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Article

Functional Kimchi Beverage Enhanced with γ-Aminobutyric Acid (GABA) Through Serial Co-Fermentation Using Leuconostoc citreum S5 and Lactiplantibacillus plantarum KS2020

by
Min-Jeong Kwon
1,
Ji-Eun Kim
2 and
Sam-Pin Lee
1,2,*
1
Department of Food Science and Technology, Keimyung University, Daegu 42601, Republic of Korea
2
The Center for Traditional Microorganism Resources, Keimyung University, Daegu 42601, Republic of Korea
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(1), 44; https://doi.org/10.3390/fermentation11010044
Submission received: 25 December 2024 / Revised: 13 January 2025 / Accepted: 17 January 2025 / Published: 19 January 2025
(This article belongs to the Special Issue Application of Lactobacillus in Fermented Food and Beverages, 2nd Edition)
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Abstract

:
A plant-based beverage enhanced with GABA was developed through serial co-fermentation using Leuconostoc citreum S5 and Lactiplantibacillus plantarum KS2020. The first lactic acid fermentation was performed by Leu. citreum S5 with a vegetable mixture consisting of sliced radish, ginger, garlic, red pepper, bell pepper, and sucrose. The viable cell count of Leu. citreum S5 increased to 9.11–9.42 log CFU/mL with higher sucrose contents, indicating the highest value of 9.42 log CFU/mL at 5% sucrose on day 1. Mannitol and dextran production levels in the first fermented vegetable mixture were 6.66–14.54 mg/mL and 0.44–2.26%, respectively. A higher sucrose content produced more dextran, resulting in a concomitant increase in viscosity of 49.4 mPa·s. The second co-fermentation for the kimchi beverage base was performed by Lb. plantarum KS2020 for 5 days, resulting in 8.22–9.60 log CFU/mL. The pH of the co-fermented kimchi beverage base increased to 6.19–9.57 with an increasing monosodium glutamate (MSG) content (3–7%), while titratable acidity significantly decreased to 0.0–0.8%. The final co-fermented kimchi beverage base was enriched with 2.6% GABA. Consequently, a GABA kimchi beverage base with probiotics, a red pigment, and a pleasant flavor was developed using only vegetable ingredients by serial co-fermentation using lactic acid bacteria.

1. Introduction

Functional beverages are currently being actively studied in the field of functional foods [1]. Among functional foods, beverages in the liquid form are particularly appealing and desirable due to their convenience and ability to facilitate easy ingestion [2]. Furthermore, the global demand for dairy-free, plant-based probiotic beverages is increasing, driven by their nutritional benefits, which include minerals, dietary fibers, antioxidants, and vitamins [3]. At the same time, with the rising incidence of stress-related disorders, environmental changes, and irregular lifestyles, there is an increasing interest in products offering functional benefits such as sleep enhancement and stress relief. This trend underscores the need for further development of plant-based beverages, coupled with research into novel probiotic formulations and functionalities.
Lactic acid bacteria (LAB) as probiotics play an important role in the fermentation of traditional Korean kimchi, a health food that has gained global recognition. The commercialization of different types of kimchi has led to an increase in its consumption abroad [4]. Various kimchi are prepared by fermenting primary vegetables such as cabbage, radish, and cucumber with seasonings containing red pepper, garlic, ginger, and salted fish. During fermentation, LAB can produce metabolites including lactic acid and carbon dioxide, and enhance nutrient acquisition as well as animal immune system stimulation [5]. Furthermore, the fermentation process involves distinct stages driven by different types of LAB. The initial fermentation of kimchi produces CO2, ethanol, and mannitol by heterofermentative LAB containing Leuconostoc spp. The more acidic environment is generated in the late stage of kimchi fermentation, allowing homofermentative LAB such as Lactobacillus spp. [6,7]. In the acid fermentation of vegetables, it is well known that the heterotype and homotype LAB play a role in the production of palatable kimchi, accompanied by typical co-fermentation.
In particular, LAB are known for their ability to produce gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter in mammals [8]. GABA is a non-proteinogenic amino acid that is synthesized from glutamic acid through decarboxylation by glutamate decarboxylase (GAD) produced by LAB [9]. It is reported that GABA, as a bioactive compound, exhibits numerous physiological functions, including improving sleep, depression, stress, and blood pressure. Although GABA is naturally present in several foods, including tea, soybeans, germinated rice, and kimchi, its content level in these foods is relatively low [10]. A comparison study of GABA production between germinated brown rice and probiotics, such as the Lactobacillus strain, reported that the bacteria produce higher amounts of GABA [11,12].
Recently, consumers including vegetarians have increasingly preferred plant-based beverages as alternatives to dairy milk due to their health and environmental benefits [13]. This growing preference has contributed to the global expansion of the plant-based beverage market [3]. In line with this trend, kimchi is being reimagined as a plant-based beverage by modifying its raw materials. To globalize traditional kimchi as a health food, it is necessary to develop a functional kimchi beverage that not only offers health benefits, but also has an appealing taste and flavor. For developing a functional kimchi beverage with palatability, the lactic acid bacteria fermentation could be optimized with a selected combination of raw materials that excludes salted fish. In particular, a GABA-enriched kimchi beverage base using LAB can provide a potential functional beverage as a lactic acid-fermented food.
In this study, we developed a functional kimchi beverage base, enhanced with higher GABA through serial co-fermentation using Leu. citreum S5 and Lb. plantarum KS2020. Our findings highlight novel co-fermentation techniques for developing GABA-enriched kimchi beverages with palatability, reduced sourness, indigenous flavor, and probiotics.

2. Materials and Methods

2.1. Starter Culture of Lactic Acid Bacteria

Leuconostoc citreum S5 (Supplementary Table S1 and Figure S1) and Lactiplantibacillus plantarum KS2020 (unpublished data), both evaluated as safe probiotics, were utilized in this study. Leu. citreum S5 was used as the starter culture for the first fermentation of the vegetable mixture. Leu. citreum S5 was isolated from dongchimi and registered as KCCM 10778P at the Korean Culture Center of Microorganisms (KCCM, Seoul, Republic of Korea). This strain was cultured on a de Man, Rogosa and Sharpe (MRS) agar plate at 25 °C for 48 h. After that, the strain was inoculated in MRS broth (BD Difco, Franklin Lakes, NJ, USA) and incubated at 25 °C for 24 h.
Lb. plantarum KS2020, isolated from dongchimi, was deposited as KCCM 12782P. Lb. plantarum KS2020 has a high production ability for GABA, and this strain was used as the second fermentation starter culture for a kimchi beverage base. The strain was cultivated on an MRS agar plate at 30 °C for 48 h, and then cultured in MRS broth at 30 °C for 24 h.

2.2. Preparation of Kimchi Beverage Base

The radish, garlic, and red pepper powder used for making the kimchi beverage base were purchased from E-mart in Daegu (Republic of Korea). Ginger and bell pepper powder were sourced from Bioworks (Gyeongbuk, Republic of Korea) and MY (Seoul, Republic of Korea). The radish was washed, peeled, and cut into pieces, and combined with 0.5% (w/v) garlic and 0.5% (w/v) ginger. These ingredients were mixed with 1% (w/v) red pepper powder and 1% (w/v) bell pepper powder. Nutrients such as sucrose (Q1, Seoul, Republic of Korea) and yeast extract (Choheung, Gyeonggi, Republic of Korea) were then added at concentrations optimized for LAB fermentation. The final volume was adjusted with sterile water. The vegetable mixture without salted fish was fermented in 1 L of glass bottles.
For the first fermentation, 0.75% (w/v) yeast extract as a nitrogen source and 0–5% (w/v) sucrose as a carbon source were added to the vegetable mixture. Subsequently, 1% (v/v) of Leuconostoc citreum S5 starter was inoculated and cultured in a constant temperature incubator at 25 °C for 1 day. For the serial co-fermentation for GABA production, 3–7% (w/v) of MSG (CJ Cheil Jedang, Seoul, Republic of Korea) was incorporated as a precursor in the first fermented vegetable mixture. A 1% (v/v) of Lactiplantibacillus plantarum KS2020 starter, a GABA-producing strain, was inoculated and incubated at 30 °C for 5 days.

2.3. Measurement of pH, Titratable Acidity, and Viable Cell Count

The pH of the kimchi beverage base was measured using a pH meter (SevenEasy pH, Mettler-Toledo AG, Schwerzenbach, Switzerland). The titratable acidity was titrated with 0.1 N NaOH (Samchun, Gyeonggi, Republic of Korea) until the endpoint pH of 8.3 was reached, and the value was calculated as the percentage of lactic acid produced.
The viable count of bacterial cells in a kimchi beverage base with two LAB strains was determined by plating in 10-fold serial dilutions using 0.85% sterile saline. To distinguish between the two types of strain, Leu. citreum was plated on a medium supplemented with 2% (w/v) sucrose, 0.5% (w/v) yeast extract, 0.25% (w/v) tryptone (BD Difco, Franklin Lakes, NJ, USA), and 0.25% (w/v) K2HPO4 (Duksan, Gyeonggi, Republic of Korea) dedicated to this strain, and incubated at 25 °C. While Lb. plantarum was plated on an MRS agar medium and incubated at 30 °C for 48 h, the colonies formed on the plates were counted and expressed as log CFU/mL. 

2.4. Analysis of Free Sugars and Organic Acids

Free sugars analysis of sucrose, glucose, fructose, and mannitol in the kimchi beverage base was performed by a high-performance liquid chromatography (HPLC) system (Knauer K-501b system). The sample was centrifuged at 15,710× g for 15 min (Eppendorf AG, Hamburg, Germany), and filtered through a 0.45 μm membrane filter. The supernatant was then diluted with deionized water, and 20 uL of the solution was injected into the injector. During HPLC analysis, the RI detector (Knauer K-2301) was used as the detector, and the sugar analysis column NH2P-50 4E 4.6 × 250 mm (Shodex, Tokyo, Japan) was employed. The mobile phase (75% acetonitrile) was run for 25 min with a flow rate of 1.0 mL/min and oven temperature of 30 °C.
The organic acids content of acetic acid, lactic acid, oxalic acid, citric acid, and malic acid in the kimchi beverage base was analyzed using HPLC (Waters 1695, Waters Co., Milford, MA, USA) with a Capcell Pak C18 column (UG120, 250 × 4.6 mm, 5 µm). Samples were filtered using a 0.45μm filter. The injected sample was monitored with a PDA detector (Waters series 2487) at 210 nm. The mobile phase consisted of equal amounts of solvent A (50 mM sodium hexanesulfonate, 20 mM H3PO4, pH 2.3) and solvent B (distilled water). The flow rate was 0.6 mL/min, with a sample injection volume of 10 µL, and the column temperature was set at 40 °C. The calculation of organic acid was derived using the following formula,
Organic acid (mg/100 g) = (C × V × D)/(S × 10)
C: concentration of sample (µg/mL), V: total volume of solution (mL), D: dilution factor, S: sample (g)

2.5. Measurement of Dextran and Viscosity

The dextran content in the vegetable mixture was measured for both water-soluble and insoluble dextran during the first LAB fermentation. To measure soluble dextran, the culture broth was diluted three times with distilled water and centrifuged at 24,948× g for 20 min (Hanil, Daejeon, Republic of Korea). Then, twice the volume of chilled 99.9% ethanol (Duksan, Gyeonggi, Republic of Korea) was added to the supernatant for the aggregation of the dextran polymer, and the supernatant was removed after centrifugation. For insoluble dextran, the method of Kim et al. [14] was used. The previously three-fold diluted sample was centrifuged, and the remaining precipitate was dissolved in 10% (w/v) KOH solution (Duksan, Gyeonggi, Republic of Korea). To separate the dextran from the cells, the solution was centrifuged as with the soluble dextran. The supernatant was treated with chilled ethanol to precipitate the dextran, and then decanted after centrifugation. The precipitates were dried in a dry oven at 105 °C, weighed, and the soluble and insoluble dextran content was calculated.
The apparent viscosity of the vegetable mixture was measured during the first fermentation using a viscometer (LVT; Brookfield, Middleboro, MA, USA). A total of 30 mL of the sample was placed into the cylinder, ensuring that the LV spindle was completely submerged in the sample. Apparent viscosity was determined at room temperature using spindle No. 01 at speeds of 6, 12, 30, and 60 rpm. All determinations were performed in five replicates and the results were expressed as mPa·s.

2.6. Qualitative Analysis of GABA and Glutamic Acid

MSG consumption and GABA production were determined by thin layer chromatography (TLC) using a silica gel 60 F254 plate (Merck KGaA, Darmstadt, Germany). The supernatant of the sample obtained after centrifugation (Eppendorf AG, Hamburg, Germany; 13,000 rpm, 15 min) was diluted three-fold with distilled water. A total of 2 µL of each sample solution was spotted onto the TLC plate and eluted using a developing solvent (organic layer of n-butyl alcohol/glacial acetic acid/distilled water (3:1:1) mixture). After drying, the silica gel plate was treated with a coloring reagent (0.2% ninhydrin; Samchun, Gyeonggi, Republic of Korea) and dried at 100 °C. The standard solutions utilized 0.5% MSG and 0.5% GABA.

2.7. Quantitative Analysis of Free Amino Acids

Quantitative analysis of free amino acids in the kimchi beverage base was conducted using HPLC (Alliance 2695, Milford, MA, USA). The sample was hydrolyzed with 6 N HCl at 110 °C in a dry oven for 22 h, and the filtrate obtained through a 0.45 µm syringe filter was used as the test solution. For derivatization, 20 µL of AccQ-Fluor reagent was added to 10 µL of the test solution, left for 1 min, heated in a 55 °C oven for 10 min, and used as the final test solution. An AccQ ∙ TaqTM C18 column (3.9 × 150 mm) was used. The mobile phase (A, Waters AccQ Taq Eluent; B, 60% acetonitrile; C, tertiary distilled water) was run for 25 min. The flow rate was 1 mL/min. The amino acid content was determined by measuring the absorbance at excitation (250 nm) and emission (395 nm) using a Waters 2475 fluorescence detector. The free amino acid content was calculated using the following formula,
GABA (mg/100 g) = (S × V × D × 100)/(W × 1000)
S: concentration of GABA in test solution (µg/mL), V: total volume of test solution (mL), D: dilution factor, W: sample (g).

2.8. Measurement of Color

The color of the kimchi beverage base was assessed using a colorimeter (Minolta Chroma Meter CR-400, Osaka, Japan; calibrated with a white plate, L* = +93.65, a* = +0.31, b* = +0.31) during the fermentation periods. The color is expressed in terms of L* (lightness), a* (redness), b* (yellowness), and C* (chroma) value. The C* value was calculated using the following equation: C* = [(a*)2 + (b*)2]1/2 [15]. All determinations were performed in five replicates.

2.9. Statistical Analysis

All experiments were replicated at least three times for statistical analysis. The data were expressed as mean value ± standard deviation. Statistical significance was determined using a two-way analysis of variance (ANOVA) employing SPSS Ver. 27.0 (SPSS INC., Chicago, IL, USA). Duncan’s multiple range test (p < 0.05) was performed to determine significant differences among the samples.

3. Results and Discussion

3.1. Physicochemical Properties of Vegetable Mixture with Different Sucrose Contents

The pH and total titratable acidity levels of kimchi are important quality parameters influencing LAB growth and metabolite accumulation [16]. There is a correlation between the pH value and the titratable acidity, with the pH decreasing from the start of fermentation, while the titratable acidity increases significantly during lactic acid fermentation [17]. During the first lactic acid fermentation using Leu. citreum S5, the initial pH of the vegetable mixture was 6.43–6.47, with a titratable acidity of 0.18–0.19% (Table 1). After the first LAB fermentation for 1 day, the pH decreased to 5.14–5.20 as the titratable acidity increased to 0.80–0.94%. Compared to the vegetable mixture fermented without sucrose (S0), a higher sucrose content of 5% (S5) resulted in greater acid production, which was measured as 0.94%.
The viable cell counts of Leu. citreum S5 in the first fermented vegetable mixture was measured based on the sucrose content (0–5%) (Table 1). The initial viable cell count of Leu. citreum S5 was 6.96 log CFU/mL, which increased in vegetable mixture with S0 (0% sucrose), S1 (1% sucrose), S3 (3% sucrose), and S5 (5% sucrose) to 9.11, 9.31, 9.37, and 9.42 log CFU/mL, respectively, after the first lactic acid fermentation for 1 day. These results indicate that the viable cell count of Leu. citreum S5 increased with higher sucrose content. It was reported that the carbon sources for LAB growth in fermentation play an important role [18]. This study confirmed that the S0 had a lower viable cell count on the first day of fermentation compared to the other conditions (S1–S5) due to insufficient carbon sources. Therefore, at least 1% sucrose was required for LAB growth and higher viable cell counts.
The viable cells were determined in the second co-fermentation for the production of the kimchi beverage base (Figure 1). Leu. citreum S5 cells gradually decreased, showing 7.70–8.49 log CFU/mL, except the kimchi beverage base without sucrose (S0) (7.12 log CFU/mL) on day 5. It showed that the viable cells of Leu. citreum S5 cultured without sucrose decreased significantly compared to those cultured with sucrose. However, during the second co-fermentation, the initial viable cell count of Lb. plantarum KS2020 was 7.52 log CFU/mL, which increased to 8.94–9.33 log CFU/mL of all the co-fermented kimchi beverage bases on day 1. Additionally, the kimchi beverage base containing sucrose (S1, S3, and S5) grew to approximately 9.60 log CFU/mL on day 3. In contrast, the kimchi beverage base without sucrose (S0) decreased from day 3, and reached 8.22 log CFU/mL on day 5. The kimchi beverage base co-fermented in the presence of sucrose (S1, S3, and S5) showed viable cell counts of 9.01, 9.52, and 9.60 log CFU/mL, respectively. These results indicate that LAB viable cell counts vary depending on the sucrose content added to the kimchi beverage base during the first lactic acid fermentation. In a report by Tkesheliadze et al. [17], Lb. plantarum increased by approximately 1 log CFU/mL within 24 h of fermentation, followed by a decline as sugar was depleted in the fermented apple juice. Similarly, Li et al. [19] observed rapid growth of Lb. plantarum during the first 24 h of fermentation under adequate nutrient content and suitable growth conditions, after which their growth slowed down. However, in this study, the viable cell counts of the kimchi beverage base co-fermented by Lb. plantarum KS2020 remained high for 5 days, highlighting its potential as a probiotic beverage with higher viable LAB cell counts.
The sucrose, fructose, glucose, and mannitol in the kimchi beverage base were quantified by HPLC based on sucrose content during fermentation (Figure 2). In the unfermented kimchi beverage base, 6.06–6.76 mg/mL of fructose, 4.57–5.08 mg/mL of glucose, and 1.71 mg/mL of sucrose were detected. The sucrose content of the kimchi beverage base (S1, S3, and S5) was also confirmed (S1, 13.51 mg/mL; S3, 46.91 mg/mL; S5, 65.59 mg/mL). As lactic acid fermentation progressed, sucrose content decreased rapidly in the early stage of fermentation. Sucrose was not detected in the kimchi beverage base without sucrose (S0) on day 1, and in the other conditions with adding sucrose (S1, S3, S5) on day 5 of the second co-fermentation. In contrast, mannitol, which was not detected at the beginning of fermentation, was produced by Leu. citreum S5 on day 1 (S0, 6.66 mg/mL; S1, 9.85 mg/mL; S3, 14.10 mg/mL; S5, 14.54 mg/mL). Mannitol, a six-carbon sugar alcohol that imparts a refreshing taste, is synthesized by Leuconostoc strains in kimchi through the reduction in fructose [20]. After the second co-lactic acid fermentation was completed for 5 days, mannitol was not measured in all samples except for S5 (5% sucrose), suggesting that Lb. plantarum can utilize mannitol [21]. Fructose and glucose were also consumed alongside sucrose decomposition by Leu. citreum S5 during day 1 of the first fermentation. Interestingly, Leu. citreum S5 utilized fructose more efficiently than glucose. Similarly to sucrose, fructose and glucose were not detected at the end of co-fermentation.
Overall, on day 1 of the first fermentation, sucrose was decomposed into fructose and glucose, while Leu. citreum S5 produced mannitol from the decomposed sucrose. Similarly to findings from other studies, Leuconostoc spp., as heterofermentative LAB, produced significant amounts of mannitol through the reductive action of fructose, glucose, and sucrose [22,23]. The fermented vegetable mixture with 5% sucrose (S5) produced the highest amount of mannitol (15 mg/mL), which remained even after co-fermentation for 5 days. The results demonstrated that LAB consumed four types of free sugars, of which mannitol was consumed last. According to the reported data [17], various bacterial strains ferment sugars in different ways depending on the substrate and fermentation time. For instance, in a probiotic cupuassu beverage, fructose was the most consumed sugar during fermentation (84.76%), followed by sucrose (62.10%) and glucose (34.52%) [24]. During LAB fermentation, glucose concentrations were reduced significantly compared to fructose. The consumption patterns also varied among strains, with Lb. plantarum and Lactobacillus delbrueckii showing a greater affinity for glucose, while Lacticaseibacillus paracasei exhibited the least ability to consume sucrose [25].
The biopolymer dextran is primarily formed by Leuconostoc strains via the fermentation of sucrose-rich media [26]. Excess hexoses are utilized by various LAB groups to produce exopolysaccharides such as dextran and levan [27]. The dextran content of the unfermented vegetable mixture was initially measured to be at 0.44% soluble and 0.04% insoluble, which are presumed to be small amounts of solid components in the vegetable mixture that had not completely precipitated (Figure 3A). In the vegetable mixture fermented without sucrose (S0), no dextran was produced until the 2 days of the first fermentation, whereas in vegetable mixture fermented with sucrose (S1, S3, and S5), soluble dextran increased to 0.65%, 1.55%, and 2.26% by the first day, respectively. Subsequently, these levels were generally maintained, although S5 continued to increase until day 2, likely due to residual fructose. Insoluble dextran also showed a slight increase on day 1 of fermentation, to 0.12%, 0.19%, and 0.20% for all other samples except S0. Overall, the results indicate that dextran production was dependent on the sucrose content of the vegetable mixture. In the study reported by Zhang et al. [28], dextran produced by various LAB exhibited desirable water-holding capacity and rheology in food. It was reported that the Leu. mesenteroides N5 strain, isolated from Borassus flabellifer sap, produced 14.53 g/L of dextran in an MRS medium with 15% sucrose [29]. Thus, it was found that dextran production was superior in the minimal vegetable mixture in this study. It is implied that the production of dextran as a biopolymer was dependent upon the nutrition composition of medium and microbial strains.
Dextran increases the viscosity of the solution due to its triple helical structure [30]. The viscosity increased noticeably as the sucrose content was higher during the first fermentation (Figure 3B). In addition, the viscosity of the vegetable mixture fermented with 5% sucrose (S5) increased to 49.4 mPa·s on day 1, and it slightly increased until day 2. These results were similar to the trend of dextran content; however, no significant difference across fermentation days was noted in other vegetable mixtures with lower sucrose content within the error range. In particular, the apparent viscosity of fermented vegetable mixture decreased as the shear rate (rotational speed) increased, indicating the pseudoplastic fluid as a non-Newtonian fluid. Similarly, the dextran solution exhibited the characteristics of a pseudoplastic non-Newtonian fluid, whose viscosity decreased with an increasing shear rate [31,32]. Thus, a higher sucrose content produced more dextran, resulting in a concomitant increase in viscosity. The dextran produced at sucrose contents above 3% could affect the viscosity of the kimchi beverage base.
In conclusion, although mannitol imparts a refreshing taste, it is viewed unfavorably in this study due to its potential to remain after fermentation and contribute to acid formation, leading to possible quality changes [33]. Additionally, GABA qualitative analysis indicated that 1% sucrose alone is sufficient to support GABA conversion (Supplementary Figure S2) without significantly affecting the viscosity. Taken together, the vegetable mixture with 1% sucrose (S1) which was fermented by Leu. citreum S5 showed a viable cell count of 9.31 log CFU/mL and a titratable acidity of 0.87% on day 1. It also exhibited 9.85 mg/mL mannitol, which was completely consumed after co-fermentation for 5 days. Therefore, the fermented vegetable mixture (S1) with 0.65% dextran was selected as the optimal condition for the first fermentation owing to its minimal use of sucrose and storage stability by limiting fermenting sugar.

3.2. Physicochemical Properties of Kimchi Beverage Base Co-Fermented with Different MSG Contents

For higher GABA production, serial co-fermentation was conducted by Lb. plantarum using a vegetable mixture first fermented by Leu. citreum S5 with 1% sucrose. GABA production was sufficiently achieved in the presence of monosodium glutamate (MSG) with a nutrient composition containing only 1% fermentable sugar. The pH and titratable acidity of the co-fermented kimchi beverage base were determined when MSG was added (M3, 3% MSG; M5, 5% MSG; M7, 7% MSG) as shown in Figure 4. As the second co-fermentation began, the pH was measured at 6.56–6.99 for day 0, depending on the content of MSG added. After 1 day, as organic acid was produced, the pH of the kimchi beverage base decreased to 6.12, 6.37, and 6.51 for M3, M5, and M7, respectively. By day 5, as MSG was converted to GABA, the pH increased slightly to 6.19 in the kimchi beverage base (M3), but increased significantly to 8.84 and 9.57 in the kimchi beverage base (M5 and M7), respectively. This is related to the fact that GABA is synthesized from glutamate by glutamate decarboxylase (GAD). This enzyme participates in controlling the acidification of the cytosolic environment by decarboxylating glutamate (an acid substrate) into a neutral compound (GABA) by consuming H+ ions [34,35]. Following the same trend as pH, the titratable acidity increased to 1.01%, 1.03%, and 1.09% for M3, M5, and M7 after the second fermentation for day 1. The decreased pH value could be attributed to sugar consumption and subsequent acid production [19]. By day 5, the titratable acidity of the kimchi beverage base with 3% MSG (M3) decreased to 0.79%, but the titratable acidity of the kimchi beverage base with 5% MSG (M5) and 7% MSG (M7) was not measured. It is concluded that the MSG in the culture medium during lactic acid fermentation using GABA-producing LAB influenced the reduction in titratable acidity due to the proton consumption.
During the second co-fermentation, Lb. plantarum capable of producing GABA can use MSG as a precursor and convert it into GABA. The conversion rate varied depending on the content of MSG and the fermentation period (Figure 5). In all kimchi beverage bases fermented, a small amount of GABA began to appear on day 1. For the kimchi beverage base co-fermented with 3% MSG (M3), the MSG exhaustion through GABA conversion occurred on day 3. However, 5% MSG in the kimchi beverage base (M5) was completely utilized for 5 days. In contrast, a large amount of MSG remained in the kimchi beverage base co-fermented with 7% MSG (M7) on day 5. The complete bioconversion of 5% MSG in the kimchi beverage base co-fermented by Lb. plantarum KS2020 was identified as the optimal condition for producing a GABA-enriched kimchi beverage base. Thus, a kimchi beverage base with higher GABA could be suitable as a fermented ingredient for functional kimchi beverages and health foods. Conclusively, a GABA-enriched kimchi beverage base could be developed by serial co-fermentation using LAB in the minimal vegetable mixture with 5% MSG as a precursor.
Quantitative analysis of GABA in the kimchi beverage base co-fermented with 5% MSG (M5) is shown in Table 2. On day 5 of the second co-fermentation, the end date of fermentation, the GABA content of the co-fermented kimchi beverage base with 5% MSG was measured at 26.38 mg/g, and the glutamic acid content was 1.49 mg/g. Compared to previous studies, this study achieved higher GABA production. It is reported that GABA production (25.2 mg/100 mL) was observed in a whey protein drink containing banana concentrate stored at 25 °C on the 15th day [12]. Additionally, brown rice milk had a GABA content of 30.2 mg/100 mL [36], and cherry kefir beverage had 3.8 mg/mL of GABA [37]. Recently, GABA production in rice wine lee vinegar was reported at 22.61 mg/g [38]. Kimchi fermented with 1% (w/w) MSG showed a GABA content of 120.3 mg/100 g after 8 weeks [39]. In this study, the higher GABA-enriched kimchi beverage base could be developed for a short period. It is also implied that GABA production was facilitated by serial co-fermentation using novel lactic acid bacteria with GABA-producing ability.
Organic acids in kimchi are important quality factors. It was produced from various substrates of vegetables and other additives by various microorganisms involved in ripening. Therefore, the type and amount of organic acids depend significantly on raw materials, ripening temperature, and time [40]. The contents of organic acids such as acetic acid, lactic acid, oxalic acid, citric acid, and malic acid in the kimchi beverage base co-fermented with 5% MSG (M5) were determined using HPLC (Table 3). Except for acetic acid, the other organic acids were contained in small amounts before LAB fermentation. During the first fermentation, acetic acid (2.29 mg/g), lactic acid (4.70 mg/g), and oxalic acid (4.80 mg/g) were produced by Leu. citreum S5, which is characteristic of heterofermentative LAB. During the second co-fermentation, lactic acid was mainly produced by Lb. plantarum KS2020, a homofermentative LAB [6], greatly increasing to 22.83 mg/g after fermentation. Acetic acid was reduced during the second co-fermentation and was not detected by the end of co-fermentation. These findings indicate that LAB either produced or consumed organic acids during LAB fermentation. Among them, Lb. plantarum KS2020 prominently released lactic acid, related to the conversion of glucose to lactic acid via the Embden–Meyerhof–Parnas pathway (EMP) in the metabolism of homofermentative LAB [41]. These results were consistent with those for kimchi reported by Lee et al. [23]. The titratable acidity of the co-fermented kimchi beverage base showed 0% titratable acidity, but the lactate content using HPLC was measured at 2.3%. This indicates that the protons of lactic acid were utilized in GABA conversion by Lb. plantarum KS2020, leaving lactic acid in the form of lactate, which contributed to an increase in the pH of the kimchi beverage base. Conclusively, the co-fermented kimchi beverage base showed no titratable acidity as it exists as lactate.
Color is an important property that directly affects visual appeal and quality evaluation, influencing consumers’ sensory acceptance of food [42]. The color measurements during co-fermentation of the kimchi beverage base co-fermented with 5% MSG are depicted in Table 4 and Figure S3. The CIE L*, a*, and b* values after fermentation were 29.32, 14.78, and 21.99, respectively, with the a* value decreasing in the second fermentation due to material dilution. Notably, the C* value ranged from 23.18 to 26.49 before and after co-fermentation, indicating a red hue of the kimchi beverage base. Similarly, in the study reported by Park et al. [43], the a* value of baechu kimchi before and after fermentation was around 15, showing a red color similar to the kimchi beverage base in this study.

4. Conclusions

This study presented the development of a higher GABA-enriched kimchi beverage base by serial co-fermentation using Leu. citreum S5 and Lb. plantarum KS2020. A GABA-enriched kimchi beverage was developed with minimal vegetables and nutrients, including yeast extract without salted fish. During the first fermentation with radish, ginger, garlic, red pepper, and bell pepper powder, the sucrose added was utilized by Leu. citreum S5 to produce mannitol (6.66–14.54 mg/mL) and dextran (0.44–2.26%), and the apparent viscosity greatly increased with 5% sucrose content. Acid production led to a pH of 5.18, a titratable acidity of 0.94%, and a viable cell count of 9.42 log CFU/mL. In the presence of 5% MSG, the functional kimchi beverage base was manufactured by co-fermentation with a vegetable mixture first fermented with 1% sucrose by Lb. plantarum KS2020. During the second co-fermentation by Lb. plantarum KS2020, the conversion to GABA resulted in an increase in pH and a decrease in titratable acidity. After the second fermentation with 5% MSG for 5 days, MSG was completely converted to GABA in a final kimchi beverage base with 26.38 mg/g GABA and 22.83 mg/g lactate. The final kimchi beverage base has a red color, low acidity, and probiotics (Leu. citreum S5; 8.45 log CFU/mL, Lb. plantarum KS2020; 9.01 log CFU/mL). Thus, it has potential as a multi-functional ingredient for developing healthy beverages.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation11010044/s1, Table S1: Antibiotic susceptibility of Leu. citreum S5; Figure S1: Hemolytic activity and LDH release from Caco-2 cells by Leu. citreum S5; Figure S2: TLC of kimchi beverage base during co-fermentation based on sucrose content; Figure S3: Photo of the kimchi beverage base samples.

Author Contributions

Conceptualization, M.-J.K. and S.-P.L.; methodology, S.-P.L.; software, M.-J.K.; formal analysis, M.-J.K.; investigation, M.-J.K.; data curation, M.-J.K.; writing—original draft preparation, M.-J.K.; writing—review and editing, S.-P.L. and J.-E.K.; visualization, M.-J.K.; supervision, S.-P.L.; project administration, S.-P.L.; funding acquisition, S.-P.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Fermentation 11 00044 g001
Figure 1. Viable cell count of the kimchi beverage base during co-fermentation. Changes in Leu. citreum S5 and Lb. plantarum KS2020 according to sucrose content. S0, sucrose-free; sucrose content added: S1 (1% sucrose); S3 (3% sucrose); S5 (5% sucrose). The results present the mean ± standard deviation (n = 3); Statistically significant differences were observed between the groups (p < 0.05).
Figure 1. Viable cell count of the kimchi beverage base during co-fermentation. Changes in Leu. citreum S5 and Lb. plantarum KS2020 according to sucrose content. S0, sucrose-free; sucrose content added: S1 (1% sucrose); S3 (3% sucrose); S5 (5% sucrose). The results present the mean ± standard deviation (n = 3); Statistically significant differences were observed between the groups (p < 0.05).
Fermentation 11 00044 g001
Fermentation 11 00044 g002
Figure 2. Free sugar content in the kimchi beverage base during co-fermentation. Changes in sucrose, fructose, glucose, and mannitol content according to sucrose content. S0, sucrose-free; sucrose content added: S1 (1% sucrose); S3 (3% sucrose); S5 (5% sucrose). The results present the mean ± standard deviation (n = 3); Statistically significant differences were observed between the groups (p < 0.05).
Figure 2. Free sugar content in the kimchi beverage base during co-fermentation. Changes in sucrose, fructose, glucose, and mannitol content according to sucrose content. S0, sucrose-free; sucrose content added: S1 (1% sucrose); S3 (3% sucrose); S5 (5% sucrose). The results present the mean ± standard deviation (n = 3); Statistically significant differences were observed between the groups (p < 0.05).
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Figure 3. Dextran content and apparent viscosity in the vegetable mixture fermented by Leu. citreum S5. (A) Differences in soluble and insoluble dextran content depending on sucrose content. (B) Changes depending on sucrose content and rotational speed; viscosity = dial reading × factor (speed 6, 10; 12, 5; 30, 2; 60, 1). S0, sucrose-free; sucrose content added: S1 (1% sucrose); S3 (3% sucrose); S5 (5% sucrose). The results present the mean ± standard deviation (n ≥ 3); Different letters (A–D) and (a–d) indicate statistically significant differences between the conditions and rotational speed, respectively, (p < 0.05).
Figure 3. Dextran content and apparent viscosity in the vegetable mixture fermented by Leu. citreum S5. (A) Differences in soluble and insoluble dextran content depending on sucrose content. (B) Changes depending on sucrose content and rotational speed; viscosity = dial reading × factor (speed 6, 10; 12, 5; 30, 2; 60, 1). S0, sucrose-free; sucrose content added: S1 (1% sucrose); S3 (3% sucrose); S5 (5% sucrose). The results present the mean ± standard deviation (n ≥ 3); Different letters (A–D) and (a–d) indicate statistically significant differences between the conditions and rotational speed, respectively, (p < 0.05).
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Figure 4. pH and titratable acidity in the kimchi beverage base during co-fermentation. (A) Change in pH according to MSG content. (B) Change in titratable acidity according to MSG content. MSG content added: M3 (3% MSG); M5 (5% MSG); M7 (7% MSG). The results present the mean ± standard deviation (n = 3); Different letters (A–C) and (a–d) indicate statistically significant differences between the conditions and fermentation times, respectively, (p < 0.05).
Figure 4. pH and titratable acidity in the kimchi beverage base during co-fermentation. (A) Change in pH according to MSG content. (B) Change in titratable acidity according to MSG content. MSG content added: M3 (3% MSG); M5 (5% MSG); M7 (7% MSG). The results present the mean ± standard deviation (n = 3); Different letters (A–C) and (a–d) indicate statistically significant differences between the conditions and fermentation times, respectively, (p < 0.05).
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Figure 5. Thin-layer chromatography (TLC) of kimchi beverage base during co-fermentation. MSG content added: M3 (3% MSG); M5 (5% MSG); M7 (7% MSG).
Figure 5. Thin-layer chromatography (TLC) of kimchi beverage base during co-fermentation. MSG content added: M3 (3% MSG); M5 (5% MSG); M7 (7% MSG).
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Table 1. Changes in viable cell count, pH, and titratable acidity in the vegetable mixture fermented by Leu. citreum S5.
Table 1. Changes in viable cell count, pH, and titratable acidity in the vegetable mixture fermented by Leu. citreum S5.
SampleViable Cell Count
(log CFU/mL)		pHTitratable Acidity (%)
0 Day1 Day0 Day1 Day0 Day1 Day
S0 (1)6.96 ± 0.07 (2)9.11 ± 0.07 b(3)6.43 ± 0.035.20 ± 0.01 a0.19 ± 0.010.80 ± 0.05 b
S19.31 ± 0.09 a6.46 ± 0.005.14 ± 0.01 d0.19 ± 0.020.87 ± 0.05 ab
S39.37 ± 0.06 a6.45 ± 0.015.17 ± 0.01 c0.18 ± 0.020.89 ± 0.04 ab
S59.42 ± 0.04 a6.47 ± 0.025.18 ± 0.00 b0.19 ± 0.030.94 ± 0.05 a
(1) S0, sucrose-free; sucrose content added: S1 (1% sucrose); S3 (3% sucrose); S5 (5% sucrose). (2) The results present the mean ± standard deviation (n = 3); (3) Different letters within a column indicate statistically significant differences between the groups (p < 0.05).
Table 2. Quantitative analysis of GABA after co-fermentation of kimchi beverage base.
Table 2. Quantitative analysis of GABA after co-fermentation of kimchi beverage base.
Second Co-Fermentation (1)GABA (mg/g)Glutamic Acid (mg/g)
M5 (2)26.38 ± 0.84 (3)1.49 ± 0.05
(1) Co-fermentation was carried out for 5 days. (2) M5, 5% MSG added. (3) The results present the mean ± standard deviation (n = 3).
Table 3. Organic acids content of kimchi beverage base during co-fermentation.
Table 3. Organic acids content of kimchi beverage base during co-fermentation.
Organic Acids (mg/g)First Fermentation TimeSecond Fermentation Time
0 Day1 Day5 Days
Acetic acidN.D. (1)b2.29 ± 0.54 (2)aN.D.b(3)
Lactic acid1.39 ± 0.02 c4.70 ± 0.03 b22.83 ± 0.30 a
Oxalic acid0.76 ± 0.01 c4.80 ± 0.01 b5.04 ± 0.02 a
Citric acid1.64 ± 0.04 a0.61 ± 0.03 bN.D. c
Malic acid0.90 ± 0.01 a0.66 ± 0.02 b0.18 ± 0.01 c
(1) Not detected. (2) The results present the mean ± standard deviation (n = 3); (3) Different letters within a row indicate statistically significant differences between the groups (p < 0.05). Co-fermentation was performed using vegetable mixture first fermented with 1% sucrose and 5% MSG.
Table 4. Color of co-fermented kimchi beverage base.
Table 4. Color of co-fermented kimchi beverage base.
Color ValueFirst Fermentation TimeSecond Fermentation Time
0 Day1 Day5 Days
CIE L*26.12 ± 0.16 (1)c29.83 ± 0.15 a(2)29.32 ± 0.04 b
CIE a*14.83 ± 0.06 b16.09 ± 0.10 a14.78 ± 0.03 b
CIE b*17.82 ± 0.21 c21.76 ± 0.09 b21.99 ± 0.05 a
C*23.18 ± 0.18 c27.06 ± 0.13 a26.49 ± 0.06 b
(1) The results present the mean ± standard deviation (n = 5); (2) Different letters within a row indicate statistically significant differences between the groups (p < 0.05). Co-fermentation was performed using a vegetable mixture first fermented with 1% sucrose and 5% MSG.
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Kwon, M.-J.; Kim, J.-E.; Lee, S.-P. Functional Kimchi Beverage Enhanced with γ-Aminobutyric Acid (GABA) Through Serial Co-Fermentation Using Leuconostoc citreum S5 and Lactiplantibacillus plantarum KS2020. Fermentation 2025, 11, 44. https://doi.org/10.3390/fermentation11010044

AMA Style

Kwon M-J, Kim J-E, Lee S-P. Functional Kimchi Beverage Enhanced with γ-Aminobutyric Acid (GABA) Through Serial Co-Fermentation Using Leuconostoc citreum S5 and Lactiplantibacillus plantarum KS2020. Fermentation. 2025; 11(1):44. https://doi.org/10.3390/fermentation11010044

Chicago/Turabian Style

Kwon, Min-Jeong, Ji-Eun Kim, and Sam-Pin Lee. 2025. "Functional Kimchi Beverage Enhanced with γ-Aminobutyric Acid (GABA) Through Serial Co-Fermentation Using Leuconostoc citreum S5 and Lactiplantibacillus plantarum KS2020" Fermentation 11, no. 1: 44. https://doi.org/10.3390/fermentation11010044

APA Style

Kwon, M.-J., Kim, J.-E., & Lee, S.-P. (2025). Functional Kimchi Beverage Enhanced with γ-Aminobutyric Acid (GABA) Through Serial Co-Fermentation Using Leuconostoc citreum S5 and Lactiplantibacillus plantarum KS2020. Fermentation, 11(1), 44. https://doi.org/10.3390/fermentation11010044

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