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Home , Escherichia coli, Helicobacter pylori

Original Article J. Glin. Biochem.Nutr., 34, 85-87, 2003

The Antibacterial Effects of Coffee on coli and pylori

Yuki Okabe,1,* Yutaka Yamamoto,1,* Kayo Yasuda,1 Kazunori Hochito,2 Katsumi Kawano,3 and Naoaki Ishiti1,**

1Department ofMolecular Life Science , Tokai UniversitySchool Medicine,of Bobseidai, Isebara 259-1193, Japan 2 Department of Bacteriology,SBS, Inc., 1-3-14 Tanashiota,Sagamihara 229-1125, Japan 3Department of TechnologyDevelopment and Assessment, IVD, SRL, Inc., Hachioji Laboratory, 153 Komiya-cho, Hachioji 192-0031, Japan

Received 1 August, 2003; Accepted 31 May, 2004

Summary Here we report that coffee showed a strong antibacterial action on (E. coli) and Helicobacter pylon (H. pylon). This action against H. pylon suggests that coffee may have action as a useful natural inhibitor of and gastric ulcers.

Key Words:coffee, antibacterial effects, Escherichia coli,

tilled water) for E. coli. Coffee was extracted directly Introduction in the liquid medium (LB medium without agar

powder for E. coli) at 90℃ after autoclaving. The Coffee attracts attention as not only a table luxury supernatant of the extract was used after centrifuga- but also a with variety of side effects, both ben- tion. eficial and harmful. For example, Tam et al. [1] The test for H. pylori was determined using the found that coffee has antibacterial effects. This sug- agar dilution-culture method. The culture medium gests that coffee may be useful as a natural inhibitor was Helicobacterpylori Selective Supplement (Dent) against . Here we also show that coffee offers (peptone 23.0 g, starch 1.0 g, sodium chloride, 5.0 g, to antibacterial action against two important intesti- and agar 10.0 g per 1,000 ml distilled water; after nal , Escherichia coli (E. coli) and autoclaving, vancomycin, 10.0 mg, lac- Helicob acter pylori (H pylori). tate, 5.0 mg, cefsulodin, 5.0 mg and amphotericin B, 5.0 mg are added in order). Coffee from the stock Materials and Methods solution was diluted with a saline-salt solution, and added to the culture medium (final concentrations of Coffeeand mediumpr epar ations 1 and 10%). Solid medium for E. coli: 12 g of commercially available 2 roasted coffee powders and one soluble Antib acterial effects coffee was added to 140 ml of water and the E. coli K12 was cultivated overnight at 37℃. extract was centrifuged as a stock solution. A portion 1) 106-109 of E. cofi was cultured on each LB agar of these stock solutions was added to autoclaved LB plate containing various concentration of coffee. Sur- agar medium (Bactopeptone 10 g, yeast extract 5 g, vival was determined by counting the number of col- NaCl 10 g and agar powder 15 g per 1,000 ml dis- onies next day. 2) 108 of E. cofi was inoculated in 100 ml of LB liquid medium containing various con- *These authors contributed equally to this work . centration of coffee and incubated at a rotatory shak- **To whom correspondence should be addressed . er. The medium (2 ml) was centrifuged at 1,500×g Tel: +81-463-93-1121 ext.2650:Fax:+81-463-94-8884 for 15 min and resuspended in 2 ml of saline-salt E-mail:[email protected] solution every hour. Growth was determined with a

85 86 Y. Okabe et al.

Fig. 2. Toxic effects of coffee to E. coli. on liquid medium. Fig. 1. Toxic effects of coffee to E. coli. on agar medium. The roasted coffee powder 1 in Fig. 1 was used. A hundred percents coffee is defined as the com- 100% coffee is defined as the commercially sug- mercially suggested concentration for daily drink- gested concentration for daily drinking. Each ing. Each point showed an average of two separate point shows an average of two separate experi- experiments. ments.

spectrophotometer (OD550). Two microliters containing about 108 of H. pylori was inoculated on the . They are cultured for 72 h under slightly aerobic conditions and exam- ined for growth ability.

Results and Discussion

E. coli: Survival by roasted coffee in the solid medium decreased as coffee concentration was Fig. 3. Sensitivity of H. pylori to a roasted coffee. Growth increased (Fig. 1). The soluble coffee needed about inhibition effect; -, none (normal growth); +, two times the concentration to obtain the effect as mild effect (slight growth); + +, strong effect (no the same amount of killing as by roasted coffee (Fig. growth). Each closed circles shows independent experimental result. 1). In the liquid medium, the growth in 12.5% of concentration of coffee was slightly delayed com- pared with control and returned to the control con- antibacterial effects may be due to oxidative damage centrations after 5 h (Fig. 2). At a concentration of from coffee, because there are some reports of reac- 25%, it was also delayed but also returned to the tive spices (ROS) generator in coffee [2, 3]. control values after 7 h. In the other hand, a concen- On the other hand, coffee is also reported to act as tration of 50% completely inhibited growth (Fig. 2). an antioxidant [4, 5]. Interestingly, it is also possible Thus, at concentrations less 50%, coffee inhibits the that coffee possesses concommitant oxidant and growth of E. coli but does not kill it (Fig. 2). antioxidant activities [6] . It is increasingly apparent H Pylori: At 1% and 10% concentrations of cof- that ROS contribute significantly to cellular and fee, growth was mildly and strongly inhibited, organismal damage, resulting in aging and death [7]. respectively (Fig. 3). ROS are produced not only during aerobic respira- These results show that coffee offers to antibacte- tion, but also by some oxidants such as radiation and rial action two important intestinal microorganisms, chemicals. To combat the effects of , E. coli and H pylori. One possibility is that these cells have antioxidant systems that include small

J. Clin.Biocbem. Nutr. Antibacterial Effects of Coffee on Bacteria 87 antioxidant molecules mostly derived from dietary [6] Stadler, R. H., Turesky, R.J., Muller, O., Markovic, J., fruits or vegetables such as coffee as as and Leong-Morgenthaler, P.M.: The inhibitory effects that scavenge ROS [8-10]. To learn whether the of coffee on -mediated oxidation and mutage- nicity. Mutat. Res., 308, 177-190, 1994. cytotoxicity to bacteria results from ROS generation [7] Harman, D.: Aging: a theory based on free radical and by coffee, we used (C. elegans), radiation chemistry. J. Geront., 2, 298-300, 1956. which has received much attention as a model to dis- [8] Yu, B. P.: Cellular defenses against damage from reac- sect the [11]. The mev-1 mutant is an opti- tive oxygen species. Physiol. Rev., 74, 139-162, 1994. mal experimental mutant to study oxidative stress, [9] Ames, B.N., Gold, L. S., and Willett, W. C.: The because it shows hypersensitivity to oxygen [12-17]. causes and prevention of . Proc. Natl. Acad. Sci. As there is no difference between wild type and the USA, 92, 5258-5265, 1995. mev-1 mutant in medium including coffee under [10] Beckman, K.B. and Ames, B.N.: The free radical the- ory of aging matures. Physiol.Rev., 78, 547-581, 1998. hyperoxia (data not shown), it is unlikely that oxida- [11] Brenner, S.: The genetics of Caenorhabditis elegans. tive stress mediate the deleterious effects including Genetics,77, 71-94, 1974. antibacterial effect of coffee. [12] Hosokawa, H., Ishii, N., Ishida, H., Ichimori, K., In conclusion, coffee inhibits the growth of bacte- Nakazawa, H., and Suzuki, K.: Rapid accumulation of ria, E. cofi and H. pylori. The antibacterial effect of fluorescent material with aging in an oxygen-sensitive coffee, especially against H pylori, may contribute to mutant mev-1 of Caenorhabditis elegans. Mech. Dev., 74, 161-170, 1994. preventing gastritis, digestive gastric ulcer or gastric cancer of which H pylori could play an important [13] Adachi, H., Fujiwara, Y., and Ishii, N.: Effects of oxy- role in the episode [18, 19], even if coffee does not gen on carbonyl and aging in Caenorhabditis elegans mutants with long (age-1) and short (mev-1) offer to oxidant action. life spans. J. Gerontol. Ser. A Biol. Sci. Med. Sci., 53, B240-B244, 1998. Acknowledgments [14] Ishii, N., Takahashi, K., Tomita, S., Keino, T., Honda, S., Yoshino, K., and Suzuki, K.: A methyl viologen- This research was supported by the Research Aid in the sensitive mutant of the nematode Caenorhabditis ele- All Japan CoffeeAssociation. We are indebted to Mr. Naoji gans. Mutat. Res., 237, 165-171, 1990. Suzuki of "The CAFE Suzuki" for his kind instruction of [15] Honda, S., Ishii, N., Suzuki, K., and Matsuo, M.: coffee preparation and Dr. Philip S. Hartman at Texas Oxygen-dependent perturbation of life span and aging Christian Universityfor reading this manuscript. rate in the nematode. J. Gerontol. Biol. Sci., 48, B57- B61, 1993. References [16] Ishii, N., Fujii, M., Hartman, P.S.,Tsuda, M., Yasuda, K., Senoo-Matsuda,N., Yanase, S., Ayusawa,D., and [1] Tam, J. P., Lu, Y.A., Yang, J. L., and Chiu, K. W.: An Suzuki, K.: A in succinate dehydrogenase unusual structural motif of peptides con- cytochrome b causes oxidative stress and ageing in taining end-to-end macrocycle and cystine-knot disul- nematodes.Nature, 394, 694-697, 1998. fides. Proc. Natl. Acad. Sci. USA, 96, 8913-8918, 1999. [17] Melov, S., Ravenscroft, J., Malik, S., Gill, M. S., [2] Long, L. H., Lan, A. N., Hsuan, F. T., and Halliwell, Walker, D. W., Clayton, P.E., Wallace, D.C., Malfroy, B.: Generation of peroxide by "antioxidant" B., Doctrow, S.R., and Lithgow, G. J.: Extension of beverages and the effect of addition. Is cocoa the life-span with / mimetics. best beverage? Free Radic. Res., 31, 67-71, 1999. Science,289, 1567-1569, 2000. [3] Halliwell, B., Clement, M. V., and Long, L. H.: [18] Parsonnet, J., Friedman, G.D., Vandersteen, D.P., Hydrogen peroxide in the human body. FEBS Lett., Chang, Y., Vogelman, J. H., Orentreich, N., and Sibley, 486, 10-13, 2000. R. K.: Helicobacterpylori and the risk of gas- [4] George, K. C., Hebbar, S.A., Kale, S.P., and Kesavan, tric carcinoma. N Engl. J. Med., 325, 1127-1131, P.C.: Caffeine protects mice against whole-body lethal 1991. dose of γ-irradiation. J Radiol. Prot. 19, 171-176, [19] Wotherspoon, A. C., Ortiz-Hidalgo, C., Falzon, M. R., 1999. and Isaacson, P.G.: Helicobacterpylori-associated gastri- [5] Devasagayam, T. P., Kamat, J. P., Mohan, H., and Kesa- tis and primary B-cell . Lancet, 338, van, P.C.: Caffeine as an antioxidant: inhibition of 1175-1176, 1991. lipid peroxidation induced by . Biochim.Biophys. Acta, 1282, 63-70, 1996.

Vol.34, No.3, 2003