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Annals of Microbiology, 57 (3) 329-335 (2007) Ability of intestinal lactic bacteria to bind or/and metabolise phenol and p-cresol Adriana NOWAK*, Zdzislawa LIBUDZISZ Institute of Fermentation Technology and Microbiology, Department of Biotechnology and Food Sciences, Technical University of Lodz, Wolczanska 171/173, 90-924 Lodz, Poland Received 26 February 2007 / Accepted 27 June 2007 Abstract - Intestinal microflora can contribute to colon cancer by the production of substances playing a role in carcinogenesis. Metabolites of protein fermentation in the colon, such as ammonia, H S, indole, phenol, skatole are toxic. Lactic bacteria existing in the colon may exert an anti-carcinogenic action, but the mechanism is poorly understood. In the present study the ability of intestin- al lactobacilli to bind or metabolise phenol and p-cresol in vitro was determined. Lactobacillus strains were cultivated in MRS and in a modified MRS broth with reduced concentrations of carbon source. Phenol and p-cresol content in the media were from 2 to 10 µg/ml. In MRS medium lactobacilli could decrease the concentration of phenol and p-cresol and it was 0.2-5.8 µg/ml for phenol and 0.2-1.4 µg/ml for p-cresol. After cultivation in a modified MRS broth, the decrease was 0.5-2.0 µg/ml for phenol and 0.5-2.4 µg/ml for p- cresol. The binding capacity of bacterial cells was rather low. After incubation of non-growing bacteria the decrease of phenol con- centration was 0.1-0.5 µg/ml and p-cresol 0.1-2.8 µg/ml. But the ability of growing lactobacilli to metabolise the compounds cannot be excluded. After interaction of lactobacilli with 10 µg/ml of phenol they displayed a lower genotoxicity, as evaluated by the alkaline comet assay. The phenomenon not always depended on the decrease of phenol concentration, but on the medium, the strain of bac- teria and for phenol it ranged from 32 to 48%. Lactobacillus strains tested did not lower the genotoxicity of p-cresol. Key words: phenol; p-cresol; intestinal microflora; Lactobacillus; DNA damage. INTRODUCTION are rather toxic. Diet reach in meat is conducive to undi- gested proteins reaching the colon, especially in its distal The digestive tract of humans harbours a large and com- part. Toxic metabolites such as ammonia, H S, amines, plex collection of microbes, which forms a part of normal indole, phenol, skatole and their derivatives are formed microflora. The colon is the most dynamic microbial ecosys- during deamination, decarboxylation, fermentation or α- and β-elimination (Roberfroid et al., 1995; Smith and tem in human with high densities of living bacteria, achiev- 11 12 ing concentration up to 10 -10 cells/g of luminal con- Macfarlane, 1996; Hughes et al., 2000). Colonic bacteria tents with up to 300-1000 different species (Roberfroid et engaged in these processes belong to Escherichia coli, al., 1995; Jansen et al., 1999; Spanggaard et al., 2000; Proteus sp., Enterococcus faecalis, Staphylococcus sp., Guarner and Malagelada, 2003). Bacteroides fragilis, Fusobacterium sp. and Clostridium sp. (Smith and Macfarlane, 1996; Hughes et al., 2000; Saikali The well-balanced intestinal microflora play a crucial role in the preventing many diseases in human. It carries et al., 2004). These bacteria can contribute to colon can- out a variety of essential metabolic reactions, such as pro- cer by the activation of genotoxic and carcinogenic sub- duction of organic acids - short chain fatty acids (SCFA - stances and converting procarcinogens to electrophiles, e.g. propionic, butyric, acetic) which are beneficial for which can easily react with DNA (Burns and Rowland, 2000). Phenol, p-cresol and phenolic compounds (phenyl humans (Roberfroid et al., 1995; Priebe et al., 2002), vita- mins synthesis (e.g. vitamins K, B , riboflavin), helps in propionate and phenyl acetate) are products of metabo- lism of aromatic amino acids (tyrosine, phenylalanine, “resistant starch” and fibre metabolism (Roberfroid et al., 1995) and protects tissues from invasion and colonisation tryptophan) (Rowland et al., 1985; Goldin, 1986; Smith by pathogenic bacteria (Guarner and Malagelada, 2003). and Macfarlane, 1996; Saikali et al., 2004). Phenols are believed to act as co-carcinogens (Chung et al., 1975; Carbohydrates and proteins are fermentative substrates present in the large intestine (Macfarlane et al., 1986). Bone et al., 1976). It was shown that N-nitrosation of sec- ondary amines (dimethylamine) by nitrite is enhanced in While products of carbohydrates fermentation are usually beneficial for the host, metabolites of protein degradation the presence of phenol and chemical reaction between phenol and nitrite produce the mutagen diazoquinone (Kikugawa and Kato, 1986; Seltzer, 1986; Shephard et * Corresponding author. Phone: +48-426313475; al., 1987). Fax: +48-426365976; E-mail: firstname.lastname@example.org 330 A. Nowak and Z. Libudzisz The potential mechanisms underlying anti-carcinogenic Modified MRS broth. In order to evaluate the impact of action of lactic acid bacteria (LAB) living in the colon may microbial growth phase on binding and/or metabolism of include: inhibition of colonic enzymes activity, control of phenol and p-cresol and to “enforce” bacteria to use the growth, potentially harmful bacteria, interaction with tested compounds as a carbon source, the medium, MRS colonocytes, stimulation of immune system, production of broth, was modified. The amount of yeast extract was physiologically active metabolites (e.g. SCFA), binding or reduced from 4 g/l (0.4%) to 2 g/l (0.2%), glucose from 20 degradation of carcinogens and toxins (Burns and Rowland, g/l (2%) to 5 g/l (0.5%), meat extract, peptone, sodium 2000; Rafter, 2003; Commane et al., 2005). acetate and ammonium citrate were removed. In this study, the ability of four strains of intestinal lac- The modified medium was inoculated with 3% inoculum tic bacteria (growing and non-growing) to bind or degrade and phenol or p-cresol was added in the concentration of 10 phenol and p-cresol, human colonic toxic metabolites, was µg/ml. The cultures were incubated at 37 C for 168 h in determined. As was previously estimated, the strains sur- anaerobic conditions. Negative and positive controls were vival and growth were slightly affected by phenol and p- prepared as previously described. The concentration of phe- cresol during 48-72 h incubation, so it could be supposed nol and p-cresol in supernatants was controlled every 4 h that bacteria are able to grow and live in the presence of (from 0 to 24 h) and every 48 h (from 24 to 168 h) with the compounds in colon during the transit time (Nowak and HPLC. Simultaneously, in order to achieve the growth curves Libudzisz, 2006). of lactobacilli, the number of living cells was controlled using Koch’s plate method (for each point the standard deviation and the variability coefficient were calculated). Bacterial cul- MATERIALS AND METHODS tures were diluted in sterile saline (0.85% NaCl), plated using MRS agar and incubated at 37 C for 48 h in anaero- Bacterial strains. The following strains of Lactobacillus bic conditions. Every dilution of the culture was fourfold plat- were employed: L. casei LOCK 0919, L. casei LOCK 0908, ed. After incubation time the colonies were counted and the L. casei LOCK 0900, L. plantarum LOCK 0945 obtained results reported as log CFU/ml (colony forming units/ml) from the collection of Institute of Fermentation Technology and the curves of growth of bacteria in the presence of phe- and Microbiology (LOCK 105), Technical University of Lodz, nol and p-cresol were obtained. The average variability coef- Poland. All bacterial strains are resistant to low pH and bile ficient (V) for Koch’s plate method was 8%. salts, so they can survive in gastrointestinal tract during the transit time (Motyl, 2002 - unpublished data). Incubation of the bacteria in phosphate buffer. In To maintain the strains activity, 24 h cultures in MRS order to estimate if non-growing Lactobacillus cells can broth were frozen in the medium at –20 C with the addi- decrease the concentrations of phenol and p-cresol the cells tion of 20% of glycerol. Before using the bacteria were were separated from MRS medium by centrifugation (10000 twice activated in MRS broth (3% inoculum) and incubated rpm, 10 min, at 4 C), washed twice with 20 ml of sterile o o at 37 C for 24 h. The stock cultures were stored at 4-5 C. phosphate buffer (pH 6.2-6.3) and centrifuged again. The As an inoculum (3%) 24 h culture of bacteria in MRS broth cells were suspended in 20 ml of the buffer with 2 and 20 9 o was used, with the cell density 10 CFU/ml. µg/ml of phenol or p-cresol and incubated at 37 C for 168 Phenol and p-cresol were purchased from Sigma-Aldrich h in anaerobic conditions. The cell concentration was 10 (St. Louis, USA). To obtain stock solutions, each compound CFU/ml. A control sample was cell suspension without the was diluted in water, to a final concentration of 0.5%. compounds. Negative and positive controls were prepared as previously described. The concentration of phenol and p- Culture conditions. cresol in all samples was determined at the beginning (at MRS broth. To determine if growing lactobacilli decrease “0” time) and after 168 h of incubation with HPLC. the concentration of phenol and p-cresol in the medium they were incubated in MRS broth (BTL, Poland) containing High performance liquid chromatography. The phenol glucose (2%) with 2 and 10 µg/ml of phenol or p-cresol. and p-cresol concentrations in all samples were quantified The cultures were incubated for 168 h at 37 C in anaero- using HPLC apparatus (Thermo Separation Products, USA), bic conditions. After that time, the cells were centrifuged equipped with UV 6000 LP detector (photodiode array), col- (10000 rpm, 15 min), washed twice with sterile distilled umn Ace 5 C18 (4.6 mm x 15 cm) and precolumn Ace 5 water, suspended in water and disintegrated by ultrasonic C18. The mobile phase contained water and acetonitrile vibrations for 5 min (impulse length 6 s, amplitude 50) at (50:50, v/v) and the flow rate was 0.5 ml/min. The 0 °C (ice bath). The cell debris were separated by centrifu- absorbance was measured at 220 nm at room tempera- gation and the concentrations of phenol and p-cresol ture. released from the cell walls were measured (the bound fraction). The comet assay. The alkaline (pH < 13) single cell gel- The choice of phenol and p-cresol concentrations was electrophoresis (comet assay) allows to detect single and justified previously (Nowak and Libudzisz, 2006). Control double strand breaks in DNA molecule as well as alkali cultures for each strain were grown in the same medium labile sites. Cells with damaged DNA display an increased without phenol or p-cresol. Additionally the positive control migration of DNA towards the anode and the tail intensity was medium without bacteria but with appropriate concen- of the comet is positively correlated with the amount of tration of phenol or p-cresol (the standard). The concen- DNA damage in a cell. trations of phenol and p-cresol in supernatants of all cul- Human promyelocytic leukaemia cell line HL60 as tar- tures were determined after inoculation (at “0” time) and get cells were used. The cells were cultivated in RPMI 1640 after 24 and 168 h incubation using HPLC. medium (Sigma-Aldrich) with addition of 10% foetal bovine serum, 1% of L-glutamine, 100 IU/ml of penicillin and 100 Ann. Microbiol., 57 (3), 329-335 (2007) 331 µg/ml of streptomycin. The cells were incubated in a 5% In case of p-cresol the concentration of the compound CO atmosphere at 37 C. The final concentration of the cell decreased, but the decrease depended on the strain and in each sample was adjusted to 1 x 10 cells/ml. Cells were the amount of p-cresol in the medium. L. casei 0919 did incubated with 10 µg/ml of phenol or p-cresol at 37 C for not decrease it in any case. For 2 µg/ml the decrease was 1 h. The positive control was sample without lactobacilli. characteristic for two strains (0900 and 0908) and it was The controls for each strain were lactobacilli in MRS broth 0.15 µg/ml and 0.65 µg/ml. For 10 µg/ml the decrease was without phenol or p-cresol. After the incubation the cells from 0.31 µg/ml to 1.36 µg/ml (Table 1). were centrifuged (1400 rpm, 15 min, 4 C) and the comet Additionally, the impact of growth phase of bacteria on assay was performed in alkaline conditions according to the their ability to use phenol and p-cresol as a carbon source procedure of Singh et al. (1988) with some modifications. was estimated. For that reason lactobacilli were cultivated The cells were suspended in 0.75% Low Melting Point for 168 h in modified MRS broth with reduced sources of (LMP) agarose and layered onto slides precoated with 0.5% the element and with phenol and p-cresol concentration of agarose and lysed at 4 C for 1 h in buffer consisting of 2.5 10 µg/ml. In these conditions, the ability to decrease phe- M NaCl, 1% Triton X-100, 100 mM EDTA and 10 mM Tris. nol or p-cresol concentration depended on the growth After lysis the slides were placed in an electrophoresis unit phase of the bacteria (Fig.1 and 2). The first decrease in and DNA was allowed to unwind for 20 min in an elec- phenol and p-cresol level was observed after 8-12 h of trophoretic solution containing 300 mM/l NaOH and 1 mM/l incubation, so at the end of the logarithmic phase of EDTA. Electrophoresis was conducted at 4 C for 20 min at growth. The decrease in phenol content was at about 1.1- electric field strength 0.73 V/cm (30 mA). Then, the slides 1.55 µg/ml what depended on the strain (Fig. 1). Till 24 h, were neutralised with 0.4 mol/l Tris and stained with 1 the end of stationary phase, the amount of phenol in medi- µg/ml 4’,6-diamidino-2-phenylindole (DAPI) and covered um increased for all strains. For L. casei 0900 it achieved with cover slips. The objects were observed at 200x mag- the initial amount (10 µg/ml) and it was the same up to 72 nification in a fluorescence microscope (Nikon, Japan) h of incubation, but for the rest of the strains it increased attached to a video camera and connected to a personal to 7.3 µg/ml and 9.3 µg/ml. Along with reaching the death computer-based image analysis system Lucia-Comet v. phase slight decrease in phenol concentration was 4.51 (Laboratory Imaging, Prague, The Czech Republic). observed for two strains and in 168 h it was from 2.0 µg/ml Fifty images were selected from each sample and the per- to 2.8 µg/ml. L. casei 0908 and 0919 showed the least abil- centage of DNA in the tail of comet was measured. Two ity to decrease the concentration of phenol during incuba- parallel tests with aliquots of the same sample were per- tion in a modified MRS broth (Fig. 1B, C). formed for a total of 100 cells and the mean of percentage For p-cresol the decrease after 8-12 h of incubation was of DNA in the tail was calculated. The results were esti- at about 0.5-2.3 µg/ml (Fig. 2). The decrease after 24 h mated as percentage of DNA in the tail of the comet and cultivation was observed for two strains and it was 1.3 they were shown as a difference between the sample and µg/ml (0900) and 2 µg/ml (0919) (Fig. 2A and C). L. casei the control. Differences were calculated by one-way analy- (0908) did not decrease p-cresol concentration at all and sis of variance (ANOVA). for L. casei 0945 the decrease was not significant (Fig. 2B and D). A slight decrease was observed during the death phase of growth (in 168 h incubation) for L. casei 0919 RESULTS (Fig. 2C). Lactobacillus strains decrease phenol and p-cresol Absorption of phenol and p-cresol by Lactobacillus concentration cells To check if intestinal lactic bacteria can decrease the con- After 168 h cultivation of lactobacilli in MRS broth all test- centration of phenol and p-cresol, Lactobacillus strains ed strains seemed to adsorb phenol and p-cresol to the cell were cultivated in MRS broth for 24 h. wall, but the ability was insignificant. The bound quantity of It was found, that after 24 h cultivation of lactobacilli in phenol was very low and it was at about 7-9% (0.7-0.9 MRS broth with 2 µg/ml of phenol, the concentration of the µg/ml) (Table 2). Only L. plantarum 0945 seemed to compound decreased for three strains (0908, 0919, 0945) metabolise phenol, because there was a lack of 4.8 µg/ml from 0.19 µg/ml to 0.42 µg/ml (Table 1). In case of 10 of the compound in total amount of the bound fraction µg/ml of phenol the decrease was characteristic for all (Table 2). For p-cresol the bound quantity was from 0.07 strains and it was from 0.87 µg/ml (0919) to 5.79 µg/ml µg/ml to 1.32 µg/ml. (0945). TABLE 1 - Phenol and p-cresol concentration after 24 h cultiva- TABLE 2 - Ability of LAB to bind/adsorb phenol and p-cresol after tion of Lactobacillus strains in MRS broth 168 h cultivation in MRS broth (the initial concentra- tion 10 µg/ml) 332 A. Nowak and Z. Libudzisz (C) (A) 10 10 10 10 9 9 9 9 8 8 8 8 7 7 7 7 6 6 6 6 5 5 5 5 4 4 4 4 0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 160 Time (h) Time (h) (D) (B) 10 10 10 9 9 9 9 8 8 8 8 7 7 7 7 6 6 6 5 5 5 5 4 4 4 4 0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 160 Time (h) Time (h) FIG. 1 - Decrease in phenol concentration during 168 h cultivation of lactobacilli with 10 µg/ml of phenol in a modified MRS broth. A: Lactobacillus casei 0900, B: L. casei 0908, C: L. casei 0919, D: Lactobacillus plantarum 0945. ●: phenol concentration (µg/ml), ▼: CFU/ml (control), ■: CFU/ml (10 µg/ml of phenol); error bars denote SD. (C) (A) 10 10 10 10 9 9 9 9 8 8 8 8 7 7 7 7 6 6 6 6 5 5 5 5 4 4 4 4 0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 160 Time (h) Time (h) (D) (B) 10 10 10 10 9 9 9 9 8 8 8 8 7 7 7 7 6 6 6 6 5 5 5 5 4 4 4 4 0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 160 Time (h) Time (h) FIG. 2 - Decrease in p-cresol concentration during 168 h cultivation of lactobacilli with 10 µg/ml of p-cresol in a modified MRS broth. A: Lactobacillus casei 0900, B: L. casei 0908, C: L. casei 0919, D: Lactobacillus plantarum 0945. ●: p-cresol concentration (µg/ml), ▼: CFU/ml (control), ■: CFU/ml (10 µg/ml of p-cresol); error bars denote SD. Log CFU/ml Log CFU/ml Log CFU/ml Log CFU/ml Log CFU/ml Log CFU/ml Log CFU/ml Log CFU/ml Ann. Microbiol., 57 (3), 329-335 (2007) 333 Decrease in phenol and p-cresol concentration dur- DISCUSSION ing 168 h incubation by non-growing cells of lacto- bacilli The aim of this study was to evaluate if intestinal lacto- Ability to decrease the phenol and p-cresol content by non- bacilli (growing and non-growing) are able to bind or growing bacterial cells (10 cfu/ml) was very slight. The metabolise – phenol and p-cresol and to estimate the rate decrease of phenol concentration in phosphate buffer was of detoxification of the compounds. In this research two observed for three strains (not for 0919) and in the pres- culture media (MRS broth and its modified version), and ence of 2 µg/ml it was from 0.1 µg/ml to 0.4 µg/ml. In the suspension of biomass in phosphate buffer were applied. presence of 20 µg/ml of the compound, the decrease was Lactobacilli appeared to reveal the possibility to decrease about 0.5-0.6 µg/ml. The decrease of p-cresol was charac- phenol and p-cresol concentration, but the ability was teristic for all strains and for 2 µg/ml of p-cresol in buffer it slight. It depended on the strain, the growth phase, phys- was at about 0.2 µg/ml and for 20 µg/ml at about 2.6 iological state of the bacteria, the medium used and the µg/ml (Table 3). concentration of phenol and p-cresol tested. Referring to growth phases of bacteria in a modified MRS broth, the decrease of phenol and p-cresol concentra- tion appeared at the end of logarithmic, during and at the TABLE 3 - Phenol and p-cresol concentration after 168 h incuba- end of the stationary phase of growth (up to 24 h), and tion of Lactobacillus strains in phosphate buffer slight during the death phase. Additionally, during the sta- tionary phase of growth a slight resorption of the com- pounds to the medium was observed and it was more char- acteristic for p-cresol. Prolonged incubation of bacteria with phenol or p-cresol, caused the lowering concentration of the compounds in the culture medium, but the differences in the concentrations were not so significant and charac- teristic for a few strains. The possibility of metabolism of the compounds can not be excluded. During cultivation of lactobacilli in the modified MRS broth on result of decay of microorganisms, some enzymes metabolising phenol and p-cresol could be released from the cells. But the binding capacity of bacteria was rather poor. The Comet assay After incubation of bacteria with phenol or p-cresol in In the comet assay it was shown, that lactobacilli reduced phosphate buffer (168 h) the decrease in their concentra- the genotoxicity of phenol and p-cresol. The degree of tion was nearing to that during cultivation in MRS broth, detoxification depended on the strain, time of incubation either in case of phenol or p-cresol, depending on the and the medium used. strain. The differences could be correlated with pH of the L. casei 0908 and 0919 showed the highest reduction of environment. The pH range in phosphate buffer (6.2-6.3) genotoxicity of phenol after cultivation in MRS broth (Fig. does not change in contrary to growth of bacteria in MRS 3A and Fig. 4). For p-cresol a slight reduction of genotoxi- and in a modified MRS broth, where microorganisms make city after cultivation in MRS broth was observed for L. casei the medium more acidic. The correlation between the bind- 0908 and 0919, but the reduction was not statistically sig- ing capacity and pH was displayed by Bolognani et al. nificant (Fig. 3B). (1997). In the studies heterocyclic aromatic amines (PhIP, IQ, MelQ, MelQx, Trp-P-1) were bound by cell walls of Phenol 0900 0908 0919 0945 p-Cresol 0900 0908 0919 0945 FIG. 3 - Effect of lactobacilli on phenol (A) and p-cresol (B) genotoxicity in the comet assay after 24 h cultivation in MRS broth. The results displayed are the difference between the samples and the control. Values marked with an asterisk are significantly different from the control (phenol or p-cresol), ANOVA (P < 0.05). DNA damage DNA damage 334 A. Nowak and Z. Libudzisz FIG. 4 - Comet tail lengths of DAPI stained HL60 cells incubated at 37 °C for 1 h with 10 µg/ml of phenol (A) and cells after incuba- tion of Lactobacillus casei 0908 with 10 µg/ml of phenol (B). Lactobacillus acidophilus and Bifidobacterium longum the The ability of lactic acid bacteria to bind or metabolise most effectively at pH 5 (about 80%), while in more (pH 3) different colon carcinogens is still a challenge for scientists. and less acidic conditions (pH 7-8) the capacity was not so It has been estimated, that 30-40% of all cancers could be efficient (about 30-50%) and it depended on the mutagen prevented by lifestyle and appropriate diet. Probiotics could and the strain (Bolognani et al., 1997). be a protective element in colon cancer prevention. It is Even so slight binding and metabolising capacity of phe- necessary to take the achieved results into consideration nol and p-cresol by bacteria could be an important proper- during selection of strains to producing probiotics. ty. Physiological level of phenol and p-cresol in colonic con- tents of healthy human is low. Significant differences in the Acknowledgements amount of these compounds are observed in the proximal The comet assay was conducted thanks to Professor Janusz and distal colon. Apparent rate of production of the com- Blasiak from Department of Molecular Genetics, University pounds in proximal colon is 1.0 µmol/h/g of gut contents of Lodz in Poland. I would like to thank Dr Michal Arabski for phenol and 0.32 µmol/h/g of gut contents for p-cresol. for help during the comet assay procedure. 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