ANTIMICROBIAL ACTIVITY OF FERMENTED GREEN LIQUID You-Ying Tu'", Hui-Long Xia

ABSTRACT made of sugar and single was investigated for its antimicrobial activity and composition changes during . Individual and total tea polyphenols, , sucrose, reducing sugar, total sugar, pH, acetic acid and total acids, concentration, superoxide dismutase and hexokinase activities, yeast yield and antimicrobial activity were measured systematically at the intervals of 12 to 24 hours of fermentation. Results shown that a 36-hour sample had total growth inhibition on Escherichia coli and Staphylococcus aureus, but almost no effects on Bifidobacterium and Lactobacillus delbrueckii subspecies bulgaricus. In order to see the effects of the main components on the microbial growth, a mixture of tea polyphenols, a mixture of acids and ethanol were tested respectively and showed various degrees of antimicrobial activities which were much less than the activity of the 36-hour kombucha, suggesting that there was a synergy effect among the components.

Keywords:Antimicrobial activity; Composition change; Fermentation; Green tea kombucha; Tea polyphenols.

INTRODUCTION Kombucha is a fermented tea beverage which is usually used for kombucha origin is traceable in more than two preparation, green tea and herbs are also used. thousand years ago. It is produced from It has been shown that green tea has a more fermentation of sugared tea by a symbiosis of stimulating effect on the kombucha fermentation and (Dufresne and Farnworth than black tea, yielding the fermentation in a short 2000, Teoh et a/. 2004). It has been reported time frame (Greenwalt, et a/. 1998). It is not known many health benefits mainly based on personal how the composition of the tea itself is affected observation and testimonials (Greenwalt, et a/. during the fermentation or how it is transformed 1998, Dufresne and Farnworth 2000) and getting (Dufresne and Farnworth 2000). quite popular today in the West. Kombucha has Kombucha is a home- product which been studied intensively since 1852, few of the means the preparation conditions are not sterile. health properties have been demonstrated by Majority of the tests for kombucha indicated that scientific and experimental studies (Dufresne a low rate of contamination from spoilage and and Farnworth 2000). pathogenic , suggesting that kombucha has antimicrobial properties to 'You-Ying Tu, Correspondence pathogenic and other "bad" microorganisms Email: youvtu@,ziu.edu.cn aDepartment of Tea Science, Zhejiang University, Hangzhou, China (Mayser et a/. 1995). Some studies reported that bCollege of Environmental Science and Engineering, Zhejiang antimicrobial activity of kombucha against a Gongshang University, Hangzhou, China range of bacteria, made with a low tea usage Antimicrobial Activity of Fermented Green Tea Liquid level (4.4 g/L), was attributable to the acetic-acid and stored at 4 OC until analysis. The residual content (Steinkraus et a/. 1996, Greenwalt etal. was collected and disrupted with methylbenzene 1998). While Sreeramulu etal. (2001) revealed (1 :0.8) for 2 hours at 37 OC. After being that the antimicrobial components of kombucha centrifuged at 15000g for 20 min, the aqueous are compounds other than organic acids, ethanol, phase was filtered through a 0.45 pm filter and proteins or tannins in tea or their derivates after stored at 4 OC until analysis. systematic investigation. 'Therefore, it is necessary to conduct further study on 2.3. ANALYSIS OF TEA POLYPHENOLS antimicrobial components of kombucha. The concentrations of tea polyphenols were The objective of this study was to investigate analyzed on a Diamonsil C18 column (4.6 x 250 compositional changes of sugared green tea mm, 5 pm particle size, ) using a Shimadzu kombucha and its antimicrobial activities during LC-2010 HPLC (Shimadzu, Japan) equipped fc-rmation with a systematic approach. with a UV detector. Wavelength was set at 280 nm. Mobile phase Aand B were all made of acetic 2. MATERIALS AND METHODS acid/acetonitrile/waterbut with different ratios (A: 2.1. PREPARATION OF SUGARED GREEN 0.5:3:96.5 and B: 0.5:30:69.5, by vol.). The flow TEA INFUSlQN rate used was 1 mUmin and 10 pL sample was Green tea (40 g, produced in Zhejiang Province, injected into the column. The column was PR China) was infused in 3000 mL boiling equilibrated initially with 100%A, and then eluted distilled water for 10 min with stirring, and then with a gradient up to 100% B in 45 min. The vacuum filtered with a paper filter. The tea residue column temperature was 35 "C. Peaks were was washed twice with 500 mL boiling water. identified by comparison with the retention time Sucrose (600 g) was added into the combined of authentic standards of (-)-epicatechin (EC), (- tea . The sugared tea infusion (STI) was )-epigallocatechin (EGC), (-)-epicatechin gallate adjusted to 5000 mL, which made 120 g/L of the (ECG), (-)- (EGCG), (-)- final sugar concentration and 8 g dry green tea (C), (-)-gallocatechin gallate (GCG), (-)- per liter. The ST1 was transferred into 36 x 250 catechin gallate (CG), and (-)-gallocatechin (GC) mL conic flasks, with each flask containing 120 (Mitsui Norin Co. Ltd, Japan). Concentration of mL STI. They were sterilized at 121 OC for 15 the individual polyphenol was calculated using min. the corresponding standard curve.

2.2.YEAST AND FERMENTATION 2.4. DETERMINATION OF ORGANIC ACIDS A yeast strain, Saccharomycodes ludwigii sp. The same HPLC and column as described (provided by Shanghai Institute of Microbiology above were used to determine the concentrations Research, PR China) was used for the ST1 of organic acids in the supernatant of the fermentation. The yeast was added into 100 mL fermenting STI. The mobile phase was 2.5% sterilized ST1 and developed over 24 hours as a NH4H,P04 (pH 2.5). Flow rate was 0.9 mL/min pre-culture. The pre-culture was used for and 10 pL sample was injected. Wavelength was inoculating ST1 at the level of 6-8 x 1O8 cells/mL. set at 280 nm. Peaks were identified by ST1 fermentation was carried out at 25 "C for up comparison with the retention time of authentic to 156 hours. At an interval of I2 hours, three standards of organic acids (Sigma). flasks of the fermenting ST1 were random Concentration of the individual organic acid was sampled for analysis. The samples were calculated using the corresponding standard centrifuged at 15000g for 20 min. The cuwe. supernatant was filtered through a 0.45 pm filter You-Ying Tu*", Hui-Long Xia 2.5. ANTIMICROBIAL ACTIVITY The assay for superoxide dismutase (SOD, EC ST1 samples were centrifuged at 40000g 1.15. 1.1) activity was based on the method of (Beckman J2-HS, USA) for 15 min to remove Beauchamp and Fridovich (1 971 ). The activity cell debris. Sterile supernatant was obtained by of hexokinase in ferment solution were filtering the supernatant through a sterile spectrophotometrically measured at 37 "C microfilter (0.22 pm, Millipore). The antimicrobial according to the method of Uyeda and Racker activity of the ST1 was tested at their natural pH. (1965). A mixture of tea polyphenol solution was prepared according to tea polyphenol 2.7. MEASUREMENT OF ETHANOL composition and individual concentrations of the CONTENT ST1 fermented for 36 hours. The mixture was The fermented ST1 (0.5 mL) was mixed with 1 made of 136 mg/L GC, II I mg/L EGC, 15 mg/L mL n-butanol and voltaxed for 1 min. Then 0.5 C, 39 mg/L EC, 249 mg/L EGCG, 52 mg/L GCG, mL trichloroacetic acid was added in and 86 mg/L ECG, 17 mg/L CG with total polyphenols voltaxed for another 1 min. After centrifuged at of 705 mg/L. 5000g for 10 min, the supernatant was used for Organic acid solution was made of 10.5 g/L GC assay. The ethanol was analyzed using a acetic acid, 0.36 g/L succinic acid, 0.69 g/L Shimadzu GC 14-B gas chromatograph isocitric acid, and 0.39 g/L L-malic acid, which equipped with flame ionization detector and a was the individual concentrations of the ST1 PEG-Wax capillary column (0.25 mm ID x 50 m). fermented for 72 hours. The GC conditions were as follows: Nitrogen was Escherichia coli, staphylococcus a ureus, a carrier gas (200 kPa). Injector and detector Bifidobacterium and Lactobacillus delbrueckii temperatures were 220 and 250 "C, subspecies bulgaricus were purchased from respectively. Injection volume was 1 pL. Ethanol Shanghai Institute of Microbiology Research, PR was identified by comparison with the retention China. They were grown and maintained on MRS time of authentic standard and the concentration medium containing agar (I5 glkg). MRS medium was calculated using a corresponding standard with agar (20 mL) was poured into each Petri curve. dish (90 mm diameter). The tea polyphenol solution, organic acid solution, 1.97 % ethanol 2.8. MEASUREMENT OF SUGAR CONTENT (the highest concentration of the fermented ST1 Total sugar and reducing sugar contents were at 120 hr) or the sterile ST1 sampling at different determined by the phenol sulfuric acid method fermentation time (0.9 mL) was mixed with the (Dubois et al. 1956) and the dinitrosalicylicacid target strain culture (0.1 mL) and settled for 30 method, respectively (Miller 1959, Roesser et al. min before spreading on the agar plates 1996). uniformly. Unfermented ST1 was used as a control. The plates of E. coliand S. aureus were 2.9. DATAANALYSIS then incubated at 37 "C for 24 hours. The plates Data were analysed and all figures were of Bifidobacterium and L. bulgaricus were generated using MS Excel 2000. incubated anaerobically at 37 "C for 24 hours. Antimicrobial activity was determined by 3. RESULTS AND DISCUSSION counting the average numbers of colonies of the Green tea infusion, sugar and yeast were the target strain grown on five agar plates. three components in the sugared green tea kombucha and subjected to a series of 2.6. THE ACTIVITIES OF SUPEROXIDE compositional changes during fermentation. The DISMUTASE AND HEXOKINASE composition of tea polyphenols in sugared tea Antimicrobial Activity of Fermented Green Tea Liquid the rest time of fermentation. Reducing sugar infusion (STI) decreased rapidly in 24 hours of which includes fructose and glucose increased fermentation (Table I), with GC, EGC, C, EC, rapidly in 48 hours from 11.5 mg/mL to 83.9 mgl EGCG, GCG, ECG, and CG decreased 33.6%, mL and then decreased to 6.7 mglmL at the end 47.6%, 40.7%, 35.4%, 36.5%, 30.1%, 32.8%, of fermentation. Total sugar content decreased and 29.6%, respectively. Total polyphenols linearly during fermentation. Hexokinase (ATP: decreased 37%. This suggests that all the tea D-hexose 6-phospho-transferase)is an enzyme polyphenols are sensitive to acidic pH. By the responsible for the catalysis of the end of the formation, the individual polyphenol phosphorylation of glucose, the first step in decreased 51.2%, 58.1%, 55.6%, 55.4%, glycolysis. It also has the ability to phosphorylate 58.1 Oh, 59%, 95.5%, and 59.3%, respectively. different hexoses. The activity of hexokinase in Total polyphenols decreased 60.9%. ECG was the yeast cells increased rapidly to maximum in the only tea polyphenol decreased 95.5% and the first two days of fermentation and then all the others decreased less than 60%. Caffeine decreased rapidly to a very low level at 108 decreased 33.7% in 24 hours of fermentation and hours. 64.3% at the end of fermentation.

Total (Time (hr) GC' EGCb C' ECd EGCG' GCG' ECGP CGGatechingr Caffeine 1 64 7 426 6 82 9 133 9 27 05 1253 5 392 2 :::: : ;;: ;;;: ;;; :;; ;;: 7930 26021 0 .r i - 3 1363 1113 745 3 244 2 1 g. 126 9 149 349 2477 546 835 701 9 251 0 4 11.0 1182 866 140 324 2336 492 787 659 9 246 9 g 0 1133 881 132 245 2004 360 648 118 5764 2048 S 5 120 1057 85 1 11 8 306 1780 352 595 11 4 5479 1964 0 .A 156 1057 876 124 789 1791 335 60 114 4934 1939 0.05 I' o yeast 0,s %C (-)-gallocatech~n EGC (-)-eptgallocatechtn,' C (-)-catechln EC (-)-eptcatechln, 1 . . .k..elhanol EGCG (-)-ep~gallocatechtngallale. ' GCG (-)-gallocatechtn gallate ECG (-)- eptcatechln 1 gallate and TG(-)-catechtn gallate Table 1 Changes of lndlv~dualtea polypheols, toal polyphenols and caffe~ne durlng ferrnentat~onof the sugared green tea (pglrnl) 0 40 80 120 160 Fermentation Time (hr) Figure 2. Changes of yeast as wet weight and ethanol concentration during +tolal sugar fermentation of the sugared green tea. 120 Yeast yield as wet weight increased from 5.4 mg/L to 18.4 mg/L in 72 hours and then decreased slightly during the rest of fermentation. The final yield was 14.7 mg/L. Ethanol increased 5: 8 rapidly after one day of fermentation, reached to 30 3 maximum at day five (1.9%) and decreased a little at the rest of the fermentation. 0 40 80 120 160 The pH value of ST1 dropped rapidly from FermentationTime (hr) 5.1 6 to 2.48 within 48 hours, then changed slightly Figure 1. Change of sucrose, reducing sugar, total sugar and hexokinase during fermentation of the suygared green tea. during the rest of the fermentation (Figure 3). The pH change was correlated to the increase of Yeast cells convert sucrose into fructose acids in STI. Acetic acid was the main acid and and glucose. Glucose is primarily used up by the its concentration increased rapidly to 12 g/L in yeast to produce ethanol and carbon dioxide. The ST1 in 84 hours and increased a little at the rest sucrose in ST1 decreased rapidly from 107 mg/ of the fermentation. Total acids showed a similar mL to 15 mg/mL during the first two days of pattern of change during fermentation. fermentation and then decreased slightly during You-Ying Tuha,Hui-Long Xia Fermentation hour of SGT Organic I POI-^ Ethanol 0 I 12 1 24 / 36 / 48 1 108 acids phenols -~ Lbulgancus 165 160 163 181 165 99 162 168 159 1 Blhdobac 254 249 245 243 247 183 146 / 226 221 I E colr 318 247 15 3 0 0 164 1 154 79 iIIIl!I t S aureus 1 6921 5161 68) 01 01 0 1 376 / 1-1 I I I I 2- 1- Table 2 Antlm~croblaleffects of the main components and the sugared green A A acetlc acld tea (SGT) at different fermentation time on S aureus E colf Bffidobactenum 1 I / and L bulgancus The unlts llsted In the table were the average numbers of o Total aclds I colonles of the target strain grown on flve agar plates 0 C' ------10 with different degrees of fermentation on

Fermentation Time (hr) microbial growth, ST1 samples were tested Figure 3. Change of pH, acetic acid and total acids during fermentation of the systematically. Unfermented ST1 (0 hr) was used sugared green tea. Aerobic organisms possess antioxidant defense as a control. The antimicrobial activities of the systems that deal with reactive oxygen species fermenting ST1 samples increased with (ROS) produced by aerobic respiration and fermentation time. The 36-hour ST1 showed total substrate oxidation. Superoxide dismutase inhibition on the growth of S. aureus, almost total (SOD) is an antioxidant enzyme to prevent cell inhibition on E. coli, but only slightly on and tissue damage initiated by ROS. Yeast Bifidobacterium, and had no effect on L. produced a large amount of SOD within 12 hours, bulgaricus. of which a large proportion of SOD was secreted After 108-hour fermentation, the ST1 showed outside the yeast. SOD activity in the fermenting 40% inhibition on Bifidobacterium and 28% on ST1 increased from 14.5 to 25 unitlmL in 72 hours L. bulgaricus. Mayser et a/. (1995) noted a low and then decreased to 20 unitlml in 108 hours rate of contamination from harmful and kept at this level until the end of the microorganisms (spoilage and pathogenic) and fermentation. The change of SOD activity in the concluded that kombucha can be prepared safely fermenting ST1 had the same pattern as the at home without pathogenic health risk. The growth curve of the yeast (Figure 4), which antimicrobial resutts from this study support that reached maximum at 72 hours. While SOD conclusion that the green tea kombucha inhibits activity in yeast cells increased continuously the growth of pathogenic bacteria. during fermentation. The green tea used in this study was 8 g dry tea per literwhich is about a normal consumption level of green tea. Since the 36-hour fermented ST1 had total growth inhibition to pathogenic bacteria, a mixture of tea polyphenols prepared according to the same concentrations of the individual tea polyphenols as the 36-hour fermented ST1 was used to test its antimicrobial activity. The tea polyphenol mixture had no inhibition on the growth of L. bulgaricus, light inhibition on Bifidobacterium (11 %), but stronger Fermentation Time (hr) Figure 4. Changes of superoxide dismutase (SOD) in fermented green tea and inhibition on S. aureus (28.6%) and E. coli in yeast cells as wet weight during fermentation of the sugared green tea (51.6%). Unfermented ST1 (0 hr) had sucrose The antimicrobial activities of tea (120 g/L) and higher concentration of tea polyphenols are well documented, while the tea polyphenols (1.25 g/L) than the tea polyphenol concentration used in those studies usually mixture (0.71 g/L). This could only be explained exceeded normal human consumption levels that sucrose in the ST1 might have some (see review of Dufresne and Farnworth 2000). In protective effects on microbial against the order to see the effects of green tea kombucha antimicrobial activity of tea polyphenols. Antimicrobial Activity of Fermented Green Tea Liquid Ethanol, using the highest concentration of the balance of "friendly" bacteria by inhibiting the 1.97% in the fermented STI, had strong inhibition growth of pathogenic bacteria in our digestive on S. aureus (72.4%) and E. coli (75.2%), but system after consumption of the kombucha. light inhibition on Bifidobacterium (13%), no In summary, a systematic approach to investigate inhibition on L. bulgaricus. AS the ethanol composition changes and antimicrobial activity concentration of the 36-hour ST1 was 0.07%, it of the green tea kombucha shown that there was would have much less antimicrobial effect. a series of composition changes during Some studies reported that antimicrobial fermentation, the 36-hour fermented ST1 had total activity of kombucha against a range of bacteria, growth inhibition on the pathogenic bacteria, and made with a low tea usage level (4.4 g/L), was the antimicrobial activity was a synergy effect of attributable to the acetic-acid content (Greenwalt the kombucha components. et a/. 1998, Steinkraus et al. 1996). Acetic acid at as little as 1 g/L concentration can inhibit ACKNOWLEDGEMENTS pathogenic and spore-forming bacteria (Adams This study was supported by Natural Science 1985). Greenwalt etal. (1998) demonstrated that Foundation of Zhejiang Province, PR China fermented kombucha at about 7 g/L acetic acid (0201075). (33 g/L total acid) had antimicrobial activity against most of the test organisms in all green REFERENCES and black tea kombucha. The antimicrobial Adams, M. R., 1985. Vinegar. In: WOOD, B. J. activity observed was due to the organic acids, B. (Ed.), Microbiology of Fermented Foods, primarily acetic acid, and was eliminated when Vol. 1. New York, Elsevier, pp. 1-48. samples were neutralized. In this study, a mixture of organic acids, the same concentrations of the Beauchamp, C., Fridovich, I., 1971. Superoxide individual acids as the 72-hour STI, had a similar dismutase: improved assays and an assay inhibitory effect on Bifidobacterium (42.5%), S. applicable to acrylamide gels, Anal. aureus (45.7%) and E. coli (48.4%) except L. Biochem. 44,276-287. bulgaricus which was not affected. This tested mixture of the acids had higher acid Dubois, M., Gilles, K.A., Hamilton, J. K., Rebers, concentration (11.9 g/L) than that at 36-hour ST1 P. A., Smith, F., 1956. Colorimetric method (7.7 g/L). While the 36-hour ST1 had total for determination of sugars and related inhibition on S. aureus and E. coli, indicating that substances. Anal. Chem. 28,350-356. acetic acid plays an important role on the Dufresne, C., Farnworth, E., 2000. Tea, antimicrobial activity of the kombucha, but it is kombucha and health: a review. Food Res. not the sole contributor to the antimicrobial Int. 33,409- 42 1. activity of the kombucha. These results in this study shown that the Greenwalt, C. J., Ledford, R. A., Steinkraus, K. main components along had much less H., 1998. Determination and antimicrobial activities on S. aureus and E. coli characterisation of the antimicrobial activity than the 36-hour STI, suggesting that there was of the fermented tea Kombucha. Lebensm. a synergy effect among the components in the Wiss. -Techno/. 31,291-296. green tea kombucha. L. bulgaricus and Bifidobacterium are Greenwalt, C. J., Steinkraus, K. H., Ledford, R. "friendly" bacteria found in human gastrointestinal A., 2000. Kombucha, the fermented tea: tract. The fermented ST1 had no or light inhibition microbiology, composition, and claimed on their growth, suggesting that it will benefit to health effects. J. Food Prot. 63,976-981. You-Ying Tu*", Hui-Long Xia Mayser, P., Fromme, S., Leitzmann, C., Grunder, Sreeramulu, G., Zhu, Y., Knol, W., 2001. K., 1995. The yeast spectrum of the 'tea Characterization of antimicrobial activity in fungus Kombucha'. Mycoses. 38,289-95. kombucha fermentation. Acta Biotechnol. 21, 49-56. Miller, G. L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Steinkraus, K. H., Shapiro, K. B., Hotchfiss, J. Anal. Chem. 31,426-428. H., Mortlock, R. P. 1996. Investigation into the antibiotic activity of tea fungus/kombucha Roesser, D. S., McCarthy, S. P., Gross, R. A., beverage. Acta Biotechnologica, 16,199-205. Kaplan, D. L., 1996. Effects of substitution site on acetyl amylose biodegradability by Teoh, A. L., Heard, G., Cox. J., 2004. Yeast amylase enzymes. Macromolecules, 29,19. ecology of kombucha fermentation. Int. J. Food Microbial. 95, 119- 126. Sreeramulu, G., Zhu, Y., Knol, W., 2000. Kombucha fermentation and its Uyeda, K., Racker, E., 1965. Regulatory antimicrobial activity. J. Agric. Food Chem. mechanisms in carbohydrate metabolism. VII. 48,2589-2594. hexokinase and phosphofructokinase. J. Biol. Chem., 240,4682 4688.