The chemical composition of kombucha is dependent on different substrate compositions, source and microbial community in the SCOBY, fermentation methods, and detection methods.

Kombucha is composed of sugars (including sucrose, glucose, and fructose), organic acids [including acetic acid, gluconic acid, glucuronic acid, lactic acid, DSL, citric acid, oxalic acid, and pyruvic acid (Jayabalan et al., 2007)], B-vitamins and vitamin C (Bauer-Petrovska and Petrushevska-Tozi, 2000), theophyllines, tea polyphenols, flavonoids, various amino acids and proteins, ethanol, biogenic amines, purine bases, hydrolytic enzymes, minerals (primarily Cu, Fe, Zn, Ni, and Mn), and metabolites secreted by yeast and bacteria (Table 2) (Malbaša et al., 2011).

Traditional kombucha fermentation utilises tea and white sugar as the main substrates. These substrates are transformed through fermentation by microorganisms, such as yeast and bacteria, which involves the gradual breakdown of sugar and a metabolic cascade that produces CO₂ and an acidic, slightly alcoholic beverage (Chakravorty et al., 2016). In a bacteriophage fermentation environment, yeasts are the primary ethanol producers, as they produce hydrolytic enzymes (of the fructosidase class) that hydrolyse sucrose to glucose and fructose, which subsequently undergo metabolic processes leading to the production of ethanol, glycerol, and carbon dioxide. The involvement of different bacterial strains during fermentation leads to metabolic differentiation. The yeast genus Saccharomyces spp. can utilise glucose and produce ethanol through the glycolytic pathway, whereas the jointed yeast Zygosaccharomyces spp. can efficiently ferment fructose to produce ethanol. Furthermore, specific yeast strains, such as the corn wine fission yeast (Schizosaccharomyces pombe), have the capability to generate ethanol from malic acid or Brettanomyces bruxellensis in the presence of elevated levels of acetic acid in aerobic environments (Villarreal-Soto et al., 2018).

During fermentation, microorganisms interact with each other, and the ethanol produced by yeast fermentation can be used by Acetobacter as a metabolic substrate for oxidation to acetic acid. In addition to acetic acid, Acetobacter can further metabolise glucose in the fermentation broth to produce glucuronic acid, which is later metabolised to gluconic acid and converted to glucuronic acid (Villarreal-Soto et al., 2019; Martínez-Leal et al., 2020). In addition, B. gluconii strains have the capability to enzymatically produce L-ascorbic acid, commonly known as vitamin C, utilizing D-sorbitol as a precursor compound, which is itself derived from glucose (Mamlouk and Gullo, 2013). Depending on the specific strain, certain lactic acid bacteria have the capability to utilize glucose in either the glycolytic pathway, resulting in the production of lactic acid as the primary metabolite, or the pentose phosphate pathway, leading to the synthesis of lactic acid, ethanol, and carbon dioxide. However, when fructose is present, the production of acetic acid occurs instead of ethanol (Laureys et al., 2020).

Bacterial cellulose film is a distinctive byproduct of kombucha fermentation, which is generated by acetate bacteria through alcohol metabolism and can be removed as waste. Komagataeibacter spp. use glucose to synthesise bacterial cellulose, and this anabolic process involves sucrose, ethanol, and glycerol (Chawla et al., 2009; Villarreal-Soto et al., 2019). The fermentation and metabolic processes of kombucha are accomplished through mutual facilitation or constraints within each flora. The synergistic effects of the flora allow for the synthesis of certain antimicrobial metabolites, the accumulation of organic acids leading to low pH, and the production of physical barriers (cellulose membranes), and other factors contributed to the hindrance of bacterial growth among competitors (Villarreal-Soto et al., 2019).

As depicted in Figure 1, the intricate phenolic compounds present in kombucha have the potential to undergo degradation or conversion into smaller biological molecules through the process of fermentation occurring within an acidic milieu, or through the enzymatic activities released by bacteria and yeast. For example, the observed elevation in overall catechin content in green and black tea kombucha subsequent to fermentation can be ascribed to the biotransformation process wherein epigallocatechin-3-gallate (EGCG) undergoes conversion into epigallocatechin gallate (ECG) and epicatechin (EC), by enzymes released by microbial communities in an acidic environment. EGCG is hydrolysed into smaller molecules and converted into epigallocatechin (EGC), EGCG, and EC (Jayabalan et al., 2008). Theaflavins and thearubigin are complex polyphenol derivatives found in black tea and are associated with colour changes in tea (Martínez-Leal et al., 2018). The lighter colour of a fully fermented kombucha may be attributable to the conversion of theaflavins to theobromine (Jayabalan et al., 2007).

Traditional kombucha is prepared from black tea and white sugar, which are the primary raw materials used in microbial fermentation. However, in the last three decades, considerable research has resulted in the replacement of black tea and white sugar with other teas, herbs, plant materials, and sugars. The pharmacological products of fermentation depend on the active substances and their amount in the broth. The metabolites generated during kombucha fermentation of alternative substrates exhibit a strong correlation with the characteristics of the initial raw materials (Table 3).

Kombucha beverages produced through the fermentation of alternative raw material extracts are known to have antibacterial potential. The antibacterial profile of various substrates after fermentation with kombucha (Table 4) is influenced by different factors, including fermentation time, raw materials, temperature, and kombucha strains. The fermentation of kombucha is often considered a key factor contributing to its antibacterial activity. The competitive growth advantage of the dominant flora of traditional kombucha and the production of secondary metabolites, such as tea polyphenols, organic acids, and ethanol, can impede the growth and proliferation of pathogenic bacteria and fungi. Ayed et al. found that the growth of certain microbes, such as Staphylococcus aureus, Bacillus cereus, and Staphylococcus epidermidis, was found to be inhibited by fermented cactus pear juice; whereas unfermented pear fruit cactus juice had no antibacterial effect, and this inhibitory property was lost when the samples were neutralized (Ayed and Hamdi, 2015). The organic acids, specifically acetic acid, were identified as the primary contributors to the observed antimicrobial activity (Ayed and Hamdi, 2015). Četojević-Simin et al. employed an agar diffusion method to investigate the antimicrobial properties of lemon balm tea. The results indicated that unfermented lemon balm tea, with a dry weight concentration of 5 g/L, did not demonstrate any antimicrobial activity. However, the fermented lemon balm tea kombucha exhibited significant antimicrobial activity against prokaryotic microorganisms (G+ bacteria and G− bacteria) (Četojević-Simin et al., 2012). While no inhibitory activity was observed against fungi, yeast, or mould, the observed inhibition had similar activity to an acetic acid solution, which inferred that acetic acid served as the primary inhibitory agent within the kombucha beverage. The presence of other heat-resistant antibacterial components was verified by neutralizing with heating (Četojević-Simin et al., 2012). Vitas et al. (2018) identified significant antimicrobial properties in subcritical aqueous extracts of yarrow after fermentation; the potential antimicrobial substances were likely organic acids, phenolic compounds of plant origin, enzymes, proteins, and bacteriocins produced through fermentation.

The primary antimicrobial agent found in kombucha is acetic acid. The phenomenon encompasses the capacity of the undissociated acid to undergo unhindered diffusion across the lipid bilayer and subsequently release protons from the cytoplasm, thereby reducing the pH of the cytoplasm. This process also involves the integration of the undissociated acid within the lipid bilayer under conditions of low external pH, ultimately resulting in the accumulation of anions. The antimicrobial activity of kombucha is primarily attributed to two mechanisms: cytoplasmic acidification and the accumulation of free acid anions at toxic concentrations (Jayabalan et al., 2008). The acidification of the bacterial cytoplasm can impede bacterial growth through the inhibition of glycolysis, hindrance of active transport, or disruption of signal transduction (Roe et al., 2002).

In addition to organic acids, other components introduced by alternative raw materials cannot be ignored in bacterial inhibition. Some phenolic compounds with antimicrobial activity can affect the hyperacidification of the plasma membrane interface.

The integrity of this membrane can be compromised either by the H+-ATPase enzyme necessary for ATP production or through interaction with bacterial DNA, resulting in modifications to bacterial physiology and impeding cellular proliferation. The presence of Lycium barbarum polysaccharides in the fermentation broth exerted a pronounced inhibitory impact on both gram-positive and gram-negative bacteria. This effect was attributed to the rapid disruption of cellular membranes, thereby impeding the passage of essential nutrients and metabolites across the bacterial cells, ultimately leading to their inhibition (Zhang et al., 2022). Zubaidah et al. (2021c) revealed that the antibacterial activity of turmeric kombucha increased with the turmeric concentration and decreased after reaching a certain level. The antibacterial components of turmeric kombucha differ from turmeric concentrations; the curcuminoid compounds in turmeric and its essential oil act as antimicrobial agents by inhibiting the metabolism of microorganisms. In addition, terpene derivatives (sesquiterpenes and monoterpenes) in essential oils can disrupt the structure of bacterial cell membranes (Zubaidah et al., 2021b). Shahbazi et al. (2018) observed a notable reduction in the minimal inhibitory concentration (MIC) of cinnamon fermentation broth with decreasing pH; cinnamon components also disrupted the cytoplasmic membranes of gram-positive and gram-negative bacteria and reduced intracellular ATP concentrations. Pure and Pure (2016b) reported that gram-positive bacteria were more sensitive to garlic extracts than gram-negative bacteria. This phenomenon can be attributed to the influence of the lipid constituents present in the cell wall, which hinder the penetration of garlic’s active compounds into the cells.

In summary, fermented broth often combines the antibacterial properties of the raw material and the microbial strain, with organic acids being the active components. Fermentation creates an acidic environment for drugs and influences the antibacterial properties of raw materials, and the active substances of the raw materials also contribute to the antibacterial power. However, owing to the differences in strains, the intricate composition of strains, and diverse culture environments, it is difficult to evaluate the chemical composition and core flora associated with the inhibitory effects reported in each study, making it difficult to achieve consistent inhibitory activity across different studies. Pure and Pure (2016b) tested various samples using the disk diffusion method against S. typhimurium, S. aureus, E. coli, S. saprophytic, B. saprophyticus, and Pseudomonas aeruginosa. The authors found that the infused and fermented samples of common nettles, banana peel, and black tea did not demonstrate any antibacterial efficacy against the tested bacteria; this was thought to be attributed to the differences in the concentration of the aqueous extracts and the assay method (Pure and Pure, 2016b). Therefore, the development of kombucha fermentation broth as a natural biological preservative requires further work. More in-depth research on the antibacterial mechanisms of kombucha is required to optimise the strain composition, regulate the fermentation process, and actively develop kombucha products that are suitable for consumption and have clear health benefits.

The fermentation process can enhance the antioxidant properties of Kombucha, thereby rendering it an excellent source of antioxidants (Table 5). Gamboa-Gómez et al. (2016) evaluated the effect of kombucha fermentation on the antioxidant activities of Litsea glaucescens and Eucalyptus camaldulensis, using three antioxidant activity indicators, namely thiobarbituric acid reactive substances (TBARS), α, α-diphenyl-ß-picrylhydrazyl (DPPH), and nitric oxide (NO). The researchers discovered that the process of kombucha fermentation enhanced the capacity of natural herbal infusions to effectively scavenge free radicals and inhibit lipid peroxidation (Gamboa-Gómez et al., 2016). Sun et al. used traditional kombucha as a control to test against a 1:1 mixture of wheatgrass juice and brown sugar tea and found that the modified fermentation broth exhibited higher levels of phenolic acid content and oxidative radical absorption capacity compared to the traditional kombucha. Notably, the DPPH scavenging rate reached 90% (Sun et al., 2015).

Polyphenols are the main antioxidants in the kombucha fermentation broth, and these entities possess the ability to readily donate hydroxyl hydrogen due to their resonance stabilization (Miller et al., 1993). This hydrogen supply enhances the DPPH-scavenging ability of the phenolic compounds. The increase in antioxidant properties of alternative raw materials for fermentation is usually associated with increased total phenol content, which is attributable to enzymatic synthesis and acid hydrolysis (Ayed et al., 2017). During the process of fermentation, microorganisms, specifically Acetobacter and Saccharomyces, present in kombucha, secrete enzymes that break down polyphenols into smaller compounds possessing potent antioxidant properties. Enzymes secreted by the kombucha SCOBY cleave flavonoid ring structures and/or phenolic conjugation sites, leading to structural rearrangements. Common rearrangements include the depolymerisation of theaflavins and theobromine and the cleavage of gallic acid into gallic acid (Chen and Liu, 2000). The potential outcome of these rearrangements is an enhancement in the bioavailability of phenolic compounds, specifically gallic acid, EC, and ECG 32.

The antioxidant properties of alternative raw materials stem from the differences in their total phenol contents and the specific substrates that generate antioxidant components during fermentation. Ayed et al. (2017) used red grape juice as an alternative substrate, and the fermentation process generated beneficial compounds that enhanced the antioxidant properties of the beverage. Ulusoy and Tamer (2019) discovered that the predominant contributors to the antioxidant activity of raspberries were anthocyanins and ellagitannins, accounting for 25% and 52% of the overall antioxidant activity, respectively. In addition, Zhang et al. (2022) found that the kombucha SCOBY has the potential to alter the composition of polysaccharides and the ratio of monosaccharides to polysaccharides after fermentation with Lycium barbarum, which affected its antioxidant activity.

As depicted in Figure 2, the anti-inflammatory effect of kombucha can be attributed to the beneficial components, including flavonoids and phenolic acids (gallic acid, catechins, and theaflavins), produced by the biotransformation and metabolism of the flora. The ameliorating effect of kombucha (15 mg/kg) on gastric ulcers was comparable to that of the positive control drug, omeprazole (3 mg/kg). Theaflavin was the main anti-inflammatory component, and the healing rate of theaflavin (1 mg/kg) in mice over 7 days was 81.4% (Banerjee et al., 2010). Many kombucha fermentation broths prepared using alternative raw materials exhibit anti-inflammatory properties. Kanno et al. (2006) used tea made from oak leaves as a substrate for preparing kombucha fermentation broth, and the broth effectively downregulated NO production and exhibited a substantial decrease in the expression of tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated macrophages (THP-1). The presence of naringin in oak demonstrated a significant inhibition of LPS-induced nitric oxide (NO) production, as well as the expression of inflammatory gene products, including inducible nitric oxide synthase (iNOS), TNF-α, and IL-6 (Kanno et al., 2006); moreover, (+)-catechin present in oak inhibited LPS-stimulated NO and TNF-α production in macrophages (Guruvayoorappan and Kuttan, 2008). The phenolic content of Kombucha, which is produced through the fermentation of oak leaf tea, contributes to its notable anti-inflammatory and antioxidant properties (Vázquez-Cabral et al., 2017). Ziemlewska et al. (2021) found that yerba mate extracts used as an alternative raw fermentation material showed potent inhibition of lipoxygenase (LOX) after 14 and 21 days of fermentation. The enzyme LOX plays a crucial role in the synthesis of leukotrienes, which are known as mediators of the pro-inflammatory response. Therefore, the regulation of LOX activity holds significant importance in the treatment of inflammation (Nworu and Akah, 2015; Oguntibeju, 2018).

Quercetin and saponins from yerba mate extract are the main components responsible for their anti-inflammatory effects, and these substances can reduce the production of IL-6, cyclooxygenase-2 (COX-2), and NO—the main mediators of the inflammatory process. Additionally, the tannins in yerba mate extracts have an inhibitory effect on LOX activity (Ziemlewska et al., 2021).

Diabetes mellitus is a metabolic disorder characterized by inadequate insulin secretion or islet insufficiency. As shown in Figure 3, studies on the hypoglycaemic effects of kombucha have mainly focused on the analysis and histological observations of the biological activities of key enzymes in the glucose metabolic pathway; only a few in-depth studies have been reported on the associated molecular mechanisms. Zubaidah et al. (2019) demonstrated that the fermentation process of snake fruit extracts into kombucha effectively mitigated oxidative stress and provided stability to fluctuations in fasting blood glucose levels within a streptozotocin-induced diabetic model. This glucose-lowering effect was comparable to that of metformin hydrochloride—the “king” glucose-lowering drug (Zubaidah et al., 2019). Watawana et al. (2016) found that king coconut water fermented into kombucha resulted in the enhanced inhibition of starch hydrolases. The inhibitory activity of both α-amylase and glucosidase was enhanced by the fermentation process, with a greater enhancement observed in α-amylase inhibitory activity compared to α-glucosidase inhibitory activity. The enzyme α-amylase exerts its action prior to α-glucosidase, thus hindering the reaction rate of α-glucosidase and impeding the release of glucose into the physiological system. This outcome proves advantageous as it demonstrates a beneficial anti-hyperglycemic effect (Watawana et al., 2016). Aung and Eun (2021) showed that the fermentation broth of laver, as an alternative raw material, inhibited amylase activity in vitro. Fermented laver kombucha, which contained important flavonoid compounds, enhanced α-amylase inhibitory activity at 25°C (Aung and Eun, 2021). Al-Dulaimi et al. (2018) explored the effects of kombucha fermentation with skim milk as a substrate on serum glucose concentrations, total lipid profiles, and body weight in male rats. Skim milk fermentation reduced blood glucose, total lipid, alanine aminotransferase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) in the rats and exerted beneficial effects on the human liver and overall health. This was attributable to the combined effects of polyphenols (such as flavonoids and catechins), vitamin E, and organic acids in the fermentation broth (Al-Dulaimi et al., 2018). Permatasari et al. (2022) found that butterfly pea flower kombucha (KBPF) inhibited α-amylase and α-glucosidase activities, achieving similar levels of inhibition to the acarbose control at concentrations of 50–250 μg/mL. In vivo, KBPF administration (130 mg/kg BW) significantly alleviated the metabolic disturbances induced by a high-fat diet through the modulation of glucose level, oxidative stress markers (superoxide dismutase), metabolic enzymes (lipases and amylases), inflammatory markers (PGC-1α, TNF-α, and IL-10), and lipids, such as total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL), and high-density lipoprotein cholesterol (HDL). In addition, KBPF had a positive effect on the phyla Bacteroidetes and Firmicutes (Permatasari et al., 2022).

As shown in Figure 4, Ziemlewska et al. (2021) determined the skin care effects of fermented yerba mate kombucha on keratinocytes and fibroblast cell lines. They found that fermentation strongly inhibited lipoxygenase, collagenase, and elastase activities. Furthermore, the researchers noted a sustained moisturizing effect subsequent to the topical administration. The fermentation solution comprised of phenolic acids, methylxanthines, and flavonoids, which actively contributed to the inhibition of enzymatic activity associated with skin aging. The primary compounds possessing moisturizing properties are antioxidants (polyphenols and flavonoids), proteins, amino acids, and carbohydrates, which feature hydroxyl groups capable of forming hydrogen bonds with water (Ziemlewska et al., 2021). Zofia et al. (2020) showed that kombucha with coffee beans as a substrate could inhibit collagenase and elastase activities, improve skin hydration, and exert sunscreen effects. The concentrations of polyphenols, flavonoids, and caffeine in the fermentation broth considerably increased the inhibition of collagenase and elastase activities. The active ingredients in the coffee fermentation product, such as monosaccharides, amino acids, vitamins, polyphenols, and flavonoids, showed nourishing and soothing effects. The sunscreen properties of fermentates are mainly attributable to substances derived from flavonoids, polyphenols, anthocyanins, proteins, amino acids, and vitamins (Zofia et al., 2020).

The consumption of kombucha can improve the immune system and enhance the ability of the body to fight cancer (Figure 5). Salafzoon et al. (2017) obtained samples from a 10-day kombucha fermentation of ginger infusion containing ginger bioactive components, such as gingerol and shogaol, which have anti-inflammatory and anti-tumour activities. These samples could inhibit tumour proliferation and stimulate apoptosis. Fermented ginger infusions reduced the activities of peroxidase, glutathione, and malondialdehyde in tumours, liver, and kidney homogenates (Salafzoon et al., 2017). Vitas et al. (2018) found that the fermentation broth of kombucha with yarrow as a substrate has antiproliferative activity against human rhabdomyosarcoma cells, human cervical cancer Hep2 cells (HeLa), murine fibroblasts (L2OB), and other tumour cells. Uțoiu et al. found that the fermentation broth of kombucha with bee pollen as a substrate exerted different levels of antitumour activity in Hep-2 and Caco-2 cells (Uțoiu et al., 2018). The fermented pollen exhibited elevated concentrations of bioactive compounds, including polyphenols, soluble silica substances, and short-chain fatty acids, thereby augmenting the health-promoting properties of pollen through kombucha fermentation (Uțoiu et al., 2018). Furthermore, the parameters for Kombucha fermentation were optimized to enhance the cytotoxic efficacy of the n-hexane fruit extract derived from Solanum nigrum L. against MCF-7 breast cancer cells (Ziska et al., 2019).

The fermentation process of African mustard leaves by kombucha resulted in a notable enhancement in the overall phenolic and ethyl acetate levels, as well as the antioxidant capacity, when compared to the unfermented samples (Rahmani et al., 2019). In addition, kombucha fermentation increased anti-acetylcholinesterase activity; moreover, it reduced the cytotoxicity of Brassica tournefortii leaves and its inhibitory effect on xanthine oxidase (Rahmani et al., 2019). Zubaidah et al. conducted a study to examine the immunomodulatory properties of turmeric and black tea kombucha on BALB/c mice. The results of their investigation revealed that turmeric kombucha exhibited a significant enhancement in the adaptive immune response, mainly in the form of increased expression levels of CD4+, TNF-α, and IFN-γ, while enhancing the innate immune response, mainly in the form of decreased expression levels of CD68 and IL-6 (Zubaidah et al., 2021c). L. barbarum (Zhang et al., 2022), rose, and jujube kernels (Zhang et al., 2020) can contribute to cellulase activity when used as alternative fermentation substrates. With the use of winter savoury peppermint, stinging nettle, wild thyme, elderberry, quince (Vitas et al., 2020), and milk (Elkhtab et al., 2017), kombucha fermentation broth showed inhibitory activity against angiotensin-converting enzyme, and the resulting beverage showed antihypertensive potential.

As shown in Figure 6, there have been numerous studies on the functions of kombucha. However, most have focused on functional efficacy, with minimal investigation into the underlying fermentation process.

Instances of health issues resulting from the consumption of kombucha have been documented in scholarly literature, encompassing symptoms such as dizziness, headaches, allergic reactions, acidosis, and gastrointestinal ailments (Villarreal-Soto et al., 2018; Batista et al., 2022). These complications primarily arise from various factors, including excessive consumption, inadequate sanitation practices at home, contamination by pathogenic microorganisms, and susceptibility of immunocompromised individuals. For instance, pregnant women are advised against consuming kombucha due to its ethanol content, substantial acetic acid concentration, and potential presence of substances like heparin (Martínez-Leal et al., 2018; de Miranda et al., 2022). The excessive ingestion of kombuella in individuals with a history of excessive alcohol consumption has the potential to result in significant liver necrosis. Findings from pathological examination suggest a correlation between kombuella and liver damage in these patients, as evidenced by markedly elevated serum levels of aspartate aminotransferase and alanine aminotransferase, surpassing what would be expected solely from alcohol consumption (Sannapaneni et al., 2023).