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Fermentation characteristics and radical scavenging capacities of ginseng berry kombucha fermented by Saccharomyces cerevisiae and Gluconobacter oxydans

Fermentation characteristics and radical scavenging capacities of ginseng berry kombucha... Kombucha is a healthy carbonated beverage made by fermenting tea extracts such as green tea and black tea through symbiotic culture of bacteria and yeast. In this study, fermentation characteristics and radical scavenging activity of ginseng berry kombucha (GBK) by Saccharomyces cerevisiae M‑5 and Gluconobacter oxydans were meas‑ ured. As fermentation time increased, pH decreased and titratable acidity increased. Reducing sugars decreased rap‑ idly on day 3. Alcohol content increased dramatically during this period and then decreased. GBK showed increased radical scavenging activity and increased total flavonoid content on day 18 of fermentation compared to before fermentation. In particular, during GBK fermentation, the content of phenolic compounds such as gallic acid (2.09‑ fold) and chlorogenic acid (2.11‑fold) increased, contributing to antioxidant activity. In addition, the major ginseno ‑ sides of GBK were identified as Rg2 (10.1 μg/mg) and Re (6.59 μg/mg), and the content of minor ginsenosides, which are easily absorbed forms, increased 2.19‑fold by fermentation. GBK also extended survival in a Drosophila model treated with 15% hydrogen peroxide. GBK also reduced reactive oxygen species (p < 0.001) through upregulation of gene expression of antioxidant enzymes such as catalase (p < 0.001), superoxide dismutase (p < 0.05), and glutathione peroxidase (p < 0.001). Therefore, GBK can be presented as a functional food that inhibits oxidative stress by increasing radical scavenging activity during fermentation. Keywords Ginseng berry kombucha, Saccharomyces cerevisiae, Gluconobacter oxydans, ROS Introduction Kombucha is a non- or low-alcohol carbonated bever- age that is fermented by adding sugar to tea extracts such as black or green teas and fermenting it using a symbi- otic culture of bacteria and yeast (SCOBY), which con- *Correspondence: Hyung Joo Suh tains yeast and acetic acid bacteria [1, 16]. As kombucha suh1960@korea.ac.kr fermentation progresses, the organisms in the SCOBY Yejin Ahn produce cellulose to form a flat, soft gel or a microbial ahnyj708@gmail.com Department of Integrated Biomedical and Life Science, Graduate biofilm film, such as a mushroom cap [13]. The yeasts School, Korea University, Seoul 02841, Republic of Korea in the SCOBY include Saccharomyces sp., Zygosaccha- Transdisciplinary Major in Learning Health Systems, Department romyces kombuchaensis, Torulopsis sp., Pichia sp., Bret- of Healthcare Sciences, Graduate School, Korea University, Seoul 02841, Republic of Korea tanomyces sp., Z. bailii, Schizosaccharomyces pombe, Department of Food Science and Nutrition, Jeju National University, S. ludwigii, and Candida kefyr. Acetic acid producing Jeju 63243, Republic of Korea bacteria in SCOBY comprise Acetobacter xylinum, A. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Choi et al. Applied Biological Chemistry (2023) 66:27 Page 2 of 9 xylinoides, A. aceti, A. pasteurianus, Gluconobacter oxy- Fermentation of ginseng berry kombucha (GBK) dans, and Bacterium gluconicum [8, 32, 33]. Ginseng berries purchased from a ginseng farm in Goe- In addition to the taste of kombucha, fermentation san (Chungcheonbuk-do, Republic of Korea) were used metabolites and physiological activities are determined to prepare the GBK. For the fermentation, 2% each of the by the fermentation substrates, SCOBY constituent S. cerevisiae and G. oxydans precultures were inoculated microorganisms, additive materials, and fermentation into sterilized ginseng berry-containing broth (GBB) methods. The various types of fermented substrates and incubated at 30  °C. The change in components dur - used in the manufacture of kombucha include green ing fermentation was measured using samples collected tea, green tea, black tea, lemon balm, oak leaves, tan- at regular intervals during the preparation. GBB was gerines, coffee, Vitis coignetiae, wheat sprout juice [16]. prepared using 20.0  g of ginseng berry, 2.0  g of sucrose, Kombucha exhibits various physiological activities such 0.02  g of ascorbic acid, and 20  mL of tea infusion dis- as anti-inflammatory, antioxidant, cholesterol reduc - solved in a total of 200  mL, and adjusted to pH 6.0. A tion, lowering blood pressure, cancer cell reduction, liver solution containing 5.4  g of black tea leaves per liter of function improvement, immune modulation, intestinal water was extracted at 85 °C for 20 min and used as a tea health, antimicrobial, and antidiabetic. Moreover, it has infusion. been reported that these physiological activities are due to the presence of polyphenols in the raw material used Analysis of component changes during fermentation to manufacture kombucha. Ginsenosides, the active pH was measured using a pH meter (Orion Star A211; ingredients in ginseng, are found in higher amounts in Thermo Fisher Scientific, Waltham, MA, USA). Titratable Panax ginseng berries than in the roots and have a unique acidity was measured using the neutralization titration ginsenoside profile [14, 18]. In addition, the leaves and method [7]. The titratable acidity was converted to citric fruits of ginseng, which contain large amounts of poly- acid (%) by adding 10  mL of the sample and neutralized phenolic substances, have been reported to have excel- with 0.1 N NaOH using phenolphthalein as the indicator. lent antioxidant activity [6, 28]. Reducing sugar was analyzed using the 3,5-dinitrosali- The use of ginseng berry (ginseng fruit) as a substrate cylic acid method and was calculated using glucose as a in this study for the preparation of kombucha has not standard [22]. Total polyphenol content was determined been reported earlier. In addition, for quality control using the Folin-Ciocalteu reagent and calculated as gallic and industrial production, kombucha was manufactured acid equivalents [31]. using only yeast and acetic acid bacteria, which have been procured safely. Therefore, we inoculated S. cerevi - Microbial analysis during fermentation of GBK siae and G. oxydans from sugar-preserved ginseng and Acetogenic bacteria were isolated by plating on a medium kombucha to ferment ginseng berry kombucha (GBK). containing 3% glucose, 0.5% yeast extract, 1% C aCO , The fermentation characteristics of GBK prepared using 3% ethanol, and 2% agar. GBK diluted to an appropri- ginseng berries and isolates that differed from those ate concentration was spread on an acetic acid bacteria of traditional kombucha were evaluated. Further, the separation medium and cultured at 30 °C for 3 days. The effects of GBK on the survival rate and gene expression number of acetic acid bacteria was calculated based on of enzymes related to reactive oxygen species (ROS) the strains that produced a transparent ring [3]. removal were measured in Drosophila under hydrogen- ™ ™ For yeast analysis, 3  M Petrifilm Rapid Yeast and peroxide (H O )-induced oxidative stress. 2 2 Mold Count (RYM) (St. Paul, MN, USA) was used. After inoculating the dried films with diluted GBK and incu - bating at 30 °C for 3 days, pale pink to cyan colonies with Methods clearly distinguished edges were counted [5]. All experi- Strains ments were repeated three times, and the number of The strains used to produce kombucha in this study was microorganisms was expressed as log colony forming S. cerevisiae M-5 and G. oxydans, which were isolated and units (CFU)/mL. stored at the Nutraceuticals laboratory of Korea Univer- sity (Seoul, Korea). In a previous study, S. cerevisiae M-5, Estimation of radical scavenging activity a strain with high β-glucosidase activity, was isolated during fermentation from sugar-preserved ginseng. Also, G. oxydans is an To measure the antioxidant activity of GBK, 2,2ʹ-azino- acetic acid-producing bacterium isolated from commer- bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and cial kombucha. For the preparation of GBK, S. cerevisiae 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging M-5 and G. oxydans were pre-cultured in potato glucose activities and ferric reducing antioxidant power (FRAP) and mannitol media for 48 h at 30 °C with shaking. Choi  et al. Applied Biological Chemistry (2023) 66:27 Page 3 of 9 were measured as previously described [23]. The scav - two groups (GBK-L and GBK-H) were fed 5% sucrose enging ability of GBK was expressed as the IC value, (m/m) and 2.5 and 5.0 mg GBK, respectively, containing which was the sample concentration that reduced the 15% H O (m/m). The number of surviving Drosophila in 2 2 generated radicals by 50%. The FRAP value was extrapo - each culture tube was recorded for 24 h. lated from a standard curve, using iron (II) sulfate hep- tahydrate as the standard. Oxidation level in Drosophila (mRNA expression of related oxidative factors) Analysis of flavonoid and ginsenoside composition in GBK The Drosophila whole body was homogenized with extracts 50 mM Tris–HCl buffer (pH 7.4) in an ice bath and cen - To analyze the flavonoid composition of GBK, 100% trifuged at 4  °C (10,000 ×g for 15  min). Next, the super- ethanol was added to the GBK sample in an amount of natant was collected, and ROS production was measured 4 times the total volume (v/v = 1:4), followed by extrac- using the 2,7-dichlorofluorescin diacetate (DCF-DA) tion under reflux twice for 2 h at 90 °C. The GBK extract method as previously described [2]. Following addition was filtered, concentrated using a rotary evaporator, and of 10  μM DCF-DA to the supernatant and incubating lyophilized. The flavonoid content of the GBK extract for 30  min at 37  °C in the dark, the fluorescence (exci - was analyzed using high-performance liquid chromatog- tation, 485  nm; emission, 535  nm) was measured. Total raphy (HPLC). The HPLC instrument (Agilent, Wald - RNA from Drosophila whole bodies was extracted using bronn, Germany) equipped with a UV detector was used. TRIzol reagent (Invitrogen, Carlsbad, CA, USA) accord- YMC-Triart C 18 (250 × 4.6  mm, 5  μm) was used as the ing to the manufacturer’s protocol [12]. RQ1 RNase-free column, and 0.2% formic acid in water for mobile phase DNase I (Promega, WI, USA) treated the RNA samples A and 0.2% formic acid in acetonitrile for mobile phase were reverse-transcribed using SuperScript III reverse B were used for component separation. The gradient transcriptase (Invitrogen), and the mRNA expression of conditions of the solvent (A:B) were 95%:5% (0–2  min), superoxide dismutase (SOD), catalase (CAT), and glu- 75%:25% (10 min), 60%:40% (30 min), 50%:50% (40 min), tathione peroxidase (GPx) was analyzed by quantitative 40%:60% (50 min), and 95%:5% (55–60 min). The solvent real-time PCR using the StepOne Plus Real-time PCR flow rate was 0.8  mL/min, the column temperature was system (Applied Biosystems, CA, USA) and a Power maintained at 35 °C, and the injection volume was 10 μL. TaqMan PCR Master Mix Kit (Applied Biosystems) with The wavelengths of the UV detector were analyzed at RpL32 (NM_001144655.3). The target genes used in qRT- 260, 292, 310 and 365  nm. The ginsenoside content was PCR were SOD (NM_057387.5), CAT (NM_080483.3), also measured using a Cadenza CD-C18 (75 × 4.6  mm, and GPx (NM_168024.2). 3 μm) column in an HPLC system (Agilent). For the analysis of ginsenoside, (A) 10% acetonitrile Statistical analysis in water and (B) 90% acetonitrile in water were used The data are presented as the mean ± standard deviation as mobile phases, and the flow rate was maintained from three repeated measurements. The statistical signif - at 1.2  mL/min. The gradient conditions of the solvent icance of the experimental group was verified at P  ˂ 0.05, were 90–76%:10–24% (0–44  min), 76–60%:24–40% (44– using Tukey’s multiple range test. The analysis was car - 56  min), 60–50%:40–50% (56–79  min), 50–90%:50–10% ried out using SPSS (version 12.0; Statistical Analysis Sys- (79–82  min) and 90%:10% (82–85  min). The wavelength tem, SPSS, Inc., Chicago, IL, USA). of the UV detector was measured at 203 nm, the column temperature was 40  °C, and the injection amount was Results 5 μL. Component changes during the fermentation of ginseng berry kombucha (GBK) Estimation of survival rate in Drosophila model Changes in pH, titratable acidity, reducing sugars, poly- Canton-S wild-type Drosophila melanogaster Meigen phenols, and flavonoid contents were measured during was a gift from the Department of Food Science and the fermentation of GBK by S. cerevisiae and G. oxydans Nutrition Jeju National University. Drosophila were (Fig.  1). During kombucha fermentation, the pH gradu- reared at 25 °C with 60% humidity and a 12-h light–dark ally decreased, with a slight change being noted 3  days cycle. Adult fruit flies were reared in a standard corn after fermentation. From pH 3.92 on the 3rd day of fer- meal medium. For H O treatment, male Drosophila mentation to pH 3.68 on the 18th day, only a marginal 2 2 were transferred to empty culture tubes and fasted for decrease was observed. Titratable acidity increased with 2  h, and filter paper soaked in different solutions (200 the incubation period and ranged from 0.05% before fer- μL) was placed inside. The control (CON) was fed 5% mentation to 0.90% after 18 days (Fig. 1A). sucrose (m/m) containing 15% H O (m/m). The other 2 2 Choi et al. Applied Biological Chemistry (2023) 66:27 Page 4 of 9 Fig. 1 Changes of titratable acidity and pH (A), and components (B) during the fermentation of ginseng berry kombucha (GBK). Values are the means ± SD for each group; different letters indicate significant differences at p < 0.05 vs. day 0 in each group based on Tukey’s test. GE gallic acid equivalent The content of reducing sugars content in GBK rapidly end of the fermentation process, indicating an increase in decreased to 190.14  μg/mL on the 3rd day of fermenta- the scavenging ability after the fermentation. In addition, tion and then gradually increased to 241.49–279.85  μg/ in comparison to before fermentation, the FRAP value mL from day 6 to day 18 of fermentation (Fig.  1B). The for reducing power increased after the process (34.89 vs. alcohol content which was 0.17  mg/mL before fermen- 46.75 mM). Collectively, this implies that during the fer- tation, increased rapidly to 6.26  mg/mL on the 3rd day, mentation of GBK, there is a simultaneous increase in the and eventually declined to 2.15  mg/mL on the 18th day reducing power of FRAP and radical scavenging activity. (Fig. 1B). There was no significant change in the polyphe - nol content of GBK during fermentation, but it was high- Changes in flavonoid and ginsenoside contents est on the 12th day of fermentation (Fig. 1B). before and after fermentation As regards the measured changes in microorganisms The changes in flavonoid contents in the GBK extract, during kombucha fermentation, acetic acid bacteria presumed to be the active constituents, before and after showed a tendency to increase as the fermentation pro- fermentation, are shown in Tables  2. The total content th gressed, and the yeast numbers increased until the 9 of flavonoids, which are active ingredients, before fer - day of fermentation and gradually decreased thereafter mentation was 0.78  μg/mg but increased to 1.32  μg/mg (Table  1). The total number of bacteria also increased after fermentation (Table  2). After fermentation of GBK, until the 9th day and then gradually reduced. the major flavonoids in the extract were gallic acid, 3,4 dihydroxy benzoic acid, and chlorogenic acid, which Changes in radical scavenging activity during fermentation increased significantly after fermentation. The radical scavenging ability and reducing power were As shown in Table  3, the main ginsenosides of GBK measured before and after GBK fermentation (Fig. 2). As after fermentation were Rg2 (sum of Rg2s and Rg2r) compared to before the fermentation, the I C value for and Re, with concentrations of 10.10  μg/mg and radical scavenging of ABTS and DPPH decreased at the 6.59 μg/mg, respectively. The total ginsenoside content, Table 1 Content of different microorganisms during the fermentation of ginseng berry kombucha (GBK) CFU (× 10 /mL) Day 0 3 6 9 12 18 Acetic‑acid producing bacteria 0 1.33 ± 0.03 1.29 ± 0.01 1.98 ± 0.12 2.20 ± 0.01 2.24 ± 0.64 Yeast 0 1.52 ± 0.12 1.87 ± 0.04 3.29 ± 0.11 3.07 ± 0.17 2.81 ± 0.11 Total microorganisms 0 2.89 ± 0.13 3.28 ± 0.23 5.37 ± 0.22 5.06 ± 0.36 5.09 ± 0.51 Values are the means ± SD for each group CFU colony forming unit Choi  et al. Applied Biological Chemistry (2023) 66:27 Page 5 of 9 Fig. 2 IC value on A ABTS and B DPPH radical and C FRAP value before and after fermentation of ginseng berry kombucha (GBK). Values are the means ± SD for each group; *** indicates significant difference (p < 0.001) after fermentation as compared to before. ABTS 2,2′‑azino ‑bis(3‑ ethylbenz othiazoline‑6‑sulfonic acid), DPPH 2,2‑ diphenyl‑1‑picrylhydrazyl, FRAP ferric reducing antioxidant power Table 2 Changes in contents of flavonoids before and after Table 3 Changes in content of ginsenosides before and after ginseng berry kombucha (GBK) fermentation ginseng berry kombucha (GBK) fermentation Flavonoids Contents (μg/mg of extract) Ginsenoside Contents (μg/mg of extract) 0 day 18 day 0 day 18 day Gallic acid 0.067 ± 0.004 0.140 ± 0.011 Rg1 0.53 ± 0.02 1.04 ± 0.03 3,4‑Dihydroxybenzoic acid 0.120 ± 0.002 0.420 ± 0.032 Re 3.40 ± 0.08 6.59 ± 0.01 Rutin 0.050 ± 0.001 0.063 ± 0.002 Rf 0.21 ± 0.02 0.37 ± 0.01 Quercetin‑3‑ glucuronide 0.023 ± 0.003 0.068 ± 0.002 Rb1 0.32 ± 0.03 0.79 ± 0.05 Rc 0.59 ± 0.01 1.04 ± 0.05 Chrysin 0.082 ± 0.002 0.092 ± 0.002 Rb2 0.36 ± 0.02 0.59 ± 0.01 Chlorogenic acid 0.095 ± 0.001 0.20 ± 0.006 Rd 0.77 ± 0.01 1.36 ± 0.01 Caffeic acid 0.042 ± 0.001 0.076 ± 0.003 ρ‑ Coumaric acid 0.026 ± 0.001 0.028 ± 0.001 Rh 1 (s) 0.27 ± 0.02 1.03 ± 0.02 Trans ferulic acid 0.037 ± 0.001 0.056 ± 0.001 Rg2 (s) 0.72 ± 0.10 3.41 ± 0.04 Apigenin 0.015 ± 0.001 0.011 ± 0.001 Rg2 (r) 1.46 ± 0.10 6.69 ± 0.04 Quercetin 0.140 ± 0.001 0.100 ± 0.001 Rg6 0.140 ± 0.001 0.100 ± 0.001 Kaempferol 0.094 ± 0.001 0.072 ± 0.001 Rk3 1.02 ± 0.15 2.00 ± 0.03 Total flavonoids 0.78 ± 0.006 1.32 ± 0.024 Rh4 0.22 ± 0.04 0.59 ± 0.01 Rg3 (s) 0.75 ± 0.03 2.28 ± 0.01 Values are the means ± SD for each group Rg3 (r) 0.97 ± 0.03 0.95 ± 0.02 CK 0.35 ± 0.03 0.49 ± 0.03 Rk1 0.24 ± 0.06 0.44 ± 0.02 which was 13.34  μg/mg at the beginning of fermenta- Rg5 0.52 ± 0.11 1.23 ± 0.04 tion, increased to 32.58  μg/mg after fermentation. Rh2 (s) 0.21 ± 0.03 0.31 ± 0.00 In addition, the content of the major ginsenoside and Major ginsenosides 6.19 ± 0.19 11.78 ± 0.17 minor ginsenoside increased from 6.19 to 11.78  μg/ Minor ginsenosides 7.16 ± 0.72 20.80 ± 0.26 mg and from 7.16 to 20.80  μg/mg compared to before Total ginsenosides 13.34 ± 0.91 32.58 ± 0.43 fermentation, respectively. Therefore, it was confirmed that the content of easily absorbed ginsenosides in Values are the means ± SD for each group. Major ginsenosides: Rg1, Re, Rf, Rb1, Rc, Rb2, and Rd; minor ginsenosides: Rh1, Rg2, Rk3, Rh4, Rg3, Rk1, and Rh2 GBK increased through fermentation. In the analysis Choi et al. Applied Biological Chemistry (2023) 66:27 Page 6 of 9 of flavonoids and ginsenosides in GBK extracts, com - on the increase in oxidative stress caused by H O treat- 2 2 pounds that did not show differences were not included ment was attributed to an increase in the expression level in the results. of oxidative stress-related enzymes. Discussion Eec ff ts of ginseng berry kombucha (GBK) on survival Despite the various physiological activities of kombu- rate and relief of oxidative stress in male fruit flies cha, its application and utility in the industry are lim- under oxidative stress conditions ited. Since the fermentation of kombucha is performed The survival rate after treatment with low concentration by a microbial community, the species and distribu- (2.5%) and high concentration (5%) of GBK was meas- tion of microorganisms involved in the process are not ured in male fruit flies treated with 15% H O to induce 2 2 confirmed, and there are concerns about the safety and oxidative stress (Fig.  3). The GBK-administered groups combination of microorganisms, such as SCOBY, using showed an increase in the survival rate compared to the sugar and extracts of green or black tea as fermentation CON group treated with H O alone. A high concen- 2 2 substrates. Quality control and industrial production of tration of GBK resulted in a significant increase in the kombucha should be carried out using officially recog - survival rate compared to CON (p < 0.001). The low con - nized microorganisms that are safe for use. To achieve centration of GBK did not show a significant increase in various physiological activities to kombucha, attempts survival rate compared to the CON group. There was no should be made to use substrates other than green or significant difference in survival rates between high and black teas. Therefore, in this study, ginseng berries were low GBK concentrations. used as a fermentation substrate to prepare kombucha Figure  4 shows the effect of GBK on ROS production using S. cerevisiae and G. oxydans, and the fermentation and gene expression of SOD, CAT, and GPx in the Dros- characteristics were measured. ophila model, wherein oxidative damage was induced There was an increase in acidity and a decrease in pH by H O treatment. Treatment with low and high con- 2 2 as a result of the organic acid acetic acid produced dur- centrations of GBK significantly reduced ROS produc - ing the fermentation process by acetic acid bacteria tion by 44.5% and 65.4%, respectively, compared to the among the strains used for fermentation of kombucha control (p < 0.001). As the GBK treatment concentration [29]. Kombucha is known to release organic acids such increased, the gene expression of CAT, GPx, and SOD, as acetic, gluconic, glucuronic, citric, l-lactic, malic, tar - enzymes related to oxidative stress removal, tended taric, malonic, oxalic, succinic, and pyruvic acid through to decrease in a concentration-dependent manner. fermentation [25]. In this study, the organic acid pro- Both CAT and GPx expression levels were significantly duced by G. oxydans resulted in a decrease in pH and an increased compared to CON by both GBK concentra- increase in titratable acidity (Fig.  1). Organic acids are tions (p < 0.001). Therefore, the inhibitory effect of GBK produced due to the metabolism of the yeast and ace- tic acid bacteria during the fermentation process. These organisms utilize carbohydrates as the main substrate to produce alcohol and organic acids [34]. We found that during the fermentation of GBK, the content of reducing sugars decreased, the ethanol content initially increased and then declined, and the titratable acidity increased continuously (Fig. 1). The ethanol content generated dur - ing kombucha fermentation is reported to be 0.7% to 1.3% [4]. When the alcohol content remains above 1%, it is proposed to initiate processes such as oxygen exposure and microfiltration to lower the concentration [15]. How - ever, in this study, because the final alcohol content was 0.25%, an additional process for lowering the alcohol was not required. Kombucha largely contains polyphenols, organic acids, Fig. 3 Eec ff ts of ginseng berry kombucha (GBK) on the survival sugars, and proteins [10], and its composition varies rate in Drosophila treated with 15% H O . Significant differences 2 2 depending on the fermentation substrate used. Kom- in percent survival (%) among groups were analyzed by log‑rank statistical method (n = 100/group). CON control, 15% H O ‑treated bucha, which uses black tea (black tea kombucha) and 2 2 group, GBK-L Group treated with 2.5 mg/g of GBK and 15% H O , 2 2 green tea (green tea kombucha) as the fermentation sub- GBK-H Group treated with 5 mg/g of GBK and 15% H O 2 2 strates for kombucha, contains polyphenols, including Choi  et al. Applied Biological Chemistry (2023) 66:27 Page 7 of 9 Fig. 4 Eec ff t of ginseng berry kombucha (GBK) on A ROS production and B–D antioxidant enzyme ‑related mRNA expression in Drosophila treated with 15% H O . Values are the means ± SD for each group; different levels of significance have been indicated *p < 0.05, **p < 0.01, ***p < 0.001 in 2 2 each group based on Tukey’s test. CON: control, 15% H O ‑treated group, GBK ‑L: Group treated with 2.5 mg/g of GBK and 15% H O , GBK‑H: Group 2 2 2 2 treated with 5 mg/g of GBK and 15% H O ; ROS reactive oxygen species, CAT catalase, SOD superoxide dismutase, GPx glutathione peroxidase 2 2 catechins, as active constituents. It has been reported ginseng and converts it into a non-glycoside ginseno- that in both black and green tea kombucha, the amount side, leading to an improvement in the absorption rate of catechin decreased until the 9th day of fermentation of ginsenoside [30]. Rg2, which is produced by the de- and increased 12th day onward [11]. As shown in Table 2, glycosylation of ginsenoside Re [26], is abundantly pre- the flavonoid content increased after fermentation, and sent in ginseng berries [17]. In the GBK, not only was 3,4-dihydrobenzoic acid and chlorogenic acid were found Re increased upon fermentation, but also Rg2. The con - to be the main flavonoids. In addition, the content of centration of Rg6 produced by the dehydration reaction ginsenosides, both major and minor (which are easily of Rg2 also increased in GBK (Table  3). Ginsenosides absorbed) increased by upon fermentation (Table 3). Rb1, Rc, Rb2, Rd, Ra1, Ra2, and Ra3 are converted to The changes described above were due to the ginsenoside Rg3 by heating during the manufactur- β-glucosidase activity of S. cerevisiae used for kom- ing process of red ginseng [19]. In ginsenoside Rg3, a bucha fermentation. β-glucosidase cleaves the sugar double bond is formed by dehydration at position 20 of chain site bound to the glycoside ginsenoside of red the dammarane backbone and is converted into three Choi et al. Applied Biological Chemistry (2023) 66:27 Page 8 of 9 structural isomers of ginsenosides Rk1, Rg5, and Rz1 which has improved radical-scavenging capabilities along [20]. Ginseng berry kombucha also exhibited increased with consumer preferences, can aid in improving physi- levels of Rg3, Rk1, and Rg5. The sugar moiety attached cal health. to C-3, C-6, or C-20 ginsenosides is deglycosylated Acknowledgements and converted to minor ginsenosides, contributing to Not applicable. an increase in bioavailability and physiological activity Author contributions [27]. Therefore, minor ginsenosides produced by the Data curation: EJC, HHS, and KYK, formal analysis: HHS, and KBH, visualization: fermentation of GBK, increase its bioavailability and KYK, and KBH, methodology: HHS, and KYK, investigation: KYK, and KBH, vali‑ contribute to an increase in its physiological functions. dation: HHS, and KBH, conceptualization: HJS, and YA, project administration: HJS, and YA, supervision: HJS, and YA, writing—original draft: EJC, HJS, and YA. The physiological activity of kombucha is closely writing—review and editing: EJC, HHS, KYK, KBH, HJS, and Y A. All authors read related with its antioxidant activity. Through fermen - and approved the final manuscript. tation, kombucha prepared using green or black tea Funding increases polyphenol components, which are known This research did not receive any specific grant from funding agencies in the to be associated with an increase in antioxidant activ- public, commercial, or not‑for ‑profit sectors. ity [8]. When the antioxidant activities were compared Availability of data and materials before and after fermentation, it was noted that the All data analyzed during this study are included in this published article and its FRAP activity increased along with ABTS and DPPH supplementary information files. after fermentation (Fig.  2). The content of chlorogenic acid [24] and 3,4-dihydroxybenzoic acid [35] increases Declarations during GBK fermentation, which is involved in the Competing interests increase in antioxidant activity. The authors declare that they have no competing interests. Free radicals generated in intracellular metabolic processes lead to oxidative damage to DNA, proteins, Received: 1 February 2023 Accepted: 18 April 2023 and lipids, the major cell components. Accumulated oxidative damage affects cellular function and, fur - thermore, leads to deterioration of tissue function, and this ultimately is responsible for increasing promoting References aging, or decreasing an individual’s lifespan [9]. The 1. Antolak H, Piechota D, Kucharska A (2021) Kombucha tea—A double most abundant free radicals are ROS, which are mainly power of bioactive compounds from tea and symbiotic culture of bacte‑ ria and yeasts (SCOBY ). Antioxidants 10:1541 produced in the electron transport chain of the mito- 2. Anupama KP, Shilpa O, Antony A, Raghu SV, Gurushankara HP (2022) chondria. Free radicals, including superoxide (O ), Jatamansinol from Nardostachys jatamansi (D.Don) DC. protects Aβ42‑ hydroxyl radicals ( OH), and hydrogen peroxide (H O ) induced neurotoxicity in alzheimer’s disease Drosophila model. Neuro‑ 2 2 Toxicology 90:62–78 can be eliminated by the cellular antioxidant defense 3. Baek C‑H, Baek S‑ Y, Lee SH, Kang J‑E, Choi H‑S, Kim J‑H, Yeo S‑H (2015) mechanisms which include the use of enzymes such as Characterization of Acetobacter sp. strain CV1 isolated from a fermented SOD and CAT and a process that involves antioxidants vinegar. Microbiol Biotechnol Lett 43:126–133 4. Bishop P, Pitts ER, Budner D, Thompson‑ Witrick KA (2022) Chemical com‑ such as flavonoids, vitamins, and ginsenosides [21]. The position of kombucha. Beverages 8:45 radical-scavenging activity of GBK can be attributed to 5. Cho M‑H, Bae E‑K, Ha S‑D, Park Y ‑S, Mok C‑K, Hong K ‑P, Kim S‑P, Park J‑ Y the presence of various flavonoids, in addition to the (2005) Evaluation of dry rehydratable film method for enumeration of microorganisms in meat, dairy and fishery products. Korean J Food Sci minor ginsenosides. Administration of GBK to Dros- Technol 37:294–300 ophila induced to undergo oxidative stress by treatment 6. Chung I‑M, Lim J‑ J, Ahn M‑S, Jeong H‑N, An T ‑ J, Kim S‑H (2016) Compara‑ with H O appears to decrease ROS production due tive phenolic compound profiles and antioxidative activity of the fruit, 2 2 leaves, and roots of Korean ginseng (Panax ginseng Meyer) according to to increased gene expression of SOD, CAT, and GPx, cultivation years. J Ginseng Res 40:68–75 enzymes involved in oxidative stress removal (Figs. 3 & 7. Decarvalho VD, Chagas SJD, Chalfoun SM, Botrel N, Juste ESG (1994) 4). Relationship between the physical‑ chemical and chemical‑ composition of green coffee and the quality of coffee beverage.1. Polyphenoloxidase Oxidative stress, a product of the interaction of genetic, and peroxidase‑activity, color index and titratable acidity. Pesqui Agro ‑ environmental, and lifestyle factors, increases the risk of pecu Bras 29:449–454 aging, disease, and death. u Th s, it is necessary to identify 8. Greenwalt C, Steinkraus K, Ledford R (2000) Kombucha, the fermented tea: microbiology, composition, and claimed health effects. J Food Prot favorable foods that have the ability to suppress or elimi- 63:976–981 nate the generation of ROS and halt the acceleration of 9. Hajam YA, Rani R, Ganie SY, Sheikh TA, Javaid D, Qadri SS, Pramodh S, aging in the human body and lead to numerous diseases. Alsulimani A, Alkhanani MF, Harakeh S (2022) Oxidative stress in human pathology and aging: molecular mechanisms and perspectives. Cells The quality control of ginseng berry kombucha can be 11:552 easily achieved, and its safety can be ensured by using 10. Jayabalan R, Malbaša RV, Lončar ES, Vitas JS, Sathishkumar M (2014) A strains isolated from food. In addition, the use of GBK, review on kombucha tea—microbiology, composition, fermentation, Choi  et al. Applied Biological Chemistry (2023) 66:27 Page 9 of 9 beneficial effects, toxicity, and tea fungus. Compr Rev Food Sci Food 34. Woo H‑ G, Lee C‑M, Jeong J‑H, Choi B‑K, Huh C‑K (2021) Quality character ‑ Safety 13:538–550 istics of kombucha made with different mixing ratios of green tea extract 11. Jayabalan R, Marimuthu S, Swaminathan K (2007) Changes in content of and yuzu juice during fermentation. Korean J Food Preserv 28:646–653 organic acids and tea polyphenols during kombucha tea fermentation. 35. Yang Y‑R, Cho J‑ Y, Park Y‑K (2012) Isolation and identification of antioxida‑ Food Chem 102:392–398 tive compounds 3, 4‑ dihydroxybenzoic acid from black onion. Korean J 12. Jo K, Kim S, Ahn Y, Suh HJ (2021) Eec ff ts of green lettuce leaf extract on Food Preserv 19:229–234 sleep disturbance control in oxidative stress‑induced invertebrate and vertebrate models. Antioxidants 10:970 Publisher’s Note 13. Kapp JM, Sumner W (2019) Kombucha: a systematic review of the empiri‑ Springer Nature remains neutral with regard to jurisdictional claims in pub‑ cal evidence of human health benefit. Ann Epidemiol 30:66–70 lished maps and institutional affiliations. 14. Kim C‑K, Cho DH, Lee K ‑S, Lee D ‑K, Park C‑ W, Kim WG, Lee SJ, Ha K‑S, Goo Taeg O, Kwon Y‑ G (2012) Ginseng berry extract prevents atherogenesis via anti‑inflammatory action by upregulating phase II gene expression. Evid Based Complement Alternat Med 2012:4903301 15. Kim J, Adhikari K (2020) Current trends in kombucha: marketing perspec‑ tives and the need for improved sensory research. Beverages 6:1–19 16. Kim JY, Shin HJ, Kim HL, Park H, Kim PK, Park S, Kim SH (2020) The compo‑ sitional and functional properties of Kombucha: a literature review. Food Eng Prog 24:1–14 17. Kim S‑ J, Kim J‑D, Ko S‑K (2013) Changes in ginsenoside composition of ginseng berry extracts after a microwave and vinegar process. J Ginseng Res 37:269 18. Kim YK, Yoo DS, Xu H, Park NI, Kim HH, Choi JE, Park SU (2009) Ginseno‑ side content of berries and roots of three typical Korean ginseng (Panax ginseng) cultivars. Nat Prod Commun 4:903–906 19. Lee H‑ J, Jung E‑ Y, Lee H‑S, Kim B‑ G, Kim J‑H, Yoon T ‑ J, Oh S‑H, Suh H‑ J (2009) Bioavailability of fermented Korean red ginseng. Prev Nutr Food Sci 14:201–207 20. Lee SM (2020) Three hydroxylated ginsenosides from heat treatmented ginseng. Korean J Pharmacogn 51:255–263 21. Liu Z‑ Q (2022) Why natural antioxidants are readily recognized by biologi‑ cal systems? 3D architecture plays a role! Food Chem 380:132143 22. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428 23. Moon JK, Shibamoto T (2009) Antioxidant assays for plant and food components. J Agric Food Chem 57:1655–1666 24. Naveed M, Hejazi V, Abbas M, Kamboh AA, Khan GJ, Shumzaid M, Ahmad F, Babazadeh D, FangFang X, Modarresi‑ Ghazani F (2018) Chlorogenic acid (CGA): a pharmacological review and call for further research. Biomed Pharmacother 97:67–74 25. Neffe ‑Skocinska K, Sionek B, Scibisz I, Kolozyn‑Krajewska D (2017) Acid contents and the effect of fermentation condition of Kombucha tea beverages on physicochemical, microbiological and sensory properties. Cyta J Food 15:601–607 26. Pei J, Wu T, Yao T, Zhao L, Ding G, Wang Z, Xiao W (2017) Biotransforma‑ tion of Ginsenosides Re and Rg 1 into Rg 2 and Rh 1 by thermosta‑ ble β‑ glucosidase from Thermotoga thermarum. Chem Nat Compd 53:472–477 27. Piao XM, Huo Y, Kang JP, Mathiyalagan R, Zhang H, Yang DU, Kim M, Yang DC, Kang SC, Wang YP (2020) Diversity of ginsenoside profiles produced by various processing technologies. Molecules 25:4390 28. Ryu H‑ J, Jung C‑ J, Beik G‑ Y (2020) Antioxidant activities of flower, berry and leaf of Panax ginseng CA Meyer. Korean J Food Sci Technol 52:342–349 29. Ryu SJ, Lee CY, Kang GS, Kim SG, Kim SH, Seo DH (2021) Optimization of compositions for kombucha with Rubus coreanus. Food Eng Prog 25:118–124 30. Shim K‑S, Park G‑ G, Park Y‑S (2014) Bioconversion of puffed red ginseng extract using β‑ glucosidase‑producing lactic acid bacteria. Food Eng Prog 18:332–340 31. Singleton VL, Orthofer R, Lamuela‑Raventós RM (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of folin‑ ciocalteu reagent. Methods Enzymol 299:152–178 32. St‑Pierre DL (2019) Microbial diversity of the symbiotic colony of bacteria and yeast (SCOBY ) and its impact on the organoleptic properties of kombucha, Food Science and Human Nutrition. The University of Maine, Orono, pp 1–94 33. Vīna I, Semjonovs P, Linde R, Deniņa I (2014) Current evidence on physi‑ ological activity and expected health effects of kombucha fermented beverage. J Med Food 17:179–188 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied Biological Chemistry Springer Journals

Fermentation characteristics and radical scavenging capacities of ginseng berry kombucha fermented by Saccharomyces cerevisiae and Gluconobacter oxydans

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Springer Journals
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Copyright © The Author(s) 2023
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2468-0834
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10.1186/s13765-023-00785-3
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Abstract

Kombucha is a healthy carbonated beverage made by fermenting tea extracts such as green tea and black tea through symbiotic culture of bacteria and yeast. In this study, fermentation characteristics and radical scavenging activity of ginseng berry kombucha (GBK) by Saccharomyces cerevisiae M‑5 and Gluconobacter oxydans were meas‑ ured. As fermentation time increased, pH decreased and titratable acidity increased. Reducing sugars decreased rap‑ idly on day 3. Alcohol content increased dramatically during this period and then decreased. GBK showed increased radical scavenging activity and increased total flavonoid content on day 18 of fermentation compared to before fermentation. In particular, during GBK fermentation, the content of phenolic compounds such as gallic acid (2.09‑ fold) and chlorogenic acid (2.11‑fold) increased, contributing to antioxidant activity. In addition, the major ginseno ‑ sides of GBK were identified as Rg2 (10.1 μg/mg) and Re (6.59 μg/mg), and the content of minor ginsenosides, which are easily absorbed forms, increased 2.19‑fold by fermentation. GBK also extended survival in a Drosophila model treated with 15% hydrogen peroxide. GBK also reduced reactive oxygen species (p < 0.001) through upregulation of gene expression of antioxidant enzymes such as catalase (p < 0.001), superoxide dismutase (p < 0.05), and glutathione peroxidase (p < 0.001). Therefore, GBK can be presented as a functional food that inhibits oxidative stress by increasing radical scavenging activity during fermentation. Keywords Ginseng berry kombucha, Saccharomyces cerevisiae, Gluconobacter oxydans, ROS Introduction Kombucha is a non- or low-alcohol carbonated bever- age that is fermented by adding sugar to tea extracts such as black or green teas and fermenting it using a symbi- otic culture of bacteria and yeast (SCOBY), which con- *Correspondence: Hyung Joo Suh tains yeast and acetic acid bacteria [1, 16]. As kombucha suh1960@korea.ac.kr fermentation progresses, the organisms in the SCOBY Yejin Ahn produce cellulose to form a flat, soft gel or a microbial ahnyj708@gmail.com Department of Integrated Biomedical and Life Science, Graduate biofilm film, such as a mushroom cap [13]. The yeasts School, Korea University, Seoul 02841, Republic of Korea in the SCOBY include Saccharomyces sp., Zygosaccha- Transdisciplinary Major in Learning Health Systems, Department romyces kombuchaensis, Torulopsis sp., Pichia sp., Bret- of Healthcare Sciences, Graduate School, Korea University, Seoul 02841, Republic of Korea tanomyces sp., Z. bailii, Schizosaccharomyces pombe, Department of Food Science and Nutrition, Jeju National University, S. ludwigii, and Candida kefyr. Acetic acid producing Jeju 63243, Republic of Korea bacteria in SCOBY comprise Acetobacter xylinum, A. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Choi et al. Applied Biological Chemistry (2023) 66:27 Page 2 of 9 xylinoides, A. aceti, A. pasteurianus, Gluconobacter oxy- Fermentation of ginseng berry kombucha (GBK) dans, and Bacterium gluconicum [8, 32, 33]. Ginseng berries purchased from a ginseng farm in Goe- In addition to the taste of kombucha, fermentation san (Chungcheonbuk-do, Republic of Korea) were used metabolites and physiological activities are determined to prepare the GBK. For the fermentation, 2% each of the by the fermentation substrates, SCOBY constituent S. cerevisiae and G. oxydans precultures were inoculated microorganisms, additive materials, and fermentation into sterilized ginseng berry-containing broth (GBB) methods. The various types of fermented substrates and incubated at 30  °C. The change in components dur - used in the manufacture of kombucha include green ing fermentation was measured using samples collected tea, green tea, black tea, lemon balm, oak leaves, tan- at regular intervals during the preparation. GBB was gerines, coffee, Vitis coignetiae, wheat sprout juice [16]. prepared using 20.0  g of ginseng berry, 2.0  g of sucrose, Kombucha exhibits various physiological activities such 0.02  g of ascorbic acid, and 20  mL of tea infusion dis- as anti-inflammatory, antioxidant, cholesterol reduc - solved in a total of 200  mL, and adjusted to pH 6.0. A tion, lowering blood pressure, cancer cell reduction, liver solution containing 5.4  g of black tea leaves per liter of function improvement, immune modulation, intestinal water was extracted at 85 °C for 20 min and used as a tea health, antimicrobial, and antidiabetic. Moreover, it has infusion. been reported that these physiological activities are due to the presence of polyphenols in the raw material used Analysis of component changes during fermentation to manufacture kombucha. Ginsenosides, the active pH was measured using a pH meter (Orion Star A211; ingredients in ginseng, are found in higher amounts in Thermo Fisher Scientific, Waltham, MA, USA). Titratable Panax ginseng berries than in the roots and have a unique acidity was measured using the neutralization titration ginsenoside profile [14, 18]. In addition, the leaves and method [7]. The titratable acidity was converted to citric fruits of ginseng, which contain large amounts of poly- acid (%) by adding 10  mL of the sample and neutralized phenolic substances, have been reported to have excel- with 0.1 N NaOH using phenolphthalein as the indicator. lent antioxidant activity [6, 28]. Reducing sugar was analyzed using the 3,5-dinitrosali- The use of ginseng berry (ginseng fruit) as a substrate cylic acid method and was calculated using glucose as a in this study for the preparation of kombucha has not standard [22]. Total polyphenol content was determined been reported earlier. In addition, for quality control using the Folin-Ciocalteu reagent and calculated as gallic and industrial production, kombucha was manufactured acid equivalents [31]. using only yeast and acetic acid bacteria, which have been procured safely. Therefore, we inoculated S. cerevi - Microbial analysis during fermentation of GBK siae and G. oxydans from sugar-preserved ginseng and Acetogenic bacteria were isolated by plating on a medium kombucha to ferment ginseng berry kombucha (GBK). containing 3% glucose, 0.5% yeast extract, 1% C aCO , The fermentation characteristics of GBK prepared using 3% ethanol, and 2% agar. GBK diluted to an appropri- ginseng berries and isolates that differed from those ate concentration was spread on an acetic acid bacteria of traditional kombucha were evaluated. Further, the separation medium and cultured at 30 °C for 3 days. The effects of GBK on the survival rate and gene expression number of acetic acid bacteria was calculated based on of enzymes related to reactive oxygen species (ROS) the strains that produced a transparent ring [3]. removal were measured in Drosophila under hydrogen- ™ ™ For yeast analysis, 3  M Petrifilm Rapid Yeast and peroxide (H O )-induced oxidative stress. 2 2 Mold Count (RYM) (St. Paul, MN, USA) was used. After inoculating the dried films with diluted GBK and incu - bating at 30 °C for 3 days, pale pink to cyan colonies with Methods clearly distinguished edges were counted [5]. All experi- Strains ments were repeated three times, and the number of The strains used to produce kombucha in this study was microorganisms was expressed as log colony forming S. cerevisiae M-5 and G. oxydans, which were isolated and units (CFU)/mL. stored at the Nutraceuticals laboratory of Korea Univer- sity (Seoul, Korea). In a previous study, S. cerevisiae M-5, Estimation of radical scavenging activity a strain with high β-glucosidase activity, was isolated during fermentation from sugar-preserved ginseng. Also, G. oxydans is an To measure the antioxidant activity of GBK, 2,2ʹ-azino- acetic acid-producing bacterium isolated from commer- bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and cial kombucha. For the preparation of GBK, S. cerevisiae 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging M-5 and G. oxydans were pre-cultured in potato glucose activities and ferric reducing antioxidant power (FRAP) and mannitol media for 48 h at 30 °C with shaking. Choi  et al. Applied Biological Chemistry (2023) 66:27 Page 3 of 9 were measured as previously described [23]. The scav - two groups (GBK-L and GBK-H) were fed 5% sucrose enging ability of GBK was expressed as the IC value, (m/m) and 2.5 and 5.0 mg GBK, respectively, containing which was the sample concentration that reduced the 15% H O (m/m). The number of surviving Drosophila in 2 2 generated radicals by 50%. The FRAP value was extrapo - each culture tube was recorded for 24 h. lated from a standard curve, using iron (II) sulfate hep- tahydrate as the standard. Oxidation level in Drosophila (mRNA expression of related oxidative factors) Analysis of flavonoid and ginsenoside composition in GBK The Drosophila whole body was homogenized with extracts 50 mM Tris–HCl buffer (pH 7.4) in an ice bath and cen - To analyze the flavonoid composition of GBK, 100% trifuged at 4  °C (10,000 ×g for 15  min). Next, the super- ethanol was added to the GBK sample in an amount of natant was collected, and ROS production was measured 4 times the total volume (v/v = 1:4), followed by extrac- using the 2,7-dichlorofluorescin diacetate (DCF-DA) tion under reflux twice for 2 h at 90 °C. The GBK extract method as previously described [2]. Following addition was filtered, concentrated using a rotary evaporator, and of 10  μM DCF-DA to the supernatant and incubating lyophilized. The flavonoid content of the GBK extract for 30  min at 37  °C in the dark, the fluorescence (exci - was analyzed using high-performance liquid chromatog- tation, 485  nm; emission, 535  nm) was measured. Total raphy (HPLC). The HPLC instrument (Agilent, Wald - RNA from Drosophila whole bodies was extracted using bronn, Germany) equipped with a UV detector was used. TRIzol reagent (Invitrogen, Carlsbad, CA, USA) accord- YMC-Triart C 18 (250 × 4.6  mm, 5  μm) was used as the ing to the manufacturer’s protocol [12]. RQ1 RNase-free column, and 0.2% formic acid in water for mobile phase DNase I (Promega, WI, USA) treated the RNA samples A and 0.2% formic acid in acetonitrile for mobile phase were reverse-transcribed using SuperScript III reverse B were used for component separation. The gradient transcriptase (Invitrogen), and the mRNA expression of conditions of the solvent (A:B) were 95%:5% (0–2  min), superoxide dismutase (SOD), catalase (CAT), and glu- 75%:25% (10 min), 60%:40% (30 min), 50%:50% (40 min), tathione peroxidase (GPx) was analyzed by quantitative 40%:60% (50 min), and 95%:5% (55–60 min). The solvent real-time PCR using the StepOne Plus Real-time PCR flow rate was 0.8  mL/min, the column temperature was system (Applied Biosystems, CA, USA) and a Power maintained at 35 °C, and the injection volume was 10 μL. TaqMan PCR Master Mix Kit (Applied Biosystems) with The wavelengths of the UV detector were analyzed at RpL32 (NM_001144655.3). The target genes used in qRT- 260, 292, 310 and 365  nm. The ginsenoside content was PCR were SOD (NM_057387.5), CAT (NM_080483.3), also measured using a Cadenza CD-C18 (75 × 4.6  mm, and GPx (NM_168024.2). 3 μm) column in an HPLC system (Agilent). For the analysis of ginsenoside, (A) 10% acetonitrile Statistical analysis in water and (B) 90% acetonitrile in water were used The data are presented as the mean ± standard deviation as mobile phases, and the flow rate was maintained from three repeated measurements. The statistical signif - at 1.2  mL/min. The gradient conditions of the solvent icance of the experimental group was verified at P  ˂ 0.05, were 90–76%:10–24% (0–44  min), 76–60%:24–40% (44– using Tukey’s multiple range test. The analysis was car - 56  min), 60–50%:40–50% (56–79  min), 50–90%:50–10% ried out using SPSS (version 12.0; Statistical Analysis Sys- (79–82  min) and 90%:10% (82–85  min). The wavelength tem, SPSS, Inc., Chicago, IL, USA). of the UV detector was measured at 203 nm, the column temperature was 40  °C, and the injection amount was Results 5 μL. Component changes during the fermentation of ginseng berry kombucha (GBK) Estimation of survival rate in Drosophila model Changes in pH, titratable acidity, reducing sugars, poly- Canton-S wild-type Drosophila melanogaster Meigen phenols, and flavonoid contents were measured during was a gift from the Department of Food Science and the fermentation of GBK by S. cerevisiae and G. oxydans Nutrition Jeju National University. Drosophila were (Fig.  1). During kombucha fermentation, the pH gradu- reared at 25 °C with 60% humidity and a 12-h light–dark ally decreased, with a slight change being noted 3  days cycle. Adult fruit flies were reared in a standard corn after fermentation. From pH 3.92 on the 3rd day of fer- meal medium. For H O treatment, male Drosophila mentation to pH 3.68 on the 18th day, only a marginal 2 2 were transferred to empty culture tubes and fasted for decrease was observed. Titratable acidity increased with 2  h, and filter paper soaked in different solutions (200 the incubation period and ranged from 0.05% before fer- μL) was placed inside. The control (CON) was fed 5% mentation to 0.90% after 18 days (Fig. 1A). sucrose (m/m) containing 15% H O (m/m). The other 2 2 Choi et al. Applied Biological Chemistry (2023) 66:27 Page 4 of 9 Fig. 1 Changes of titratable acidity and pH (A), and components (B) during the fermentation of ginseng berry kombucha (GBK). Values are the means ± SD for each group; different letters indicate significant differences at p < 0.05 vs. day 0 in each group based on Tukey’s test. GE gallic acid equivalent The content of reducing sugars content in GBK rapidly end of the fermentation process, indicating an increase in decreased to 190.14  μg/mL on the 3rd day of fermenta- the scavenging ability after the fermentation. In addition, tion and then gradually increased to 241.49–279.85  μg/ in comparison to before fermentation, the FRAP value mL from day 6 to day 18 of fermentation (Fig.  1B). The for reducing power increased after the process (34.89 vs. alcohol content which was 0.17  mg/mL before fermen- 46.75 mM). Collectively, this implies that during the fer- tation, increased rapidly to 6.26  mg/mL on the 3rd day, mentation of GBK, there is a simultaneous increase in the and eventually declined to 2.15  mg/mL on the 18th day reducing power of FRAP and radical scavenging activity. (Fig. 1B). There was no significant change in the polyphe - nol content of GBK during fermentation, but it was high- Changes in flavonoid and ginsenoside contents est on the 12th day of fermentation (Fig. 1B). before and after fermentation As regards the measured changes in microorganisms The changes in flavonoid contents in the GBK extract, during kombucha fermentation, acetic acid bacteria presumed to be the active constituents, before and after showed a tendency to increase as the fermentation pro- fermentation, are shown in Tables  2. The total content th gressed, and the yeast numbers increased until the 9 of flavonoids, which are active ingredients, before fer - day of fermentation and gradually decreased thereafter mentation was 0.78  μg/mg but increased to 1.32  μg/mg (Table  1). The total number of bacteria also increased after fermentation (Table  2). After fermentation of GBK, until the 9th day and then gradually reduced. the major flavonoids in the extract were gallic acid, 3,4 dihydroxy benzoic acid, and chlorogenic acid, which Changes in radical scavenging activity during fermentation increased significantly after fermentation. The radical scavenging ability and reducing power were As shown in Table  3, the main ginsenosides of GBK measured before and after GBK fermentation (Fig. 2). As after fermentation were Rg2 (sum of Rg2s and Rg2r) compared to before the fermentation, the I C value for and Re, with concentrations of 10.10  μg/mg and radical scavenging of ABTS and DPPH decreased at the 6.59 μg/mg, respectively. The total ginsenoside content, Table 1 Content of different microorganisms during the fermentation of ginseng berry kombucha (GBK) CFU (× 10 /mL) Day 0 3 6 9 12 18 Acetic‑acid producing bacteria 0 1.33 ± 0.03 1.29 ± 0.01 1.98 ± 0.12 2.20 ± 0.01 2.24 ± 0.64 Yeast 0 1.52 ± 0.12 1.87 ± 0.04 3.29 ± 0.11 3.07 ± 0.17 2.81 ± 0.11 Total microorganisms 0 2.89 ± 0.13 3.28 ± 0.23 5.37 ± 0.22 5.06 ± 0.36 5.09 ± 0.51 Values are the means ± SD for each group CFU colony forming unit Choi  et al. Applied Biological Chemistry (2023) 66:27 Page 5 of 9 Fig. 2 IC value on A ABTS and B DPPH radical and C FRAP value before and after fermentation of ginseng berry kombucha (GBK). Values are the means ± SD for each group; *** indicates significant difference (p < 0.001) after fermentation as compared to before. ABTS 2,2′‑azino ‑bis(3‑ ethylbenz othiazoline‑6‑sulfonic acid), DPPH 2,2‑ diphenyl‑1‑picrylhydrazyl, FRAP ferric reducing antioxidant power Table 2 Changes in contents of flavonoids before and after Table 3 Changes in content of ginsenosides before and after ginseng berry kombucha (GBK) fermentation ginseng berry kombucha (GBK) fermentation Flavonoids Contents (μg/mg of extract) Ginsenoside Contents (μg/mg of extract) 0 day 18 day 0 day 18 day Gallic acid 0.067 ± 0.004 0.140 ± 0.011 Rg1 0.53 ± 0.02 1.04 ± 0.03 3,4‑Dihydroxybenzoic acid 0.120 ± 0.002 0.420 ± 0.032 Re 3.40 ± 0.08 6.59 ± 0.01 Rutin 0.050 ± 0.001 0.063 ± 0.002 Rf 0.21 ± 0.02 0.37 ± 0.01 Quercetin‑3‑ glucuronide 0.023 ± 0.003 0.068 ± 0.002 Rb1 0.32 ± 0.03 0.79 ± 0.05 Rc 0.59 ± 0.01 1.04 ± 0.05 Chrysin 0.082 ± 0.002 0.092 ± 0.002 Rb2 0.36 ± 0.02 0.59 ± 0.01 Chlorogenic acid 0.095 ± 0.001 0.20 ± 0.006 Rd 0.77 ± 0.01 1.36 ± 0.01 Caffeic acid 0.042 ± 0.001 0.076 ± 0.003 ρ‑ Coumaric acid 0.026 ± 0.001 0.028 ± 0.001 Rh 1 (s) 0.27 ± 0.02 1.03 ± 0.02 Trans ferulic acid 0.037 ± 0.001 0.056 ± 0.001 Rg2 (s) 0.72 ± 0.10 3.41 ± 0.04 Apigenin 0.015 ± 0.001 0.011 ± 0.001 Rg2 (r) 1.46 ± 0.10 6.69 ± 0.04 Quercetin 0.140 ± 0.001 0.100 ± 0.001 Rg6 0.140 ± 0.001 0.100 ± 0.001 Kaempferol 0.094 ± 0.001 0.072 ± 0.001 Rk3 1.02 ± 0.15 2.00 ± 0.03 Total flavonoids 0.78 ± 0.006 1.32 ± 0.024 Rh4 0.22 ± 0.04 0.59 ± 0.01 Rg3 (s) 0.75 ± 0.03 2.28 ± 0.01 Values are the means ± SD for each group Rg3 (r) 0.97 ± 0.03 0.95 ± 0.02 CK 0.35 ± 0.03 0.49 ± 0.03 Rk1 0.24 ± 0.06 0.44 ± 0.02 which was 13.34  μg/mg at the beginning of fermenta- Rg5 0.52 ± 0.11 1.23 ± 0.04 tion, increased to 32.58  μg/mg after fermentation. Rh2 (s) 0.21 ± 0.03 0.31 ± 0.00 In addition, the content of the major ginsenoside and Major ginsenosides 6.19 ± 0.19 11.78 ± 0.17 minor ginsenoside increased from 6.19 to 11.78  μg/ Minor ginsenosides 7.16 ± 0.72 20.80 ± 0.26 mg and from 7.16 to 20.80  μg/mg compared to before Total ginsenosides 13.34 ± 0.91 32.58 ± 0.43 fermentation, respectively. Therefore, it was confirmed that the content of easily absorbed ginsenosides in Values are the means ± SD for each group. Major ginsenosides: Rg1, Re, Rf, Rb1, Rc, Rb2, and Rd; minor ginsenosides: Rh1, Rg2, Rk3, Rh4, Rg3, Rk1, and Rh2 GBK increased through fermentation. In the analysis Choi et al. Applied Biological Chemistry (2023) 66:27 Page 6 of 9 of flavonoids and ginsenosides in GBK extracts, com - on the increase in oxidative stress caused by H O treat- 2 2 pounds that did not show differences were not included ment was attributed to an increase in the expression level in the results. of oxidative stress-related enzymes. Discussion Eec ff ts of ginseng berry kombucha (GBK) on survival Despite the various physiological activities of kombu- rate and relief of oxidative stress in male fruit flies cha, its application and utility in the industry are lim- under oxidative stress conditions ited. Since the fermentation of kombucha is performed The survival rate after treatment with low concentration by a microbial community, the species and distribu- (2.5%) and high concentration (5%) of GBK was meas- tion of microorganisms involved in the process are not ured in male fruit flies treated with 15% H O to induce 2 2 confirmed, and there are concerns about the safety and oxidative stress (Fig.  3). The GBK-administered groups combination of microorganisms, such as SCOBY, using showed an increase in the survival rate compared to the sugar and extracts of green or black tea as fermentation CON group treated with H O alone. A high concen- 2 2 substrates. Quality control and industrial production of tration of GBK resulted in a significant increase in the kombucha should be carried out using officially recog - survival rate compared to CON (p < 0.001). The low con - nized microorganisms that are safe for use. To achieve centration of GBK did not show a significant increase in various physiological activities to kombucha, attempts survival rate compared to the CON group. There was no should be made to use substrates other than green or significant difference in survival rates between high and black teas. Therefore, in this study, ginseng berries were low GBK concentrations. used as a fermentation substrate to prepare kombucha Figure  4 shows the effect of GBK on ROS production using S. cerevisiae and G. oxydans, and the fermentation and gene expression of SOD, CAT, and GPx in the Dros- characteristics were measured. ophila model, wherein oxidative damage was induced There was an increase in acidity and a decrease in pH by H O treatment. Treatment with low and high con- 2 2 as a result of the organic acid acetic acid produced dur- centrations of GBK significantly reduced ROS produc - ing the fermentation process by acetic acid bacteria tion by 44.5% and 65.4%, respectively, compared to the among the strains used for fermentation of kombucha control (p < 0.001). As the GBK treatment concentration [29]. Kombucha is known to release organic acids such increased, the gene expression of CAT, GPx, and SOD, as acetic, gluconic, glucuronic, citric, l-lactic, malic, tar - enzymes related to oxidative stress removal, tended taric, malonic, oxalic, succinic, and pyruvic acid through to decrease in a concentration-dependent manner. fermentation [25]. In this study, the organic acid pro- Both CAT and GPx expression levels were significantly duced by G. oxydans resulted in a decrease in pH and an increased compared to CON by both GBK concentra- increase in titratable acidity (Fig.  1). Organic acids are tions (p < 0.001). Therefore, the inhibitory effect of GBK produced due to the metabolism of the yeast and ace- tic acid bacteria during the fermentation process. These organisms utilize carbohydrates as the main substrate to produce alcohol and organic acids [34]. We found that during the fermentation of GBK, the content of reducing sugars decreased, the ethanol content initially increased and then declined, and the titratable acidity increased continuously (Fig. 1). The ethanol content generated dur - ing kombucha fermentation is reported to be 0.7% to 1.3% [4]. When the alcohol content remains above 1%, it is proposed to initiate processes such as oxygen exposure and microfiltration to lower the concentration [15]. How - ever, in this study, because the final alcohol content was 0.25%, an additional process for lowering the alcohol was not required. Kombucha largely contains polyphenols, organic acids, Fig. 3 Eec ff ts of ginseng berry kombucha (GBK) on the survival sugars, and proteins [10], and its composition varies rate in Drosophila treated with 15% H O . Significant differences 2 2 depending on the fermentation substrate used. Kom- in percent survival (%) among groups were analyzed by log‑rank statistical method (n = 100/group). CON control, 15% H O ‑treated bucha, which uses black tea (black tea kombucha) and 2 2 group, GBK-L Group treated with 2.5 mg/g of GBK and 15% H O , 2 2 green tea (green tea kombucha) as the fermentation sub- GBK-H Group treated with 5 mg/g of GBK and 15% H O 2 2 strates for kombucha, contains polyphenols, including Choi  et al. Applied Biological Chemistry (2023) 66:27 Page 7 of 9 Fig. 4 Eec ff t of ginseng berry kombucha (GBK) on A ROS production and B–D antioxidant enzyme ‑related mRNA expression in Drosophila treated with 15% H O . Values are the means ± SD for each group; different levels of significance have been indicated *p < 0.05, **p < 0.01, ***p < 0.001 in 2 2 each group based on Tukey’s test. CON: control, 15% H O ‑treated group, GBK ‑L: Group treated with 2.5 mg/g of GBK and 15% H O , GBK‑H: Group 2 2 2 2 treated with 5 mg/g of GBK and 15% H O ; ROS reactive oxygen species, CAT catalase, SOD superoxide dismutase, GPx glutathione peroxidase 2 2 catechins, as active constituents. It has been reported ginseng and converts it into a non-glycoside ginseno- that in both black and green tea kombucha, the amount side, leading to an improvement in the absorption rate of catechin decreased until the 9th day of fermentation of ginsenoside [30]. Rg2, which is produced by the de- and increased 12th day onward [11]. As shown in Table 2, glycosylation of ginsenoside Re [26], is abundantly pre- the flavonoid content increased after fermentation, and sent in ginseng berries [17]. In the GBK, not only was 3,4-dihydrobenzoic acid and chlorogenic acid were found Re increased upon fermentation, but also Rg2. The con - to be the main flavonoids. In addition, the content of centration of Rg6 produced by the dehydration reaction ginsenosides, both major and minor (which are easily of Rg2 also increased in GBK (Table  3). Ginsenosides absorbed) increased by upon fermentation (Table 3). Rb1, Rc, Rb2, Rd, Ra1, Ra2, and Ra3 are converted to The changes described above were due to the ginsenoside Rg3 by heating during the manufactur- β-glucosidase activity of S. cerevisiae used for kom- ing process of red ginseng [19]. In ginsenoside Rg3, a bucha fermentation. β-glucosidase cleaves the sugar double bond is formed by dehydration at position 20 of chain site bound to the glycoside ginsenoside of red the dammarane backbone and is converted into three Choi et al. Applied Biological Chemistry (2023) 66:27 Page 8 of 9 structural isomers of ginsenosides Rk1, Rg5, and Rz1 which has improved radical-scavenging capabilities along [20]. Ginseng berry kombucha also exhibited increased with consumer preferences, can aid in improving physi- levels of Rg3, Rk1, and Rg5. The sugar moiety attached cal health. to C-3, C-6, or C-20 ginsenosides is deglycosylated Acknowledgements and converted to minor ginsenosides, contributing to Not applicable. an increase in bioavailability and physiological activity Author contributions [27]. Therefore, minor ginsenosides produced by the Data curation: EJC, HHS, and KYK, formal analysis: HHS, and KBH, visualization: fermentation of GBK, increase its bioavailability and KYK, and KBH, methodology: HHS, and KYK, investigation: KYK, and KBH, vali‑ contribute to an increase in its physiological functions. dation: HHS, and KBH, conceptualization: HJS, and YA, project administration: HJS, and YA, supervision: HJS, and YA, writing—original draft: EJC, HJS, and YA. The physiological activity of kombucha is closely writing—review and editing: EJC, HHS, KYK, KBH, HJS, and Y A. All authors read related with its antioxidant activity. Through fermen - and approved the final manuscript. tation, kombucha prepared using green or black tea Funding increases polyphenol components, which are known This research did not receive any specific grant from funding agencies in the to be associated with an increase in antioxidant activ- public, commercial, or not‑for ‑profit sectors. ity [8]. When the antioxidant activities were compared Availability of data and materials before and after fermentation, it was noted that the All data analyzed during this study are included in this published article and its FRAP activity increased along with ABTS and DPPH supplementary information files. after fermentation (Fig.  2). The content of chlorogenic acid [24] and 3,4-dihydroxybenzoic acid [35] increases Declarations during GBK fermentation, which is involved in the Competing interests increase in antioxidant activity. The authors declare that they have no competing interests. Free radicals generated in intracellular metabolic processes lead to oxidative damage to DNA, proteins, Received: 1 February 2023 Accepted: 18 April 2023 and lipids, the major cell components. Accumulated oxidative damage affects cellular function and, fur - thermore, leads to deterioration of tissue function, and this ultimately is responsible for increasing promoting References aging, or decreasing an individual’s lifespan [9]. The 1. Antolak H, Piechota D, Kucharska A (2021) Kombucha tea—A double most abundant free radicals are ROS, which are mainly power of bioactive compounds from tea and symbiotic culture of bacte‑ ria and yeasts (SCOBY ). Antioxidants 10:1541 produced in the electron transport chain of the mito- 2. Anupama KP, Shilpa O, Antony A, Raghu SV, Gurushankara HP (2022) chondria. Free radicals, including superoxide (O ), Jatamansinol from Nardostachys jatamansi (D.Don) DC. protects Aβ42‑ hydroxyl radicals ( OH), and hydrogen peroxide (H O ) induced neurotoxicity in alzheimer’s disease Drosophila model. Neuro‑ 2 2 Toxicology 90:62–78 can be eliminated by the cellular antioxidant defense 3. Baek C‑H, Baek S‑ Y, Lee SH, Kang J‑E, Choi H‑S, Kim J‑H, Yeo S‑H (2015) mechanisms which include the use of enzymes such as Characterization of Acetobacter sp. strain CV1 isolated from a fermented SOD and CAT and a process that involves antioxidants vinegar. Microbiol Biotechnol Lett 43:126–133 4. Bishop P, Pitts ER, Budner D, Thompson‑ Witrick KA (2022) Chemical com‑ such as flavonoids, vitamins, and ginsenosides [21]. The position of kombucha. Beverages 8:45 radical-scavenging activity of GBK can be attributed to 5. Cho M‑H, Bae E‑K, Ha S‑D, Park Y ‑S, Mok C‑K, Hong K ‑P, Kim S‑P, Park J‑ Y the presence of various flavonoids, in addition to the (2005) Evaluation of dry rehydratable film method for enumeration of microorganisms in meat, dairy and fishery products. Korean J Food Sci minor ginsenosides. Administration of GBK to Dros- Technol 37:294–300 ophila induced to undergo oxidative stress by treatment 6. Chung I‑M, Lim J‑ J, Ahn M‑S, Jeong H‑N, An T ‑ J, Kim S‑H (2016) Compara‑ with H O appears to decrease ROS production due tive phenolic compound profiles and antioxidative activity of the fruit, 2 2 leaves, and roots of Korean ginseng (Panax ginseng Meyer) according to to increased gene expression of SOD, CAT, and GPx, cultivation years. J Ginseng Res 40:68–75 enzymes involved in oxidative stress removal (Figs. 3 & 7. Decarvalho VD, Chagas SJD, Chalfoun SM, Botrel N, Juste ESG (1994) 4). Relationship between the physical‑ chemical and chemical‑ composition of green coffee and the quality of coffee beverage.1. Polyphenoloxidase Oxidative stress, a product of the interaction of genetic, and peroxidase‑activity, color index and titratable acidity. Pesqui Agro ‑ environmental, and lifestyle factors, increases the risk of pecu Bras 29:449–454 aging, disease, and death. u Th s, it is necessary to identify 8. Greenwalt C, Steinkraus K, Ledford R (2000) Kombucha, the fermented tea: microbiology, composition, and claimed health effects. J Food Prot favorable foods that have the ability to suppress or elimi- 63:976–981 nate the generation of ROS and halt the acceleration of 9. Hajam YA, Rani R, Ganie SY, Sheikh TA, Javaid D, Qadri SS, Pramodh S, aging in the human body and lead to numerous diseases. Alsulimani A, Alkhanani MF, Harakeh S (2022) Oxidative stress in human pathology and aging: molecular mechanisms and perspectives. Cells The quality control of ginseng berry kombucha can be 11:552 easily achieved, and its safety can be ensured by using 10. Jayabalan R, Malbaša RV, Lončar ES, Vitas JS, Sathishkumar M (2014) A strains isolated from food. In addition, the use of GBK, review on kombucha tea—microbiology, composition, fermentation, Choi  et al. Applied Biological Chemistry (2023) 66:27 Page 9 of 9 beneficial effects, toxicity, and tea fungus. Compr Rev Food Sci Food 34. Woo H‑ G, Lee C‑M, Jeong J‑H, Choi B‑K, Huh C‑K (2021) Quality character ‑ Safety 13:538–550 istics of kombucha made with different mixing ratios of green tea extract 11. Jayabalan R, Marimuthu S, Swaminathan K (2007) Changes in content of and yuzu juice during fermentation. Korean J Food Preserv 28:646–653 organic acids and tea polyphenols during kombucha tea fermentation. 35. Yang Y‑R, Cho J‑ Y, Park Y‑K (2012) Isolation and identification of antioxida‑ Food Chem 102:392–398 tive compounds 3, 4‑ dihydroxybenzoic acid from black onion. Korean J 12. Jo K, Kim S, Ahn Y, Suh HJ (2021) Eec ff ts of green lettuce leaf extract on Food Preserv 19:229–234 sleep disturbance control in oxidative stress‑induced invertebrate and vertebrate models. Antioxidants 10:970 Publisher’s Note 13. Kapp JM, Sumner W (2019) Kombucha: a systematic review of the empiri‑ Springer Nature remains neutral with regard to jurisdictional claims in pub‑ cal evidence of human health benefit. Ann Epidemiol 30:66–70 lished maps and institutional affiliations. 14. Kim C‑K, Cho DH, Lee K ‑S, Lee D ‑K, Park C‑ W, Kim WG, Lee SJ, Ha K‑S, Goo Taeg O, Kwon Y‑ G (2012) Ginseng berry extract prevents atherogenesis via anti‑inflammatory action by upregulating phase II gene expression. Evid Based Complement Alternat Med 2012:4903301 15. Kim J, Adhikari K (2020) Current trends in kombucha: marketing perspec‑ tives and the need for improved sensory research. Beverages 6:1–19 16. Kim JY, Shin HJ, Kim HL, Park H, Kim PK, Park S, Kim SH (2020) The compo‑ sitional and functional properties of Kombucha: a literature review. Food Eng Prog 24:1–14 17. Kim S‑ J, Kim J‑D, Ko S‑K (2013) Changes in ginsenoside composition of ginseng berry extracts after a microwave and vinegar process. J Ginseng Res 37:269 18. Kim YK, Yoo DS, Xu H, Park NI, Kim HH, Choi JE, Park SU (2009) Ginseno‑ side content of berries and roots of three typical Korean ginseng (Panax ginseng) cultivars. Nat Prod Commun 4:903–906 19. Lee H‑ J, Jung E‑ Y, Lee H‑S, Kim B‑ G, Kim J‑H, Yoon T ‑ J, Oh S‑H, Suh H‑ J (2009) Bioavailability of fermented Korean red ginseng. Prev Nutr Food Sci 14:201–207 20. Lee SM (2020) Three hydroxylated ginsenosides from heat treatmented ginseng. Korean J Pharmacogn 51:255–263 21. Liu Z‑ Q (2022) Why natural antioxidants are readily recognized by biologi‑ cal systems? 3D architecture plays a role! Food Chem 380:132143 22. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428 23. Moon JK, Shibamoto T (2009) Antioxidant assays for plant and food components. J Agric Food Chem 57:1655–1666 24. Naveed M, Hejazi V, Abbas M, Kamboh AA, Khan GJ, Shumzaid M, Ahmad F, Babazadeh D, FangFang X, Modarresi‑ Ghazani F (2018) Chlorogenic acid (CGA): a pharmacological review and call for further research. Biomed Pharmacother 97:67–74 25. Neffe ‑Skocinska K, Sionek B, Scibisz I, Kolozyn‑Krajewska D (2017) Acid contents and the effect of fermentation condition of Kombucha tea beverages on physicochemical, microbiological and sensory properties. Cyta J Food 15:601–607 26. Pei J, Wu T, Yao T, Zhao L, Ding G, Wang Z, Xiao W (2017) Biotransforma‑ tion of Ginsenosides Re and Rg 1 into Rg 2 and Rh 1 by thermosta‑ ble β‑ glucosidase from Thermotoga thermarum. Chem Nat Compd 53:472–477 27. Piao XM, Huo Y, Kang JP, Mathiyalagan R, Zhang H, Yang DU, Kim M, Yang DC, Kang SC, Wang YP (2020) Diversity of ginsenoside profiles produced by various processing technologies. Molecules 25:4390 28. Ryu H‑ J, Jung C‑ J, Beik G‑ Y (2020) Antioxidant activities of flower, berry and leaf of Panax ginseng CA Meyer. Korean J Food Sci Technol 52:342–349 29. Ryu SJ, Lee CY, Kang GS, Kim SG, Kim SH, Seo DH (2021) Optimization of compositions for kombucha with Rubus coreanus. Food Eng Prog 25:118–124 30. Shim K‑S, Park G‑ G, Park Y‑S (2014) Bioconversion of puffed red ginseng extract using β‑ glucosidase‑producing lactic acid bacteria. Food Eng Prog 18:332–340 31. Singleton VL, Orthofer R, Lamuela‑Raventós RM (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of folin‑ ciocalteu reagent. Methods Enzymol 299:152–178 32. St‑Pierre DL (2019) Microbial diversity of the symbiotic colony of bacteria and yeast (SCOBY ) and its impact on the organoleptic properties of kombucha, Food Science and Human Nutrition. The University of Maine, Orono, pp 1–94 33. Vīna I, Semjonovs P, Linde R, Deniņa I (2014) Current evidence on physi‑ ological activity and expected health effects of kombucha fermented beverage. J Med Food 17:179–188

Journal

Applied Biological ChemistrySpringer Journals

Published: May 11, 2023

Keywords: Ginseng berry kombucha; Saccharomyces cerevisiae; Gluconobacter oxydans; ROS

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