Determination and characterization of the anti-microbial activity of the fermented tea Kombucha
C.J. Greenwalt, R.A. Ledford, and K.H. Steinkraus Department of Food Science Cornell University Ithaca, New York 14853
Early reports on Kombucha, a traditional fermented tea beverage, suggested that it has anti-microbial activity against a spectrum of organisms, and that concentrates of unfermented tea components also have anti-microbial properties. Therefore, the focus of this study was to determine and characterize Kombucha’s anti-microbial activity using an absorbent disc method. Anti-microbial activity was observed in the fermented samples containing 33 g/L total acid (7 g/L acetic acid) against the tested gram positive and gram negative organisms (Agrobacterium tumefaciens, Bacillus cereus, Salmonella cholerasuisserotype typhimurium, Staphylococcus aureus, and Escherichia coli). Candida albicans was not inhibited by Kombucha. The contribution of tea itself to the anti-microbial activity of Kombucha proved to be insignificant in the tested organisms, even at the highest levels tested, 70 g/L (7%) dry tea. As a result, the anti-microbial activity of Kombucha was attributed to its acetic acid content.
Kombucha is a traditional fermented tea that has gained popularity in the United States as it is increasingly associated with health-promoting effects. Kombucha is a slightly sweet, acidic tea beverage currently consumed worldwide, but historically in China, Russia, and Germany (1). Numerous popular media features in the United States have highlighted the beverage and its uses, including The New York Times and Miami Herald, suggesting that Kombucha consumption can reduce blood pressure, relieve arthritis, increase the immune response, and cure cancer (2,3).
Kombucha lends itself to expansive consumption as a healthful beverage since it is easily and safely produced at home (4). Production of the tea is achieved by infusing tea leaves into freshly boiled water and sweetened with about 100 g/L (10%) sucrose or honey. Once the tea has cooled to room temperature the gelatinous surface growth/mat from a previous batch is added to the sweetened tea. After about a seven to ten-day incubation at room temperature, the mat is transferred to a new fermentation and the Kombucha beverage is ready. The final product is a slightly carbonated, acidic beverage comprised of sugars, organic acids, tea components, vitamins, and minerals, resembling cider.
The Kombucha colony/mat represents a symbiotic relationship between bacteria and yeasts (5). Acetobacter xylinum has been shown to be the primary bacterium in the colony (6). Hesseltine reported the presence of Acetobacter sp. (NRRL B-2357) and two yeasts; Pichia and Zygosaccharomyces (NRRL Y-4810 and 4882) in Kombucha (7). Mayseret al. demonstrated that the yeast composition of the colony is highly variable; but that,Brettanomyces, Zygosaccharomyces and Saccharomyces occurred most frequently in the German household samples studied (6). Roussin determined that the typical North American Kombucha microorganisms were Acetobacter xylinum, Zygosaccharomyces, andSaccharomyces cerevisiae (8). In addition, Mayser et al. noted a low rate of contamination from harmful microorganisms (spoilage and pathogenic) and concluded that Kombucha can safely be prepared at home without pathogenic health risk (6). The acidity of the product, at around 33 g/L total acid, is relatively high which limits the ability of many other organisms, possible contaminants, to grow (9). However, if the fermentation is allowed to continue too long, the acidity can increase to very high levels which may pose a potential health risk if consumed. This was speculated to be the cause of one woman’s death in 1995 when she died from acidosis and intestinal perforations after consuming large amounts of very acidic Kombucha (10).
Throughout the fermentation the yeasts break down sugar into glucose and fructose (8). Glucose is used by the yeasts to yield ethanol and carbon dioxide. The primary Kombucha bacterium, Acetobacter, initially oxidizes ethanol to acetaldehyde and then to acetic acid (11). The secondary biochemical activity of Acetobacter is the oxidation of glucose to gluconic acid (11). Glucose is also used by acetic acid bacteria to synthesize microbial cellulose (11). Fructose remains part of the ferment broth and is utilized by the microorganisms to a lesser degree. Acetic acid concentrations may rise to levels as high as 30 g/L (3%) if the tea is allowed to ferment for up to 30 days (12). The usual concentration of acetic acid consumed in Kombucha is 10 g/L (1%) (4). Gluconic acid is also present in substantial quantities, about 20 g/L (2%) (13).
The most recent chemical analysis to date, is the investigation conducted by Roussin and his colleagues. Roussin determined by High Performance Liquid Chromatography and Mass Spectrophotometry identification that fructose, acetic acid and gluconic acid were the primary constituents of the fermented sweetened black tea (8). These and all constituents tested were shown to vary in batches and different colonies tested (8). Roussin also noted that the vitamin content of the fermented tea was not in sufficient concentrations to assist human health (8). This investigation also revealed that no glucuronic acid was present in the samples tested (8).
Numerous studies refer to Kombucha's anti-microbial activity and suggest that it might influence the gastro-intestinal microbial flora of humans. Steinkraus et al. showed that the anti-microbial activity of Kombucha against Helicobacter pylori (primary cause of gastritis and peptic ulcer disease), Escherichia coli, Staphylococcus aureus, and Agrobacterium tumefaciens made with a low tea usage level (4.4% g/L dry w/v), was attributable to the acetic acid content (4). According to Levine and Fellers, acetic acid can inhibit and destroy microorganisms when used in sufficiently high concentrations (14). However, at as little as 1g/L (0.1%) acetic acid, pathogenic and spore forming bacteria are inhibited (15).
Steinkraus et al. stated that they could not directly compare their results to other studies on the inhibitory activity of the tea, fermented and unfermented, since they used a substantially lower level of tea (4). De Silva and Saravanapavan discussed the tea cider prepared with 10 g/L (1%) tea w/v (16). Gadd suggested preparing tea cider with 11 to 15 g/L tea w/v (17). Hesseltine investigated the anti-microbial activity of Kombucha prepared with 37 g/L (3.7%) tea w/v (7). Using this undrinkable level of tea, Hesseltine reported that Agrobacterium tumefaciens, a common plant pathogen, was inhibited in the fermented tea and neutralized samples (7). Hesseltine, did not report the effect of the unfermented substrate, therefore it is unknown if the tea components contributed to the neutralized ferment’s anti-microbial activity.
Toda et al. (18,19) demonstrated that unfermented tea at high concentrations (using 20% dry tea) inhibit Staphylococcus aureus, Staphylococcus epidermidis, Salmonella typhi, Salmonella typhimurium, Salmonella enteritidis, Shigella flexneri, Shigella dysenteriae,and Vibrio spp. At concentrations of 200 g/L w/v tea, Diker et al. (20,21) showed that black and green tea extracts (50 times the usual level of tea used for consumption) had bactericidal activity against Campylobacter jejuni, Campylobacter coli, and Helicobacter pylori.
Yokihiko and Watanabe (22) found that Clostridium botulinum spores were killed when inoculated into tea drinks. This investigation demonstrated that the inhibitory effects observed could have been due to the catechin content of the tea. Later, it was determined that most of the bactericidal activity of tea itself may be attributed to the polyphenols, specifically catechins (23,24,25). The polyphenolic group that is most reactant during the enzymatic fermentation of fresh green leaves to black tea leaves are the catechins (26). Green tea has a much higher catechin content than black tea. As a result, green tea may have more anti-microbial activity than black tea (27). Kodama et al. demonstrated that crude catechins may be useful in the prevention of some bacterial plant diseases (28). The catechin fraction of black tea, at about 0.4 mg/ml, was shown to be anti-microbial against Streptococcus mutans, related to dental carries found in human teeth (25). Clearly, high levels of tea have anti-microbial effects, however it has yet to be shown if drinkable levels have similar properties.
The focus of this study was to test the anti-microbial activity of Kombucha brews made with increasing tea concentrations (black and green), thereby characterizing the contributions of unfermented tea and fermentation components. Thus, determining if pathogenic growth can be prevented by the consumption of the fermented tea, which may be important in aiding immunity and illness prevention and could lead to better overall health.
MATERIALS AND METHODS
Kombucha was prepared by adding 100 g/L (10%) weight/volume sucrose and tea leaves of desired dry weight to boiling water. Normal drinkable tea of 4.4 g/L (0.44%) weight of dry tea per volume of boiled water, and increased levels of 8.7 g/L, 17 g/L, 35 g/L, and 70 g/L were prepared in duplicate. Both black (Lovers Leap Orange Pekoe Tea, Pure Premium Ceylon Tea) and green (Japanese Sencha Tea, Pure Premium Green Tea ) tea leaves from Metropolitan Tea Company Ltd. were tested. The leaves were steeped for 30 minutes and removed. After the tea reached room temperature (about 25° C) the colony/mat was added from the previous batch. The original colony/mat was kindly provided by Professor Keith Steinkraus, Department of Food Science, Cornell University. The fermentation continued until the desired taste and acidity was reached. The fermentation was terminated at the organoleptically pleasing total acidity of about 33 g/L (3.3%). This end-point was determined previously (data not published) by analyzing the sensory attributes, pH, and acidity of a variety of Kombucha ferments. The fermentation time averaged nine days at 25° C.
When the fermented samples reached the desired end point, the anti-microbial activities were tested in duplicate with the following organisms: Staphylococcus aureus NRRL B-1317, Staphylococcus aureus NRRL B-1318, Escherichia coli serotype H10 (non-pathogenic) NRRL B-2207, Escherichia coli serotype H48 (pathogenic) NRRL B-3704,Salmonella cholerasuis serotype typhimurium NRRL B-4420, Bacillus cereus NRRL B-14720, Bacillus cereus NRRL B-14725, Candida albicans NRRL Y-12983, Agrobacterium tumefaciens. The NRRL test organisms were kindly supplied by the Northern Regional Research Laboratory of the United States Department of Agriculture-Agriculture Research Service (USDA-ARS) in Peoria, IL. Agrobacterium was kindly provided by Dr. Stephen Winans, Section of Microbiology, Cornell University. All bacterial species were cultivated on agar plates prepared with 25g/L mannitol, 15g/L Bacto agar, 5g/L Difco yeast extract, and 3 g/L Bacto peptone as recommended by the American Type Culture Collection.Candida albicans was cultivated on YM agar plates prepared with 20 g/L Bacto agar, 10 g/L glucose, 5 g/L Bacto peptone, 3 g/L malt extract, and 3 g/L Difco yeast extract as recommended by the Northern Regional Research Laboratory of the USDA-ARS. The bacterial species were chosen to represent the most common pathogenic and undesirable organisms associated with food. Agrobacterium tumefaciens is a common plant pathogen that is not associated with food but was chosen as a test organism in this investigation because it was used in previous studies. An equal amount of gram negative and gram positive organisms were tested to observe differences among the two types of prokaryotes. Candida albicans was included because of its predominance as a common human pathogen.
Sterile cotton applicator swabs were used to inoculate the surface of the agar with the test organisms rather than a constant volume of culture because the swab method yielded a uniform mat of growth more consistently. A 2.5 cm cellulosic absorbent pad (Millipore AP 1002500) was used as the anti-microbial disc to estimate the zone of inhibition (clearing). The 2.5 cm disc was saturated with Kombucha or other test solution and placed on the freshly inoculated agar with sterile forceps. All plates were incubated at 37° C for 72 hours. The anti-microbial activity of each test solution was estimated by measuring the zone of inhibition around the disc. The diameter of the disc was subtracted from the measured clear zone.
Test solutions included duplicates of unfermented controls, samples fermented to about 33 g/L (3.3%) total acidity, and fermented samples neutralized to pH 7 with 1 g/L NaOH of both black and green tea preparations at the various concentrations (4.4 g/L, 8.7 g/L, 17 g/L, 35 g/L, and 70 g/L dry tea weight per volume of boiled water). Tested control solutions included duplicates of commercial vinegar at 50 g/L (5%) acetic acid and prepared 10 g/L (1%) acetic acid samples.
The pH and titratable acidity of each sample were checked daily to follow and characterize the fermentation progression. The pH was determined using Baxter S/P pH indicator strips or a Beckman f 10 pH Meter and the titratable acidity was determined by titrating 10 ml samples with 1 g/L NaOH using 500 m l of 10 g/L (10 g/L) phenolphthalein as the visual endpoint indicator. The total acidity was then calculated (as gluconic acid), by multiplying the volume (ml) of 1g/L NaOH needed to titrate the sample by 1.96. Gluconic acid was used as the reference acid since it was demonstrated by Roussin that gluconic is typically the primary acid component (8).
Total acidity was broken down into volatile and non-volatile acid components by boiling 10 ml samples for ten minutes to drive-off the volatiles. The boiled samples were brought back to volume and titrated with 1 g/L NaOH. The volatile acidity was calculated as the total acidity minus the non-volatile acidity.
The production of ethanol and glucose, in addition to acidity, was quantified. The Sigma 333-A alcohol test kit was used for the determination of the ethanol content. Alcohol dehydrogenase catalyzed the conversion of ethanol and the absorbance at 340 nm was recorded and used to calculate ethanol concentrations. Glucose was estimated with Stanbio’s Glucose LiquiColor 1070 enzymatic test kit from Sigma. Glucose oxidase and peroxidase developed a colored product from glucose. The absorbance of the colored product at 500 nm was used to calculate the amount of glucose present in the sample.
RESULTS AND DISCUSSION
The Kombucha colonies used in this investigation had a tendency to produce about 3.3% total acid, 0.7% acetic acid, 4.8% glucose, and 0.6% ethanol after a nine-day fermentation. There was no lactic acid produced by these colonies (verified with HPLC; 9). The average pH of the fermented samples tested was 2.5. The pH of the neutralized samples was 7.0. When the fermentation was allowed to continue beyond the desired endpoint, the acidity reached levels as high as 24 g/L (2.4%) acetic acid, with 14 g/L (1.4%) ethanol.
Tables 1 and 2 show the anti-microbial activities of the tested solutions in black and green tea preparations, respectively. The unfermented tea samples showed no anti-microbial properties against most of the test organisms even at 70 g/L dry tea, but Staphylococcus aureus was minimally inhibited when the dry weight of tea reached 35 g/L (w/v) and higher. Levels of tea above 4.4 g/L had an offensive bitter taste and were undrinkable. Therefore, drinkable levels of tea (4.4 g/L) possess no observable anti-microbial effects. In addition, the contribution of tea itself to the anti-microbial activity of Kombucha proved to be insignificant, despite the anti-microbial activity found in extracts and concentrates by researchers such as Toda et al., Ahn et al., and Kawamura and Takeo (18,22,24).
Fermented Kombucha, at about 7 g/L acetic acid (33 g/L total acid), had anti-microbial activity against all test organisms in all green and black tea preparations, except Candida albicans. Candida albicans was not inhibited by any test solutions except tested commercial vinegar (50 g/L acetic acid). The anti-microbial activity observed was due to the organic acids, primarily acetic acid, and was eliminated when samples were neutralized.
The test organisms reacted similarly in all black and green tea Kombucha preparations, even in the highest level of tea used (70 g/L). There appeared to be a proportional similarity between the 10 g/L acetic acid control and the fermented samples which contained about 7 g/L acetic acid. Therefore, the anti-microbial activity was primarily a result of the acetic acid components of the ferment, as Steinkraus et al. found in their Kombucha prepared with 4.4 g/L dry tea (4).
Hesseltine reported the inhibition of Agrobacterium tumefaciens in neutralized Kombucha at about 40 g/L dry weight/volume tea (7). However, in this investigation, Agrobacteriumtumefaciens had no vulnerability to the unfermented tea components, even at 70 g/L dry tea, but it was inhibited in all the fermented samples.
The anti-microbial activity observed in the fermented samples containing 33 g/L total acid (7 g/L acetic acid) was significant against the tested gram positive and gram negative pathogenic organisms. Candida albicans was not inhibited by Kombucha. Tea, at drinkable levels, demonstrated no anti-microbial properties. The contribution of tea itself to the anti-microbial activity of Kombucha proved to be insignificant in the tested organisms, even at the highest levels tested. As a result, the anti-microbial activity of Kombucha was from the acetic acid composition.
Kombucha may be a healthful beverage in view of its anti-microbial activity against a range of pathogenic bacteria. This may promote immunity and general well being. It is recommended that Kombucha be consumed at 33 g/L total acid, 7 g/L acetic acid, to obtain these beneficial attributes.
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