Antibacterial Activity of Tea and Coffee: Their Extracts and Preparations

Abstract

Tea, Camellia sinensis (L) O. Kuntze (Theaceae) and coffee, Coffea arabica Linn. (Rubiaceae) possess antimicrobial activity in addition to various biological properties. In this study six strains of human pathogenic bacteria were assessed for their sensitivity to aqueous extracts of tea and coffee along with their preparations i.e. supplemented with milk and sugar. Antibacterial potential of the extracts has been compared with some of the commonly employed antibiotics. Bactericidal activity of the selected extracts was assessed by viable cell count method. Both tea and coffee inhibited bacteria to a variable extent and retained their antibacterial activity even after addition of milk and sugar. Equal effectiveness of tea/coffee extracts and their preparations justifies their potential as antibacterial agents.

Keywords:

• Camellia sinensis
• Coffea arabica
• Bactericidal
• Coffee
• Tea

INTRODUCTION

Different antibiotics and their chemical formulations are used to combat many diseases caused by human pathogens. The use of antibiotics has been under constant challenge due to emergence of resistant microorganisms.[1–3] Various plants and their products have been in use for their medicinal value since ancient times.[4] Some of these have also been shown to possess antimicrobial activity [5–8] holding great potential against resistant microorganisms. Tea and coffee are the two non-alcoholic beverages consumed commonly all over the world. Tea has acquired a privileged place and popularity amongst all sections of people and it is no exaggeration to say that it is an all purpose, all time drink for all people. There are three types of tea (green, black, and oolong tea) and two types of coffee (ground and instant coffee) commercially available in the market. Tea and coffee are known to possess various biological properties e.g. their protective role in the pathogenesis of chronic diseases such as cancer,[9,[1,[2] cardiovascular diseases/ coronary heart diseases,[10–12] atherosclerosis, liver cirrhosis, and arthritis.[9–12] The biological properties of tea and coffee are accredited mainly to their polyphenolic contents (flavonoids, catechins, tannins); and caffeine, etc. The ability of these components to scavenge reactive oxygen species impart them potent antioxidant activities in vitro and in vivo.[13] Furthermore, the role of herbal teas in disease prevention and cure has been partly attributed to the antioxidant properties of phenolic compounds present in the plant extracts.[14] Tea was first reported to possess antiviral activity against influenza virus,[15] and their antimicrobial effect gained scientific attention in recent years.[16–24] In vitro bactericidal activity of coffee along with that of green and black tea opened a new area of research emphasizing their use as effective antimicrobial agents.[16] In earlier studies, tea and coffee have been tested at higher concentrations and that too in buffer saline solution or their aqueous extracts while in practice tea/coffee is consumed with milk and sugar and at relatively lower concentrations (1% on an average basis). Thus, the present study was aimed to test the antibacterial effect of low concentrations of aqueous extracts of tea, coffee, and their preparations—i.e., the way these are consumed in daily life, against some pathogenic bacteria.

MATERIALS

Bacteria

Six strains of clinically important pathogenic bacteria viz., Enterococcus faecalis (MTCC 439), Pseudomonas aeruginosa (MTCC 1035), Salmonella typhi (MTCC 531), Shigella flexneri (MTCC 1457), Staphylococcus aureus (MTCC 87) and Staphylococcus epidermidis (MTCC 435) were obtained from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India. These bacterial cultures were maintained by regular subculturing on nutrient agar slants and stored at 4°C as well as at – 80°C by making their suspension in 10% glycerol.

Collection of Tea and Coffee samples

Six samples including three black teas (BT) viz. BT A, BT B and BT C; one green tea (GT) and two instant coffee samples (CF) viz. CF A and CF B were obtained from the local market of Amritsar.

METHODS

Preparation of Aqueous Extracts of Tea and Coffee

Different concentrations (1–5%) of tea extract were prepared by adding weighed quantity of tea to known volume of boiling water and it was allowed to brew for two minutes. The sample was then filtered through muslin cloth to get its extract. However, coffee extracts were prepared by boiling the weighed amount of coffee in a known volume of water to get the desired concentrations.

Preparation of Tea and Coffee with Milk and Sugar

Average quantity of milk and sugar per cup of tea/coffee was worked out by taking five random preparations. Based on this, different concentrations of tea were prepared having 5.5% sugar (weight/volume) and 20% milk (volume/volume) whereas different concentrations of coffee were prepared in milk containing 4% sugar.

Preparation of Inoculum

A loopful of isolated colonies of a particular bacterial strain growing on nutrient agar plate was taken and inoculated into 4 ml of peptone water and then incubated at 37°C for 4 h. The concentration of the bacterial suspension was then adjusted with peptone water to obtain a turbidity visually comparable to that of 0.5 McFarland standard prepared by mixing 0.5 ml of 1.75% (w/v) barium chloride dihydrate (BaCl_2. 2H₂O) with 99.5 ml of 1% (v/v) sulfuric acid (H₂SO₄). This turbidity is equivalent to approximately 1 − 2 × 10⁸ colony forming units per ml (CFU/ml). This activated culture was then used as inoculum for further testing.

Sensitivity of Bacteria

Antibacterial activity of tea/coffee extracts and their preparations against reference strains was tested according to Bauer et al. [25] using nutrient agar medium. Each experiment was repeated thrice. The bacteria with a clear zone of inhibition of more than 12 mm were considered sensitive.

Comparison with Antibiotics

Sensitivity of reference strains of bacteria to tea and coffee was compared with four commonly employed antibiotics- ampicillin (10 μg/disc), chloramphenicol (30 μg/disc), co-trimoxazole (25 μg/disc) and tobramycin (10 μg/disc) using standard disc diffusion method.

Determination of Bactericidal Activity of Tea and Coffee

Bactericidal activity of tea and coffee was measured using their 5% concentration by viable cell count method.[16]

Statistical Analysis

All values have been expressed as mean ± standard deviation and the difference in the antibacterial activity of the samples evaluated by applying t-test (Table 1). Results were considered to indicate statistically significant difference at P value ≤ 0.05.

Table 1 Comparative sensitivity of bacteria (in mm) to standard antibiotics and aqueous extract of tea, coffee as well as their preparations (5%)

							^cStandard antibiotics
Bacteria	^aBTextract	^bBTprep	^aGTextract	^bGTprep	^aCFextract	^bCFprep	A	C	Co	Tb
Ent. faecalis	—	—	—	—	22.5	23.6	21	23	—	09
Ps. aeruginosa	17	15	18	15.5	20.4	21.4	—	16	—	19
Salm. typhi	16.5	15	16.9	15.7	—	—	10	19	16	13
Sh. flexneri	19	18	19.5	18	21.3	22	23	25	18	17
Staph. aureus	20	17	23	19	16.3	16.8	30	22	21	18
Staph. epidermidis	25	23	27.5	25	19.6	20	26	20	—	17
Mean	16.2	14.6	17.5	15.5	16.7	17.3	18.3	20.8	9.16	15.5
± SD	8.52	7.76	9.39	8.35	8.44	8.78	11.22	3.19	10.16	3.78
t-test: ^aBT Vs ^bBT^aGT Vs ^bGT^aCF Vs ^bCF ^aBT Vs ^aGT ^bBT Vs ^bGT;
t-value: 0.3401 0.3899 0.1206 0.2512 0.1021
p value: P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05
—  = no zone of inhibition; BT  =  Black tea; GT  =  Green tea; CF  =  Coffee; ^aExtract; ^bPreparation; ^cAntibiotics: A  =  Ampicillin, C  =  Chloramphenicol, Co  =  Co-trimoxazole, T  =  Tobramycin.

RESULTS AND DISCUSSION

Alternative medicine is gaining much attention to combat the problem of resistant microbes and to counter the side effects of antibiotics being used by human population. A variety of plants/plant products are getting renewed interest to be explored for their antimicrobial properties in a more systematic manner. Tea and coffee, the two commonly consumed beverages, have been considered in the present study to assess their antibacterial potential. Initial experiments were carried out on Mueller-Hinton and Nutrient agar media, which did not reveal any significant difference, thus further studies, were carried out using nutrient agar medium only.

Sensitivity of Bacteria to Tea and Coffee

Different bacteria were sensitive to both tea and coffee extracts as well as their preparations to variable extent and no single product was found to be equally effective for all the organisms. Some of the organisms were resistant to low concentrations (1–3%) of the extracts though sensitivity increased moderately with increase in concentration. Of the different black teas tested, BT A gave the best results and thus data pertaining to it only has been presented along with green tea and coffee samples (Table 1). Similarly, the two coffee samples were almost equally effective and thus the data has been presented only for one coffee sample giving comparatively better results. Ent. faecalis and Salm. typhi were resistant to tea and coffee respectively. Ent. faecalis resistant to tea was however the most sensitive organism to coffee and the sensitivity of Ent. faecalis to coffee extract and its preparation is consistent with earlier studies [18] but the resistance shown by this organism to tea samples could be due to low concentration used in the present study. The resistant nature of Salm. typhi to coffee extract as well as its preparation corroborates earlier studies of Toda et al., [16] though Mahajan et al. [17] and Shetty et al. [19] found it to be sensitive. Sh. flexneri resistant to both tea and coffee [16,[9] was found sensitive in the present study, in consonance with studies [17] carried out at a concentration of 20–25%. Gram-positive bacteria were comparatively more sensitive to tea than Gram-negative bacteria; however, for coffee no such distinction was made. Greater resistance of Gram-negative bacteria to plant extracts has been reported [16,[6] and this could be attributed to the differences in their cell wall structure. Gram-negative bacteria have an outer phospholipidic membrane, which acts as a barrier to many environmental substances, including antibiotics while Gram-positive bacteria are more susceptible having an outer peptidoglycan layer which is not an effective permeability barrier.[27] Green tea has been shown to possess better antimicrobial activity as compared to black tea.[16,[8] This difference could be attributed to their method of manufacture involving fermentation and distortion as the nature and extent of distortion of tea shoots has been shown to influence the appearance and liquoring properties of the end product.[29] It is possible that some of the components responsible for the antimicrobial activity may be degraded or altered during fermentation and cutting-tearing-curling process. Hence, non-fermented green tea has more antibacterial activity. However, no such distinction between the two tea samples could be made in the present study, which might be due to their low concentrations used. Presumably the differences observed among earlier studies and the present study are due to strain variations, the sources and infusion strength of the various samples (tea and coffee).[30] Supplementation of milk and sugar to tea and coffee slightly altered their antibacterial effect, which however was statistically insignificant at P value of 0.05 (Table 1). Marginal decrease in inhibitory effect on addition of milk and sugar to tea might be attributed to some complex formation with milk proteins with polyphenols/antimicrobial components of tea thus making them inaccessible for their inhibitory action.[31] In fact, a number of other studies have demonstrated that addition of milk to tea does not affect the bioactivity of tea flavonoids.[32]

Sensitivity of Bacteria to Commonly Employed Antibiotics

The different cultures responded to standard antibiotics in a variable manner and gave an inhibition zone ranging from 9 to 30 mm. Salm. typhi, Sh. flexneri, and Staph. aureus were sensitive to all the antibiotics while Staph. epidermidis and Ent. faecalis were resistant to co-trimoxazole and Ps. aeruginosa was resistant to ampicillin and co-trimoxazole (Table 1). Comparison of sensitivity pattern of tea and coffee with that of standard antibiotics revealed that some bacterial strains although resistant to certain antibiotics were sensitive to one or the other tea and/or coffee sample, e.g. Ent. faecalis and Staph. epidermidis resistant to co-trimoxazole; and Ps. aeruginosa resistant to ampicillin and co-trimoxazole, were inhibited by tea/coffee extracts and their preparations. Ps. aeruginosa reported to be resistant to tea and coffee[15] was found sensitive in the present study in conformity with earlier studies.[16] It further underlines the potential of these samples to be used as antibacterial agents especially against Ps. aeruginosa, one of the most common Gram-negative pathogens associated with nosocomial infections and infections in immunocompromised patients. Unfortunately, resistance to available antibiotics is on the rise and there are a limited number of antipseudomonal agents with reliable activity against Ps. aeruginosa including penicillins, cephalosporins, carbapenems and fluoroquinolones, particularly ciprofloxacin.[33]

Bactericidal Activity of Tea and Coffee Samples

The data obtained by agar diffusion assay was supported by viable cell count method. The incubation of different bacteria with tea samples (aqueous extracts as well as their preparations) resulted in a steady decline of viable cell counts. Staph. epidermidiswas the most sensitive organism, where aqueous green tea extract and its preparation resulted in a killing of 97% and 95%, respectively; while black tea caused a killing of 92% and 88%, respectively, after 24 h of incubation. Ent. faecalis was the most sensitive organism to coffee where its aqueous extract and preparation resulted in a killing of 94% and 92%, respectively, while Salm. typhi was completely resistant. Aqueous extracts as well as the preparations of tea/coffee samples gave almost similar killing pattern and thus only the data pertaining to extracts of green tea, black tea, and coffee has been presented (Fig.1, 2, 3). As shown above complete killing could not be obtained even after 24 h though in earlier studies [16,17,19] 100% killing was observed within 3–4 h, which again might be attributed to high concentrations (20–25%) of tea and coffee used.

Figure 1 Viable cell count of different bacteria with 5% aqueous green tea extract.

Figure 2 Viable cell count of different bacteria with 5% aqueous black tea extract.

Figure 3 Viable cell count of different bacteria with 5% aqueous coffee extract.

Statistical Analysis

The difference between antibacterial activity of extracts^a and preparations^b of tea as well as coffee were found to be statistically insignificant at P value 0.05. Similarly, on comparing the effect of tea and coffee with that of different antibiotics^c did not reveal any statistically significant results (Table 1). This indicated the equal potential of tea and coffee in comparison to antibiotics.

CONCLUSIONS

All the tested bacteria were sensitive to tea/coffee extracts and their preparations to a variable extent. Staph. epidermidis and Ent. faecalis, resistant to co-trimoxazole, were however found to be the most sensitive organisms to tea and coffee respectively. Similarly Ps. aeruginosa resistant to ampicillin and co-trimoxazole, was inhibited by tea and coffee. Statistically insignificant difference observed in antibacterial activity of tea/coffee extracts and their preparations suggest that tea/coffee retained their antibacterial properties even after supplementation with milk and sugar. Furthermore, equal effectiveness of tea/coffee extracts and preparations even at low concentrations (1–5%) in comparison to high concentrations (15–20%) used in earlier studies justifies their consumption by millions in daily routine all over the world. Tea and coffee preparations are equally effective with their antimicrobial effect intact in comparison to bitter concoctions of tea and coffee. The present study further supports the use of these plants as potential antibacterial agents especially against nosocomial infections.