jeong Hee Shin
Namhae Garlic Research Institute, Namhae 668-812, Republic of Korea
Jong Sang Kim
School of Applied Biosciences and Food Science and Biotechnology, Kyungpook National University, Daegu 702-701, Republic of Korea
Kye Man Cho
College of Pharmacy, Sunchon National University, Suncheon 540-950, Republic of Korea
kmcho@gntech.ac.kr

Abstract
The changes in the β-glucosidase activity, total phenolic contents, isoflavone contents, and antioxidant activities during the fermentation of cheonggukjang by Bacillus amyloliquefaciens MJ1-4 with and without garlic were investigated. The levels of total phenolic and isoflavone-malonylglycoside,-acetylglycoside, and -aglycone contents increased, whereas the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino- bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging activities and ferric reducing/antioxidant power (FRAP) assay results increased, but isoflavone-glycoside levels decreased during cheonggukjang fermentation. The levels of total phenolic and total isoflavone contents and the antioxidant activities were higher in cheonggukjang fermented without garlic (CFWOG) than in cheonggukjang fermented with garlic (CFWG) after 24 h of fermentation, but they were lower in CFWOG than in CFWG after 72h of fermentation. In particular, the highest levels of total phenolic, daidzein, glycitein, and genistein were present at concentrations of 15.18 mg/g, 264.4 μg/g, 16.4 μg/g, and 31.1 μg/g after 72h of fermentation in CFWG, showing 82.89% in DPPH radical scavenging activity, 106.32% in ABTS radical scavenging activity, and 1.47 (OD 593 nm ) in FRAP assay, respectively. From these results, we suggest that the high antioxidant activity of CFWG might be related to the markedly higher levels of total phenolic contents, isoflavone-malonylglycosides, -acetylglycosides, and -aglycones achieved during fermentation.
Keywords
Introduction
Soybeans [ Glycine max (L.) Merrill] and soy products have long been consumed as an important protein source to complement grain protein in Asian countries. Besides protein, they are notably rich in isoflavones, anthocyanins, saponins, lipids, and oligosaccharides [13] . Various products, such as cheonggukjang , doenjang , douche , tempeh , and tofu , have been produced from soybeans in Asian countries, and fermentation is one of the major processes. The chemical structure and concentration of isoflavones in soy foods are dependent on many factors, including genotype, crop year, crop location, storage period, thermal processing, processing type, and the presence of microorganisms [1 , 2 , 13 , 18 - 20 , 32] . Isoflavone-aglycones are reported to be absorbed faster and in higher amounts than their corresponding glycoside forms [2 , 13 , 19 , 20 , 32] .
The market size scale of soybean fermented foods was approximately 980 billion Korean won in 2012. Of these, gochujang , doenjang , kanjang , and others (including cheonggukjang ) occupied 30.9%, 15.7%, 15.7%, and 24.5%, respectively( http://www.aTFIS.or.kr ). Cheonggukjang is made from cooked whole soybeans fermented with microorganisms, including Bacillus sp. that is usually contained in the air or in the rice straw, at about 40℃ without adding salts over two to three days [2] . In particular, cheonggukjang is traditionally produced in the house using different processes depending on the region; thus, its physicochemical and functional properties vary according to differences in the soybeans, microorganisms, and fermentation time [21] . The incorporation of β-glucosidase has been employed in an attempt to increase the content of isoflavone-aglycones in cheonggukjang [1 , 2 , 32] . In addition, several researchers have reported that the total phenolic and isoflavone-aglycone contents increased depending on whether the antioxidant activities increased after cheonggukjang fermentation [1 , 2 , 5] .
Garlic ( Allium sativum . L) is widely used as a seasoning for food, as well as having long-standing application as a medicinal agent for the treatment of a variety of human diseases and disorders. Garlic is one of the most highly antioxidant and hypoglycemic foods, and it is used for both culinary and medicinal purposes because it contains polyphenol-based antioxidant materials [12] .
Oxidative stress, defined as an imbalance between the production of reactive oxygen species (ROS) and antioxidant defense, is associated with a number of pathological conditions, such as inflammation, carcinogenesis, aging, atherosclerosis, and reperfusion injury [11] . These “free radicals” are usually removed or inactivated in vivo by a team of antioxidants. Individual members of the antioxidant defence team are deployed to prevent generation of ROS, to destroy potential oxidants, and to scavenge ROS. Thus, oxidative stress-induced tissue damage is minimized. Antioxidants have been used for the prevention of cardiovascular disease, cancer, and diabetes, whose prevalence has been increasing sharply in recent years [3] .
The purposes of the present research were to investigate changes in the total phenolic and isoflavone contents and antioxidant effects via 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging activities and ferric reducing/ antioxidant power (FRAP) assay during cheonggukjang fermentation with and without garlic. Moreover, we investigated the possibility that the enhancing effect on the antioxidant activities observed during cheonggukjang fermentation with and without garlic may be related to the total phenolic content and isoflavone composition of this product.
Materials and Methods
- Materials, Chemicals, and Instruments
Yellow soybeans and garlics were harvested in 2011 and 2012 and were provided by the Processing Establishments of Hamyang Agricultural Cooperative in Hamyang-gun and Saenamhae Agricultural Cooperative in Namhae-gun, Korea, respectively. The tryptic soy (TS) medium was purchased from Difco (Becton Dickinson Co., Sparks, MD, USA). Three isoflavone aglycones, including daidzein, genistein, and glycitein, were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), and three isoflavone glycosides, including genistin, daidzin, and glycitin, were purchased from Indofine (Hillsborough, NJ, USA). Three malonyl- and three acetyl-isoflavone glycosides (malonylgensitin, malonylglycitin, malonyldaidzin, acetylgenistin, acetylglycitin, and acetyldaidzin) were purchased from LC Laboratories (Woburn, MA, USA). HPLC-grade H 2 O, methanol, and acetonitrile were purchased from Fisher Scientific (Fairlawn, NJ, USA). Glacial acetic acid, Folin-Ciocalteu phenol reagent, 2,2-diphenyl-2-picrylhydrazyl hydrate (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), and rutin were obtained from Sigma-Aldrich Chemical Co. All other reagents were of analytical grade.
The UV spectra were measured with a Spectronic 2D spectrophotometer (Thermo Electron Co., CL, USA). HPLC was performed using an Agilent 1200 series system (Agilent Co., Forest Hill, Victoria, Australia) equipped with a quaternary HPLC pump, a degasser, and an Agilent 1200 series diode array detector (DAD). The isoflavones were analyzed on a LiChrospher 100 RP C 18 column (4.6 × 150 mm, 5 μm; Merck, Germany), whereas the phenolic acids were analyzed on an XTerra RP C8 column (4.6 × 250 mm, 5 μm; Waters Corp., Milford, MA, USA).
- Growth Test of Bacillus sp. on Cheonggukjang with Garlic
Bacillus subtilis groups, such as B. subtilis , B. amyloliquefaciens , B. licheniformis , and B. pumilus , were previously isolated and collected from Korean cheonggukjang , doenjang , and kanjang , respectively [1 , 2 , 17 , 27] . B. subtilis groups were kindly provide by the Laboratory of Food Microbiology, Gyeongnam National University of Science and Technology and Gyeongsang National University, Jinju, Korea, respectively. The bacterial cultures were grown in TS broth at 37 ± 2℃ overnight, and garlic at 0%, 5%, 10%, and 20% (w/w) was added to the soaking soybeans, steamed, and cooled down. Then, the cooked soybeans were inoculated with 5% (w/w) of various Bacillus sp. and fermented for 72 h at 37 ± 2℃ in an incubator. The cell concentrations were checked by a viable count determination on TS agar plates after 48 h of incubation.
- Cheonggukjang Preparation
The cheonggukjang fermentation procedure described by Cho et al . [2] was followed to prepare the soybeans. Garlic was washed with running tap water before being chopped into pieces. CFWOG ( cheonggukjang fermentation without garlic): Whole soybeans (1,000 g) were washed and soaked with three volumes of tap water at 20 ± 2℃ for 12 h and steamed for 30 min at 121 ± 1℃. CFWG ( cheonggukjang fermenting with garlic): Whole soybeans (1,000 g) were washed and soaked with three volumes of tap water at 20 ± 2℃ for 12 h, and then 10% garlic was added and the soybeans were steamed for 30 min at 121 ± 1℃. The steamed soybeans with and without garlic were allowed to stand for 1 h at 37 ± 2℃ to cool down. Then, the cooked soybeans with and without garlic were inoculated with 5% (w/w) of the MJ1-4 strain (5.9 × 10 9 CFU/ml) and fermented for 72 h at 37 ± 2℃ and sampled at 0, 12, 24, 48, and 72 h.
- Viable Cell Numbers and β-Glucosidase Assay
A 1 g sample was mixed with 9 ml of 0.85% NaCl solution, and the diluted suspension (0.1 ml aliquots) was spread on tryptic soy agar (TSA) plates. The plates were incubated at 37℃ for 24 h, and then the colony counts were performed.
The method described by Cho et al . [2] was used to determine the β-glucosidase activity. Ground cheonggukjang (1.0 g) was mixed with 20 ml of 50 mM sodium phosphate buffer (pH 7.0), vortexed for 1 min, and centrifuged at 6,000 × g at 4℃ for 30 min. The supernatant was collected and filtered through a 0.45 μm filter before analysis. The β-glucosidase activity in the crude extract was assayed by determining the rate of hydrolysis of p -NPG. The crude extract (250 μl) was added to 250 μl of substrate (5 mM p -NPG) in 50 mM sodium phosphate buffer (pH 7.0). After 30 min of incubation at 37℃, the enzymatic reaction was stopped by adding 500 μl of 0 .2M glycine- NaOH (pH 10.5) and the contents were immediately measured in a spectrophotometer at 405 nm. The blank solution was composed of 2.5 ml of 50 mM glycine-NaOH, 2.0 ml of substrate solution, and 0.5 ml of 50 mM citric buffer (pH 4.5) containing 0.1 M NaCl. The p -NP released by the action of the enzyme was determined by referring to a calibration curve prepared from the p -NP in concentrations that varied from 5 to 300 mmol. One unit of β -glucosidase activity was defined as the amount of enzyme that liberated 1 μM of p -NPG.
- Extraction of Isoflavones and Analysis
The isoflavone extract and analysis were performed as previously described [2] . Briefly, each of the ground powders (10 g) was extracted with 100 ml of 50% methanol by shaking (320 rpm) at 30℃ for 12 h; the extracts were filtered through Whatman No. 2 filter paper (GE Healthcare Life Sci., Piscataway, NJ, USA) and then filtered through a 0.45 μm Millipore PVDF filter (Schleicher & Schuell, GmbH, Dassel, Germany). Samples of the filtrates were used for HPLC analysis. The rest of the filtrates were dried under a vacuum. The dried samples were stored at -70℃ in the dark until further use in antioxidant activity assays, and then the dried materials were redissolved in 50% methanol at 1 mg/ml.
The isoflavones were analyzed by HPLC. A 20 μl sample of the crude 50% methanol extracts was injected onto an analytical C 18 column with the column temperature set to 30℃. The isoflavones were detected by monitoring the elution at 254 nm using a diode array detector. The isoflavones in the samples were identified by comparing their retention times with those of standards. The mobile phase was composed of 0.1% glacial acetic acid in water (solution A) and 100% acetonitrile (solution B). The gradient conditions were as follows: 0-20 min, 10% B; 30 min, 20% B; 40min, 25% B; and 50min, 35% B. The solvent flow rate was maintained at 1 ml/min.
- Total Phenolic Contents (TPCs)
A method based on that of Cho et al . [2] , which uses gallic acid equivalents (GAE), was used to quantify the TPCs in the 50% methanol extracts. A 500 μl aliquot of each isoflavone extract was mixed with 250 μl of 2 N Folin-Ciocalteu’s reagent. After standing at room temperature for 3 min, a 500 μl aliquot of a 25% sodium carbonate (Na 2 CO 3 ) solution was added, and that mixture was allowed to stand at room temperature for 1 h. Quantification was performed using a linear regression equation on the gallic acid standard curve. Five gallic acid standard solutions of 100, 250, 500, 750, and 1,000 mg/l were prepared in deionized water, and 500 μl of each standard solution was collected and prepared using the same procedure described above. The absorbance of the solution was measured at 750 nm, and a standard curve was obtained by plotting the concentration against absorbance.
- DPPH Radical Scavenging Activity
The DPPH radical scavenging activity of the samples was measured by the method of Cho et al . [2] with a slight modification. Briefly, the various concentrations of extract samples (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.
- ABTS Radical Scavenging Activity
ABTS •+ was dissolved in methanol to a final 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 [3] . This scavenging activity (%) was expressed as a percentage using the following formula: ABTS radical scavenging activity (%) = (1 – absorbance of sample/absorbance of control) × 100.
- FRAP Assays
The FRAP assay developed by Choi et al . [3] was used. Briefly, 1.5 ml of working FRAP reagent pre-warmed to 37℃ (300 mM acetate buffer (pH 3.6):10 mM TPTZ in 40 mM HCl:20 mM FeCl3 = 10:1:1 (v/v/v)) was mixed with 50 μl of the test samples and standards. This mixture was vortexed, and the absorbance at 593 nm was read against a reagent blank at a predetermined time after the sample-reagent mixing. The test was performed at 37℃, and a 0-4 min reaction time window was used.
- Statistical Analysis
All values are means of determinations in three independent experiments. Differences in the means of each value were determined by one-way ANOVA followed by the Tukey’s multiple range tests at p < 0.05 using the Statistical Analysis System software ver. 9.0 (SAS Institute, Cary, NC, USA).
Results and Discussion
- Change in the Viable Cell Numbers and β-Glucosidase Activity During Cheonggukjang Fermentation
The bacterial cell concentrations of B. amyloliquefaciens MJ1-4 were 10.23, 10.45, 10.77, and 9.97 log CFU/ml after cheonggukjang fermentation with 0%, 5%, 10%, and 20% garlic at 37℃ for 72 h, respectively (data not shown). The bacterial population and β-glucosidase activity during CFWOG and CFWG are shown in Table 1 . The viable cell numbers and β-glucosidase activity increased with fermentation. As the results show, the viable cell numbers of bacteria in CFWOG ranged from 7.19 to 10.59 CFU/ml, while the β-glucosidase activity increased greatly to a maximum of 24.8 unit/g after 48 h and then decreased gradually during fermentation. On the other hand, the viable cell numbers of bacteria in the CFWG ranged from 7.11 to 12.52 CFU/ml, while the β-glucosidase activity increased greatly to a maximum of 24.2 unit/g after 72 h during fermentation ( Table 1 ). Bacillus species are known to be responsible for producing cheonggukjang [1 , 2] . Several studies reported that the viable cell numbers of Bacillus spp. increased depending on the increased β-glucosidase activities during cheonggukjang fermentation [1 , 2 , 32] .
Change of viable cell numbers and β-glucosidase activity duringcheonggukjangfermentation without and with garlic byBacillus amyloliquefaciensMJ1-4 starter.
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aCFWOG : Cheonggukjang fermented without garlic. bCFWG : Cheonggukjang fermented with 10% garlic. cData are presented as the mean ± SD (n = 3) of triplicate determinations. 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). dOne unit of β-glucosidase activity was defined as the amount of enzyme that liberated 1 μM of p-NPG.
- Change of Total Phenolic and Isoflavone Contents During Cheonggukjang Fermentation
The change in the total phenolic contents and isoflavone-glycoside, -malonylglycoside, and -aglycone rates during CFWOG and CFWG are shown in Figs. 1 and 2 , respectively. The CFWOG and CFWG at 37℃ for 0, 12, 24, 48, and 72 h showed 5.05 (CFWOG) and 5.49 (CFWG) mg GAE/g, 6.39 and 5.98 mg GAE/g, 10.12 and 9.64 mg GAE/g, 12.67 and 13.52 mg GAE/g, and 13.58 and 15.18 mg GAE/g dry weight, respectively ( Fig. 1 ). Phenolic compounds are known to possess antioxidant properties [3] . The phenolics are secondary plant metabolites that are present in all plants. Phenolics are usually found in conjugated forms through hydroxyl groups with sugars and glycosides in plant materials [25] . Catalyzing the release of the total phenolic contents from the soybean substrate during fermentation may thus lead to an increase in the content of those compounds, as shown in Fig. 1 . Similar previous studies reported that the total phenolic content increased during soybean fermentation in foods, such as cheonggukjang and natto [1 , 2 , 8 , 28] . Few studies have examined the phenolic acid groups in soybean and soybean-based products, and those studies were carried out from a physiological point of view [1 , 2 , 10] . Gallic, protocatechuic, p -coumaric, caffeic, chlorogenic, gentisic, p -hydroxylbenzoic, vanillic, and ferulic acids are the main components of the soybean phenolic acids, and they possess strong antioxidant activity [1 , 2 , 10 , 24 , 26] . In addition, Kim et al . [12] reported that gallic, p -coumaric, o -coumaric, m -coumaric, caffeic, and ferulic acids are the components of the garlic phenolic acids.
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Change of the total phenolic contents during cheonggukjang fermentation with Bacillus amyloliquefaciens MJ1-4 starter. Data are presented as the mean ± SD (n = 3) of triplicate determinations. Means with different lowercase letters (a, b, and c) indicate significant differences of fermentation times by Tukey’s multiple range test (p < 0.05).
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Change of β-glycoside, malonyl-β-glycoside, acetyl-β-glycoside, and aglycone rates during cheonggukjang fermentation without and with garlic by Bacillus amyloliquefaciens MJ1-4 starter. (A) Cheonggukjang without garlic. (B) Cheonggukjang with garlic.
In the case of CFWOG, the isoflavone-malonylglycoside, -acetylglycoside, and -aglycone rates increased throughout fermentation to approximately 1.3-, 16.5-, and 3.7-fold relative to their starting amounts at 72 h (22.0%, 9.9%, and 26.9%, respectively), but the isoflavone-glycoside contents decreased from 75.7% to 41.2% at the end of fermentation (72 h) ( Fig. 2 A). During CFWG, the levels of isoflavone-malonylglycoside, -acetylglycoside, and -aglycone increased throughout fermentation to approximately 1.5-, 19.6-, and 3.1-fold relative to their starting amounts at 72 h (15.3%, 11.8%, and 27.9%, respectively), but the isoflavone-glycoside contents decreased from 80.2% to 45.0% at the end of the fermentation time (72 h) ( Fig. 2 B). In particular, daidzin of the glycoside type decreased from 450.0 to 152.1 μg/g, and the corresponding daidzein of the aglycone type increased to a maximum of 264.4 μg/g at 72 h during the fermentation process ( Table 2 ). The isoflavone-glycosides decreased, while the isoflavone-aglycones increased during cheonggukjang fermentation ( Figs. 3 A- 3 D).
Change of 12 isoflavone contents duringcheonggukjangfermentation byBacillus amyloliquefaciensMJ1-4 starter.
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aCFWOG: Cheonggukjang fermented without garlic. bCFWG: Cheonggukjang fermented with 10% garlic. cData are presented as the mean ± SD (n = 3) of triplicate determinations. 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). dtr, trace < 0.002 μg/g. end, not detected.
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Profile of HPLC chromatogram from isoflavone extracts. (A) Cheonggukjang without garlic, by Bacillus amyloliquefaciens MJ1-4 (0 h), (B) Cheonggukjang without garlic, by Bacillus amyloliquefaciens MJ1-4 (72 h), (C) Cheonggukjang with garlic, by Bacillus amyloliquefaciens MJ1-4 (0 h), and (D) Cheonggukjang without garlic, by Bacillus amyloliquefaciens MJ1-4 (72 h). 1, Daidzin; 2, glycitin; 3, genistin; 4, malonyldaidzin; 5, malonylglycitin; 6, acetyldaidzin; 7, malonylgenistin; 8, acetylglycitin; 9, daidzein; 10, glycitein; and 12, genistein.
Generally, thermal conversion of isoflavone-malonylglycoside into the corresponding isoflavone-glycoside is the major change in the isoflavone profile that occurs during the cooking process. Yang et al . [32] reported that thermal cooking of raw-soaked soybeans significantly increased the isoflavone-glycoside level by 57.1% and decreased the isoflavone-malonylglycosides by 57.6%. Additionally, the isoflavone-glycoside and -malonylglycoside contents in raw soybeans were 328 and 2,277 mg/kg, which changed by 1,103 and 139 mg/kg, respectively, during the soaking and steaming process [7] . B. amyloliquefaciens MJ1-4 is capable of producing β-glycosidase in soybeans with and without garlic. The fermentation seemed to cause a significant increase in the amount of isoflavone-aglycones accompanied by a decrease in isoflavone glycosides. The phenomenon of a sharp increase in β-glucosidase activity corresponds well with the marked increase in isoflavone aglycone contents in the fermented cheonggukjang .
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 soybeancontaining foods, such as tofu , douchi , and cheonggukjang , decrease depending on the processing conditions [1 , 2 , 4 , 7 , 23 , 32] . Jang et al . [7] reported that the total isoflavone content in raw soybeans was 2.87 μg/g, and this content decreased by approximately 50% during cooking prior to cheonggukjang fermentation. The total isoflavone content decreased from 1,055 μg/g (0 h) to 870 μg/g (36 h) during cheonggukjang fermentation by B. subtilis [32] . Recently, Cho et al . [2] showed that the total isoflavone content in cheonggukjang fermentation decreased approximately 64% from an initial 2,923.21 to 1,051.59 μg/g after 60 h of fermentation. In this study, the total isoflavone content decreased by approximately 15% after steam and fermentation processing in raw soybeans from 1,182.7 to 1,001.1 μg/g at the end of fermentation (48 h). Yang et al . [32] suggested that cooking soybeans in the presence of water can greatly decrease the total isoflavone content.
In general, most isoflavones in soybean are present in glycoside form, and they are converted into aglycones during fermentation by microbial β-glucosidase activity [1 , 2 , 7 , 22 , 23 , 31 , 32] . Cho et al . [2] recently reported that the levels of isoflavone-aglycones increased, while the β-glucosidase activity and isoflavone-glycosides decreased, during cheonggukjang fermentation by the potential probiotic B. subtilis CS90. In this study, we found that the starter B. amyloliquefaciens MJ1-4 had the effect of increasing the β-glucosidase activity, and the alglycone contents increased over 48 h. In contrast, Yang et al . [32] reported that the addition of B. subtilis had no effect on β-glucosidase activity, and the aglycone contents did not increase during cheonggukjang fermentation. Shon et al . [29] showed that the isoflavone content in the cheonggukjang made of yellow soybeans, large black soybeans, and small black soybeans was 499.15, 1,278.04, and 907.85 μg/g, respectively, and the large black soybeans had higher isoflavone content than yellow soybeans. Kim et al . [13] reported that the isoflavone contents were higher in cheonggukjang of Daepung cultivar than in cheonggukjang of other cultivars, such as Nampung , Sodam , and Cheongja Recently, Hwang et al . [6] reported that total isoflavone contents were higher in cheonggukjang of Seoritae than cheonggukjang of Seomoktae at 0 and 48 h of fermentation. These contrasting results suggest that the isoflavone concentrations and profiles in cheonggukjang vary depending on the thermal processing, soybean cultivar, fermentation period, and microorganism starter.
- Change of Antioxidant Activities During Cheonggukjang Fermentation
The CFWG exhibited stronger antioxidant activities than the CFWOG during the fermentation periods of 0, 48, and 72 h, but the CFWG showed lower antioxidant activities than CFWOG during the fermentation periods of 12 and 24 h ( Fig. 4 ). To examine the hydrogen-donating activity, we performed a DPPH radical scavenging assay. In the CFWOG, the DPPH radical activities of fermented soybean at 37°C for 0, 12, 24, 48, and 72 h were 32.02%, 37.87%, 49.95%, 63.26%, and 62.44%, respectively. In the case of the CFWG, the levels of DPPH radical activity of fermented soybeans at 37℃ for 0, 12, 24, 48, and 72 h were 36.56%, 34.82%, 46.59%, 71.55%, and 82.29%, respectively ( Fig. 4 A).
To determine the hydrogen-donating antioxidants and chain-breaking antioxidants, we measured the ABTS radical scavenging ability of CFWOG and CFWG. The levels of ABTS radical activity in CFWOG increased greatly from 73.41% at 0 h of fermentation to 101.11% at 72 h of fermentation. Additionally, the ABTS radical activities in CFWG increased greatly from 75.28% at 0 h of fermentation to 106.32% at 72 h of fermentation ( Fig. 4 B).
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Change of antioxidant activities during cheonggukjang fermentation by Bacillus amyloliquefaciens MJ1-4 starter. (A) DPPH radical scavenging activity, (B) ABTs radical scavenging activity, and (C) FRAP assay. Data are presented as the mean ± SD (n = 3) of triplicate determinations. Means with different lowercase letters (a, b, and c) indicate significant differences of fermentation times by Tukey’s multiple range test (p < 0.05).
The FRAP assay is a direct test of the total antioxidant power. In the case of CFWOG, the values resulting from the FRAP assay of the fermented soybeans at 37℃ for 0, 12, 24, 48, and 72 h increased by 0.55, 0.67, 0.82, 1.04, and 1.20, respectively. During CFWG, the values resulting from FRAP assay of fermented soybeans at 37℃ for 0, 12, 24, 48, and 72 h increased by 0.59, 0.66, 0.76, 1.31, and 1.47 corresponding to increases of 15.59%, 23.33%, 29.77%, and 31.85%, respectively ( Fig. 4 C).
The total phenolic contents were measured as an overall indicator of the contents of these molecules with antioxidant properties [30] . Shon et al . [28] reported that a methanol extract of cheonggukjang exhibited a radical scavenging activity of 69-87% and total phenolic contents of 0.13- 0.27 mg/g. In contrast, Kao and Chen [9] reported that the DPPH radical scavenging activity was not affected by the various isoflavone standards, and mixtures of two and four isoflavone standards were more effective in scavenging DPPH free radicals (yielding 5.6% and 10.5% scavenging activity, respectively). Similarly, many isoflavones were reported to have low scavenging potency for DPPH free radicals, with scavenging effects only half that of α-tocopherol and one-third that of epicatechin [16] . However, isoflavones have direct free radical quenching ability, with daidzein and genistein being particularly effective [1 , 2 , 28] . In addition, Kim et al . [14] reported that the cheonggukjang extract and its constituents, genistein and daidzein, exhibited significant antioxidant activity in vitro . We recently reported that the radical scavenging activity increased from 53.6% to 93.9% depending on the total phenolic and isoflavone-aglycone (daidzein) contents during cheonggukjang fermentation by the potential probiotic B. subtilis CS90 [2] . In particular, Kwak et al . [15] suggested that the stronger antioxidant activity of cheonggukjnag might be related to the markedly higher total phenolic contents and isoflavoneaglycones and -malonylglycosides achieved during fermentation. In addition, phenolic acids, such as gallic acid and its derivatives, are also reported to have antioxidant and antimutagenic activities, and daily intake is proposed to bring various health benefits, including reducing the risk of disease [9] .
In conclusion, this study has documented for the first time that changes occurred in the total phenolic contents and in the contents of 12 isoflavones during cheonggukjang fermentation by B. amyloliquefaciens MJ1-4 with and without garlic. Among these changes, the total phenolic and isoflavoneaglycone contents were markedly increased, while the isoflavone-glycosides were decreased according to the β-glucosidase activities. The total phenolic and total isoflavone contents and antioxidant activities were higher in CFWOG than in CFWG at 24 h of fermentation, but they were lower in the CFWOG at 72 h of fermentation. These results suggest that the high antioxidant activity of cheonggukjang fermented with garlic might be related to the markedly higher total phenolic and isoflavone-aglycone contents achieved during fermentation.
Acknowledgements
This work was supported by a grant from IPET (High Value-added Food Technology Development Program, 2012, 112066-3), Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.
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