Screening of Tannase-Producing Fungi Isolated from Tannin-Rich Sources

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1 International Journal of Agricultural and Food Research ISSN Vol. 2 No. 3, pp (2013) Screening of Tannase-Producing Fungi Isolated from Tannin-Rich Sources Hamada A. Abou-Bakr *, Malak A. El-Sahn and Amr A. El-Banna Department of Food Science and Technology, Faculty of Agriculture, Alexandria University, Alexandria, Egypt Abstract Realizing the importance of the enzyme tannase, the present study aims to isolate and screen high tannase-producing fungi from tannin-rich sources. One hundred and five isolates of tannase-producing fungi were isolated from nineteen tannin-rich sources from local environment in Egypt. Isolates were identified and primary screened using tannic acid agar plate method. Eleven fungal cultures were chosen as the best tannase producers. These fungal cultures were subjected to quantitative secondary screening for tannase production using submerged fermentation technique. Accordingly, four fungal cultures, which exhibited high tannase activity were chosen to undergo final screening. A fungal culture, isolated from tannery soil sample, was considered as the most promising and identified as Aspergillus niger Van Tieghem. Tannase produced by the fungus was mainly extracellular with negligible intracellular tannase and considered as inducible enzyme. Keywords: Fungal tannase; Tannase activity; Screening; Aspergillus niger; Tannin-rich sources 1. Introduction Tannase (tannin acyl hydrolase; EC ) is one of the hydrolases and is known to catalyze the breakdown of ester and depside bonds from hydrolysable tannins and gallic acid esters. This enzyme is known to display two different activities. The first one is an esterase activity; by which it can hydrolyze ester bonds of gallic acid esters with glucose (galloyl-glucose) or alcohols (e.g. methyl gallate). The second activity is called depsidase activity; by which it can hydrolyze depside bonds of digallic acid (Haslam and Stangroom, 1996; Saxena and Saxena, 2004; Sharma et al., 2000). Tannase is an industrially important enzyme and has several applications in various industries such as foods, animal feeds, cosmetic, pharmaceutical, chemical, leather industries and so on. (Aguilar and Gutierrez-Sanchez, 2001; Aguilar et al., 2007; Dũenas et al., 2007; Jun et al., 2007) Tannase can be obtained from plants, animals, and microbial sources. Microorganisms are considered as the most important and commercial sources of tannase, that is because the produced tannases are more stable than similar ones obtained from the other sources. Moreover, microorganisms can produce tannase in high quantities in a constant way. Microbial tannase is favoured also because the microbes can be subjected to genetic manipulation more readily than plants and animals, resulting in an increase in tannase production (Aguilar and Gutierrez-Sanchez, 2001; Purohit et al., 2006). * Corresponding author: hamada.metwali@alex agr.edu.eg

2 2 Abou Bakr, El Sahn and El Banna 2013 Screening of Tannase Producing Fungi Fungi are the most studied microorganisms for tannase production. Fungi have the ability to degrade tannins as a sole carbon source (Aguilar and Gutierrez-Sanchez, 2001). The common genus used for tannase production either for research purposes or industrial production was Aspergillus and the common Aspergillus species used for tannase production was Aspergillus niger. Tannase-producing fungi were isolated from soils and tannery effluent (Enemuor and Odibo, 2009; Manjit et al., 2008). The present investigation aims to isolate and screen high tannase-producing fungi from tanninrich sources. 2. Materials and Methods 2.1 Chemicals The chemicals used throughout the study were of analytical reagent grade and obtained from Sigma, Aldrich, Merck and VEB Laborchemie Apolda (Germany); BDH and JUDEX chemicals (England); Biolife (Italy); Chemapol (Czechoslovakia) and El-Nasr Pharma-ceutical Chemicals Co. ADWIC (Egypt). 2.2 Sources for Isolation of Tannase-Producing Fungi Nineteen samples collected from various natural sources (9 samples of mouldy tannins-rich plants, 8 samples of soil down tannin-rich plants, a sample of tannery soil and a sample of tannery effluent) were used to isolate tannase-producing fungi. All samples were collected from various locations in Alexandria and El-Beheira Governorates, Egypt. Samples were transported to laboratory either for direct use or to be preserved in refrigerator at 4º C until used. 2.3 Media Tannic acid agar medium (TAA) was used for the isolation of tannase-producing fungi and for the primary screening of fungal isolates (Pinto et al., 2001). It was prepared from the following components, g/l: tannic acid,10; NaNO 3, 3; KCl, 0.5; MgSO 4 7H 2 O, 0.5; KH 2 PO 4, 1.0; FeSO 4 7H 2 O, 0.01; agar, 30. The medium was adjusted at ph 4.5± 0.2 and sterilized at 121º C for 15 min. The solution of tannic acid was sterilized separately by passing through MILLEX -OR membrane filter (33 mm diameter, 0.22 µm pore size, Millipore, France) and adjusted separately at ph 4.5± 0.2, then added to the medium. The modified Czapek-Dox s minimal medium was used for secondary and final screening of fungi (Bradoo et al., 1996). It was prepared from the following ingredients,g/l: tannic acid, 10; NaNO 3, 6; KCl, 0.52; MgSO 4 7H 2 O, 0.52; KH 2 PO 4, 1.52; FeSO 4 7H 2 O, 0.01; ZnSO 4 7H 2 O, 0.01; Cu(NO 3 ) 2 3H 2 O, 0.01; ph 4.5± 0.2. Sterilization and adjustment of ph were carried out as mentioned in preparation of TAA medium. Potato dextrose agar (PDA) supplemented with 0.01% tannic acid was used for maintaining fungal isolates (Bajpai and Patil, 1997). It was prepared from the following ingredients, g/l: glucose, 20; infusion of 200 g potatoes; agar, 15; tannic acid, 0.1; ph 5.6± Isolation of Tannase-Producing Fungi Each isolation source sample was serially diluted (10-1 to 10-4 ) with sterile distilled water. One milliliter from each dilution was plated into TAA medium using pour plate technique. Incubation was carried out at 30º C for 96 hrs under aerobic conditions. Fungi capable to grow and form clearing zone around its colonies due to the hydrolysis of tannin were selected and purified (Murugan et al., 2007).The cultures obtained were grown on PDA, supplemented with 0.01 tannic acid, slants and maintained at 4º C (working cultures). Stock cultures were maintained under paraffin oil. 2.5 Screening and Selection of Tannase- Producing Fungal Cultures Primary screening for highest tannase producers was carried out using TAA plates as described by Bradoo et al. (1996). The plates were point inoculated with the isolate and incubated at 30º C. The diameter of clear zones (including the colonies diameters) formed due to hydrolysis of tannic acid around the fungal colonies were measured after 96 hrs of incubation, then compared in order to choose the highest tannase producers. All measurements were carried out in triplicates. Fungal cultures, which exhibited high tannase activity in the primary screening, were subjected to secondary screening using submerged fermentation technique (Batra and Saxena, 2005; Bradoo et al.,

3 International Journal of Agricultural and Food Research Vol. 2 No. 3, pp ). Approximately spores of each of potent tannase-producing fungi were inoculated into 250 ml-erlenmeyer flasks containing 50 ml of sterilized modified Czapek-Dox s minimal medium (ph 4.5± 0.2). Cultures were grown at 30º C for 96 hrs and shook intermittently three times a day at 200 rpm for 2 minutes each time in an orbital incubator (Model INR-200, Gallenkamp, UK). At the end of fermentation time the extracellular and intracellular tannase activity per flask were assayed. The promising fungal cultures that were selected from the secondary screening were subjected to final screening using the above mentioned method. Samples were withdrawn at regular intervals of 24 hrs and assayed for extracellular tannase activities. Extracellular, intracellular and total tannase activities per flask were measured in triplicates. Comparison between the means of tannase activities for each tested culture was carried out statistically. The analysis of variance (one way ANOVA) was carried out by STATESTICA 7.0 software (StatSoft, Inc., USA) and differences among the means were determined for significance at p<0.05 using Fisher Least Significant Difference test (LSD 0.05 ). 2.6 Inoculum Preparation Inoculum was prepared by the method described by Ramirez-Coronel et al. (2003). The fungal isolate was cultivated on PDA slant then incubated for 4 days at 30 C until a good sporulation was obtained. Spores were then scraped into a sterile 0.02% Tween 80 solution and counted in a Neubauer chamber (Harisha, 2007). The suitable volume, which contained the needed number of spores was calculated. 2.7 Identification of Fungal Isolates The cultural characteristics of the selected fungal isolates on PDA were recorded. The fungal isolates were also examined microscopically. The slide agar method (Benson, 2001) was used for preparation of isolates. Slides were examined microscopically under low and high power after staining with lactophenol cotton blue stain. Simplified key to some selected common genera (Barnett and Hunter, 1987) was used for identification of isolates to the level of genera. Species of Aspergillus were identified according to the description of Doctorfungus database. Macroscopic and microscopic features of selected fungal isolates were compared with descriptions and photos of fungal genera and species (Barnett and Hunter, 1987; Doctor fungus, 2009). 2.8 Harvesting the Enzyme and Enzyme Assay The fermentation medium was filtered through Whatman No.1 filter paper. The obtained filtrate was used for extracellular tannase determination (Gupta et al., 1997). The method described by Sharma et al. (2000) was used for harvesting the intracellular tannase. Tannase activity was assayed using the spectrophotometric method (Sharma et al., 2000). Methyl gallate was used as substrate. The method is based on the chromogen formation between gallic acid and rhodanine. The method was modified by using 0.2 ml of 1 N instead of 0.5 N of potassium hydroxide solution for enhancing the color formation. One unit (U) of the enzyme was defined as micromoles of gallic acid formed per minute by 1 ml of enzyme extract under optimum conditions of tannase activity. 3. Results and Discussion 3.1 Isolation of Tannase Producing Fungi One hundred and five isolates of tannaseproducing fungi were isolated from nineteen samples collected from the environment at Alexandria and Beheira Governorates, Egypt. Source, number, scientific name and code number of isolates are presented in Table 1. Selection of the isolation sources was based on the fact that the presence of tannase-producing microorganisms may fairly exist in tannins containing environments. Many authors used similar isolation sources for isolating tannase-producing fungi. Tannery effluent samples were used for isolating tannaseproducing fungi (Batra and Saxena, 2005; Manjit et al., 2008; Murugan et al., 2007). Tannaseproducing Aspergillus tamarii was isolated from soil inundated by effluent of a tannery at Oji River local Government Area of Enugu State, Nigeria (Enemuor and Odibo, 2009). Tannase-producing Rhizopus oryzae was isolated from soil sample of Indian Institute of Technology campus (Hadi et al., 1994).

4 4 Abou Bakr, El Sahn and El Banna 2013 Screening of Tannase Producing Fungi Table 1 Source, number, scientific name and code number of tannase-producing isolates Isolation source Bengal fig (Ficus benghalensis ) fruits Number of isolates Scientific name and code number of isolate Aspergillus Aspergillus Aspergillus Fusarium niger flavus spp. spp. Penicillium spp. 5 N1, N5 N N3, N4 -- Unripe dates 6 J3, J4, J5, J6 -- J J1 Guava leaves 5 R1, R R5 R4 R2 Persimmon (Diospyrus kaki) fruits 7 H4, H5, H6 H H1, H7 H3 Trichoderma spp. Pomegranate (Punica granatum) leaves 5 P2, P3, P P1 P5 Pomegranate fruits 4 G3, G4, G G4 -- Pomegranate peels 4 K2 K K1 K4 River-red-gum (Eucalyptus camaldulensis Dehn) leaves 4 F2 F F4 F3 Tea (Green leaves) 3 M2, M M1 -- Soil down bengal fig tree 6 O2, O3, O6 O O5 O4 Soil down date palm trees (Muddy) Soil down date palm trees (Sandy 1) Soil down date palm trees (Sandy 2) 5 B1, B2, B B4, B D D2 D3 10 E4, E5, E6, E9 E7 E3 E8 E2, E12 E10 Soil down guava trees 8 S2, S3, S6, S7 S1, S S4, S8 -- Soil down pomegranate trees 5 Q1, Q2, Q4 Q Q3 -- Soil down river-red-gum trees 6 C2, C4 C3 -- C5 C1 C6 Soil from plant pot 6 L5, L6 L4 L3 -- L1 L2 Tannery soil 7 I5, I6, I7, I1 I3 -- I2 I4 Tannery effluent 6 A2, A4, A5 A6 A3 -- A1 --

5 International Journal of Agricultural and Food Research Vol. 2 No. 3, pp Table 2 Primary screening of tannase producers on tannic acid agar plates after 96 h Fungal culture code The sum of colony and hydrolytic clear zone diameters (mm) ± SD Fungal culture code The sum of colony and hydrolytic clear zone diameters (mm) ± SD Fungal culture code The sum of colony and hydrolytic clear zone diameters (mm) ± SD A1 9 ± 0 G1 21 ± 0.6 M3 40 ± 0.6 A2 42 ± 0 G2 26 ± 0.6 N1 42 ± 0 A3 39 ± 0.6 G3 42 ± 0.6 N2 29 ± 0 A4 46 ± 0.6 G4 47 ± 1 N3 18 ± 0 A5 36 ± 0 H1 29 ± 0 N4 9 ± 0 A6 24 ± 0.6 H2 27 ± 0 N5 38 ± 0.6 B1 38 ± 0 H3 7 ± 0 O1 28 ± 0 B2 53 ± 0 H4 42 ± 0 O2 42 ± 0 B3 40 ± 0 H5 39 ± 0 O3 44 ± 1 B4 9 ± 0.6 H6 50 ± 2.3 O4 16 ± 0 B5 10 ± 0 H7 11 ± 0 O5 10 ± 0 C1 13 ± 0.6 I1 11 ± 0 O6 45 ± 1 C2 35 ± 0.6 I2 40 ± 0 P1 11 ± 0 C3 26 ± 0 I3 44 ± 1.4 P2 49 ± 0.6 C4 16 ± 0 I4 47 ± 0.6 P3 44 ± 0 C5 7 ± 0.6 I5 52 ± 0 P4 51 ± 0 C6 10 ± 0 I6 45 ± 1.4 P5 12 ± 1.2 D1 36 ± 1.2 I7 53 ± 0 Q1 48 ± 0.6 D2 12 ± 0.6 J1 6 ± 0 Q2 52 ± 0 D3 10 ± 0 J2 15 ± 0 Q3 13 ± 0.6 E1 25 ± 0 J3 42 ± 0.6 Q4 50 ± 0 E2 11 ± 0 J4 49 ± 0.6 Q5 27 ± 0.6 E3 25 ± 0 J5 41 ± 0 R1 39 ± 0 E4 44 ± 0.6 J6 43 ± 0 R2 18 ± 0 E5 35 ± 1 K1 21 ± 2.3 R3 50 ± 0 E6 49 ± 0 K2 39 ± 0 R4 15 ± 0.6 E7 10 ± 0 K3 20 ± 0 R5 16 ± 0.6 E8 4 ± 0 K4 5 ± 0 S1 27 ± 0 E9 40 ± 1.2 L1 18 ± 0 S2 40 ± 0.6 E10 10 ± 0 L2 27 ± 0 S3 47 ±1.2 E11 6 ± 0 L3 14 ± 0.6 S4 10 ± 0 E12 34 ± 0.6 L4 8 ± 0 S5 28 ± 0 F1 25 ± 0.6 L5 47 ± 1 S6 39 ± 0.6 F2 40 ± 0.6 L6 44 ± 1.2 S7 48 ± 1.5 F3 25 ± 0.6 M1 10 ± 0 S8 9 ± 0 F4 26 ± 0 M2 45 ± 0

6 6 Abou Bakr, El Sahn and El Banna 2013 Screening of Tannase Producing Fungi Sixteen potent tannase-producing fungi were isolated from different forest soils of Midnapore District; West Bengal, India (Banerjee et al., 2001). Soil samples of Tunisian olive oil mill were used for isolation of tannase producing fungi (Kachouri et al., 2005). None of the plants studied in the present work (Table 1) was reported as a source for tannase-producing fungi. 3.2 Primary Screening of Tannase Producers Data presented in Table 2 show that the sum of colony and hydrolytic clear zone diameters vary among fungal cultures and ranged from 4 to 53 mm. The fungal cultures coded B2 and I7, I5, Q2, P4, Q4, R3, H6, P2, E6 and J4 organized in descending order had the largest sum of colonies and hydrolytic clear zone diameters, consequently, they were chosen as the best tannase- producers and were subjected to secondary screening. Pinto et al. (2001) preferred determination of the colonies diameter instead of diameter of the clear zones because the observation of the clear zones was very difficult. 3.3 Secondary and Final Screening of Selected Fungal Isolates Eleven selected fungal cultures from the primary screening step were subjected to secondary screening for tannase production. Data presented in Table 3 show that there were significant differences (p<0.05) between total tannase production of the tested isolates. Culture No. I5 exhibited the highest tannase productivity (313.7 total tannase units/ flask) followed by cultures Q2, P4 and then B2. Significant correlation (r =0.75) between the sum of colony and hydrolytic clear zone diameters (primary screening) and mean total tannase activity (secondary screening) was found. Data are illustrated in Figure 1. Similar results were reported by other investigators (Bradoo et al., 1996; Murugan et al., 2007; Pinto et al., 2001). All tested cultures produced tannase mainly extracellularly (97.4, 97.8, 96.9 and 98.1 % of total tannase units/flask for isolates I5,Q2, P4 and B2, respectively), in addition to negligible intracellular amounts. Therefore, it can be concluded that tannase produced by tested cultures is extracellular enzyme. This result seems to be logical because it is well known that the tannic acid molecules have great molecular size and mass that prevent crossing of such compounds through the cell membrane of microorganisms. Therefore, the microorganism has to excrete the tannin-hydrolytic enzyme (tannase) extracellularly in order to breakdown such big molecules outside its cells into smaller derivatives that can cross through its membranes as nutrients (Bradoo et al., 1997). Table 3 Secondary screening of promising fungal isolates selected from the primary screening step Isolate code Mean of extracellular tannase (U/flask ± SD) Mean of intracellular tannase (U/flask ± SD) Mean of total tannase activity (U/flask ± SD) B ± ± ± 13.8 c E ± ± ± 2.9 de H ± ± ± 7.3 d I ± ± ± 7.9 a I ± ± ± 7.4 c J ± ± ± 8.6 f P ± ± ± 9.5 de P ± ± ± 2.1 bc Q ± ± ± 14.7 b Q ± ± ± 5.1 d R ± ± ± 53.6 e The same superscript letters indicate that there is no significant difference at p < The experiment was carried out in 250 ml-conical flasks containing 50 ml of the modified Czapek-Dox s minimal medium containing 1% filter sterilized tannic acid. Fermentation conditions: initial ph, 4.5±0.2; Temperature, 30º C; inoculum size 5x10 7 spores/flask; shaking, intermittently 3 times a day at 200 rpm for 2 min each time. Aguilar et al. (2001) found that Aspergillus niger Aa-20 produced extra- and intracellular tannase but most of tannase activity was expressed extracellularly and the extracellular: intracellular tannase ratio ranged from 8 to 16. Also Aspergillus niger was found to produce 4.7 times more extracellular tannase (16.8 U/ml) than intracellular

7 International Journal of Agricultural and Food Research Vol. 2 No. 3, pp tannase (3.6 U/ml) (Murugan et al., 2007). In addition, it was found that Aspergillus aculeatus DBF9 produced five times more intracellular than extracellular tannase in a liquid medium (Banerjee et al., 2001). Figure 1: Correlation between the sum of colony and hydrolytic clear zone diameters and total tannase activity Many investigators reported the production of only extracellular tannase by fungi such as Aspergillus japonicus (Gupta et al., 1997), Penicillium variabile (Saxena and Saxena, 2004), Aspergillus awamori (Mahapatra et al., 2005), co-culture of Aspergillus foetidus and Rhizopus oryzae (Banerjee et al., 2005; Purohit et al., 2006) and Aspergillus fumigatus (Manjit et al., 2008). Others reported that tannase was produced mainly intracellularly by fungi such as Penicillium chrysogenum (Rajakumar and Nandy,1983), Aspergillus niger, Aspergillus fischerii, Fusarium solani and Trichoderma viride (Bajpai and Patil, 1997) and Aspergillus niger Van Tieghem MTCC 2425 (Sharma et al., 1999; Sharma et al., 2000). The four selected fungal cultures from the secondary screening were subjected to a final screening which was carried out by the same technique of the secondary screening. In addition, a comparison between extracellular tannase activities was carried out at each 24 hrs intervals. The objective of this step was to confirm the obtained results from secondary screening and to select the most promising fungal culture that produce the highest tannase in shortest time. Because of the negligible amounts of intracellular tannase that was produced by the promising fungal cultures, the final screening was carried out by comparing the extracellular tannase of the tested cultures while intracellular tannase was ignored. Data presented in Table 4 confirm the previous results of secondary screening. Tannase productivity of the Isolate I5 (305.4 extracellular tannase units/flask) was significantly higher than the other three cultures Q2, P4 and B2 after 48, 72 and 96 hours. Consequently, the fungal culture No. I5 was considered as the most promising isolate. Table 4 Final screening of promising fungal isolates Fermentation time (h) Mean of extracellular tannase (U/flask ± SD) B2 I5 P4 Q ± 4.1 b ± 3.6 a ± 11.9 b ± 2.2 a ± 3.5 c ± 2.0 a ± 6.1 d ± 8.8 b ± 29.3 b ± 6.8 a ± 5.3 b ± 29.1 b ± 14.3 c ± 3.9 a ± 1.4 c ± 14.8 b The same superscript letters in each row indicate that there is no significant difference at p < The experiment was carried out in 250 ml-conical flasks containing 50 ml of the modified Czapek-Dox s minimal medium containing 1% filter sterilized tannic acid. Fermentation conditions: initial ph, 4.5±0.2; Temperature, 30º C; inoculum size 5x10 7 spores /flask; shaking, intermittently 3 times a day for 2 min at 200 rpm.

8 Table 5 Macroscopic and microscopic characteristics of fungal isolates No. of Isolates Characteristics used in identification to genera level Characteristics used in identification to species level Scientific name of isolates Hyphae Conidiophores Conidia Macroscopic features Microscopic features 5 Septate Separate, distinct, Single celled - - Aspergillus spp. 48 non- septate, spherical conidia Culture colonies are black Smooth conidiophores Aspergillus niger 14 terminating in a globose swelling (vesicle), bearing metulae and phialides radiating from the entire vesicle surface. remaining together in one chain with the youngest at the base of chain. and reverse white to yellow Culture colonies are yellowgreen and reverse goldish to red brown to yellow Rough conidiophores, sclerotia are present Aspergillus flavus 23 Septate Separate, distinct, septate, bearing a clusters of branches, phialides born on cylinder branches and arranged in brush-like head 12 Septate Separate, distinct, septate, Irregularly branched, not verticillate Single celled spherical conidia remaining together in one chain with the youngest at the base of chain. Single celled, not remaining together in one chain, grouped in small clusters held together by slime 3 Septate Very short Macroconidia have more than 3cells typically Canoeshaped without appendages, one celled conidia also present. - - Penicillium spp. - - Trichoderma spp Fusarium spp. 8 Abou Bakr, El Sahn and El Banna 2013 Screening of Tannase Producing Fungi

9 International Journal of Agricultural and Food Research Vol. 2 No. 3, pp Table 6 Cultural and microscopic characteristics of the most promising isolate (I5) Characteristic Cultural characteristics Microscopic characteristics Result Colony* Shape Circular Appearance Powdery Margin Filamentous Colour Initially white, quickly becoming black with conidial production Reverse Reverse is pale yellow and growth may produce radial fissures in the agar Spores Type Conidia typically one celled and remaining together in chains arising from the tips of the phialides with the youngest at the base of chain. Colour Black Shape Spherical Diameter Ranged from 4 to 5 μm Conidiophores Length Ranged from 1200 to 1800 μm Diameter 12 μm Appearance Hyaline Other characteristics Hyphae Type Septate Appearance Hyaline *Colonies grown on PDA medium at 30º C for 96 h Distinct, separate, non-septate, unbranched, arising from thick walled foot cell and terminating in a globose vesicle (diameter μm). Metulae and phialides cover the entire vesicle and radiating from the entire vesicle surface. 3.4 Identification of Fungal Isolates Identification of fungal isolates was carried out according to their macro- and micro morphology (Table 5). Identified isolates belong to the following genera: Aspergillus, Fusarium, Penicillium and Trichoderma. Several fungal genera have been reported to produce tannase: Aspergilli (Batra and Saxena, 2005; Pinto et al., 2001; Murugan et al., 2007), Penicilli (Batra and Saxena, 2005; Murugan et al., 2007), Fusarium and Trichoderma (Bajpai and Patil, 1997). Full description of most promising isolate (isolate No. I5) including cultural and microscopic characteristics is presented and illustrated in Table 6 and Figure 2, respectively. This fungal isolate was identified as Aspergillus niger Van Tieghem. The fungus Aspergillus niger Van Teighem was discovered in 1867 by Van Teighem as tannase producer. He was the first to demonstrate that the formation of gallic acid during fermentation of gall nut (which contains high levels of tannins) was due to the action of Aspergillus niger tannase (Knudson, 1913). Aspergillus niger was used by many researchers as tannase producer (Bajpai and Patil, 1997; Gupta et al., 1997; Knudson, 1913; Murugan et al., 2007; Pinto et al., 2001; Ramirez- Coronel et al., 2003). 3.5 Nature of Aspergillus niger Van Tieghem Tannase Data illustrated in Figure 3 show that, in the presence of glucose as sole carbon source, Aspergillus niger Van Tieghem couldn t produce tannase whereas, it produced tannase in the presence of tannic acid as sole carbon source. These results confirm that Aspergillus niger Van Tieghem tannase is an inducible enzyme as it was produced only in the presence of its substrate (tannic acid). These results are in agreement with that of Knudson (1913) who confirmed that Aspergillus niger tannase is an inducible enzyme as it is induced only when the Aspergillus niger is grown in the presence of tannic acid.

10 10 Abou Bakr, El Sahn and El Banna 2013 Screening of Tannase Producing Fungi Figure 2: Microscopic photos of Aspergillus niger Van Tieghem. (A) on PDA medium; (B) arising condiophore from a thick walled foot cell, 250X; (C) septate hyphae, 250X; (D) detailed structure of conidiophores, 400X. substrate. Whereas, it couldn t be produced when sucrose was the only carbon source (Bajpai and Patil, 1997). On the other hand, some investigators revealed that Aspergillus japonicus tannase is a constitutive enzyme (Gupta et al., 1997; Bradoo et al., 1997). Figure 3: Extracellular tannase production in the presence of tannic acid or glucose. Other investigators also confirmed that tannase produced by Aspergillus niger is inducible enzyme as it was produced only in the presence of its 4. Conclusion One hundred and five isolates of tannaseproducing fungi isolated from 19 tannin-rich sources belonging to Aspergillus, Fusarium, Pinicillium and Trichoderma were screened to select the most promising one. The promising fungus isolated from tannery soil sample, was identified as Aspergillus niger Van Tieghem and capable to produce inducible extracellular tannase.

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