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Tannin Acyl Hydrolase Production by Citrobacter sp. isolated from Tannin rich Environment, using Tamarindus indica seed powder
Abstract
Bacterial isolate, Citrobacter sp., from tannery effluent loaded sites has proved as a potent producer of tannase. Production of tannase was compared in solid-state and submerged fermentation using tamarind seed as sole carbon source. Two times increase in tannase activity was seen in solid-state fermentation (90 U) than submerged fermentation (50 U) at 48 h from 5 g substrate.
... Kumar et al. (2010) used a combination of nutrient agar and tannic acid (% w/v: beef extract, 0.3; peptone, Deschamps et al. (1983) isolated five organisms by enrichment culture using a mineral medium having condensed tannin as the sole carbon source. Kumar et al. (1999) and Wilson et al. (2009) isolated Citrobacter freundii and Citrobacter sp. using mineral medium having tannic acid as sole carbon source. isolated number of tannase producing bacterial strains by plate dilution technique using selective media (% w/v: tannic acid, 1; K 2 HPO 4 , 0.05; KH 2 PO 4 , 0.05; MgSO 4 , 0.05; NH 4 NO 3 , 0.3 and agar, 3). ...
... Recent literature on SSF for tannase production claim increased productivity, high activity titres and increased stability to pH and temperature changes than SmF. Previously, Wilson et al. (2009) and Jana et al. (2013a) recorded approximately 45 and 7 times higher productivity of bacterial tannase in SSF than SmF, respectively. However, unlike the submerged tannase production, a perusal of literature showed that, production of bacterial tannase through SSF has been scarcely reported. ...
... In addition to that, tamarind seed is also most commonly used as solid substrate in SSF as an alternative tannin (carbon) source (Wilson et al., 2009;Jana et al., 2013a). Other natural tannin that has been used for the SSF is coffee husk, palm kernel cake, wheat bran (Sabu et al., 2006;Natarajan and Rajendran, 2012). ...
... The commercial importance of enzyme tannase is well established at industrial level. There are several reports on tannase production by bacteria isolated from their natural habitats including Bacillus sphaericus from general soil 11 , Enterobacter cloacae from compost 23 , Pseudomonas aeruginosa from tannery soil 12 , Bacillus subtilis from sal forest soil 24 , Pantoea sp. from olive wastes 17 , Citrobacter sp. from tannery effluent 16 , Serratia ficaria from tea processing factory 19 , Lactobacillus apodemi from mouse faeces 25 , Staphylococcus lugdunensis from human faeces 14 , Enterobacter asburiae from fish gut 15 , Enterobacter ludwigii from rumen of goats 26 , Bacillus sp. from rhizospheric soil of Cycas species 27 , Klebsiella pneumoniae from rhizospheric soil of Acacia species 28 etc. In the present research study, isolation and identification of a potent tannase producing bacteria from the rhizospheric soil of Casia species were accomplished. ...
... Earlier research investigators on bacterial tannase have reported enzyme activities of 0.356 U/ml for Bacillus licheniformis tannase 31 , 0.6 U/ml for E. cloacae tannase 23 , 1.87 U/ml for C. freundii tannase 32 , 0.4 U/ml for Citrobacter sp. 16 Identification of potent tannase producing bacterial isolate: Morphological and microscopic study of the bacterial isolate SSRY4 revealed that it is a gram positive, rod shaped bacterium (Table 1). A single band (size=1.5kb) of 1372 bp was obtained upon amplification of 16s-rRNA gene of genomic DNA of isolate SSRY4 (Fig. 3). ...
... The commercial importance of enzyme tannase is well established at industrial level. There are several reports on tannase production by bacteria isolated from their natural habitats including Bacillus sphaericus from general soil 11 , Enterobacter cloacae from compost 23 , Pseudomonas aeruginosa from tannery soil 12 , Bacillus subtilis from sal forest soil 24 , Pantoea sp. from olive wastes 17 , Citrobacter sp. from tannery effluent 16 , Serratia ficaria from tea processing factory 19 , Lactobacillus apodemi from mouse faeces 25 , Staphylococcus lugdunensis from human faeces 14 , Enterobacter asburiae from fish gut 15 , Enterobacter ludwigii from rumen of goats 26 , Bacillus sp. from rhizospheric soil of Cycas species 27 , Klebsiella pneumoniae from rhizospheric soil of Acacia species 28 etc. In the present research study, isolation and identification of a potent tannase producing bacteria from the rhizospheric soil of Casia species were accomplished. ...
... Earlier research investigators on bacterial tannase have reported enzyme activities of 0.356 U/ml for Bacillus licheniformis tannase 31 , 0.6 U/ml for E. cloacae tannase 23 , 1.87 U/ml for C. freundii tannase 32 , 0.4 U/ml for Citrobacter sp. 16 Identification of potent tannase producing bacterial isolate: Morphological and microscopic study of the bacterial isolate SSRY4 revealed that it is a gram positive, rod shaped bacterium (Table 1). A single band (size=1.5kb) of 1372 bp was obtained upon amplification of 16s-rRNA gene of genomic DNA of isolate SSRY4 (Fig. 3). ...
A potent tannase producing bacterial strain was isolated from the rhizospheric soil of Casia species and was identified as Bacillus haynesii strain SSRY4 (Genbank Accession Number MN031245). Various environment samples were inoculated in enrichment liquid media containing 1% (w/v) tannic acid for 48 hrs. Upon plating of appropriately diluted culture aliquots on tannic acid agar plates, only few of them developed bacterial colonies showing clearly visible zone of tannic acid hydrolysis around them. Zones of hydrolysis were measured in mm. Eight bacterial colonies showing maximum tannic acid hydrolysis zone around them were selected. Pure cultures were developed by repeated streaking on nutrient agar plates. The isolates were then screened for their ability to produce tannase in submerged conditions. Upon quantitative estimation of tannase production, only three isolates SSRY1, SSRY2 and SSRY4 showed considerably good enzyme activities of 1.37 U/ml, 1.29 U/ml and 1.56 U/ml respectively. Various morphological and microscopic features of the bacterial isolate SSRY4 were studied and a gram positive, rod shaped bacterium was confirmed. 16s rRNA sequencing results confirmed its identity as Bacillus haynesii strain SSRY4 (Genbank Accession Number MN031245).
... Over the last 25 years, the interest in bacterial tannases has risen owing to their widespread applications, ability to undergo genetic manipulations, and capability to live under extreme temperature conditions. Thus, in this regard, several tannaseproducing bacteria have been identified till date among which bacterial strains belonging to genera such as Lonepinella (Goel et al. 2007), Staphylococcus (Noguchi et al. (2007), Lactobacillus (Guzman-Lopez et al. (2009), Pseudomonas (Selwal et al. 2010), Serratia (Belur et al. (2010), Bacillus (Raghuwanshi et al. (Raghuwanshi et al. 2011;Muhammad et al. 2016), Azobacter (Gauri et al. (2012), Klebsiella (Sivashanmugam and Jayaraman 2013), Citrobacter (Wilson et al. (2009) Pantonea (Pepi et al. 2010, and Enterobacter (Mandal and Ghosh 2013) are predominant. The molecular weights of tannase of bacterial origin usually lie within 46.5-90 kDa (Jana et al. 2013). ...
... Several natural tannin containing substrates like wheat bran, coffee pulp and tea residue, tamarind seed powder, and rice bran have been efficiently utilized for maximal tannase production via SSF. Polyurethane foam has been the most commonly used natural support amongst various other supports (Rodrıguez-Duran et al. 2011); Wilson et al. (2009) and Jana et al. (2013) documented as high as 45 times and 7 times enhanced bacterial tannase production via SSF in comparison to SMF. However the original research studies utilizing SSF for bacterial tannase production are scanty. ...
The outburst of green biotechnology has facilitated a substantial upsurge in the usage of enzymes in a plethora of industrial bioconversion processes. The tremendous biocatalytic potential of industrial enzymes provides an upper edge over chemical technologies in terms of safety, reusability, and better process control. Tannase is one such enzyme loaded with huge potential for bioconversion of hydrolysable tannins to gallic acid. Tannins invariably occur in pteridophytes, gymnosperms, and angiosperms and predominately cumulate in plant parts like fruits, bark, roots, and leaves. Furthermore, toxic tannery effluents from various tanneries are loaded with significant levels of tannins in the form of tannic acid. Tannase can be principally employed for debasing the tannins that predominately occur in the toxic tannery effluents thus providing a relatively much cheaper measure for their biodegradation. Over the years, microbial tannase-catalyzed tannin degradation has gained momentum. The plentious availability of tannin-containing agro- and industrial waste paves a way for efficient utilization of microbial tannase for tannin degradation eventually resulting into gallic acid production. Gallic acid has received a great deal of attention as a molecule of enormous therapeutic and indusrial potential. The current worldwide demand of gallic acid is 8000 t per annum. As a matter of fact, bioconversion of tannins into gallic acid through fermentation has not been exploited completely. This necessitates further studies for development of more efficient, economical, productive processes and improved strains for gallic acid production so as to meet its current demand.
... The strains having positive test for tannase production was further subjected to quantitative estimation and results revealed that Bacillus amyloliquefaciens showed highest enzyme production (1.269 U/mL) followed by Enterobacter aerogenes, Roultella ornithinolytica and Klebsiella oxytoca (Table 4) after 24 h of incubation at 37 °C. For the tannase production, first strain Lactobacillus plantarum was isolated from the waste of olive mill (Wilson et al., 2009;Ayed and Hamdi, 2002). Bacterial strain capable of degrading tannin was isolated from goat and sheep feces (Mosleh et al., 2014). ...
Tannase (Tannin acyl hydrolase) is an intracellular/extracellular enzyme produced by microorganisms. Tannase has high market demand due to its important role in different industries. The optimization of different parameters for each microorganism is necessary for obtaining maximum tannase yield. The aim of present study was to optimize the medium components and their concentration employing applications of response surface methodology (RSM). Ten bacterial strains isolated from fish gut content were screened for tannase producing potential. Among these, four strains, Klebsiella oxytoca, Roultella ornithinolytica, Bacillus amyloliquefaciens and Enterobacter aerogenes expressed greenish zones around their colonies. B. amyloliquefaciens showed highest tannase production (1.27 IU/mL) under un-optimized conditions and was selected for further work. During one factor at a time optimization of physical parameters, incubation temperature 37 °C, pH 5, inoculum size 1% and incubation period of 24 h yielded maximum tannase. To screen the significant medium components, 12 experimental runs of Plackett-Burman design for six variables (tannic acid, K 2HPO4, CaCl2, MgSO4, NH4NO3 and yeast extract) were carried out. From these experimental runs, the enzyme assay results were analyzed using multiple regression. Three variables i.e., tannic acid, CaCl 2 and yeast extract showed significant impact on tannase production. Concentrations of these variables were optimized using BoxBehnken design (BBD). Results of 15 experimental runs of BBD showed maximum tannase production corresponding to 0.5% tannic acid, 0.1% CaCl 2 and 0.275% yeast extract. The highest tannase activity was recorded at pH 7, 0.5% substrate concentration and 40 °C.
... Red gram husk [3] . sugar cane bagasse [4] and also using different microbial strains like bacteria [5,6] and other fungal strains [7,8,9] . The major crop waste produced in India are straws of paddy, wheat, millet, sorghum, pulses, oil seed crops, maize stalks, jute sticks, sugar cane trash, mustard stalks, etc. ...
... Thereafter, the fall in the activity may be due to competitive or non-competitive inhibition to the tannase active sites (Kar et al., 1999). In a comparison of this article, Citrobacter sp (Wilson, Rojan, Kumar, and Sabu, 2009), Pseudomonas aeruginosa IIIB 8914 (Selwal et al., 2010), Lactobacillus sp. ASR-S1 (Sabu, Augur, Swati, and Pandey, 2006), Lactobacillus plantarum MTCC1409 (Natarajan and Rajendran, 2012), and bacterial strain B2.2 (Nandini, Nandini, and Sundari, 2015) showed tannase activity of 18 U/gds, 13.65 U/mL, 0.5 U/gds, 5.319 and 9.15 U/g with tamarind seed powder, Amla leaves, wheat bran, coffee husk and combination of pomegranate peel and spent tea powder respectively. ...
In this present research, tannase producing bacteria were isolated from the samples of the gastrointestinal tract of a Goat. The liquid enrichment and spread plate technique were adopted and 6 distinct bacterial colonies were isolated on a nutrient agar plate. Based on qualitative and quantitative methods of screening, one isolate among six was found promising and identified as Bacillus cereus M1GT by using 16S rRNA gene sequencing technique. The cost-effective substrate Triphala was used as a tannin and energy source for tannase production through solid-state fermentation in shake flask independently. Process factors were screened by a two-step optimization process i.e. one factor at a time method and central composite design. The study of statistical experimental design with Triphala at optimized conditions showed 6.1-fold (0.116 U/gds) higher in tannase activity than that obtained in submerged fermentation. Further, the kinetics of Bacillus cereus M1GT under optimum process conditions were studied. The Logistic equation (growth, ‘µ’), Luedeking Piret equation (Tannase, ‘α’, and ‘β’) and Substrate utilization equation (Tannic acid, ‘m’ and ‘n’) of unstructured kinetic models were evaluated with the MATLAB program. The experimental and simulated values exhibited a good correspondence, indicating that models will define tannase production process.
... Red gram husk [3] . sugar cane bagasse [4] and also using different microbial strains like bacteria [5,6] and other fungal strains [7,8,9] . The major crop waste produced in India are straws of paddy, wheat, millet, sorghum, pulses, oil seed crops, maize stalks, jute sticks, sugar cane trash, mustard stalks, etc. ...
Tannin acyl hydrolase produced extra-cellularly by Aspergillus niger in solid state fermentation of Rice husk. The enzyme was purified from the cell-free extract by ammonium sulphate precipitation followed by diethylaminoethyl-cellulose column chromatography. The protein content of crude and purified was found to be 7·6 and 0·45 mg/ml respectively. The specific activity of crude tannase was found to be 28·5 U/mg of protein while that of purified tannase was 173·0 U/mg of protein. The optimum temperature and pH of the crude sample was analysed and found to be 35 to 40 ºC and pH 5 respectively. The enzyme obtained was applied for declarification of pomegranate juice. In the declarification of fruit juice, a 56% decrease in tannin content was observed after 2 h of incubation at 37 ºC with 1 ml of purified tannase (173 U/mg).
... Many researchers have reported tannase production with coffee husk from Lactobacillus plantarum 5,19 ; tamarind seed powder from Trichoderma harzianum MTCC 10841 20 , Citrobacter sp. 21 , Lactobacillus plantarum 4,19 whereas tea leaves from R.oryzae showed tannase production 18 . Since the substrate triphala is rich in tannin content, hence showed maximum tannase production (Fig. 2), therefore for further optimization studies to develop media for tannase production by Bacillus gottheilii M2S2, triphala powder was considered. ...
Production of tannase was performed in packed bed reactor filled with an inert support polyurethane foam (PUF) using Bacillus gottheilii M2S2. The influence of process parameters such as fermentation time (24–72 h), tannic acid concentration (0.5–2.5% w/v), inoculum size (7–12% v/v), and aeration rate (0–0.2 L/min) on tannase production with PUF were analyzed using one variable at a time (OVAT) approach. The outcome of OVAT was optimized by central composite design. Based on the statistical investigation, the proposed mathematical model recommends 1% (w/v) of tannic acid, 10% (v/v) of inoculum size and 0.13 L/min of aeration rate for maximum production (76.57 ± 1.25 U/L). The crude enzyme was purified using ammonium sulfate salt precipitation method followed by dialysis. The biochemical properties of partially purified tannase were analyzed and found the optimum pH (4.0), temperature (40 °C) for activity, and Km (1.077 mM) and Vmax (1.11 µM/min) with methyl gallate as a substrate. Based on the SDS-PAGE analysis, tannase exhibited two bands with molecular weights of 57.5 and 42.3 kDa. Briefly, the partially purified tannase showed 4.2 fold increase (63 ± 1.60 U/L) in comparison to the submerged fermentation and the production of tannase was validated by using NMR spectrometer.
... The Sequencing PCR was done according to manufacturer (Beckman Coulter). The amplified product was sequenced on Beckman coulter CEQ 8000 DNA sequencer.[1][13]Antibiotic sensitive test was done at LALA Ram Sarup Institute of Tuberculosis and Respiratory disease, New Delhi. ...
Tannase is a commercial important enzyme used to catalyze the hydrolysis of ester and depside bond of tannin rich material which has yielded industrially valuable products. Keeping the significance of this enzyme, it has been always isolated from tannin-rich source and showed its potential in degrading it. The present study deals with isolating bacteria producing tannase enzyme from non-tannin rich source. Six potential bacteria were isolated from virgin soils which had enzyme activity equally to reported bacteria. These bacteria were identified on basis of antibiotic sensitivity test and 16s rRNA. Klebsiella pneumonia isolated from garlic field soil showed 1.40 U/ml activity and Enteroabacter cloacaeshowed 1.23 U/ml which is more than reported. Hence this enzyme has potential in various food and pharmaceutical industries.
... Many researchers have reported tannase production with coffee husk from Lactobacillus plantarum 5,19 ; tamarind seed powder from Trichoderma harzianum MTCC 10841 20 , Citrobacter sp. 21 , Lactobacillus plantarum 4,19 whereas tea leaves from R.oryzae showed tannase production 18 . Since the substrate triphala is rich in tannin content, hence showed maximum tannase production (Fig. 2), therefore for further optimization studies to develop media for tannase production by Bacillus gottheilii M2S2, triphala powder was considered. ...
Tannase is an important enzyme which finds commercial applications in food industry to reduce the level of tannins in fruit juices, preparation of instantaneous tea and production of gallic acid. Various low cost tannin rich residues such as coffee husk, tamarind seed powder, tea leaves and Triphala powder were studied in semi-solid state fermentation process. Triphala was found to be a prominent substrate which has exhibited maximum tannase activity of 29 ± 0.35 U/L. Thereafter, sequential statistical approach was used to optimize tannase production with Triphala in shake flask. The classical one-variable-at-a-time approach determined moistening media, tannic acid and inoculum volume which significantly influenced the tannase production. A central composite design showed that the optimal values of these factors were 6.2 mL, 1% (w/v) and 6.4 mL respectively. Subsequently, a 7-fold increase in corresponding tannase yield (106 ± 0.61 U/L) was obtained, compared with that produced in the submerged fermentation. The crude tannase showed optimum activity at 40°C and pH 4.0. Vmax and Km values were 1.404 µmol/ml.min and 1.24 mM respectively.
... Red gram husk [3] . sugar cane bagasse [4] and also using different microbial strains like bacteria [5,6] and other fungal strains [7,8,9] . The major crop waste produced in India are straws of paddy, wheat, millet, sorghum, pulses, oil seed crops, maize stalks, jute sticks, sugar cane trash, mustard stalks, etc. ...
Tannin acyl hydrolase produced extra-cellularly by Aspergillus niger in solid state fermentation of Rice husk. The enzyme was purified from the cell-free extract by ammonium sulphate precipitation followed by diethylaminoethyl-cellulose column chromatography. The protein content of crude and purified was found to be 7·6 and 0·45 mg/ml respectively. The specific activity of crude tannase was found to be 28·5 U/mg of protein while that of purified tannase was 173·0 U/mg of protein. The optimum temperature and pH of the crude sample was analysed and found to be 35 to 40 ºC and pH 5 respectively. The enzyme obtained was applied for declarification of pomegranate juice. In the declarification of fruit juice, a 56% decrease in tannin content was observed after 2 h of incubation at 37 ºC with 1 ml of purified tannase (173 U/mg).
... Incubation time, (X 1 ), h Inoculum volume, (X 2 ), %vv À 1 Initial pH (X 3 ) Tannase activity (U L À 1 ) After two more transfers, 1 mL of turbid broth was diluted appropriately and then spread plated on screening medium (Nutrient agar plates), incubated for 24 h. Separate and morphological distinct colonies obtained on nutrient agar plates were screened for tannase activity by growing them in M1 medium (Ajay et al., 1999;Murugan et al., 2007;Wilson et al., 2009). The strains showing maximum tannase activity were considered for further tannase production and were identified using 16S rRNA gene sequence homology at Agharkar research Institute (Pune, India) as described elsewhere (Thivaharan and Vytla, 2013). ...
Tannin acyl hydrolase (E.C. 3.1.1.20), commonly referred as "tannase", breaks hydrolysable tannins into gallic acid and glucose. It finds commercial applications in food industry to reduce the level of tannins in fruit juices, preparation of instantaneous tea and production of gallic acid. In this work, a tannase producing Bacillus gottheilii M2S2 was isolated from the tannery effluent soil and a sequential optimization strategy was used to determine the effect of carbon source and inducer-tannic acid, nitrogen source NH4NO3, inoculum volume, initial pH, incubation time and mineral salts (KH2PO4, MgSO4, NaCl and CaCl2·2H2O) in the production of tannase from Bacillus gottheilii M2S2 in solid state fermentation. Polyurethane foam was used as an inert support for growth of the microorganism. The microorganism was fermented at 32 °C in an optimized liquid medium composed of tannic acid (4%), NH4NO3 (2%), KH2PO4 (0.1%), MgSO4 (0.2%), NaCl (0.1%) and CaCl2·2H2O (0.05%) adjusted to initial media pH 4.74 then inoculated with 7.035% vv⁻¹ inoculum and impregnated on polyurethane foam for 26.45 h under static condition A final activity of 49.32±0.30 U L⁻¹ was achieved, which represents an increase of 328% in relation to the initial unoptimized conditions.
Microorganisms have been used for the production of various enzymes, including inducible tannase for various industrial and environmental applications. Tannases have lot of potential to convert hydrolysable tannins to gallic acid, which is one of the important industrial and therapeutic significant molecules whose demand is over 10000 tons per year. Tannins invariably occur in angiosperms, gymnosperms and pteridophytes, and predominantly present in various parts of plants such as, leaves, roots, bark and fruit. Furthermore, tannery effluents are frequently loaded with significant levels of tannic acid. Tannase can be effectively used to decrease tannin load in the toxic tannery effluent thus providing the opportunity to minimize the operational cost. Over the past three decades, tannase from microbial sources has been proposed for the degradation of natural tannins. The availability of various agro-industrial residues paves a way for maximum utilization of tannase production for the degradation of tannin and eventually the production of gallic acid. In this review, an illustrative and comprehensive account on tannase from microbial source for current day applications is presented. The present review emphasises on up-to-date microbial sources of tannases, biochemical properties, optimization of tannase production in solid state and submerged fermentation and its industrial and environmental applications.
Globally, around five billion metric tons of agro waste is produced every year. In India alone, solid wastes exceeding 960 million tons is produced annually as by‐products of municipal, agricultural, mining, industrial activities, etc. Several industries utilizing plant constituents as raw and processing materials generate extensively high volumes of wastewater having plentiful tannins. Toxic tannery effluent wastes from tannery industries also possess superabundant levels of tannins as tannic acid. Thus, there is a continuously growing apprehension about these stockpiling wastes which are creating pollution and health hazards. A worthy and sustainable approach to deal with these accumulating wastes can be to utilize them as alternates of high cost raw materials for economic production of high value products like gallic acid. The biocatalytic potential of bacterial tannase in hydrolyzing natural tannins and tannic acid may be exploited for biotransformation of tannins into gallic acid.
Tannase has different benefits in food, chemical and pharmaceutical fields. Seventeen Serratia marcescens isolates were collected from septicemia, wound infections and hospital environment(babies incubators).These isolates were identified by biochemical tests and Vitek 2 system that contained Vitek GNI card then conformed by16S rRNA gene products(amplified size 179 bp) for genotypic detection. After that, they screened for higher tannase production and Serratia marcescens b9 was a better producer of tannase with a larger diameter of a dark green zone. The tannase activity was increased to 63U/ml when this isolate was cultivated under the optimal conditions which consisted of using nutrient broth supplemented with ber leaves at pH value 5.5 and a temperature equals to 37°C for 72 hours. In the partial purification of tannase, ammonium sulfate was more efficient than organic solvents, since it was found that 70% saturation of ammonium sulfate led to precipitate of tannase with tannase activity of 80U/ml. In contrast, 30% of ethanol, acetone, and isopropanol led to precipitate of tannase with different levels of activity ranged between 45-47U/ml. Consequently, ber leaves have a potential as an effective and much cheaper (economical) substrate for tannase production in comparison with traditionally used substrates like tannic acid.
Lactobacillus plantarum produced an extracellular tannase after 24 h growth on minimal medium of amino acids containing 2 g tannic acid l–1. Enzyme production (6 U ml–1) was optimal at 37 C and pH 6 with 2 g glucose l–1 and 7 g tannic acid l–1 in absence of O2.
The aim of this work was to select strains of Aspergillus niger for tannase production. Growth of colonies in plates with tannic acid-containing medium indicated their ability to synthesize tannase. Tannase activity was also measured in solid-state fermentation. A. niger 11T25A5 was the best tannase producer (67.5 U.g-1/72 hours of fermentation).
Lactobacillus casei was grown at 37 degrees C on sugarcane bagasse (5 g) soaked with cassava starch hydrolysate (final moistening volume 34 ml) containing 3 g reducing sugar in a solid-state condition. The maximum yield of L-lactic acid after various process optimisations was 2.9 g/5 g initial substrate corresponding to 97% conversion of sugar to lactic acid with initial substrate moisture of 72%.
Bacillus licheniformis KBR 6 produced maximum extracellular tannase activity at 0.21U ml–1 with 1.5% (w/v) tannic acid either in the absence or presence of glucose (1gl–1) after 18–21h growth though the organism did not attain maximum growth until 36h.
A tannase yielding bacterial strain was isolated from sheep excreta. It was identified as Lactobacillus sp. ASR S1. The bacterial strain produced extracellular tannase under solid-state fermentation (SSF) using tamarind seed powder (TSP), wheat bran (WB), palm kernel cake (PKC) and coffee husk (CH). Among different substrates, coffee husk resulted maximal extra-cellular production of tannase. To optimize the extracellular yield of tannase under SSF various physico-chemical and nutritional parameters were studied. Supplementation of tannic acid was found useful for enzyme synthesis by the bacterial culture selectively depending up on the substrate. Maximum tannase production (0.85 U/gds) was obtained when SSF was carried out using coffee husk, supplemented with 0.6% tannic acid and 50% (w/v) moisture, inoculated with 1 mL cell suspension and incubated at 33 °C for 72 h.
A method for assay of microbial tannase (tannin acyl hydrolase) based on the formation of chromogen between gallic acid and rhodanine is reported. Unlike the previous protocols, this method is sensitive up to gallic acid concentration of 5 nmol and has a precision of 1.7% (relative standard deviation). The assay is complete in a short time, very convenient, and reproducible.
A tannase producing bacterial strain KBR 6 has been isolated from lateritic soil and identified as Bacillus licheniformis. It is capable of producing tannase in the medium containing only tannic acid. The rapid degradation of tannic acid and production of extracellular tannase was observed in three different media containing tannic acid (M1), tannic acid + basal salt (M2) and tannic acid + basal salt + glucose (M3). Maximum enzyme production and growth of the organism was obtained at 18-21 h and 30-36 h, respectively. The increased order of enzyme production in relation to different media is as per the following sequence, M3 > M2 > M1. The maximum growth and enzyme production was observed at pH 5.0. The pH and temperature optima of the enzyme activity were found to be at 5.75 and 60 degrees C respectively. Paper chromatographic analysis indicates that gallic acid is the enzymatic degradative product of tannic acid.
We examined a range of oenological lactic acid bacteria species and reference strains for their potential to degrade tannins. Bacterial tannase activity was checked by a spectrophotometric and a visual reading method. None of the strains belonging to the oenological species of the genus Lactobacillus, Leuconostoc, Oenococcus or Pediococcus were tannase producers, with the exception of Lactobacillus plantarum. All the L. plantarum strains analyzed were positive for tannase activity and their identities were reconfirmed by L. plantarum PCR-specific assay or by sequencing the 16S rDNA. Tannase activity could be considered an important criterion for the selection of malolactic starter cultures since it might confer advantages in the winemaking process by reducing astringency and haze in wine.
Palm kernel cake (PKC), the residue obtained after extraction of palm oil from oil palm seeds and tamarind seed powder (TSP) obtained after removing the fruit pulp from tamarind fruit pod were tested for the production of tannase under solid-state fermentation (SSF) using Aspergillus niger ATCC 16620. The fungal strain was grown on the substrates without any pretreatment. In PKC medium, a maximum enzyme yield of 13.03 IU/g dry substrate (gds) was obtained when SSF was carried out at 30 degrees C, 53.5% initial substrate moisture, 33 x 10(9) spores/5 g substrate inoculum size and 5% tannic acid as additional carbon source after 96 h of fermentation. In TSP medium, maximum tannase yield of 6.44 IU/gds was obtained at 30 degrees C, 65.75% initial substrate moisture, 11 x 10(9) spores/5 g substrate inoculum, 1% glycerol as additional carbon source and 1% potassium nitrate as additional nitrogen source after 120 h of fermentation. Results from the study are promising for the economic utilization and value addition of these important agro residues, which are abundantly available in many tropical and subtropical countries.
Tannase activity of bacteria capable of degrading tannin-protein complexes was determined by a newly developed visual reading method. The method is based on two phenomena: (i) the ability of tannase to hydrolyze methyl gallate to release free gallic acid and (ii) the green to brown coloration of gallic acid after prolonged exposure to oxygen in an alkaline condition. The method has been successfully used to detect the presence of tannase in the cultures of bacteria capable of degrading tannin-protein complexes.
The present study was aimed at finding the optimal conditions for immobilization of Bacillus licheniformis KBR6 cells in calcium-alginate (Ca-alginate) beads and determining the operational stability during the production of tannin-acyl-hydrolase (tannase) under semicontinous cultivation.
The active cells of B. licheniformis KBR6 were immobilized in Ca-alginate and used for the production of tannase. The influence of alginate concentration (5, 10, 20 and 30 g l(-1)) and initial cell loading on enzyme production were studied. The production of tannase increased significantly with increasing alginate concentration and reached a maximum enzyme yield of 0.56 +/- 0.03 U ml(-1) at 20 g l(-1). This was about 1.70-fold higher than that obtained by free cells. The immobilized cells produced tannase consistently over 13 repeated cycles and reached a maximum level at the third cycle. Scanning electron microscope study indicated that the cells in Ca-alginate beads remain in normal shape.
The Ca-alginate entrapment is a promising immobilization method of B. licheniformis KBR6 for repeated tannase production. Tannase production by immobilized cells is superior to that of free cells because it leads to higher volumetric activities within the same period of fermentation.
This is the first report of tannase production from immobilized bacterial cells. The bacterium under study can produce higher amounts of tannase with respect to other fungal strains within a short cultivation period.
Bergey's Manual of Determinative Bacteriology
- D H Bergey
- F C Harrison
- R S Breed
- B W Hammer
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