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ORIGINAL ARTICLE æ æ 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. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 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. REFERENCES 21. 1. Apajalahti JH, Sarkilahti LK, Maki BR, Heikkinen JP, Nurminen PH, Holben WE. Effective recovery of bacterial DNA and percent guanine-plus-cytosine-based analysis of community structure in the gastrointestinal tract of broiler chickens. Appl Environ Microbiol 1998; 64: 4084 /8. 2. Apajalahti JH, Kettunen A, Bedford MR, Holben WE. Percent G/C profiling accurately reveals diet-related differences in the 19. 22. 23. gastrointestinal microbial community of broiler chickens. Appl Environ Microbiol 2001; 67: 5656 /67. Apajalahti JH, Kettunen H, Kettunen A, Holben WE, Nurminen PH, Rautonen N, Mutanen M. Culture independent microbial community analysis reveals that inulin in the diet primarily affects previously unknown bacteria in the mouse caecum. Appl Environ Microbiol 2002; 68: 4986 /95. Madigan MT, Marlinko JM, Parkar J. Host /parasite relationship. In: Brock Biology of Microorganism, 9th edn. PrenticeHall, Inc, Upper Saddle River, New Jersey, 2000: 773 /9. Bernasconi P. The Intestinal Flora and Ecosystem. France: Scientifiques des Laboratories Biocodes, 1984: 16 /50. Schlegel HG. Microorganisms and the environment. In: General Microbiology, 7th edn. Cambridge: Cambridge University Press, 1995: 568 /97. Jankauskiene R. Defence mechanisms in fish: Lactobacillus genus bacteria of intestinal wall in feeding and hibernating carps. Ecology 2000; 1: 3 /6. Swain T. Plant Biochemistry. New York: Academic Press, 1965: 552. Rao BSN, Prabhavathi T. Tannin content of some Indian foods and feeds. J Sci Food Agric 1982; 33: 89 /94. Bhat TK, Singh B. Sharma OP. Microbial degradation of tannins / a current perspective. Biodegradation 1998; 9: 343 / 57. Toranzos GA, McFeters GA. Detection of indicator microorganisms in environmental freshwaters and drinking waters. In: Manual of Environmental Microbiology. Washington DC: ASM Press, 1997: 184 /97. McLeod NM. Plant tannins / their role in forage quality. Nutr Abstr Rev 1974; 44: 803 /15. Chesson A, Stewart CS, Wallace RJ. Influence of plant phenolic acids on growth and cellulolytic activity of rumen bacteria. Appl Environ Microbiol 1982; 44: 597 /603. Makkar HPS, Singh B, Bawra RK. Effect of tannin-rich leaves of oak (Quercus incana ) on various microbial enzyme activities of the bovine rumen. Br J Nutr 1988; 60: 287 /96. Martin SA, Akin DE. Effect of phenolic monomers on the growth and glucosidase and xylanase activity of Bacteroides ruminicola and on the carboxymethyl cellulase, b-glucosidase and xylanase activities of Bacteroides succinogenes. Appl Environ Microbiol 1988; 54: 3600 /4. Osawa R, Sly L. Occurrence of tannin-protein complex degrading Streptococcus sp. in feces of various animals. Syst Appl Microbiol 1992; 15: 144 /7. Mondal KC, Pati BR. Studies on the extracellular tannase from newly isolated Bacillus licheniformis KBR 6. J Basic Microbiol 2000; 40: 223 /32. Mondal KC, Banerjee D, Banerjee R, Pati BR. Production and characterization of tannase from Bacillus cereus KBR9. J Gen Appl Microbiol 2001; 47: 263 /9. Kumar R, Singh M. Tannins: their adverse role in ruminant nutrition. J Agric Food Chem 1984; 32: 447 /53. Reddy NR, Pierson MD, Sathe SK, Salunkhe DK. Dry bean tannins: a review on nutritional implications. JAOCS 1985; 62: 540 /9. Prinz JF, Lucas PW. Saliva /tannin interactions. J Oral Rehabit 2000; 27: 991 /4. Glahn RP, Wortley GM, South PK, Miller DD. Inhibition of iron uptake by phytic acid, tannic acid, and ZnCl2; studies using an in vitro digestion/CaCo-2 cell model. J Agric Food Chem 2002; 50: 390 /5. Makkar HPS. Tannins / bane or boon? Sci Reporter 1988; Jan: 18 /23.