Research Unit of Alcohol Diseases, University Central Hospital of Helsinki and
1 Anaerobe Reference Laboratory, National Public Health Institute, Helsinki, Finland
Received 23 December 1999; in revised form 23 May 2000; accepted 7 June 2000
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ABSTRACT |
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INTRODUCTION |
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Microaerophilic lactobacilli and anaerobic bifidobacteria are important members of the human indigenous flora of the large intestine, levels up to 1010 and 1012 colony forming units (CFU)/g respectively of intestinal contents have been recovered in human faeces (Goldin and Gorbach, 1984). Both lactobacilli and bifidobacteria have been suggested to have probiotic or beneficial effects in humans and are therefore used in probiotic dairy products (Fuller and Gibson, 1997
). Some lactobacilli (Coconnier et al., 1992
), especially Lactobacillus GG ATCC 53103 (Elo et al., 1991
), and some strains of bifidobacteria (Bernet et al., 1993
) adhere in vitro to human cell lines. It has been shown that Lactobacillus GG while colonizing the human gastrointestinal tract decreases the level of faecal-glucuronidase (Goldin et al., 1992
; Saxelin et al., 1995
) the activity of which may be associated with the production of potentially carcinogenic substances. In addition, feeding of Lactobacillus GG to rats has been demonstrated to diminish the incidence of procarcinogen-induced colon tumours (Goldin et al., 1996
), and also to reduce both the endotoxaemia and the severity of alcohol-induced experimental liver injury in chronically ethanol-fed rats (Nanji et al., 1994
). Some lactic acid bacteria, such as group N streptococci and Leuconostoc cremoris, are able to form small amounts of acetaldehyde and ethanol from glucose (Lees and Jago, 1976
). However, to date, little is known about the capacity of indigenous lactobacilli, bifidobacteria, and probiotic Lactobacillus GG ATCC 53103 to metabolize ethanol and its first metabolite, acetaldehyde.
The aim of this study was to elucidate in vitro the capacity of human lactobacilli and bifidobacteria to metabolize ethanol and acetaldehyde under conditions that may prevail in the human large intestine after moderate alcohol intake. To this end, we examined whether aerobically grown lactobacillus strains possess either ADH or ALDH activity. Furthermore, we studied whether aerobically and anaerobically grown lactobacillus and anaerobically grown bifidobacterium strains can metabolize ethanol to carcinogenic and toxic acetaldehyde and further metabolize acetaldehyde to non-toxic compounds, such as acetate, in vitro. In addition, we evaluated the effect of growth media (lactobacillus selective medium MRS or non-selective brucella agar) and bacterial concentration on the ability of lactobacilli to metabolize acetaldehyde to acetate. Finally, we determined the ability of lactobacilli and bifidobacteria to metabolize acetaldehyde in vitro in the presence of ethanol, i.e. under conditions more closely resembling those in the human large intestine after alcohol intake.
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MATERIALS AND METHODS |
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Acetaldehyde production
The capacity of bacteria (Table 1) to produce acetaldehyde from ethanol was determined by incubation of duplicated samples containing 450 µl of intact bacterial suspension in closed vials with 50 µl of 22 mM ethanol (final concentration) for 1 h at 37°C. Samples incubated without ethanol were used as controls and were processed simultaneously as described above.
Acetaldehyde metabolism
The capacity of bacteria (Table 1) to metabolize acetaldehyde was determined by incubation of duplicate samples containing 450 µl of the intact bacterial suspension with 50 µl of 0.05 mM or 0.5 M acetaldehyde (final concentrations) in closed vials for 1 h at 37°C. Samples incubated without ethanol were used as controls, and acetaldehyde standards prepared by incubating the same final concentrations of acetaldehyde (0.05 mM or 0.5 M) but without bacteria were processed simultaneously as described above.
In addition, the capacity of bacteria (Table 2) to metabolize acetaldehyde in vitro in the presence of ethanol was determined by incubation of duplicate samples containing 350 µl of the intact bacterial suspension with 50 µl of acetaldehyde (final concentration 0.5 mM) with or without 50 µl of ethanol (final concentration 22 mM) for 1 h at 37°C. Bacterial samples incubated without ethanol and acetaldehyde were used as controls and were processed simultaneously. Acetaldehyde standards, prepared by using the same final concentrations of acetaldehyde (0.05 mM or 0.5 M) and/or ethanol (22 mM), but without bacteria, were processed as described above. To control for artefactual acetaldehyde production, the reaction was stopped by addition of 50 µl of 6 M perchloric acid. Ethanol and acetaldehyde were analysed by head-space gas chromatography at 37°C as described previously (Jokelainen et al., 1996a
).
Acetate production
To study acetate production, duplicate suspensions of lactobacilli (Table 1) were incubated with 5 mM acetaldehyde (final concentration) for 1 h at 37°C. Samples incubated without acetaldehyde were used as controls and were processed simultaneously. Acetate was then measured spectrophotometrically in bacterial supernatants obtained by centrifugation at 1000 g for 10 min, as described previously (Nosova et al., 1996
).
Calculations
The amount of acetaldehyde produced was calculated by subtracting the amount of acetaldehyde produced by controls from the amount of acetaldehyde of the samples incubated with ethanol. The amount of acetaldehyde metabolized was calculated by subtracting the amount of acetaldehyde produced by the bacterial suspension after the addition of acetaldehyde, or acetaldehyde and ethanol, from the sum of the amount of acetaldehyde produced by controls and the appropriate acetaldehyde standard. The amount of acetate produced was calculated by subtracting the amount of acetate produced by the controls from the amount of acetate of the samples incubated with acetaldehyde.
Statistics
Results are expressed, unless otherwise mentioned, as the means of duplicate determination with bacteria. The statistical significance of the difference (Fig. 4) was analysed by the t-test using InStat software (version 2.0).
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RESULTS |
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Lactobacillus strains metabolized ethanol only weakly to acetaldehyde. Among the lactobacillus and bifidobacterium strains, Lactobacillus GG AN ATCC 53103 (80 nmol/109 CFU/h) had the highest acetaldehyde-producing capacity (Fig. 1). The capacity of Lactobacillus acidophilus AHP 6509 and Lactobacillus fermentum AHP 6412 to metabolize ethanol was negligible. In contrast, nearly all aerobically and anaerobically processed bifidobacterium strains metabolized ethanol to acetaldehyde more actively, with Bifidobacterium spp. AN AHP 16467 (72 nmol/109 CFU/h) showing the highest acetaldehyde-producing capacity among the bifidobacteria (Fig. 1
).
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Acetaldehyde consumption was not influenced by the type of growth medium, MRS or brucella agar (data not shown).
All bacterial strains were able to metabolize 500 µM acetaldehyde both under aerobic and anaerobic conditions (Fig. 5). Lactobacillus GG AN ATCC 53103 showed the highest capacity to metabolize acetaldehyde (359 nmol/109 CFU/ml), even in the presence of 22 mM ethanol (130 nmol/109 CFU/ml). The addition of ethanol to the acetaldehyde medium, however, diminished the acetaldehyde-consuming capacity of all bacterial strains (Fig. 6
). Moreover, the addition of ethanol abolished totally the acetaldehyde metabolism of Lactobacillus brevis AHP 6281 and that of Bifidobacterium bifidum ATCC 15696 (Fig. 6
).
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DISCUSSION |
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Acetaldehyde, a highly reactive and toxic metabolite of ethanol, has been shown in experimental animals to be carcinogenic (International Agency for Research on Cancer, 1985) and has been linked to several toxic effects of ethanol on organs (Lieber et al., 1980
; Yokoyama et al., 1998
). In addition, acetaldehyde produced via microbial ethanol oxidation has been proposed to be a pathogenetic factor in the development of alcohol-associated gastrointestinal injury (Seitz et al., 1990
; Salaspuro, 1997
).
Most of the ingested ethanol is absorbed during the first hour after its intake in the stomach and small intestine. Alcohol is evenly distributed into the water phase of the human body, so ethanol concentrations in the large intestine are comparable to that found in the blood (Halstedt et al., 1973; Levitt et al., 1982
). The ethanol concentration of 22 mM closely corresponds to 100 mg/dl in the blood that is useful as reference in studies on alcohol drinking. For example, after alcohol intake of 25% v/v by men (0.8 g/kg body wt) at 1 h after alcohol ingestion, the ethanol level in the blood as well as in the large intestine was 100 mg/dl (Jones and Jönsson, 1994
). In rats, after ethanol administration of a dose of 1.5 g/kg body wt, the mean intracolonic ethanol level determined at 2 h was 20 mM and the corresponding acetaldehyde level was 480 µM (Visapää et al., 1998
). In this study, we used 22 mM ethanol and 500 µM acetaldehyde concentrations, corresponding to concentrations that can be found in the large intestine of humans after drinking alcohol.
Results of the present study show that both lactobacilli and bifidobacteria have a very weak capacity to metabolize ethanol to acetaldehyde in vitro at concentrations that may prevail in the human large intestine after normal social alcohol drinking. For example, Lactobacillus GG ATCC 53103 and Bifidobacterium spp. AHP 16467 produced nearly 20-fold lower acetaldehyde concentrations than did the facultative anaerobic strain Escherichia coli IH 13369, which so far has been shown to have the highest capacity to produce acetaldehyde from ethanol under the same conditions as used in this study (Jokelainen et al., 1996a). However, the capacity of lactobacilli and bifidobacteria to metabolize acetaldehyde at a concentration of 50 µM, close to the endogenous intracolonic acetaldehyde level (Jokelainen et al., 1996b
), was comparable to the acetaldehyde-metabolizing capacity of some of the facultative anaerobic and aerobic bacterial strains isolated from the human gastrointestinal tract (Nosova et al., 1996
). For example, the acetaldehyde-metabolizing capacity of Lactobacillus GG ATCC 53103 was about half of that of Escherichia coli IH 50763, which so far has been shown to have the highest acetaldehyde-consuming capacity among the facultative anaerobic and aerobic bacterial strains tested (Nosova et al., 1996
).
The capacity of lactobacillus strains to metabolize acetaldehyde to acetate was also comparable to that of the facultative anaerobic and aerobic bacterial strains isolated from the human gastrointestinal tract (Nosova et al., 1996). Because acetate is an intermediate product in the metabolism of acetaldehyde by bacterial ALDHs, its substantial production by lactobacilli further illustrates their capacity for acetaldehyde consumption. For example, acetate production by Lactobacillus GG ATCC 53103 was only one-fifth of that of aerobic Pseudomonas aeruginosa ATCC 27853, which has been shown earlier to have the highest acetate production capacity from acetaldehyde among the facultative anaerobic and aerobic gastrointestinal bacteria (Nosova et al., 1996
). The capacity of Lactobacillus GG AN ATCC 53103 to metabolize acetaldehyde in vitro was detectable starting from the bacterial concentration of 108 CFU/ml and this capacity increased significantly with rising bacterial concentrations up to 109 CFU/ml. Recently, a 1010-CFU oral dose of Lactobacillus GG ATCC 53103 to volunteers has been shown to result in faecal recovery of Lactobacillus GG up to 107 CFU/g faeces (Saxelin et al., 1995
). Accordingly, our data suggest that sufficient acetaldehyde consumption in vivo may require a higher total daily oral dose of Lactobacillus GG.
Most lactobacilli are microaerophilic, which explains the poor growth and subsequent insufficient counts of aerobically processed lactobacilli. Bifidobacteria are anaerobic; thus, they were not suitable for the aerobically performed study of ADH and ALDH activity. Moreover, we did not find any measurable ADH and ALDH activity in the supernatants of aerobically grown lactobacilli. In addition, the acetaldehyde-consuming capacity of anaerobically processed Lactobacillus GG ATCC 53103 was significantly higher than that of the aerobically processed one (Fig. 4). Acetate production from acetaldehyde by anaerobically processed Lactobacillus GG ATCC 53103 was also four times higher than that of the aerobically processed strain. These data suggest that, for sufficient acetaldehyde consumption in vivo by Lactobacillus GG ATCC 53103, anaerobic conditions such as those prevailing in the lumen of the large intestine are required in order to promote this activity.
All lactobacillus and bifidobacterium strains were effectively able to metabolize the 500 µM acetaldehyde solution, i.e. the concentration that may occur in the large intestine after alcohol administration (Visapää et al., 1998). The addition of ethanol to the acetaldehyde media in vitro decreased the ability of these bacterial strains to metabolize acetaldehyde. Under anaerobic conditions, acetaldehyde consumption by bacteria may occur either by alcoholic fermentation or via ALDH to acetate (Zeikus, 1980
). Accordingly, because the excess of ethanol inhibited alcoholic fermentation, acetaldehyde metabolism under this condition most probably occurred via the ALDH pathway. In the present study, acetaldehyde metabolism by Lactobacillus brevis AHP 6281 and Bifidobacterium bifidum ATCC 16596 was totally blocked by the addition of ethanol, which suggests that acetaldehyde consumption by these bacterial strains occurs predominantly via alcoholic fermentation. Other bacterial strains were able to use both pathways for acetaldehyde consumption. Lactobacillus GG AN ATCC 53103 possessed the highest capacity to metabolize acetaldehyde in the presence of ethanol as well.
In conclusion, lactobacillus and bifidobacterium strains are weak producers of toxic and carcinogenic acetaldehyde from ethanol, but have a relatively better capacity to remove acetaldehyde at least in vitro. Their capacity to metabolize endo-genous acetaldehyde is comparable to that of the facultative anaerobic or aerobic gastrointestinal bacteria. In the presence of ethanol, the capacity of all bacterial strains for acetaldehyde consumption diminishes. Anaerobically processed Lactobacillus GG ATCC 53103 had the highest capacity to metabolize acetaldehyde both with and without the presence of ethanol in vitro. A positive correlation existed between rising bacterial concentrations and an increase in the acetaldehyde-metabolizing capacity of Lactobacillus GG AN ATCC 53103. These data suggest that lactobacilli and bifidobacteria and, especially, Lactobacillus GG ATCC 53103, may have a beneficial regulatory effect on the intracolonic endogenous and exogenous acetaldehyde levels. It remains to be established whether these probiotics could be useful in the prevention of acetaldehyde-associated gastrointestinal morbidity associated with excessive alcohol intake.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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