Effects of Tea and Fruits Juice on Bacterial Proliferation

CHUGOKUGAKUEN J. 2007 Vol. 6, pp. 5-1 0 Copyright02007 by Chugokugakuen Original Article Effects of Tea and Fruits Juice on Bacterial Proliferation Hideo Hayashi, Masako Iizuka, Tomoko Katsuta, Tomoko Shimada, Atsuko Mishima, Norisuke Sakaeda and Yoshie Manabe Department of Human Nutrition, Faculty of Contemporary Lge Science, Chugokugakuen University, Ohyama 701-01 97, Japan Effect of tea and fruits on the proliferation of opportunistic pathogens were examined. Three kind of teas: green tea, oolong tea and black tea and four pathogens: Bacillus subtilis, Staphylococctus aureus, E. coli and Pseudomonas aeruginosa were subjected to the experiments. The three kinds of tea leaf extracts (25g leaves in 500ml at 95C for 5min) inhibited the proliferation of S. aureus, B. subtilis and E. coli, but'not of P. aerugz'nosa, The effectiveness was correlated with the catechin content of the teas and MIC of catechin against S. aureus was lmg/ml, against B. subtilis and E. coli were 1-2mg/ml. The effect was bactericidal and the three bacterial strains tested were all killed at the concentration of above MIC, but P. aeruginosa was resistant. Juice of lemon and citrus (Sudachi), inhibited the proliferation of all the four organisms within 60min soaking, but those of grapefruit, kiwi and apple were less effective in inhibiting the proliferation. The effect was bactericidal and the results suggest the major factor was the lower ph. Key Words: Tea, fruit, bactericidal, effect, catechin Introduction Tea is the most common beverage in the world [I]. People drink tea as a snack, to refresh themselves during work time, or as an after meal beverage. Tea drinking has universal history of promoting comfortable human relationships. Tea contains many kinds of favorable components for human health and over 70 chemical components have been identified [2-41. Among those components, tannic acids, cathechin, and caffeine are common in most teas [5I. Some of the biologically active components such as caffeine and tunic acids are applied for medical use and some 'Corresponding author. Hideo Hayashi Department of Human Nutrition, Faculty of Contemporary Life Science, Chugokugakuen University, Niwase 83, Okayama 701-0197, Japan e-mail: hihayash@cjc.ac.jp are used as supplements for astringent skin care toners. In some countries in South Asia, they traditionally eat the boiled or pickled tea leaves after meals for detoxication of food that might be toxic. It is generally accepted that tea leaves and the hot water extracts act as antibiotics beside the psychoactive substances such as caffeine and tunic acid. There are some reports that catechin in tea leaves showed bactericidal activities against a certain kind of pathogenic bacteria [61. If this is the case, eating tea leaves or drinking tea after meal are reasonable methods to protect from some bacterial food poisoning or toxication. Fruits are taken as snacks and desserts. Fruits provide us with a variety of nutrients such as vitamins, fibers, and polysaccharides. Recently some fruit extracts have been used as supplements in some kinds of skin care astringents, citing that the extract was effective in preventing bacterial infection.

6 Hayashi et al. CHUGOKUGAKUEN J. Vol. 6 Tea and fruits generally contain some kind of antibacterial agents that play an important role in the regulation of balancing of normal bacterial flora in the intestine. The major effective component was suggested to be catechins which may inhibit growth of some harmful bacterial or accelerate growth of favorable ones in the human intestine [6]. In this study, we examined the effect of tea leave extracts and fruit juices on the proliferation of some pathogenic bacteria that tends to cause nosocominal infection. We found polyphnol was a major component in inhibiting bacterial proliferation and the low ph of the fruit juice was a major factor in inhibiting bacterial growth. Material and Methods Three different kinds of tea leaves were used according to the different manufacturing processes: Green tea [7, 81 ; dry tea leaves, Oolong tea; semi-fermented leaves, and Black tea [9] ; fermented tea leaves. All the teas were purchased at a tea house. Twenty five grams of tea leaves were soaked in 500ml of hot water (95OC) for 5 minutes. The liquid was then filtrated with a filter paper to remove the leaves. The filtrated tea leaf extracts were used for preparation of a broth medium to be used instead of water; namely, 18g of the broth powder was dissolved in 500ml of the filtrate, and then 5ml was placed in test tubes, followed by autoclaving. Agar plates containing the leaf extracts were prepared in the same manner with broth media and 20ml was placed in plastic Petri dishes (diameter 9cm). The broth powder and the agar powder were purchased from Nissui Co. Tokyo. Five kinds of fruit were subjected to the experiments: Apples, lemons, grapefruit, kiwi and citrus (Sudachi). Each fruit was smashed or squeezed and the juice was filtrated through filter paper followed by a second filtering process using a membrane filter (pore size 0.22mm) for sterilization. The bacterial strains used were: Bacillus subtilis, Pseudm aerugimsa, Staphylococcus auras, and Escherzchia coli. All strains had been kept in our laboratory as reference strains under the control of the safety law and licensed personnel. Effect of tea on the bacterial proliferation [lo]. Each bacterial strain was pre-cultured overnight in normal broth (concentration of bacteria approximately 108-9/ml) and each of 0.05ml of the culture was inoculated into the culture media then incubated at 37 C for 12 hours. The growth rate was monitored by turbidity assay at 600nm using the tea containing broth as the control. Because of the turbidity of the control tube, it was difficult to express the growth rates in numerical terms. The growth rates were described by (+) for positive proliferation and (-) for negative proliferation. From the above cultured tubes, 50pl of the each culture was taken and inoculated evenly onto nutrient agar plates (9cm diameter), then cultured at 37 C for colony forming unit (CFU) counting. The results were expressed as - : no colony per plate, + : less than 20 colonies per plate, + + : 20-50 colonies per plate and + + + : over 50 colonies per plate. Catechin content was measured with absorbance at 270nm using a commercially available pure catechin hydrate (MP Biochemicals, France) as the standard [I I]. Minimum Inhibitory Concentration (MIC), and Minimum Bactericidal Concentration (MBC), of the catechin were measured by standard serial dilution method [5]. Efiect of fruit juice on bacterial proliferation. Two milliliters of each fruit juice, which was sterilized with filtration through a 0.22mm pore size membrane filter, was placed in sterilized test tubes and 0.05ml of the over night culture of which bacterial concentration was approximately 108-g/ml, was mixed and kept for one hour and 24 hours at room temperature. The CFU of the mixture was assayed by the same method described above. The effect of ph only on the growth of bacteria was measured using the following buffers: 50mM acetic acid buffer for ph2, 50mM phthalic acid buffer for ph 3.0, 3.5, 4.0, respectively [121. All of the fruit juices used for the assay were clear and transparent liquid and no specific absorbance was detected by the scanning from 230nm to 700nm. The concentration of vitamin C in the juices was measured by the Shimada method [131. Result Eflect of tea Effects of the tea were examined in the liquid

2007 tea and fruits juice on bacterial 7 media containing each tea leave extract. The result is shown in Table 1. Oolong tea and green tea showed an inhibitory effect on the proliferation of S. aureus, while black tea was effective on S. aureus, B. subtilis and E. coli. P. amgiwsa was resistant to these teas. Gram positive organisms seemed to be more susceptible than gram negative ones, and E. coli is more susceptible than P. amgiwsa. It is clear that susceptibility or resistance to the tea extracts is different from one bacterial species to another. Because the major component of such bactericidal effect in the tea leaves was suggested to be catechin, the content of catechin of the extracts was measured as shown in Table 1. Black tea contained the highest amount of catechin among the three teas, which may correlate with the inhibitory effect on the proliferation. The effect of catechin was examined using purified commercially available catechin. As shown in Table 2, S. aum was more susceptible to catechin than the other organisms, and E. coli and B. subtilis showed susceptibility at the concentration of 2-3mg/ Table 1 media Bacterial proliferation in tea extracts containing broth Tea extract containing broth media was prepared as described in material and method. -: no bacterial growth after 12 h incubation at 37C. + : positive growth in the media. Catechin content was measure with absorbance at 270nm using pure catechin for the calibration. Table 2 Effect of catechin on bacterial growth in liquid media Each organism was cultured in catechin containing broth for 12 hours, then 50pl of the culture was inoculated on agar plate followed by 12 hour incubation at 37C. Indicator for viability. - : no colonies, + : less than 20 colonies, + + : over 20 but less than 50 colonies, + + + : over 50 colonies on an nutrient agar plate (4 9 cm). Table 3 Minimum Inhibitory concentration and minimum bactericidal concentration of catechin on the bacteria S. aureus 8. subtilis E. coli P. aeruginosa Catechin concentration (mg/ml) MIC < 1.0 > 1.O --< 2.0 > 1.O --< 2.0 > 3.0 MBC < 1.0-3.0-3.0 > 3.0 ml. P. aeruginosa was resistant to catechin even at the higher concentration of over 3mg/ml, which was the maximum solubility of catechin in the water phase. The result showed a good agreement with that of the effect of the tea extracts (Table 1). Minimum inhibitory concentration and minimum bactericidal concentration of catechin against these organisms were examined by the stepwise dilution method as shown in Table 3. MIC for S. aureus was less than lmg/ml, and that for E. coli and B. subtilis were 2-3mg/ml. P aerugiwsa was resistant to high content of catechin. After treatment of these organisms with the extracts, without addition of broth, for one hour, 0.05ml were placed onto agar plates for the CFU checking. The result was almost the same as that of Table 1 (date not shown). These results indicated that the tea extract is effective in inhibiting bacterial proliferation at lower concentrations and killing them at the higher concentrations. The effectiveness seems to be correlated with the concentration of catechin, suggesting that when we drink tea, less diluted is better than more diluted in terms of bactericidal effect. Eflect of fruit juice It was rather difficult to prepare the fruit juice containing culture media because of the method for sterilization. The effect was examined by a method of viability checking after exposing the fruit juice for a certain period of time. Each fruit juice was sterilized by filtration and the organisms were mixed with the juice, and then kept for 60min or 24h. Fifty micro liter of the mixture was injected onto nutrient agar plate to check the CFU. Vitamin C content and the ph of each fruit juice were examined as shown in Tables 4 and 5. Although the content and the ph of the fruit juice were inconsistent because of the quality of the fruits, we tried to purchase them as close to the same freshness as possible and used the concentration of Vitamin C and the ph as the standard

8 Hayashi et al. CHUGOKUGAKUEN J. Vol. 6 Table 4 Effect of fruit juice on the proliferation of bacteria PH Vitamin C (mg/ml) 2.2 12.52 2.27 4.99 3.08 7.4 - indicates negative growth on the normal agar plates of bacteria after the treatments, + indicates positive growth. ph and vitamin C content were measured before the incubation with bacteria. Aqua=distilled water. 3.22 22.55 3.62 3.13 3.94 0 Table 5 Effect of fruit juice on the proliferation of bacteria PH Vitamin C (mg/ml) 2.4 12.52 2.35 4.99 3.08 7.4 - indicates negative growth on the normal agar plates of bacteria after the treatments, + indicates positive growth. The ph and vitamin C content were measured after the incubation with bacteria. Incubation with bacteria did not change the ph. Aquazdistilled water. 3.5 22.55 3.85 3.13 4.2 0 Table 6 Effect of ph on the proliferation of bacteria - : no growth of colony, + : positive growth of colony. of quality. The effectiveness of the juice was also inconsistent from time to time probably due to the quality of the fruit used in the examination. Tables 4 and 5 show average data of experiments repeated more than 3 times. Lemon juice and citrus juice killed S. aureus and P. aemgimsa effectively within 60min, but grapefruit, kiwi, and apple juice were less effective (Table 4). Keeping the organisms in the juice for 24h did not make a significant difference when compared with 60min incubation (Table 5). The ph and vitamin C content remained the same even after 24h incubation at room temperature, which might indicate these organisms could not proliferate in the fruit juice at all. It is not surprising that different strain showed different susceptibility to the juice. If we look for any common factor that kill bacteria in fruits, the low ph seemed to be the common factor of the killing effect. Since the killing effect of the juice seemed to correlate with the low ph, effect of ph alone was examined by using a buffer prepared by acetic acid or phthalic acid. The result was shown in Table 6. It was clear that all the four organisms examined were killed at a ph below 3.5 for 60min treatment with the buffer. This result indicates that the major factor of the killing effect was the low ph and that grapefruit and kiwi juice might contain some components that protected the organisms from killing by low ph. Each fruit juice may contain some other small factors that may affect bacterial growth, but we indicated the low ph, in other words, the acidity, is an important factor for killing those organisms tested in this study.

tea and fruits juice on bacterial 9 Discussion Tea is the most common prepared beverage throughout the world but the way of serving tea and the preparation of tea leaves for drinking vary depending upon geographical, ethnic, climatic, and cultural differences. It is interesting to investigate the culture of tea and actually many valuable papers have been published on the subject, although tea does not contain major essential nutrients for maintaining health. Teas used in this study were manufactured by three different processes; these are rawdried green tea, semi-fermented oolong tea and fermented black tea. The common favorable components of those teas are caffeine, tannic acids, and catechin. According to the official publication Standard Tables of Food composition in Japan, 5th ed. [14], the green tea extract (log of leaves in 500m1, at 90 C for I min.) contains 0.02 g of caffeine, 0.07g of tannic acids, oolong tea extract (15g of leaves in 650m1, at 90C for 30sec.) contains 0.02g of caffeine and 0.03g of tannic acids, and black tea extract (5g leaves in 360ml for 1.5min to 4min) contains 0.039 of caffeine and 0.10g of tannic acidsg. We soaked the three kinds of tea leaves, 25g each, in 500ml of hot water at the same temperature 95 C. The content of catechin in the extract we measured was, oolong tea: 0.35g, black tea: 0.74g, and green tea: 0.55g. This indicated catechin is a major component in the teas compared to caffeine or tunic acids We focused on the catechin content [15I that varied among the teas as shown in Table 1 and indicated that the higher the catechin content, the more effective the inhibition of bacterial proliferation. The effectiveness on bacterial proliferation is different depending upon bacterial species. Gram positive organisms tended to be more susceptible. In fact, there are some reports mentioning that catechin was effective for the prevention of tooth cavities and periodontal diseases that are caused by gram positive cocci, and also for the inhibition of proliferation of Clostridium perfringens in the human intestine. Another report showed the catchin enhanced the effect of antibiotics that affect on the bacterial cell wall [3]. Catechin is a complex compound, but contains a phenol base as the common active residue. The active phenol residue and the polymer may play a role in killing gram positive organisms by interfer- ing with the cell wall structure and/or the synthesis, most probably by competing with peptideglycan components in the cell wall. The beneficial medical effects of catechin are that it is said to be good for lowering high blood pressure, preventing stomach ulcers, and anti-viral infection, allergy treatment, aging protection, etc [I]. Those effects should not be exaggerated much to the public, because there remain many points that need to be clarified by scientific research in terms of the safe dosage and side effects. The custom of drinking tea has been accepted by people throughout history, so that there should be good reasons for it to keep people healthy. Tea contains not only catechin but many other components like tannic acid, caffeine, and some other polyphenol, but as far as bactericidal effects are concerned, it seems that catechin is the major bactericidal component. Fruits contain many nutritional components and some bactericidal components like polyphenol, flavonoids and citric acid. In the fruit juice we used for this study, the content of polyphenol was below the level necessary for detection by absorbance assay. This study showed that the ph of fruit juice is most correlated with the bactericidal effect. The ph of lemon and citrus juice was 2--3 and killed the organism in 60min. The same effect was observed with the buffer only, which indicated the ph is an effective factor of the juice. On the other hand, the buffer of ph 3.0 and 3.5 killed all the four organisms but grapefruit and kiwi were not so effective although the ph the juice were below 3.5. This result can be explained by the experimental fact that the buffers were hypotonic while the juices were isotonic. But another reason is that fruit juices contained some factors that may protect organisms from dying. Tea and fruits have been favored by humans for centuries, without scientific evidence of their benefits to health. It is very interesting to know that such traditional foods and beverages have played a very important role in protecting humans from infectious agents. Acknowledgement. This study was carried out by the fourth-year students of the Department of Human Nutrition, Faculty of Contemporary Life Science Chugoku-gakuen University as their 'Graduation Research Program'. The period of time devoted to this study was so limited that some data were not complete and more experiments should be done. However, we wished the data to be published in

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