Cell surface Hydrophobicity Contributes to Lactobacillus Tolerance to Antibacterial Actions of Catechins

Abstract

Although most Gram-positive bacteria are sensitive to epigallocatechin gallate (EGCg), some species of lactic acid bacteria (LAB) are highly tolerant. The mechanism of LAB tolerance to the antibacterial action of EGCg was investigated. LAB strains with three different cell wall composition types were used: Lactobacillus plantarum NBRC15891 (meso-DAP-type), Lactobacillus fermentum NBRC15885 (Orn-type), and Lactobacillus delbrueckii NBRC3073 (Lys-type). The minimum inhibitory concentration of EGCg for L. plantarum NBRC15891, L. fermentum NBRC15885, and L. delbrueckii NBRC3073 were >1000, >1000, and 500 µg/mL at pH 6.5, respectively. The cell surface hydrophobicity (CSH), and contents of extracellular polymeric substances (EPS) and teichoic acid of these strains suggested that strains with low CSH and producing greater amounts of EPS are highly resistant to EGCg at pH 6.5. After EGCg treatment, the membrane potential decreased in strains with high susceptibility to EGCg. Our findings suggested that LAB characterized by high EPS level and low CSH are resistant to EGCg at pH 6.5.

Introduction

Catechins, which are phenolic compounds found in green tea, exhibit antibacterial activity (Cabrera et al., 2006; Cushnie and Lamb, 2011; Daglia, 2012; Nakayama et al., 2012; Nakayama et al., 2013), in addition to antioxidant and anticarcinogenic activity (Cabrera et al., 2006; Halliwell, 1996; McKay and Blumberg, 2002; Rice-Evans et al., 1996; Yang et al., 2001). The functional properties and safety of catechins for human consumption (catechins have been ingested by humans since ancient times) make them highly applicable to a variety of food processes.

The major catechins found in green tea include epigallocatechin gallate (EGCg), epigallocatechin (EGC), epicatechin gallate (ECg), and epicatechin (EC) (Singh et al., 2011). The mechanism of antibacterial action of these compounds differs depending on their structure (Akiyama et al., 2001; Cabrera et al., 2006; Mabe et al., 1999). The antibacterial action of catechins partly arises from damage to the cell membrane or oxidization of cellular components by hydrogen peroxide, generated from catechins. EGCg and EGC, which contain a galloyl moiety with three hydroxyl groups, produce hydrogen peroxide above pH 7.0 (Akagawa et al., 2003; Arakawa et al., 2004; Ikigai et al., 1993; Nakayama et al., 2002). Galloyl moieties of EGCg and ECg are involved in absorption on cell membrane and disturbance of cell membrane function; however, the detailed mechanisms underlying these activities remain unclear (Kajiya et al., 2002; Kumazawa et al., 2004; Sirk et al., 2008; Sirk et al., 2009).

In previous studies, we demonstrated that certain species of lactic acid bacteria (LAB), despite being Gram-positive, exhibit a high tolerance to catechins (Nakayama et al., 2008). LAB have played an important role in food production and in the maintenance of good health in humans since ancient times. For example, LAB strains such as Lactobacillus delbrueckii and Lactococcus lactis are used in the fermentation of dairy products (Vinderola et al., 2002), while other species, such as Lactobacillus plantarum, Leuconostoc mesenteroides, and Pediococcus pentosaceus, are used for the fermentation of Japanese pickles (Shinagawa et al., 1996). By ingesting these foods, LAB are taken into the intestine, where they produce secondary metabolites beneficial for preventing the proliferation and growth of pathogens (Doron and Gorbach, 2006; Round and Mazmanian, 2009). LAB may also degrade food quality, e.g., spoilage of wine or beer, and the production of off-flavors in juice (Stiles and Holzapfel, 1997). As such, LAB must be carefully controlled in food manufacturing processes. The combined intake of useful LAB and catechins might be beneficial to health because of their individual advantageous functionalities. In such a case, useful LAB strains must be resistant to the antibacterial actions of catechins. Thus, understanding the susceptibility of LAB to catechins and the antibacterial mechanisms of catechins, not only for microbial control in foods and but also for the development of new functional foods, is important for the food industry.

EGCg is the most abundant catechin in green tea and it exhibits strong antibacterial activity compared with other catechins (Mabe et al., 1999; Nakayama et al., 2013). To investigate the different susceptibilities of LAB to catechins, we evaluated the physical and chemical properties of cell surfaces and changes in membrane potential after EGCg treatment of various LAB strains.

Materials and Methods

Bacterial strains and culture conditions    LAB strains with three different cell wall composition types (Kleerebezem et al., 2010; Schleifer et al., 1972) were used. L. plantarum subsp. plantarum NBRC15891 (meso-DAP-type cell wall), L. fermentum NBRC15885 (Orn-type cell wall), and L. delbrueckii subsp. lactis NBRC3073 (Lys-type cell wall) were purchased from Biological Resource Center (NBRC), National Institute of Technology and Evaluation, Chiba, Japan. Lactobacillus brevis IOK-1 (Lys-type cell wall) isolated from onion by our company was also used in the experiment to image membrane potential. These strains were cultured on de Man-Rogosa-Sharpe agar (MRS agar, Oxoid Ltd., Basingstoke, UK) plates under anaerobic conditions using the Anaero-Pack system (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan) for 48 h at 30°C. Escherichia coli NBRC 3972 and Staphylococcus aureus NBRC 13276 were purchased from NBRC and cultured on tryptic soy agar (TSA; Becton, Dickinson & Co, Franklin Lakes, NJ, USA), and TSA with 2% NaCl, respectively, for 24 h at 37°C.

Measurements of Minimum Inhibitory Concentration (MIC)    LAB cells cultured on MRS agar plates were inoculated into 10 mL of MRS broth and cultured for 24 h at 30°C under anaerobic conditions using the Anaero-Pack system. Culture fluid was diluted with sterile water (OD₆₆₀ = 0.10) to prepare bacterial suspensions with approximate concentrations of 10⁸ CFU/mL. These suspensions were further diluted (×10), and the resulting bacterial suspensions (final concentration approximately 10⁷ CFU/mL) were used in the antibacterial experiments.

EGCg (Teavigo; DSM, Heerlen, Netherlands) was dissolved in pure water to a concentration of 10 mg/mL, and the pH was adjusted to 5.0 with 1N NaOH. This stock solution was serially diluted with water to concentrations of 5.0, 2.5, 1.25, and 0.625 mg/mL, which were used for antibacterial experiments after filter sterilization.

Three mL of 50% LB Broth (Luria-Bertani (Miller); Becton, Dickinson & Co) medium adjusted to pH 5.0, 6.5 or 8.0, 300 µL of individual EGCg solutions, and 30 µL of the bacterial suspensions (10⁷ CFU/mL) were mixed thoroughly. These tubes were incubated under anaerobic conditions at 30°C without shaking, and the viable cell count was measured by plating at 0, 24, and 48 h after the start of incubation. The concentration at which the viable cell count decreased to one-tenth of that at the beginning was defined as the MIC.

Evaluation of bacterial cell surface hydrophobicity (CSH)    To determine CSH, the modified method of Geertsema-Doornbusch et al. (1993) was used. After culturing 48 h on agar plates at 30°C, as described above, bacterial cells were washed twice with saline solution, and the cells were suspended with 10 mM HEPES buffer (pH 7.0) to attain OD₆₅₀ = 0.15. To 7 mL of the bacterial suspension in a glass test tube, 3 mL of hexane was added. The mixture was then agitated vigorously for 1 min at room temperature. After standing the tube at room temperature for 20 min, the OD₆₅₀ of the aqueous phase was measured.

CSH was calculated using the following equation:

CSH (%) = (1-turbidity after agitation and 20 min / turbidity prior to agitation) × 100

CSH was also determined using S. aureus NBRC13276, to which catechins readily adsorb, and E. coli NBRC3972, to which catechins slightly adsorb. After incubation in tryptic soy broth (TSB; Becton, Dickinson & Co) for 48 h at 30°C, CSH was determined as described above.

Evaluation of amount of extracellular polymeric substances (EPS)    LAB cells cultured on MRS agar plates under the conditions described above were transferred to 400 mL of MRS broth in 500-mL Duran reagent bottles. Then, the EPS of LAB were extracted and purified by the modified Orsod's method (2012). The bacterial cells were cultured for 24 h at 30°C, washed twice with saline, and then resuspended in 20 mL of 1.5 mM NaCl solution containing 10 mM EDTA. After heating at 50°C for 10 min, the cells were removed by centrifugation at 8000×g for 10 min and the supernatant was collected. A 3x volume of 99.5% ethanol (4°C) was added to the collected supernatant, and the resulting solution was stored for 48 h at 4°C. The precipitates recovered after centrifuging the solution at 8000×g for 20 min were resuspended in 50 mL of sterilized distilled water. The suspensions were then dialyzed at 4°C using an 8-kDa cut-off dialysis tube (Spectrum Laboratories, Inc., Rancho Dominguez, CA, USA) for 24 h. The post-dialysis samples were freeze-dried and EPS concentrations were measured after dissolving the freeze-dried samples in distilled water. EPS concentrations were measured using the phenol- sulfuric acid method (Dubois et al., 1956) and expressed as equivalent D-glucose mass per bacterial cell mass (dried).

Table 2. The MIC and CSH of E. coli NBRC3972 and S. aureus NBRC13276

	MIC (µg/mL) of EGCg				CSH (%)
	pH 5.0	pH 6.0	pH 7.0	pH 8.0
E. coli NBRC3972	>1000	1000	250	250	1.1
S. aureus NBRC13276	500	500	250	63	49.8

These data were cited from our previous paper (Nakayama et al., 2011).

Evaluation of amount of teichoic acid    The amount of teichoic acid was measured by the modified method of Armstrong et al. (1958). The three LAB strains were transferred to MRS agar plates and cultured under anaerobic conditions using the Anaero-Pack system for 1 day at 30°C. The cultured cells were then transferred to 400 mL of MRS broth in 500-mL Duran reagent bottles. The bacterial suspensions were cultured statically for 24 h at 30°C. Bacterial cells recovered by centrifuging at 16,500×g for 10 min were resuspended in 30 mL of 0.05 M phosphate-buffered saline (PBS, pH 7.2) and disrupted by sonication (Ultrasonic Disruptor; Tomy Seiko Co. Ltd., Tokyo, Japan) at approximately 97 watts for 2 to 3 h on ice. The precipitates resulting from centrifugation at 14,800×g for 10 min were designated as cell-wall suspensions and used in the following experiment. Cell-wall suspensions were washed twice with 5 mL of 0.05 M PBS (pH 7.2) by centrifugation at 47,400×g for 10 min and once with 4 mL of 0.05 M PBS (pH 7.6). Each 4-mL suspension was digested with 200 µL of 1 mg/mL protease E solution (Merck, Whitehouse Station, NJ, USA) for 2 h at 37°C. The protease E was then removed by centrifugation at 20,000 rpm for 10 min, and the precipitate was washed twice with 4 mL of 0.05 M PBS (pH 7.6) to yield the cell-wall components. After freeze-drying, the cell wall components (approximately 0.10 g) were suspended in 10 mL of 10% trichloroacetic acid. Then teichoic acid was extracted from the cell wall components by overnight gentle shaking at 37°C. These cell wall suspensions were centrifuged at 8000×g for 15 min, a 4x volume of cold acetone was added to the supernatant under agitation, which was allowed to stand overnight at 4°C. The resulting precipitates were collected by centrifugation at 5000×g for 10 min, washed with cold acetone and ether, and freeze-dried. The dry weights were determined and used to calculate the teichoic acid content per gram (dry weight) of cell wall.

Evaluation of hydrogen peroxide resistance    Cells of the three LAB strains were suspended in HEPES buffer (pH 8.0) containing 0.9% hydrogen peroxide to the final concentrations of approximately 10⁵ to 10⁶ CFU/mL. Aliquots (100 µL) were withdrawn from these suspensions after incubation for 0, 5, 10, 15, 20, 30, 40, and 60 min at 30°C and plated onto MRS agar plates. Viable cell counts were quantified based on the number of colonies formed after culturing for 48 h at 30°C. Survival rates (%) were calculated by dividing the counts of each time by that at 0h after addition of the bacterial cells.

Imaging membrane potential using voltage-sensitive dyes    L. plantarum NBRC15891, L. delbrueckii NBRC3073 and L. brevis IOK-1 were used in this experiment. After exposure to 250 µg/mL of EGCg for 1 h, color changes of these bacterial strains stained with the cationic fluorophore JC-1 were observed at pH 5.5, 6.5, and 8.0. Accumulation of JC-1 is dependent on membrane potential. If the cell membrane potential is high, the fluorophore will accumulate in the bacterial cells in high concentrations. At high concentrations, the fluorophore forms “J-aggregates” and emits red light and not green light. If membrane potential is low, accumulation of the fluorophore is low and the emitted light is green (Aguiar et al., 2012; Reers, 1991; Smiley et al., 1991). Cells of these three LAB strains cultured on MRS agar plates were inoculated to 50% LB broth (at pH 5.0, 6.5, or 8.0) containing 250 µg/mL EGCg at a final concentration of 10⁸ CFU/mL. After incubation for 1 h at 25°C, cells were recovered by centrifugation at 10,000 rpm for 10 min at 25°C and resuspended in 500 µL of membrane permeabilization buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10 mM D-glucose). Each cell suspension was mixed with 2 µL of 5,5′,6,6′- tetrachloro - 1,1′,3,3′- tetraethylbenzimidazoyl carbocyanine iodide (JC-1, Thermo Fisher Scientific Inc., Waltham, MA, USA) solution, mixed, and incubated in the dark for 5 min. Aliquots (10 µL) of the bacterial suspensions were pipetted onto poly-L-lysine- coated glass slides and allowed to stand in the dark for 5 min. PBS was used to remove any excess JC-1 liquid. After placing a coverslip on the sample, the membrane potential was visualized using an epifluorescence microscope (Eclipse E600; Nikon, Tokyo, Japan), fitted with a 40× objective lens, at an excitation wavelength of 450 to 490 nm and a long-pass 520-nm optical filter.

Calculation of partition-coefficient (LogP) value for different catechin compounds    LogP values were calculated using CS Chem Draw Ultra ver. 6.0 (PerkinElmer, Inc., Waltham, MA, USA) (Maffezzoni and Girelli, 1998).

Results

MIC of catechins against LAB and CSH, EPS masses, and teichoic acid content of LAB    The MICs of catechins and CSH, EPS masses, and teichoic acid content of LAB are shown in Table 1.

Table 1. The CSH, and EPS and teichoic acid contents of LAB with different susceptibilities to EGCg

	MIC (µg/mL) of EGCg			CSH (%)	EPS (mg)*1	Teichoic acid (g)*2
	pH 5.0	pH 6.5	pH 8.0
L. plantarum NBRC15891	>1000*3	>1000	>1000	15.5	15.9	0.58
L. fermentum NBRC15885	>1000	>1000	250	8.2	28.8	0.31
L. delbrueckii NBRC3073	500	500	N.D.*4	51.7	1.49	0.32

*1  Amount per g - dry weight of cells.

*2  Amount per g - dry weight of cell wall.

*3  Bacteria grew in the presence of EGCg at 1000 µg/mL, the highest concentration tested.

*4  Not Detected (No growth in 50% LB medium without EGCg)

We have shown that EGCg susceptibility was proportional to the amount of EGCg absorbed on the cell surface (Nakayama et al., 2012; Nakayama et al., 2013). To clarify the relationship between LAB cell surface properties and EGCg susceptibility, CSH, EPS masses, and teichoic acid content were determined. The CSHs were determined to be as follows: L. plantarum NBRC15891, 15.6%; L. fermentum NBRC15885, 8.2%; and L. delbrueckii NBRC3073, 51.7% (Table 1). Given their low CSHs, it is considered that the surfaces of L. plantarum NBRC15891 and L. fermentum NBRC15885 have similar hydrophilicity to that of E. coli NBRC3972. Conversely, the CSH value of L. delbrueckii NBRC3073 was greater than 50, indicating that its surface hydrophobicity is similar to that of S. aureus NBRC13276.

Hydrogen peroxide resistance of LAB    L. delbrueckii NBRC3073 exhibited superior hydrogen peroxide resistance compared to the two other LAB strains, with 40% cell survival after 60 min of exposure (Fig. 1.). The viable cell count for the other two LAB strains after 60 min of exposure was below the detection limit, indicating that the hydrogen peroxide resistance of these two LAB strains was low.

Fig. 1.

Susceptibility of LAB to hydrogen peroxide Viable cell counts of L. plantarum NBRC15891 (●), L. fermentum NBRC15885 (■), and L. delbrueckii NBRC3073 (▲) were determined after treatment with hydrogen peroxide at pH 8.0.

Change in membrane potential after EGCg treatment    After treatment with 250 µg/mL of EGCg, L. plantarum NBRC15891 was found to maintain its membrane potential (emission of red light) at all pHs, indicating that the cell membrane was not damaged by EGCg exposure. In contrast, the membrane potential of L. delbrueckii NBRC3073 and L. brevis IOK-1 declined at pH 5.0, 6.5, and 8.0 (emission of green light) indicated that the cell wall was damaged. Images of L. brevis IOK-1, which exhibited the most obvious color change among the three strains, are shown in Fig. 2. The MIC of EGCg against L. brevis IOK-1 in 50% LB medium was 500 µg/mL, but 250 µg/mL of EGCg damaged the membrane.

Fig. 2.

Membrane potential of L. brevis IOK-1 estimated by JC-1 staining

L. brevis IOK-1 cells were stained with JC-1 after the treatment with (B) and without (A) 250 µg/mL of EGCg at pH 8.0. Bar: 10 µm

LogP values of the different catechin compounds    CSH and LogP are values of the hydrophobicity of bacterial cell surfaces and single substance, respectively. Gallated catechins showed higher antibacterial activity than non-gallated catechins, and EGCg was observed to absorb onto the cell surface in our previous research (Nakayama et al., 2008; Nakayama et al., 2011). CSHs of catechin-susceptible LAB were high, so it was predicted that gallated catechins, which have a high LogP value, easily interacted via hydrophobic interaction. The LogP values of EGCg, EGC, ECg, and EC are shown in Table 3. It was demonstrated that the values were higher for the gallated catechins. These results are in agreement with those reported in a prior study (Kajiya et al., 2002).

Table 3. Structures and LogP values of catechins

Substance	EGCg	EGC	ECg	EC
Structure
LogP	1.43	0.13	2.1	0.04

Discussion

Nishiyama and Kozaki (1991) reported that polyphenol tolerance is related to the cell wall structure of LAB. They elucidated that LAB with a meso-DAP-type cell wall has higher polyphenol resistance than those with a Lys-type cell wall; however, they did not describe polyphenol resistance of those with an Orn-type cell wall. On the other hand, in the previous paper, we found that EGCg was more heavily adsorbed onto the cell-surface structure of S. aureus, which is sensitive to catechins, than onto E. coli, which is tolerant to catechins. Here, we investigated whether the catechin tolerance of LAB is correlated with the amount of catechins adsorbed to their cell-surface.

EGCg and ECg, which exhibited higher anti-bacterial activity than EGC and EC, also had higher LogP values. On the other hand, S. aureus had high CSH and large amounts of EGCg adsorbed onto its surface, whereas E. coli, the surface of which was covered with LPS, had low CSH and EGCg did not adsorb to its cell surface. These findings indicated that catechins absorbed on the cell surface due to hydrophobic interactions, and the cell surface hydrophobicity was important for the absorption of catechins. A major component of the EPS of LAB is hydrophilic polysaccharides (Kleerebezem et al., 2010). These facts suggested that CSH of bacterial strains with high EPS content is low. Our findings indicated that LAB with low CSH had high catechin tolerance at pH5.0, a condition under which no hydrogen peroxide was generated from EGCg. Moreover, at pH6.5, a condition in which hydrogen peroxide is slightly generated from EGCg, L. delbrueckii NBRC3073 with high CSH had low catechin tolerance, and other species with low CSH exhibited high catechin tolerance. The results of this report suggest that CSH is dependent on the amount of EPS rather than teichoic acid.

Membrane damage of EGCg-treated cells was estimated from the membrane potential. The membrane potential of L. plantarum NBRC15891, which had high EGCg tolerance, did not decrease after treatment with 250 µg/mL EGCg, suggesting no damage or rapid recovery from damage in the membrane. On the other hand, the membrane potential of L. delbrueckii, which was sensitive to EGCg, decreased soon after treatment with EGCg, suggesting membrane damage. MICs of EGCg against L. plantarum NBRC15891 and L. delbrueckii NBRC3073 were above 250 µg/mL (>1000 and 500 µg/mL, respectively). These findings suggest that adequate EGCg reached the cell membrane in a short period of time, damaged the cell membrane and exhibited antibacterial activity.

MICs of EGCg against L. plantarum NBRC15891, L. fermentum NBRC15885, and L. delbrueckii NBRC3073 differed from each other. It was considered that this difference was attributable not to species or cell wall composition type but to CSH or the amount of EPS. To clarify the basis of differences in MIC of EGCg between L. plantarum NBRC15891 and L. fermentum NBRC15885, despite the equally low CSHs, the scavenging ability of reactive oxygen species and cell-surface constituents, especially glucolipids and secreted viscous substances of these strains are under investigation.

References

•  Aguiar,  J.S.,  Araújo,  R.O.,  Rodrigues,  M.D.,  Sena,  K.X.F.R.,  Batista,  A.M.,  Guerra,  M.M.P.,  Oliveira,  S.L.,  Tavares,  J.F.,  Silva,  M.S.,  Nascimento,  S.C., and  da Silva,  T.G. (2012). Antimicrobial, antiproliferative and proapoptotic activities of extract, fractions and isolated compounds from the stem of Erythroxylum caatingae Plowman. Int. J. Mol. Sci., 13, 4124-4140.
•  Akiyama,  H.,  Fujii,  K.,  Yamasaki,  O.,  Oono,  T., and  Iwatsuki,  K. (2001). Antibacterial action of several tannins against Staphylococcus aureus. J. Antimicrob. Chemoth., 48, 487-491.
•  Akagawa,  M.,  Shigemitsu,  T., and  Suyama,  K. (2003). Production of hydrogen peroxide by polyphenols and polyphenol-rich beverages under quasi-physiological conditions. Biosci. Biotech. Biochem., 67, 2632-2640.
•  Arakawa,  H.,  Maeda,  M.,  Okubo,  S., and  Shimamura,  T. (2004). Role of hydrogen peroxide in bactericidal action of catechin. Biol. Pharm. Bull., 27, 277-281.
•  Armstrong,  J.J.,  Baddiley,  J.,  Buchanan,  J.G.,  Carss,  B., and  Greenberg,  G.R. (1958). Isolation and structure of ribitol phosphate derivatives (teichoic acids) from bacterial cell walls. J. Chem. Soc., 4344-4354.
•  Cabrera,  C.,  Artacho,  R., and  Giménez,  R. (2006). Beneficial effects of green tea - A review. J. Am. Coll. Nutr., 25, 79-99.
•  Cushnie,  T.P. and  Lamb,  A.J. (2011). Recent advances in understanding the antibacterial properties of flavonoids. Int. J. Antimicrob. Ag., 38, 99-107.
•  Daglia,  M. (2012). Polyphenols as antimicrobial agents. Curr. Opin. Biotech., 23, 174-181.
•  Doron,  S. and  Gorbach,  S.L. (2006). Probiotics: their role in the treatment and prevention of disease. Expert Rev. Anti. Infect. Ther., 4, 261-275.
•  Dubois,  M.,  Gilles,  K.A.,  Hamilton,  J.K.,  Rebers,  P.A., and  Smith,  F. (1956). Colorimetric method for determination of sugars and related substances. Anal. Chem., 28, 350-356.
•  Geertsema-Doornbusch,  G.I.,  Van der Mei,  H.C., and  Busscher,  H.J. (1993). Microbial cell surface hydrophobicity. The involvement of electrostatic interactions in microbial adhesion to hydrocarbons (MATH). J. Microbiol. Meth., 18, 61-68.
•  Halliwell,  B. (1996). Antioxidants in human health and disease. Annu. Rev. Nutr., 16, 33-50.
•  Ikigai,  H.,  Nakae,  T.,  Hara,  Y., and  Shimamura,  T. (1993). Bactericidal catechins damage the lipid bilayer. Biochim. Biophys. Acta, 1147, 132-136.
•  Kajiya,  K.,  Kumazawa,  S., and  Nakayama,  T. (2002). Effects of external factors on the interaction of tea catechins with lipid bilayers. Biosci. Biotech. Biochem., 66, 2330-2335.
•  Kleerebezem,  M.,  Hols,  P.,  Bernard,  E.,  Rolain,  T.,  Zhou,  M.,  Siezen,  R.J., and  Bron,  P.A. (2010). The extracellular biology of the lactobacilli. FEMS Microbiol. Rev., 34, 199-230.
•  Kumazawa,  S.,  Kajiya,  K.,  Naito,  A.,  Saitô,  H.,  Tuzi,  S.,  Tanio,  M.,  Suzuki,  M.,  Nanjo,  F.,  Suzuki,  E., and  Nakayama,  T. (2004). Direct evidence of interaction of a green tea polyphenol, epigallocatechin gallate, with lipid bilayers by solid-state nuclear magnetic resonance. Biosci. Biotech. Biochem., 68, 1743-1747.
•  Mabe,  K.,  Yamada,  M.,  Oguni,  I., and  Takahashi,  T. (1999). In vitro and in vivo activities of tea catechins against Helicobacter pylori. Antimicrob. Agents Chemother., 43, 1788-1791.
•  Maffezzoni,  C. and  Girelli,  R. (1998). MOSES: Modular modeling of physical systems in an object-oriented database. Math. Comp. Model. Dyn., 4, 121-147.
•  McKay,  D.L. and  Blumberg,  J.B. (2002). The role of tea in human health: An update. J. Am. Coll. Nutr., 21, 1-13.
•  Nakayama,  M.,  Shigemune,  N.,  Tokuda,  H.,  Furuta,  K.,  Matsushita,  T.,  Yoshizawa,  C., and  Miyamoto,  T. (2008). Antibacterial activity of green tea extracts and the effect of pH on that activity. Bokin Bobai, 36, 439-448 (in Japanese).
•  Nakayama,  M.,  Shigemune,  N.,  Tsugukuni,  T.,  Hitomi,  J.,  Matsushita,  T.,  Mekada,  Y.,  Kurahachi,  M., and  Miyamoto,  T. (2012) Mechanism of the combined anti-bacterial effect of green tea extract and NaCl against Staphylococcus aureus and Escherichia coli O157:H7. Food control, 25, 225-232.
•  Nakayama,  M.,  Shigemune,  N.,  Tsugukuni,  T.,  Tokuda,  H., and  Miyamoto,  T. (2011). Difference of EGCg adhesion on cell surface between Staphylococcus aureus and Escherichia coli visualized by electron microscopy after novel indirect staining with cerium chloride. J. Microbiol. Methods, 86, 97-103.
•  Nakayama,  M.,  Shimatani,  K.,  Ozawa,  T.,  Shigemune,  N.,  Tsugukuni,  T.,  Tomiyama,  D.,  Kurahachi,  M.,  Nonaka,  A., and  Miyamoto,  T. (2013). A study of the antibacterial mechanism of catechins: Isolation and identification of Escherichia coli cell surface proteins that interact with epigallocatechin gallate. Food control, 33, 433-439.
•  Nakayama,  T.,  Ichiba,  M.,  Kuwabara,  M.,  Kajiya,  K., and  Kumazawa,  S. (2002). Mechanisms and structural specificity of hydrogen peroxide formation during oxidation of catechins. Food Sci. Technol. Res., 8, 261-267.
•  Nishiyama,  R. and  Kozaki,  M. (1991) Studies on phenol related compounds inhibiting the growth of microorganisms. Teikyo Junior College Kiyou, 8, 49-71 (in Japanese).
•  Orsod,  M.,  Joseph,  M., and  Huyop,  F. (2012). Characterization of exopolysaccharides produced by Bacillus cereus and Brachybacterium sp. isolated from Asian sea bass (Lates calcarifer). Mal. J. Microbiol., 8, 170-174.
•  Reers,  M. (1991). J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry, 30, 4480-4486.
•  Rice-Evans,  C.A.,  Miller,  N.J., and  Paganga,  G. (1996). Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Bio. Med., 20, 933-956.
•  Round,  J.L. and  Mazmanian,  S.K. (2009). The gut microbiota shapes intestinal immune responses during health and disease. Nat. Rev. Immunol., 9, 313-323.
•  Shinagawa,  H.,  Nishiyama,  R., and  Okada,  S. (1996). Function of lactic acid bacteria during fermentation of Japanese pickles “Shibazuke”. Nippon Shokuhin Kagaku Kogaku Kaishi, 43, 582-585 (in Japanese).
•  Singh,  B.N.,  Shankar,  S., and  Srivastava,  R.K. (2011) Green tea catechin, epigallocatechin-3-gallate (EGCG): Mechanisms, perspectives and clinical applications. Biochem. Pharmacol., 82, 1807-1821.
•  Sirk,  T.W.,  Brown,  E.F.,  Sum,  A.K., and  Friedman,  M. (2008). Molecular dynamics study on the biophysical interactions of seven green tea catechins with lipid bilayers of cell membranes. J. Agric. Food Chem., 56, 7750-7758.
•  Sirk,  T.W.,  Brown,  E.F.,  Friedman,  M., and  Sum,  A.K. (2009). Molecular binding of catechins to biomembranes: relationship to biological activity. J. Agric. Food Chem., 57, 6720-6728.
•  Schleifer,  K.H. and  Kandler,  O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol. Rev., 36, 407-477.
•  Smiley,  S.T.,  Reers,  M.,  Mottola-Hartshorn,  C.,  Lin,  M.,  Chen,  A.,  Smith,  T.W.,  Steele Jr.,  G.D., and  Chen,  L.B. (1991). Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc. Natl. Acad. Sci. USA, 88, 3671-3675.
•  Stiles,  M.E. and  Holzapfel,  W.H. (1997). Lactic acid bacteria of foods and their current taxonomy. Int. J. Food Microbiol., 36, 1-29.
•  Vinderola,  C.G.,  Mocchiutti,  P., and  Reinheimer,  J.A. (2002). Interactions among lactic acid starter and probiotic bacteria used for fermented dairy products. J. Dairy Sci., 85, 721-729.
•  Yang,  C.S.,  Landau,  J.M.,  Huang,  M.-T., and  Newmark,  H.L. (2001). Inhibition of carcinogenesis by dietary polyphenolic compounds. Annu. Rev. Nutr., 21, 381-406.