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Screening of tannase-producing fungi isolated from tannin-rich sources

Tannase (tannin acyl hydrolase; EC 3.1.1.20) is one of 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, 1966; Sharma et al., 2000).

Tannase is an industrially important enzyme. It has several applications in foods, animal feeds, cosmetic, pharmaceutical, chemical,leather industries etc. (Aguilar and Gutierrez-Sanchez,2001).

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 microorganisms 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). Fungi are the most studied microorganisms for tannase production. Fungi have the ability to degrade tannins as a sole carbon source (Aguilar et al., 2007).

ABSTRACT

A. A. El-Banna*, H. A. Abou-Bakr, M. A. El-Sahn Department of Food Science and Technology, Faculty of Agriculture, Alexandria University, Aflaton St., El-Shatby, P.O. Box 21545, Alexandria, Egypt.

Realizing the importance of the enzyme tannase, the present study aimed 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 the highest 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.

INTRODUCTION

Figure 1: Aspergillus niger Van Tieghem colony surrounded by a hydrolytic clear zone. (Fungus grown on TAA medium at 30º C for 96h) 

REFERENCES

CONTACTS

RESULTS

AIM OF INVESTIGATION

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).The modified Czapek-Dox’s minimal medium was used for secondary and final screening of fungi (Bradoo et al., 1996).

Screening and selection of tannase-producing fungal cultures

Fungal cultures which exhibited the highest tannase activity in the primary screening were subjected to secondary screening using submerged fermentation technique described by Bradoo et al. (1996) and Batra and Saxena (2005).

Tannase activity was assayed using the spectrophotometric method of Sharma et al. (2000). Methyl gallate was used as a substrate. The method is based on the chromogen formation between gallic acid and rhodanine.

Identification of fungal isolates

Simplified key to some selected common genera reported by 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 Doctor fungus database.

The present work aimed to isolate and screen high tannase-producing fungi from tannin-rich sources.

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.

MATERIALS & METHODSSources 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.

Table 1 Secondary screening of promising fungal isolates selected from the primary screening step.

The same superscript letters indicate that there is no significant difference at p < 0.05.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 5x107 spores/flask; shaking, intermittently 3 times a day at 200 rpm for 2 min each time.

Table 2 Final screening of promising fungal isolates. 

Fermentation time (h)

Mean of extracellular tannase (U/flask ± SD)

B2 I5 P4 Q2

24 140.7 ± 4.1 b 159.3 ± 3.6 a 128.7 ± 11.9 b 155.4 ± 2.2 a

48 161.2 ± 3.5 c 199.1 ± 2.0 a 143.3 ± 6.1 d 178.3 ± 8.8 b

72 198.4 ± 29.3 b 256.8 ± 6.8 a 189.1 ± 5.3 b 195.6 ± 29.1 b

96 211.5 ± 14.3 c 305.4 ± 3.9 a 217.4 ± 1.4 c 243.7 ± 14.8 b

The same superscript letters in each row indicate that there is no significant difference at p < 0.05.

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 5x107 spores /flask; shaking, intermittently 3 times a day at 200 rpm for 2 min each time.. 

Isolate code

Mean of extracellular tannase

(U/flask ± SD)

Mean of intracellular tannase

(U/flask ± SD)

Mean of total tannase activity

(U/flask ± SD)

B2 211.5 ± 14.3 4.1 ± 0.8 215.6 ± 13.8 c

E6 149.2 ± 2.8 9.7 ± 0.4 158.9 ± 2.9 de

H6 164.3 ± 7.1 6.9 ± 0.2 171.2 ± 7.3 d

I5 305.4 ± 7.8 8.3 ± 0.7 313.7 ± 7.9 a

I7 203.4 ± 7.8 7.6 ± 0.5 211 ± 7.4 c

J4 62.3 ± 8.3 6.3 ± 0.3 68.6 ± 8.6 f

P2 143.3 ± 9.8 7.6 ± 0.4 150.9 ± 9.5 de

P4 217.4 ± 1.4 7.0 ± 0.7 224.4 ± 2.1 bc

Q2 243.7 ± 14.8 5.6 ± 0.1 249.3 ± 14.7 b

Q4 171.2 ±5.7 8.7 ± 0.6 179.9 ± 5.1 d

R3 127.4 ± 5.4 6.1 ± 0.6 133.5 ± 53.6 e

Figure 3: Extracellular tannase production in the presence of tannic acid or glucose.

The experiment was carried out using SmF technique in 250 ml-Erlenmeyer flasks containing 50 ml of modified Czapek-Dox’s minimal medium containing either 1% filter sterilized tannic acid or glucose.Fermentation conditions: initial pH, 4.5±0.2; incubation temperature, 30º C; inoculum size 5x107 spores /flask; flasks was incubated under intermittent shaking 3 times a day at 200 rpm for 2 min each time.

CONCLUSION

One hundred and five isolates of tannase-producing fungi isolated from 19 tannin-rich sources were screened to select the most promising one. The promising fungus isolated from tannery soil sample, identified as Aspergillus niger Van Tieghem and capable to produce extracellular and inducible tannase. To the best of our knowledge we report, for the first time, the isolation of tannase-producing fungi from the following plants: Bengal fig fruits; unripe date fruits; guava leaves; persimmon fruits; pomegranate leaves, fruits and peels; river-red gum leaves and green tea. Tannin-rich sources in various countries should be considered as a good source for isolation of tannase-producing microorganisms. A collaborative study is needed.

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Aguilar C N, Rodriguez R, Gutiérrez-Sanchez G, Augur, C, Favela-Torres, E, Prado-Barragan L, Ramirez-Coronel A and Contreras-Esquivel J C (2007). Microbial tannases: advances and perspectives. Applied Microbiology and Biotechnology 76: 47-59.

Barnett HL and Hunter BB (1987). Illustrated Genera of Imperfect Fungi, 4th ed. pp. 35-39, 92,94,130. Prentice Hall, Inc. New Jersey.

Batra A and Saxena RK (2005). Potential tannase producers from the genera Aspergillus and Penicillium. Process Biochemistry 40: 1553-1557.

Bradoo S, Gupta R and Saxena RK (1996). Screening of extracellular tannase- producing fungi: Development of a rapid and simple plate assay. Journal of General and Applied Microbiology 42: 325-329.

Haslam E and Stangroom JE (1966). The esterase and depsidase activities of tannase. The Biochemical journal 99: 28-31.

Pinto GAS, Leite SGF, Terzi SC and Couri S (2001). Selection of tannase-producing Aspergillus niger strains. Brazilian Journal of Microbiology 32: 24-26.

Sharma S, Bhat TK and Dawra RK (2000). A spectrophotometric method for assay of tannase using rhodanine. Analytical Biochemistry 279: 85-89.

Purohit JS, Dutta JR, Nanda RK and Banerjee R (2006). Strain improvement for tannase production from co-culture of Aspergillus foetidus and Rhizopus oryzae. Bioresource Technology 97: 795-801.

• Corresponding author. Tel.: +2 03 5902608; Cell phone: +2 010 9063416; fax: +2 03

5922780.E-mail address: amrbanna47@gmail.com.