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Mechanism of inhibition of tannic acid and related compounds on the growth of intestinal bacteria
Food and Chemical Toxicology 36 (1998) 1053±1060
Mechanism of Inhibition of Tannic Acid and Related Compounds on the Growth of Intestinal Bacteria K.-T. CHUNG1*, Z. LU1 and M. W. CHOU2 Department of Microbiology and Molecular Cell Sciences, The University of Memphis, Memphis, TN 38152 and 2Division of Biochemical Toxicology, National Center for Toxicology Research, Jeerson, AR 72079, USA
1
(Accepted 1 June 1998) AbstractÐTannic acid, propyl gallate and methyl gallate, but not gallic acid, were found to be inhibitory to the growth of intestinal bacteria Bacteroides fragilis ATCC 25285, Clostridium clostridiiforme ATCC 25537, C. perfringens ATCC 13124, C. paraputri®cum ATCC 25780, Escherichia coli ATCC 25922, Enterobacter cloacae ATCC 13047, Salmonella typhimurium TA98 and S. typhimurium YG1041 at 100±1000 mg/ml in culture broth. Neither Bi®dobacterium infantis ATCC 15697 nor Lactobacillus acidophilus ATCC 4356 was inhibited by any of the above compounds up to 500 mg/ml. Tannic acid has a much greater relative binding eciency to iron than propyl gallate, methyl gallate or gallic acid. The inhibitory eect of tannic acid to the growth of intestinal bacteria may be due to the strong iron binding capacity of tannic acid; whereas the eect of propyl gallate and methyl gallate probably occurs by a dierent mechanism. The growth of E. coli was restored by the addition of iron to the medium after the precipitate caused by tannic acid was removed. Neither B. infantis nor L. acidophilus require iron for growth. This probably contributes to their resistance to tannic acid. Because tannins are abundant in the human diet, tannins may aect the growth of some intestinal bacteria and thus may have an impact on human health. # 1998 Elsevier Science Ltd. All rights reserved Keywords: growth; intestinal bacteria; inhibition; iron deprivation; tannic acid. Abbreviations: GI = gastrointestinal; LIC = lowest inhibitory concentration; TSB = tryptic soy broth.
INTRODUCTION
Vegetable tannins are water-soluble phenolic compounds having a molecular weight between 500 and 3000 Daltons. Vegetable tannins can be classi®ed into hydrolysable and condensed tannins. Hydrolysable tannins contain either gallotannins or ellagitannins. Gallotannins yield glucose and gallic acid on hydrolysis by acids, bases or certain enzymes. Ellagitannins contain one or more hydroxydiphenoyl residues which are linked to glucose as a diester in addition to gallic acid. Upon hydrolysis, the hydrodiphenoyl residue undergoes lactonization to produce ellagic acid. Condensed tannins are the polymerized products of ¯avan-3-ols and ¯avan-3,4-diols, or a mixture of the two. The polymers, referred to as ``¯avolans'' are popularly called condensed tannins ( more recently called proanthocyanidins) (Chung et al., 1998). Tannins are present in a variety of fruits and vegetables *Author for correspondence.
(Deshpande et al., 1984; Salunkhe et al., 1989). Wines and teas also contain some tannins (Ho and Singleton, 1977; Sanderson et al., 1975). It is estimated that people in the United States ingest 1 g of tannic acid, the most simple form of hydrolysable tannin, each day (IARC, 1976; Sanyal et al., 1997). Tannins may serve as a natural antimicrobial agent to protect fruits and vegetables against microbial infection (Chung and Murdock, 1991; Scalbert, 1991). When food tannins are ingested, the initial contact is the gastrointestinal (GI) tract, which is one of the most active metabolic sites in the human body (Chung, 1996). Microorganisms in the GI tract may play a role in many disease processes including cancer (Chung, 1996). How tannins interact with the intestinal microbiota and aect metabolism in the GI tract may be of important to health. Tannins have been reported to have various health eects such as antinutritional, carcinogenic, mutagenic, anticarcinogenic and antimutagenic activities (Butler and Rogler, 1992; Chadwick et al.
0278-6915/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(98)00086-6
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1992; Chung et al. 1996; Horikawa et al. 1994; Marzo et al. 1990; Oterdoom, 1985). How these eects are related to intestinal microbiota is not clearly known. The inhibitory eects of tannins from dierent sources on various microorganisms have been demonstrated (Chang et al., 1994; Chung et al., 1993, 1995; Scalbert, 1991). Bae et al. (1993) showed that condensed tannins from birdfoot trefoil (Lotus corniculatus L.) were inhibitory to the endoglucanase activity of cellulose digesting Fibrobacter succinogenes S85 in the rumen. The growth of other rumen bacteria such as Butyrivibrio ®brisolvens A38, Streptococcus bovis 45S1 were also reported to be inhibited by condensed tannins from sain leaf (Onobrychis viciifolia Scop.) (Jones et al., 1994). However, very limited information is available related to the inhibitory eect of tannins on the growth of human intestinal bacteria. The aim of this paper is to examine whether tannic acid and its hydrolytic product, gallic acid, have any inhibitory eects on the growth of some intestinal bacteria, and to identify potential mechanisms of inhibition. Methyl gallate and propyl gallate are also included since they are esters forms of gallic acid. These results will further our understanding as to how food tannins may impact human health.
MATERIALS AND METHODS
Organisms Bacteroides fragilis ATCC 25285, Bi®dobacterium infantis ATCC 15697, Clostridium clostridiiforme ATCC 25537, C. perfringens ATCC 13124, C. paraputri®cum ATCC 25780, Escherichia coli ATCC 25922, Enterobacter cloacae ATCC 13047 and Lactobacillus acidophilus ATCC 4356 were obtained from the American Type Culture Collection (Rockville, MD, USA). Salmonella typhimurium TA98 was provided by Dr B. N. Ames of the Department of Biochemistry, University of California, Berkeley, California, USA. S. typhimurium YG1041 was provided by Dr Masahiko Watanabe, Division of Genetics and Mutagenesis, National Institute of Hygienic Sciences, Tokyo, Japan. These bacteria were chosen because they represent dierent groups of intestinal bacteria (Chadwick et al., 1992; Holdeman and Moore 1972). Culturing methods Lactobacilli MRS broth, modi®ed BHI, reinforced clostridial medium and tryptic soy broth (TSB) were obtained from Difco (Detroit, MI, USA). Nutrient broth no. 2 was obtained from Oxoid (Hampshire, UK). The anaerobic techniques of Holdeman and Moore (1972) were followed. The modi®ed chopped-meat medium was prepared anaerobically according to American Type Culture
Collection Media Handbook (ATCC, 1989). Bacteroides fragilis was grown in modi®ed chopped meat medium; Bi®dobacterium infantis was on reinforced clostridial medium; Clostridium clostridiiforme, C. perfringens and C. paraputri®cum were on modi®ed BHI. The above bacteria were grown anaerobically (Holdeman and Moore, 1972). Enterobacter cloacae and Escherichia coli were on TSB; Lactobacillus acidophilus was on Lactobacillus MRS broth; S. typhimurium TA98 and YG1041 were on nutrient broth. The latter ®ve bacteria were grown aerobically. Chemicals Agar, ammonium chloride, calcium chloride, PIPES, gallic acid, methyl gallate, propyl gallate and tannic acid were purchased from Sigma Chemical Co. (St Louis, MO, USA). Chrome azurol S, hexadecyltrimethyl-ammonium bromide, piperzine, 5-sulfosalicyclic acid, 8-hydroxyquinoline and thiamine hydrochloride were purchased from Aldrich Chemical Company, Inc. (Milwaukee, WI, USA). Casamino acid was from Difco Laboratory (Detroit, MI, USA). Gallic acid, methyl gallate, propyl gallate, tannic acid and ferrous sulfate were freshly prepared by dissolving in deionized water at proper concentrations followed by ®lter-sterilization. When anaerobic cultures were used, these solutions were prepared anaerobically under nitrogen (Holdeman and Moore, 1972). Sensitivity testing Gallic acid, methyl gallate or tannic acid were added to the growth media of each bacteria aerobically or anaerobically, depending on the type of culture. For cultures containing gallic acid, methyl gallate or propyl gallate, bacterial growth was measured at 600 mm using a Bausch & Lomb Spectronic 20 spectrophotometer (Milton Roy Company, Rochester, NY, USA). The growth of bacterial cultures containing tannic acid was measured by plate count method (FDA, 1992). Results reported were the average of three independent experiments. Estimation of the lowest inhibitory concentration (LIC) The method for estimating the lowest eective inhibitory concentration for bacterial growth was adopted following the determination of the detection limit of the analytical instrument (Ingle and Crouch, 1988). Brie¯y, for each bacterial species the growth response at stationary phase (i.e. absorbance when using a spectrophotometer) was plotted against the concentrations of the compound. Based on the method of Ingle and Crouch (1988), the response from the test data must be at least twice the standard deviation (SD) lower than that from the corresponding control set in order to show
Tannic acid inhibition of intestinal bacteria
signi®cant dierence between them. Therefore the concentration corresponding to the absorbance on the regression curve, which is just twice the SD smaller than the control was de®ned as the LIC for the bacterial growth. The LIC for each bacterial strain for each compound was separately determined on each ®gure. Determination of iron concentration The iron concentration in the media was determined using an atomic spectrophotometer, SpectrAA-30/40 Zeeman (Varian Techtrom Pty., Mulgrava, Victoria, Australia) equipped with Zeeman background correction (SpectraAA-30/40 Zeeman Operation Manual, 1986). The measured iron concentration of various media were between 1.18 and 1.62 mM. Iron withdrawing experiments (A) Plate assay. The iron withdrawing experiment was carried out on agar plates containing low-iron medium, ferrous sulfate (1 mM) and agar (1.5%). The low-iron medium was prepared with MM9 (Maniatis et al., 1982; Schwyn and Nielands, 1987) to which was added casamino acids (0.3%), thiamine±HCl (2 ppm), Tris buer (0.87%) and succinic acid (0.2%). The succinic acid and casamino acids were neutralized with sodium hydroxide and deferrated by extraction with 3% (w/w) 8hydroxyquinoline in chloroform before being added to the medium. Aliquots (1 ml) of an overnight E. coli ATCC 25922 culture in TSB (378C) was added to 100 ml of the above agar medium maintained at 458C. Tannic acid, propyl gallate or methyl gallate was added to the medium to give a ®nal concentration of 1000, 100 and 100 mg/ml, respectively. Approximately 20 ml was poured into a petri dish and allow to solidify at 48C for 2 hr. Wells (8 mm) were cut into the agar and ®lled with 50 ml 2 mM iron solution (FeSO47H2O, freshly prepared and ®lter-sterilized) or sterile distilled water (control). Bacterial growth around the well was observed after incubation of the plates at 378C for 48 hr. (B) Broth assay. Tannic acid was added to TSB to give the ®nal concentration of 1 mg/ml. The precipitates were removed by centrifugation. Sterilized FeSO47H2O solution was aseptically added to the supernatants to give the ®nal concentration of 0.63, 1.25 or 2.5 mM and use as the growth broth. After inoculation, the bacterial growth was measured spectrophotometrically as described above. Determination of iron bindings by tannins and siderophore (A) Production of siderophore. A 10-ml volume of low-iron medium (Schwyn and Nielands, 1987) was inoculated with E. coli and incubated at 378C for 12 hr. 0.1 ml of this culture was inoculated into 10 ml of the same medium. As a control, a ¯ask
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with 2 mM added iron (FeSO47H2O) was also inoculated with E. coli. The cultural density during incubation was monitored at 600 mm using a Spectronic 20 to verify iron starvation, which was manifested by a slower growth rate and lower absorbance values. After 24 hr, when the stationary phase was reached (plate count 2.5 107/ml), the cells were removed by centrifugation at 3000 g. The supernatants were used as siderophores (Maniatis et al., 1982). (B) Iron binding assay. The iron binding assay was conducted according to a Chrome Azurol S (CAS) method (Schwyn and Neilands, 1987) in which the colour change of the blue iron±dye complex at 630 mm indicates the amount of the iron bound with the compound. The standard curve of iron±dye solution containing dierent concentrations of iron was used for determination of those iron-compound complexes. Molar ratio of the initial concentration of the compounds in the solution to the iron±compound complexes formed indicates amount of compound required to form a unit of iron±compound complex. The relative binding eciency was calculated according to the following equation: Relative Binding Efficiency Molar ratio tannic acid to iron Molar ratio test compound to iron The initial concentration of siderophore produced by E. coli was calculated according to Harris et al. (1979). RESULTS
The eect of gallic acid, methyl gallate, propyl gallate and tannic acid on the growth of Clostridium perfringens ATCC 13124 is illustrated in Fig. 1. Similar inhibitory patterns were found when these compounds were present in the growth medium with Bacteroides fragilis, Enterobacter cloacae, C. clostridiiforme, C. paraputri®cum, E. coli and S. typhimurium (data not shown). At the concentrations tested, Lactobacillus acidophilus was not inhibited by methyl gallate or propyl gallate. A much higher concentration of tannic acid was required to inhibit the growth of this bacterium (Fig. 2). Similarly, Bi®dobacterium infantis was not inhibited by tannic acid at 1 mg/ml, nor by methyl gallate and propyl gallate (data not shown). The estimated LICs of these compounds towards these bacteria were listed in Table 1. Gallic acid showed no inhibitory eect to any of the bacteria tested. When tannic acid (1 mg/ml) was added to agar plates with low iron content, no visible growth of E. coli was observed. However, when a small amount (50 ml) of iron solution (FeSO47H2O, 2 mM) was added to the well (8 mm in diameter) of the plate, a heavy smear of E. coli growth
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Fig. 1(a,b).
occurred around the well. Similar experiments were done with propyl gallate and methyl gallate. No bacterial growth occurred around the well (data not shown). The eect of iron removal on bacterial growth was further tested using TSB culture. After the precipitates (tannic acid and iron complex) were removed and a known amount of iron (0.63 to 2.5
mM) added to the medium, the growth of E. coli resumed (Fig. 3). The same experiment was performed with propyl gallate and methyl gallate, but no resumption of bacterial growth was detected. Similar experiments were conducted using C. perfringens, S. typhimurium TA98 and Bacteroides fragilis, with similar results.
Tannic acid inhibition of intestinal bacteria
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Fig. 1. Eect of tannic acid and related compounds on the growth of Clostridium perfringens. Each data point is an average of three measurements. (a) tannic acid, (b) propyl gallate, (c) methyl gallate and (d) gallic acid. Tannic acid, propyl gallate and methyl gallate show signi®cant inhibition at concentrations greater than 10 mg/ml (P < 0.05). Gallic acid shows no signi®cant inhibition at concentrations up to 1000 mg/ml.
The relative binding eciencies of siderophores produced by E. coli, tannic acid, methyl gallate, propyl gallate and gallic acid towards iron were determined and shown in Table 2. It was found that it took only 0.9 molecules of tannic acid to chelate one molecule of iron. Harris et al. (1979) reported that it took 3.0 molecules of siderophore
prepared from the crude supernatant of E. coli culture on low iron medium to chelate one molecule of iron. If we standardized the relative binding eciency of tannic acid as one, the relative binding eciency of siderophore is 0.3. Using similar calculations, the relative binding eciency of gallic acid, methyl gallate and propyl gallate were 1.1 10ÿ4,
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Fig. 2. Eect of tannic acid on the growth of Lactobacillus acidophilus. Each of the data points is an average of three measurements. Tannic acid shows signi®cant inhibition only at concentrations greater than 500 mg/ml.
1.2 10ÿ4 and 1.4 10ÿ4, respectively. Thus, the binding of tannic acid is equivalent to that of the natural siderophores; whereas propyl gallate, methyl gallate and gallic acid have negligible binding capacities to iron. DISCUSSION
All bacteria studied were originally isolated from human intestine. It is of interest to note that tannic acid, propyl gallate and methyl gallate, but not gallic acid, are inhibitory to the growth of all the tested intestinal bacteria except the lactic acid bacteria L. acidophilus and Bi®dobacterium infantis. Tannic acid has a stronger inhibitory eect than propyl gallate or methyl gallate. The inhibitory eect occurs to both aerobes and anaerobes, Gram
(+) and Gram (ÿ) (Table 2). This pattern of inhibition is consistent with the previous ®ndings of Chung et al. (1993, 1995). Many of the bacteria tested have been reported to carry important metabolic activation enzymes such as nitroreductase and azoreductase (Chadwick et al., 1992; Ra®i et al., 1991). Food tannins may also aect these enzymes of intestinal bacteria. This needs to be further explored. Many microorganisms produce siderophoresÐ low molecular weight chelating agents that bind and solubilize iron (Crowley et al., 1991; Lewin, 1984; Neilands, 1995). Antimicrobial activity of tannins through iron deprivation has been suggested (Haslam, 1996; Mila et al., 1996; Scalbert, 1991). Tannic acid may work like a siderophore to chelate iron from the medium and make iron unavailable
Table 1. Estimated lowest inhibitory concentration (LIC) of methyl gallate, propyl gallate and tannic acid with various test microorganisms LIC (mM) Name of organism Bacteroides fragilis ATCC 25285 Bi®dobacterium infantis ATCC 15697 Clostridium clostridiiforme ATCC 25537 Clostridium paraputri®cum ATCC 25780 Clostridium perfringens ATCC 13124 Enterobacter cloacae ATCC 13047 Escherichia coli ATCC 25922 Lactobacillus acidophilus ATCC 4356 Salmonella typhimurium TA98 Salmonella typhimurium YG1041 *No inhibition. No inhibition was observed with gallic acid.
Methyl gallate
Propyl gallate
Tannic acid
883.8 Ð* 272.7 261.9 7.1 66.6 92.9 Ð 23.7 23.5
575.7 Ð 351.2 265.4 24.4 33.7 17.3 Ð 17.4 6.3
91.8 Ð 144.9 56.3 3.1 9.5 27.0 181.6 14.7 8.5
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Fig. 3. Growth resumption of E. coli by adding dierent amount of iron in the tannic acid present medium. For the control, E. coli was grown in low iron medium resulting from tannic acid binding. Stimulation of bacterial growth was observed at added iron concentration greater than 0.63 mM (P < 0.05). Each of the data points is an average of three measurements.
to the microorganisms. The growth of the tannic acid inhibited E. coli culture was restored by the supplementation of additional iron ( Fig. 3). Propyl gallate and methyl gallate are esters of gallic acid, which is the basic structural unit of hydrolysable tannins. But both propyl and methyl gallates are unable to bind iron eciently. Their inhibitory eect on the growth of these bacteria is, therefore, not through the deprivation of iron from the medium. Other mechanisms must be considered. Microorganisms growing under aerobic conditions need iron for a variety of functions including reduction of ribonucleotide precursor of DNA, for formation of heme, and for other essential purposes. Neilands (1995) reported that a level of at least one micromolar iron is needed for optimal growth. Iron does not stimulate the growth of some lactic acids because they have no heme enzymes, and the crucial iron-containing ribonucleotide reductase has been replaced with an enzyme using Table 2. Relative binding eciency of gallic acid, methyl gallate, propyl gallate, tannic acid and siderophore to iron Compound
Relative binding eciency*
Tannic acid Siderophore Propyl gallate Methyl gallate Gallic acid
1.0 0.3 1.4 10ÿ4 1.2 10ÿ4 1.1 10ÿ4
*Relative binding eciency was calculated based on the measured molar ratio of tannic acid to iron divided by the measured molar ratio of the test compound to iron.
adenosylcobalamine (Bruyneel et al., 1989; Pandey et al., 1994; Reichard, 1993). This explains the resistance of L. acidophilus and Bifodobacterium infantis to tannic acid. The present ®ndings indicate that tannic acid may aect the GI microbial population by inhibiting the growth of some bacterial ¯ora but not lactic acid bacteria. Lactic acid bacteria are generally considered to be bene®cial to human health (Bruyneel et al., 1989). Therefore, tannic acid may serve as a regulator of microbial populations in the intestinal tract, which is bene®cial to human health. Whether other types of tannins would have the similar eect is of interest. The overall eects of dietary tannins to human health requires further extensive investigation.
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Mechanism of Inhibition of Tannic Acid and Related Compounds on the Growth of Intestinal Bacteria K.-T. CHUNG1*, Z. LU1 and M. W. CHOU2 Department of Microbiology and Molecular Cell Sciences, The University of Memphis, Memphis, TN 38152 and 2Division of Biochemical Toxicology, National Center for Toxicology Research, Jeerson, AR 72079, USA
1
(Accepted 1 June 1998) AbstractÐTannic acid, propyl gallate and methyl gallate, but not gallic acid, were found to be inhibitory to the growth of intestinal bacteria Bacteroides fragilis ATCC 25285, Clostridium clostridiiforme ATCC 25537, C. perfringens ATCC 13124, C. paraputri®cum ATCC 25780, Escherichia coli ATCC 25922, Enterobacter cloacae ATCC 13047, Salmonella typhimurium TA98 and S. typhimurium YG1041 at 100±1000 mg/ml in culture broth. Neither Bi®dobacterium infantis ATCC 15697 nor Lactobacillus acidophilus ATCC 4356 was inhibited by any of the above compounds up to 500 mg/ml. Tannic acid has a much greater relative binding eciency to iron than propyl gallate, methyl gallate or gallic acid. The inhibitory eect of tannic acid to the growth of intestinal bacteria may be due to the strong iron binding capacity of tannic acid; whereas the eect of propyl gallate and methyl gallate probably occurs by a dierent mechanism. The growth of E. coli was restored by the addition of iron to the medium after the precipitate caused by tannic acid was removed. Neither B. infantis nor L. acidophilus require iron for growth. This probably contributes to their resistance to tannic acid. Because tannins are abundant in the human diet, tannins may aect the growth of some intestinal bacteria and thus may have an impact on human health. # 1998 Elsevier Science Ltd. All rights reserved Keywords: growth; intestinal bacteria; inhibition; iron deprivation; tannic acid. Abbreviations: GI = gastrointestinal; LIC = lowest inhibitory concentration; TSB = tryptic soy broth.
INTRODUCTION
Vegetable tannins are water-soluble phenolic compounds having a molecular weight between 500 and 3000 Daltons. Vegetable tannins can be classi®ed into hydrolysable and condensed tannins. Hydrolysable tannins contain either gallotannins or ellagitannins. Gallotannins yield glucose and gallic acid on hydrolysis by acids, bases or certain enzymes. Ellagitannins contain one or more hydroxydiphenoyl residues which are linked to glucose as a diester in addition to gallic acid. Upon hydrolysis, the hydrodiphenoyl residue undergoes lactonization to produce ellagic acid. Condensed tannins are the polymerized products of ¯avan-3-ols and ¯avan-3,4-diols, or a mixture of the two. The polymers, referred to as ``¯avolans'' are popularly called condensed tannins ( more recently called proanthocyanidins) (Chung et al., 1998). Tannins are present in a variety of fruits and vegetables *Author for correspondence.
(Deshpande et al., 1984; Salunkhe et al., 1989). Wines and teas also contain some tannins (Ho and Singleton, 1977; Sanderson et al., 1975). It is estimated that people in the United States ingest 1 g of tannic acid, the most simple form of hydrolysable tannin, each day (IARC, 1976; Sanyal et al., 1997). Tannins may serve as a natural antimicrobial agent to protect fruits and vegetables against microbial infection (Chung and Murdock, 1991; Scalbert, 1991). When food tannins are ingested, the initial contact is the gastrointestinal (GI) tract, which is one of the most active metabolic sites in the human body (Chung, 1996). Microorganisms in the GI tract may play a role in many disease processes including cancer (Chung, 1996). How tannins interact with the intestinal microbiota and aect metabolism in the GI tract may be of important to health. Tannins have been reported to have various health eects such as antinutritional, carcinogenic, mutagenic, anticarcinogenic and antimutagenic activities (Butler and Rogler, 1992; Chadwick et al.
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1992; Chung et al. 1996; Horikawa et al. 1994; Marzo et al. 1990; Oterdoom, 1985). How these eects are related to intestinal microbiota is not clearly known. The inhibitory eects of tannins from dierent sources on various microorganisms have been demonstrated (Chang et al., 1994; Chung et al., 1993, 1995; Scalbert, 1991). Bae et al. (1993) showed that condensed tannins from birdfoot trefoil (Lotus corniculatus L.) were inhibitory to the endoglucanase activity of cellulose digesting Fibrobacter succinogenes S85 in the rumen. The growth of other rumen bacteria such as Butyrivibrio ®brisolvens A38, Streptococcus bovis 45S1 were also reported to be inhibited by condensed tannins from sain leaf (Onobrychis viciifolia Scop.) (Jones et al., 1994). However, very limited information is available related to the inhibitory eect of tannins on the growth of human intestinal bacteria. The aim of this paper is to examine whether tannic acid and its hydrolytic product, gallic acid, have any inhibitory eects on the growth of some intestinal bacteria, and to identify potential mechanisms of inhibition. Methyl gallate and propyl gallate are also included since they are esters forms of gallic acid. These results will further our understanding as to how food tannins may impact human health.
MATERIALS AND METHODS
Organisms Bacteroides fragilis ATCC 25285, Bi®dobacterium infantis ATCC 15697, Clostridium clostridiiforme ATCC 25537, C. perfringens ATCC 13124, C. paraputri®cum ATCC 25780, Escherichia coli ATCC 25922, Enterobacter cloacae ATCC 13047 and Lactobacillus acidophilus ATCC 4356 were obtained from the American Type Culture Collection (Rockville, MD, USA). Salmonella typhimurium TA98 was provided by Dr B. N. Ames of the Department of Biochemistry, University of California, Berkeley, California, USA. S. typhimurium YG1041 was provided by Dr Masahiko Watanabe, Division of Genetics and Mutagenesis, National Institute of Hygienic Sciences, Tokyo, Japan. These bacteria were chosen because they represent dierent groups of intestinal bacteria (Chadwick et al., 1992; Holdeman and Moore 1972). Culturing methods Lactobacilli MRS broth, modi®ed BHI, reinforced clostridial medium and tryptic soy broth (TSB) were obtained from Difco (Detroit, MI, USA). Nutrient broth no. 2 was obtained from Oxoid (Hampshire, UK). The anaerobic techniques of Holdeman and Moore (1972) were followed. The modi®ed chopped-meat medium was prepared anaerobically according to American Type Culture
Collection Media Handbook (ATCC, 1989). Bacteroides fragilis was grown in modi®ed chopped meat medium; Bi®dobacterium infantis was on reinforced clostridial medium; Clostridium clostridiiforme, C. perfringens and C. paraputri®cum were on modi®ed BHI. The above bacteria were grown anaerobically (Holdeman and Moore, 1972). Enterobacter cloacae and Escherichia coli were on TSB; Lactobacillus acidophilus was on Lactobacillus MRS broth; S. typhimurium TA98 and YG1041 were on nutrient broth. The latter ®ve bacteria were grown aerobically. Chemicals Agar, ammonium chloride, calcium chloride, PIPES, gallic acid, methyl gallate, propyl gallate and tannic acid were purchased from Sigma Chemical Co. (St Louis, MO, USA). Chrome azurol S, hexadecyltrimethyl-ammonium bromide, piperzine, 5-sulfosalicyclic acid, 8-hydroxyquinoline and thiamine hydrochloride were purchased from Aldrich Chemical Company, Inc. (Milwaukee, WI, USA). Casamino acid was from Difco Laboratory (Detroit, MI, USA). Gallic acid, methyl gallate, propyl gallate, tannic acid and ferrous sulfate were freshly prepared by dissolving in deionized water at proper concentrations followed by ®lter-sterilization. When anaerobic cultures were used, these solutions were prepared anaerobically under nitrogen (Holdeman and Moore, 1972). Sensitivity testing Gallic acid, methyl gallate or tannic acid were added to the growth media of each bacteria aerobically or anaerobically, depending on the type of culture. For cultures containing gallic acid, methyl gallate or propyl gallate, bacterial growth was measured at 600 mm using a Bausch & Lomb Spectronic 20 spectrophotometer (Milton Roy Company, Rochester, NY, USA). The growth of bacterial cultures containing tannic acid was measured by plate count method (FDA, 1992). Results reported were the average of three independent experiments. Estimation of the lowest inhibitory concentration (LIC) The method for estimating the lowest eective inhibitory concentration for bacterial growth was adopted following the determination of the detection limit of the analytical instrument (Ingle and Crouch, 1988). Brie¯y, for each bacterial species the growth response at stationary phase (i.e. absorbance when using a spectrophotometer) was plotted against the concentrations of the compound. Based on the method of Ingle and Crouch (1988), the response from the test data must be at least twice the standard deviation (SD) lower than that from the corresponding control set in order to show
Tannic acid inhibition of intestinal bacteria
signi®cant dierence between them. Therefore the concentration corresponding to the absorbance on the regression curve, which is just twice the SD smaller than the control was de®ned as the LIC for the bacterial growth. The LIC for each bacterial strain for each compound was separately determined on each ®gure. Determination of iron concentration The iron concentration in the media was determined using an atomic spectrophotometer, SpectrAA-30/40 Zeeman (Varian Techtrom Pty., Mulgrava, Victoria, Australia) equipped with Zeeman background correction (SpectraAA-30/40 Zeeman Operation Manual, 1986). The measured iron concentration of various media were between 1.18 and 1.62 mM. Iron withdrawing experiments (A) Plate assay. The iron withdrawing experiment was carried out on agar plates containing low-iron medium, ferrous sulfate (1 mM) and agar (1.5%). The low-iron medium was prepared with MM9 (Maniatis et al., 1982; Schwyn and Nielands, 1987) to which was added casamino acids (0.3%), thiamine±HCl (2 ppm), Tris buer (0.87%) and succinic acid (0.2%). The succinic acid and casamino acids were neutralized with sodium hydroxide and deferrated by extraction with 3% (w/w) 8hydroxyquinoline in chloroform before being added to the medium. Aliquots (1 ml) of an overnight E. coli ATCC 25922 culture in TSB (378C) was added to 100 ml of the above agar medium maintained at 458C. Tannic acid, propyl gallate or methyl gallate was added to the medium to give a ®nal concentration of 1000, 100 and 100 mg/ml, respectively. Approximately 20 ml was poured into a petri dish and allow to solidify at 48C for 2 hr. Wells (8 mm) were cut into the agar and ®lled with 50 ml 2 mM iron solution (FeSO47H2O, freshly prepared and ®lter-sterilized) or sterile distilled water (control). Bacterial growth around the well was observed after incubation of the plates at 378C for 48 hr. (B) Broth assay. Tannic acid was added to TSB to give the ®nal concentration of 1 mg/ml. The precipitates were removed by centrifugation. Sterilized FeSO47H2O solution was aseptically added to the supernatants to give the ®nal concentration of 0.63, 1.25 or 2.5 mM and use as the growth broth. After inoculation, the bacterial growth was measured spectrophotometrically as described above. Determination of iron bindings by tannins and siderophore (A) Production of siderophore. A 10-ml volume of low-iron medium (Schwyn and Nielands, 1987) was inoculated with E. coli and incubated at 378C for 12 hr. 0.1 ml of this culture was inoculated into 10 ml of the same medium. As a control, a ¯ask
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with 2 mM added iron (FeSO47H2O) was also inoculated with E. coli. The cultural density during incubation was monitored at 600 mm using a Spectronic 20 to verify iron starvation, which was manifested by a slower growth rate and lower absorbance values. After 24 hr, when the stationary phase was reached (plate count 2.5 107/ml), the cells were removed by centrifugation at 3000 g. The supernatants were used as siderophores (Maniatis et al., 1982). (B) Iron binding assay. The iron binding assay was conducted according to a Chrome Azurol S (CAS) method (Schwyn and Neilands, 1987) in which the colour change of the blue iron±dye complex at 630 mm indicates the amount of the iron bound with the compound. The standard curve of iron±dye solution containing dierent concentrations of iron was used for determination of those iron-compound complexes. Molar ratio of the initial concentration of the compounds in the solution to the iron±compound complexes formed indicates amount of compound required to form a unit of iron±compound complex. The relative binding eciency was calculated according to the following equation: Relative Binding Efficiency Molar ratio tannic acid to iron Molar ratio test compound to iron The initial concentration of siderophore produced by E. coli was calculated according to Harris et al. (1979). RESULTS
The eect of gallic acid, methyl gallate, propyl gallate and tannic acid on the growth of Clostridium perfringens ATCC 13124 is illustrated in Fig. 1. Similar inhibitory patterns were found when these compounds were present in the growth medium with Bacteroides fragilis, Enterobacter cloacae, C. clostridiiforme, C. paraputri®cum, E. coli and S. typhimurium (data not shown). At the concentrations tested, Lactobacillus acidophilus was not inhibited by methyl gallate or propyl gallate. A much higher concentration of tannic acid was required to inhibit the growth of this bacterium (Fig. 2). Similarly, Bi®dobacterium infantis was not inhibited by tannic acid at 1 mg/ml, nor by methyl gallate and propyl gallate (data not shown). The estimated LICs of these compounds towards these bacteria were listed in Table 1. Gallic acid showed no inhibitory eect to any of the bacteria tested. When tannic acid (1 mg/ml) was added to agar plates with low iron content, no visible growth of E. coli was observed. However, when a small amount (50 ml) of iron solution (FeSO47H2O, 2 mM) was added to the well (8 mm in diameter) of the plate, a heavy smear of E. coli growth
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Fig. 1(a,b).
occurred around the well. Similar experiments were done with propyl gallate and methyl gallate. No bacterial growth occurred around the well (data not shown). The eect of iron removal on bacterial growth was further tested using TSB culture. After the precipitates (tannic acid and iron complex) were removed and a known amount of iron (0.63 to 2.5
mM) added to the medium, the growth of E. coli resumed (Fig. 3). The same experiment was performed with propyl gallate and methyl gallate, but no resumption of bacterial growth was detected. Similar experiments were conducted using C. perfringens, S. typhimurium TA98 and Bacteroides fragilis, with similar results.
Tannic acid inhibition of intestinal bacteria
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Fig. 1. Eect of tannic acid and related compounds on the growth of Clostridium perfringens. Each data point is an average of three measurements. (a) tannic acid, (b) propyl gallate, (c) methyl gallate and (d) gallic acid. Tannic acid, propyl gallate and methyl gallate show signi®cant inhibition at concentrations greater than 10 mg/ml (P < 0.05). Gallic acid shows no signi®cant inhibition at concentrations up to 1000 mg/ml.
The relative binding eciencies of siderophores produced by E. coli, tannic acid, methyl gallate, propyl gallate and gallic acid towards iron were determined and shown in Table 2. It was found that it took only 0.9 molecules of tannic acid to chelate one molecule of iron. Harris et al. (1979) reported that it took 3.0 molecules of siderophore
prepared from the crude supernatant of E. coli culture on low iron medium to chelate one molecule of iron. If we standardized the relative binding eciency of tannic acid as one, the relative binding eciency of siderophore is 0.3. Using similar calculations, the relative binding eciency of gallic acid, methyl gallate and propyl gallate were 1.1 10ÿ4,
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Fig. 2. Eect of tannic acid on the growth of Lactobacillus acidophilus. Each of the data points is an average of three measurements. Tannic acid shows signi®cant inhibition only at concentrations greater than 500 mg/ml.
1.2 10ÿ4 and 1.4 10ÿ4, respectively. Thus, the binding of tannic acid is equivalent to that of the natural siderophores; whereas propyl gallate, methyl gallate and gallic acid have negligible binding capacities to iron. DISCUSSION
All bacteria studied were originally isolated from human intestine. It is of interest to note that tannic acid, propyl gallate and methyl gallate, but not gallic acid, are inhibitory to the growth of all the tested intestinal bacteria except the lactic acid bacteria L. acidophilus and Bi®dobacterium infantis. Tannic acid has a stronger inhibitory eect than propyl gallate or methyl gallate. The inhibitory eect occurs to both aerobes and anaerobes, Gram
(+) and Gram (ÿ) (Table 2). This pattern of inhibition is consistent with the previous ®ndings of Chung et al. (1993, 1995). Many of the bacteria tested have been reported to carry important metabolic activation enzymes such as nitroreductase and azoreductase (Chadwick et al., 1992; Ra®i et al., 1991). Food tannins may also aect these enzymes of intestinal bacteria. This needs to be further explored. Many microorganisms produce siderophoresÐ low molecular weight chelating agents that bind and solubilize iron (Crowley et al., 1991; Lewin, 1984; Neilands, 1995). Antimicrobial activity of tannins through iron deprivation has been suggested (Haslam, 1996; Mila et al., 1996; Scalbert, 1991). Tannic acid may work like a siderophore to chelate iron from the medium and make iron unavailable
Table 1. Estimated lowest inhibitory concentration (LIC) of methyl gallate, propyl gallate and tannic acid with various test microorganisms LIC (mM) Name of organism Bacteroides fragilis ATCC 25285 Bi®dobacterium infantis ATCC 15697 Clostridium clostridiiforme ATCC 25537 Clostridium paraputri®cum ATCC 25780 Clostridium perfringens ATCC 13124 Enterobacter cloacae ATCC 13047 Escherichia coli ATCC 25922 Lactobacillus acidophilus ATCC 4356 Salmonella typhimurium TA98 Salmonella typhimurium YG1041 *No inhibition. No inhibition was observed with gallic acid.
Methyl gallate
Propyl gallate
Tannic acid
883.8 Ð* 272.7 261.9 7.1 66.6 92.9 Ð 23.7 23.5
575.7 Ð 351.2 265.4 24.4 33.7 17.3 Ð 17.4 6.3
91.8 Ð 144.9 56.3 3.1 9.5 27.0 181.6 14.7 8.5
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Fig. 3. Growth resumption of E. coli by adding dierent amount of iron in the tannic acid present medium. For the control, E. coli was grown in low iron medium resulting from tannic acid binding. Stimulation of bacterial growth was observed at added iron concentration greater than 0.63 mM (P < 0.05). Each of the data points is an average of three measurements.
to the microorganisms. The growth of the tannic acid inhibited E. coli culture was restored by the supplementation of additional iron ( Fig. 3). Propyl gallate and methyl gallate are esters of gallic acid, which is the basic structural unit of hydrolysable tannins. But both propyl and methyl gallates are unable to bind iron eciently. Their inhibitory eect on the growth of these bacteria is, therefore, not through the deprivation of iron from the medium. Other mechanisms must be considered. Microorganisms growing under aerobic conditions need iron for a variety of functions including reduction of ribonucleotide precursor of DNA, for formation of heme, and for other essential purposes. Neilands (1995) reported that a level of at least one micromolar iron is needed for optimal growth. Iron does not stimulate the growth of some lactic acids because they have no heme enzymes, and the crucial iron-containing ribonucleotide reductase has been replaced with an enzyme using Table 2. Relative binding eciency of gallic acid, methyl gallate, propyl gallate, tannic acid and siderophore to iron Compound
Relative binding eciency*
Tannic acid Siderophore Propyl gallate Methyl gallate Gallic acid
1.0 0.3 1.4 10ÿ4 1.2 10ÿ4 1.1 10ÿ4
*Relative binding eciency was calculated based on the measured molar ratio of tannic acid to iron divided by the measured molar ratio of the test compound to iron.
adenosylcobalamine (Bruyneel et al., 1989; Pandey et al., 1994; Reichard, 1993). This explains the resistance of L. acidophilus and Bifodobacterium infantis to tannic acid. The present ®ndings indicate that tannic acid may aect the GI microbial population by inhibiting the growth of some bacterial ¯ora but not lactic acid bacteria. Lactic acid bacteria are generally considered to be bene®cial to human health (Bruyneel et al., 1989). Therefore, tannic acid may serve as a regulator of microbial populations in the intestinal tract, which is bene®cial to human health. Whether other types of tannins would have the similar eect is of interest. The overall eects of dietary tannins to human health requires further extensive investigation.
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