PRODUCTION OF TANNASE BY ISOLATED ASPERGILLUS TERREUS UNDER SUBMERGED FERMENTATION

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1 International Journal of Science, Technology & Management PRODUCTION OF TANNASE BY ISOLATED ASPERGILLUS TERREUS UNDER SUBMERGED FERMENTATION M. Narsi Reddy 1 N. L. N Reddy 2 1 Department of Biotechnology, CMR College of Engineering & Technology, Medchal, Hyderabad, Telangana, (India) 2 Department of Chemical Engineering, Chaitanya Bharathi Institute of Technology, Gandipet Hyderabad, Telangana, (India) ABSTRACT Tannins are water-soluble polyphenolic compounds found in plants as secondary metabolites. Study on optimization of submerged fermentative production of tannase by Aspergillus terrus was carried out. Aspergillus terrus was selected out of eight fungal isolates have the ability to grow on the presence of tannic acid. Tannase enzyme production by Submerged Fermentation (SmF) For Aspergillus terrus, the optimum Tannic Acid concentration1% and fermentation conditions were found to be ph, temperature 3 C,rpm 1,aeration ml,carbon.1-1%, and fermentation period of72 h. Keywords: Aspergillus Terrus, Carbon Source, Nitrogen source, Submerged Fermentation, Tannase. I. INTRODUCTION Tannins are naturally occurring secondary metabolites found in plants and are considered as the fourth most abundant plant constituent after cellulose, hemicellulose and lignin. They are water-soluble polyphenolic compounds with molecular masses ranging from.3 to kda (Haslam, 1989). In nature, tannins are widely distributed worldwide among different families of higher plants including tara, gall, oak, sumach, trillo, myrobalan and so on. They are also found in common foods such as tea, cashew nut, hazelnut, walnut, grape, mango, strawberry, raspberry, blackberry, etc. and high concentrations of tannins are present in different plant parts such as leaves, fruits, bark, wood, seeds and roots (Li et al., 6). One of the major characteristic of tannins is its ability to form strong complexes with carbohydrates and proteins. Tannins are classified into two major groups, hydrolysable and condensed tannins (Lekha and Lonsane, 1997; Auguilar and Gutierrez-Sanchez, 1; Belmares et al., 4), which is based on two differences such as the sugar content and the availability as substrates to tannase. Condensed tannins do not have sugar residues comprising of only flavan-3-ol or flavan-3-4-diol polymers, while hydrolysable tannins are polyesters of a sugar moiety (or other non-aromatic polyhydroxy compounds) and organic acids. These compounds undergo hydrolytic cleavage on treatment with dilute acids to release the respective sugar and acid moiety. If the acid component is gallic acid or ellagic acid, the compounds are known as gallotannins or ellagitannins (Serrano et al., 9). Most ellagitannins are mixed esters comprising of both hexahydroxydiphenic acid (forms ellagic acid when hydrolyzed with elimination of water) and gallic acid. 12 P a g e

2 International Journal of Science, Technology & Management It was earlier established that high molecular mass tannins have a stronger anti-nutritional effects and lower biological activities. The plausible reason is that high molecular mass tannins form complexes with proteins, carbohydrates and digestive enzymes such as amylase, lipase, protease, pectinase and cellulase, and thus reduce the nutritional values of feeds (Chung et al., 1998). Thus, tannins have toxic or anti-nutritional implications on ruminants, which reduce their feed intake with lower nutrient digestibility and protein bioavailability (Barry et al., 1986; Lowry et al., 1996). In contrast, small molecular mass tannins including monomeric, dimeric and trimeric tannins are considered less anti-nutritional and are readily absorbed by the animal (Butler and Rogler, 1992). It was demonstrated that medicinal herbs exhibited noticeable biological and pharmacological activities such as anti-carcinogenic, antitumor, antiviral and inhibition of lipid peroxidation due to the presence of small molecule tannins (Okuda et al., 1992). Further, in vitro and in vivo investigations indicated that ellagic acid significantly inhibits cancer formation in the colon, oesophagus, liver, lung, and skin of rats and mice, and is thus identified as a possible chemotherapeutic agent against human carcinogenesis (Lee, 1992). Tannic acid and ellagic acid sulfate were earlier demonstrated to exhibit anti-hiv activity (Mizumo et al., 1992). Considering the benefits of small molecule tannins, the biodegradation of gallotannins and ellagitannins can be explored using microbial tannases. Tannase (tannin acyl hydrolase, EC ), is an industrially important inducible enzyme produced by different filamentous fungi like Aspergillus, Penicillium, Fusarium and Trichoderma species (Iibuchi et al., 1967; Rajakumar and Nandy, 1983; Lekha and Lonsane, 1994; Bajpai and Patil, 1996) along with bacteria (Deschamps et al., 1983; Skene and Brooker, 199) and yeasts (Aoki et al., 1976). It possess both esterase and depsidase activities and catalyzes the hydrolysis reactions of the ester bonds, in particular hydrolyzes the ester and depside bonds present in galloyl groups of gallotannins and hexahydroxydiphenoyl groups of ellagitannins (Daniel et al., 1991). Tannase finds a wide range of applications in the production of gallic acid, an intermediate for trimethoprim, used in pharmaceutical industry, substrate for chemical synthesis of pyrogallol or ester galates which are used as food preservatives, manufacture of instant tea, clarification of beer and beverages, reduction of anti-nutritional effects of tannins in animal feed and in decontamination of tannery effluents. The present investigation was aimed at isolation of fungal cultures from soil samples collected from different biosphere zones of India and screening of different isolates available in the lab culture collection for identifying a promising tannase producing culture. Further, the optimization of production conditions for a promising culture was standardized under both submerged fermentation. II. MATERIALS AND METHODS 2.1 Chemicals All chemicals were of analytical grade purchased from Himedia Biosciences Mumbai and all the substrates for SSF were procured from the local dhal mills. 2.2 Microorganism Aspergillus terreus was isolated from soil collected from different biospheare zones of India. The culture was maintained on a Potato Dextrose Agar plates containing tannic acid and maintained at 4 C and sub-cultured at monthly intervals. 13 P a g e

3 International Journal of Science, Technology & Management 2.3 Screening for Tannase Producers Chloramphenicol Rose Bengal Agar and Potato dextrose agar plates were used to isolate fungal cultures from soil samples collected from different biosphere zones of India. Czapek Dox agar plates supplemented with tannic acid (1% w/v) and bromocresol blue indicator (.2%, w/v) were used to screen tannase producers. Assay plates were stabbed with fungal isolates and incubated at 28 C / 72 h. Plates were checked for zones of hydrolysis around the colonies. Positive cultures were screened in mineral salts medium with tannic acid (1% w/v) as C source 2.4 Tannase Activity Estimation Tannase activity produced by different fungal isolates was assessed using the rhodanine method (Sharma et al., ). The method is based on the formation of a pink colored chromogen with gallic acid released by the action of tannase on methyl gallate., which was read at nm using a spectrophotometer (Shimadzu UV 16A, Japan). One unit of tannase activity was defined as the amount of enzyme required to liberate one micromole of gallic acid per minute under the defined reaction conditions. 2. Preparation of Spore Suspension Since tannase is an inducible enzyme, pre-induced inoculum was used which was prepared using potato dextrose agar medium where 2% tannic acid was used as a sole carbon source. Induced inoculum was prepared with 2 ml spore suspension and was incubated at 3 C under shaking conditions for 72 h. 2.6 Optimization of Submerged Fermentation Conditions for Tannase Production Based on the screening studies, the Aspergillus terreus culture was used for further studies on the optimization of tannase production under submerged fermentation (SmF) conditions. Parametric optimization of tannase production conditions were assessed in mineral salts medium (per liter): NaNO 3, 3 g; KH 2 PO 4, 1 g; MgSO 4,. g; KCl,. g; FeSO 4.7H 2 O,.1 g with tannic acid addition at a concentration of 1 g l -1 under submerged fermentation conditions with respect to ph, temperature, inoculum level, incubation period, concentration of tannic acid substrate, aeration, agitation, carbon and nitrogen sources, and surfactants as inducers. The effect of ph and temperature on tannase production was evaluated under ph conditions ranging from ph 3. to 8. and temperature conditions ranging from 24 C to 34 C. The effect of inoculum level and incubation period on tannase production was examined with a spore inoculum at a level ranging from.-6. ml and different time durations ranging from h. The influence of agitation on tannase production was studied from 6 to 18 rpm. The effect of tannic acid as substrate was tested at different concentrations ranging from. to 2.%. Various carbon sources including glucose, fructose, sucrose, maltose, and corn starch, and nitrogen sources comprising of yeast extract, peptone, beef extract, NaNO 3, NH 4 NO 3 and casein were examined at concentrations ranging from.1 1 % (w/v) level in production medium. Different surfactants such as Tween 8, Tween, Triton X-1, SDS as inducers tested at concentrations ranging from.1 1 % (w/v). Experiments were performed in ml baffled flasks containing 1 ml of medium. A 1% spore inoculum (concentration of spores) of 4 days grown old culture was inoculated into each flask and the flasks were incubated at 28 C for 3 days with agitation at 1 rev min -1 on a multi-stack shaking incubator (Model LSI-4M, Daihan LabTech Co., Ltd., Kyonggi-Do, South Korea). Samples were withdrawn periodically at one day interval, the fungal biomass was separated by filtration through Whatman filter paper No. 1 and the cell-free supernatant was used for assaying the extracellular tannase activity. 14 P a g e

4 International Journal of Science, Technology & Management III. RESULTS AND DISCUSSION 3.1 Screening for Tannase Producers About 1 fungal strains isolated from soil samples collected from different biosphere zones of India were screened for tannase activity on petri plates supplemented with tannic acid as substrate. About eight fungal isolates were short-listed which exhibited positive tannase activity (Figure 1). The zone diameter of the tannase activity exhibited by the fungal isolates on tannic acid supplemented plates is shown in Figure 2. P a g e

5 Diameter of clearing zone (mm) International Journal of Science, Technology & Management 1 Aspergillus niger Sclerotium rolfsii Aspergillus terreus Penicillium crysogenum Rhizopus oryzae Aspergillus flavus Fusarium oxysporum Trichoderma viride Figure 2: Zone Diameter of Tannase Producing Fungal Strains These eight short-listed fungal strains were further subjected to screening for checking tannase activity by submerged fermentation in mineral salts medium. The enzyme activity shown by the individual fungal isolates is shown in Figure Effect of ph on tannase production The effect of ph on tannase production from fungal species under study was determined by growing each species in fermentation medium contained NaNO 3, 3 g/l; KH 2 PO 4, 1 g/l; MgSO 4,. g/l; KCl,. g/l; FeSO 4.7H 2 O,.1 g/l and Tannic acid, 1 g/l. The medium was adjusted to different ph levels i.e., from ph 3. to 8.. Tannase production was measured and monitored at an interval of 12 h over a fermentation period of 72 h. The maximum activity of the enzyme was recorded at ph. (see Figure 4). 16 P a g e

6 Relative tannase activity (%) Relative tannase activity (%) International Journal of Science, Technology & Management Aspergillus niger Aspergillus terreus Rhizopus oryzae Fusarium oxysporum Sclerotium rolfsii Penicillium crysogenum Aspergillus flavus Trichoderma viride ph Figure 4: Comparative Tannase Production from Fungal Species at Different Ph 3.3 Effect of Temperature on Tannase Production To observe the effect of initial temperature of tannase production, the fungal isolates cultivated in the production medium was incubated at various temperatures i.e., 24 C to 34 C. The maximum activity of the enzyme recorded for all the fungal isolates was close to 3 C (Figure ) and a declining trend in enzyme production was observed at higher temperatures Aspergillus niger Aspergillus terreus Rhizopus oryzae Fusarium oxysporum Sclerotium rolfsii Penicillium crysogenum Aspergillus flavus Trichoderma viride Temperature o C Figure : Comparative Tannase Production from Fungal Species at Different Temperatures 17 P a g e

7 Tannase activity (U/ml) Tannase activity (U/ml) International Journal of Science, Technology & Management 3.4 Effect of Inoculum Level on Tannase Production To study the effect of different inoculum levels on tannase production, spore suspension was prepared from 72 h old cultures grown on PDA slants by adding 1 ml of sterile distilled water containing.1% of Tween 8 and the spores were suspended with a sterile loop. One millilitre of the spore suspension containing spores/ml was used as source of inoculum. The flasks containing the medium was inoculates with a spore inoculum at a level range from.-6. ml. Maximum tannase activity was observed at a inoculum level of 2. ml where in the maximum enzyme activity recorded was 28.4 U/ml (Figure 6), and a higher inoculum level showed a decrease in the production ability of the Aspergillus terreus isolate Inoculum volume (ml) Figure 6: Effect of Inoculum Level on Tannase Production 3. Effect of Agitation on Tannase Production To study the effect of different agitation levels on tannase production, the agitation of the medium was varied from 6-18 rpm. The maximum enzyme production was observed when the culture was agitated at 1 rpm which favored an enzyme activity of 3.6 U/ml (Figure 7) Agitation (rpm) Figure 7: Effect of Agitation on Tannase Production 18 P a g e

8 Tannase activity (U/ml) Tannase activity (U/ml) International Journal of Science, Technology & Management 3.6 Effect of Incubation Period (H) On Tannase Production To evaluate the effect of incubation period on tannase production, the Aspergillus terreus isolate was incubated for different time durations ranging from h. Maximum tannase production (32. U/ml) was observed when the culture was cultivated for 72 h (Figure 8). With a further increase in the incubation period over 72 h, a decreased enzyme activity was observed Incubation time (h) Figure 8: Effect of Incubation Period (H) On Tannase Production 3.7 Effect of Aeration on Tannase Production To study the effect of aeration on the tannase production, the amount of medium volume was varied in each flask in the range of 17 ml. A volume level of ml medium in the Erylenmeyer flask favoured a maximum tannase activity of 31.6 U/ml (Figure 9) Medium level (ml) Figure 9: Effect of Aeration on Tannase Production 19 P a g e

9 Tannase activity (U/ml) Tannase activity (U/ml) International Journal of Science, Technology & Management 3.8 Effect of Substrate Level on Tannase Production When tannic acid at various concentrations (.,.,.7, 1., 1., 1., 1.7, and 2.%) were added to the production medium and incubated at 28 C for 72 h. The maximum enzyme production was observed at 1.% level of tannic acid with a tannase activity of 32. U/ml (Figure 1) Substrate level (%) Figure 1: Effect of Substrate Level on Tannase Production 3.9 Effect of Carbon Sources on Tannase Production To study the effect of different carbon sources on tannase production, different carbon sources such as glucose, sucrose, maltose, fructose, and corn starch were supplemented to the production medium at.1-1 % level. The results showed that sucrose and maltose favored maximum enzyme production of 33.4 U/ml and 29.3 U/ml and corn starch showed an enzyme activity of 19.8 U/ml. (Figure 11) Glucose Fructose Sucrose Maltose Corn starch Carbon sources (%) Figure 11: Effect of Carbon Sources on Tannase Production 11 P a g e

10 Tannase activity (U/ml) Tannase activity (U/ml) International Journal of Science, Technology & Management 3.1 Effect of Nitrogen Sources on Tannase Production To study the effect of nitrogen requirement, different nitrogen sources such as yeast extract, peptone, beef extract, NaNO 3, NH 4 NO 3 and casein were added to the production medium and incubated at 28 C for 72 h. The results indicated that complex organic nitrogen sources supported better tannase production over the inorganic nitrogen compounds. Beef extract (3.6 U/ml) showed maximum influence in enhancement of enzyme production followed by NaNO 3 (31.2 U/ml) (Figure 12) Peptone Yeast extract Beef extract NH4NO3 NaNO3 Casein Nitrogen source (%) Figure 12: Effect of nitrogen sources on tannase production 3.11 Effect of Surfactants on Tannase Production The effect of various surfactants on tannase production was studied by addition of Tween 8, Tween, TritonX-1, SDS to the production medium. Tween (38.4 U/ml) was observed as better surfactants for tannase production and followed by TritoX-1 (3.1 U/ml) (Figure 13) Tween 8 Tween TritonX1 SDS Surfactants (%) Figure 13: Effect of Surfactants on Tannase Production 111 P a g e

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