Metagenomic analysis and bioactive profiling of kombucha fermentation: antioxidant, antibacterial activities, and molecular docking insights into gastric cancer therapeutics

Abstract

Kombucha is fermented and produced with a biofilm called a symbiotic culture of bacteria and yeast, which is drunk all over the world for its beneficial effects on human health and energy levels. The metagenomic study of kombucha frequently detected microorganisms in proteobacteria, firmicutes, and actinobacteria. And also, yeast and fungi are Ascomycota and Basidiomycota is present in green leaf and sugarcane juice fermented kombucha. The kombucha extracts’ biological activities were assessed using pH, total phenolic content, antioxidant, antibacterial, and anticancer activity. Fermentation may enhance biological activity and the generation of bioactive substances. These results showed the pH −3.1 ± 0.2 and TPC −0.721 μg/mL of gallic acid equivalent. The antioxidant radicals scavenging activity of kombucha was evaluated by DPPH, ABTS, H₂O₂ and TAC. The bioactive chemicals identified by FT-IR and HR-LC/MS analysis of Kombucha totaled 45 components. The identified compounds were further move on to perform molecular docking study against gastric cancer target proteins 4H9M, 2DQ7 and 1TVO are binding with Nequinate compounds showing best LibDock scores 105.12, 114.49, and 108.97. So, this study suggests that knowledge can potentially active bioactive compounds are present in kombucha and it’s stimulated the mechanism of gastrointestinal transit. Additionally, the metagenomic analysis gives strength to understand the bacterial and fungal distribution and its molecular mechanism from Kombucha.

Introduction

Foods play a crucial role in a specific position as they provide the energy and essential nutrients for life maintenance. Bioactive food factors, which are not considered nutrients but can have beneficial health effects, are being substantiated.¹ Kombucha, a Manchurian beverage, is made from sweet tea infusions, primarily black and green tea, and other varieties that can be used as a base. Which are fermented by a mixture of sugar as a substrate and a microbial consortium composed of bacteria and yeast.² Kombucha, originating from the Japanese words “Dr Kombu” and “cha”, was introduced to Russia as “Tea Kvass” and later spread to Eastern Europe in the 20^th century to treat digestive issues.³ Kombucha tea is a non-alcoholic beverage fermented from sweetened green or black tea using a symbiotic culture of bacteria and yeast (SCOBY). The drink, known as Manchurian tea fungus, Indian tea fungus, mushroom tea, grib tea kvass, Manchu Fungus, tea wass, and tea beer, is considered beneficial and pro-healthy for consumers globally. Kombucha, a popular fermented drink with low alcohol content, has experienced rapid growth in functional beverages among consumers due to its slightly acidic and carbonated taste.^4,5

Kombucha fermentation begins with sugar-infused tea inoculation and is left to stand at room temperature for two weeks.⁶ During fermentation, the cellulose layer and acidic broth produce natural acids, lowering the pH of kombucha. The aging time of kombucha is determined by the starter culture and maturation apportion, which includes sugar, tea, starter culture, yeast, time, number of microbes, and aging temperature. Other than being affected by pH, temperature, and bacterial movement, SCOBY development in the maturation handle is also impacted by the aging time.^7,8 The aging time is more often than not 7–60 days, but the normal aging time of 15 days is ideal.⁹ Fermented food products like kombucha are renowned for their high content of probiotics, which are known to have a positive impact on health. Probiotics have potential benefits such as suppressing harmful bacteria, supporting beneficial gut microbial populations, engaging with intestinal lining, metabolizing nutrients, and regulating immune system signaling.^10–15 This fermented tea drink is widely consumed due to its potential health benefits, including potential protection against cancer, cardiovascular diseases, and neurodegenerative diseases.⁴ It has been found to have a beneficial impact on digestion and intestinal microbiota, provide relief from arthritis, exhibit antimicrobial properties, alleviate hemorrhoids, detoxify the body, offer hepatoprotective benefits, help reduce insomnia, alleviate headaches, and have a positive effect on mood.^1,5,16 The production of various bioactive metabolites, such as amino acids, vitamins and ethanol, which result from the complex interactions between bacteria and yeasts utilizing sugar, tea bases, and other substrates.¹⁷

Gastric cancer, also known as stomach cancer, is aggressive and has various clinical manifestations.¹⁸ It is influenced by several receptors, including Src family tyrosine kinase (FYN) (PDB ID: 2DQ7) and Mitogen-activated protein kinase (MAPK) (PDB ID: 1TVO), which is crucial in pancreatic cancer metastasis.¹⁹ These receptors contribute significantly to the development and progression of stomach cancer.²⁰ In cancer biology, inhibiting the extracellular signal-regulated kinases ERK1 and ERK2, which are part of the Ras-MAPK signal transduction pathway complexed with the histone deacetylases HDAC4 protein, is of great therapeutic interest.²¹ Thus, the present study identifies the drug compounds from the fermented Kombucha drink and performs the molecular docking study against gastric cancer. Also, we performed the metagenomic analysis based on V3-V4 for bacterial distribution, and ITS region for fungal distribution in the Kombucha drink. These outcomes provide a clear avenue for the presence of beneficial microbial communities for the treatment of gastric cancer.

Methodology

Material and reagent

Water, green tea (Lipton clear & light loose green tea leaves), Sugarcane juice, kombucha SCOBY. Folin–Ciocalteu reagent, sodium hydroxide, sodium carbonate, 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), Hydrogen peroxide (H₂O₂), 2,2- diphenyl-1-picrylhydrazyl (DPPH), Ascorbic acid, etc.

Collection of samples

The kombucha starter culture and SCOBY was obtained by Amazon Peepal farm products store in Himachal Pradesh and it was maintained in sugared black tea under aseptic conditions.

Preparation of green tea

Lipton Clear & Light Loose Green Tea Leaves were used in this preparation. All containers and equipment were sterilized at 121 °C for 15 minutes before use. Ten grams of tea leaves were added to 1 liter of boiling water, maintaining 1:100 ratio. The tea was then allowed to infuse at 80 °C for 15 minutes. After the infusion, the tea was filtered through a sterile sieve.²²

Preparation of kombucha

The kombucha used in this study was commercially sourced and ready for consumption. The SCOBY was inoculated into green tea, with 250 mL of sugarcane juice and 10 mL of starter culture added to sterilized 2.5 L glass jars (sterilized at 121 °C for 20 min). The jars were covered with cheesecloth and incubated in darkness at 25 °C for 14 days, as depicted in Supplementary Fig. S1. After fermentation, the newly formed SCOBY was preserved in 300 mL of kombucha within sterile containers and refrigerated at 5 °C for further analysis.²

Metagenomic analysis

Metagenomic sequencing was done on all kombucha samples for the V3-V4 Region and ITS region using the universal primers for the respective sequencing in illumina Miseq platform (Illumina, San Diego, CA, USA), the primer used for amplification was represented in Supplementary Table S1. Sequencing was conducted using the MiSeq Reagent Kit v3, employing 2 × 300 bp paired-end reads. The sequencing data were processed to remove low quality regions based upon the phred score and clustered them to 100% to remove false hits. The taxonomic classification was done using BaseSpace 16S metagenomics app (Illumina, San Diego, CA, USA) with 97% sequecne clustering against Ribosomal Database Project (RDP) using naive Bayesian classifier algorithm,²³ Sequences were taxonomically assigned with at least 99% identity in the reference database after being compared to the UNITE Fungal ITS Database v7.2 and a custom RefSeq 16S v3 rRNA DADA2 Database. In order to assess changes in microbial communities, the relative abundance of each genus was compared across samples. The sample with raw sequence reads were deposited in NCBI-SRA portal, with corresponding accession PRJNA1126837 for bacterial reads and fungal ITS reads.

Titratable acidity (TA)

The TA value of a kombucha sample was determined using the Chen and Liu method.²⁴ The titration involved a 100 mL sample of kombucha and 0.1 mol/L NaOH, with the endpoint at 7 pH. The TA was expressed as the volume consumed per 100 ml sample. The endpoint was reached when the pH stabilized at 7.0, indicating the neutralization of the sample's acids. This value represents the total acidity of the kombucha, primarily acetic acid and other organic acids present in the beverage.²⁴

Determination of total phenolic concentration (TPC)

The TPC of kombucha samples was determined using the Folin–Ciocalteu colorimetric method.²⁵ 1 mL sample of diluted kombucha was mixed with distilled water and Folin–Ciocalteu reagent, followed by a 5-min incubation at room temperature. After incubation, 3 mL of Na₂CO₃ solution (75 g/L) was added to neutralize acids and facilitate the formation of a blue-colored complex. The mixture was incubated for 30 min to allow complete color development. The absorbance was then measured at 760 nm using a UV–Vis spectrophotometer, indicating the concentration of phenolic compounds in the sample.

Antioxidant assay

DPPH scavenging activity

The free radical scavenging activity was measured by the method of Blois.²⁶ The various concertation (10, 25, 50, 75, and 100 μg/mL) of kombucha extracts were made into distilled H₂O with 100 μL of 0.1 mM DPPH in ethanol solution added in 96 well plates. The mixture was incubated at 25 °C for 30 min, and the reduction of DPPH free radicals was measured using a Microplate reader absorbance at 517 nm, with ascorbic acid as the standard by applying the following equation:

Hydroxyl radical scavenging activity (HRS)

The HRS activity of the extracts was evaluated using the method described by Klein et al. and Nguyen et al.^27,28 Kombucha extracts (100 μL) at concentrations ranging from 10 to 100 μg/mL were mixed with 45 μL of 8 mM ferrous sulfate heptahydrate (FeSO₄·7H₂O), 63 μL of 5.7 mM salicylic acid (HOC₆H₄COOH), and 72 μL of 6 mM hydrogen peroxide (H₂O₂). The reaction mixtures were incubated at 37 °C for 30 min, after which the intensity of the resulting yellow color was measured by absorbance at 562 nm. Ascorbic acid served as the positive control, and the percentage of HRS activity was calculated.

ABTS radical scavenging activity

The kombucha extracts were prepared following the procedure outlined by Re et al.²⁹ and followed by Kozłowska et al.³⁰ ABTS was dissolved in 7.8 mM deionized water and mixed with 2.5 mM potassium persulphate. The mixture was stored in the dark for 12–16 h to prevent incomplete oxidation before use. Subsequently, 100 μL of kombucha extract at varying concentrations (10, 25, 50, 75, and 100 μg/mL) was combined with 40 μL of ABTS solution and incubated in the dark for 15 min. Ascorbic acid was used as a positive control. The absorbance of the samples was recorded at 734 nm. The radical scavenging activity of the kombucha extract was calculated using the following equation:

Ferric reducing antioxidant power assay (TAC)

The antioxidant capacity of kombucha extract was determined using the FRAP assay, as previously described by Benzie and Strain.³¹ The assay relies on the reduction of the ferric tripyridyl triazine complex (Fe³⁺-TPTZ), which is colorless, to the blue-colored ferrous tripyridyl triazine complex (Fe²⁺-TPTZ). The FRAP reagent was prepared by combining 300 mmol/L acetate buffer (pH 3.6), 20 mmol/L ferric chloride, and 10 mmol/L 2,4,6-tripyridyl-s-triazine dissolved in 40 mmol/L hydrochloric acid, in a ratio of 10:1:1 (v:v). Kombucha extracts at various concentrations (10, 25, 50, 75, and 100 μg/mL) were mixed with the reagent and ascorbic acid (used as a positive control). A 100 μL aliquot of each sample was combined with 100 μL of the FRAP reagent and mixed thoroughly. After incubation at 37 °C for 30 minutes, the absorbance was measured at 593 nm. The total antioxidant activity of the kombucha extract was determined using the following equations.

Kirby Bauer assay

The antibacterial activity of kombucha extracts was evaluated using the Kirby-Bauer disc diffusion method.³² The assay was performed against five bacterial strains: Salmonella typhi, Escherichia coli, Actinobacteria, Pseudomonas sp. and Klebsiella pneumoniae. Bacterial cultures were grown in Mueller-Hinton broth at 37 °C with continuous shaking at 200 rpm. The cell density of each culture was determined spectrophotometrically by measuring the optical density (OD) at 600 nm. Bacterial suspensions were adjusted to a concentration of 10⁸ CFU/mL and evenly spread on Mueller-Hinton agar plates using sterile cotton swabs. Sterile 6 mm discs were soaked with kombucha extract and placed onto the inoculated agar plates. The plates were incubated at 37 °C for 16–18 h, with Ampicillin serving as the positive control. After incubation, the antibacterial activity was evaluated by measuring the zone of inhibition (ZOI) around each disc.

Fourier transform infrared spectra (FT-IR) analysis

FTIR analysis is useful tool for the identification of functional groups present in the kombucha sample and recorded in (FT-IR-4600, JASCO) ranging from 4,000 to 550 cm⁻¹. To compare the spectra data was recorded and functional groups were identified standard chart.³³

High-resolution liquid chromatography-mass spectrometry (HR-LC/MS) analysis

The HR-LC/MS analysis of kombucha extract was conducted to profile bioactive compounds using an HR-LCMS system from Agilent Technologies such as Thermo Fisher, Q-Exactive plus Biopharma – High-Resolution Orbitrap, and Waltham, MA. The analysis was performed at the SAIF, IIT-B, India. To separate compounds, a C18 column (3 μm, 50 × 2.1 mm) at 40 °C was utilized. The mobile phase contained 0.1% formic acid and acetonitrile in water, with a flow rate of 0.2 mL/min. The injection volume for each sample was 3 μL. Gradient elution was performed with acetonitrile ranging from 5% to 95% over 30 min. Detection was done in the wavelength range of 280–350 nm, and compound identification was based on retention time, mass spectrometric data, and matching scores. The mass spectrometer, equipped with an electrospray ionization source, operated in both positive and negative ion modes (35 V, 300 °C), with a mass range of 110–2,000 m/z. The total run time for each analysis was 20 min. Compounds were identified based on their unique mass spectra and fragmentation patterns, with phytochemical compounds confirmed using PubChem.³⁴

Molecular docking studies

Molecular docking analysis was conducted using Dassault Systèmes BIOVIA Discovery Studio (v22.1.100, licensed version). The 3D structures of the target proteins (PDB IDs: 4H9M, 2DQ7, and 1TVO) were retrieved from the Protein Data Bank (PDB). Ligands for the docking studies were chosen based on the HR-LC/MS analysis of the kombucha extract. Protein preparation included the removal of water molecules, ligands, and ions, assignment of bond orders, addition of hydrogen atoms, and verification of the protonation states of amino acids. The docking grid was optimized to identify the most favorable binding sites, and molecular docking was performed using the LibDock algorithm.³⁵

Statistical analysis

The results were expressed as mean ± standard deviation. The differences between the means were analyzed by a using a one-way ANOVA test at the P < 0.05 level. The results were statistically analyzed by Microsoft Excel and GraphPad Prism 9.5.0 software.

Results and discussion

The physicochemical analysis revealed significant changes in the acidity of kombucha compared to the initial green tea infusion. The pH decreased from 6.2 ± 0.2 to 3.1 ± 0.2, and titratable acidity increased from 0.38 ± 0.3 to 0.55 ± 0.2 (Table 1). These results are consistent with typical fermentation effects, which are known to lower pH and increase acidity due to the production of organic acids.^36–38 The reduction in pH is crucial as it creates an environment that inhibits the growth of unwanted microorganisms and supports the development of beneficial bacteria and yeast during fermentation.³

Table 1.

Values of pH and Titratable acidity for green tea and 10 days of fermented kombucha..

Sample	pH	Titratable acidity (%)
Green tea	6.2 ± 0.2	0.38 ± 0.3
Fermented kombucha	3.1 ± 0.2	0.55 ± 0.2

Note: Values are expressed as mean ± standard deviation (n = 3).

The total phenolic content was higher in kombucha (0.721 μg/mL) compared to green tea (0.591 μg/mL) (Table 2). This increase in phenolic content is attributed to the fermentation process, which enhances the release and stability of phenolic compounds.^2,22,39 Phenolic compounds are known for their antioxidant properties, which contribute to the health benefits of kombucha. The results align with previous study that show similar ranges of phenolic content in kombucha extracts, reinforcing the idea that fermentation improves the phenolic profile of tea.⁴⁰

Table 2.

Various antioxidant activity of Kombucha extract compared to standard ascorbic acid.

Concentration (μg/mL)	DPPH		ABTS		H 2 O 2		TAC
	Ascorbic acid (%)	Kombucha (%)	Ascorbic acid (%)	Kombucha (%)	Ascorbic acid (%)	Kombucha (%)	Ascorbic acid (%)	Kombucha (%)
10	12.05 ± 0.1	23.83 ± 0.4	24.62 ± 0.5	30.50 ± 0.6	2.77 ± 0.5	7.70 ± 0.6	47.78 ± 0.5	56.53 ± 0.6
25	18.84 ± 0.4	33.93 ± 0.2	28.60 ± 0.7	34.57 ± 0.7	7.05 ± 0.6	9.83 ± 0.4	52.93 ± 0.5	58.82 ± 0.5
50	26.56 ± 0.5	42.55 ± 0.8	30.96 ± 0.5	37.71 ± 0.7	9.56 ± 0.7	11.67 ± 0.7	55.85 ± 0.5	60.84 ± 0.5
75	30.89 ± 0.3	49.56 ± 0.6	33.69 ± 0.3	39.86 ± 0.3	11.67 ± 0.7	13.75 ± 0.7	59.66 ± 0.7	62.63 ± 0.7
100	37.61 ± 0.7	60.67 ± 0.6	35.82 ± 0.5	43.66 ± 0.4	14.73 ± 0.5	15.64 ± 0.5	61.91 ± 0.6	64.62 ± 0.6

Note: value is expressed as (n = 3) mean ± Standard division.

Kombucha demonstrated significant antioxidant activity across various assays. DPPH inhibition at 60.67 ± 0.6% at 100 μg/mL exceeded that of ascorbic acid (37.61 ± 0.7%). This indicates a potent antioxidant capacity, which is consistent with the strong antioxidant effects reported for fermented kombucha.^9,41 ABTS scavenging activity of kombucha was 43.66 ± 0.4%, higher than vitamin C (35.82 ± 0.5%). The hydroxyl radical scavenging activity was 15.64 ± 0.5%, slightly higher than ascorbic acid (14.73 ± 0.5%), as depicted in Fig. 1A–D. These results confirm that kombucha exhibits robust antioxidant activity, which is essential for combating oxidative stress and related diseases. However, while kombucha showed strong antioxidant activity, green tea maintained a greater inhibitory capacity of the DPPH radical in some studies.⁴²

Fig. 1.

Antioxidant activity of DPPH, ABTS, H2O2, and TAC of kombucha extract.

The antibacterial activity of kombucha was significant, with the highest inhibition against Klebsiella pneumoniae (38 mm) and notable effects against E. coli, Salmonella typhi, Actinobacteria, and Pseudomonas (Fig. 2; Table 3). Kombucha’s antibacterial properties are attributed to its bioactive compounds, which can disrupt bacterial cell membranes and inhibit bacterial growth. The effectiveness against various pathogens underscores kombucha’s potential as a natural antimicrobial agent, supporting its traditional use for health benefits.

Fig. 2.

Antibacterial activity of Kombucha extract against (A) E. coli, (B) S. typhi, (C) K. pneumonia, (D) Actinobacteria and (E) Pseudomonas sp.

Table 3.

Antibacterial activity of kombucha.

S. No.	Strain	Zone of inhibition (mm)
		Kombucha extract	Ampicillin 10mcg
1	E. coli	27	15
2	S. typhi	29	17
3	K. pneumonia	38	21
4	Actinobacteria	22	19
5	Pseudomonas sp.	23	19

FTIR analysis of kombucha extract identified several key functional groups, including O-H stretching, C-H stretching, N-O stretching, and C-N stretching (Fig. 3; Table 4). These functional groups indicate the presence of various compounds such as alcohols, alkenes, nitro compounds, sulfonyl chlorides, and amines. The results are consistent with the findings of Kaashyap et al.,⁴³ who reported differential peaks in kombucha extracts indicative of specific functional groups. The identification of these functional groups provides insights into the chemical composition of kombucha and its potential bioactive properties.

Fig. 3.

FT-IR analysis of Kombucha extract.

Table 4.

FT-IR analysis of kombucha extract.

S. No.	Absorption (cm −1 )	Group	Compound class
1	3847.2915	O-H stretching	Alcohol
2	3774.9739	O-H stretching	Alcohol
3	3664.0872	O-H stretching	Alcohol
4	3455.8127	O-H stretching	Alcohol
5	2921.6274	C-H stretching	Alkane
6	1549.5232	N-O stretching	Nitro compound
7	1408.7451	S=O stretching	Sulfonyl chloride
8	1095.3693	C-N stretching	Amine
9	669.178	C-Br stretching	Halo compound

HR-LC/MS analysis identified 45 bioactive compounds in kombucha, including Celereoin and Fucofuroeckol B (Fig. 4; Tables 5 and 6). The high retention times of these compounds suggest their significant presence in the extract. The diversity of identified compounds highlights kombucha’s rich bioactive profile and supports its potential therapeutic applications. These findings are important for understanding the complex chemical composition of kombucha and its bioactivity.

Fig. 4.

HR-LC/MS analysis of kombucha (A) positive and (B) negative ionization chromatogram.

Table 5.

HR-LC/MS analysis of Kombucha for positive ionization mode.

S. No.	RT	Compound name	CID	Compound Structure	MF	MW	M/Z [+] value	H-D	H-A	X log P-value
1	1.031	(+/−)-3-[(2-methyl-3furyl)thio]-2-butanone	12980878		C9H12O2S	184.0554	185.0632	0	3	2.1
2	1.037	1-Pyrenylsulfate	9543290		C16H10O4S	298.0333	299.0415	1	4	4.1
3	1.566	Cyclopiazonic acid	54682463		C20H20N2O3	336.1485	337.1559	2	3	2.2
4	1.774	Alpha-Viniferin	196402		C42H30O9	678.1939	362.0859	6	9	6.8
5	1.815	Beta-Rhodomycin	3037123		C28H33NO10	543.2101	566.1995	6	11	2
6	1.893	Medicanine	101409750		C7H13NO3	159.0886	182.0779	2	4	−2.6
7	1.9	Adefovir Dipivoxil	60871		C20H32N5O8P	501.2	524.1893	1	12	1.8
8	1.937	N-(1-Deoxy-1-fructosyl)serine	131752245		C9H17NO8	267.0921	268.0995	7	9	−5.4
9	2.214	N-Hexanoylglycine methyl ester	338209		C9H17NO3	187.1196	210.1088	1	3	1.4
10	2.526	Diethanolamine	8113		C4H11NO2	105.0792	128.0684	3	3	−1.4
11	2.715	Dinitramine	34468		C11H13F3N4O4	322.087	345.0762	1	9	3.2
12	3.476	3beta,6beta- Dihydroxynortropane	22297531		C7H13NO2	143.0939	166.083	3	3	−0.7
13	4.006	L-Sorbose	439192		C6H12O6	180.0615	181.0688	5	6	−2.8
14	4.008	Feruloyl-2-hydroxyputrescine	131751430		C14H20N2O4	280.138	281.1454	4	5	0.1
15	4.746	Phenylethylamine	1001		C8H11N	121.0885	144.0777	1	1	1.4
16	4.844	Isoleucyl-Hydroxyproline	25227053		C11H20N2O4	244.1405	267.1296	3	5	−2.8
17	5.054	Methyl N-methyl anthranilate	6826		C9H11NO2	165.0779	188.0671	1	3	2.3
18	5.109	D-Vacciniin	12444645		C13H16O7	284.0874	307.0766	4	7	−0.9
19	5.279	Para-Trifluoromethylphenol	67874		C7H5F3O	162.0289	163.0361	1	4	2.8
20	5.495	1-[2-(4-Nitrophenyl)ethenylsulfonyl]-5-propan-2-yloxypyrrolidin-2-one	3087425		C15H18N2O6S	354.0897	355.0969	0	6	2
21	5.98	L-Capreomycidine	135471152		C6H12N4O2	172.095	195.086	4	4	−4.5
22	6.2	Nequinate	26383		C22H23NO4	365.1637	366.1711	1	5	5
23	6.274	8-Azaadenosine	96410		C9H12N6O4	268.0927	291.0819	4	9	−2.2
24	6.343	Orotidine	92751		C10H12N2O8	288.0586	289.0659	5	8	−2.6
25	6.645	Homomangiferin	5491388		C20 H20 O11	436.0963	459.0856	7	11	0
26	7.383	7a- Hydroxy -O-Carbamoyl-deacetylcephalosporin C	194116		C15H20N4O8S	416.4	433.1063	5	10	−5
27	7.39	2-(Arabinosylamino)-3-(glucosylamino)propanenitrile	131752666		C14H25N3O9	379.1595	402.1489	9	12	−4.5
28	7.418	Tyromycic acid	12444570		C30H44O3	452.3293	453.3368	1	3	7
29	7.42	3-Oxo-12,18-ursadien-28-oic acid	14707579		C30H44O3	452.3287	475.3181	1	3	6.2
30	7.552	Catechin 7-O-gallate	471393		C22H18O10	442.0833	443.0907	7	10	1.1
31	8.167	6-Chloropurine riboside	93003		C10H11ClN4O4	286.0431	287.0503	3	7	0.3
32	8.986	Celereoin	5315768		C14H14O5	262.0818	285.0711	2	5	1.6

Note: RT: Retention time; CID: Compound ID; MF: Molecular formula; MW: Molecular weight; H-D: Hydrogen donor; H-A: Hydrogen Acceptor.

Table 6.

HR-LC/MS analysis of Kombucha for negative ionization mode.

S. No.	RT	Compound name	CID	Compound Structure	MF	MW	M/Z [+] value	H-D	H-A	X log P-value
1	2.663	Oxadiazon	29732		C15H18Cl2N2O3	344.0652	343.0577	0	4	4.8
2	2.67	Chartreusin	5281394		C32H32O14	640.1728	685.1712	5	14	2.1
3	5.48	3-O-alpha-L Arabinopyranosylproanthocya nidin A5'	14841174		C35H32O16	708.1777	707.1707	11	16	0.9
4	5.661	Flazine	5377686		C17H12N2O4	308.0802	353.0784	3	5	2.1
5	5.724	Cicerin 7-(6malonylglucoside)	74413691		C26H26O15	578.1309	577.124	5	15	0.6
6	5.966	Bisisodiospyrin	169144		C44H26O12	746.1355	745.1284	4	12	7.2
7	6.268	6-Methoxy-3-(1,3-thiazolyl-2yl)-1H-indole	11160579		C12H10N2OS	230.0496	289.0633	1	3	2.7
8	6.365	(−)-Epigallocatechin 3-gallate 7-glucoside 4″-glucuronide	102025303		C34H36O22	796.1727	795.1656	14	22	−1.1
9	6.685	Assamicain A	14284578		C44H36O22	916.1563	915.149	17	22	4.3
10	6.691	11-O-Demethylpradinone II	441168		C24H16O11	480.0647	479.0578	8	11	2.3
11	7.327	Quercetin 3-(2-p-coumaroylglucoside)	44259191		C30H26O14	610.1406	609.1337	8	14	2.7
12	7.589	Beta-Alanyl-CoA	11966133		C24H41N8O17P3S	838.1617	883.1595	10	23	−8.9
13	7.591	Fucofuroeckol B	442681		C24H14O11	478.0537	477.0487	7	11	3.8

Note: RT: Retention time; CID: Compound ID; MF: Molecular formula; MW: Molecular weight; H-D: Hydrogen donor; H-A: Hydrogen Acceptor.

Molecular docking studies revealed that kombucha-derived compounds, especially Nequinate, exhibit strong binding affinities with key target proteins. Nequinate showed high LibDock scores of 105.12, 114.49, and 108.97 for jack bean urease (PDB ID: 4H9M), MAPK (PDB ID: 1TVO) and FYN (PDB ID: 2DQ7) respectively. Specifically, Nequinate interacted with 4H9M through one carbon-hydrogen bond with Val640, one pi-donor hydrogen bond with Cme592, and four alkyl interactions with Arg439, Arg639, Pro573, and Met588. For 2DQ7, it formed one hydrogen bond with Leu17, three carbon-hydrogen bonds with Met85, Gly88, and Asn86, and six alkyl interactions with Lys39, Val25, Tyr84, Leu137, Ala37, and Ala147, as depicted in Fig. 5; Tables 7 and 8. In the case of 1TVO, Nequinate established two carbon-hydrogen bonds with Arg67 and Tyr36, and one alkyl interaction with Val39. These interactions indicate that kombucha compounds, particularly Nequinate, may possess significant therapeutic potential by modulating the function of these target proteins, aligning with previous studies that emphasize the therapeutic promise of kombucha’s bioactive compounds.⁴⁴

Fig. 5.

Representation of 3D and 2D view of macromolecular interaction of target receptor (A) 4H9M, (B) 2DQ7, and (C) 1TVO against Nequinate.

Table 7.

LibDock score value of target protein with all bioactive compounds from kombucha.

S. No.	Compound name	CID	LibDock score
			4H9M	2DQ7	1TVO
1	(+/−)-3-[(2-methyl-3furyl)thio]-2-butanone	12980878	60.58	68.25	68.79
2	1-Pyrenylsulfate	9543290	-	104.168	91.307
3	Cyclopiazonic acid	54682463	-	102.37	97.43
4	Alpha-Viniferin	196402	-	-	-
5	Beta-Rhodomycin	3037123	-	-	-
6	Medicanine	101409750	68.16	72.36	72.96
7	Adefovir Dipivoxil	60871	-	-	-
8	N-(1-Deoxy-1-fructosyl)serine	131752245	-	-	-
9	N-Hexanoylglycine methyl ester	338209	62.49	81.14	81.43
10	Diethanolamine	8113	53.85	61.23	58.95
11	Dinitramine	34,468	-	36.93	46.89
12	3beta,6beta- Dihydroxynortropane	22297531	64.01	58.05	69.23
13	L-Sorbose	439192	-	-	-
14	Feruloyl-2-hydroxyputrescine	131751430	102.83	119.28	107.84
15	Phenylethylamine	1001	55.48	57.92	61.22
16	Isoleucyl-Hydroxyproline	25227053	76.36	88.97	83.89
17	Methyl N-methyl anthranilate	6826	66.59	62.34	60.81
18	D-Vacciniin	12444645	-	106.15	104.44
19	Para-Trifluoromethylphenol	67874	58.63	61.13	64.96
20	1-[2-(4-Nitrophenyl)ethenylsulfonyl]-5-propan-2-yloxypyrrolidin-2-one	3087425	-	92.11	98.208
21	L-Capreomycidine	135471152	69.25	84.75	76.08
22	Nequinate	26383	105.12	114.49	108.97
23	8-Azaadenosine	96410	-	-	-
24	Orotidine	92751	-	-	-
25	Homomangiferin	5491388	-	-	-
26	7a- Hydroxy -O-Carbamoyl-deacetylcephalosporin C	194116	-	-	-
27	2-(Arabinosylamino)-3-(glucosylamino)propanenitrile	131752666	-	-	-
28	Tyromycic acid	12444570	-	-	-
29	3-Oxo-12,18-ursadien-28-oic acid	14707579	-	98.81	93.46
30	Catechin 7-O-gallate	471393	-	-	-
31	6-Chloropurine riboside	93003	-	93.52	94.15
32	Celereoin	5315768	-	80.67	83.38
33	Oxadiazon	29732	-	85.78	84.73
34	Chartreusin	5281394	-	-	-
35	3-O-alpha-L Arabinopyranosylproanthocya nidin A5'	14841174	-	-	-
36	Flazine	5377686	-	107.59	108.70
37	Cicerin 7-(6malonylglucoside)	74413691	-	-	-
38	Bisisodiospyrin	169144	-	-	-
39	6-Methoxy-3-(1,3-thiazolyl-2yl)-1H-indole	11160579	-	85.507	81.55
40	(−)-Epigallocatechin 3-gallate 7-glucoside 4″-glucuronide	102025303	-	-	-
41	Assamicain A	14284578	-	-	-
42	11-O-Demethylpradinone II	441168	-	-	-
43	Quercetin 3-(2-p-coumaroylglucoside)	44259191	-	-	-
44	Beta-Alanyl-CoA	11966133	-	-	-
45	Fucofuroeckol B	442681	-	-	-

Note: CID: Compound ID.

Table 8.

Interaction of amino acid residues for Nequinate with target proteins.

S. No.	Target protein	LibDock score value	Bonding category	Interacted residues
1	4H9M	105.12	Carbon hydrogen bond	Val640
			Pi-Donor hydrogen bond	Cme592
			Alkyl interaction	Arg 439, pro573, Met588, Arg 639
2	2DQ7	114.49	Hydrogen bond	Leu17
			Carbon hydrogen bond	Met85, Gly88 & Asn86
			Alkyl interaction	Lys39, Val25, Tyr84, Leu137, Ala37&147
3	1TVO	108.97	Carbon hydrogen bond	Tyr36, Arg67
			Alkyl interaction	Val39

Metagenomic analysis revealed that Proteobacteria were the most abundant bacterial phylum, with genera such as Agromyces, Kocuria, Komagataeibacter, and Stenotrophomonas being prominent (Fig. 6A–C). The fungal analysis identified Ascomycota as the predominant phylum, with Aspergillaceae and Saccharomycetales as the most abundant families (Fig. 7A–C). Phylogenetic analysis confirmed the dominance of Proteobacteria and Ascomycota in kombucha (Fig. 8). Understanding these microbial communities is crucial for evaluating the health benefits and safety of kombucha, as well as for optimizing fermentation processes.

Fig. 6.

Represents a percentage of the most relative abundance of bacterial distribution present in Kombucha at different levels (A) Phylum, (B) Family, (C) Genus using the V3-V4 region.

Fig. 7.

Represents a percentage of the most relative abundance of fungal distribution present in Kombucha at different levels (A) Phylum, (B) Family, (C) Genus using the ITS region.

Fig. 8.

Demonstrates the phylogenetic distribution of species found in bacterial (A), and fungal (B) domain in Kombucha.

The presence of Proteobacteria and therapeutic potential of Ascomycota fungi highlight the dual aspects of kombucha’s microbiome. Proteobacteria can contribute to antibiotic production and vaccine development,^45,46 while Ascomycota fungi offer potential for immune modulation and therapeutic applications.^47–50 This diverse microbial composition underscores the complexity of kombucha’s health benefits and its potential for further research and application.

Conclusion

This study thoroughly evaluates the physicochemical, antioxidant, antibacterial, and molecular characteristics of kombucha and green tea. The results demonstrate that fermentation enhances the phenolic content, antioxidant activity, and antibacterial properties of kombucha. The identification of bioactive compounds and their strong binding affinities with target proteins suggest potential therapeutic applications. The metagenomic analysis highlights the diverse microbial composition of kombucha, emphasizing its potential health benefits. Further research is needed to optimize fermentation processes and fully explore the therapeutic potential of kombucha.

Acknowledgments

The authors are thankful to Prof. V. Rajesh Kannan, Rhizosphere Biology Laboratory, Department of Microbiology, Bharathidasan University, Tiruchirappalli for providing their laboratory facilities.

Authors contribution

TS Shalini: Data collection, draft manuscript writing, reviewing and editing; R Prathiviraj: Conceptual, reviewing, editing, analysis and approved for final manuscript; P Senthilraja: Funding conceptual, reviewing, editing and approve for final manuscript.

Funding

Not Applicable.

Conflict of interest statement: The authors declare that no conflicts of interest in the present manuscript.