Increased Anticancer Activity of Organic Kimchi with Starters Demonstrated in HT-29 Cancer Cells

study


Introduction
Kimchi is a traditional Korean fermented food characterized by both the physiological activity of the raw materials and the probiotic function of lactic acid bacteria (LAB), the microorganisms used for fermenting it. Kimchi is a food made of vegetables, such as cabbage, radish, and green onions, with other seasonings, and is reported to have functional health properties, such as antioxidative, immune-enhancement, anti-obesity, and anti-diabetes The purpose of this study is to use the HT-29 human colon cancer cell line to detect the effect of organic anticancer kimchi (OAK) on the growth rate inhibition of cancer cells and the expression of mRNA and the proteins of genes associated with cell cycle arrest and apoptosis. Kimchi used in this study was prepared using organic ingredients, with starter cultures LP + LM and nF1 with enhanced anticancer activity, and improved taste and quality similar to or better than commercial products.

Kimchi Preparation
The common and organic ingredients used to make kimchi were supplied by Damchaewon Co. (Taean, Chungcheongnam-do, Korea), and production was also conducted by the same company. Organic ingredients were grown without the use of chemical fertilizers and pesticides and were certified by the Ministry of Agriculture, Food and Rural Affairs of the Republic of Korea. The organic ingredients used in OAK were cabbage, red pepper powder, garlic, ginger, radish, green onion, pear, and leaf mustard. The starter strains Lactiplantibacillus plantarum PNU (KCCM 11352P) and Leuconostoc mesenteroides PNU (KCCM 11353P) were isolated from kimchi in our laboratory. These two starters were supplied as live bacteria from Mediogen Co. Ltd., which can manufacture food probiotic starters, and the starters were mixed in 0.9% physiological saline at a concentration of 1 × 10 6 CFU per 1 g of baechu cabbage. Dead nano-sized Lactiplantibacillus plantarum nF1 was provided by Immunobiotech Co., Ltd. (Seoul, Korea). Commercial kimchi (CK) was prepared using organic kimchi from Damchaewon Co. The cabbage was cut in 1/2 and pickled with sea salt for 24 h, then the water was discarded, and it was mixed with the other ingredients to prepare the different kimchi samples ( Table 1). The standard kimchi (SK), CK, anticancer kimchi (AK), and organic anticancer kimchi (OAK) samples produced were fermented for 4 weeks at 5 • C. As the 3rd week was the optimal maturity period, the kimchi samples from that period were used for the experiment. Table 1. Kimchi recipes of standard, commercial, anticancer, and organic anticancer kimchi.

Preparation of Kimchi Extracts
The kimchi samples at week 3 were freeze-dried in a freeze dryer (FD5512, Ilshin BioBase Co., Gyeonggi-do, Korea) and ground into powder in a mill. Subsequently, the powder was then extracted twice for 24 h with 20 times its weight of methanol and filtered. Then, the extract was concentrated using a rotary vacuum evaporator (EYELA, Rikakikai Co., Tokyo, Japan), and the concentrated extract was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) and used in the subsequent experiments [8].

Measurements of pH Value, Acidity, and Salinity of Kimchi
Kimchi juice was collected weekly, and pH and acidity levels were measured using a pH meter (M220, Corning Co., Lowell, MA, USA). The acidity of the sample was diluted 20-fold according to the standard test method of the Association of Official Agricultural Chemists (AOAC) and titrated by adding 0.1 N NaOH until the pH reached 8.4. The amount of 0.1 N NaOH used was measured. To measure salinity, 300 uL of kimchi juice was placed into the salt meter (PAL-ES2, ATAGO Co., Tokyo, Japan) and the measurement was obtained [5]. Acidity was measured as follows: Acidity (%) = mL of 0.1N NaOH × normality of NaOH × 0.09 weight of sample (g) × 100

Sensory Evaluation of Kimchi
This sensory evaluation was approved by the research review board of CHA University, Gyeonggi, Seongnam, South Korea. A total of 10 trained sensory panelists evaluated the samples, and the evaluation was conducted through quantitative descriptive analysis (QDA). There were total of 11 evaluation items, and for all the items, extremely disliked or weak was evaluated using the number 1, and extremely good or strong was evaluated using the number 9. The evaluation parameters were appearance, sour smell, green smell, moldy smell, salty flavor, bitter flavor, sour flavor, moldy flavor, refreshing flavor, hardness, and overall acceptability [19].

Assessment of Total Phenol (TP) Content
The content of total phenolic (TP) compound content was measured using the gallic acid equivalence method. A total of 75 µL of Folin-Ciocalteu reagent (Sigma-Aldrich Co., St. Louis, MO, USA) was mixed with 25 µL of the methanol extract of kimchi of a certain concentration (250 mg/mL) and reacted at room temperature for 5 min. Then, 200 µL of 7.5% Na 2 CO 3 solution and 700 µL of distilled water (D.W.) were added, and the light was blocked for 40 min at room temperature. The absorbance was measured at 765 nm with a Wallac Victor3 1420 Multilabel Counter (Perkin-Elmer, Wellesley, MA, USA). The standard curve was drawn using gallic acid as the reference (standard concentration was 0.03125-1 mg/mL), and the standard curve was used to calculate the TP content of the kimchi [21].

Assessment of the Total Flavonoid (TF) Content
A total of 1 mL of diethylene glycol was added to 100 µL of the kimchi extract and left at room temperature for 5 min. Then, 100 µL of 1N NaOH was added, the mixture was heated to 37 • C for 60 min, and the absorbance was measured at 420 nm. The flavonoid content was calculated from the standard calibration curve obtained using Quercetin (Sigma-Aldrich Co., St. Louis, MO, USA) as the reference (standard concentration is 0-1280 µg/mL) to draw the standard curve [22].

HT-29 Cell Culture
An HT-29 human colon carcinoma cell line was purchased from the Cell Line Bank in Seoul, Korea. The cells were incubated at 37 • C in a 5% CO 2 incubator with Roswell Park Memorial Institute (RPMI) 1640 medium (Welgene Inc., Daegu, Korea) containing a 1% penicillin-streptomycin solution (Gibco BRL., Rockville, MD, USA) and 10% inactivated fetal bovine serum (FBS) (Welgene Inc., Daegu, Korea). The cultured cancer cells were refed 2-3 times a week, and after washing with phosphate-buffered saline (PBS), 0.05% trypsin-0.02% ethylenediaminetetraacetic acid (EDTA) were detached and centrifuged. After centrifugation, the accumulated cancer cells and medium were mixed well using a pipette, and 10 mL of each solution was injected into a 75T cell culture flask, subcultured every 2-3 days, and used for the experiment. The MTT assay is commonly used to measure cellular metabolic activity as an indicator of the proliferation and death rates of cells. Cultured HT-29 cells were seeded in a 96-well plate at a concentration of 5.0 × 10 4 cells/mL. After incubation for 24 h, the kimchi extract samples were used to treat the cells at concentrations of 0.5, 1, 1.5, 2, and 2.5 mg/mL. After 48 h of incubation, the MTT solution (500 µg/mL) was dissolved in the medium and 100 µL was added to each well. Further incubation for 4 h was performed under the same culture conditions. After dissolving the formed formazan crystals in DMSO, the absorbance was measured at 540 nm using a Wallac Victor3 1420 Multilabel Counter [23].

Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
The cultured cells of the HT-29 human colon cancer cell line were counted using a cell counter (Luna automated cell counter; Logos Biosystems, Anyang, Korea), and cells at 1.0 × 10 5 cells/mL per well were dispensed into a 6-well plate and cultured for 24 h. Subsequently, the HT-29 cancer cells were treated for 48 h with the addition of a medium containing SK 2, CK 2, AK 2, and OAK 2 mg/mL. After the removal of the medium, RNA was isolated from the cells using Trizol (Invitrogen, Carlsbad, CA, USA) and dissolved in 0.1% diethyl pyrocarbonate (DEPC, Bioneer Co., Daejeon, Korea). The total dissolved RNA was quantified using a NanoDrop ND-1000 (NanoDrop Technologies Inc., Wilmington, DE, USA), and the quantified RNA was synthesized into cDNA using Superscript II reverse transcriptase (Invitrogen). The synthesized cDNA was analyzed for gene expression using a thermal cycler BioRad CFX-96 real-time system (Bio-Rad, Hercules, CA, USA) [24]. The genes Bcl-2, p21, p53, Bim, Bak, Bad, caspases 3 9, and GAPDH were used, and their primer sequences are shown in Table 2.

Western Blot
Cell proteins were reacted with 1 mL radioimmunoprecipitation assay (RIPA, Invitrogen, USA) buffer, and the proteins were separated by centrifugation at 13,000 rpm for 15 min at 4 • C. The separated protein was quantified by the Bradford assay method. The extracted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred into a polyvinylidene fluoride (PVDF, Bio-Rad, Hercules, CA, USA) membrane, and non-specific proteins were blocked with 5% skimmed milk containing phosphate-buffered saline with Tween 20 (PBST). After blocking, the PVDF membrane was washed three times with PBST and once with PBS, and the primary antibody was reacted overnight at 4 • C. The PVDF membrane was then washed three times with PBST and once with PBS, and the secondary antibody was reacted for 2 h at room temperature. For Bcl-2, p53, Bax, caspases 3 9, and α-tublin, Santa Cruz (Dallas, TX, USA) primary antibodies were used, and the bands of the proteins were detected using an Amersham Imager 680 (GE Healthcare Life Sciences, Chicago, IL, USA) [25]. Table 2. Primer sequences of RT-qPCR assay.

Statistical Analysis
The data were analyzed using Graph Pad Prism 9.4.1 (GraphPad, San Diego, CA, USA), and the data of all experiments were expressed as mean ± standard deviation. Twoway and one-way ANOVAs (analysis of variances) were used to confirm the significance between each group using Duncan's multiple-range test. The results were considered significant when p < 0.05 or less, and all experimental data were analyzed using the SPSS v18 statistical software package (SPSS Inc., Westlands, Hong Kong).

Analysis of Physicochemical Characteristics of Kimchi Samples
When the kimchi was fermented, pH, acidity, salinity, and sensory evaluations were used as indicators to check the quality. Acidity, pH value, and salinity were evaluated during the fermentation period of a total of 4 weeks, and acidity was found to be at the most optimal ripening stage when the fermentation was conducted at 5 • C for 2~3 weeks [26]. As shown in Table 3, when fermented under these conditions, the acidity levels of SK, CK, AK, and OAK were 0.83 ± 0.04, 0.87 ± 0.01, 0.84 ± 0.01, and 0.85 ± 0.00%, respectively. This increased from 0.34 ± 0.01, 0.49 ± 0.01, 0.42 ± 0.01, and 0.43 ± 0.02%, respectively, at week 0. The pH values of 4.11 ± 0.02, 4.11 ± 0.00, 4.16 ± 0.02, and 4.19 ± 0.03, respectively, were measured at week 3, which tended to decrease from 5.43 ± 0.04, 5.48 ± 0.04, 5.38 ± 0.06, and 5.49 ± 0.05, respectively, at week 0. Salinity levels were 1.83 ± 0.03, 1.70 ± 0.05, 1.73 ± 0.02, 1.66 ± 0.03 at week 0 for SK, CK, AK, and OAK, respectively. In the third week of fermentation, the pH values measured were 1.40 ± 0.03, 1.26 ± 0.03, 1.49 ± 0.02, and 1.47 ± 0.04, respectively. A sensory evaluation was conducted in the 3rd week, when the ripeness of the kimchi was the best, and a total of 11 parameters were evaluated using a 9-point scale method (Figure 1). In the assessment of overall acceptability, SK scored 4.9, CK scored 5.7, and AK and OAK both scored 5.5. In the assessment of appearance, AK (6.1), SK and CK (6.4), and OAK (6.5) received high scores; however, there was no statistical significance to the variation in scores (p > 0.05). In the measurement of sour smell and flavor, SK scored 5.9 and 6.3 points, and CK scored 6.4 and 5.9 points, respectively, which were higher scores than AK and OAK, which scored 5.1 and 4.4 points and 4.6 and 5.4 points, respectively (p < 0.05). Other than these evaluation parameters, there was no variation in statistical significance among the kimchi samples with respect to green appearance, moldy smell, salty flavor, bitter flavor, moldy flavor, refreshing flavor, and hardness (p > 0.05). A sensory evaluation was conducted in the 3rd week, when the ripeness of the kimchi was the best, and a total of 11 parameters were evaluated using a 9-point scale method (Figure 1). In the assessment of overall acceptability, SK scored 4.9, CK scored 5.7, and AK and OAK both scored 5.5. In the assessment of appearance, AK (6.1), SK and CK (6.4), and OAK (6.5) received high scores; however, there was no statistical significance to the variation in scores (p > 0.05). In the measurement of sour smell and flavor, SK scored 5.9 and 6.3 points, and CK scored 6.4 and 5.9 points, respectively, which were higher scores than AK and OAK, which scored 5.1 and 4.4 points and 4.6 and 5.4 points, respectively (p < 0.05). Other than these evaluation parameters, there was no variation in statistical significance among the kimchi samples with respect to green appearance, moldy smell, salty flavor, bitter flavor, moldy flavor, refreshing flavor, and hardness (p > 0.05).

Antioxidant Capacities of Kimchi Samples
The evaluation of the antioxidant capacities of the kimchi samples was performed using DPPH free-radical scavenging activity, and the total phenol and flavonoid contents were ascertained and are shown in Figure 2. When the extracted kimchi sample was treated with 2 mg/mL, the free-radical scavenging activity increased in the order of SK, CK, AK, and OAK. The total phenolic content of OAK was highest among the kimchi samples. OAK showed a significant difference at 0.5 (p < 0.05) and 1 (p < 0.01) mg/mL versus the corresponding concentrations of the other kimchi samples, and the phenol content increased in the order of SK, CK, AK, and OAK at 2 mg/mL. With regard to the total flavonoid content, there was no difference between the kimchi samples up to 0.5 mg/mL; however, at 1 mg/mL, the flavonoid contents of AK and OAK increased compared to SK, and at 2 mg/mL, OAK showed the highest flavonoid content.

Antioxidant Capacities of Kimchi Samples
The evaluation of the antioxidant capacities of the kimchi samples was performed using DPPH free-radical scavenging activity, and the total phenol and flavonoid contents were ascertained and are shown in Figure 2. When the extracted kimchi sample was treated with 2 mg/mL, the free-radical scavenging activity increased in the order of SK, CK, AK, and OAK. The total phenolic content of OAK was highest among the kimchi samples. OAK showed a significant difference at 0.5 (p < 0.05) and 1 (p < 0.01) mg/mL versus the corresponding concentrations of the other kimchi samples, and the phenol content increased in the order of SK, CK, AK, and OAK at 2 mg/mL. With regard to the total flavonoid content, there was no difference between the kimchi samples up to 0.5 mg/mL; however, at 1 mg/mL, the flavonoid contents of AK and OAK increased compared to SK, and at 2 mg/mL, OAK showed the highest flavonoid content. Figure 2. DPPH free-radical scavenging, total phenol, and total flavonoid contents of kimchi at different concentrations. SK: standard kimchi; CK: commercial kimchi; AK: anticancer kimchi; OAK: organic anticancer kimchi. *, **, ***, **** symbols mean significantly different (p < 0.05), (p < 0.01), (p < 0.001), (p < 0.0001), respectively, by one-and two-way ANOVA tests.

Inhibition Rate of HT-29 Human Colon Carcinoma Cells by Kimchi
The HT-29 cells were treated with kimchi extract in increasing concentrations from 0.5 to 2.5 mg/mL in units of 0.5 mg/mL ( Figure 3). As the concentration increased, the inhibition rate of HT-29 cancer cell growth also increased. At all concentrations, OAK showed the highest rate of inhibition of HT-29 cell growth, followed by AK. A concentration of 2 mg/mL was chosen for the follow-up experiment, during which SK showed an inhibition rate of 18.99 ± 0.55%, CK 22.37 ± 2.09%, AK 25.63 ± 5.65%, and OAK 35.49 ± 2.17%.

Inhibition Rate of HT-29 Human Colon Carcinoma Cells by Kimchi
The HT-29 cells were treated with kimchi extract in increasing concentrations from 0.5 to 2.5 mg/mL in units of 0.5 mg/mL ( Figure 3). As the concentration increased, the inhibition rate of HT-29 cancer cell growth also increased. At all concentrations, OAK showed the highest rate of inhibition of HT-29 cell growth, followed by AK. A concentration of 2 mg/mL was chosen for the follow-up experiment, during which SK showed an inhibition rate of 18.99 ± 0.55%, CK 22.37 ± 2.09%, AK 25.63 ± 5.65%, and OAK 35.49 ± 2.17%.

mRNA Expression of Genes Associated with Cell Cycle Arrest
SK, CK, AK, and OAK significantly increased the mRNA expression levels of p21 and p53 genes compared to the control group (Figure 4). AK and OAK increased the mRNA expression levels of p21 1.70 and 2.33 times, respectively, compared to the control group. AK increased the expression levels of p53 1.59 times and OAK 2.65 times. Among all the groups, the highest increase in mRNA expression levels of p21 and p53 was observed with OAK. Thus, it was confirmed that OAK increases the expression of genes related to cell cycle arrest.

mRNA Expression of Genes Associated with Cell Cycle Arrest
SK, CK, AK, and OAK significantly increased the mRNA expression levels of p21 an p53 genes compared to the control group ( Figure 4). AK and OAK increased the mRNA expression levels of p21 1.70 and 2.33 times, respectively, compared to the control group AK increased the expression levels of p53 1.59 times and OAK 2.65 times. Among all th groups, the highest increase in mRNA expression levels of p21 and p53 was observed wit OAK. Thus, it was confirmed that OAK increases the expression of genes related to ce cycle arrest.

mRNA Expression of Genes Associated with Apoptosis
The mRNA expression levels of Bim, Bad, Bak, Bcl-2, and caspases 3 9 related to apoptosis are presented in Figure 5. AK and OAK increased the mRNA expression levels of Bim related to pro-apoptosis 5.07 times compared to the control group. AK increased the mRNA expression levels of Bad and Bak by 4.74 and 5.03 times, and OAK by 5.63 and 5.41 times, respectively. AK reduced the mRNA expression levels of Bcl-2, an antiapoptosis-related gene by 0.34 times and OAK by 0.20 times. It was confirmed that OAK significantly increased the expression levels of pro-apoptosis genes and decreased the expression levels of anti-apoptosis-related genes. In addition, the mRNA expression levels of caspases 3 and 9 were increased 2.59 and 2.01 times, respectively, by OAK compared to the control group, showing the greatest increase. These results confirm that kimchi samples, especially OAK, promote the apoptosis of HT-29 cells by regulating the expression levels of apoptosis-related mRNA.

mRNA Expression of Genes Associated with Apoptosis
The mRNA expression levels of Bim, Bad, Bak, Bcl-2, and caspases 3 9 related to apoptosis are presented in Figure 5. AK and OAK increased the mRNA expression levels of Bim related to pro-apoptosis 5.07 times compared to the control group. AK increased the mRNA expression levels of Bad and Bak by 4.74 and 5.03 times, and OAK by 5.63 and 5.41 times, respectively. AK reduced the mRNA expression levels of Bcl-2, an anti-apoptosis-related gene by 0.34 times and OAK by 0.20 times. It was confirmed that OAK significantly increased the expression levels of pro-apoptosis genes and decreased the expression levels of anti-apoptosis-related genes. In addition, the mRNA expression levels of caspases 3 and 9 were increased 2.59 and 2.01 times, respectively, by OAK compared to the control group, showing the greatest increase. These results confirm that kimchi samples, especially OAK, promote the apoptosis of HT-29 cells by regulating the expression levels of apoptosis-related mRNA. Figure 5. Effects of kimchi on the mRNA expression levels of Bim, Bad, Bak, Bcl-2, and caspases 3 and 9 in HT-29 cancer cells. SK: standard kimchi, 2 mg/mL; CK: commercial kimchi, 2 mg/mL; AK: anticancer kimchi, 2 mg/mL; OAK: organic anticancer kimchi, 2 mg/mL. Fold ratio: gene expression/GAPDH×control numerical value (control fold ratio = 1). Means with different letters (a-d) above the bars are significantly different (p < 0.05) by Duncan's multiple range test. GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Expression Levels of Proteins Associated with Cell Cycle Arrest and Apoptosis
Western blot analysis was performed to further confirm the mechanisms of cell cycle arrest and apoptosis of HT-29 cells using kimchi extracts ( Figure 6). These extracts significantly reduced Bcl-2 protein expression and the lowest expression level was observed for OAK. Expressions of p53 and Bax proteins increased significantly in the order of SK, CK, AK, and OAK. OAK showed the highest expression levels of caspases 3 and 9, followed by AK. The Western blot results are almost consistent with the mRNA analysis, suggesting that OAK, an organic functional kimchi, inhibits the growth of HT-29 cancer cells through cell cycle arrest and apoptosis pathways. Figure 5. Effects of kimchi on the mRNA expression levels of Bim, Bad, Bak, Bcl-2, and caspases 3 and 9 in HT-29 cancer cells. SK: standard kimchi, 2 mg/mL; CK: commercial kimchi, 2 mg/mL; AK: anticancer kimchi, 2 mg/mL; OAK: organic anticancer kimchi, 2 mg/mL. Fold ratio: gene expression/GAPDH×control numerical value (control fold ratio = 1). Means with different letters (a-d) above the bars are significantly different (p < 0.05) by Duncan's multiple range test. GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Expression Levels of Proteins Associated with Cell Cycle Arrest and Apoptosis
Western blot analysis was performed to further confirm the mechanisms of cell cycle arrest and apoptosis of HT-29 cells using kimchi extracts ( Figure 6). These extracts significantly reduced Bcl-2 protein expression and the lowest expression level was observed for OAK. Expressions of p53 and Bax proteins increased significantly in the order of SK, CK, AK, and OAK. OAK showed the highest expression levels of caspases 3 and 9, followed by AK. The Western blot results are almost consistent with the mRNA analysis, suggesting that OAK, an organic functional kimchi, inhibits the growth of HT-29 cancer cells through cell cycle arrest and apoptosis pathways.

Discussion
Kimchi is a fermented food that is prepared using LAB at 5 °C for 2 to 3 weeks to reach the optimal ripening stage. At this time, the pH range is 4.2~4.5 and the acidity level is 0.6~0.9% [27]. LAB produce organic acid through the fermentation process in food while lowering the pH level and increasing acidity to inhibit the growth of other bacteria, thus preserving the product's shelf life and quality [3]. The salinity level of kimchi distributed in Korea is 1.4~2.5%, and it is known that, as kimchi matures, the salinity reduces from the initial salinity level following a certain storage period due to the osmotic pressure inside and outside the cabbage [5]. According to the results of this study, the pH levels between kimchi samples in the initial week 0 ranged from 5.4 to 5.54, acidity ranged from 0.33 to 0.50%, and salinity ranged from 1.63 to 1.86%. As the fermentation progressed, CK tended to decrease in pH level more rapidly than AK and OAK. These results are similar to those observed by Choi et al. [28], in which the fermentation rate decreased when mistletoe extract was mixed in. In the sensory evaluation, CK showed the highest score for sour smell compared to the other samples, probably due to the production of a large amount of organic acid as the fermentation rapidly progressed.
Kimchi contains natural antioxidants, such as carotenoids, vitamins, flavonoids, and other phenolic compounds, because it basically composed of cabbage, radish, red pepper powder, garlic, and ginger [29]. In addition, the characteristics of kimchi change depending on the cultivation method of these ingredients. According to a study by Seong et al. [30], organic cabbage has higher levels of dietary fiber, ascorbic acid, chlorophyll, and carotenoids than conventional cabbage. This is because organic plants do not benefit from pesticides and must endure and survive pests and diseases on their own, thus increasing their phytochemical content [31]. In addition, Jung et al. [32] confirmed that the total phenol and flavonoid contents in organically grown young radish kimchi increased by 1.5 to 3 times compared to general young radish kimchi. Therefore, in this study, an antioxidant evaluation was conducted for the kimchi samples, and free-radical scavenging activity, total phenolic content, and total flavonoid content were measured. Oxygen free radicals are known to be involved in the development of systemic inflammatory response syndrome due to oxidative stress when free radicals exceed the antioxidant capacity of the body, and it has been confirmed that antioxidants that remove these free radicals can reduce inflammatory response [33]. In this study, OAK showed the highest free-radical scavenging effect, followed by AK. In addition, OAK was found to have the highest total

Discussion
Kimchi is a fermented food that is prepared using LAB at 5 • C for 2 to 3 weeks to reach the optimal ripening stage. At this time, the pH range is 4.2~4.5 and the acidity level is 0.6~0.9% [27]. LAB produce organic acid through the fermentation process in food while lowering the pH level and increasing acidity to inhibit the growth of other bacteria, thus preserving the product's shelf life and quality [3]. The salinity level of kimchi distributed in Korea is 1.4~2.5%, and it is known that, as kimchi matures, the salinity reduces from the initial salinity level following a certain storage period due to the osmotic pressure inside and outside the cabbage [5]. According to the results of this study, the pH levels between kimchi samples in the initial week 0 ranged from 5.4 to 5.54, acidity ranged from 0.33 to 0.50%, and salinity ranged from 1.63 to 1.86%. As the fermentation progressed, CK tended to decrease in pH level more rapidly than AK and OAK. These results are similar to those observed by Choi et al. [28], in which the fermentation rate decreased when mistletoe extract was mixed in. In the sensory evaluation, CK showed the highest score for sour smell compared to the other samples, probably due to the production of a large amount of organic acid as the fermentation rapidly progressed.
Kimchi contains natural antioxidants, such as carotenoids, vitamins, flavonoids, and other phenolic compounds, because it basically composed of cabbage, radish, red pepper powder, garlic, and ginger [29]. In addition, the characteristics of kimchi change depending on the cultivation method of these ingredients. According to a study by Seong et al. [30], organic cabbage has higher levels of dietary fiber, ascorbic acid, chlorophyll, and carotenoids than conventional cabbage. This is because organic plants do not benefit from pesticides and must endure and survive pests and diseases on their own, thus increasing their phytochemical content [31]. In addition, Jung et al. [32] confirmed that the total phenol and flavonoid contents in organically grown young radish kimchi increased by 1.5 to 3 times compared to general young radish kimchi. Therefore, in this study, an antioxidant evaluation was conducted for the kimchi samples, and free-radical scavenging activity, total phenolic content, and total flavonoid content were measured. Oxygen free radicals are known to be involved in the development of systemic inflammatory response syndrome due to oxidative stress when free radicals exceed the antioxidant capacity of the body, and it has been confirmed that antioxidants that remove these free radicals can reduce inflammatory response [33]. In this study, OAK showed the highest free-radical scavenging effect, followed by AK. In addition, OAK was found to have the highest total phenol and flavonoid contents at 1 and 2 mg/mL sample treatment concentrations, respectively, suggesting that the antioxidant effect was significantly increased with the use of organic ingredients.
According to Park and Hong [29], kimchi has been confirmed to present anticancer, anti-aging, antioxidant, and anti-obesity effects due to the complex action of its ingredients, fermentation products, and LAB. Furthermore, Kim et al. [34] and Cui et al. [35] confirmed that kimchi presents anticancer and anti-obesity effects through in vivo experiments. In the case of mistletoe extracts added to AK and OAK, it was confirmed that they had significant anticancer effects on HT-29 and A549 cells when mixed with kimchi [36]. In addition, it was also confirmed that kimchi mixed with mistletoe extract, pear, mustard leaf, mushroom, and sea tangle juice was confirmed to present higher anticancer activity than standardized kimchi on HT-29 cells [4,37]. In the case of LP and LM, which were used as starter cultures LAB isolated from kimchi, they presented all the basic properties of probiotics, such as acid resistance, bile resistance, intestinal adhesion, and heat stability [38]. LP has antimutagenic effects, phagocytosis, and inhibits the growth of pathogenic bacteria, and LM is mainly used as a starter for kimchi and imparts a refreshing taste with non-carcinogenic properties [39,40]. When these two were used as starters for kimchi, Lee et al. [8] confirmed that they had an anti-obesity effect, and Bong et al. [9] found that starter-added kimchi had highly effective anticancer effects on HT-29 and HCT-116 cells. nF1 is a heat-treated LP biogenic consisting of non-viable Lactiplantibacillus plantarum cells. In a recent study by Choi et al. [41], it was shown to have an immune-enhancing effect on immunosuppressed animal models, and in the study by Kim et al. [42], it was confirmed that nF1-added kimchi improved the symptoms of irritable bowel syndrome (IBS). As seen in Figure 3, AK and OAK display a higher HT-29 cancer cell death rate than SK, which appears to be due to the addition of sub-materials, starter strains, and nF1 included in AK and OAK, as mentioned above. In addition, OAK was manufactured with the same organic ingredients as CK; however, there were differences in the contents of ingredients and sub-ingredients. Specifically, mushroom and sea tangle juice, mistletoe extract, starters, and nF1 are believed to have increased the death rate of HT-29 cells in OAK.
Uncontrolled cell proliferation and abnormal gene function and expression are the hallmarks of cancer, and tumor cells usually result from damage to genes that regulate cell cycle arrest [43]. To inhibit the growth of these cancer cells, cell cycle arrest and apoptosis are achieved by regulating the expression of transcription factors and suppressing growth signaling pathways [44]. The tumor suppressor gene p53 is involved in cell cycle arrest and apoptosis and is activated by signals, such as DNA damage, oncogene activation, and stress [45]. When p53 is activated, it plays an important role in determining the duration and outcome of cell cycle arrest at the G2 checkpoint, an important cell cycle checkpoint, and it can activate pro-apoptosis genes, such as Bax, or down-regulate anti-apoptosis genes, such as Bcl-2 [46]. p21, a downstream mediator of p53, induces G1 arrest in the cell cycle by binding to cyclin E/cyclin-dependent kinases (Cdk)2 and cyclin D/Cdk4 complexes. It also inhibits DNA replication by promoting the transcriptional silencing of transcription factor E2F1 targets important for DNA replication and cell cycle progression [47]. The p53 and p21 genes are involved in cell division throughout the G1, S, and G2/M phases of the cell cycle, and by measuring the expression of these genes, it is possible to determine how much a substance, such as an anticancer drug, inhibits cancer cell division [48]. Therefore, we investigated the effect of kimchi samples on the expression levels of p21 and p53. Both AK and OAK significantly increased the mRNA expression levels of p21 and p53 in HT-29 cells (Figure 4), and also increased the protein expression level of p53 ( Figure 6). Between them, OAK was observed to increase the mRNA and protein expression levels the most. Previous studies have reported that kimchi inhibits the growth of cancer cells by up-regulating the expression of cell cycle arrest-related genes [49]. Similarly, in this study, the expression levels of p21 and p53 were increased the most by OAK, suggesting that functional recipes, starters, and organic ingredients induce the up-regulation of cell cycle arrest-related factors in HT-29 cells.
Apoptosis is a type of programmed cell death observed in multicellular organisms. This is summarized as cell death due to cell morphology and internal biochemical changes, which include cell rupture, cell membrane changes, nuclear fragmentation, chromatin condensation, and chromosome cleavage, and ends with the cells being phagocytosed and processed by other cells [50]. The first link between apoptosis and cancer began in the late 1980s, when it was understood that the Bcl-2 gene specifically blocks B-cell apoptosis in lymphomas [51]. The mitochondrial pathway of apoptosis is the major mechanism of physiological cell death, which is initiated by the pro-apoptotic members of the Bcl-2 family that cause mitochondrial outer-membrane permeabilization (MOMP). Pro-apoptosis, which induces the release of cytochrome c from mitochondria, and anti-apoptosis, which inhibits the release, play a role in apoptosis by interacting with each other [52]. Anti-apoptosis genes include Bcl-2 and Bcl-xL, which inhibit apoptosis and pro-apoptosis genes, such as Bax and Bak, which are downstream effectors that directly induce and promote apoptosis [53]. In addition, the pro-apoptotic BH3-only protein is an upstream member of the chain reaction, and when activated, suppresses anti-apoptosis and activates pro-apoptosis factors, such as Bax and Bak, to induce mitochondrial apoptosis [54]. Yu et al. [4] reported that kimchi supplemented with functional additives down-regulated Bcl-2 and up-regulated pro-apoptotic protein expression in HT-29 cells. Furthermore, Bong et al. [9] confirmed that kimchi with starters increased the mRNA expression of Bax and decreased the expression of Bcl-2, making it more effective in regulating apoptosis-related factors. In this study, the mRNA expression of anti-apoptotic Bcl-2 was decreased in HT-29 cells by treatment with OAK, an organic functional kimchi, and the mRNA expressions of pro-apoptotic Bak and BH3-only proteins Bim and Bad were up-regulated ( Figure 5). In addition, the measurement of protein expression levels ( Figure 6) confirmed that the expression of Bcl-2 decreased and the expression of Bax increased.
The caspase-cascade system plays an important role in the induction, transduction, and amplification of intracellular apoptotic signals. Caspases are classified according to their roles into initiator (caspases 2, 8, 9, and 10), executioner (caspases 3, 6, and 7), and inflammatory caspases (caspases 1, 4, and 5) [55]. Caspases 3, 7, and 9 have been reported to play a central role in the apoptotic process. When caspase 9 is activated, it cleaves and activates caspases 3/7 to further cleave other endogenous substrates and induce apoptosis [56]. Caspase 3 degrades intracellular structural and functional proteins and causes apoptosis [57]. In this study, the expression levels of caspases 3 and 9 were the highest in OAK, and OAK regulated the expression levels of caspase family members, resulting in high apoptotic activity in HT-29 cells. These results are similar to those of Kim et al. [37], in which the significant up-regulation of caspases 3 and 9 was observed in aged kimchi. These results show that kimchi supplemented with a functional recipe of nF1, starters, and organic ingredients can play an important anticancer role by regulating the expression of cell cycle arrest, apoptosis, and caspase-related genes. Considering the difference between kimchi samples prepared with different ingredients and lactic acid bacteria, it seems that the functional recipe of OAK prepared with the abovementioned ingredients improves the fermentation quality and antioxidant and anticancer effects of kimchi. The kimchi recipes prepared in this way were the best anticancer kimchi recipes confirmed after 20 years of research in this laboratory. Therefore, OAK seems to be an ideal kimchi for the development of premium organic anticancer kimchi. The kimchi prepared with the previous recipes has also been confirmed to have anti-obesity and antiinflammatory effects through various in vitro and in vivo experiments; however, the kimchi made with this recipe has not yet been confirmed to display the various effects. Therefore, it is necessary to confirm the functionality of the kimchi made with this recipe and conduct a follow-up experiment.

Conclusions
These results confirm that OAK, an organic anticancer functional kimchi prepared in this laboratory, shows an increased antioxidant capacity (DPPH inhibition rate and total phenol and flavonoid contents), while maintaining the physicochemical characteristics and taste of the existing kimchi. In addition, OAK significantly inhibited the growth of HT-29 human colon cancer cells and up-regulated the mRNA expression of cell cycle arrest-related genes p53 and p21 and apoptosis-promoting genes Bim, Bad, Bak, and caspases 3 and 9. At the same time, the mRNA expression of Bcl-2, an anti-apoptosis gene, was decreased. Similarly, the protein expression levels of Bak, p53, and caspases 3 and 9 increased, and the expression of Bcl-2 decreased. In conclusion, kimchi produced using organic ingredients, starters, and nF1 maintains the taste and quality of existing kimchi and maintains constant fermentation to produce kimchi with a uniform quality. In addition, there is a possibility of developing a functional food with high anticancer activity using organic ingredients and LAB probiotics components.