Pestalotiopsis mangiferae isolated from cocoa leaves and concomitant tannase and gallic acid production

The enzyme tannase is of great industrial and biotechnological importance for the hydrolysis of vegetable tannins, reducing their undesirable effects and generating products for a wide range of processes. Thus, the search for new microorganisms that permit more stable tannase production is of considerable importance. A strain of P. mangiferae isolated from cocoa leaves was selected and investigated for its capacity to produce tannase enzymes and gallic acid through submerged fermentation. The assessment of the variables affecting tannase production by P. mangiferae showed that tannic acid, ammonium nitrate and temperature were the most significant (8.4 U/mL). The variables were analyzed using Response Surface Methodology - RSM (Box-Behnken design), with the best conditions for tannase production being: 1.9% carbon source, 1% nitrogen source and temperature of 23 °C. Tannase activity doubled (16.9 U/mL) after the optimization process when compared to the initial fermentation. A pH of 7.0 was optimal for the tannase and it presented stability above 80% with pH between 4.0 and 7.0 after 2h of incubation. The optimal temperature was 30 °C and activity remained at above 80% at 40–60 °C after 1 h. Production of gallic acid was achieved with 1% tannic acid (0.9 mg/mL) and P. mangiferae had not used up the gallic acid produced by tannic acid hydrolysis after 144 h of fermentation. A 5% tannic acid concentration was the best for gallic acid production (1.6 mg/mL). These results demonstrate P. mangiferae’s potential for tannase and gallic acid production for biotechnological applications.     

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.     

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.     

Microbial transformation of tannin-rich substrate to gallic acid through co-culture method

Modified solid-state fermentation (MSSF) of tannin-rich substrate yielding tannase and gallic acid was carried out using a co-culture of the filamentous fungi, Rhizopus oryzae (RO IIT RB-13, NRRL 21498) and Aspergillus foetidus (GMRB013 MTCC 3557). Powdered fruits of Terminalia chebula and powdered pod cover of Caesalpinia digyna was used in the process and the different process parameters for maximum production of tannase and gallic acid by co-culture method were optimized through media engineering. MSSF was carried out at the optimum conditions of 30 degrees C and 80% relative humidity. The optimal pH and incubation period was 5.0 and 48 h respectively. Through the co-culture technique the maximum yield of tannase and gallic acid was found to be 41.3 U/ml and 94.8% respectively.     

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.     

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.     

Optimization of culture medium for novel cell-associated tannase production from Bacillus massiliensis using response surface methodology

Naturally immobilized tannase (tannin acyl hydrolase, E.C. 3.1.1.20) has many advantages, as it avoids the expensive and laborious operation of isolation, purification, and immobilization, plus it is highly stable in adverse pH and temperature. However, in the case of cell-associated enzymes, since the enzyme is associated with the biomass, separation of the pure biomass is necessary. However, tannic acid, a known inducer of tannase, forms insoluble complexes with media proteins, making it difficult to separate pure biomass. Therefore, this study optimizes the production of cell-associated tannase using a "protein-tannin complex" free media. An exploratory study was first conducted in shake-flasks to select the inducer, carbon source, and nitrogen sources. As a result it was found that gallic acid induces tannase synthesis, a tryptose broth gives higher biomass, and lactose supplementation is beneficial. The medium was then optimized using response surface methodology based on the full factorial central composite design in a 3 l bioreactor. A 2(3) factorial design augmented by 7 axial points (alpha = 1.682) and 2 replicates at the center point was implemented in 17 experiments. A mathematical model was also developed to show the effect of each medium component and their interactions on the production of cell-associated tannase. The validity of the proposed model was verified, and the optimized medium was shown to produce maximum cell-associated tannase activity of 9.65 U/l, which is 93.8% higher than the activity in the basal medium, after 12 h at pH 5.0, 30 degrees C. The optimum medium consists of 38 g/l lactose, 50 g/l tryptose, and 2.8 g/l gallic acid.     

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.     

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     

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.     

利用黑曲霉单宁酶酶法制取没食子酸的研究

利用已有的 10株高单宁酶活性的菌株为起始菌 ,经活化分离选择 ,借助Ⅱ级发酵培养程序、生物转化、结合TLC分析进行筛选实验。最后选出具有高单宁酶活性的 1号和 5 0号菌株 ,开展了没食子酸 (GA)克量级生物转化法制备实验 ,结果表明 ,本酶法工艺是可行的 ,在发酵液中GA的浓度分别达到2 0 .6mg/ml和 2 1 3mg/ml,产品产率 (以从五倍子提取的单宁酸计 )达到 41 2 %和 42 6 % ,具有潜在的工业开发价值     

Biotransformation of hydrolysable tannin to ellagic acid by tannase from Aspergillus awamori

Madhuca indica, locally known as mahua in India is a multipurpose tree species. Mahua, particularly bark contains a significant amount of hydrolysable tannin (17.31%) which can be utilized for ellagic acid production through biotransformation. In the present study, mahua bark utilized not only as a raw material for tannase production but also for ellagic acid a well-known therapeutic compound. After prior confirmation of hydrolysable tannin content in bark, it has been supplemented, as a substrate for tannase production through solid state fermentation of Aspergillus awamori. Tannase production, as well as biodegradation of the hydrolysable tannin reached a maximum at 72?h of incubation time. The optimum conditions for tannase production are solid to liquid ratio of 1:2, 35?°C, pH 5.5 and 72h incubation time which resulted 0.256?mg/mL of an extract of ellagic acid. Maximum tannase activity of 56.16?IU/gds at 35?°C and 72h of incubation time is recorded. It seems that tannase production and biotransformation of hydrolysable tannins using bark powder of mahua can be considered as an appropriate alternative to the existing procedures of ellagic acid production.     

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.     

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.     

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     

Lipase production by Aspergillus terreus using mustard seed oil cake as a carbon source

An extracellular lipase-producing fungus was isolated from the garden soil of the Post Graduate Department of Botany, Utkal University, Bhubaneswar, Odisha, India and identified as Aspergillus terreus. The A. terreus strain isolated was found to be capable of producing lipase in both solid state culture and liquid static surface culture. Experiments aimed at evaluating and improving the production of lipase and at studying the culture conditions revealed that of the many different materials tested as substrates, mustard oil cake (MoC) was the best substrate for extracellular lipase production. A correlation was found between the lipase production profile and biomass development. In a study aimed at continuing this line of research, we have investigated the influence of various culture conditions, such as environmental (i.e. temperature and pH), nutritional (i.e. carbon, nitrogen, metal ions, vitamins, combined agro-wastes and growth regulators) and other factors (inoculum size and initial moisture content) on the production of lipase by A. terreus in solid state and liquid static surface cultures. We observed that optimum lipase biosynthesis occurred under the following conditions: initial pH of 6.0, 30 °C, a 96-h incubation, lactose and ammonium persulphate as the carbon and nitrogen source respectively and 80 % moisture content. Changes in the vitamins (vitamin C, riboflavin, folic acid and vitamin E) and growth regulators (gibberellic acid, kinetin, 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid) did not support enhanced lipase production. MoC and neem oil cake (NoC) added to the media at a ratio of 9:1 respectively, supported maximum lipase production. Based on these results, we concluded that controlling the various culture conditions, supplementing MoC as a substrate and nutrient source modification of the medium can spectacularly enhance lipase biosynthesis by A. terreus.     

Strain improvement for tannase production from co-culture of Aspergillus foetidus and Rhizopus oryzae

Spores from the co-culture of Aspergillus foetidus and Rhizopus oryzae were subjected to UV, heat and NTG (3-nitro,5-methylguanidine) mutagenesis. A few colonies were screened from the selected media for tannase study. Amongst all, the best mutant isolated from the heat treatment (60 degrees C for 60 min) was SCPR 337. The maximum yield of gallic acid and tannase in case of mutant strain was 95.2% and 53.6 U/ml with an incubation period of 30 h as compared to wild strain where the incubation period was 48 h with an enzyme activity of 44.2 U/ml and gallic acid yield of 94%, respectively. The mutant was sensitive to tetracycline and was also an over-producer of protease and amylase.     

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.     

Use of coconut coir fibers as an inert solid support for production of cyclosporin A

In the present study, coconut coir was evaluated as an inert support for the production of cyclosporin A (CyA) using Tolypocladium inflatum MTCC 557 by solid state fermentation. Initially, four different inert supports such as coconut coir, polyurethane foam, polystyrene beads, and sugarcane baggase were screened using different production media as moistening agents for the maximum production of CyA. Different parameters such as fermentation time, carbon sources, moisture content, pH, and inoculum size were optimized. It was observed that coconut coir impregnated with medium modified with glycerol as carbon source, pH 6, at 80% moisture content, and inoculum size of 2.5 mL/2.5 g support produced 2641 mg/kg of CyA after 12 days as compared to 998 mg/kg before optimization. The yields were further increased to 3597 mg/kg substrate with addition of combination of amino acids after 48 h of fermentation.     

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.