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Small Ruminant Research 69 (2007) 217–220

Technical note

Phenotypic characterization of tannin–protein complexdegrading bacteria from faeces of goat

Gunjan Goel ∗, Anil Kumar Puniya, Kishan SinghDairy Microbiology Division, National Dairy Research Institute, Karnal 132 001, India

Received 24 June 2005; received in revised form 19 December 2005; accepted 29 December 2005Available online 7 February 2006

bstract

Tannin–protein complex degrading bacteria after enrichment were isolated from unadapted goat faecal samples. Based on theorphological, hemolytic and biochemical characters, the isolates were categorized in two groups comprising GF1–GF4 and

F5–GF6. All the isolates were gram-positive cocci, catalase negative belonging to different strains of Group D streptococci,nterococcus faecalis. Among six isolates, GF1 was the most resistant that could tolerate up to 4% of tannic acid in the mediumith no significant change in the morphology. Tannase activity was detected in all the isolates, indicating their tannin degradingotential while gallate decarboxylase activity was detected only in three isolates GF1, GF2 and GF6.

2006 Elsevier B.V. All rights reserved.

ase

eywords: Tannin; Goat; Enterococcus; Tannase; Gallate decarboxyl

. Introduction

Tannins, the polyphenolic compounds of higherlants have the ability to bind polymeric substrates likeroteins, carbohydrates and are reported to be toxico gastrointestinal microorganisms (Bhat et al., 1998).he inhibitory effect of tannins has been shown toe due to the reduction of enzyme activity, dysfunc-ioning of cell membrane and deprivation of substrate

etal ions and minerals (Goel et al., 2005a). Basedn their molecular structure, tannins are classified asydrolyzable (HT) and condensed (CT, proanthocyani-

ins) tannins. Reports are available that microorgan-sms can degrade HTs but there are evidences thathese have the ability to tolerate CTs (Krause et al.,

∗ Corresponding author. Fax: +91 1842250042;obile: +91 98960 25877.

E-mail address: gunjangoelndri@rediffmail.com (G. Goel).

921-4488/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.smallrumres.2005.12.015

2005). A number of gastrointestinal microorganismsfrom adapted domesticated and wild animals (e.g. sheep,goat, elk and deer) can play an important role in deriv-ing dietary proteins from tannin rich foliage. Osawa(1990) first reported the presence of tannin–protein com-plex (TPC) degrading Streptococcus sp. from koalas fedon Eucalyptus leaves. Odenyo et al. (1999) reportedthe existence of tannin tolerant or degrading microor-ganisms in the ruminal fluid of different African rumi-nants. Many other bacterial species of animal originsuch as Streptococcus gallolyticus (Osawa et al., 1995a),Selenomonas ruminantium (Skene and Brooker, 1995),Lonepinella koalarum (Osawa et al., 1995b), Strepto-coccus sp. (Goel et al., 2005b) have been reportedto degrade TPC. The present investigation was car-ried out to detect TPC degrading microbes in diges-

tive tract of small ruminants; goats that were not fedwith tannin rich forages. The effect of tannic acid onthe growth and morphology of TPC degraders was alsostudied.

nant Research 69 (2007) 217–220

Table 2Sugar fermentation pattern of the isolates

Carbohydrates GF1 GF2 GF3 GF4 GF5 GF6

Dextrose + + + + + +Maltose + + + + + +Sorbitol − − + + − +Rhamnose − − +/− − − −Xylose − − + +/− − +Lactose + + + + + +Fructose + + + + + +Raffinose + + − +/− + +Inulin − − − − − −Sucrose + + − + + +Mannitol − + +/− + + +Mannose + + + + + +Inositol − − − − − −Galactose + + +/− + + +Trehalose + + + + + +Arabinose − − − − − −Cellobiose + + − − + +Mellibiose +/− +/− + + + +Adonitol − − − − − −Dulcitol − +/− − − − +/−N-acetylglucosamine + + + + +/− +Methyl glucoside − − − − − −

218 G. Goel et al. / Small Rumi

2. Materials and methods

Faecal samples were collected in sterile specimenbottle directly from rectum of 10 goats maintained atthe National Dairy Research Institute, Karnal, havingno previous history of tannin rich forages in diet. Theenrichment of each sample was carried out in an enrich-ment broth consisting of mineral solution supplementedwith 1% tannic acid for 24 h. Thereafter, the isolationof bacteria was carried out on tannic acid overlaid brainheart infusion (BHI) agar plates at 37 ◦C for 48–96 h asper the method described by Osawa (1990). After incu-bation, the plates were observed for tannin hydrolyzingzone around the colonies. The isolates showing visi-ble zone of hydrolysis were subcultured in BHI brothand identified by morphological examination, hemoly-sis, sugar fermentation profile and standard biochemicaltests (Tables 1 and 2). The tests used for identificationwere based on the previous reports of tannin degrad-ing organisms (Osawa, 1990; Osawa and Sly, 1991;Ephraim et al., 2005). Utilization of xylan and cellulosewas determined qualitatively by Congo-red method by

streaking the cultures on BHI agar plates supplementedwith 0.5% of each polysaccharide. After incubation, theplates were flooded with 0.3% Congo-red solution andobserved for zone of hydrolysis. Utilization of tannic

Table 1Biochemical tests used for characterization of isolates

Tests GF1 GF2 GF3 GF4 GF5 GF6

Zone on T-BHI + + + + + +Colonies on blood agar � � � � � �

Utilization of cellulose − − + + + +Utilization of xylan + + + + + +Catalase − − − − − −Gelatinase − − − − − −Tannase + + + + + +Gallate decarboxylase + + − − − +Arginine dihydrolase + + + + + +�-Galactosidase − − + + + +�-Glucouronidase − − + − − +Esculin hydrolysis + + + + + +Voges-Proskauer + + + + + +Reduction of 0.01%tetrazolium

+ + + + + +

Reduction of 0.04%tellurite

+ + + + + +

Hippurate hydrolysis − − − − − −Clots on litmus milk + + + − + +Growth at 10 ◦C + + + + + +Growth at 45 ◦C + + + + + +Growth at 6.5% NaCl + + + + + +Resistance to pH 9.6 + + + + + +

+, isolate is able to produce enzyme/utilize of substrate; −, not able toproduce enzyme/utilize the substrate.

Salicin + + − − +/− −Erythritol − − − − − −Glycerol − − − − − −Amygdalin + + + + + +

+, the isolate is able to ferment the sugar; −, the isolate is not able toferment the sugar; +/−, the isolate is weakly fermenting the sugar.

acid was determined by swarm plate assay on T-BHIplates (Chowdhury et al., 2004). Tannic acid resistanceability of the isolates was determined in BHI broth sup-plemented with (0–4% TA). Because of the interferenceof tannin–protein complex with optical density, standardviable cell count was used to determine the growth of theculture. The cultures were examined under oil immersionfor morphological variations (Goel et al., 2005a). Tan-nase and gallate decarboxylase activity was determinedby visual assay method (Osawa and Walsh, 1993, 1995)

3. Results and discussion

Six isolates having capability of degradingtannin–protein complex were obtained from goatswithout any tannin rich feeding habitat. The colonieswith zone of degradation appeared after 3 days ofincubation suggesting that the bacteria preferentiallyutilizes other substrates first followed by tannic acid

and thereafter, resulting in a zone of hydrolysis. Threedifferent types of colonies were obtained on T-BHIagar plates viz.: (i) pin pointed, circular colonies with alarge zone of the tannin degradation, (ii) large, mucoid

G. Goel et al. / Small Ruminant Research 69 (2007) 217–220 219

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densed tannins. Anim. Feed Sci. Technol. 121, 59–75.

ig. 1. Tannin–protein complex degrading bacteria from goat faecesn T-BHI plate showing zone of hydrolysis.

olonies with a small zone around their periphery andiii) medium sized glistening colonies without anyone of tannin degradation (Fig. 1). Six representativeolonies with distinct characteristics named GF1–GF6ere subcultured. Morphologically all the isolates wereram-positive diplococci or cocci in chain of threeo four cells. Tables 1 and 2 depicts the biochemicalroperties and difference in the sugar fermentationattern of the isolates. All the isolates were catalaseegative and showed � (GF1–GF4) and � (GF5 andF6) hemolysis on blood agar. During Congo-red assay

or enzyme activity the isolates GF3–GF6 showed bothhe xylanase and cellulase activity while the isolatesF1 and GF2 showed only xylanase activity. The

solates showed some differences in sugar fermentationattern. Biochemically, all the isolates were negativeor hippurate hydrolysis and gelatin liquefication.ll the isolates were able to grow at pH 9.6, sodium

hloride at concentration of 6.5% and at a temperaturef 10 and 45 ◦C. The isolates were also able to reduce.01% tetrazolium, 0.04% tellurite and found to possessrginine dihydrolase and esculin hydrolysis activity.n the basis of biochemical activities, all the isolatesere found to belong to different strains of Group

streptococci, Enterococcus faecalis (Manero andlanch, 1999).

The bacteria on T-BHI moved from the center toeriphery indicating positive test result in swarm platessay. The isolate GF1 was the most resistance organ-

sm (up to 4% of TA) compared to 3% resistance ofsolate GF2 and rest were resistant to only 1% of TA. Anxtended lag phase of 3 and 6 h observed at a tannic acidoncentration of 3 and 4% indicated the inhibitory effect

Fig. 2. Tannic acid resistance ability of the isolate GF1.

of tannic acid on the growth of isolate GF1 (Fig. 2).The increased tannic acid in the medium resulted indecreased growth rate of the isolates. There were no sig-nificant variations in the morphology in comparison tothe control, when the isolates were grown in increasedtannic acid concentration of 0–4%. However, formationof long chains was not observed as reported by Goel etal. (2005b) for ruminal streptococcal isolate C-4. Tan-nase activity was detected in all the isolates, indicatingconversion of tannic acid into gallic acid. Gallate decar-boxylase activity was detected only in three isolates GF1,GF2 and GF6 indicating their potential to convert gallicacid into pyrogallol.

From the above finding, it can be stated thattannin–protein complex degrading enterococci arepresent in the faeces of non-adaptive goats, which canbe further exploited for their ability to degrade tanninsin the tannin rich forages.

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