BR and kimchi ingredients (kimchi cabbage, red pepper powder, garlic, ginger, carrots, dropworts, sesame, white sugar, glutinous rice powder, white radishes, and salted anchovy sauce) were purchased in a local market. Folin-Ciocalteu reagent, sodium carbonate, gallic acid, sodium nitrite, aluminum chloride, sodium hydroxide, catechin, DPPH, ABTS, potassium ferricyanide, trichloroacetic acid, and thioacetamide were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other reagents used in this study were of analytical grade.

BR were stored at −40℃ before processing. Frozen BR were thawed at room temperature for 12 h and then pulverized using a blender (Hurom slow juicer; Quezon City, Philippines). The extracted juices were freeze-dried using a PVTFD 500R device (ilShinBioBase, Gyeonggi-do, Korea) and then powdered using an electric mixer (HMF-3260S; Hanil Co., Seoul, Korea). White radishes, carrots, and dropworts were cut into small pieces, and garlic and ginger were ground together using an electric mixer. Glutinous rice powder and water were mixed (1:10, w/w) and heated in a saucepan until gelatinization, after which the cooled gelatinized rice powder was mixed with the other kimchi ingredients. The composition (%, w/w) of kimchi seasoning (KS) was as follows: 27.0% white radish, 23.0% glutinous rice paste, 12.2% red pepper powder, 9.0% salted shrimp sauce, 5.4% garlic, 5.4% dropwort, 4.5% salted anchovy sauce, 4.0% chives, 4.0% carrot, 2.7% ginger, 1.4% sesame, and 1.4% white sugar. To prepare BJP-added kimchi (BAK), 1 kg of brined kimchi cabbage was combined with 370 g of KS, and BJP was added at 0.5% (BAK-0.5), 1% (BAK-1), and 2% (BAK-2) (w/w) concentrations relative to the total weight of the brined kimchi cabbage. As a control, the same method was used, except in the absence of BJP. Kimchi fermentation proceeded at 4℃ for 4 weeks. Characteristics of the fermented kimchi samples were investigated by measuring the pH, acidity, and microbial count in half of each sample, while the other half was freeze-dried, powdered, and stored at −70℃ for subsequent measurement of antioxidant effects. The kimchi powders (30 g) used for antioxidant analysis were extracted with boiled distilled water (600 mL) for 2 h and 20 min. The extracts were centrifuged at 13,000 g for 20 min and filtered through Whatman No. 1 filter paper (GE Life Sciences, Pittsburgh, PA, USA). The extraction step was repeated twice. The filtrate was concentrated in a rotary evaporator (R-210; Buchi, Flawil, Switzerland), and the residual water was removed via lyophilization. The dried samples were stored at −70℃ until further use.

The BAK samples (300 g each) were blended using a hand blender (HHM-800; Hanil, Gyeonggi-do, Korea), filtered through sterilized gauze, and used as kimchi filtrates. The pH values of the kimchi filtrates were determined with a pH meter (MP120; Mettler Toledo, Greifensee, Switzerland). Diluted BAK samples were further diluted 2-fold, and the total acidity was titrated to pH 8.3 with 0.1 N NaOH and expressed as the percentage of lactic acid. To determine total bacterial counts, samples were serially diluted in saline (0.85% NaCl), spread onto plate-count agar (Merck, Darmstadt, Germany), and incubated at 37℃ for 24 h. LAB were plated on MRS agar (Difco, Detroit, MI, USA) and incubated at 30℃ for 24 h. Microbial counts were determined for each population as the mean values from triplicate measurements and expressed as the log colony forming units (CFU)/mL.

The TPC and TFC were measured using a modification of a previously described method [15,16]. The samples (200 µL) were oxidized with 5 mL of Folin-Ciocalteu reagent, and the mixtures were neutralized with 3 mL of 5% sodium carbonate. After a 30 min incubation at room temperature in the dark, the absorbance of each sample at 725 nm was measured in a 96-well plate using a plate reader (SpectraMax i3x; Molecular Devices, Wals-Siezenheim, Austria). The TPC in each sample was determined using a standard curve for gallic acid, and the results are expressed as the µg of gallic acid equivalents (GAE) per mg of BAK extract powder. The TFC was measured by performing an aluminum chloride-colorimetric assay. Briefly, 500 µL of each sample was mixed with 75 µL of 5% sodium nitrite and incubated for 5 min at room temperature, after which 150 µL of 10% aluminum chloride was added to the mixture, followed by 0.5 mL of 1 M NaOH and 275 µL of deionized water. The absorbance of each sample was measured at 510 nm in a 96-well plate using the plate reader. The TFC was expressed as the µg of catechin equivalents (CE) per mg of BAK extract powder.

DPPH free radical scavenging activity [DPPH half maximal effective concentration (EC₅₀)] was measured according to a previously described method [17], with slight modifications. Briefly, 0.2 mL of each diluted BAK sample was added to 2.8 mL of 60 µM DPPH solution, and the mixture was shaken and allowed to stand for 30 min in the dark, followed by measurement of the absorbance at 515 nm in a 96-well plate using a plate reader. ABTS cation radical scavenging activity (ABTS EC₅₀) was measured using a modified version of the method described by Re et al. [18]. The stock solution was prepared by stirring 7 mM ABTS (5 µL) and 150 mM potassium persulfate (88 µL) at room temperature for 12 h to 15 h, after which the ABTS solution was diluted with ethanol to achieve an absorbance of 0.70 at 734 nm. The ABTS solution (1 mL) was then added to 0.1 mL of the diluted sample solution, which was vortexed and incubated for 10 min in the dark. ABTS EC₅₀ values were measured at 734 nm using the plate reader. The percentage of radical scavenging was calculated from the following formula: [1 - (A sample/A control) × 100]. The EC₅₀ value was calculated from a curve of the radical-scavenging percentage versus the extract concentration.

Reducing power was determined according to the method described by Oyaizu [19], with slight modifications. BAK sample extracts (1 mL) were mixed with 2.5 mL of 200 mM sodium phosphate buffer (pH 6.6), after which 2.5 mL of 1% potassium ferricyanide was added, and the mixture was incubated at 50℃ for 20 min. After cooling, 2.5 mL of 10% trichloroacetic acid (w/w) was added, and the mixture was centrifuged at 3,000 rpm for 10 min. A 2.5 mL sample of the supernatant was mixed with 500 µL of 0.1% ferric chloride. The absorbance of the sample was measured at 700 nm. The reducing power was presented as the difference in absorbance between samples with and without extract addition. The EC₅₀ was calculated as described for radical scavenging activity.

Six-week-old male Sprague-Dawley rats were purchased from Koatech (Pyeongtaek, Korea). After 1 week of adaptation, the rats were divided into 4 groups (n = 9/group; average weight: ~150 g) and separately housed in stainless-steel-bottomed cages. All animals were housed at room temperature (22 ± 2℃) and 50 ± 5% humidity, with a 12-h light/dark cycle (7:00–19:00). To induce LC in rats, thioacetamide (TAA) was dissolved in physiological saline and administered intraperitoneally twice weekly based on a dose of 200 mg/kg body weight for 4 weeks. The 4 groups included a normal group (rats fed a normal chow diet), the TAA group (LC rats fed a normal chow diet), the TAA + CTL group (LC rats fed chow diets containing 0.1% kimchi without BJP), and the TAA + BAK-1 group (LC rats fed chow diets containing 0.1% kimchi with 1% added BJP). The study was designed to provide 210 mg wet weight of kimchi to each rat daily, which is equivalent to the amount of kimchi consumed by healthy Korean adults (96.3 g/day). Chow was provided daily for 4 weeks, and body weights were measured once weekly. Dietary intake was measured at a fixed time daily, and dietary intake efficiency was calculated by dividing the weight gain over the entire period by the dietary intake during the same period. The experimental protocols in this study were approved by the institutional animal care and use committee (IACUC) of Berry & Biofood Research Institute (approval number: BBRIIACUC-15002).

Rats were fasted for 12 h before being sacrificed by anesthetization with diethyl ether. Blood was collected from the abdominal vein immediately after laparotomy, and serum was obtained by centrifuging the heparin tube at 3,000 rpm for 15 min in order to separate the supernatant. Serum glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) enzyme levels were analyzed using a Fuji DRI-CHEM 4000 instrument (Fuji Film, Tokyo, Japan) to identify liver damage. The activity of superoxide dismutase (SOD), an antioxidant enzyme, was measured using a colorimetric assay kit (BioVision, Milpitas, CA, USA).

All data are expressed as the mean ± standard deviation (SD) of 3 replicates. Statistical analysis was performed using SPSS software (v23.0 for windows; IBM Corp., Armonk, NY, USA). Data were subjected to Student's t test, one-way analysis of variance (ANOVA), and two-way ANOVA, and the mean values were separated using Duncan's multiple-range test, with statistical significance defined as P < 0.05. The differences in the antioxidant activities of kimchi according to the addition of BJP (same fermentation state) and fermentation state (same BJP amount) were verified using one-way ANOVA. The effects of BJP addition and fermentation period on kimchi antioxidant effects were evaluated by two-way ANOVA. Differences in serum levels and antioxidant enzyme activity between BAK and control groups were evaluated using Student's t test.

BAK with various concentrations of BJP was fermented at 4℃ for 4 weeks, and changes in the chemical (pH and acidity) and microbiological (total bacterial and LAB counts) properties of the 4 groups were monitored (Fig. 1 and 2). During the fermentation period, the pH and acidity changes in the 4 groups showed similar patterns. The pH decreased from a range of 5.14–5.74 to 3.95–4.11, and the acidity increased from a range of 0.28%–0.39% to 0.93%–0.99%. As the BJP content of the BAK groups increased, the pH decreased and the acidity increased (P < 0.05). The total bacterial and LAB counts in the BAK groups ranged from 9.11 log CFU/mL to 9.53 log CFU/mL and 9.11 log CFU/mL to 9.60 log CFU/mL, respectively, with the highest levels observed at 2 weeks of fermentation (P < 0.05). However, there was no significant difference according to the amount of BJP added.

Changes in the TPC and TFC during fermentation of the 4 groups were monitored (Table 1). BAK groups showed higher TPC and TFC relative to those of the control group, regardless of fermentation time. During the fermentation period, the TPC and TFC in the BAK groups ranged from 4.82 µg GAE/mg to 6.63 µg GAE/mg and 3.61 µg GAE/mg to 5.36 µg CE/mg, respectively, which were higher than those in the control group (P < 0.05). In the BAK groups, TPC and TFC increased along with the concentration of BJP until the first week of fermentation. However, there was no significant difference in TPC or TFC according to the amount of BJP added after 2 weeks of fermentation. The TPC and TFC in the BAK groups increased up to 2 weeks of fermentation, reaching their highest levels, followed by decreases. The BAK-1 group exhibited the highest TPC (6.63 µg GAE/mg) and TFC (5.36 µg CE/mg) levels at 2 weeks of fermentation.

The changes in DPPH free radical and ABTS cation radical scavenging activities and reducing power observed during the fermentation period in each of the 4 groups are shown in Table 2. The changes in DPPH and ABTS scavenging activities in the BAK groups were similar during fermentation, although the ABTS EC₅₀ values were lower. The DPPH and ABTS scavenging activities of the BAK groups were significantly higher than that of the control group, regardless of fermentation time (P < 0.05). The DPPH and ABTS EC₅₀ values of the BAK groups ranged from 9.41 mg/mL to 30.00 mg/mL and 8.26 mg/mL to 23.52 mg/mL, respectively, with the lowest and highest values observed in the BAK-1 group fermented for 2 weeks and the BAK-0.5 group immediately after creation (0 weeks).

Similar to the DPPH and ABTS scavenging activities, the reducing power observed in the BAK groups was significantly higher than that of the control group, showing the highest activity at 2 weeks of fermentation (P < 0.05). The EC₅₀ values of the BAK groups ranged from 3.54 mg/mL to 6.01 mg/mL, with the highest reducing power observed in the BAK-2 group. In contrast to the DPPH and ABTS scavenging activities, the reducing power significantly increased depending on the amount of BJP added (P < 0.05). Based on these results, the BAK-1 group exhibiting excellent antioxidant activities was selected for analysis of antioxidant activity in vivo.

Changes in serum GOT and GPT activities following ingestion of BAK-1 are shown in Fig. 3. GOT and GPT activities were significantly elevated in the liver of rats treated with TAA as compared with the normal group (P < 0.001). The TAA + BAK-1 group displayed significantly reduced GOT and GPT activities compared with those of the TAA group (P < 0.001), whereas the TAA + CTL group showed no significant difference. Compared with the TAA + CTL group, the GOT and GPT activities of the TAA + BAK-1 group were significantly decreased by 14.1% and 60.5%, respectively (P < 0.01).

The effects of dietary supplementation with BAK-1 on hepatic SOD activity reduced by TAA treatment were assessed. As shown in Fig. 4, hepatic SOD activity was significantly decreased in the TAA-treated group as compared with that observed in the normal group (P < 0.01). BAK-1 supplementation remarkably restored the hepatic SOD activity reduced by TAA (P < 0.001), and these effects were more pronounced than those of kimchi without BJP. Additionally, compared with the TAA + CTL group, SOD activity in the TAA + BAK-1 group was higher (P < 0.01), increasing by 48.2%.