EVALUATION OF ANTIOXIDANT ACTIVITIES OF KOMBUCHA GREEN TEA.

Shimaa S. Ibrahim 1 , Soheir S. Abd-El Salam 1 , Mahmoud M. Amer 1 , Aly F. Mohamed 2 , Sherin M.Emam 3 and Reyad M. El-Sharkawy 1 . 1. Department of Botany and Microbiology, Faculty of Science, Benha University, Egypt. 2. International Center for Training and Advanced Research, Egypt. 3. Departments of Medical Microbiology and Immunology, Faculty of Medicine, BenhaUniversity, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 14 November 2018 Final Accepted: 16 December 2018 Published: January 2019

The yeast cells convert the sugar into ethanol by the glycolysis pathway (Sievers et al., 1995). Acetic acid bacteria convert glucose to gluconic acid and fructose into acetic acid. Acetic acid stimulates the yeast to produce ethanol which facilitates the growth of acetic acid bacteria and then the production of acetic acid (Liu et al., 1996). Both ethanol and acetic acid have been reported to have antimicrobial activity against pathogenic bacteria (Liu et al., 1996).
The benefits that have been attributed to KGT is also mainly due to green tea catechins content which is polyphenol derivative. These substances act as potent antioxidants and protect against the development of diseases. The beneficial effects of tea mentioned below have been studied mainly in vitro while others are based on clinical and epidemiological evidence (Valenzuela, 2004). Tea catechins act as antioxidants, due to the fact that they are molecules with a high capacity to scavenge free radicals and even metals which is also known as redox potential.
Kombucha green tea is claimed to help digestion, prevent microbial infection, help in combating stress, give relief from arthritis, cure cancer and AIDS, and increase immunity (Ram et al., 2000). Some of these claims effects have been proven; this drink does exert antimicrobial activity against a range of bacteria (Greenwalt et al., 1998(Greenwalt et al., , 2000Sreeramulu et al., 2000), prevent the angiogenesis of prostate cancer cells (Srihali et al., 2013), prohibits paracetamol induced hepatotoxicity (Pauline et al., 2001), blocks pancreatic alpha-amylase in the small intestine (Kallel et al., 2012) and the activity of glucuronidase an enzyme indirectly related with cancers (Wang et al., 2010).
Kombucha green tea has been ascribed to the presence of phenolic antioxidants (Vijayaraghavan et al. 2000) which depend primarily on the phenolic content of the substrate and secondarily on the normal microbiota of the Kombucha culture which in turn determine the nature of produced metabolites (Jayabalan et al. 2014). These compounds act as antioxidants in the body by scavenging harmful free radicals implicated in degenerative diseases.
The analyses of the fermented liquid proved the presence of acetic, lactic and gluconic acids as the major chemical compounds . They have been suggested to be the major antimicrobial agent (Greenwaltet al. 1998) and other compounds such as bacteriocins and teaderived phenolic compounds may be involved (Sreeramuluet al. 2001). Systematic investigation of the antimicrobial activity of KGT the presence of antimicrobial compounds other than organic acids or proteins (enzymes) produced during fermentation process (Sreeramuluet al. 2001).

Materials and Methods:-
Chemicals:-All chemicals and solvents were purchased from Sigma-Aldrich, USA.

Collection of sample:-
Lipton green tea and Sucrose were purchased from local market, Kombucha culture starter (Botany and Microbiology Department, Faculty of Science, Benha University, Egypt).

Preparation of kombucha green tea (KGT):-
Kombucha green tea was prepared according to the method by Malbaša et al. (2011). Sugared water was prepared with 100 grams sucrose per liter of boiled drinkable water. The boiled sugared-water was then infused with 10 grams of Lipton green tea (thread hanging outside) and boiled again for 5 minutes after that the tea bag was 949 removed. The boiled sugared green tea was transferred into a clean transparent glass jar that had been previously sterilized at 121°C for 20 min and then allowed to cool to room temperature. It was inoculated with 5% (w/v) of freshly grown tea fungus that had been cultured in the same medium for 14 days and 10% (v/v) of previously fermented liquid tea broth aseptically. The fermentation container was then covered with Gauze for aeration that is required for fermentation of the green tea and kept in a clean environment and left to grow at 30°C a period of fermentation. Where Ao is the absorbance of the control, and A1 is the absorbance in the presence of the kombucha green tea at 7 th , 14 th , 21 th and 28 th day of fermentation.

DPPH scavenging activity assay:-
The electron donating ability of KGT were determined from bleaching of purple colored ethanol solution of DPPH. This spectrophotometric assay uses the stable radical 2, 2-diphenyl-1-picrylhydrazyl as a reagent. DPPH was prepared at a concentration of 0.002%. Different of concentrations of KGT (0.05, 0.1, 0.2, 0.4, 0.8, 1.2 and 1.6) were taken in separate test tubes and volumes were made up to 2 ml using distilled water. Then, 2 ml of DPPH solution was added in each test tube and these solutions were kept in darkness for 30 minutes. Later optical density was recorded at 517 nm using spectrophotometer D250 plus, Analytic Jena at 7 th , 14 th , 21 th and 28 th days of fermentation. Distilled water with DPPH was used as control (lawrence R and lawrence K, 2011; Ebrahimi et al., 2016).

Inhibition of DPPH activity (%) = (A-B/A) × 100
Where, A=optical density of control; B=optical density of sample. IC 50 factor of samples were also evaluated; IC 50 factor indicates the concentration or amount of KGT that can inhibit 50% of free radical agent. Samples with smaller needed amount to inhibit 50% of DPPH have higher radical scavenging activity (Ebrahimi et al., 2016). The IC 50 is calculated by graphpad prism 7.

Antimicrobial activity of kombucha green tea:-
The antimicrobial activity of the kombucha green tea (KGT) was determined by the disk diffusion method which is based on the spread of antimicrobial compound in solid medium (Lesueur et al., 2007). The Mueller-Hinton agar was poured in sterile petri dishes (90 mm diameter). The sterilized filter paper discs (6 mm diameter) were impregnated with 50µ of KGT against certain bacterial strains included as Escherichia coli (C95), Salmonella typhimurium (ST74), klebsiella pneumonia (K55) and shigelladysenteriae (S151) obtain from the Faculty of Medicine, Benha University at different days of fermentation to determine the best fermentation day that gives highest inhibition zone using unfermented tea as negative control. Petri dishes were allowed to stand for 30 min at room temperature before incubation at 37 °C for 24 h. The effect of kombucha was reflected by the appearance around disc with a transparent circular zone corresponding to the absence of growth. The diameter of inhibition zone was measured in mm. The larger the diameter of the area, the more susceptible the strain was determined (Choi et al., 2006).

Results and Discussion:-
Antioxidant activity:-There has been a global trend toward the use of phytochemical present in natural resources as antioxidants and functional foods.Kombucha green tea (KGT) is also attributed to its antioxidant activity. Antioxidants are known to decrease or prevent disorders and metabolic diseases made by free radicals (Jayabalan et al. 2014). Hydrogen peroxide assay is one of methods used for determination of antioxidant activities. Kombucha green tea exhibited higher hydrogen peroxide scavenging activity at 14 th day of fermentation by 38.8% while 35.59% at 7 th day of fermentation slightly decrease 21 th and 28 th by 37.3% and 36% and this illustrated in (figure 1). The differences in 950 H 2 O 2 scavenging capacities may be attributed to active components which determine their electron donating abilities.The results had a statistically significantly (p < .05) The second method for determination of antioxidant activity is DPPH free radical scavenging that was used to determine the concentrations of KGT at which they scavenge the 50% of the DPPH solution termed as IC 50 values. The lower the IC 50 value of an antioxidant the higher would be its free radical scavenging power. The data showing increasing of KGT concentration lead to increasing % of inhibition within fermentation time that reach to maximum at 14 th day of fermentation with slight decrease at 2 1th and 28 th days of fermentation and this showed at ( Figure 2  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 molecularweight components and structural modifications of tea polyphenols by enzymes produced by bacteria and yeast during fermentation.The extent of the antioxidant activity in the Kombucha green tea beverage depended on many factors such as fermentation period, substrate types and the normal flora of the KGT culture that determined the nature of the secondary metabolites that developed during the fermentation process (Jayabalan et al., 2014).We considered the scavenging effects to be attributable to the sum of the antioxidant ability of many compounds present in the KGT and to the synergistic effects between certain metabolic produced during the fermentation process.

Antimicrobial activity:-
In the present study, the antimicrobial activity of kombucha green tea was screened against various pathogens in vitro by disk diffusion method. The result shown at table (1) and figure (4) is the antibacterial activity of KGT against gram negative entrobacteriaceae. The table indicates that the best antimicrobial activity occurred at 14 th day of fermentation. KGT has effect on klebsiellapneuonimae (K55) by 21 mm at 14 th day of fermentation with no change at 2 nd period of fermentation. In addition, appear an effect on Escherichi coli (C95) (10 mm) at 14 th day of fermentation. And also there is no effect of KGT on shigelladysenteriae (S151) and Salmonella typhimurium (ST74). Unfermented kombucha green tea on tested bacteria showing no activity.  Enteric bacterial infections are a major disease burden in human population in developing countries. Earlier these infections could be treated with low-priced antibiotics. However, recently the treatment has become higher cost with less successful due to the presence of multi-drug resistance among the enteric strains such as E. coli, Salmonella spp and Shigella spp (Barman et al., 2010). Hence, it has become imperative that the problem of multidrug resistance is battled at the earnest by exploring medicines or products from natural sources as a potential source of novel antimicrobial agents against the resistant bacteria (Ross et al., 2001).
The inhibition to E. coli and klebsiella pneuonimaeare important because this enteropathogenic microorganism is responsible for diarrheal disease and nosocomial infections that still the most prevalent and important public health problem in developing countries which is mainly cause death in children with less than 5 years of age, despite advances in knowledge, understanding and management that have occurred over recent years (Fagundes Neto and Scaletsky, 2000).
The Kombucha green tea showed also remarkable antimicrobial activity against E. coli (C95) and klebsiella pneuonimae (K55) where antimicrobial activity increased with fermentation time with slightly changes at 2 nd period of fermentation. There are numerous reports that antimicrobial activity of kombucha tea against pathogenic microorganisms is largely attributable to acetic acid (Greenwalt et al., 1998 ; and antibiotics present in the kombucha tea (Chen and Liu, 2000;Jayabalan et al., 2007). More, Sreeramulu et al. (2001) reported that the metabolites produced by the bacteria and/or yeasts during the fermentation of kombucha tea are responsible for its antimicrobial activity. Our results are similar to previous studies.

Conclusion:-
This study revealed that the kombucha green tea (KGT) possess great with antioxidant and antimicrobial activities.

Recommendation:-
These findings provide additional support for the traditional use of Kombucha green tea (KGT) in the treatment of metabolic diseases and different types of cancer.