Tej K. Bhat

Microbial degradation of tannins – A current perspective

Tannins are water-soluble polyphenolic compounds having wide prevalence in plants. Hydrolysable and condensed tannins are the two major classes of tannins. These compounds have a range of effects on various organisms – from toxic effects on animals to growth inhibition of microorganisms. Some microbes are, however, resistant to tannins, and have developed various mechanisms and pathways for tannin degradation in their natural milieu. The microbial degradation of condensed tannins is, however, less than hydrolysable tannins in both aerobic and anaerobic environments. A number of microbes have also been isolated from the gastrointestinal tract of animals, which have the ability to break tannin-protein complexes and degrade tannins, especially hydrolysable tannins. Tannase, a key enzyme in the degradation of hydrolysable tannins, is present in a diverse group of microorganisms, including rumen bacteria. This enzyme is being increasingly used in a number of processes. Presently, there is a need for increased understanding of the biodegradation of condensed tannins, particularly in ruminants.

Thin-layer chromatography of gallic acid, methyl gallate, pyrogallol, phloroglucinol, catechol, resorcinol, hydroquinone, catechin, epicatechin, cinnamic acid, p-coumaric acid, ferulic acid and tannic acid

Six solvent systems of varying suitability are reported for the thin-layer chromatographic separation of simple phenolics and related compounds such as gallic acid, methyl gallate, pyrogallol, phloroglucinol, catechol, resorcinol, hydroquinone, catechin, epicatechin, cinnamic acid, p-coumaric acid, ferulic acid and tannic acid. The solvent system chloroform-ethyl acetate-acetic acid (50:50:1) facilitated the separation of all the compounds except pyrogallol and ferulic acid; resorcinol and hydroquinone, which were in turn resolved in benzene-dioxane-acetic acid (85:15:1). Detection was carried out using iodine vapour, ferric chloride reagent, ferric chloride-ferricyanide reagent and vanillin-sulfuric acid reagent.

Biodegradation of tannic acid in an in vitro ruminal system

Abstract The microbial degradation of tannic acid by ruminal fluid of cattle which had no prior exposure to tannin-rich diets, was studied in an in vitro syringe system. Tannic acid caused an increase in the gas volume and a decrease in the ammonia concentration of the buffered rumen liquor during incubation for 24–72 h. The fermentation products from tannic acid were gallic acid, pyrogallol and resorcinol. This is the first report on the ability of ruminal fluid taken from cattle unexposed and unadapted to tanniniferous diets to degrade tannic acid into simpler products.

Value Addition of Feed and Fodder by Alleviating the Antinutritional Effects of Tannins

Tannins are one of the important plant secondary metabolites having wide prevalence in the plant kingdom. They are a prominent constituent of various types of feed, fodder and agro-industrial wastes. The intake of tannins at a low level has recently been found to have some positive effects in ruminants. However, the use of tannin-rich biomass as animal feed, having high content of tannins, is limited by the antinutritional effects of tannins at this level in an animal system. A number of physical, chemical, biological and miscellaneous approaches have been developed for inactivation or removal of tannins for enhancement of the feeding value of tannin-rich biomass. However, none of the individual method is successful in total inactivation or removal of tannins without loss of nutritive value, and this limits the utilization of a vast amount of plant resource. A cohesive and an integrated detanninification strategy is required for alleviating the antinutritional effects of tannins in animals and upgrading the feeding value of tanniniferous biomass.

The Environmental and Human Effects of Ptaquiloside-Induced Enzootic Bovine Hematuria: A Tumorous Disease of Cattle

In this review, we address the major aspects of enzootic bovine hematuria and have placed special emphasis on describing the etiology, human health implications, and advanced molecular diagnosis of the disease.Enzootic bovine hematuria (EBH) is a bovine disease characterized by the intermittent presence of blood in the urine and is caused by malignant lesions in the urinary bladder. This incurable disease is a serious malady in several countries across many continents. Accurate early-stage diagnosis of the disease is possible by applying advanced molecular techniques, e.g., detection of genetic mutations in the urine of cows from endemic areas. Use of such diagnostic approaches may help create an effective therapy against the disease.There is a consensus that EBH is caused primarily by animals consuming bracken fern (P. aquilinum) as they graze. The putative carcinogen in bracken is ptaquiloside(PT), a glycoside. However, other bracken constituents like quercetin, isoquercetin,ptesculentoside, caudatoside, astragalin, and tannins may also be carcinogenic.Studies are needed to identify the role of other metabolites in inducing urinary bladder carcinogenesis.The bovine papilloma virus is also thought to be an associated etiology in causing EBH in cattle. There is growing alarm that these fern toxins and their metabolites reach and contaminate the soil and water environment and that the carcinogen (PT)is transmitted via cows milk to the human food chain, where it may now pose a threat to human health. An increased incidence of gastric and esophageal cancer has been recorded in humans consuming bracken ferns, and among those living for long periods in areas infested with bracken ferns.Although preliminary therapeutic vaccine trials with inactivated BPV-2 against EBH have been performed, further work is needed to standardize and validate vaccine doses for animals.

An improved method for thin layer chromatographic analysis of saponins.

Analysis of saponins by thin layer chromatography (TLC) is reported. The solvent system was n-butanol:water:acetic acid (84:14:7). Detection of saponins on the TLC plates after development and air-drying was done by immersion in a suspension of sheep erythrocytes, followed by washing off the excess blood on the plate surface. Saponins appeared as white spots against a pink background. The protocol provided specific detection of saponins in the saponins enriched extracts from Aesculusindica (Wall. ex Camb.) Hook.f., Lonicera japonica Thunb., Silene inflata Sm., Sapindusmukorossi Gaertn., Chlorophytum borivilianum Santapau & Fernandes, Asparagusadscendens Roxb., Asparagus racemosus Willd., Agave americana L., Camellia sinensis [L.] O. Kuntze. The protocol is convenient, inexpensive, does not require any corrosive chemicals and provides specific detection of saponins.

Biotransformation of lantadenes, the pentacyclic triterpenoid hepatotoxins of lantana plant, in guinea pig.

Oral administration of lantana (Lantana camara var. aculeata) leaf powder to guinea pigs at a dose of 6 g/ kg body weight elicited cholestasis. The animals were euthanized 48 h after dosing. Liver homogenates, bile, gall bladder, blood, urine, contents of gastrointestinal tract (GIT) and faeces were analysed for the principal hepatotoxin in lantana leaves viz. lantadene A (LA), its congeners and biotransformation products, using high performance liquid chromatographic technique. Lantadenes could not be detected in liver, bile, gall bladder, blood and urine samples. LA and lantadene B (LB), their derivatives reduced lantadene A (RLA), reduced lantadene B (RLB) and two unidentified metabolites could be detected in the contents of lower GIT and faeces. In vitro incubation of lantana leaf powder with guinea pig caecal contents under anaerobic conditions elicited biotransformation of LA and LB to RLA and RLB, respectively. On the other hand, incubation of lantana leaf powder with cattle rumen liquor under anaerobic conditions did not elicit biotransformation of lantadenes.

Disposition of lantadene A, the pentacyclic triterpenoid hepatotoxin, orally administered to guinea pigs.

Lantadene A (LA) administered orally to guinea pigs elicited cholestasis. LA could not be detected in liver, bile, gall bladder, blood and urine. LA and its biotransformation product reduced lantadene A (RLA) could be detected in caecum, large intestine, and faeces. In vitro incubation of LA with liver homogenates under aerobic and anaerobic conditions did not elicit its biotransformation to RLA. On the other hand, in vitro incubation of LA with guinea pig caecal and large intestinal contents under anaerobic conditions elicited conversion of LA to RLA. This is the first report of the biotransformation of LA in the animal system.

Metagenomics in animal gastrointestinal ecosystem: a microbiological and biotechnological perspective.

Metagenomics- the application of the genomics technologies to nonculturable microbial communities, is coming of age. These approaches can be used for the screening and selection of nonculturable rumen microbiota for assessing their role in gastrointestinal (GI) nutrition, plant material fermentation and the health of the host. The technologies designed to access this wealth of genetic information through environmental nucleic acid extraction have provided a means of overcoming the limitations of culture-dependent microbial genetic exploitation. The molecular procedures and techniques will result in reliable insights into the GI microbial structure and activity of the livestock gut microbes in relation to functional interactions, temporal and spatial relationships among different microbial consortia and dietary ingredients. Future developments and applications of these methods promise to provide the first opportunity to link distribution and identity of rumen microbes in their natural habitats with their genetic potential and in situ activities.

Fungal degradation of lantadene A, the pentacyclic triterpenoid hepatotoxin of lantana plant

Seven white-rot fungi, known to degrade complex biomolecules and xenobiotics were used for investigation on biodegradation of lantadene A (LA), the bioactive pentacyclic triterpenoid of Lantana camara. Pleurotus sajor-caju and Merulius tremellosus PRL-2845 did not degrade LA. Trametes versicolor MTCC-138, Heterobasidion annosum MTCC-146, Phellinus pini RAB-83-19, Pleurotus ostreatus MTCC-142 and Phlebia radiata 2 utilized LA to the extent of 11-15.7%. This is the first report on the fungal degradation of a pentacyclic triterpenoid.