Antibacterial action on pathogenic bacterial spore by green tea catechins

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1 Journal of the Science of Food and Agriculture J Sci Food Agric : () DOI:./jsfa.9 Antibacterial action on pathogenic bacterial spore by green tea catechins Yukiko Hara-Kudo, Akiko Yamasaki, Miho Sasaki, Tsutomu Okubo, Yuji Minai, Minoru Haga, Kazuo Kondo and Yoshiko Sugita-Konishi Department of Microbiology, National Institute of Health Sciences, Setagaya-ku, Tokyo -, Japan Laboratory of Biochemistry, Department of Agriculture, Tamagawa University, -- Tamagawa-gakuen, Machida, Tokyo 9-, Japan Institute of Environmental Science for Human Life, Ochanomizu University, Bunkyo-ku, Tokyo -, Japan NF Division Research and Development, Taiyo Kagaku o, Ltd, Yokkaichi -, Mie, Japan Abstract: Antibacterial effects of catechins, the major green tea polyphenols, were studied using lostridium and Bacillus spores. with crude catechins decreased the number of botulinum and butyricum spores but not B cereus spores. Furthermore, the effects of six catechin derivatives on spores were investigated. ( )-Epicatechin gallate (Eg), ( )-epigallocatechin (EG), ( )- epigallocatechin gallate (E) and (+)-gallocatechin gallate () were more effective in decreasing botulinum and butyricum spore numbers than (+)-catechin () and ( )-epicatechin (E). The vegetative growth of botulinum and B cereus was inhibited by crude extracts of the catechins. Specifically, purified and E inhibited the vegetative growth of botulinum and B cereus. The inhibitory effect of Eg on B cereus was similar to that of. However, toxin-production by B cereus was not inhibited by catechin. Damage to the membrane of butyricum spores by catechin derivatives was shown using fluorescent microscopy. This study shows that low concentrations of catechins, although requiring a long exposure time, inhibited the growth of bacterial spores. However, the effects of the purified derivatives of the catechins were not the same and and E were found to be the most potent. Spores that are generally resistant to many disinfectants were sensitive to catechins. Society of hemical Industry Keywords: green tea; catechins; spore; lostridium; Bacillus INTRODUTION Green tea extracts contain catechins along with caffeine, vitamins, amino acids and theanine. The catechins are known for their effects on viruses, intestinal microflora, oral diseases, cancer, and oxidation. 9 The antibacterial activities of catechins have been well studied. However, these reports describe non-spore-forming bacteria. Bacteria such as lostridium and Bacillus form spores that are resistant to many physical and chemical treatments. To kill spores of these species requires high temperatures ( ) and pressure for min or strong chemical disinfectants such as formalin. However, the milder conditions found in foods can affect spores. lostridium botulinum spores decreased in numbers after a long incubation with the tannin fraction of black tea and tea components including catechins and theaflavin were shown to decrease the spores of lostridium and Bacillus in agar media. However, there are few detailed data on the bactericidal effects of tea catechins and their pure derivatives incubated with lostridium and Bacillus spores and this paper reports studies on this topic. atechin inhibits bacterial toxin production and activity. atechin inhibits the activity of the α- toxin of Staphylococcus aureus and the hemolytic activity of TDH. Entero-hemorrhagic Escherichia coli O:H failed to produce Vero cytotoxin with low concentrations of catechin. However, other studies on toxin production in the presence of catechins with other pathogens are limited. In this study, it was found that low concentrations of catechins inhibit bacterial spore viability and some purified derivatives of the catechins strongly inhibit viability. MATERIALS AND METHODS Strains lostridium botulinum (Type B, Sb-), lostridium butyricum (BP 9) and enterotoxin-producing Bacillus cereus (JM ) were used. Because lostridium butyricum is not highly pathogenic, the orrespondence to: Yukiko Hara-Kudo, Department of Microbiology, National Institute of Health Sciences, Setagaya-ku, Tokyo -, Japan ykudo@nihs.go.jp ontract/grant sponsor: Japan Human Sciences Foundation; contract/grant number: KH (Received July ; revised version received November ; accepted March ) Published online June Society of hemical Industry. J Sci Food Agric //$.

2 Effects of catechins on bacterial spores organism has been used in many experiments including elemental studies. butyricum, lostridium botulinum, known to have high toxigenicity and difficult to control, and Bacillus cereus, a pathogen widely distributed in the environment, were used as targets in this study. atechins hemical analysis of the crude catechin fraction from green tea used in this study showed that it contained many polyphenols,.% being comprised of (+)-catechin () at.%, ( )- epicatechin (E) at.9%, (+)-gallocatechin (G) at.%, ( )-epigallocatechin (EG) at.%, (+)-gallocatechin gallate () at.9%, ( )- epigallocatechin gallate (E) at.9% and ( )- epicatechin gallate (Eg) at.%. The crude catechins also contained sugars at.% and amino acids at.%. After the crude catechin had been suspended in distilled water at a concentration of mg ml,the solution was sterilized using a disk filter (pore size. µm) (Millipore, Bedford, MA, USA)., E, G, EG,, E and Eg were purified from the crude catechins using the method reported previously. After purification, the catechins were suspended in distilled water at mg ml and (a). botulinum µg/ml µg/ml µg/ml µg/ml µg/ml (b). butyricum µg/ml µg/ml µg/ml µg/ml µg/ml (c) B. cereus µg/ml µg/ml µg/ml µg/ml µg/ml Figure. The antibacterial activity of crude catechins on the spores of botulinum (a), butyricum (b) and B cereus (c). The bar in the graph is the standard error. p <.; p <.. J Sci Food Agric : ()

3 Y Hara-Kudo et al the solution was sterilized with a disk filter (pore size. µm) (Millipore). Preparation of spores botulinum and butyricum were cultured on GAM agar medium (Nissui, Tokyo, Japan) at for h using anaerobic conditions using the gas pack system (Becton Dickinson, Sparks, MD, USA). B cereus was cultured on nutrient agar medium (Nissui) at for h using aerobic conditions. olonies of each strain were suspended in sterilized distilled water. After sonication twice for min, the suspension was centrifuged at g for min. The pellet was resuspended in sterilized distilled water. This procedure was repeated, and the product was used as the spore suspension in this study. The number of spores were counted by culture on GAM agar medium, anaerobically, or nutrient agar medium, aerobically, at forh. The presence of spores in each spore suspension was confirmed using Wirtz s stain method with light microscopy observation. Sporicidal effects of catechins for butyricum, botulinum and B cereus Spores of botulinum, butyricum and B cereus were suspended in phosphate-buffered saline (PBS) (Nal,.g; Kl,.g; NaHPO,.g; and KH PO,. g; in l of distilled water) containing crude catechins at,,, and µgml and incubated at. The number of spores after day and,,, and weeks of incubation was counted using culture on GAM agar medium or nutrient agar medium. For each of the purified catechin derivatives, µgml was used and the experiments were carried out in the same way as using crude catechins. All experiments were performed in triplicate. Vegetative growth of botulinum and B cereus in crude catechins and purified catechin derivatives botulinum and B cereus were cultured in sensitivity test broth (casamino acids,. g; heart extract,. g; starch,. g; glucose,. g; L-tryptophan,. g; L- cystine,. g; and biotin, µg; in litre of distilled water) (Nissui) at for h anaerobically and aerobically, respectively. The culture was diluted to cfu ml with buffered peptone water (peptone;. g; Nal,. g; Na PO,.g; and KH PO,. g; in litre of distilled water.) (Oxoid, Basingstoke, Hampshire, UK). This dilution (. ml) was inoculated into sensitivity test broth (9.9 ml) containing crude catechins at,,, and µgml. The inoculated broth containing botulinum and B cereus was incubated at for h anaerobically and aerobically, respectively. After incubation at for,, and h, the number of botulinum and B cereus spores were counted on GAM agar medium and nutrient agar medium, respectively. After h, the production of the toxin of B cereus in the culture was determined using the method described below. For the purified catechin derivatives µgml wasusedandtheexperimentswereperformedinthe way same as using the crude catechins. All of these experiments were performed in triplicate. Measurement of toxin produced by B cereus The enterotoxin of B cereus in culture was measured using a reversed passive latex agglutination kit (RET- RPLA, Denka Seiken, Tokyo, Japan). After h of incubation, the B cereus broth culture containing the catechins was centrifuged at g for min. The supernatants were sterilized by filtration (pore size,. µm) and then ml of the filtrates and their two-fold serial dilutions were mixed with the same volume of the reaction medium containing latex beads coated with the anti-enterotoxin of B cereus antibody. Titers were expressed as the reciprocal of the last dilution at which the agglutination of the latex beads was observed after incubation for h at room temperature. These experiments were performed in triplicate. Observation of spores with fluorescence microscopy Spores of butyricum with each catechin derivative after weeks of incubation at were stained with a Live/Dead BacLight bacterial viability kit (Molecular Probes, Eugene, OR, USA). Statistics Significant differences between the bacterial numbers incubated with catechins and without catechins were analyzed using Student s t-test. RESULTS AND DISUSSION Antibacterial effects of crude catechins on the spores of botulinum, butyricum and B cereus The initial spore counts of botulinum, butyricum and B cereus were.,. and. cfu ml, respectively. The number of botulinum spores in PBS using crude catechins at and µgml decreased to undetectable levels ( cfu ml ) after weeks of incubation (Fig a). The number of butyricum spores in PBS with crude catechins at µgml was undetectable ( cfu ml ) after weeks and for and µgml the spores were undetectable after weeks (Fig b). omparison of the sensitivity of butyricum with that of botulinum showed that butyricum was more sensitive to the catechins than botulinum (Fig a and b). The number of B cereus spores decreased only slightly over the weeks of incubation in the J Sci Food Agric : ()

4 Effects of catechins on bacterial spores (a) botulinum ontrol E Eg EG E (b) butyricum ontrol E Eg EG E Figure. The antibacterial effects of catechin derivatives on the spores of botulinum (a) and butyricum (b). The concentration of each catechin derivative was µgml. The bar in the graph is the standard error. p <.; p <.. J Sci Food Agric : ()

5 Y Hara-Kudo et al (a) botulinum µg/ml µg/ml µg/ml µg/ml µg/ml (b) B cereus µg/ml µg/ml µg/ml µg/ml µg/ml Figure. Inhibitory growth effect of crude catechins on vegetative botulinum and Bcereus. The bar in the graph is the standard error. p <.; p <.. crude catechins and did not reach undetectable levels (Fig c). However, the decrease in spores was significant at concentrations at > µgml. The effect of crude catechins on botulinum, butyricum and B cereus depended on the concentration of the former. In this study, B cereus spores were almost resistant to catechins whereas the lostridia were highly sensitive. This may be related to the bacteria being either anaerobic or aerobic, suggesting that the oxidative state may be involved in the sensitivity to catechins. Antibacterial effects of purified catechin derivatives on the spores of botulinum and butyricum Six purified catechin derivatives at µgml were tested. With botulinum, the antibacterial effect of each catechin derivative took longer to show (Fig a) than when using the crude catechins (Fig a), the spores incubated with Eg, EG, E and t taking weeks to reach undetectable numbers ( cfu ml ). Because of the differences in sensitivity of the purified components compared with the effect of crude catechins ( µgml ), the results shown in Fig b were possibly due to Eg, EG, E and because and E show less sporicidal activity. With butyricum, the antibacterial effect of each catechin took longer to show than that of crude catechins and was the same as for botulinum. Because all of the purified catechins were sporicidal for butyricum, this suggests why the crude tea-derived catechins were more sporicidal with butyricum than botulinum. E and are reported to have high antibacterial activity against non-spore-forming vegetative bacteria.,,9 Also, Eg and E have been found to be more effective in inhibiting lostridium difficile and lostridium perfringens growth than, E, G and EG using a paper disc method. In this study, it was found that catechins affect bacterial spores with a longer period of incubation being necessary as compared with the effects on vegetative bacteria. onsidering the component parts of crude catechins, the antibacterial activity (Fig a) was probably due to E and as they are the main components of crude catechins and the most potent. With butyricum, Eg, E and rapidly decreased the population within weeks (Fig b). On the other hand, in the presence of and E bacterial numbers were maintained for weeks. This suggests that the antibacterial effect of crude catechins (Fig b) was due to Eg, E and because of their higher concentrations in crude catechins. J Sci Food Agric : ()

6 Effects of catechins on bacterial spores (a) botulinum E Eg EG E ontrol (b) B cereus E Eg EG E ontrol Figure. Inhibitory growth effect of catechin derivatives on vegetative botulinum and Bcereus. The concentration of each catechin derivative was µgml. The bar in the graph is the standard error. p <.; p <.. J Sci Food Agric : () 9

7 Y Hara-Kudo et al The gallic acid esters of catechins (Eg, E and ) show higher activities although the reason has not been identified. Likewise in this study, the antibacterial activities and growth-inhibitory activities of E and EG against bacterial spores were generally lower than those of their corresponding gallic acid esters. Inhibition of the growth of vegetative botulinum and B cereus by crude catechins The numbers of botulinum spores in the presence of > µgml of catechins quickly decreased (Fig a); but botulinum survived at µgml of catechins and grew similarly to when no catechins were present. The growth of vegetative B cereus was inhibited with > µgml of catechins (Fig b) and the numbers decreased below the detection limit ( cfu ml ) at h. Vegetative B cereus was more sensitive to the catechins than botulinum whereas B cereus spores were more resistant. The effect of crude catechins against vegetative botulinum and B cereus was dependent on the concentration of crude catechins, similar to botulinum sporicidal effects. alcium is absorbed during spore germination, the calcium-binding protein on the surface of the spore being important for this uptake. Because catechins are known to interact with proteins, they might affect the calcium-binding protein and thus possibly inhibit the germination of the spores. Inhibition of the growth of vegetative botulinum and B cereus by purified catechin derivatives The numbers of botulinum spores did not increase in the presence of E and ; however, the bacterium grew with, E and Eg (Fig a). Vegetative B cereus was more sensitive than botulinum (Fig b). B cereus did not grow with Eg, and E. The populations incubated with Eg and decreased to undetectable numbers at h. The most effective catechins were the gallic acid esters. Both E and are generally more effective for bacterial sporicide and vegetative killing, as shown here. It is known that spore-forming bacteria are present in the soil and the environment. atechins found in the foliage of many plant species may be useful for killing the spores of pathogens in the soil and the environment. Green tea contains larger quantities of E than black tea and coffee. Therefore, in the anaerobic intestine of humans, it is possible that green tea and its extracts may be useful for the disinfection of spore-forming bacteria in the gut. Effect of catechins on toxin production by B cereus At µgml of crude catechins, B cereus grew well after h of incubation (Fig b) and the enterotoxin production was not inhibited (Fig a). Vero cytotoxin production of E. coli O in culture supernatant was (a) rude catechins Percent inhibition oncentration of catechin (µg/ml) (b) atechin derivatives Percent inhibition E Eg EG E Figure. Effect of catechins on toxin production of B cereus. inhibited with µgml of and E although the bacterium grew well. In this study, growth was inhibited by Eg, E and. However, enterotoxin production following growth of B cereus was not inhibited by catechin derivatives even at µgml (Fig b). We have previously studied the effects of crude catechins on E. coli O. Growth of E. coli O and toxin production were inhibited at a concentration of µgml. In this study, it was demonstrated that vegetative B cereus was more sensitive than E. coli O. On the other hand, the production of difficile cytotoxin in culture supernatant was completely inhibited with a crude catechin concentration of µgml, although difficile in culture supernatant grew well in a preliminary experiment (data not shown), showing that whereas the toxin of Bcereus was not affected, the toxin of a lostridium was. Effect of catechins on the spore membrane Although the number of spores exposed to catechins decreased as shown in Figs and, we did not know if the spores died or the germination was inhibited. Therefore, the changes in the characteristics of the spore bacterial membrane were investigated using fluorescence microscopy after double staining with SYTO 9 and propidium iodide (Live/Dead BacLight bacterial viability kit). SYTO 9 and propidium iodide stain nucleic acids, with green and red fluorescence, respectively. As a result of the difference in permeability between the stains, intact cells and damaged cells are visualized with green fluorescence (alive) and with red fluorescence (dead), respectively. Damage to the spore membrane of butyricum was observed using fluorescence microscopy (Plate ). Many spores of butyricum at a concentration of µgml of each derivative showed a red color, which indicated they had a lethally damaged membrane (Plate a), whereas those staining green were not damaged in the absence of catechins (Plate b). We previously determined that catechins damage the membrane of E. coli O. Ikigai et al studied the damage of the lipid membrane by catechins ontrol J Sci Food Agric : ()

8 Y Hara-Kudo et al Effects of catechins on bacterial spores (a) (b) Plate. Observation of butyricum spores using vital dye fluorescence microscopy after weeks of incubation with catechin derivatives. (a) E and (b) PBS control. Society of hemical Industry J Sci Food Agric : ()

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