Determination of antibacterial activity of tea water concentrates against E. coli and S. aureus

Tea is one of the most common beverages consumed since centuries. It has antimicrobial activity against various pathogens. Current research study was designed to evaluate and compare the antibacterial activity of different commercially available tea (green and black) concentrates against pathogenic bacterial strains of gram-negative Escherichia coli (E. coli) and gram-positive Staphylococcus aureus (S. aureus). Modified broth micro dilution method was used for the evaluation and analysis of tea samples. For this purpose, stock solutions of green and black tea concentrates, mannitol broth and bacterial suspensions were prepared. E. coli and S. aureus 10 μl suspension was added according to labelled wells and incubated. Minimum inhibitory concentration was confirmed by well diffusion method. The results suggest that green tea water concentrate is effective against both strains of these pathogenic bacteria, inhibiting their growth at 6.2 mg/ml. The black tea water concentrate is found more efficient against E. coli than S. aureus, with the minimum inhibitory concentration at 6.2 mg/ml and 12.5 mg/ml respectively. The findings of the current study encourage and recommend the use of both green and black tea in normal routines as well as a traditional medicinal remedy for the treatment of various human ailments.


Introduction
In beverages, tea (Camellia sinensis) is most commonly consumed in the world. Tea contains ingredients that refresh mind by stimulating and producing good feelings [1]. Tea has various types and flavours such as oolong tea, green tea and black tea. Green tea is most commonly used due to its antioxidant and antimicrobial activities. In the modern world, green tea flavour is used in various items such as candies, soft drinks and ice creams [2]. Tea is well known as a therapeutic agent for different maladies [3,4]. According to research studies, tea have antimicrobial, anti-inflammatory, antioxidant, anti-cancer and antibacterial activities against number of pathogens [5,6]. In ancient India and China, human ailments were cured with tea as natural remedy [7]. Green tea is product of C. sinensis dried leaves, however black tea require fermentation process that produce thearubigins and theaflavins. These oligomeric polyphenolic compounds derived from flavanol tea units are biologically active components of tea [8]. Main flavonoids found in tea are tannins, catechins and theaflavins. Catechin is subdivided into epigallocatechin gallate (EGCG), epicatechin (EC), epigallocatechin (EGC) and epicatechin gallate (ECG) [9]. Polyphenols quantity in black and green tea is about 10 % and 40% respectively [10]. Green tea has antimicrobial activity against gram-positive and gram-negative bacteria [11]. EGCG have maximum radical scavenging activity and is the most abundant polyphenol, due to popularity of tea and absence to toxicity. Tea and caffeine might contribute to and promote anticarcinogenic effects [12,13]. Animal models and cell line studies have shown antiangiogenic, anticarcinogenic and antiproliferative effects of tea flavonoids [12,13]. According to literature, in presence of tea, various pathogens (Aeromonas sobria, Clostridium perfringens, Bacillus cereus, Staphylococcus aureus (S. aureus), Pleisomonas shigelloides and Vibrio parahaemolyticus), fail to grow [13,14]. The objective of current research study was based on determination of antimicrobial activity of green tea and black tea against gram-positive bacteria (Staphylococcus aureus) and gramnegative bacteria (Escherichia coli).

Materials and Methods Tea concentrates preparation
Tea samples (20g) were collected in flasks (250ml), distilled water (150ml) was added and kept on shaking incubator (ES-20) at (26-30°C) for three days. After incubation, the samples solution was filtered with the help of Whatman filter paper. The filtrates were separated in two different screw capped flasks. For solvent evaporation Rotavapor Buchi Rotavapor (R-200) equipment was used.

Mannitol broth preparation
Mannitol broth (50 ml) was prepared in (250 ml) flask and screw capped tubes. The broth was poured and stored at 5 °C. The test microorganisms (E. coli and S. aureus) were inoculated at 10 6 colony forming units into tubes and vortexed gently.

Determination of minimum inhibitory concentration (MIC)
Green tea and black tea stock solutions were prepared for each sample by adding 200 mg concentrate in 1 ml distilled water (200 mg/m1). To remove debris, the stock was centrifuged at 1000 rpm. Sterile micro-titer plate was used for determination of MIC. The plate first 4 rows were labelled, 1 st row: s-g-w-1 to s-g-w-8, 2 nd row: egw1 to e-g-w-8, 3 rd row: s-b-w-1 to s-b-w-8 and 4 th row: eb-w-1 to e-b-w-8 respectively. Mannitol broth 40 μl was added through micropipette in to each well respectively. 50 μl mannitol broth was added to positive-control to 9 th and 10 th well and negative-control to 9 th and 10 th wells of microtiter plate (96U-MS-9096UZ) respectively. For green tea, stock solution 40 μl was added to 1 st row, well (s-g-w-1), the concentrates were properly dispensed than 40 μl solution was transferred to well (s-g-w-2). The process was repeated up to 8 th well (s-g-w-8) respectively. Finally, 40 μl solution was discarded. Similar practice was repeated to 8 th (s-g-w-8) well respectively and 40 ml solution was discarded from 8 th (s-g-w-8) well. Similar steps was done for 2 nd row (eg-w-1 to e-g-w-8) wells. For dilutions of black tea, similar practice was done to 3 rd row (s-b-w-1 to s-b-w-8) wells of plate and 4 th row (e-b-w-1 to e-b-w-8). The concentrates of tea (green and black) were (0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 mg / ml) respectively. Suspension of Staphylococcus aureus (S. aureus) 10 µl through micropipette was added to 1 st and 3 rd row of plate. 10 µl suspension of E.coli was added to 2 nd and 4 th row of microtiter plate and kept on incubation for one day at temperature (35 °C). Methyl red dye 15 µl solution was added to every well of microtiter plate respectively. With the help of plate-reader at (450 nm), optical density of wells was noted.

MIC (minimum inhibitory concentration)
Each concentrate stock solution (100 mg/ml) was prepared by adding 100 mg in 1 ml distilled water and centrifuged at 2000 rpm. Test microorganisms (S. aureus and E. coli), suspensions (10 6 colony forming units) were inoculated on warmed mueller-hinton agar plates. Uniform size of wells was made on agar plates and (50 µl) tea concentrates was added to each well respectively and incubated at 37 °C for 24 hours.

Results Green tea water concentrates antibacterial activity
The minimum inhibitory concentration value of green tea concentrate for both the test organism was found at the concentration of 6.2 mg/ml. The growth of test microorganism was inhibited at 100, 50, 25, 12.5 and 6.25 mg/m1. Lower inhibition was found at 6.2 mg/m1 (Fig. 1). Well diffusion method and confirmatory test resulted that both test microorganisms are susceptible to green tea concentrations up to 10 mg/ml and resistant at 1 mg/ml (Table 1). S. aureus is susceptible up to 10 mg/ml concentrates of green tea with MIC 6.2 mg/ml, slightly resistant to concentrates of black tea with MIC 12.5 mg/ml. Black tea concentrates antibacterial activity By modified broth micro-dilution method, the MIC value of black tea concentrate was found at 12.5 and 6.25 mg/ml, for S. aureus and E. coli, respectively. Growth of S. aureus concentrates at 12.5 mg/ml at dilutions of 100, 50, 25 was inhibited whereas the growth of E. coli was inhibited at 6.25 mg/ml. At concentration of 100 mg/ml, maximum inhibition was observed and at concentrations of 6.25 and 12.5 mg/ml, E. coli and S. aureus was resistant respectively (Fig. 2). Well diffusion method indicated that S. aureus have shown resistance at 10.0 mg/ml concentration and E. coli have shown resistant at 1.0 mg/ml concentration as shown in (Table 1). The results show that E. coli is more susceptible to black and green tea concentrates having MIC 6.25 mg/ml.

Discussion
This research study conducted for antimicrobial activity of black tea and green tea at different concentrates resulted effects against pathogenic bacteria (Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli)). Green tea concentrates resulted better antibacterial activity against E. coli and S. aureus that was confirmed by well diffusion method as indicated in (Fig. 1, 2 & Table 1). The effects of black tea concentrates as antibacterial agent is comparatively less pronounced than green tea [15]. The basic explanation behind this difference in antibacterial activity is due to presence of various bio-active secondary metabolite components [16]. The present analysis demonstrates that consumption of green tea and black tea is healthy in constrained amount as it has potential to inhibit the growth of various pathogenic microorganisms. According to literature, herbal plants tested for antimicrobial activity is widely accessible. Previous research studies have also reported that black tea and green tea solvents have potential of antioxidant properties and antimicrobial activity [16]. Black tea and green tea have strong antimicrobial activity against various pathogens i.e. E. coli and S. aureus. Tea (black and green) can also be used as alternate of antibiotics against bacterial infections [15]. Tea extracts are used as antiadhesive agent to stop pathogenic bacteria adhesion to host cell membrane [17,18]. It is confirmed that tea has been proven safe, less costly, and easily available compound [19].

Conclusion
According to current research study, green tea has potential to inhibit growth of E. coli and S. aureus. However, E. co1i is relatively more susceptible to black tea than green tea concentrations. S. aureus indicates slight resistance to concentrates of black tea. Therefore, current research study supports the recommended use of tea (black and green tea) in folk medicines and beverages in satisfactory quantity.