Abstract
This study was executed to develop probiotics producing S-adenosyl-L-methionine (SAMe), a methyl group donor in the 5-methyltetrahydrofolate methylation reaction in animal cells. SAMe is an essential substance in the synthesis, activation, and metabolism of hormones, neurotransmitters, nucleic acids, phospholipids, and cell membranes of animals. SAMe is also known as a nutritional supplement for improving human brain function. In this study, SAMe-producing strains were identified in six kinds of Cheonggukjang, and strains with excellent SAMe production were identified, with one strain in the Enterococcus genus and six strains in the Bacillus genus. Strains with a large amount of SAMe production included lactic acid bacteria, such as Enterococcus faecium, Enterococcus durans, and Enterococcus sanguinicola, as well as various strains in the Bacillus genus. The SAMe-overproducing strains showed antibacterial activity against some harmful microbes, in addition to weak acid resistance and strong bile resistance, indicating characteristics of probiotics. Cheonggukjang-originated beneficial bacterial strains overproducing SAMe may be commercially useful for manufacturing SAMe-rich foods.
Key Words: : Cheonggukjang, probiotics, S-adenosyl-L-methionine
Introduction
S-adenosyl-L-methionine (SAMe) was first discovered by Cantoni in 1952. SAMe is a substance primarily present in tissues and body fluids of animals, and it plays an important role in the 5-methyltetrahydrofloate (5-MTHF) methylation reaction as a methyl group donor.1 SAMe is an important bioregulator made from essential amino acids, L-methionine, and ATP by methionine adenosyltransferase.2 It is mostly synthesized in the liver at a rate of about 8 g a day and is involved in many biochemical metabolism processes.3
Many studies have confirmed that SAMe content varies depending on the content of L-methionine in foods.4 Its functionality has been recognized in western societies, including Europe and the USA, and SAMe has been reported to be an essential substance in processes, including in polyamine synthesis in cells and the synthesis, activation, and metabolism of hormones, neurotransmitters, nucleic acids, phospholipids, and cell membranes.5
Also, it is required in the production of important brain compounds such as neurotransmitters and phospholipids, including phosphatidylcholine and phosphatidylserine, and is known to be effective in improving brain functions.6 The metabolic reactions of SAMe in the body include transmethylation, transsulfuration, and polyamine.7 In the methylation reaction, the methyl group of SAMe is donated to various acceptor substrates such as DNA, phospholipids, and proteins; in the transsulfuration reaction, SAMe is converted to taurine, a major antioxidant in cells, and cysteine, a precursor of glutathione, through several enzymatic reactions.8
Finally, SAMe is used in the synthesis of polyamines that are essential to the growth of normal cells.9 It also donates the methyl group to the propolyamine group in the synthesis of spermine and spermidine.10 It has been reported that SAMe has pharmacological effects in the restoration of hepatic functions in alcoholic liver disease, in reducing muscle fatigue and rigidity in patients with fibromyalgia syndrome, and in improvement in patients with depression with about a 66% clinical improvement.11,12 The pharmacological effects of SAMe appear to be due to the metabolic reactions of SAMe in the body, including transmethylation, transsulfuration, and polyamine.6
SAMe is also converted to taurine (a major antioxidant in cells) and cysteine (a precursor of glutathione, another well-known antioxidant in cells) through several enzymatic steps of the transsulfuration reaction.6 This reaction can remove reactive oxygen species that are detrimental to the body. SAMe is used in the synthesis of polyamines that are essential to the growth of normal cells6
Up to now, studies on SAMe primarily reported its effects in the treatment of depression, arthritis, and hepatic cirrhosis, and it has been recognized as a healthful functional food.13 Foods containing SAMe include Cheonggukjang, one of our traditional fermented foods. Thus, this study was performed to isolate Cheonggukjang-originated strains that produce physiologically functional material, SAMe, select and identify strains that produce large amounts of SAMe in foods, and find strains producing SAMe with probiotics through antibacterial activity to function against harmful microbes such as food poisoning bacteria.
Materials and Methods
Cheonggukjang sample preparation
Naturally existing strains in six kinds of Cheonggukjang were isolated and their colonies were analyzed to select strains that improve SAMe production. Cheonggukjang samples used in the study were purchased in September, 2012. A various type of Chenoggukjang samples were purchased and collected to isolation of their bacterial strains, which were mixed in a different ratio of ingredients as C1 (soybean 95%, salt 5%; Sunchang-gun, Korea), C2 (soybean 70%, water 28%, salt 2%; Sunchang-gun, Korea), C3 (soybean 98%, salt 2%; Cheongsong-gun, Korea), C4 (soybean 100%; Changwon-si, Korea), C5 (soybean 95%, salt 5%; Gwangju-si, Korea), and C6 (soybean 97%, salt 2%, red pepper powder 1%; Anseong-si, Korea).
Characteristics of selected strains
Cheonggukjang samples were diluted 10 times (0.85% NaCl 225 mL+sample 25 g) under a sterilized environment and then homogenized using a stomacher (speed level 5, 1 min). Then, the homogenate was smeared on each selective medium and incubated to separate total microbes, lactic acid bacteria, anaerobic bacteria, yeast, and mold. Microbial separation and collection were performed depending on the colony types of microbes produced. Microbes grown in different selective media were identified. The separation of microbes used tryptic soy agar (TSA) for analysis of the number of total microbes. In addition, for the analysis of total lactic acid bacteria, MRS agar was adjusted to pH 5.5. For a detailed analysis of the lactic acid bacteria, m-LBS (for Lactobacillus species), KF-Streptococcus (for Enterococcus and Pediococcus species), and phenylethyl alcohol with 2% sucrose agar (PES, for Leuconostoc species) were used.14,15 Also, potato dextrose agar with an adjusted pH using tartaric acid was used for the analysis of the yeast and mold.
Culture of strains isolated from Cheonggukjang
To examine SAMe production ability, strains were inoculated onto a nutrient medium TSB, incubated at 35°C for 24–48 h to maintain 108–109 CFU/mL, and centrifuged (3,000 g, 4°C, 10 min) to eliminate microbial cells and collect the supernatant for analysis.
SAMe production
The production of SAMe was analyzed using high-performance liquid chromatography (HPLC).16,17 Prepared samples were filtered using a 0.45 μm syringe filter and stored at −20°C for analysis. The resultant solution was analyzed using the HPLC column (C18-4.6×205 nm, 5 μm; Shiseido, Tokyo, Japan). The mobile phases were 100% methanol and 0.25 M ammonium acetate (pH 5.5) with acetic acid at a flow rate of 1.5 mL/min, an oven temperature of 40°C. An injection volume was 20 μL with UV detection (UV-2075; Jasco, Tokyo, Japan) at 210 nm. The concentration of SAMe was measured based on peak areas. The standard for SAMe was purchased from Sigma-Aldrich (St. Louis, MO, USA), and the ammonium acetate and methanol used in the analysis and all other reagents were purchased for HPLC use.
Analysis of sugar fermentation by isolated strains
Among the isolated strains, the Enterococcus genus strains used an API 20 Strep system kit (bioMérieux, Marcy l'Etoile, France) and the Bacillus genus strains used an API 50 CHB system kit (bioMérieux) for the analysis of the sugar fermentation of the strains. According to the API kit manual, diluted strains from Cheonggukjang samples were transferred to each selective medium and incubated at 37°C for 24–48 h to identify the presence/absence of various sugar fermentations.
Acid resistance and bile resistance of strains
For the resistance to pH, strains were activated in TSB (108–109 CFU/mL), washed with PBS or 0.85% NaCl, and then centrifuged. After TSA medium preparation with pH levels of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, and 5.5 (control pH 7.0—medium pH), strain samples were spotted on TSA, and changes of the colony after 18–24 h were measured. Similarly, for the bile resistance of strains, the resistance of each strain was measured in the medium with or without Oxgall. After strain activation on TSB (108–109 CFU/mL), samples were washed with PBS or 0.85% NaCl (centrifuge use), 1% of the washed strain was inoculated onto the medium with or without 0.3% Oxgall and spotted on TSA, and the changes of the colony were measured after 18–24 h.
Antibacterial activity of strains
The activity of strains that inhibited five of the indicators, including Escherichia coli O157:H7, Enterococcus faecalis, Salmonella choleraesuis, Staphylococcus aureus, and Listeria monocytogenes, were measured. Similarly, in the soft agar method, strains were activated and spotted on TSA and incubated for 12 h in an incubator. Then, 10 mL of soft agar with 0.75% agar concentration was prepared and indicators were added to the agar at 50°C to reach 105 CFU/mL before it was hardened. It was evenly spotted onto TSA with grown strains, and the circle around the colony was observed every 4 h.
Identification of isolated strains by 16S rRNA sequence analysis
Strains with a higher production of SAMe were identified by Macrogen, Inc. (Seoul, Korea). Genomic DNA was extracted from each strain and was used for the PCR amplification of 16S rRNA gene. We performed PCR using two primers, 27F (forward primer, 5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (reverse primer, 5′-TACGGYTACCTTGTTACGACTT-3′), for 16S rRNA sequencing.18
Results
Isolation of Cheonggukjang-originated colonies
Colonies were analyzed from six kinds of Cheonggukjang. Microbes were grouped by shape, color, and characteristics and a total of 36 different colonies were classified. Six strains producing the highest SAMe for each type of Cheonggukjang were selected as the experimental group. The Bacillus genus, which is known as the major fermentation strain in Cheonggukjang, was isolated using a method for all the microbes with TSA medium; although yeast and molds, in addition to lactic acid bacteria, were separated using a selective medium. In the Cheonggukjang, most of the dominant microbes were in the Bacillus genus and the Enterococcus genus. The amount of SAMe production was measured in the 36 strains isolated from the 6 kinds of Cheonggukjang as shown in Table 1. As a result, a high level of SAMe-producing strain C4 isolated from Cheonggukjang produced 2.23% of SAMe. Two C4-producing stains produced the most SAMe (Table 1). To utilize these strains in future studies, it was considered necessary to examine the characteristics and identification of these 36 strains and thus, these strains were sent to Macrogen Inc. for the determination of 16S rRNA sequencing and identification through NCBI's blast search, as shown in Table 1. Population sizes of strains producing high levels of SAMe were greater than the other strains.
Table 1.
SAMe Production and Identification of Strains Isolated from Cheonggukjang
| Name | Isolation medium | SAMe production (mM) | Population size | Identification result |
|---|---|---|---|---|
| C1-1 | TSA | 0.31 | 2.1×103 | Bacillus licheniformis |
| C1-2 | PES | 0.27 | 2.7×102 | Bacillus subtilis |
| C1-3 | PES | 0.46 | 4.1×104 | Bacillus subtilis |
| C1-4 | mLBS | 0.55 | 1.6×102 | Bacillus licheniformis |
| C1-5* | MRS | 0.63 | 1.7×105 | Enterococcus faecium |
| C1-6 | KF | 0.19 | 2.3×102 | Enterococcus faecium |
| C2-1 | TSA | 0.63 | 3.5×106 | Bacillus licheniformis |
| C2-2 | TSA | 0.38 | 1.2×104 | Bacillus licheniformis |
| C2-3 | PES | 0.40 | 1.9×103 | Bacillus cytotoxicus |
| C2-4 | mLBS | 0.21 | 3.2×104 | Bacillus subtilis |
| C2-5 | MRS | 0.32 | 8.1×103 | Bacillus subtilis |
| C2-6 | KF | 0.39 | 7.7×104 | Bacillus licheniformis |
| C3-1 | TSA | 0.48 | 9.6×102 | Bacillus licheniformis |
| C3-2 | PES | 0.66 | 4.7×105 | Bacillus amyloliquefaciens subsp. plantarum |
| C3-3 | PES | 0.59 | 3.4×104 | Bacillus subtilis |
| C3-4 | mLBS | 0.21 | 5.7×103 | Bacillus subtilis |
| C3-5 | mLBS | 0.36 | 9.7×104 | Enterococcus faecium |
| C3-6 | MRS | 0.19 | 4.2×103 | Bacillus subtilis |
| C4-1 | TSA | 1.575 | 1.3×105 | Bacillus amyloliquefaciens |
| C4-2 | TSA | 0.48 | 3.4×103 | Bacillus amyloliquefaciens |
| C4-3 | PES | 2.23 | 6.0×104 | Bacillus subtilis |
| C4-4 | mLBS | 0.36 | 1.4×104 | Bacillus amyloliquefaciens |
| C4-5 | KF | 0.39 | 3.7×103 | Bacillus sp. |
| C4-6 | KF | 0.34 | 4.4×104 | Pediococcus acidilactici |
| C5-1 | TSA | 0.83 | 2.2×106 | Bacillus subtilis |
| C5-2 | TSA | 0.29 | 8.9×104 | Bacillus subtilis |
| C5-3 | PES | 0.49 | 9.5×104 | Enterococcus faecium |
| C5-4 | MRS | 0.35 | 7.2×103 | Bacillus subtilis |
| C5-5 | MRS | 0.49 | 2.4×104 | Bacillus amyloliquefaciens |
| C5-6 | KF | 0.38 | 2.3×103 | Bscillus subtilis |
| C6-1 | TSA | 0.51 | 2.4×102 | Bacillus subtilis |
| C6-2 | TSA | 0.34 | 4.3×104 | Bacillus amyloliquefaciens |
| C6-3 | PES | 0.66 | 2.5×104 | Bacillus licheniformis |
| C6-4 | mLBS | 0.86 | 1.9×106 | Bacillus licheniformis |
| C6-5 | mLBS | 0.40 | 3.0×104 | Bacillus subtilis |
| C6-6 | KF | 0.25 | 2.4×104 | Enterococcus faecium |
Top seven samples shown in bold.
KF, KF-streptococcus agar; mLBS, modify-lactobacillus selective agar; MRS, modify-rogesa agar; PES, phenylethyl alcohol with 2% sucrose agar; SAMe, S-adenosyl-L-methionine; TSA, tryptic soy agar.
Sugar fermentation of strains
Substrate utilization is one of the important characteristics of microbes. Information regarding sugar utilization can be useful when we introduce SAMe-producing microbial strains into food. It is also known that substrate utilization is important for producing antibacterial agents. For the six strains identified as Bacillus genera, an experiment was performed using an API 50 CHB system kit and for the C1-5 strain identified as Enterococcus genus, the sugar fermentation was measured using an API 20 Strep kit (Table 2). As a result, the Enterococcus faecium genus strain exhibited sugar fermentation ability with ribose, lactose, trehalose, and raffinose, among 10 carbohydrates in the API 20 Strep kit. Each of the remaining six Cheonggukjang-originated strains in the Bacillus genus had different substrate preferences for fermentation depending on the strain, but they also showed strong fermentation in substrates such as glucose, fructose, mannose, glycerol, sucrose, trehalose, maltose, and N-acetyl-glucosamine. Although the difference among fermentable substrates was not great for each strain, it is considered that these strains seem to have enzymes that use many sugars and can ferment various sugars available in the body through food intake.
Table 2.
Sugar Fermentation Characteristics of Strains with Excellent SAMe Production
| Carbohydrate | C1-5 | C2-1 | C3-2 | C4-1 | C4-3 | C5-1 | C6-4 |
|---|---|---|---|---|---|---|---|
| Control | − | − | − | − | − | − | − |
| Glycerol | ND | + | ++ | + | ++ | ++ | ++ |
| Erythritol | ND | − | − | − | − | − | + |
| D-arabinose | ND | − | − | − | − | − | + |
| L-arabinose | − | − | − | − | − | ++ | ++ |
| Ribose | + | − | + | + | − | + | + |
| D-xylose | ND | − | + | − | − | − | − |
| L-xylose | ND | − | − | − | − | − | − |
| Adonitol | ND | − | − | − | − | − | − |
| Methyl-B-xylopyranoside | ND | − | − | − | − | + | − |
| Galactose | ND | + | + | + | − | + | + |
| Glucose | ND | ++ | ++ | ++ | ++ | ++ | ++ |
| Fructose | ND | ++ | ++ | ++ | ++ | ++ | ++ |
| Mannose | ND | ++ | ++ | ++ | ++ | ++ | ++ |
| Sorbose | ND | − | − | − | − | + | − |
| Rhamnose | ND | − | − | − | − | − | − |
| Dulcitol | ND | − | − | − | − | − | − |
| Inocitol | ND | − | ++ | − | + | − | ++ |
| Mannotol | − | − | ++ | − | − | ++ | ++ |
| Sorbitol | − | − | ++ | − | − | ++ | ++ |
| Methyl-α-D-mannopyranoside | ND | + | − | − | − | − | − |
| Methyl-α-D-glucoside | ND | + | + | − | − | ++ | + |
| N-acetyl-glucosamine | ND | ++ | + | + | + | ++ | + |
| Amygdalin | ND | ++ | + | + | + | ++ | − |
| Arbutin | ND | ++ | − | − | − | ++ | − |
| Esculin | ND | ++ | ++ | ++ | ++ | ++ | − |
| Salicin | ND | ++ | + | − | + | ++ | − |
| Cellobiose | ND | ++ | ++ | ++ | ++ | ++ | − |
| Maltose | ND | ++ | + | + | + | ++ | ++ |
| Lactose | + | − | + | − | + | ++ | − |
| Melibiose | ND | − | − | + | − | − | + |
| Sucrose | ND | ++ | ++ | ++ | ++ | ++ | ++ |
| Trehalose | + | ++ | + | ++ | + | ++ | ++ |
| Inulin | − | − | − | − | − | − | + |
| Melezitose | ND | − | − | − | − | − | − |
| Raffinose | + | − | + | − | + | − | − |
| Starch | − | − | − | − | − | − | − |
| Glycogen | − | − | − | − | − | − | − |
| Xylitol | ND | − | − | + | − | − | − |
| Gentiobiose | ND | − | ++ | − | + | − | − |
| D-turanose | ND | − | − | − | − | − | + |
| D-lyxose | ND | − | − | − | − | − | − |
| D-tagatose | ND | + | − | − | − | + | − |
| D-fucose | ND | − | − | − | − | − | − |
| L-fucose | ND | − | − | − | − | − | − |
| D-arabitol | ND | − | − | − | − | − | − |
| L-arabitol | ND | − | − | − | − | − | − |
| Gluconate | ND | − | − | − | − | − | − |
| 2-keto-gluconate | ND | − | − | − | − | + | − |
| 5-keto-gluconate | ND | − | − | − | − | − | − |
ND, not detected; ++ , strong positive; +, positive; −, negative.
Acid resistance and bile resistance
Probiotics need to have strong acid resistance and bile resistance to survive in acidic gastric environments. Among the probiotic characteristics of the seven strains isolated from Cheonggukjang, acid resistance and bile resistance were examined. It was difficult to screen strains with strong acid resistance because strains could not grow below pH 5.0, but grew above pH 5.0. Most strains showed resistance to weak acids. All strains could grow at pH 5.5 (Table 3). As for bile resistance, seven strains showed similar growth compared with the control group, suggesting that these strains have strong bile resistance.
Table 3.
Weak Acid and Bile Resistance of SAMe-Producing Strains
| Strain | pH 4.0 | 4.5 | 5.0 | 5.5 | 7.0 (con) | Oxgall |
|---|---|---|---|---|---|---|
| C1-5 | − | − | − | + | + | + |
| C2-1 | − | − | − | + | + | + |
| C3-2 | − | − | − | + | + | + |
| C4-1 | − | − | − | + | + | + |
| C4-3 | − | − | − | + | + | + |
| C5-1 | − | − | − | + | + | + |
| C6-4 | − | − | − | + | + | + |
Antibacterial activity
The antibacterial activity was measured for five indicators such as E. coli O157:H7, E. faecalis, S. choleraesuis, S. aureus, and L. monocytogenes using Cheonggukjang-originated strains, which showed that the antibacterial activity was not observed when using the paper disc method. Instead, the antibacterial activity for the five indicators was partially identified in seven strains with the exception of one strain, C6-4, through a soft agar method (Table 4). There was a strain that inhibited all five indicators, C3-2 and C5-1. C1-5 strains inhibited at least four indicator strains, showing bacteria-inhibiting ability for several indicators. C3-2 and C5-1 could grow at the lowest pH and showed a strong inhibition to indicators, suggesting that these may be considered the most beneficial strains with a strong efficacy of probiotics.
Table 4.
Antibacterial Activity of Strains with a High Level of SAMe Production
| Strains | Escherichia coli O157:H7 | Enterococcus faecalis | Salmonella choleraesuis | Staphylococcus aureus | Listeria monocytogenes |
|---|---|---|---|---|---|
| C1-5 | + | + | + | + | − |
| C2-1 | − | + | − | + | − |
| C3-2 | + | + | + | + | + |
| C4-1 | − | − | + | + | − |
| C4-3 | − | − | − | + | − |
| C5-1 | + | + | + | + | + |
| C6-4 | − | − | − | − | − |
Discussion
Cheonggukjang-originated strains that produce physiologically the functional material, SAMe, were isolated as well as selected strains that could produce large amounts of SAMe in foods. The production of SAME was measured in 36 strains from 6 kinds of Cheonggukjang and, after the identification of these strains, mostly belonged to the Bacillus genus. Particularly among strains isolated from Cheonggukjang, two strains showed over 1.0 mM of SAMe production. The strain with the maximum production of SAMe was a C4-3 strain isolated from the C4 Cheonggukjang sample, which produced 2.23 mM. The mechanism of SAMe production by the strains from Cheonggukjang was not clear. However, the results were similarly shown in a previous study as SAMe product strains from Kimchi. The strains isolated from the fermented Kimchi products, produced the amount of SAMe 1.22–1.58 mM.13 To utilize these strains in future studies, it was considered necessary to examine the characteristics and identification of these seven strains.
Among probiotic characteristics, acid resistance and bile resistance were examined in seven strains. The gastric pH in healthy humans can be as low as about 2–2.519 or high as pH 6 or above after food intake.20 Most strains could grow in the weak acidic condition over pH 5.5. On the other hand, for bile resistance, all strains showed similar growth compared with the control group. This result suggested that these strains have bile resistance and, thus, probiotic characteristics. To be utilized as probiotics, lactic acid bacteria are required to have strong resistance against the acidic gastric environment and bile containing various digestive enzymes.21 It was also reported that the surviving bacteria should colonize on surfaces of the animal gut with the extreme environment to be able to promote immune activity and to show anticancer activity.21 In line with this, Lactobacillus acidophilus RMK567 isolated from raw milk is well-known as probiotics. As compared to this strain, the SAMe-producing strains screened in this study displayed weak acid resistance, but strong bile resistance surviving the medium containing 0.3% Oxgall.22 Similarly, a screened E. faecalis OA18 strain producing functional material ornithine was utilized for a fermented milk drink made with kefir grains.23
The results of antibacterial activity for five indicators such as E. coli O157:H7, E. faecalis, S. choleraesuis, S. aureus, and L. monocytogenes in the strains originating from the Cheonggukjang showed the antibacterial activity. These strains are considered to be strains that can be involved in the regulation of the intestines. More importantly, Bacillus subtilis MP56 strains showed negligible effect on gram negative bacteria, but showed antibacterial activity against gram-positive bacteria such as S. aureus.24 As compared to the B. subtilis MP56 strain, however, the Bacillus genus screened in this study, deployed strong antibacterial activity against both gram-negative and gram-positive bacteria. The Bacillus species screened in this study can be useful as probiotics having antibacterial activity against both gram-negative and gram-positive bacteria. Similarly, Doenjang fermented with a B. subtilis strain exhibited antibacterial activity, suppressed harmful bacteria, and promoted the growth of beneficial yeasts and fungi.25 Therefore, we may utilize the screened Bacillus strains for other fermented foods exhibiting antibacterial activity against both gram-negative and gram-positive bacteria.
In terms of fermentation characteristics, the results of the substrate utilization of six strains showed that fermentable substrates vary depending on the strains. Thus, strains seem to have enzymes for using sugars and can ferment various sugars that enter the body through food intake. It has been considered that utilization of carbon and nitrogen sources is an important characteristic for producing antibacterial agents.26 Rye et al. isolated the B. subtilis producing antibacterial agent from traditional fermented food, Doenjang. Interestingly, the B. subtilis strain producing antibacterial agent showed similar sugar utilization to the strains screened in this study, suggesting that the screened strain may be employed for production of antibacterial agents.26 In addition, sugar utilization information is important when we implement SAMe-producing microbial strains for food. Yu et al. utilized E. faecalis for fermenting milk. Therefore, we may apply the screened SAMe-producing strains to various foods more efficiently based on the information of substrate utilization. In the previous research, the Bacillus spp. strains producing functional materials were used for making Doenjang and preparing soy ice cream with fermented soybean powder.27 The screened strains can be customized as starters in various fermented foods to provide SAMe and probiotics in food based on the substrate utilization.
Acknowledgment
This study was supported by a grant from the Korea Food Research Institute (project no. E0131901).
Author Disclosure Statement
No competing financial interests exist.
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