Optimisation of extracellular tannase production from <Emphasis Type="Italic">Paecilomyces variotii</Emphasis>

The tannase production by Paecilomyces variotii was confirmed by high performance thin layer chromatography (HPTLC), and substrate specificity of the tannase was determined by zymogram analysis in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE). A clear band of activity observed after electrophoresis of culture filtrate in non-denaturing gels indicated the production of extracellular tannase by P. varoitii. HPTLC analysis revealed that gallic acid was the enzymatic degradation product of tannic acid during the fermentation process. The optimum condition for tannase production was at 72 h of incubation in shaking condition and addition of 1.5% tannic acid, 1% glucose and 0.2% sodium nitrate at temperature of 35°C and pH of 5–7. The production of extracellular tannase from Paecilomyces variotii was investigated under optimized conditions in solid-state fermentation (SSF), submerged fermentation (SmF) and liquid surface fermentation (LSF) processes. The maximum extracellular tannase production was obtained within 60 h of incubation under SSF followed by SmF and LSF.     

Co-production of tannase and gallic acid by a novel Penicillium rolfsii (CCMB 714)

Abstract

A novel tannase and gallic acid-producing Penicillium rolfsii (CCMB 714) was isolated from cocoa leaves from the South of Bahia. The influence of nutritional sources and the simultaneous effect of parameters involved in the fermentation process were available. Tannase (9.97 U?mL^?1) and gallic acid (9?mg mL^?1) production were obtained in 48?h by submerged fermentation in non-optimized conditions. Among the carbon sources, tested gallic acid and tannic acid showed the highest tannase production (p<.05) when compared with methyl gallate and glucose. After optimization using the temperature and tannic acid concentration as variables with the Central Compound Rotational Design (CCRD), the maximal tannase production (25.6?U mL^?1) was obtained at 29.8?°C and 12.7%, respectively, which represents an increase of 2.56 times in relation to the initial activity. The parameters optimized for the maximum production of gallic acid (21.51?mg mL^?1) were 30?°C and 10% tannic acid. P. rolfsii CCMB 714 is a new strain with a high tannase and gallic acid production and the gallic acid produced is very important, mainly for its applications in the food and pharmaceutical industry.     

Production and Partial Purification of Tannase from Aspergillus ficuum Gim 3.6

A novel fungal strain, Aspergillus ficuum Gim 3.6, was evaluated for its tannase-producing capability in a wheat bran-based solid-state fermentation. Thin-layer chromatography (TLC) analysis revealed that the strain was able to degrade tannic acid to gallic acid and pyrogallol during the fermentation process. Quantitation of enzyme activity demonstrated that this strain was capable of producing a relatively high yield of extracellular tannase. Single-factor optimization of process parameters resulted in high yield of tannase after 60 hr of incubation at a pH of 5.0 at 30°C, 1 mL of inoculum size, and 1:1 solid–liquid ratio in the presence of 2.0% (w/v) tannic acid as inducer. The potential of aqueous two-phase extraction (ATPE) for the purification of tannase was investigated. Influence of various parameters such as phase-forming salt, molecular weight of polyethylene glycol (PEG), pH, and stability ratio on tannase partition and purification was studied. In all the systems, the target enzyme was observed to preferentially partition to the PEG-rich top phase, and the best result of purification (2.74-fold) with an enzyme activity recovery of 77.17% was obtained in the system containing 17% (w/w) sodium citrate and 18.18% (w/w) PEG1000, at pH 7.0.     

A new process for simultaneous production of tannase and phytase by <Emphasis Type="Italic">Paecilomyces variotii</Emphasis> in solid-state fermentation of orange pomace

The production of enzymes such as tannases and phytases by solid-state fermentation and their use in animal feed have become a subject of great interest. In the present work, Paecilomyces variotii was used to produce tannase and phytase simultaneously. Solid-state fermentation, a process initially designed for tannase production, was implemented here using orange pomace as substrate. Orange pomace is the waste product of the large orange juice industry in Brazil, and it has also been used as an ingredient in animal feed. In addition to enzymatic production, biotransformation of the phenolic content and antioxidant capacity of the orange pomace were analyzed after fermentation. Fermentation conditions, namely moisture level and tannic acid concentration rate, were studied using CCD methodology. The response surface obtained indicated that the highest tannase activity was 5,000 U/gds after 96 h at 59% (v/w) and 3% (w/w) and that of phytase was 350 U/gds after 72 h at 66% (v/w) and 5.8% (w/w) of moisture level and tannic acid concentration, respectively. The amount of tannase production was similar to the levels achieved in previous studies, but this was accomplished with a 7% (w/w) reduction in the amount of supplemental tannic acid required. These results are the first to show that P. variotii is capable of producing phytase at significant levels. Moreover, the antioxidant capacity of orange pomace when tested against the free radical ABTS was increased by approximately tenfold as a result of the fermentation process.     

Tannase production by Aspergillus aculeatus DBF9 through solid-state fermentation

Tannase an industrially important enzyme was produced by Aspergillus aculeatus DBF9 through a solid-state fermentation (SSF). The organism produced good amount of enzyme and gallic acid in wheat bran among the solid substrate used in SSF. Maximum enzyme and gallic acid production occurred in 5% tannic acid after 72 h. Eighty percent initial substrate moisture and 30 degrees C temperature was found suitable for tannase production.     

Purification,structural characterization and biotechnological potential of tannase enzyme produced by Enterobacter cloacae strain 41

Tannase production by Enterobacter cloacae strain 41 was investigated under submerged fermentation which was optimized at various circumstances such as pH, temperature, substrate, and agitation, carbon, and nitrogen sources. Tannase was purified by a two-step approach comprising of ion exchange and size exclusion chromatography, respectively. The maximum tannase production was achieved at 1.0% tannic acid concentration, incubation temperature of 50 °C, and initial pH 6.0. The molecular weight of purified tannase was 45 kDa on 10% SDS-PAGE, and it was confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). The enzymatic products of purified tannase were characterized by HPLC, TLC and FT-IR spectroscopy which showed the functional groups such as OH, CO, and CC. The purified tannase retained the activity up to 90% under the condition at 50 °C and pH 6.0 after 1 h incubation. Enzyme kinetics and inhibition studies were also investigated. Cytotoxicity studies showed that the tannase has no cytotoxic effects on Vero cell line. The results indicated the E. cloacae strain 41 would give a potential source for the efficient production of tannase and can be used in tannery effluent degradation, food, and pharmaceutical industrial applications.     

Purification and characterization of tannase from Paecilomyces variotii: hydrolysis of tannic acid using immobilized tannase

An extracellular tannase (tannin acyl hydrolase) was isolated from Paecilomyces variotii and purified from cell-free culture filtrate using ammonium sulfate precipitation followed by ion exchange and gel filtration chromatography. Fractional precipitation of the culture filtrate with ammonium sulfate yielded 78.7% with 13.6-folds purification, and diethylaminoethyl–cellulose column chromatography and gel filtration showed 19.4-folds and 30.5-folds purifications, respectively. Molecular mass of tannase was found 149.8 kDa through native polyacrylamide gel electrophoresis (PAGE) analysis. Sodium dodecyl sulphate–PAGE revealed that the purified tannase was a monomeric enzyme with a molecular mass of 45 kDa. Temperature of 30 to 50°C and pH of 5.0 to 7.0 were optimum for tannase activity and stability. Tannase immobilized on alginate beads could hydrolyze tannic acid even after extensive reuse and retained about 85% of the initial activity. Thin layer chromatography, high performance liquid chromatography, and ¹H-nuclear magnetic resonance spectral analysis confirmed that gallic acid was formed as a byproduct during hydrolysis of tannic acid.     

Catechine biotransformation by tannase with sequential addition of substrate

This work studied the effect of a sequential addition of substrate on tannase reaction for the increase of epigallocatechin (EGC) and gallic acid. The addition of 0.5–1% GTE increased the production of gallic acid during 2 h in a single tannase reaction, while the addition of more than 2% in GTE rather showed a decrease in gallic acid level with an increase of EGCG level compared with 1% GTE addition group, suggesting that GTE addition of 2% and over inhibits the reaction of tannase. Examination of sequential addition of 1% GTE on tannase reaction showed that second addition of 1% GTE at 2 h promoted tannase reaction by increasing production of gallic acid, but further addition (2 and 3 h) rather inhibited tannase reaction with lowered gallic acid and enhanced EGCG levels. This result showed that one additional treatment of 1% GTE during tannase reaction is effective in an increase of gallic acid production. Moreover, levels of degallated products including EGC, EC, and GC were increased by 7.3, 4.5, and 3.5-fold, respectively in sequential addition of GTE at 2 h. pH change derived from gallic acid production was not shown to related to tannase activity. Therefore, our study suggests that one sequential addition is a suitable process for desirable production of green tea extracts enriched in active components such as gallic acid and EGC.     

Biosynthesis of tannin acyl hydrolase from tannin-rich forest residue under different fermentation conditions

Caesalpinia digyna, a tannin-rich forest residue, was used as substrate for production of tannase and gallic acid. Media engineering was carried out under solid-state fermentation, submerged fermentation and modified solid state fermentation conditions for optimum synthesis of tannase and gallic acid (based on 58% tannin content in the raw material). Tannase vis-à-vis gallic acid recovery under modified solid-state fermentation condition was maximum. Conversions of tannin to gallic acid under solid-state fermentation, submerged fermentation and modified solid-state fermentation conditions were 30.5%, 27.5% and 90.9%, respectively. Journal of Industrial Microbiology & Biotechnology (2000) 25, 29–38. Received 02 November 1999/ Accepted in revised form 12 February 2000     

Microbial production of gallic acid by modified solid state fermentation

Bioconversion of tannin to gallic acid from powder of teri pod (Caesalpinia digyna) cover was achieved by the locally isolated fungus, Rhizopus oryzae, in a bioreactor with a perforated float for carrying solid substrate and induced inoculum. Modified Czapek-Dox medium, put beneath the perforated float, with 2% tannic acid at pH 4.5, temperature 32°C, 93% relative humidity, incubated for 3 days with 3-day-old inoculum was optimum for the synthesis of tannase vis-à-vis gallic acid production. Conversion of tannin to gallic acid was 90.9%. Diethyl ether was used as the solvent for extraction of gallic acid from the fermented biomass. Received 14 December 1998/ Accepted in revised form 17 June 1999     

Optimization of tannase production by Aureobasidium pullulans DBS66

Tannase production by Aureobasidium pullulans DBS66 was optimized. The organism produced maximum tannase in the presence of 1% tannic acid after 36 h. Maximum gallic acid accumulation was observed within 36 h and tannic acid in the fermented broth was completely degraded after 42 h of growth. Glucose had a stimulatory effect on tannase synthesis at 0.1% (w/v) concentration. The organism showed maximum tannase production with (NH4)2HPO4 as nitrogen source. Shaking speed of 120 rpm and 50-ml broth volume have been found to be suitable for maximum tannase production.     

Production of tannase from wood-degrading fungus using as substrate plant residues: purification and characterization

In the present study Lenzites elegans, Schizophyllum commune, Ganoderma applanatum and Pycnoporus sanguineus (wood-degrading fungi) were assayed for their tannase producing potential in culture media containing plant residues or/and tannic acid as carbon source. Aspergillus niger was used as positive control for tannase production. We also carried out the isolation, purification and characterization of the enzyme from the fungi selected as the major productor. The highest fungal growth was observed in A. niger and L. elegans in the media containing tannic acid + glucose + plant residues (Fabiana densa). A. niger and L. elegans reached the highest extracellular tannase production in a medium containing tannic acid + F. densa and in a medium supplemented with glucose + tannic acid + F. densa. The produced enzyme by L. elegans was purified by DEAE-Sepharose. Km value was 5.5 mM and relative molecular mass was about 163,000. Tannase was stable at a pH range 3.0–6.0 and its optimum pH was 5.5. The enzyme showed an optimum temperature of 60°C and was stable between 40 and 60°C. This paper is the first communication of tannase production by wood-degrading fungi. Fermentation technology to produce tannase using plant residues and xylophagous fungi could be very important in order to take advantage of plant industrial waste.     

Statistical optimization for tannase production from <Emphasis Type="Italic">Aspergillus niger</Emphasis> under submerged fermentation

Statistically based experimental design was employed for the optimization of fermentation conditions for maximum production of enzyme tannase from Aspergillus niger. Central composite rotatable design (CCRD) falling under response surface methodology (RSM) was used. Based on the results of ‘one-at-a-time’ approach in submerged fermentation, the most influencing factors for tannase production from A. niger were concentrations of tannic acid and sodium nitrate, agitation rate and incubation period. Hence, to achieve the maximum yield of tannase, interaction of these factors was studied at optimum production pH of 5.0 by RSM. The optimum values of parameters obtained through RSM were 5% tannic acid, 0.8% sodium nitrate, 5.0 pH, 5 × 10⁷ spores/50mL inoculum density, 150 rpm agitation and incubation period of 48 h which resulted in production of 19.7 UmL⁻¹ of the enzyme. This activity was almost double as compared to the amount obtained by ‘one-at-a-time’ approach (9.8 UmL⁻¹).     

Fungal cultures of tar bush and creosote bush for production of two phenolic antioxidants (Pyrocatechol and Gallic acid)

‘Tar bush’ and ‘creosote bush’ were substrates of fungal cultivation for tannase production and gallic acid and pyrocatechol accumulation. Aspergillus niger GH1 grew similarly on both plant materials under solid state culture conditions, reaching maximal levels after 4 d. Fungal strain degraded all tannin content of creosote bush after 4 d of fermentation and >75 % of tar bush after 5 d. Higher level of tannase activity was detected in tar bush fermentation. Biotransformation of tannins to gallic acid was high (93 % in creosote bush and 89 % in tar bush). Pyrocatechol was released poorly. Kinetic parameters of tannin conversion were calculated.     

Effects of inoculum concentration,temperature, and carbon sources on tannase production during solid state fermentation of cashew apple bagasse

In this study, the optimization of tannase production by solid state fermentation was investigated using cashew apple bagasse (CAB), an inexpensive residue produced by the cashew apple agroindustry, as a substrate. To accomplish this, CAB was enriched with 2.5% (w/w) tannic acid and 2.5% (w/w) ammonium sulphate and then moistened with water (60 mL/100 g of dry CAB). The influence of inoculum concentration (10⁴ to 10⁷ spores/g), temperature (20, 25, 30, and 35°C) and several additional carbon sources (glucose, starch, sucrose, maltose, analytical grade glycerol, and glycerol produced during biodiesel production) on enzyme production by Aspergillus oryzae was then evaluated. Supplementation with maltose and glycerol inhibited tannase synthesis, which resulted in lower enzyme activity. Starch and sucrose supplementation increased enzyme production, but decreased the enzyme productivity. The maximum tannase activity (4.63 units/g of dry substrate) was obtained at 30°C, using 10⁷ spores/g and 1.0% (w/v) sucrose as an additional carbon source.     

Production of tannase by Aspergillus niger Aa-20 in submerged and solid-state fermentation: influence of glucose and tannic acid

Tannase production by Aspergillus niger Aa-20 was studied in submerged (SmF) and solid-state (SSF) fermentation systems with different tannic acid and glucose concentrations. Tannase activity and productivity were at least 2.5 times higher in SSF than in SmF. Addition of high tannic acid concentrations increased total tannase activity in SSF, while in SmF it was decreased. In SmF, total tannase activity increased from 0.57 to 1.03 IU/mL, when the initial glucose concentration increased from 6.25 to 25 g/L, but a strong catabolite repression of tannase synthesis was observed in SmF when an initial glucose concentration of 50 g/L was used. In SSF, maximal values of total tannase activity decreased from 7.79 to 2.51 IU when the initial glucose concentration was increased from 6.25 to 200 g/L. Kinetic results on tannase production indicate that low tannase activity titers in SmF could be associated to an enzyme degradation process which is not present in SSF. Tannase titers produced by A. niger Aa-20 are fermentation system-dependent, favoring SSF over SmF. Journal of Industrial Microbiology & Biotechnology (2001) 26, 296–302. Received 07 July 2000/ Accepted in revised form 15 February 2001     

Tannase production by Paecilomyces variotii

Surface response methodology was applied to the optimization of the laboratory scale production of tannase using a lineage of Paecilomyces variotii. A preliminary study was conducted to evaluate the effects of variables, including temperature ( degrees C), residue (%) (coffee husk:wheat bran), tannic acid (%) and salt solutions (%) on the production of tannase during 3, 5 and 7 days of fermentation. Among these variables, temperature, residues and tannic acid had significant effects on tannase production. The variables were optimized using surface response methodology. The best conditions for tannase production were: temperature (29-34 degrees C); tannic acid (8.5-14%); % residue (coffee husk:wheat bran 50:50) and incubation time of 5 days. The supplementation of external nitrogen and carbon sources at 0.4%, 0.8% and 1.2% concentration on tannase production were studied in the optimized medium. Three different nitrogen sources included yeast extract, ammonia nitrate and sodium nitrate along with carbon source (starch) were studied. Only ammonia nitrate showed a significant effect on tannase production. After the optimization process, the tannase activity increased 8.6-fold.     

Degradation of tannic acid and purification and characterization of tannase from Enterococcus faecalis

Tannins, present in various foods, feeds and forages, have anti-nutritional activity; however, presence of tannase in microorganisms inhabiting rumen and gastrointestinal tract of animals results in detoxification of these tannins. The present investigation was carried out to study the degradation profile of tannins by Enterococcus faecalis and to purify tannase. E. faecalis was observed to degrade tannic acid (1.0% in minimal media) to gallic acid, pyrogallol and resorcinol. Tannase from E. faecalis was purified up to 18.7 folds, with a recovery of 41.7%, using ammonium sulphate precipitation, followed by DEAE-cellulose and Sephadex G-150. The 45 kDa protein had an optimum activity at 40 °C and pH 6.0 at substrate concentration of 0.25 mM methyl gallate.     

Enhancement of tannase production by Lactobacillus plantarum CIR1: validation in gas-lift bioreactor

The optimization of tannase production by Lactobacillus plantarum CIR1 was carried out following the Taguchi methodology. The orthogonal array employed was L18 (2¹ × 3⁵) considering six important factors (pH and temperature, also phosphate, nitrogen, magnesium, and carbon sources) for tannase biosynthesis. The experimental results obtained from 18 trials were processed using the software Statistical version 7.1 using the character higher the better. Optimal culture conditions were pH, 6; temperature, 40 °C; tannic acid, 15.0 g/L; KH₂PO₄, 1.5 g/L; NH₄Cl, 7.0 g/L; and MgSO₄, 1.5 g/L which were obtained and further validated resulting in an enhance tannase yield of 2.52-fold compared with unoptimized conditions. Tannase production was further carried out in a 1-L gas-lift bioreactor where two nitrogen flows (0.5 and 1.0 vvm) were used to provide anaerobic conditions. Taguchi methodology allowed obtaining the optimal culture conditions for the production of tannase by L. plantarum CIR1. At the gas-lift bioreactor the tannase productivity yields increase 5.17 and 8.08-fold for the flow rates of 0.5 and 1.0 vvm, respectively. Lactobacillus plantarum CIR1 has the capability to produce tannase at laboratory-scale. This is the first report for bacterial tannase production using a gas-lift bioreactor.     

Production of tannase through solid state fermentation using Indian Rosewood (Dalbergia Sissoo)sawdust—a timber industry waste

The tannase producing strain Aspergillus heteromorphus MTCC 8818 was used in the present study for the production of tannase under solid state fermentation using Rosewood (Dalbergia sissoo) sawdust—a timber industry waste—as substrate. Various physico-chemical parameters were optimized for extracellular yield of tannase. Maximum tannase (1.84 U/g dry substrate) and gallic acid (5.4 mg/g ds) was observed at 30 °C after 96 h of incubation. Czapek dox medium was found to be the best moistening agent, with pH and relative humidity of 5.5 and 70 %, respectively. The constituents of Czapek dox medium were varied to enhance enzyme production. The optimum concentration of modified Czapek dox constituents contained 0.2 % NaNO₃, 0.05 % K₂HPO₄ and MgSO₄, 0.15 % KCl. Among the additional salts supplemented to Czapek dox medium, ZnSO₄ and CuSO₄ were found to have a stimulating effect, with a relative tannase activity of 116 and 111 %, respectively. Glucose as an external carbon source was found to be a repressor of enzyme production.