What is kombucha

Kombucha tea is a fermented drink made with tea, sugar, bacteria and yeast ¹. Although it’s sometimes referred to as kombucha mushroom tea, kombucha is not a mushroom — it’s a colony of bacteria and yeast. Kombucha tea is made by adding the colony (bacteria and yeasts) to sugar and tea, and allowing the mix to ferment ². The resulting liquid contains vinegar, B vitamins and a number of other chemical compounds. A floating cellulosic pellicle layer and the sour liquid broth are the 2 portions of kombucha tea (Figure 1). It tastes like sparkling apple cider and can be produced in the home by fermentation using mail order or locally available tea fungus. Though green tea can be used for kombucha preparation, black tea and white sugar are considered the finest substrates.

Traditional substrate for the kombucha fermentation is black or green tea extract sweetened with 5% to 8% sucrose. Different tea leaf varieties, amounts of sugar, fermentation time, and composition of tea fungus may account for differences in composition and therefore also the biological activities of kombucha tea.

Figure 1. Kombucha black tea having fermented broth and tea fungus

Proponents claim kombucha tea helps prevent and manage serious health conditions, from blood pressure to cancer. These claims are not backed by scientific evidence ³. Limited evidence suggests kombucha tea may offer benefits similar to probiotic supplements, including promoting a healthy immune system and preventing constipation. All the biological activities have been investigated using animal experimental models. At present, however, valid medical studies of kombucha tea’s role in human health are very limited — and there are risks to consider.

There have been reports of adverse effects, such as stomach upset, infections and allergic reactions in kombucha tea drinkers. Kombucha tea is often brewed in homes under nonsterile conditions, making contamination likely. When improperly manufactured ceramic pots have been used for brewing, lead poisoning has occurred — the acids in the tea can leach lead from the ceramic glaze.

In short, there isn’t enough evidence that kombucha tea delivers on its health claims. At the same time, several cases of harm have been reported. Therefore, the prudent approach is to avoid kombucha tea until more definitive information is available.

Microorganisms of kombucha tea

Tea fungus or kombucha is the common name given to a symbiotic growth of acetic acid bacteria and osmophilic yeast species in a zoogleal mat which has to be cultured in sugared tea. According to Jarrell and others ⁴, kombucha is a consortium of yeasts and bacteria. The formal botanical name Medusomyces gisevii was given to it by Lindau (Hesseltine 1965). Tea fungus is not a mushroom. That name is wrongly given due to the ability of bacteria to synthesize a floating cellulose network which appears like surface mold on the undisturbed, unshaken medium.

Similarly to milk-derived kefir, the exact microbial composition of kombucha cannot be given because it varies. It depends on the source of the inoculum for the tea fermentation. One of the clearer accounts of the microbes found in kombucha starter is from Hesseltine ⁵. He isolated an Acetobacter sp. (NRRL B-2357) and 2 yeasts (NRRL YB-4810, NRRL YB-4882) from a kombucha sample received from Switzerland and used these microorganisms to produce kombucha tea.

The most abundant prokaryotes in this culture belong to the bacterial genera Acetobacter and Gluconobacter. The basic bacterium is Acetobacter xylinum ⁶. It produces a cellulosic floating network on the surface of the fermenting liquid. The network is the secondary metabolite of kombucha fermentation but also one of the unique features of the culture ⁷. Sievers and others ⁸ reported that the microflora embedded in the cellulose layer was a mixed culture of A. xylinum and a Zygosaccharomyces sp. The predominant acetic acid bacteria found in the tea fungus are A. xylium, A. pasteurianus, A. aceti, and Gluconobacter oxydans ⁹. Gluconacetobacter sp. A4 (G. sp. A4), which has strong ability to produce D-saccharic acid-1,4-lactone (DSL), was the key functional bacterial species isolated from a preserved kombucha by Yang and others ¹⁰. Strains of a new species in the genus Acetobacter, namely Acetobacter. intermedius sp. nov., were isolated from kombucha beverage and characterized by Boesch and others ¹¹. Dutta and Gachhui ¹² isolated the novel nitrogen-fixing Acetobacter nitrogenifigens sp. nov., and the nitrogen-fixing, cellulose-producing Gluconacetobacter kombuchae sp. nov., from kombucha tea. An investigation by Marsh and others ¹³ indicated that the dominant bacteria in 5 kombucha samples (2 from Canada and one each from Ireland, the United States, and the United Kingdom) belong to Gluconacetobacter (over 85% in most samples) and Lactobacillus (up to 30%) species. Acetobacter was determined in very small number (lower than 2%).

In addition to acetic acid bacteria there are many yeast species in kombucha. A broad spectrum of yeasts has been reported including species of Saccharomyces, Saccharomycodes, Schizosaccharomyces, Zygosaccharomyces, Brettanomyces/Dekkera, Candida, Torulospora, Koleckera, Pichia, Mycotorula, and Mycoderma. The yeasts of Saccharomyces species were identified as Saccharomyces sp. ¹⁴ and as Saccharomyces cerevisiae ¹⁵, Saccharomyces bisporus ⁷, Saccharomycoides ludwigii ¹⁶, Schizosaccharomyces pombe ¹⁷, Zygosaccharomyces sp. ¹³, Zygosaccharomyces rouxii ¹⁸, and Zygosaccharomyces bailii ¹⁹. The genus Brettanomyces was isolated by several workers. Herrera and Calderon-Villagomez ¹⁸ isolated Brettanomyces intermedius, Liu and others ⁹ and Teoh and others ¹⁷ isolated Brettanomyces bruxellensis, and Jayabalan and others ¹⁹ isolated B. claussenii. An examination of 2 commercial kombucha and 32 cultures from private households in Germany ²⁰ showed variable compositions of yeasts. The predominant yeasts were Brettanomyces, Zygosaccharomyces, and Saccharomyces spp. Roussin ⁶ determined Zygosaccharomyces and S. cerevisiae as the typical yeasts in North American kombucha. Kurtzman and others ²¹ isolated an ascosporogenous yeast, Zygosaccharomyces kombuchaensis sp. n. (type strain NRRL YB-4811, CBS 8849), from kombucha. An investigation of the physiology of Z. kombuchaensis sp. n., related to the spoilage yeasts Zygosaccharomyces lentus, clearly showed that these 2 species were not same ²².

Candida sp. is included in a great number of kombucha beverages. Kozaki and others ¹⁴ isolated Candida famata, Candida guilliermondii, and Candida obutsa. In kombucha samples from Mexico, Herrera and Calderon-Villagomez ¹⁸ detected C. famata. Teoh and others ¹⁷ identified Candida stellata. From a local kombucha in Saudi Arabia, Ramadani and Abulreesh ¹⁶ isolated and identified 4 yeasts: Candida guilliermondi, Candida colleculosa, Candida kefyr, and Candida krusei. C. krusei were identified in kombucha from a district of Ankara ¹⁵.

Chemical composition of kombucha tea

Chemical analysis of kombucha showed the presence of various organic acids, such as acetic, gluconic, glucuronic, citric, L-lactic, malic, tartaric, malonic, oxalic, succinic, pyruvic, usnic; also sugars, such as sucrose, glucose, and fructose; the vitamins B1, B2, B6, B12, and C; 14 amino acids, biogenic amines, purines, pigments, lipids, proteins, some hydrolytic enzymes, ethanol, antibiotically active matter, carbon dioxide, phenol, as well as some tea polyphenols, minerals, anions, DSL, as well as insufficiently known products of yeast and bacterial metabolites ²³.

Yeasts and bacteria in kombucha are involved in such metabolic activities that utilize substrates by different and in complementary ways. Yeasts hydrolyze sucrose into glucose and fructose by invertase and produce ethanol via glycolysis, with a preference for fructose as a substrate. Acetic acid bacteria make use of glucose to produce gluconic acid and ethanol to produce acetic acid. The pH value of kombucha beverage decreases due to the production of organic acids during fermentation ²⁴.

The results presented in Table 1 indicate the predominant components of traditional kombucha beverage. These data suggest the heterogeneity of investigations performed on kombucha. The main differences in the investigated components are related to the duration of fermentation and the content of black tea. The researchers from different parts of the world used the same initial content of sucrose (10%). Researchers used different amounts of kombucha tea broth for the initial inoculation: 20% ²⁵ and 10% ²⁶. The fermentation process was performed in small volume reactors (glass jar or beaker), up to 1 L. The measured values of components propose that applied parameters (fermentation temperature, fermentation time, and initial content of sucrose and black tea), as well as the composition of kombucha culture have impact on the metabolic activity of kombucha, and therefore, on the end products of the metabolism.

Table 1. Predominant components in kombucha tea at the end of the fermentation on sugared black tea infusion

Component	Component content (g/L)	Initial sucrose (%)	Black tea	Fermentation temperature (°C)	Fermentation time (d)	Reference
Acetic acid	8	10	2 bags	24 ± 3	60	Chen and Liu 25
	4.69	10	12 g/L	24 ± 3	18	Jayabalan and others 26
Glucuronic acid	0.0031	5	1.5 g/L	28	21	Lončar and others 27
	0.0026	7	1.5 g/L	28	21	Lončar and others 28
	0.0034	10	1.5 g/L	28	21	Lončar and others 29
	1.71	10	12 g/L	24 ± 3	18	Jayabalan and others 26
Gluconic acid	39	10	2 bags	24 ± 3	60	Chen and Liu 25
Glucose	179.5	7	1.5 g/L	28	21	Malbaša and others 30
	24.59	7	1.5 g/L	28	21	Lončar and others 27
	12	10	2 bags	24 ± 3	60	Chen and Liu 25
Fructose	76.9	7	1.5 g/L	28	21	Malbaša and others 30
	5.40	7	1.5 g/L	28	21	Lončar and others 27
	55	10	2 bags	24 ± 3	60	Chen and Liu 25
Remained sucrose	192.8	7	1.5 g/L	28	21	Malbaša and others 30
	11	10	2 bags	24 ± 3	60	Chen and Liu 25
	2.09	7	1.5 g/L	28	21	Lončar and others 27

Acetic acid bacteria from kombucha produce acetic acid, as one of the main metabolites, when sucrose is used as a carbon source. Many authors determined the content of acetic acid in the beverage obtained after cultivation of kombucha on traditional substrate. Chen and Liu ²⁵ followed extended kombucha fermentation and determined the highest rate of 11 g/L after 30 d. The trend of acetic acid content was slow, increased with time, and then gradually decreased to 8 g/L, at the end of fermentation (60 d; Table 1). The same pattern was established by Jayabalan and others²⁶ who monitored the fermentation until the 18th day on green tea (12 g/L) sweetened with 10% sucrose. The highest content was 9.5 g/L on the 15th day. Molasses was used in place of sucrose by Malbaša and others ³⁰. Kombucha fermentation on molasses produced only 50% of acetic acid in comparison with sucrose at the same stage of fermentation. This might be due to the poor growth of acetic acid bacteria on molasses.

Glucuronic and gluconic acids are also major organic acids that are produced as a result of the kombucha fermentation process on traditional substrate. Lončar and others ²⁷ determined the glucuronic acid after kombucha fermentation on sweetened black tea. The highest amount was measured after 7, and 21 d (0.0034 g/L; Table 1). Jayabalan and others ²⁶ established the maximum value of 2.33 g/L D-glucuronic acid after 12 d of fermentation. Chen and Liu ²⁵ determined that gluconic acid was not produced until the 6th day of fermentation. The ending concentration amounted the about 39 g/L after 60 d (Table 1).

Yavari and others ³¹ cultivated kombucha on sour cherry juice sweetened with 0.6%, 0.8%, and 1% sucrose. Glucuronic acid was produced in very large amounts of 132.5 g/L which was determined on the 14th day of fermentation, in substrate with 0.8% sucrose. The fermentation process was conducted at 37 °C. Yavari and others ³² used response surface methodology to predict the value of glucuronic acid content in kombucha beverage obtained after fermentation on grape juice sweetened with 0.7% sucrose, and the highest value was achieved after 14 d of fermentation at 37 °C. Franco and others ³³ established the presence of glucuronic (0.07 to 9.63 g/L) and gluconic (0.04 to 1.16 g/L) acids in a product obtained after kombucha cultivation on black tea sweetened with glucose (0.062% to 1.51%). Yang and others ¹⁰ also determined the presence of gluconic acid and 2-keto gluconic acid, after cultivation of Gluconacetobacter sp. A4 isolated from kombucha and a strain of lactic acid bacteria, on 5 g/L black tea sweetened with 10% glucose.

L-lactic acid is not a characteristic compound for traditional kombucha beverage, but it is detected and determined. Jayabalan and others ²⁶ examined kombucha prepared with green tea to have a higher concentration of lactic acid than kombucha prepared from black tea and tea waste material. The maximum value of 0.54 g/L was established on the 3rd day. Malbaša and others ³⁴ measured the content of L-lactic acid after kombucha fermentation on molasses and established that it is a metabolic product present in large amounts. The presence of L-lactic acid after kombucha fermentation on molasses can be correlated to the L-lactic content of molasses itself which can be produced as a result of degradation of invert sugar in molasses. Molasses also contains amino nitrogen and biotin, which affect the intensity of kombucha fermentation.

The composition of kombucha beverage indicates the presence of numerous compounds and it depends on cultivation substrate, time and temperature of fermentation process, as well as the microorganisms present in the culture, but also on the applied method of analysis.

How to make kombucha

The amounts of tea, sugar, and tea fungus differ in different places. The standard procedure is as follows:

1. Tap water (1 L) is boiled and during boiling 50 g sucrose is stirred in.
2. Then 5 g tea leaves is added and removed by filtration after 5 min.
3. After cooling to room temperature (20 ºC) the tea is inoculated with 24 g tea fungus (the culture) and poured into a beaker (1 L) previously sterilized with boiling water.
4. The growth of undesirable microorganisms is inhibited by the addition of 0.2 L previously fermented kombucha, thus lowering the pH.
5. The beaker is covered with a paper towel to keep insects, especially Drosophila fruit flies away.
6. The incubation is carried out at 20 ºC to 22 ºC. The optimal temperature is in the wide range of 18 ºC and 26 ºC.
7. In the next few days, the newly formed daughter culture will start to float and form a clear thin gel-like membrane across the available surface. This is the newly formed tea fungus available as a new layer above the old tea fungus which was inoculated to begin the fermentation. At this time, the tea will start to smell fermented and there will be gas bubbles appearing from the carbonic acid produced during the fermentation. The mother culture will remain at its original volume as it sinks to the bottom of the tea broth where it remains under the newly forming daughter culture.
8. After 10 to 14 d, a new tea fungus will have developed on the surface of the tea as a disc of 2-cm thickness covering the whole diameter of the beaker. The newly formed tea fungus is removed with a spoon and kept in a small volume of fermented tea. The remaining beverage is filtered and stored in capped bottles at 4 ºC ³⁵.
9. The taste of the kombucha changes during fermentation from a pleasantly fruity sour-like sparkling flavor after a few days to a mild vinegar-like taste after a long incubation period. The 50 g sucrose/L provide the optimal concentrations of ethanol and lactic acid, and this sugar concentration has been used in traditional recipes for the preparation of “teakwass” (another name for kombucha) for a long time ³⁵. An optimum fermentation time is required for the production of kombucha with pleasant flavor and taste. Longer fermentation produces high levels of acids (like mild vinegar) that may pose potential risks when consumed ³⁶.

Kombucha side effects

Although kombucha tea has been reported to have curative effects, there is some evidence of toxicity associated with it. Some individuals have reported dizziness and nausea after consuming certain kombucha products. Two cases of unexplained severe illness have also been reported following kombucha consumption ³⁷. Kombucha tea is contraindicated in pregnant and lactating women. It has been found to cause lead poisoning and gastrointestinal toxicity in 2 individuals. The presence of anthrax Bacillus in kombucha tea fermented in unhygienic condition was reported by Sadjadi ³⁸. Further, Gamundi and Valdivia ³⁹ stated the risks of consuming kombucha beverage by HIV-positive patients. Side effects like allergic reactions, jaundice, nausea, vomiting, and head and neck pain related to consumption of kombucha were reported in 4 patients ⁴⁰. A married couple who had been drinking kombucha tea for 6 months, which was brewed in a ceramic pot, was reported to have symptomatic lead poisoning requiring chelation therapy ⁴¹. It was postulated that acids in the drink eluted lead from the glaze pigment used in the ceramic pot. Sabouraud and others ⁴² reported cases of lead poisoning in adults identified as anemia due to the lead-glazed earthenware jug which was used to store kombucha. A case of acute renal failure with lactic acidosis and hyperthermia within 15 h of kombucha tea ingestion by a 22-y-old HIV-positive male with a blood lactate level of 12.9 mmol/L and serum creatinine of 2.1 mg/dL was recorded ⁴³. However, all of these cases were very isolated and involved only a small number of individuals. Moreover, there is no substantial evidence to confirm the toxicity of any kombucha tea or the occurrence of illness by earlier studies ⁴⁴.

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