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Radical Scavenging Activities of Tannin Extracted from Amaranth (Amaranthus caudatus L.)
Radical Scavenging Activities of Tannin Extracted from Amaranth (Amaranthus caudatus L.)
Journal of Microbiology and Biotechnology. 2015. Jun, 25(6): 795-802
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : September 29, 2014
  • Accepted : January 22, 2015
  • Published : June 28, 2015
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About the Authors
Hyeon-Ju Jo
Department of Food Science and Technology, Seoul National University of Science & Technology, Seoul 139-743, Republic of Korea
Kang-Hyun Chung
Department of Food Science and Technology, Seoul National University of Science & Technology, Seoul 139-743, Republic of Korea
Jin A Yoon
Department of Food & Nutrition, Baewha Women’s University, Seoul 110-735, Republic of Korea
Kwon-Jai Lee
Department of Advanced Materials Engineering, Daejeon University, Daejeon 300-716, Republic of Korea
Byeong Chun Song
Division of Food Bioscience, Konkuk University, Chungju 380-701, Republic of Korea
Jeung Hee An
Division of Food Bioscience, Konkuk University, Chungju 380-701, Republic of Korea
anjhee@hanmail.net

Abstract
This study investigates the bioactivity of tannin from amaranth ( Amaranthus caudatus L.) extracts. The antioxidant activities of the extracts from amaranth leaves, flowers, and seeds were evaluated. Tannin from leaves of amaranth has been evaluated for superoxide scavenging activity by using DPPH and ABTS + analysis, reducing power, protective effect against H 2 O 2 -induced oxidative damage in L-132 and BNL-CL2 cells, and inhibition of superoxide radical effects on HL-60 cells. At a concentration of 100 µg/ml, tannin showed protective effects and restored cell survival to 69.2% and 41.8% for L-132 and BNL-CL2 cells, respectively. Furthermore, at the same concentration, tannin inhibited 41% of the activity of the superoxide radical on HL-60 cells and 43.4% of the increase in nitric oxide levels in RAW 264.7 cells. The expression levels of the antioxidant-associated protein SOD-1 were significantly increased in a concentration-dependent manner in RAW 264.7 cells treated with tannin from amaranth leaves. These results suggest that tannin from the leaves of Amaranthus caudatus L. is a promising source of antioxidant component that can be used as a food preservative or nutraceutical.
Keywords
Introduction
Tannins are a unique group of water-soluble phenolic metabolites of relatively high molecular weight that have the ability to form strong complexes with carbohydrates and proteins [13] . Tannins are of great interest from the perspective of nutrition and medicine because of their potent antioxidant capacity and possible protective effects on human health [17] . Numerous studies have demonstrated potentially significant biological effects of tannins, including antioxidant and radical scavenging activities as well as inhibition of lipid peroxidation and lipoxygenases in vitro [13] . Tannins have been shown to have antimicrobial and antiviral [13] , antimutagenic [6] , anticarcinogenic [1] , and antidiabetic properties [13] . Moreover, the antioxidant activity of tannins results from their free radical- and reactive oxygen species-scavenging properties, as well as from their ability to chelate transition metal ions that initialize the oxidation process [13] . Interestingly, tannic acid inhibited skin, lung, and forestomach tumors caused by polycyclic aromatic hydrocarbon carcinogens and N -methyl- N -nitrosourea in mice [8] .
Amaranthus is a fast-growing crop cultivated mainly in Latin America, Africa, and Asia [21] . Amaranth ( Amaranthus spp.) seeds contain high-quality protein with an amino acid composition close to ideal protein; therefore, many different protein concentrates of amaranth have been developed [10] . Amaranth flour represents a suitable foodstuff for patients on a gluten-free diet and amaranth consumption has positive effects on plasma lipids [4] . Amaranth oil is useful as a natural antioxidant supplement because of its high content of unsaturated fatty acids and unique presence of squalene [15] . Amaranth is an important source of natural food colorants, betacyanins, which may also be useful natural antioxidants [15] . Amaranth pectin, a gel-forming component that binds toxic and radioactive metals and removes them from the human body, can be utilized in food production [10] . However, the biological activity of tannin from amaranth extract has not been reported.
The aim of this study was to investigate the antioxidant and radical-scavenging properties of tannic acid extracted from amaranth. The antioxidant potential of tannic acid was assessed as the total antioxidant and scavenging activity of free radicals such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino- bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS + ), and by the reducing antioxidant power assay. Tannin was also evaluated for its protective effects against oxidative damage in L-132 and HL-60 cells and for its ability to inhibit nitric oxide generation in RAW 264.7 cells. Thus, newly discovered antioxidant properties of tannins are demonstrated.
Materials and Methods
- Materials
We used red and green leaves, and red and purple flowers from Amaranthus caudatus L. The leaves, flowers, and seeds of amaranth were supplied by Q&A Korea Corporation of Pyeongcang, Korea. Dried material (30 g) was extracted with methanol (300 ml) in a shaking incubator (SI-600R; Lab Companion, Daejeon, Korea) for 24 h at 25℃. The water extracts were boiled for 15 min at 121℃ (1.23 bar) by using an autoclave (SJ-220A100; Sejong Scientific Co., Ltd., Bucheon, Korea).
Red amaranth leaves extracted with methanol (RLM, 3.88 g), green amaranth leaves extracted with methanol (GLM, 3.97 g), red amaranth leaves extracted with hot water (RLHW, 10 g), green amaranth leaves extracted with hot water (GLHW, 9.14 g), red amaranth flower extracted with methanol (RFM, 4.32 g), purple amaranth flower extracted with methanol (PFM, 5.21 g), red amaranth flower extracted with hot water (RFHW, 6.43 g), purple amaranth flower extracted with hot water (PFHW, 7.21 g), seeds extracted with methanol (SM, 9.4 g), and seeds extracted with hot water (SHW, 4.69 g) were frozen, lyophilized, and stored at -70℃ ( Table 1 ).
Yield, percentage yield, and RC50values of ABTS+scavenging activities, DPPH scavenging activities, and reducing power of leaves, flower, and seed extracts from amaranth.
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1) Each value is the mean ± SD of triplicate determinations (n = 3). 2) Means with different letters (a-j) within a column are significantly different at p < 0.05. RLM (red leaves extracted with methanol), GLM (green leaves extracted with methanol), RFM (red flower extracted with methanol), PFM (purple flower extracted with methanol), SM (seeds extracted with methanol), RLHW (red leaves extracted with hot water), GLHW (green leaves extracted with hot water), RFHW (red flower extracted with hot water), PFHW (purple flower extracted with hot water), SHW (seeds extracted with hot water).
A human lung epithelial cell line (L-132), a human promyelocytic leukemia cell line (HL-60), an embryonic murine hepatocyte cell line (BNL-CL2), and a macrophage cell line (RAW 264.7) were purchased from the Korea Cell Line Bank (Seoul, Korea). L-132, BNL-CL2, and RAW 264.7 cells were grown in Dulbecco’s minimum Eagle’s medium (DMEM; Welgene, Daegu, Korea), and HL-60 cells were grown in RPMI 1640 (Welgene) culture medium. Media were supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin (GIBCO, Grand Island, NY, USA). The cells were incubated in a humidified atmosphere with 5% CO 2 at 37℃.
- Free Radical Scavenging Activity
ABTS + radical scavenging measurements were obtained according to the method described by Re et al. [19] with modifications. DPPH radical scavenging measurements were obtained according to the method described by Blois [2] with modifications. The reducing power of tannic acid was determined by the method described by Oyaizu [18] with slight modification.
- Determination of Total Phenolic and Flavonoid Contents
Total phenolic content was determined colorimetrically using Folin–Ciocalteau reagent, as described previously [9] . Total flavonoid content was calculated by the AlCl 3 colorimetric method [23] .
- Identification and Purification of Tannin
The extract was combined with 5.0 ml of Folin–Denis reagent and Na 2 CO 3 solution in a volume of 100 ml and mixed well, and after 30 min, absorbance was read at 725 nm using a spectrophotometer (Biochrom). Tannic acid (Sigma-Aldrich Co.) was used for preparing the standard curve. Total tannin content is expressed as mg tannic acid equivalents (TAE, dry basis)/g of sample. High-performance liquid chromatography (HPLC) was performed with a reversed-phase column (SunFire C18, 4.6 × 250 mm, 5-µm diameter; Waters, Milford, MA, USA) and analyzed with HPLC Empower Software (Waters). The mobile phase was water–acetonitrile 80/20% (v/v). The flow rate was 1 ml/min, and the injection volume was 5 µl. The chromatograms were detected at 230 nm and collected at 30℃. Tannic acid was purchased from Sigma-Aldrich and used as a standard.
- Protective Effect Against H2O2-Induced Oxidative Damage
The effects of tannin on cancer cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Genetrone, Seoul, Korea) assay.
- Superoxide Anion Radical (O2-) Generation and Nitric Oxide (NO) Assay
For this assay, superoxide anion inhibition was measured in 12- O -tetradecanoyl-phorbol-13-acetate (TPA)-induced cultured HL-60 cells [11] . RAW 264.7 cells (1 × 10 6 cells/well) were seeded in 96-well plates and incubated at 37℃ for 24 h [7] . Cell viability was assessed by the MTT assay.
- Western Blot Analysis
Cells were lysed in ice-cold lysis buffer (RIPA, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na 2 EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na 3 VO 4 , 1 µg/ml leupeptin, and 1 mM PMSF). The membranes were then incubated with SOD-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and β-actin (Cell Signaling Technology, Beverly, MA, USA) antibodies, followed by the secondary antibody, goat anti-rabbit IgG (H+L) HRP conjugate (Zymax, San Francisco, CA, USA). The blots were detected by chemiluminescence using X-ray film (AGFA, Mortsel, Belgium).
- Statistical Analyses
All determinations were performed in triplicate. Data are expressed as means ± standard deviation (SD). The data were subjected to an analysis of variance (ANOVA, p < 0.05) and Duncan’s multiple range test (SPSS 18; SPSS Inc., Chicago, IL, USA).
Results
- Free Radical Scavenging Activity of Extracts from Amaranth
Table 1 shows the extraction yield, percentage yield, ABTS + scavenging activities, DPPH scavenging activities, and reducing power of leaves, flower, and seed extracts from amaranth. The hot water extracts (RLHW, GLHW, RFHW, and PFHW) of leaves and flowers showed a higher extraction yield in comparison with that of methanol extracts (RLM, GLM, RFM, and PFM) of leaves and flowers. However, the yield of SM was higher than that of SHW. The amaranth extracts had significant ABTS + radical scavenging effects. The RC 50 (50% reduction concentration) of the extracts RLM, PFM, RLHW, and PFHW was 166.91, 195.66, 303.39, and 321.72 µg/ml, respectively. The results of the DPPH radical scavenging assay showed that extracts from amaranth leaves, flowers, and seeds have antioxidant activity. Although only slight antioxidant effects of the other extracts were found, RLM (RC 50 = 189.90 µg/ml) and PFM (RC 50 = 155.06 µg/ml) showed significant antioxidant activity. The extract from amaranth exhibited potent reducing ability. The methanol extracts from amaranth leaves had a more powerful reducing power of 1.14 ± 0.263 (RLM) and 0.868 ± 0.037 (GLM) than the other extracts (0.1~0.63) at a 1 mg/ml concentration. These studies indicate that RLM is the most powerful extract among the extracts from the leaves, flowers, and seeds of amaranth. The ABTS + , DPPH, and radical scavenging of GLM, RFM, SM, GLHW, RFHW, and SHW were found to be slight (>1000 µg/ml). The activity of purified tannin-ABTS + radical scavenging was evaluated as 87.1%, and the purified tannin from amaranth had a powerful effect compared with RLM (42.9%) at a concentration of 100 µg/ml ( Fig. 1 A). The DPPH radical scavenging activities of purified tannin and RLM were 66.3% and 31.3% at the concentration of 100 µg/ml, respectively ( Fig. 1 B). Despite the 10-fold concentration between purified tannin and RLM, the reducing power showed similar activities ( Fig. 1 C). These results revealed that purified tannin from leaves of amaranth has stronger radical scavenging and reducing power activities than that of RLM. The total phenol, flavonoid, and tanin contents of the extraxts from amaranth are presented in the Table 2 . Interestingly, the RLM fraction showed the highest tannin content. Thus, RLM fractions were purified by HPLC for further analysis.
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Free radical scavenging activity of amaranth extracts. (A) ABTS+ scavenging activities of RLM (100 mg/ml) and tannin (100 mg/ml) from amaranth. (B) DPPH scavenging activities of RLM (100 mg/ml) and tannin (100 mg/ml) from amaranth. (C) Reducing power of RLM (1 mg/ml) and tannin (100 mg/ml) from amaranth. (D) HPLC profiles of RLM derived from amaranth and tannin. RLM: red leaves extracted with methanol and purified with methanol:acetonitrile (8:2) solution by HPLC analysis. (E) Chemical structure of tannin. RLM: red leaves extracted with MeOH. Each value is the mean ± SD (n = 3).
Comparison of total polyphenol and flavonoid contents of RLM from amaranth flowers.
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1) Total phenolic content was expressed as mg/g gallic acid equivalent (GAE). 2) Total flavonoid content was expressed as mg/g catechin equivalent (CE). 3) Tannic acid content was expressed as mg/g tannic acid equivalent (TAE). 4) Each value is the mean ± SD of triplicate determinations (n = 3). 5) Means with different letters (a-j) within a column are significantly different at p < 0.05. RLM (red leaves extracted with methanol), GLM (green leaves extracted with methanol), RFM (red flower extracted with methanol), PFM (purple flower extracted with methanol), SM (seeds extracted with methanol), RLHW (red leaves extracted with hot water), GLHW (green leaves extracted with hot water), RFHW (red flower extracted with hot water), PFHW (purple flower extracted with hot water), SHW (seeds extracted with hot water).
- Total Phenol, Flavonoid, and Tannin Content of Extracts from Amaranth
The total phenol, flavonoid, and tannin contents of the extracts from amaranth are presented in the Table 2 . The total phenol content of the hot water extracts, RLHW and GLHW, were found to be equivalent to 6.82 ± 0.84 and 6.43 ± 0.52 mg of gallic acid, respectively, higher than that of the other extracts (1.69 ± 0.12~6.07± 0.64 mg GAE/g). GLM exhibited the highest total flavonoid content at 3.57± 0.17mg CE/g, followed by RLM (2.88 ± 0.27mg CE/g), PFM (2.55 ± 0.16 mg CE/g), and RFM (1.71 ± 0.24 mg CE/g). Interestingly, the total phenol content of the hot water extracts of leaves was higher than the other extracts; however, the flavonoid content of the methanol extracts of leaves was higher than that of the hot water extracts. The tannin contents of RLM, GLM, RLHW, and GLHW were 0.827± 0.02, 0.516 ± 0.02, 0.526 ± 0.01, and 0.579 ± 0.00 mg tannic acid equivalent/g, respectively. Interestingly, the RLM fraction showed the highest tannin content. Thus, RLM fractions were purified by HPLC for further analysis.
- Identification and Purification of Tannin in the RLM Extract
The HPLC fingerprint of the RLM from amaranth had a profile representing the triterpene constituents, with tannic acid as a major component, identified by comparison with the standard ( Fig. 1 D).
- Protective Effect of Tannin Against H2O2-Induced Oxidative Damage of Normal Cells
The cell death caused by H 2 O 2 treatment was significantly inhibited by the addition of amaranth extracts ( Fig. 2 ). Cytotoxicity of tannin from RLM was not shown at a concentration of 25-100 µg/ml. The cell viability after H 2 O 2 treatment of L-132 cells decreased to 14.9% and BNLCL2 cells to 17.3%. H 2 O 2 has been extensively used as an inducer of oxidative stress in L-132 and BNL-CL2 cells. It readily crosses the cellular membrane, giving rise to the highly reactive hydroxyl radical that then reacts with macromolecules, including DNA, proteins, and lipids, and ultimately damages the cell [12 , 22] . Cell viability was significantly increased by the addition of tannin. At a concentration of 100 µg/ml, the tannin increased cell viability to 69.2% for L-132 cells and 41.8% for BNL-CL2 cells after H 2 O 2 treatment. Therefore, it is possible that the protective effect of tannin against H 2 O 2 -induced cell death is due to a direct reaction of tannin with H 2 O 2 , or with free radicals derived from H 2 O 2 , before they could react with the cells.
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Protective effects of the tannin from amaranth on cell viability against H2O2 -induced oxidative damage in L-132 (A) and BNL-CL2 cells (B). Cell viability was measured by MTT assay. Cells were incubated for 24 h before the addition of H2O2. Oxidative damage was induced with 2 mM H2O2 for 30 min. PC: positive control; NC: negative control. *p-value of <0.05 was considered statistically significant compared with the control group. Each value is the mean ± SD (n = 3).
- Superoxide Anion Radical Inhibitory Activity and Nitric Oxide Inhibiting Activity of Tannin
Tannins of RLM were tested for their inhibitory effects on tumor promoter TPA-induced O 2 - generation in human promyelocytic HL-60 cells being differentiated into granulocyte-like cells by treatment with 1.25% DMSO for 6 days. The extracellular O 2 - production was detected by measuring the levels of the reduced form of cytochrome c . As shown in Fig. 3 A, cytotoxicity of tannin was not observed at a concentration of 25-100 µg/ml. The tannin of RLM inhibited cytochrome c reduction by 0.1% and 41% at concentrations of 25 and 100 µg/ml, respectively, after stimulation with TPA. The present study showed for the first time that tannin is a potential phytochemical, showing not only free radical scavenging activity, but also suppression of O 2 - generation from inflammatory leukocytes.
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Inhibitory effects of tannin on superoxide anion radical generation and nitric oxide production. (A) Inhibitory effect of the tannin (25, 50, and 100 µg/ml) from amaranth on TPA-induced O2- generation in differentiated HL-60 cells. PC: positive control; NC: negative control. LPS: cells treated with LPS (20 µg/ml), tetrahydrobiopterin (10 µg/ml), 200 mM L-arginine, and IFN-γ (100 U/ml). (B) Inhibitory effects of the tannin (25, 50, and 100 µg/ml) from amaranth on nitric oxide production in RAW 264.7 macrophages cells. *p-value of <0.05 was considered statistically significant compared with the untreated control group. Each value is the mean ± SD (n = 3).
To determine the effects of tannin from RLM on NO production, different concentrations of tannin (25-100 µg/ml) were incubated with LPS/IFN -activated RAW 264.7 macrophages. As shown in Fig. 3 B, no cytotoxicity of tannin alone was shown at a concentration of 25-100 µg/ml. The effects of tannin on NO generation were quite variable (inhibitory rates ranged from 8.7% to 43.3%).
- Expression of SOD-1 Protein by Tannin
We examined the effect of tannin on Cu/Zn superoxide dismutase (SOD-1) protein expression ( Fig. 4 ). Western blots were analyzed by calculating the relative density of the immunoreactive bands using Image J software. The relative densities of the bands were normalized to β-actin bands (arbitrarily set at 1) and expressed as the fold change as compared with β-actin. In RAW 264.7 cells, tannin (25-100 µg/ml) induced a dose-dependent increase in SOD-1 protein expression levels (1.38-1.64), as demonstrated by western blot analysis. Specifically, tannin induced a 1.64-fold increase in the expression of SOD-1 over control at a concentration of 100 µg/ml ( Fig. 4 B).
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Dose-dependent effect of the tannin on the expression of antioxidant protein in RAW 264.7 cells. Cells were treated with 25, 50, and 100 µg/ml tannin for 24 h. (A) The expression of SOD-1 was determined by western blot analysis. Equal loading of protein was confirmed by β-actin quantification. (B) For each protein, the relative density was measured and normalized to β-actin bands (arbitrarily set at 1). *denotes a statistically significant difference (p < 0.05) from the control group.
Discussion
Tannin, flavonoids, and phenolic acids are the most predominant components of amaranth. These compounds have radical scavenging and antioxidant activities. Amaranth extracts have phenolic hydroxyl groups in their structures. Antioxidants of phenolic groups have been recognized to function as electron or hydrogen donors [20] . The role of antioxidants has attracted much interest with respect to their protective effect against free radical damage that may be the cause of many diseases, including cancer [5] . The antioxidant effect of amaranth extract is due mainly to the tannin components. Some tannin compounds have been reported to also show alkylperoxyl radical scavenging activity, thus reducing radical-mediated pathogenesis, such as carcinogenesis. The present study showed for the first time that tannin is a potential phytochemical showing not only free radical scavenging ability, but also suppressing O 2 - and NO generation from inflammatory leukocytes, including neutrophils and macrophages. The free radical scavenging activity and the powerful inhibition activity of nitric oxide in RAW 264.7 cells were observed with tannin from amaranth. NO has diverse physiological roles and contributes to the immune defense against viruses, bacteria, and other parasites [3 , 10] . The inhibitory activity of tannin was 43.77% at a 100 µg/ml concentration. This study shows that tannin from extracts of the leaves of amaranth has a more powerful effect than the extracts of Amaranthus caudatus L. seeds, Strychnos ignatii Semen , and Glycyrrhizae Radix to inhibit NO generation. Extracellular O 2 - production was detected by measuring the levels of the reduced form of cytochrome c . Our results demonstrate that the tannin from amaranth shows powerful protective effects against oxidative stress, inhibitory effects on TPAinduced O 2 - generation in human promyelocytic HL-60 cells, and inhibition effect of NO production. Our results suggest that the antioxidant activity of tannin is associated with its free radical inhibitory activity in oxidative damaged cells. Tannin has a powerful effect to inhibit O 2 - generation, comparable to that of ascorbic acid, a wellknown water-soluble radical scavenger [16] .
We determined the effect of tannin on the level of SOD-1 protein by western blotting to gain insight into the mechanism of the inhibitory effect of tannin on inflammation in RAW 264.7 cells. As can be seen in Fig. 4 , treatment of the cells with tannin increased the level of SOD-1. SOD-1 is a key enzyme in the dismutation of superoxide radicals resulting from cellular oxidative metabolism into hydrogen peroxide [14] . Because inflammation is characterized by macrophage activation, we examined the possibility that altered SOD-1 activity would affect the inflammatory process. We speculated that up-regulation of SOD-1 would increase the ability of macrophages to confront an increased level of ROS during inflammation. In contrast, inhibition of SOD-1 would diminish this ability, resulting in inhibition of the immune response. Here, we have demonstrated that the tannin-induced inhibitory effect of inflammation in RAW 264.7 cells was caused by increasing SOD-1 protein. These results indicate that tannin is an active component of the leaves of Amaranthus caudatus L. that contributes to its antioxidant activities. In conclusion, this study has demonstrated that extracts from the leaves, flowers, and seeds of amaranth have antioxidant activity. The extracts showed DPPH and ABTS + radical-scavenging effects and reducing power. In L-132 and BNL-CL2 cells, tannin extracted from red leaves showed a strong, concentrationdependent protective effect against oxidative stress. A powerful inhibition of nitric oxide production in RAW 264.7 cells was observed with tannin from RLM. The tannin from RLM showed high flavonoid and tannin content and more powerful protective effects against oxidative stress, inhibitory effects on TPA-induced O 2 - generation in human promyelocytic HL-60 cells, and inhibition of NO production. Tannin from amaranth possesses antioxidative activity and could be a potential antitumor agent.
Acknowledgements
This research was supported by iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries), the High Value-added Food Technology Development Program (112123-3), and the Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea and by High Value-added Food Technology Development Program (314040-03-1-HD030) Ministry of Agriculture, Food and Rural Affairs (MAFRA).
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