Kombucha tea is a slightly sweet, slightly acidic refreshing beverage consumed worldwide. It is obtained from infusion of tea leaves by the fermentation of a symbiotic association of bacteria and yeasts forming “tea fungus” (Chen and Liu 2000). 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. Kombucha is the internationally used Germanized form of the Japanese name for this slightly fermented tea beverage. It was first used in East Asia for its healing benefits. Kombucha originated in northeast China (Manchuria) where it was prized during the Tsin Dynasty (“Ling Chi”), about 220 B.C., for its detoxifying and energizing properties. In 414 A.D., the physician Kombu brought the tea fungus to Japan and he used it to cure the digestive problems of the Emperor Inkyo. As trade routes expanded, kombucha (former trade name “Mo-Gu”) found its way first into Russian (as Cainiigrib, Cainii kvass, Japonskigrib, Kambucha, Jsakvasska) and then into other eastern European areas, appearing in Germany (as Heldenpilz, Kombuchaschwamm) around the turn of the 20th century. During World War II, this beverage was again introduced into Germany, and in the 1950's it arrived in France and also in France-dominated North Africa where its consumption became quite popular. The habit of drinking fermented tea became acceptable throughout Europe until World War II which brought widespread shortages of the necessary tea leaves and sugar. In the postwar years, Italian society's passion for the beverage (called “Funkochinese”) peaked in the 1950s. In the 1960s, science researchers in Switzerland reported that drinking kombucha was similarly beneficial as eating yogurt and kombucha's popularity increased. Today, kombucha is sold worldwide in retail food stores in different flavors and kombucha culture is sold in several online shopping websites. A kombucha journal is electronically published by Gunther W. Frank and available worldwide in 30 languages (Dufresne and Farnworth 2000; Hartmann and others 2000).
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 (2000), 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 (1965). 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 (Danielova 1954; Konovalov and Semenova 1955; Sievers and others 1995; Roussin 1996). 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 (Markov and others 2001). Sievers and others (1995) 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 (Liu and others 1996). 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 (2010). 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 (1998). Dutta and Gachhui (2006, 2007) 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 (2014) 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. (Konovalov and others 1959; Kozaki and others 1972) and as Saccharomyces cerevisiae (Herrera and Calderon-Villagomez 1989; Liu and others 1996; Markov and others 2001; Safak and others 2002), Saccharomyces bisporus (Markov and others 2001), Saccharomycoides ludwigii (Reiss 1987; Markov and others 2001; Ramadani and Abulreesh 2010), Schizosaccharomyces pombe (Reiss 1987; Teoh and others 2004), Zygosaccharomyces sp. (Sievers and others 1995; Markov and others 2001; Marsh and others 2014), Zygosaccharomyces rouxii (Herrera and Calderon-Villagomez 1989), and Zygosaccharomyces bailii (Herrera and Calderon-Villagomez 1989; Liu and others 1996; Jayabalan and others 2008b). The genus Brettanomyces was isolated by several workers. Herrera and Calderon-Villagomez (1989) isolated Brettanomyces intermedius, Liu and others (1996) and Teoh and others (2004) isolated Brettanomyces bruxellensis, and Jayabalan and others (2008b) isolated B. claussenii. An examination of 2 commercial kombucha and 32 cultures from private households in Germany (Mayser and others 1995) showed variable compositions of yeasts. The predominant yeasts were Brettanomyces, Zygosaccharomyces, and Saccharomyces spp. Roussin (1996) determined Zygosaccharomyces and S. cerevisiae as the typical yeasts in North American kombucha. Kurtzman and others (2001) 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 (Steels and others 2002).
Candida sp. is included in a great number of kombucha beverages. Kozaki and others (1972) isolated Candida famata, Candida guilliermondii, and Candida obutsa. In kombucha samples from Mexico, Herrera and Calderon-Villagomez (1989) detected C. famata. Teoh and others (2004) identified Candida stellata. From a local kombucha in Saudi Arabia, Ramadani and Abulreesh (2010) 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 (Turkey; Safak and others 2002).
The presence of the following was also established: Torula (Reiss 1987), Torulopsis (Konovalov and others 1959; Herrera and Calderon-Villagomez 1989; Markov and others 2001), Torulaspora delbrueckii (Teoh and others 2004), Mycotorula (Konovalov and others 1959), Mycoderma (Konovalov and others 1959; Reiss 1987), Pichia (Reiss 1987), Pichia membranefaciens (Kozaki and others 1972; Herrera and Calderon-Villagomez 1989), Kloeckera apiculata (Danielova 1954; Kozaki and others 1972; Safak and others 2002), and Kluyveromyces africanus (Safak and others 2002).
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. The investigations of the beverage were always conducted under static conditions by the following: (Konovalov and Semenova 1955; Danielova 1957; Steiger and Steinegger 1957; Reiss 1987; Hauser 1990; Sievers and others 1995; Blanc 1996; Liu and others 1996; Roussin 1996; Petrović and others 1999; Bauer-Petrovska and Petrushevska-Tozi 2000; Chen and Liu 2000; Lončar and others 2000; Malbaša and others 2002a, 2008a, 2008b, 2011; Chu and Chen 2006; Franco and others 2006; Jayabalan and others 2007, 2008a; Kumar and others 2008; Wang and others 2010; Yang and others 2010; Yavari and others 2010, 2011; Velićanski and others 2013; Vitas and others 2013).
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 (Dufresne and Farnworth 2000).
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 (Taiwan—Chen and Liu 2000, Serbia—Lončar and others 2000, and India—Jayabalan and others 2007) used the same initial content of sucrose (10%). Researchers used different amounts of kombucha tea broth for the initial inoculation: 20% (Chen and Liu 2000), and 10% (Lončar and others 2000; Malbaša and others 2002a; Jayabalan and others 2007). 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 (2000)|
| ||4.69||10||12 g/L||24 ± 3||18||Jayabalan and others (2007)|
|Glucuronic acid||0.0031||5||1.5 g/L||28||21||Lončar and others (2000)|
| ||0.0026||7||1.5 g/L||28||21||Lončar and others (2000)|
| ||0.0034||10||1.5 g/L||28||21||Lončar and others (2000)|
| ||1.71||10||12 g/L||24 ± 3||18||Jayabalan and others (2007)|
|Gluconic acid||39||10||2 bags||24 ± 3||60||Chen and Liu (2000)|
|Glucose||179.5||7||1.5 g/L||28||21||Malbaša and others (2002a)|
| ||24.59||7||1.5 g/L||28||21||Lončar and others (2000)|
| ||12||10||2 bags||24 ± 3||60||Chen and Liu (2000)|
|Fructose||76.9||7||1.5 g/L||28||21||Malbaša and others (2002a)|
| ||5.40||7||1.5 g/L||28||21||Lončar and others (2000)|
| ||55||10||2 bags||24 ± 3||60||Chen and Liu (2000)|
|Remained sucrose||192.8||7||1.5 g/L||28||21||Malbaša and others (2002a)|
| ||11||10||2 bags||24 ± 3||60||Chen and Liu (2000)|
| ||2.09||7||1.5 g/L||28||21||Lončar and others (2000)|
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 (2000) 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 others (2007) 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 (2008a, 2008b). 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 (2000) 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 (2007) established the maximum value of 2.33 g/L D-glucuronic acid after 12 d of fermentation. Chen and Liu (2000) 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 (2010) 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 (2011) used response surface methodology (RSM) 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 (2006) 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 (2010) 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 (2007) 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 (2008a, 2008b) 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.
Citric acid is also not a characteristic metabolic product of the traditional beverage. Malbaša and others (2011) determined an average value of 25 g/L citric acid in the total acids (substrate with 1.5 g/L of black tea and 7% sucrose), and Jayabalan and others (2007) measured it only on the 3rd day of fermentation, 0.03 and 0.11 g/L, in kombucha prepared with green and black tea, respectively.
Sucrose is the most common carbon source in kombucha fermentation. Its considerable amount stays largely unfermented during the process (Malbaša and others 2002a). Investigations showed that 34.06% of sucrose stays unfermented after 7 d, and after 21 d this value is 19.28% (Table 1). Chen and Liu (2000) determined that the content of sucrose linearly decreased during the first 30 d, followed by a slow-rate decline. Malbaša and others (2008b) established that utilization of 7% sucrose from molasses reached 97%, after 14 d of fermentation. The decline of sucrose concentration is more pronounced when the concentration of sucrose in molasses is optimal (7%), compared to the systems with pure sucrose. Utilization in the samples with molasses is slow when the content of sucrose is lower (Malbaša and others 2008a, 2008b). Yavari and others (2010) concluded that sucrose utilization, after the 4th day, began to speed up and this trend continued until the 14th day when the lowest sucrose content (2.1 g/L) was determined.
Malbaša and others (2002a) measured the contents of D-glucose and D-fructose in traditional kombucha and the highest values were 19.60 (on 14th day) and 10.25% (on 10th day), respectively. Lončar and others (2000) concluded that sucrose, glucose, and fructose were not utilized entirely after 21 d of fermentation and confirmed that fructose was metabolized before glucose. Chen and Liu (2000) established that glucose was not produced analogous to fructose (0.085%/d) but in lower amount (0.041%/d). The beverage, obtained on Jerusalem artichoke tuber extract, contained sugars in lower amount in comparison to sucrose substrate, except for D-fructose (10.41% on 5th day). In addition to sucrose and D-glucose, the presence of inulooligosaccharides were also determined (Malbaša and others 2002a).
Bauer-Petrovska and Petrushevska-Tozi (2000) quantified water-soluble vitamins in kombucha made with 0.7% sucrose and 5 g/L black tea. The values were as follows: vitamin B1 74 mg/100 mL, vitamin B6 52 mg/100 mL, vitamin B12 84 mg/100 mL, and vitamin C 151 mg/100 mL. Malbaša and others (2011) measured the maximum content of vitamin B2 in samples obtained with native kombucha (10th day, 7% sucrose and 1.5 g/L), on black (8.30 mg/100 mL) and green (9.60 mg/100 mL) tea. In that investigation, the content of vitamin C increased constantly in all obtained products and reached the highest value of 28.98 mg/L on 10th day in beverage produced with combination of acetic acid bacteria and S. cerevisiae isolated from native kombucha. This value was slightly lower (27.86 mg/L) in traditional product at the same stage of fermentation (Malbaša and others 2011). Vitamin C was also quantified in an investigation by Vitas and others (2013) by RSM methodology in the fermented milk products obtained by kombucha previously cultivated on winter savory (30 mg/L) and stinging nettle extract (45 mg/L). RSM methodology predicted values of vitamin C that are much higher in comparison to values obtained for traditional kombucha products, obtained after 7-d long fermentation period (15.19 mg/L) when the beverage is usually consumed.
The contents of manganese, iron, nickel, copper, zinc, lead, cobalt, chromium, and cadmium in the usual kombucha were determined by Bauer-Petrovska and Petrushevska-Tozi (2000). The contents of the examined minerals were in range from 0.004 μg/mL for cobalt to 0.462 μg/mL for manganese. Determination of toxic elements indicated the following values: 0.005 μg/mL for lead, 0.001 μg/mL for chromium, whereas cadmium was not detected. It was concluded that essential minerals (Cu, Fe, Mn, Ni, and Zn) increased as a result of the metabolic activity of kombucha. The cobalt content did not increase, possibly because of its inclusion in vitamin B12 (Bauer-Petrovska and Petrushevska-Tozi 2000). Kumar and others (2008) established the presence of fluoride, chloride, bromide, iodide, nitrate, phosphate and sulfate in beverage with 10% sucrose and 5 g/L of black tea, after 7 d, and the highest measured value was 3.20 mg/g, for fluoride. The anionic mineral composition of kombucha and black tea was considerably different.
Chu and Chen (2006) examined a traditional beverage (4 g/L of black tea, 10% sucrose, 15 d of long fermentation period) and established that total phenol content of all kombucha samples showed a linear increase during fermentation time. Jayabalan and others (2008a) also established a highly pronounced increase of the total phenol content in all samples. Chu and Chen (2006) proved that the content was up to 7.8 mM gallic acid equivalent (GAE; 15th day of fermentation) and only around 4 mM GAE for black tea. Jayabalan and others (2007) investigated epicatechin isomers EGCG ([-]-epigallocatechin-3-gallate), EGC ([-]-epigallocatechin), ECG (-]-epicatechin-3-gallate), and EC ([-]-epicatechin) and demonstrated changeable stability during the fermentation process. Degradation of EGCG and ECG was reduced in the substrate with green tea when compared to substrates with black tea and tea waste material. Consistent degradation was observed for theaflavin and thearubigins. The highest value was measured for EC on 12th day in kombucha with green tea (around 150%), and for EGC, on the same day, in kombucha with tea waste material (around 140%) and black tea (around 115%). It is assumed that EGCG and ECG were converted to their corresponding catechin EGC and EC. The color of kombucha broth was lighter in comparison to the color of black tea and this suggested that polyphenols did undergo microbial change in the acidic environment by the enzymes liberated by bacteria and yeast (Jayabalan and others 2007).
Wang and others (2010) measured the content of DSL in kombucha, and it was in the range from 57.99 (sample from household) to 132.72 μg/mL (sample from laboratory). Yang and others (2010) established the increase of DSL content during the 8 d, when the highest value was reached followed by decrease in DSL till the end of fermentation. They concluded that lactic acid bacteria have a positive effect on DSL production, in symbiosis with Gluconacetobacter sp. A4. The optimum medium conditions for fermentation were glucose (10%) and black tea (5 g/L).
Chen and Liu (2000) established that the content of ethanol increased with time and reached the highest value at around 5.5 g/L, followed by a slow decline. The same pattern was observed by Reiss (1994) who concluded that ethanol production increased to a maximum on the 6th day of fermentation, with a subsequent decrease.
Jayabalan and others (2007) indicated that the protein content increased with fermentation time, in the range of 0.1 to 3.0 mg/mL, during 12 d of fermentation, in all samples. Afterwards, it continued to decrease because of yeast and bacterial extracellular protein decreases.
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.
Fermentation of kombucha on substrates other than tea
Traditional substrate for the kombucha fermentation is black or green tea extract sweetened with 5% to 8% sucrose. Besides traditional substrates, the possibility of use of alternative substrates has been established in various studies. Malbaša (2004) reviewed some attempts in applying nontraditional substrates for the kombucha fermentation such as Coca-Cola, red wine, white wine, vinegar, extract of Jerusalem artichoke, milk, fresh sweet whey, reconstituted sweet whey, acid whey, Echinacea, Mentha, and more.
Jayabalan and others (2007, 2008a) revealed the possibility of using tea waste material for manufacturing kombucha beverage with satisfying quality. Studies of some alternative cultivation medium have shown that green tea and lemon balm tea have more stimulating effect on the kombucha fermentation than black tea, thus providing the fermentation product in a shorter time (Greenwalt and others 1998; Velićanski and others 2007). Talawat and others (2006) prepared kombucha beverage from mulberry tea, Japanese green tea, jasmine tea, and oolong tea. Velićanski and others (2013) cultivated kombucha on sage, thyme, and peppermint teas. Some scientists attempted the kombucha fermentation on sweetened sour cherry juice (Yavari and others 2010).
A possible substrate for the kombucha fermentation is Jerusalem artichoke tuber extract which has been reported in several articles. It was found that kombucha beverage obtained on the Jerusalem artichoke tuber substrate could be appropriate as dietetic product, because of the low Dglucose and Dfructose contents, and also because of the presence of inulooligosaccharides which act as dietetic fibers and are expected to increase the population of resident bifidobacteria in the human intestinal flora (Malbaša and others 2002a; Lončar and others 2007).
The fact that fermentative liquids with Jerusalem artichoke tuber extracts contain almost the same metabolites as the beverage with sucrose, plus additional ingredients like fructooligosaccharides and inulin, which are prebiotics, contributes to the quality of the final product. Kombucha metabolism is more intensive on a substrate with Jerusalem artichoke tuber extract, with the same applied culture of microorganisms. Specifically, contents of L-lactic, L-ascorbic, and total organic acids are significantly higher (Malbaša and others 2002b).
Some investigations with molasses as a substrate for the kombucha fermentation have also been conducted. Molasses from sugar beet processing is attractive because of its low price and the presence of a number of components, including minerals, organic compounds, and vitamins, which are very useful for the fermentation process (Rodrigues and others 2006). The first results on the metabolic activity of kombucha on sugar beet molasses were published in 2001 (Lončar and others 2001). The next investigation (Malbaša and others 2008a) additionally confirmed that the molasses from sugar beet processing can be used as a low-cost carbon source in kombucha fermentation of black tea. The products obtained on these substrates were rich in lactic acid, which may be considered as an advantage compared to the product on sucrose. The content of lactic acid is related to the higher quantity of invert sugar, biotin, and amino nitrogen in the molasses (Malbaša and others 2008b). The chemical composition of the substrate with molasses is considerably richer, in comparison to the substrate with pure sucrose, but it was proved that 7% sucrose from molasses corresponded to an optimal concentration, which produced low levels of less desired acetic acid and high levels of physiologically important L-lactic acid.
Reiss (1994) proved the possibility of application of lactose as a source of carbon for the kombucha fermentation. There were also a few investigations related to kombucha fermentation on substrates containing lactose. Belloso Morales and Hernández-Sánchez (2003) successfully cultivated kombucha on cheese whey. Malbaša and others (2009) proved that fermented beverages can be produced by kombucha fermentation on cow milk. The metabolic activity of kombucha starters on milk was significantly different from the activity on sucrose. Even the texture and taste of the products obtained were similar to yogurt; the chemical compositions of the new beverages differed significantly from the composition of yogurt. The investigations of Vitas and others (2013) proved that the fermented milk beverages can be successfully produced by application of kombucha obtained by cultivation on sweetened stinging nettle and winter savory extracts.
Kombucha tea as an antimicrobial source
Kombucha tea has been studied by many researchers for its inhibitory activity on many pathogenic microorganisms. Tea containing 4.36 g of dry tea per liter and 10% sucrose and fermented with tea fungus showed no antibiotic activity in the beverage beyond that caused by acetic acid, a primary product of the fermentation (Steinkraus and others 1996). Kombucha tea containing 33 g/L total acid (7 g/L acetic acid) had antimicrobial efficacy against Agrobacterium tumefaciens, Bacillus cereus, Salmonella choleraesuis serotype Typhimurium, Staphylococcus aureus, and Escherichia coli, but not for Candida albicans (Greenwalt and others 1998). Kombucha tea could inhibit the growth of the pathogens Entamoeba cloacae, Pseudomonas aeruginosa, B. cereus, E. coli, Aeromonas hydrophila, Salmonella typhimurium, Salmonella enteritidis, Shigella sonnei, Staphylococcus epidermis, Leuconostoc monocytogenes, Yersinia enterocolitica, S. aureus, Campylobacter jejuni, Helicobacter pylori, and C. albicans (Sreeramulu and others 2000, 2001). Kombucha tea prepared from different substrates like mulberry tea, Japanese green, jasmine tea, oolong tea, and black tea was tested on pathogenic bacteria of humans and shrimp. Results revealed that black tea kombucha possessed the greatest inhibitory activity and Vibrio parahaemolytica showed the highest susceptibility to the fermented tea (Talawat and others 2006). Battikh and others (2012) reported that kombucha prepared from both black tea and green tea had antimicrobial potential against the tested human pathogenic microorganisms, except C. krusei, and kombucha green tea exhibited the highest antimicrobial potential. Afsharmanesh and Sadaghi (2013) reported that the body weight, feed intake, and protein digestibility of broiler chickens fed with a diet having 1.2 g/kg kombucha tea (20% concentration) were significantly increased compared to the control and green tea-fed broilers. They suggested that kombucha tea can be an alternative to antibiotic growth promoters in the diets of broilers.
Research on kombucha has demonstrated its antimicrobial efficacy against pathogenic microorganisms of both Gram-positive and Gram-negative origin. Antimicrobial activity of kombucha tea is largely attributable to the presence of organic acids, particularly acetic acid, large proteins, and catechins. Acetic acid and catechins are known to inhibit a number of Gram-positive and Gram-negative microorganisms (Sreeramulu and others 2000).
Kombucha tea as an antioxidant source
There has been a global trend toward the use of phytochemicals present in natural resources as antioxidants and functional foods. Bioactive molecules of natural resources are being utilized in the food industry, and there is evidence that these molecules can act as antioxidants within the human body. Antioxidant activity of Kombucha is correlated with its many claimed beneficial effects like cancer prevention, immunity enhancement, and alleviation of inflammation and arthritis. Jayabalan and others (2008a) reported on the free radical scavenging abilities of kombucha tea prepared from green tea, black tea, and tea waste material. They have shown that total phenolic compounds, scavenging activity on DPPH radical, superoxide radical, and inhibitory activity against hydroxyl radical-mediated linoleic acid were increased with an increase in fermentation time, whereas reducing power, hydroxyl radical scavenging ability (ascorbic acid-iron EDTA), and antilipid peroxidation ability were decreased. Malbaša and others (2011) studied the influence of 3 starter cultures (mixed culture of acetic bacteria and Zygosaccharomyces sp., mixed culture of acetic bacteria and S. cerevisiae, and native local kombucha) on the antioxidant activities of green tea and black tea kombucha beverage to hydroxyl and DPPH radicals. They observed the highest antioxidant activity with native kombucha on green tea beverage and acetic acid bacteria with Zygosaccharomyces sp. culture on black tea beverage. The antioxidant property of kombucha tea was tested against tertiary butyl hydroperoxide (TBHP)-induced cytotoxicity using murine hepatocytes and showed that kombucha tea neutralized the TBHP-induced changes and prevented cell death. These counter effects were also shown by the unfermented black tea, but the kombucha tea was found to be more efficient (Bhattacharya and others 2011b).
The antioxidant activity of kombucha tea is due to the presence of tea polyphenols, ascorbic acid, and DSL. Kombucha tea was observed to have higher antioxidant activity than unfermented tea and that may be due to the production of low-molecular-weight components and structural modifications of tea polyphenols by enzymes produced by bacteria and yeast during fermentation.
Kombucha exhibited increased free radical scavenging activities during fermentation. The extent of the activity depended upon the fermentation time, type of tea material, and the normal microbiota of the kombucha culture, which in turn determined the nature of their metabolites. Although free radical scavenging properties of kombucha showed time-dependent profiles, prolonged fermentation is not recommended because of accumulation of organic acids, which might reach harmful levels for direct consumption. The identification of extracellular key enzymes responsible for the structural modification of components during kombucha fermentation and potent metabolites responsible for the free radical scavenging abilities are necessary to elucidate the metabolic pathway during kombucha fermentation. Metabolic manipulations may be one of the effective methods to enhance the antioxidant activities and fermentation efficiency of kombucha.
Kombucha tea as hepatoprotective agent
Kombucha tea has been studied for its hepatoprotective property against various environmental pollutants in animal models and cell lines and it has been shown that it can prevent hepatotoxicity induced by various pollutants. Kombucha tea (prepared from black tea) was tested against paracetamol (Pauline and others 2001), carbontetrachloride (Murugesan and others 2009), aflatoxin B1 (Jayabalan and others 2010a), cadmium chloride (Ibrahim 2011), TBHP (Bhattacharya and others 2011b), and acetaminophen (Abshenas and others 2012; Wang and others 2014). It was demonstrated that it can effectively attenuate the physiological changes driven by these liver toxicants. The volume of kombucha tea, number of doses, treatment period, and the method of administration used in these studies were not same. In most of the studies, male albino rats (Pauline and others 2001; Murugesan and others 2009; Jayabalan and others 2010a; Ibrahim 2011; Wang and others 2014) were used and a few other studies were conducted with Balb/c mice (Abshenas and others 2012) and isolated murine hepatocytes (Bhattacharya and others 2011a). Hepatoprotective efficacy of kombucha tea was studied by measuring liver toxicity markers (serum glutamic pyruvate transaminase, serum glutamic oxaloacetic transaminase, malondialdehyde, alkaline phosphatase, gamma glutamyl transpeptidase), reduced glutathione, antioxidant enzymes (glutathione-S-transferase, glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase), various levels of creatinine and urea, nitric oxide levels in liver, and by histopathological analysis of liver tissue. More recently, apoptosis, reactive oxygen species generation, changes in mitochondrial membrane potential, cytochrome c release, activation of caspases (3 and 9) and Apaf-1 were studied to show the hepatoprotective property of Kombucha tea against TBHP (Bhattacharya and others 2011b).
Antioxidant activity and its ability to facilitate both antioxidant and detoxification processes in the liver were ascribed to the hepatoprotection offered by kombucha tea. Wang and others (2014) reported that hepatoprotective effects of kombucha tea against acetaminophen is largely attributed to the presence of DSL, and Gluconacetobacter sp. A4 was the primary producer of it. Most of the studies concluded that kombucha tea could be beneficial against liver diseases, for which oxidative stress is a well-known causative factor.
Tea fungus (fungal biomass) and its applications
Cellulose produced during the fermentation by A. xylinum appears as a thin membrane on the surface of tea broth where the cell mass of bacteria and yeast is attached (Figure 2A and 2B). This mixture of microorganisms and cellulose is likely why kombucha is also called “tea fungus” (Sreeramulu and others 2000). Cellulose prepared from pellicles of A. xylinum has a unique characteristic in terms of its chemical stability, molecular structure, and mechanical strength (Czaja and others 2006). A similar cellulose network floating on the surface of various fruit juices fermented by a symbiotic culture composed of A. xylinum and yeasts, and called “note,” is consumed in the Philippines as a delicacy. The cellulose network produced by a pure culture of A. xylinum is used for the treatment of skin burns and other dermal injuries in Brazil (Blanc 1996). Caffeine and related compounds (theophylline and theobromine) are identified as activators for cellulose production in A. xylinum (Lončar and others 2001). In ancient days, this cellulose biofilm was used for the treatment of wounds. Microbial cellulose synthesized in abundance by A. xylinum shows vast potential as a novel wound healing system (Czaja and others 2006).
Figure 2. (A, B)–Scanning electron microscope image of the consortia of yeasts and bacteria in a portion of tea fungus (magnification 2a = 3500× and 2b = 2700× (reproduced with prior permission; El-Taher 2011).
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Dried tea fungal biomass has been efficiently utilized as a biosorbent to remove metal pollutants from waste water by several researchers worldwide (Murugesan and others 2005; Mamisahebei and others 2007; Razmovski and Šćiban 2008). The charges possessed by the bacteria and yeasts present in the cellulose biomass were correlated with absorbent ability. Mamisahebei and others (2007) investigated the efficiency of tea fungal biomass pretreated with FeCl3 to remove arsenic from aqueous solution and found that maximum capacities of tea fungal biomass for arsenic (V) were obtained at 3.98 × 10−3 mmol/g at pH of 6 to 8. Razmovski and Šćiban (2008) studied the efficiency of waste tea fungal biomass to remove Cr(VI) and Cu(II) ions from aqueous solutions in a batch biosorption system and reported that the optimum pH values for biosorption of Cr(VI) and Cu(II) by waste tea fungal biomass were 2.0 and 4.0, respectively. Murugesan and others (2005) studied the proximate composition of tea fungal biomass and reported that it contains 179.38 g crude protein, 120 g crude fiber, 4.82 g phosphorus, 6.56 g calcium, and 8.92 MJ metabolizable energy per kilogram of biomass. They also reported that the supplementation of tea fungal biomass at 150 g/kg poultry feed increased feed consumption, body weight, performance efficiency factor (PEF), and the carcass characteristics (dressed weight, eviscerated weight, liver, heart and gizzard) of test broilers significantly over the control.
Tea fungus was found to be rich in crude fiber, crude protein, and the amino acid lysine, and an increase in fermentation time increased the biochemical components of tea fungus (Jayabalan and others 2010b). Coculturing Gluconacetobacter hansenii CGMCC 1671 and S. cerevisiae CGMCC 1670 in traditional kombucha with 10.37% inoculum, initial pH 4.96, and medium volume of 77.13 mL in a 250 mL flask resulted in 300.093 mg/g of bacterial cellulose (Tan and others 2012). The researchers concluded that coculturing pure strains of traditional kombucha can be used to provide bacterial cellulose of high grade in addition to produce the high-quality kombucha beverage. Tea broth with a sucrose concentration of 9% produced the highest yield of bacterial cellulose (66.9%), and the thickness and yield of this bacterial cellulose increased with fermentation time and surface area:depth ratio (Goh and others 2012a). Characterization of microbial cellulose produced from kombucha after 8 d of fermentation, by employing SEM, FTIR, X-ray diffractometry, adsorption isotherm, and by measuring the swelling properties, was done by Goh and others (2012b). Their results on SEM showed that an ultrafine network makes up the cellulose layer. FTIR confirmed the presence of a characteristic region of anomeric carbons and β-1,4-linkages. Cellulose was confirmed to be free from contaminants such as lignin or hemicellulose. X-ray diffraction studies showed that the overall degree of crystallinity index of dried tea fungal biomass was slightly lower than that of microbial cellulose. Hence, it can also be used for the preparation of cellulose-based chemicals like carboxymethylcellulose and can be fermented to bioethanol. Zhu and others (2013) demonstrated that kombucha cellulose had good biocompatibility with primary cultured Schwann cells (neurilemma cells), and the kombucha cellulose did not show histological and hematological toxic effects on nerve tissues in vivo.