Download Impact of Tannic Acid on the Gastrointestinal Microflora

ORIGINAL ARTICLE
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Impact of Tannic Acid on the Gastrointestinal
Microflora
S. Samanta1,2, S. Giri2, S. Parua1, D.K. Nandi1, B.R. Pati2 and K.C. Mondal1
From the 1Department of Physiology, Raja NL Khan Women’s College, Midnapur-721102, West Bengal, and
2
Microbiology Unit, Department of Botany & Forestry, Vidyasagar University, Midnapore-721102, West Bengal, India
Correspondence to: K.C. Mondal, Department of Physiology, Raja NL Khan Women’s College, Midnapur-721102, West
Bengal, India (email: [email protected])
Microbial Ecology in Health and Disease 2004; 16: 32 /34
The cultivable microflora of the intestines in rat have been examined after exogenous administration of tannic acid, a major dietary
component found in plant materials. Normal counts of total bacteria, total coliform and Escherichia coli per mg of faeces in experimental
rats were 6.1/103, 1.7/103 and 1.5/103 respectively, present at a ratio of 4:1.1:1. Ingestion of tannic acid (at a dose of 45 mg per 100g of
body weight per day) into male albino rats reduced the bacterial population in 6 days and after that their number increased. The body weight
of the animals decreased by about 22% after 21 days of tannic acid treatment. The changes in microbial population indicate that tannic acid
can impair the ecological balance of gastrointestinal flora. Key words: tannic acid, gastrointestinal flora, total coliform, E. coli .
INTRODUCTION
The symbiotic association between animals and microorganisms exists in a highly integrated ecosystem with multiple inter-relations. A large number of microorganisms
inhabit the gastrointestinal tract and they are generally
referred to as normal flora or microbiota, most of which are
bacteria. The microbial community of the gastrointestinal
tract is not well understood owing to the inadequacy of
classical culture-dependent methods. Only a few of the
organisms can be cultured under laboratory conditions, as
most of these bacterial populations require specific growth
factors which are provided by other community residents or
secretions from the host tissues. Recently phylogenetic
analysis of 16S ribosomal DNA has been used to analyse
microbial communities in the gastrointestinal tract (1 /3);
however, Apajalahli et al. (3) stated that no single technique
could give an overall view of the total bacteria and at the
same time be species-specific. The majority of the culturable
bacteria are Gram-positive, strictly anaerobic streptococci,
lactobacilli, eubacteria, clostridia, peptostreptococci,
Gram-negative rods and bacteroides (4, 5). These indigenous microorganisms perform several beneficial activities (4,
5) for the host, such as synthesis of vitamins (thiamine,
riboflavin, pyridoxine, B12, K, etc.), and help in the
metabolism of steroids (bile acid, cholesterol, sex hormones), etc. In addition, the normal gastrointestinal
microflora plays a major role in preventing pathogenic
bacterial infections and diseases through colonization
resistance (6, 7).
# Taylor & Francis 2004. ISSN 0891-060X
Intestinal microbial populations subsist on nutrients
received from the partially digested diet of the host. In
vegetables, tannins are the fourth most abundant constituent after cellulose, hemicellulose and lignin (8). Animals
take in tannin directly through their feed and fodder. Apart
from vegetables, humans consume tannins in large amounts
through tea, coffee, tobacco, catechu, etc. Daily intake of
tannins through diet by the people of different parts of
India varies from 1500 to 2500 mg (9). Tannic acid, a
popular example of plant tannins, is a polyphenolicpolyhydroxy compound. Because of this chemical nature
it can form complexes with proteins or enzymes or other
macromolecules and is generally referred to as an antinutrient as well as an antimicrobial agent (10).
Considering the beneficial role of the normal gastrointestinal flora, the present investigation evaluated the effect
of tannic acid on these groups of culturable organisms
through an in vivo experiment for the first time.
MATERIALS AND METHODS
Animals and treatment
Male albino rats were used as the model animal; they were
forced to ingest tannic acid by means of a gastric tube and
their faecal matter was examined for microbial analysis.
Rats of average weight of 90 /95 g were used in the present
study. Twenty animals were separated into two groups of 10
and accustomed to the animal house conditions for 7 days
before treatment with sufficient amounts of balanced diet.
Microbial Ecology in Health and Disease
DOI: 10.1080/08910600310026158
Tannin /gastrointestinal flora interaction
87.59/4.8
87.39/4.2
85.89/3.5
82.39/3.7
78.69/2.9
75.99/4.7
72.19/3.9
68.69/4.6
6.1/1039/0.31 4.3/1039/0.43 1.9/1029/0.22 3.9/1039/0.64 9.4/1039/0.23 8.0 /1049/0.39 2.1/1079/0.09 8.5 /10109/0.91
Body weight (g) (mean9/SE)
Total bacteria/mg of faeces
(mean9/SE)
Total coliform/mg of faeces
Number of E. coli /mg of faeces
7.2/105
4.0/105
3.1 /104
8.5 /103
2.7/103
1.9/103
1.2/103
0.9/103
0.5/102
0.3/102
0.7/103
0.4/103
15
12
9
6
3
Period of tannic acid treatment (days)
The intestinal microflora is an important part of the
digestive system of all animals. Variation in their number,
particularly of bacterial species in the gastrointestinal tract,
is due to their substrate specificity and growth requirements.
That is why only a fraction of the microflora is enumerated
during standard culturing and phenotyping analysis. Detailed information on the microbial community composition in this natural system can be achieved by the analysis of
16S rDNA sequences obtained directly from the samples by
PCR amplification, cloning and sequencing (1 /3).
In this present experiment, the action of tannic acid was
evaluated by counting culturable microflora in faecal
matter. Normally 1 mg of rat faeces contains total bacteria
6.1 /103, total coliforms 1.7 /103 and E. coli 1.5 /103,
present at a ratio of 4:1.12:1 respectively (Table I). It has
also been found that the among total microbial flora, onethird of the population is coliform. The presence of E. coli
in the faeces of experimental rats is about 25% of the total
microflora, whereas its number in humans is normally not
more than 1% (6). It is not clear to us why the number of E.
coli in rats is so high. This outnumbered population may be
due to the poor habitat and nutritional conditions of rats.
Earlier, Madigan et al. (4) noted that the quantity of normal
flora of the gastrointestinal tract varies among the species.
They also reported that in guinea pigs, 80% of the intestinal
flora are lactobacilli, whereas very negligible numbers of the
same organism are present in humans.
The numbers of all types of culturable microflora
decreased sharply up to 6 days of treatment in rats ingesting
tannic acid (Table I). This quantitative inhibition of the
microflora may be due to tannic acid toxicity. Tannic acid
can impair the growth of microorganisms by forming
complexes with the surface protein (10), or by inhibiting
the activities of important cellular enzymes (12 /15).
Table I
RESULTS AND DISCUSSION
Quantification of gastrointestinal flora after tannic acid treatment for various time periods
18
The dried faeces of both treated and control rats were first
dissolved in sterilized distilled water by homogenization.
The clear supernatant obtained from centrifugation (1000
g /5 min) was taken for microbial analysis. The total
bacterial flora of faeces was enumerated in nutrient agar
media by standard pour plate technique. The most probable
number of total coliforms and E. coli was assessed in
MacConkey and E. coli medium (E.C. broth) respectively
(11). The density of coliform bacteria was calculated using
an MPN table (11).
1.7/103
1.5/103
21
Analytical measurements
3.2 /108
6.5 /107
Control
Parameter
After adaptation, commercial tannic acid (C76H52O46; EMerck, Mumbai, India) was administered to a group of
animals at a dose of 45.0 mg per 100 g body weight daily
(LD50 /2260 mg/kg) for 21 days. Rat faeces were collected
just after dropping onto clean paper underlying the cage.
33
34
S. Samanta et al.
The most striking observation in this experiment is that
the quantities of all cultivable microbes were increased
several fold after 6 days of tannic acid treatment (Table I).
Bacterial flora, total coliforms and E. coli were increased
about 107-fold, 105-fold and 104-fold, respectively, after 21
days of experiment in comparison with their respective
normal levels. There may be several reasons for this
overgrowth of microbial population in the intestine. Osawa
and Sly (16) reported that enterobacteria could degrade
tannin /protein complex by secreting the extracellular
enzyme tannase. Tannase is an inducible enzyme in
bacteria, responsible for the breakdown of tannic acid
into gallic acid (3,4,5-trihydroxybenzoic acid) and glucose
(17, 18). These degradative products of tannic acid can
enter into the organism and induce their growth by
producing energy through the TCA (tricarboxylic acid)
cycle (10). Tannic acid also has an adverse effect on food
intake of animals by inhibiting the activities of digestive
enzymes and reducing the absorption capacity of food and
salts (19 /22). Apart from this, tannic acid can bind with the
epithelial cell layer of the alimentary canal and causes
ulceration (23). All these factors help in the availability of
nutrients for intestinal flora and induce their growth. In this
respect Bernasconi (5) noted that excessive microbial
proliferation causes digestive tract infections or systemic
infections in certain cases. The overgrowth of enterobacteria, low digestibility and overall toxicity of tannic acid
may be correlated with the decrease of body weight of rats
during treatment with tannic acid (Table I). Makkar (23)
noted that tannic acid has also hepatotoxic, nephrotoxic
and carcinogenic effects in animals.
From this experiment it can be stated that tannic acid can
disrupt the ecological balance of the gastrointestinal flora
of the rat. After 21 days of tannic acid treatment, the
excessive microbial proliferation, as well as diminution of
body weight of the experimental animal, established the
antinutrient and toxic effects of tannic acid. The average
daily intake of tannin by humans is about 1500 /2500 mg
(9), so the excess amount of tannin should be eliminated
from the food and fodder by chemical or biotechnological
means for improvement of food quality.
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ACKNOWLEDGEMENTS
The authors are grateful to Prof. P. Sengupta, Ex-principal, Raja
NL Khan Women’s College and Prof. C. Betal, Head, Department
of Physiology, Raja NL Khan Women’s College for their constant
encouragement and support regarding this work.
20.
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