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An in-depth study of any enzyme and its applications require a steady and unerring approach. An infallible assay method ensuring high accuracy in quantifying enzyme activity determines the success of enzyme catalyzed reactions. A thorough understanding of assay requirements & a sound knowledge of calculation part along with its merits and demerits must be taken into consideration before selecting an assay method. Even though a number of tannase assay methods exist in literature; however, a great majority of them deal with certain shortcomings and limitations. This review portrays some of the most widely adopted methods of assaying tannase activity; both in qualitative and quantitative manner. The main highlights and methodological limitations of the assay methods have also been discussed so as to facilitate scientific communities worldwide to select the most suitable assay method as per their requirement.

Tannase from Aspergillus niger NL112 was purified 5.1-fold with a yield of 50.44% via ultrafiltration, DEAE-Sepharose Fast Flow column chromatography, and Sephadex G-100 column chromatography. The molecular weight of the purified tannase was estimated as 45 kDa. The optimum temperature and pH for its activity were 45 °C and 5.0, respectively. The results of circular dichroism, FT-IR (Fourier transform infrared) spectroscopy, and fluorescence spectra indicated that high temperature could lead to the change of tannase secondary and tertiary structures. Tannase had a greater affinity for tannic acid at 40 °C with a Km value of 2.12 mM and the greatest efficiency hydrolysis (Kcat/Km) at 45 °C. The rate of inactivation (k) increased with the increase of temperature and the half-life (t1/2) gradually decreased. It was found to be 1.0 of the temperature quotient (Q10) value for tannic acid hydrolysis by tannase. The thermodynamic parameters of the interaction system were calculated at various temperatures. The positive enthalpy (ΔH) values and decreasing ΔH values with the increase of temperature indicated that the hydrolysis of tannase was an endothermic process. Our results indicated that elevated temperature could change the tertiary structure of tannase and reduce its thermostability, which caused a gradual decrease of tannase activity with an increase in temperature.

Partially purified tannase of Talaromyces verruculosus (305.6 U/mL) was immobilized on calcium alginate beads (8.0 ± 1.0 mm) using 1% glutaraldehyde as a cross-linking agent. Immobilized enzyme preparation retained (39.4%) 120.4 U/mL tannase activity, 79.6% immobilization yield, and 49.5% efficiency. Immobilized preparation was characterized by SEM and it revealed the changed topology of the immobilized bead. Functional groups present in immobilized beads were examined by FTIR spectra. Immobilized tannase was found optimally active at pH 8.0 and 60 °C, whereas the stability was obtained between 30 and 60 °C, but at high (9.0) and low pH (4.0), immobilized tannase was also found very stable. Beads were found stable up to 9th cycle of reuse with 49.27% relative activity. After the treatment by immobilized tannase, 78.02 ± 1.93, 72.24 ± 2.05, 65.29 ± 1.21, 59.06 ± 0.96, and 64.61 ± 1.07% tannin reduction were measured in black tea product 1, black tea product 2, black tea product 3, green tea product 1, and green tea product 2, respectively. The greatest change in pH, turbidity, and antioxidant activity was found in black tea product 2 infusion while the least change was obtained in green tea product 2 infusion after the tannase treatment.

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.

Tannase production from Bacillus velezensis TA3 was optimized by sequential statistical approach using pomegranate as a solid substrate. Easily available and low-cost substrates such as cow dung, coffee husk, apple peel and pomegranate peel were initially screened. Among these substrates, pomegranate peel was found to enhance tannase activity. Further, pomegranate peel was used as the substrate to optimize the factors by traditional methods. In one-variable-at-a-time approach optimum process conditions were, 48 h incubation (1.92 U/g), 0.3% tannic acid (1.33 U/g), 0.08% minerals (2.3 U/g) with 55% initial moisture content (2.7 U/g). To screen the impact of variables such as fermentation period, moisture content, sodium chloride level, tannic acid concentration and required minerals on tannase production under solid state fermentation, a two-level full factorial design was used. Among these factors, incubation time, moisture and tannic acid concentrations were found to have significant influence on tannase production (p

Made available in DSpace on 2022-04-28T19:47:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-11-03 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Enzyme immobilization is an important alternative to stabilize enzyme properties favoring the efficiency of derivatives (enzyme + support/matrix) for different purposes. According to this, the current study aimed to immobilize the Aspergillus fumigatus CAS21 tannase and the use of the derivatives in the treatment of the effluent produced by the tannery industry. The tannase was immobilized on sodium alginate, DEAE-Sephadex, amberlite, and glass pearls as supports. Calcium alginate was the most adequate support for tannase immobilization with 100% yield and 94.3% for both efficiency and activity. The best tannase activity for the calcium alginate derivative was obtained at 50°C–60°C and pH 5.0. Thermal and pH stabilities evaluated for 24 h at 30°C–60°C and pH 4–7, respectively, were improved if compared to the stability of the free enzyme. Considering the reuse of the calcium alginate derivative, 78% of the initial activity was preserved after 10 catalytic cycles, and after the 9-month storage at 4°C, the activity was maintained in 70%. This derivative was applied in a packed bed reactor (PBR) for the treatment of tannin-rich effluents from the tannery industry. The reduction of the tannin content was effective reaching degradation of 74–78% after 48 h of PBR operation. The concentration of total phenolic compounds was also reduced, and the color and clarity of the effluent improved. In conclusion, the calcium alginate derivative is an attractive alternative as biocatalyst for large-scale treatment of the effluents from the tannery industry. Instituto de Química de Araraquara—UNESP Departamento de Biologia Faculdade de Filosofia Ciências e Letras de Ribeirão Preto—USP Instituto de Química de Araraquara—UNESP FAPESP: 2016/11311-5

Background Herbaspirillum camelliae is a gram-negative endophyte isolated from the tea plant. Both strains WT00C and WT00F were found to hydrolyze epigallocatechin-3-gallate (EGCG) and epicatechin-3-gallate (ECG) to release gallic acid (GA) and display tannase activity. However, no tannase gene was annotated in the genome of H. camelliae WT00C. Results The 39 kDa protein, annotated as the prolyl oligopeptidase in the NCBI database, was finally identified as a novel tannase. Its gene was cloned, and the enzyme was expressed in E. coli and purified to homogeneity. Moreover, enzymatic characterizations of this novel tannase named TanHcw were studied. TanHcw was a secretary enzyme with a Sec/SPI signal peptide of 48 amino acids at the N-terminus, and it catalyzed the degradation of tannin, methyl gallate (MG), epigallocatechin-3-gallate (EGCG) and epicatechin-3-gallate (ECG). The optimal temperature and pH of TanHcw activities were 30 °C, pH 6.0 for MG and 40 °C, pH 7.0 for both EGCG and ECG. Na+, K+ Mn2+ and Triton-X100, Tween80 increased the enzyme activity of TanHcw, whereas Zn2+, Mg2+, Hg2+, EMSO, EDTA and β-mercaptoethanol inhibited enzyme activity. Km, kcat and kcat /Km of TanHcw were 0.30 mM, 37.84 s−1, 130.67 mM−1 s−1 for EGCG, 0.33 mM, 34.59 s−1, 105.01 mM−1 s−1 for ECG and 0.82 mM, 14.64 s−1, 18.17 mM−1 s−1 for MG, respectively. Conclusion A novel tannase TanHcw from H. camelliae has been identified and characterized. The biological properties of TanHcw suggest that it plays a crucial role in the specific colonization of H. camelliae in tea plants. Discovery of the tannase TanHcw in this study gives us a reasonable explanation for the host specificity of H. camelliae. In addition, studying the characteristics of this enzyme offers the possibility of further defining its potential in industrial application.

A tannase-encoding gene, AotanB, from Aspergillus oryzae RIB40 was overexpressed in A. oryzae AOK11 niaD-deficient mutant derived from an industrial strain under the control of an improved glucoamylase gene promoter PglaA142. The recombinant tannase, designated as rAoTanBO, was produced efficiently as an active extracellular enzyme. Purified rAoTanBO showed a smeared band with a molecular mass of approximately 80–100 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The rAoTanBO had a molecular mass of 65 kDa, after treatment with endo-β-N-acetylglucosaminidase H. Purified rAoTanBO exhibited maximum activity at 30–35°C and pH 6.0. The tannase activity of purified rAoTanBO towards natural and artificial substrates was 2–8 folds higher than that of the recombinant enzyme produced by Pichia pastoris, designated as rAoTanBP. N-terminus of the mature rAoTanBP had six more amino acids than the N-terminus of the mature rAoTanBO. Kinetic analyses showed that rAoTanBO had higher catalytic efficiency (kcat/Km) than rAoTanBP. rAoTanBO was stable up to 60°C and higher thermostability than rAoTanBP. N-linked oligosaccharides had no effect on the activity and stability of rAoTanBO and rAoTanBP.

Hutajulu IBE, Kulla PDK, Retnaningrum E. 2021. Diversity of lactic acid bacteria isolated during fermentation of indigenous cassava obtained from Sumba, East Nusa Tenggara, Indonesia. Biodiversitas 22: 2561-2570. Traditional fermented food products hold a great significance in the routine diet of Indonesian inhabitants. The present study investigated the diversity of lactic acid bacteria (LAB) found in fermented indigenous cassava, ubi karet busuk, produced in Sumba, East Nusa Tenggara, Indonesia. The isolated LAB was characterized for their phenotypic, biochemical, and molecular traits, which involved 16S rRNA gene sequencing, determination of lactic acid fermentation pathway, and measurement of b-glucosidase and tannase activity in the LAB strains. During fermentation, 15 LAB strains were obtained from the cassava. Among these, 93.33% strains were found to be homofermentative, whereas 6.73% strains displayed heterofermentative traits. BLASTn and phylogenetic analysis classified 15 LAB strains into six species, namely Enterococcus faecium, Enterococcus faecalis, Enterococcus casseliflavus, Leuconostoc mesenteroides, Lactococcus lactis, and Enterococcus sp. Among these, 10 LAB strains displayed b-glucosidase activity, with highest enzyme activity of 7.13 U/mL recorded in L. lactis F14IS3. Interestingly, two of these strains also showed expression of tannase, with F6IS1 strain displaying highest enzyme activity of 38.23 U/mL. Thus, the study results highlighted the suitability of these b-glucosidase and tannase-producing LAB strains to be used as a starter in the fermentation of cassava and other substrates such as vegetables, fruits, and legumes. These strains can also be utilized in animal feed, food, and beverage industries.

A total of 117 Bacillus strains were isolated from Miang, a culture relevant fermented tea of northern Thailand. These strains were collected from 16 sampling sites in north Thailand. In this collection 95 isolates were tannin-tolerant Bacillus capable of growth on nutrient agar supplemented with 0.5% (w/v) total tannins from tea leaves extract (TE). The strains were also positive for pectinase, xylanase and amylase activity, while 91 and 86 isolates were positive for cellulase and &beta, mannanase, respectively. Only 21 isolates producing extracellular tannase were selected for further characterization. Identification by 16S rRNA gene sequence analysis revealed that more than 50% (11 of 21 isolates) were Bacillus tequilensis, whereas the remaining were B. siamensis (3), B. megaterium (3), B. aryabhattai (3) and B. toyonensis (1). B. tequilensis K34.2 produced the highest extracellular tannase activity of 0.60 U/mL after cultivation at 37 &deg, C for 48 h. In addition, all 21 isolates were resistant to 0.3% (w/v) bile salt, sensitive to gentamicin, erythromycin, vancomycin and kanamycin and also tolerant to acidic condition. Cell hydrophobicity varied from 9.4 to 80.4% and neutralized culture supernatants of some Bacillus isolates showed bacteriocin producing potentiality against Samonella enterica serovar Typhimurium TISTR 292. All tested probiotic properties indicated that B. tequilensis K19.3, B. tequilensis K34.2 and B. siamensis K19.1 had high probiotic potential. This is the first report describing tannin-tolerant Bacillus and their extracellular tannase producing capability in Miang, a traditional fermented tea of Thailand.

Made available in DSpace on 2020-12-12T01:18:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-04-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The Aspergillus fumigatus CAS21 tannase was spray dried with β-cyclodextrin, Capsul® starch, soybean meal, lactose, and maltodextrin as adjuvants. The moisture content and water activity of the products ranged from 5.6 to 11.5% and from 0.249 to 0.448, respectively. The maximal tannase activity was achieved at 40–60 ºC and pH 5.0–6.0 for the powders containing β-cyclodextrin and Capsul® starch, which was stable at 40 ºC and 40–60 ºC for 120 min, respectively. For all the dried products, tannase retained its activity of over 80% for 120 min at pH 5.0 and 6.0. Salts and solvents influenced the activity of the spray-dried tannase. The activity of the spray-dried tannase was maintained when preserved for 1 year at 4 ºC and 28 ºC. Spray-dried tannase reduced the content of tannins and polyphenolic compounds of leather effluent and sorghum flour and catalyzed the transesterification reaction. The spray drying process stabilized the tannase activity, highlighting the potential of dried products for biotechnological applications. Instituto de Química de Araraquara – UNESP, Avenida Professor Mário Degni s/nº, Quitandinha Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo USP, Av. do Café s/n Faculdade de Filosofia Ciências e Letras de Ribeirão Preto – USP, Avenida Bandeirantes 3900 Instituto de Química de Araraquara – UNESP, Avenida Professor Mário Degni s/nº, Quitandinha CNPq: 142389/2017-0 FAPESP: 2016/11311-5

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.

Background High amounts of insoluble substrates exist in the traditional solid-state fermentation (SSF) system. The presence of these substrates complicates the determination of microbial biomass. Thus, enzyme activity is used as the sole index for the optimization of the traditional SSF system, and the relationship between microbial growth and enzyme synthesis is always ignored. This study was conducted to address this deficiency. All soluble nutrients from tea stalk were extracted using water. The aqueous extract was then mixed with polyurethane sponge to establish a modified SSF system, which was then used to conduct tannase production. With this system, biomass, enzyme activity, and enzyme productivity could be measured rationally and accurately. Thus, the association between biomass and enzyme activity could be easily identified, and the shortcomings of traditional SSF could be addressed. Results Different carbon and nitrogen sources exerted different effects on microbial growth and enzyme production. Single-factor experiments showed that glucose and yeast extract greatly improved microbial biomass accumulation and that tannin and (NH4)2SO4 efficiently promoted enzyme productivity. Then, these four factors were optimized through response surface methodology. Tannase activity reached 19.22 U/gds when the added amounts of tannin, glucose, (NH4)2SO4, and yeast extract were 7.49, 8.11, 9.26, and 2.25%, respectively. Tannase activity under the optimized process conditions was 6.36 times higher than that under the initial process conditions. The optimized parameters were directly applied to the traditional tea stalk SSF system. Tannase activity reached 245 U/gds, which is 2.9 times higher than our previously reported value. Conclusions In this study, a modified SSF system was established to address the shortcomings of the traditional SSF system. Analysis revealed that enzymatic activity and microbial biomass are closely related, and different carbon and nitrogen sources have different effects on microbial growth and enzyme production. The maximal tannase activity was obtained under the optimal combination of nutrient sources that enhances cell growth and tannase accumulation. Moreover, tannase production through the traditional tea stalk SSF was markedly improved when the optimized parameters were applied. This work provides an innovative approach to bioproduction research through SSF. Electronic supplementary material The online version of this article (10.1186/s13068-018-1093-0) contains supplementary material, which is available to authorized users.

Insect gut can digest food comprise of tannins, this study characterize the novel tannase genes of microflora residing in the gut of Silk worm Bombyx mori. Tannase activity in the gut suspension was examined through plate assay by supplementing different concentrations of tannic acid, such as 0.5%, 1%, and 2%. The gut flora of B. mori showed maximum zone of inhibition in all concentrations of tannic acid. This preliminary assay proved the presence of dynamic and proficient tannase producing bacteria identified as Pseudomonas-MK-JP-Bm1. In the same manner, all the variable residents in tannic treated plates were subjected to biotinylated tannase probe hybridization, which emitted high resolution signal in Pseudomonas-MK-JP-Bm1. Tannic acid degradation wasconfirmed by spectrophotometer, HPLC and FTIR analysis. The tannase gene was amplified using degenerate primers which were successfully cloned and sequenced. The phylogenetic study of obtained tannase sequence revealed

Due to the feature of high hydrolysis, tannase is widely used in food, beverage, brewing and other fields. However, high cost in producing natural tannase makes it difficult to apply tannase to industry in a large-scale. Microbial expression systems can be used for preparing numerous amount of enzyme at low cost, so in this paper Aspergillus niger N5-5 was expressed using E. coli system. Specific primers were designed based on the Aspergillus niger N5-5 sequence N3 (GenBank, No.: KP677552), and tannase gene tan was promoted to carry 6 His tag and enzyme cutting site which contains NdeI/HindIII using PCR amplification. Then, tannase gene tan was connected to expression vector by NdeI/HindIII enzyme cutting. In this way, recombinant expression vector tan-pET43.1a was formed. Then, the expression vector pET43.1a by NdeI/HindIII enzyme cutting was transformed into E. coli BL21 (DE3) to induce expression of Aspergillus niger N5-5. When the induced fungi were disrupted by the ultrasonic wave, the crude enzyme was extracted and purified by using the IMAC, and then the activity of the crude enzyme and pure enzyme was determined. According to the results of determination of the tannase activity, the tannase activity of the crude enzyme was greatly improved after the crude enzyme was purified, and the specific activity of the pure enzyme was about 8 times of that of the crude enzyme. The results of SDS-PAGE of the pure enzyme showed that the molecular mass of the pure enzyme was about 65 kDa/64–65 kDa, which was consistent with the expected result (64.2 kDa), It can be concluded that the crude enzyme solution was purified successfully. The results of pure enzyme’s protein identification by Western Blotting showed that clear protein bands pro-3 were observed. Molecular mass of clear protein bands pro-3 was about 65 kDa, which was in line with the expected results (64.2 kDa). It can be seen that the aforementioned expression protein could be specifically combined with His tag. It proved expression protein to be a recombinant fusion protein with 6 His tag.

Tannase (tannin acyl hydrolse, E.C.3.1.1.20) is an enzyme that catalyzes the hydrolysis of ester and depside bonds of hydrolysable tannins. Among the filamentous fungi the genus Penicillium is recognized as the second best producer of tannase. This enzyme presents applications in several industrial segments, such as in the production of judges and teas. The industrial production of tannase can be performed through solid state fermentation (SSF) by the use of tannin rich fruit residues, in order to minimize production costs. In the present study 31 species of Penicillium isolated from Caatinga and Atlantic Forest were tested quantitatively for the production of tannase. The species were kept in the Collection of Cultures Micoteca - URM (WDCM604) under mineral oil. The enzyme production was carried out under SSF using “caja” (Spondias lutea L.) and Manga (Mangifera indica L.) residues that are rich in tannins. Mango residue proved to be the best inducer of the production of tannase by Penicillium species, most of which presented high tannase activity, ranging from 14.48 to 89.48 U mL-1. The three best producers were Penicillium rolfssi URM6216 with 89.49 U mL-1, Penicillium janthinellum URM5993 with 85.93 U mL-1 and Penicillium glabrum URM6092 with 59.26 U mL-1. Therefore, P. rolfssi URM6216 is being indicated for optimization of tannase production by SSF, using mango residue as a substrate. This is the first study on the potential of hog-plum (caja) and mango wastes as substrates for tannase production by filamentous fungi. Key words: Penicillium rolfssi URM6216, tannin acyl hydrolase, waste, mango, caja.

Aims The gut microbiota capable of degrading plant biomass and antinutritional phytometabolites are of immense importance. This study reports isolation and characterization of tannase-producing rumen bacteria that could also degrade euptox A (9-oxo-10,11-dehydroageraphorone) present in Eupatorium adenophorum (Spreng). Methods and results Migratory Gaddi goats were selected as source of inoculums for isolating rumen bacteria with ability to produce tannase which catalyses degradation of hydrolysable tannins (HTs). Three rumen bacterial isolates producing tannase were studied, and identified as Klebsiella variicola strain PLP G-17 LC, K. variicola strain PLP S-18 and Klebsiella pneumoniae strain PLP G-17 SC. The isolates exhibited optimal tannase activity at 40°C, and pH 6·0. The bacteria could also degrade euptox A, a potent hepatotoxin in E. adenophorum Spreng, a widely distributed noxious weed. Conclusions The rumen bacteria could degrade antinutritional HTs and euptox A. Culture-independent metagenomic interventions are envisioned to completely decipher the rumen microbial ecology and exploit its genetic and metabolic potential. Significance and impact of the study The bacteria producing tannase which catalyses degradation of HTs, and concurrently degrading euptox A, may have potential as microbial feed additives to increase utilization of plant biomass containing antinutritional phytometabolites.

Tannins are polyphenolic compounds produced by plants and they are used in industrial vegetable tanning of leather. Tannins represent one of the low biodegradability substances in tannery wastewaters with high recalcitrant soluble chemical oxygen demand, furthermore high concentration of tannins can inhibit biological treatment. In the present study, four novel rotating submerged packed bed reactors were inoculated with a selected fungal strain to reach a biological degradation of tannins in non-sterile conditions. The selected fungal strain, Aspergillus tubingensis MUT 990, was immobilised in polyurethane foam cubes carriers and inserted inside a submerged rotating cage reactors. The reactors were feed with a solution composed of four tannins: Quebracho (Schinopsis spp.), Wattle (Mimosa spp.), Chestnut (Castanea spp.) and Tara (Caesalpinia spp.). Four reactors with a volume of 4 L each were used, the co-substrate was pure malt extract, the hydraulic retention time was 24 h and the pH setpoint was 5.5. The reactors configuration was chosen to allow the study of the effect of rotation and the co-substrate addition on tannins removal. The experiment lasted two months and it was achieved 80% of chemical oxygen demand and up to 90% dissolved organic carbon removal, furthermore it was detected an important tannase activity.

Aureobasidium pullulans has been observed as one of the most abundant species in freshly pressed grape juice. Despite this, little is known about the consequences for the wine-making process associated with the presence and proliferation of this fungus, including its interaction with other ferment-derived microorganisms and impact on the composition of the resulting wine. In this study, the physiology of abundant A. pullulans grape juice isolates was investigated through lab scale fermentation trials, demonstrating the ability of this species to survive in grape juice while producing polysaccharides, polymers of malic acid (poly β-malic acid) and enzymes with pectinase, β - glucosidase and tannase activity. A possible antagonistic effect against yeast through competition for metals including Fe and Zn was also observed. Overall, the data suggests this abundant species could have important implications for wine production and quality.

Made available in DSpace on 2020-12-10T17:01:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-11-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Aspergillus ochraceus biofilm, developed on an inert support, can produce tannase in Khanna medium containing 1.5% (w/v) tannic acid as the carbon source, at an initial pH of 5.0, for 72 h at 28 degrees C. Addition of 0.1% (w/v) yeast extract increased enzyme production. The enzyme in the crude filtrate exhibited the highest activity at 30 degrees C and pH 6.0. At 50 degrees C, the half-life was 60 min and 260 min at pH 6.0. In general, addition of detergents and surfactants did not affect tannase activity significantly. Tannase has potential applications in various biotechnological processes such as the production of propyl gallate and in the treatment of tannin-rich effluents. The content of tannins and total phenolic compounds in effluents from leather treatment was reduced by 56-83% and 47-64%, respectively, after 2 h of enzyme treatment. The content of tannins and total phenolic compounds in the sorghum flour treated for 120 h with tannase were reduced by 61% and 17%, respectively. Interestingly, the same A. ochraceus biofilm was able to produce tannase for three sequential fermentative process. In conclusion, fungal biofilm is an interesting alternative to produce high levels of tannase with biotechnological potential to be applied in different industrial sectors. UNESP, Inst Quim Araraquara, Ave Prof Mario Degni S-N, BR-14800900 Araraquara, SP, Brazil Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Ave Bandeirantes 3900, Ribeirao Preto, SP, Brazil UNESP, Inst Quim Araraquara, Ave Prof Mario Degni S-N, BR-14800900 Araraquara, SP, Brazil FAPESP: 2016/11311-5

In this study, three samples of olive oil wastes: Olive Rinse Water (OR), Olive Mill Wastewater (OM) and Olive Pomace (OP), which were collected from an olive oil mill located at Fez-Morocco, were analyzed for their microbiota. A total of 81 isolates were obtained and constituted a microbial bank formed of 35 bacteria, 41 yeasts and 5 fungi. The microbial communities have been compared in term of metabolic potential by testing hydrolytic enzymes activities of lipase, protease, amylase, cellulase, pectinase and tannase on agar plate media. The results reveal that among the examined microorganisms, 68 isolates were able to produce at least one of the screened enzymes. The pectinase activity was the predominant one (39.51%), followed by cellulase activity (34.57%) and by lipase activity (27.16%). However, the amylase activity was observed only for 11.11% of the studied isolates, followed by protease activity (9.88%) and by tannase activity 6.17%. Thereby, considering the isolates ability to produce enzymes, they can be considered as potential candidates for industrial and biotechnological applications. Lipases are versatile enzymes that are used widely, it’s why the mostly aim of this work was to isolate new bacterial strains producing high level of lipases with interesting biochemical criteria. Thus, two isolates OR34 and OM2 showed the maximum lipase production under the standard conditions, about 9 U/ml and 11 U/ml, respectively, using pH-stat technique. They were identified as Proteus vulgaris and Serratia marcescens, respectively, via partial 16S rDNA gene sequence analysis.

Tannase is an inducible enzyme which hydrolysate tannin to gallic acid and used to in many industries such as food and pharmaceutical. This enzyme has scooped more attention in recent times. A maximum production of tannase (ranged from 0.30 to 0.93 enzyme index and from1.05 to 1.87 U/mg specific tannase activity (STA)) and gallic acid (from 0.07 to 0.76 mg/ml gallic acid concentration (GAC)) was achieved by 8 fungal strains out of 24 fungal and yeast strains belonged to genera; Aspergillus, Rhizopus, Trichoderma, Fusarium, Penicillium, Candida and Saccharomyces. The selected fungi were grown on tannin-rich substrates (eucalyptus leaves, pomegranate peel, banana peel, guava leaves and wheat bran) and by-products (corn steep liquor (CSL) and soybean extract) as sole carbon and nitrogen sources for tannase and GA production at 28°C for 6 days under liquid-surface (LSF), submerged (SmF) and solid-state (SSF) fermentation. Results indicated that A. niger A8 and T. viride with 10% (v/v) of inoculum size gave a high STA (from 8.08 to 10.95 U/mg) and GAC (from 2.62 to 4.00 mg/ml) on pomegranate and banana peels supplemented with CSL after 4 days incubation at 28°C under SSF compared to SmF and LSF. STA (ranged from 10.68 to 12.93 U/mg) and GAC (ranged from 3.56 to 4.16 mg/ml) were increased when inoculated the medium with both A. niger A8+T. viride (with 5:5% v/v of inoculum size) more than when inoculated with each of them separately.

In this research, optimization of the production medium to enhance tannase production by Bacillus gottheilii M2S2 in laboratory-scale packed bed reactor was studied. Amount of substrate Triphala, moisture content, aeration rate, and fermentation period was chosen for optimization study. During one variable at a time optimization, the highest tannase activity of 0.226 U/gds was shown with Triphala as a substrate at the fermentation period of 32 h. Furthermore, the optimum conditions predicted by response surface methodology (RSM) and genetic algorithm (GA) were found to be 11.532 g of substrate Triphala, 47.071% of the moisture content, and 1.188 L/min of an aeration rate with uppermost tannase activity of 0.262 U/gds. In addition, the single hidden layer feedforward neural network (SLFNN) and the radial basis function neural network (RBFNN) of an artificial neural network (ANN) were adopted to compare the prediction performances of the RSM and GA. It revealed that the ANN models (SLFNN, R(2) = 0.9930; and RBFNN, R(2) = 0.9949) were better predictors than the RSM (R(2) = 0.9864). Finally, the validation experiment exhibited 0.265 U/gds of tannase activity at the optimized conditions, which is an 11-fold increase compared to unoptimized media in shake flask.

Tannase (Tannin Acyl Hydrolase EC 3.1.1.20) is an industrial inducible enzyme capable of hydrolyzing hydrolyzable tannin ester linkage gallotannin and ellagitannin, producing gallic acid and glucose. Tannase is extensively used in the pharmaceutical, chemical, cosmetics, textile, leather, food, feed, and beverage industries. In the beverage industry, tannase is used as a clarifying agent to clarify tannin present in coffee, coffee-flavored soft drinks, tea, and fruit juices by removing phenolic compounds. In the pharmaceutical industry, tannase is used to produce gallic acid, an intermediary compound in the production of antibacterial drug, trimethoprim, while in the food industry, tannase is used to synthesize crucial antioxidant food preservative propyl gallate (3,4,5-trihydroxybenzoate). Most of the tannase production utilizes bacteria such as Bacillus sp. as tannase producer under submerged fermentation, SmF. Despite the immense industrial potential of tannase, it has not fully been exploited due to lack of knowledge, and fewer studies reported filamentous fungi for tannase production. This study aimed to screen potential tannase-producing fungi from various agri-industrial by-products such as rice by-products, spent tea, spent coffee ground, banana peels, mango peels, desiccated coconut residue, soybean residue, sweet potato peels, and onions. Fungal isolate, J1 ( Aspergillus niger ) was identified as the efficient tannase-producing fungus due to the hydrolytic zone's largest diameter (60.7 ± 0.6) mm. It achieved high tannase activity with (6.86 ± 0.04) U/ml in submerged fermentation, SmF. In conclusion, filamentous fungi isolated from agri-industrial by-products have huge potential as an efficient tannase producer.

In this study, different probiotics commonly used to produce fermented dairy products were inoculated independently for Chenopodium formosanum Koidz. fermentation. The strain with the highest level of antioxidant activity was selected and the fermentation process was further optimized via response surface methodology (RSM). Lactobacillus plantarum BCRC 11697 was chosen because, compared to other lactic acid bacteria, it exhibits increased free radical scavenging ability and can produce more phenolic compounds, DPPH (from 72.6% to 93.2%), and ABTS (from 64.2% to 76.9%). Using RSM, we further optimize the fermentation protocol of BCRC 11697 by adjusting the initial fermentation pH, agitation speed, and temperature to reach the highest level of antioxidant activity (73.5% of DPPH and 93.8% of ABTS). The optimal protocol (pH 5.55, 104 rpm, and 24.4°C) resulted in a significant increase in the amount of phenolic compounds as well as the DPPH and ABTS free radical scavenging ability of BCRC 11697 products. The IC50 of the DPPH and ABTS free radical scavenging ability were 0.33 and 2.35 mg/mL, respectively, and both protease and tannase activity increased after RSM. An increase in lower molecular weight (L. plantarum can be a powerful source of natural antioxidants for preventing free radical-initiated diseases.

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 biotran...

Miang is a fermented food product prepared from the tea leaves of Camellia sinensis var. assamica, and is traditionally produced in mountainous areas of northern Thailand. Although Miang has a long history and reveals deep-rooted cultural involvement with local people in northern Thailand, little is known regarding its microbial diversity. Yeasts were isolated from 47 Miang samples collected from 28 sampling sites, including eight provinces in upper northern Thailand. A hundred and seven yeast isolates were recovered and identified within 14 species based on the comparison of the D1/D2 sequence of the large subunit (LSU) rRNA gene. Candida ethanolica was determined to be the dominant species that was frequently found in Miang together with minor resident yeast species. All yeast isolates demonstrated their tannin-tolerant capability when cultivated on yeast malt agar (YMA) containing 50g/l tannin, but nine isolates displayed clear zones forming around their colonies, e.g., Debaryomyces hansenii, Cyberlindnera rhodanensis, and Sporidiobolus ruineniae. The results obtained from a visual reading method of tannase revealed that all yeast isolates were positive for methyl gallate, indicating that they possess tannase activity. It is assumed that a tannin-tolerant ability is one of the most important factors for developing a yeast community in Miang. This research study is the first report to describe tannin-tolerant yeasts and yeast communities in traditionally fermented tea leaves.

Tannase production by Aspergillus oryzae using various agro-wastes as substrates by submerged fermentation was studied in this research. The microbe was isolated from degrading corn kernel obtained from the corn fields at Tiruchengode, India. The microbial identification was done using 18S rRNA gene analysis. The agro-wastes chosen for the study were pomegranate rind, Cassia auriculata flower, black gram husk, and tea dust. The process parameters chosen for optimization study were substrate concentration, pH, temperature, and incubation period. During one variable at a time optimization, the pomegranate rind extract produced maximum tannase activity of 138.12 IU/mL and it was chosen as the best substrate for further experiments. The quadratic model was found to be the effective model for prediction of tannase production by A. oryzae. The optimized conditions predicted by response surface methodology (RSM) with genetic algorithm (GA) were 1.996% substrate concentration, pH of 4.89, temperature of 34.91 °C, and an incubation time of 70.65 H with maximum tannase activity of 138.363 IU/mL. The confirmatory experiment under optimized conditions showed tannase activity of 139.22 IU/mL. Hence, RSM-GA pair was successfully used in this study to optimize the process parameters required for the production of tannase using pomegranate rind.

Laphet is a traditional fermented food in Myanmar, made from tea leaves (Camellia sinensis) by fermentation with limited air passage. We performed microbial diversity analyses on 14 Laphet products collected from different locations in Myanmar. Amplicon-based sequencing results revealed Lactobacillus and Acetobacter were abundant bacteria and Candida, Pichia, Cyberlindnera, and Debaryomyces were abundant yeast. Using selective media, eight species of lactic acid bacteria and nine species of yeast were isolated; Lactobacillus plantarum and L. collinoides were dominant bacteria and Pichia manshurica, Candida boidinii, and Cyberlindnera jadinii were major yeasts. PCR-DGGE analysis confirmed that most of the dominant bacterial and yeast species found in culture dependent analysis were present in Laphet samples. Microbial diversity and pH of Laphet were different between samples from tea plantation area and local markets due to possible differences in incubation time periods. When tannase activity was tested, 23 among 29 bacterial isolates and two among 36 yeast isolates showed positive activities. These findings provide new insights into microbial diversity of Laphet and increased our understanding of the core bacterial and yeast species involved in the manufacture of Laphet.

WOS: 000482118200004, In this study, 17 L. brevis strains obtained from fermented products in previous studies were purified and these selected isolates were identified by the DuPont Qualicon RiboPrinter (R) system. Spectrophotometric and visual reading method indicated that isolate L. brevis A6X showed the highest activity. It was observed that pyrogallol product with the decarboxylase activity of gallic acid was composed of tannic acid using the thin layer chromatography. The proximate molecular weights of the enzyme produced by the selected isolate of L. brevis were determined by the SDS-PAGE method, and also the effect of different temperature, pH, substrate concentration, the concentration of tannic acid and various minerals on activity were all investigated. As a result of the test, it was found that the optimum temperature was 37 degrees C, pH 5.0, methyl gallate concentration used as the substrate 7 mM and tannic acid concentration 1.75 mM. While enzyme activity increased in the presence of Ca+, Zn+ and K+ ions, it did not show any changes in the presence of surfactant (Tween 80), inhibitor (DMSO), denaturing agent (urea). In addition, in presence of Mg+, Hg+ and Zn+ ions caused the tannase activity to decrease., Anadolu University of Scientific Research Project Program, This work was financially supported by the Anadolu University of Scientific Research Project Program.

including cellulase, tannase and pectinase. Isolates were morphologically and molecularly identified as Aspergillus niger, Aspergillus tubingensis, Aspergillus japonicus and Aspergillus aculeatus. Isolates were screened for enzyme production ability on solid and in liquid media. Enzymatic activity was determined in the culture filtrate of liquid medium. A total of six isolates were found to produce multiple hydrolytic enzymes. The highest activity of cellulase was produced by A. japonicus ZGM4 and A. aculeatus ZGM6 as 40 and 35 U g-1 dry biomass, respectively. All the isolates exhibited high level of tannase activity in the range of 150-343 U g-1 dry biomass after 24 h of incubation. A. tubingensis ZGM5 and A. aculeatus ZGM6 were found to produce the highest pectinase activity at a level of 130 and 117 U g-1 dry biomass, respectively. In the light of these results, isolates can be used for multiple hydrolytic enzyme production in industry.

Made available in DSpace on 2018-12-11T16:53:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) One of the tannase isoforms produced by the fungus Aspergillus fumigatus CAS-21 under submerged fermentation (SbmF) was purified 4.9-fold with a 10.2% recovery. The glycoprotein (39.1% carbohydrate content) showed an estimated molecular mass of 60 kDa. Optimum temperature and pH for its activity were 30–40 °C and 5.0, respectively. It showed a half-life (t50) of 60 min at 45 and 50 °C, and it was stable at pH 5.0 and 6.0 for 3 h. The tannase activity was insensitive to most salts used, but it reduced in the presence of Fe2(SO4)3 and FeCl3. On contrary, in presence of SDS, Triton-X100, and urea the enzyme activity increased. The Km value indicated high affinity for propyl gallate (3.61 mmol L−1) when compared with tannic acid (6.38 mmol L−1) and methyl gallate (6.28 mmol L−1), but the best Kcat (362.24 s−1) and Kcat/Km (56.78 s−1 mmol−1 L) were obtained for tannic acid. The purified tannase reduced 89 and 25% of tannin content of the leather tannery effluent generated by manual and mechanical processing, respectively, after 2-h treatment. The total phenolic content was also reduced. Additionally, the enzyme produced propyl gallate, indicating its ability to do the transesterification reaction. Thus, A. fumigatus CAS-21 tannase presents interesting properties, especially the ability to degrade tannery effluent, highlighting its potential in biotechnological applications. Instituto de Química de Araraquara- UNESP, Avenida Professor Mário Degni s/nº, Quitandinha Faculdade de Filosofia Ciências e Letras de Ribeirão Preto USP, Avenida Bandeirantes 3900 Instituto de Química de Araraquara- UNESP, Avenida Professor Mário Degni s/nº, Quitandinha FAPESP: 2016/11311-5

Tannase (tannin acyl hydrolase EC 3.1.1.20) is an inducible enzyme that catalyzes the hydrolysis of ester and depside bonds present in hydrozable tannins. In the present study, tannase from a novel Klebsiella pneumoniae KP715242 was purified and characterized for the desirable properties. The enzyme was purified with an overall purification of 8.55-fold leading an yield of 9.5%. The molecular mass of 38.20 kDa suggested this enzyme to be one of the smallest tannase of the bacterial source. Optimum temperature and pH for tannase activity were found to be 50 °C and 5.5 respectively. The optimum temperature of 50 °C puts this enzyme in thermostable category of bacterial tannase reported till date. Km of the enzyme for tannic acid was found to be 0.00375 M with Vmax 3.64 mM/S. Effects of various conventional inhibitors, metal salts, organic solvents, surfactants and chelators on tannase activity were assessed. Methyl gallate, sodium thioglycolate and xylene inhibited the enzyme maximally. Among the metal ions Hg2+ had maximum inhibitory effect on the tannase activity whereas Zn2+, Mg2+, Mn2+ were found to be enzyme activators. Surfactants and chelators inhibited the enzyme, however, no significant effect of EDTA was observed on tannase activity.

In recent years, tannase has gained increasing interest mainly because of its potential applications. One of the most important functions of tannic acid (TA) hydrolase is the release of gallic acid (GA) from complex tannins. The aim of the study was to determine the dynamic changes in tannase activity depending on the carbon source in the culture medium. An extracellular and intracellular tannase activity analysis was carried out with the use of spectrophotometric analysis and confirmed by capillary electrophoresis in cultures of white-rot fungi: Phellinus pini, Fomes fomentarius, and Tyromyces pubescens. The inducible potential of TA and rapeseed meal on the activity of tannin acyl hydrolase was confirmed during 14 days of culturing. Different effects of the tested compounds on stimulation of tannase activity in selected fungal strains have been demonstrated. We concluded that rapeseed meal was the best inducer of tannase activity in the case of P. pini. However, the highest concentrations of GA were observed after stimulation by the TA in the cultures of F. fomentarius and T. pubescens.

Tannins, which are polyphenols present in various plants, have anti-nutritional activity; however, their negative effects are mitigated by the presence of tannin-degrading microorganisms in the gastrointestinal tract of animals. This has never been investigated in the plateau zokor (Myospalax baileyi) – the predominant small herbivore in the alpine meadow ecosystem of Qinghai Province, China – which consumes tannin-rich herbaceous plants. Tannase activity in the feces of the plateau zokor increased from June to August corresponding to the increase in hydrolyzable tannin concentrations in plants during this period, and three tannin-degrading facultative anaerobic strains (designated as E1, E2, and E3) were isolated from the cecum of these animals. Sequencing of the 16S rDNA gene identified isolates of strain E1 as belonging to the genusEnterococcus, and E2 and E3 to the genusBacillus. All of the bacteria had cellulose-degrading capacity. This study provides the first evidence of symbiotic bacterial strains that degrade tannic acid and cellulose in the cecum of plateau zokor.

The present study investigates the optimization of tannase production from Aspergillus nidulans for various physicochemical parameters and harvests tannase for its chemical characterization. The maximum tannase activity was observed on the third day of incubation at 35°C and the stability was observed at pH 5.5-6.0 by holding its 100% activity. The tannase was partially purified from A. nidulans [FT10] by ammonium sulfate precipitation at different concentrations, and it was found that at 80% of ammonium sulfate concentration, the precipitate exhibited the maximum activity for tannase of 96 U/ml. LCMS showed its M/Z value as 162.3 which was reconfirmed by SDS-PAGE. The UV spectrum and FTIR confirmed the presence of two oxy- and three hydroxyl groups in the benzene ring structure. The antibacterial activity of tannase was enhanced with antibiotics such as streptomycin and ceftazidime whereas the biofilm formation was significantly inhibited by the purified tannase. The scavenging activity was greatly increased with purified component and when the concentration of the purified tannase, FT10 was increased. To the best of our knowledge, this is one of the few reports where microbial species was used as the source for producing tannase enzyme and its role in various bioactivities such as antibacterial, anti-biofilm and antioxidant activity was evaluated.

Tannin acyl hydrolases, or tannases (EC 3.1.1.20), are enzymes with potential biotechnological applications. In this work, we describe the gene and amino acid sequences of the tannase from Aspergillus niger GH1. In addition, we engineered Pichia pastoris strains to produce and secrete the enzyme, and the produced tannase was characterized biochemically. The nucleotide sequence of mature tannase had a length of 1,686 bp, and encodes a protein of 562 amino acids. A molecular model of mature A. niger GH1 tannase showed the presence of two structural domains, one with an α/β-hydrolase fold and one lid domain that covers the catalytic site, likely being residues Ser-196, Asp-448, and His-494 the putative catalytic triad, which are connected by a disulfide bond between the neighboring cysteines, Cys-195 and Cys-495. A 120-ml shake flask culture with a constructed recombinant P. pastoris strain showed extracellular tannase activity at 48 h induction of 0.57 U/ml. The produced tannase was N-glycosylated, consisted of two subunits, likely linked by a disulfide bond, and had an optimum pH of 5.0 and optimum temperature of 20 °C. These biochemical properties differed from those of native A. niger GH1 tannase. The recombinant tannase could be suitable for food and beverage applications.

Tannin acyl hydrolase (tannase, EC3.1.1.20) catalyzes the hydrolysis of hydrolyzable tannins. It is used in the manufacture of instant tea and in the production of gallic acid. In this study, we reported that the overexpression, purification and characterization of an Aspergillus niger tannase. The tannase gene was cloned from A. niger SH-2 and expressed in the A. niger strain Bdel4 which is low-background of secreted proteins. The recombinant tannase was purified by desalting, followed by gel filtration for characterization. The tannase activity achieved 111.5 U/mL at 168 h, and the purity of the enzyme in the broth supernatant was estimated to be over 70%. The optimum temperature and pH of the recombinant tannase was ∼40 °C and 7.0, respectively. The tannase activity was inhibited by Mg2+, Ca2+, Cu2+, Ba2+, Ni2+ and EDTA, and was enhanced by Mn2+ and Co2+. Since A. niger is a GRAS microorganism, the recombinant tannase could be purification-free due to its high purity. The results of this study suggested that this recombinant strain could be subjected to large-scale production of A. niger tannase.

Fourteen isolates of Serratia marcescens were collected from patients suffering from septicemia. All theseisolates revealed different levels in tannase production. Tannase was partially purified from Serratia marcescens b9 by precipitation method at 70% saturation of ammonium sulfate. Au, Pt, SnO2 and SiO2 nanoparticles were prepared by laser ablation and examined by transmission electron microscopy (TEM), X-ray diffraction pattern and UV-Visible absorption spectroscopy. Conjugation of SiO2 nanoparticles to tannase by feeding and pulses methods were prepared and characterized by TEM, X-ray diffraction pattern and UV-Visible spectrum. SiO2 nanoparticles conjugated partially purified tannase by feeding showed the higher effectiveness and higher significant level against all tested UTI causing in comparison with ciprofloxacin antibiotic, SiO2 nanoparticles alone, partially purified tannase alone and partially purified tannase by pulses. So that we can conclude that feeding method was the best method for enhancement partially purified tannase activity to maximum level thus SiO2 nanoparticles conjugated partially purified tannase may be a useful antibacterial agent for the treatment of urinary tract infection.

Microbial life can be found over a wide range of extreme conditions like high salinity, pH, temperature, pressure, light intensity, oxygen and nutrients. Halotolerant or halophilic microorganisms, are able to live in saline environments, offer potential applications in various fields of Biotechnology. The halotolerant or halophilic microorganisms are important for maintenance of soil condition and nutrient recycling in saline environment. The environment is ideally placed at the inter-phase between the terrestrial and marine environment and support a rich and diverse group of microorganisms. In this study, attempts were made to isolate the halotolerant marine bacteria and screen them for enzymes to be used for the decolorization of textile dyes and tannery effluent. A total of 192 halotolerant marine bacteria were isolated from Kelambakkam and Marakanam Salterns and named as AMETH101 to AMETH292. The isolated strains were tested for their growth at various salt concentrations such as 0M, 0.5M, 2.5M and 5M. All were found to grow well at 0.5M NaCl concentration. These organisms were studied for their ability to produce enzymes like lipase, tannase and protease. Among these strains, 107 have exhibited lipase activity, 41 strains exhibited tannase activity and 130 strains have exhibited protease activity. Totally 192 strains were tested for IAA production using qualitative assay and found that 41 strains showed positive activity and the remaining strains showed negative activity. For morphological characterization, KOH string test and catalase tests were performed. The results indicated that among the 192 strains, 138 strains were gram positive and remaining were gram negative strains while all the strains were positive for catalase test. This preliminary study has demonstrated that bioprospecting of halotolerant marine bacteria may yield potential microorganisms to be used in various environmental and biotechnological applications. Isolated halotolerant bacteria are very good producers of extracellular enzymes and IAA. These halotolerant bacteria with the combined ability of producing bioremediation enzymes and plant growth promoting hormones enhances the potential uses of them for the treatment and reuse of industrial effluents for the irrigation of ornamental plants.

Tannin-degrading bacteria (TDGB) were isolated from the rumen of goats fed oak leaves containing diet. The isolates were screened for their ability to increase in vitro digestibility of oak leaves (Quercus semicarpifolia), and fifteen best isolates were selected for further characterisation. The isolates TDGB 19, 20, 406, 415, 417, 420, 425, 437, 446, and 450 were Gram-positive cocci with medium- and long-chains, and isolates TDGB 409, 428, 430, and 433 were Gram-positive cocco-bacilli of short chain length. All the isolates exhibited tannase activity with the maximum in TDGB 417 (72.0 units; nmol methyl gallate degraded/min/ml) and minimum in TDGB 433 (28.92 units). All the fifteen isolates utilized fructose, galactose, lactose, starch, maltose, glycerol, and mannose as carbon source. The isolates were tested for their tolerance to phenolic monomers like ferulic acid, vanillic acid, coumaric acid, pyrogallol acid, and gallic acid. All the isolates could tolerate the presence of pyrogallol and gallic acid up to 20 mM, but ferulic was toxic at 20 mM level for all the isolates. The isolates were able to degrade tannic acid to pyrogallol except isolates TDGB 409, 428, 430, and 433, which could degrade tannic acid only up to gallic acid after 24 h of incubation as assessed by thin-layer chromatography. None of the isolates degraded tannic acid to resorcinol even after 96 h of incubation. The phylogenetic analysis of the isolates using sequences of PCR amplicons generated by the three sets of primers, two primers sets targeting 16S rRNA gene and one targeting sodA gene for superoxide dismutase enzyme, revealed that the isolates TDGB 7, 19, 20, 406, 417, 446, and 450 formed a close cluster with Streptococcus gallolyticus with 99 to 100 % similarity in all the three phylogenetic trees. There was a maximum of 11 % improvement in vitro true degradability of tannin-rich feed in the presence of live culture of TDGB 406 and can be exploited as a probiotic to improve nutrient utilization of tannin-containing feed given to the livestock.

Lactobacillus plantarum is frequently isolated from the fermentation of plant material where tannins are abundant. L. plantarum strains possess tannase activity to degrade plant tannins. An L. plantarum tannase (TanBLp, formerly called TanLp1) was previously identified and biochemically characterized. In this study, we report the identification and characterization of a novel tannase (TanALp). While all 29 L. plantarum strains analyzed in the study possess the tanBLp gene, the gene tanALp was present in only four strains. Upon methyl gallate exposure, the expression of tanBLp was induced, whereas tanALp expression was not affected. TanALp showed only 27% sequence identity to TanBLp, but the residues involved in tannase activity are conserved. Optimum activity for TanALp was observed at 30°C and pH 6 in the presence of Ca2+ ions. TanALp was able to hydrolyze gallate and protocatechuate esters with a short aliphatic alcohol substituent. Moreover, TanALp was able to fully hydrolyze complex gallotannins, such as tannic acid. The presence of the extracellular TanALp tannase in some L. plantarum strains provides them an advantage for the initial degradation of complex tannins present in plant environments. © 2014, American Society for Microbiology., This work was supported by grants AGL2011-22745, BFU2010-17929/BMC, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD (MINECO), S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid), and RM2012-00004 (Instituto Nacional de Investigación Agraria y Alimentaría). N. Jiménez is the recipient of an FPI fellowship from the MINECO.

Gut microorganisms are thought to play a role in the degradation of plant secondary metabolites in herbivores. Here, we examined the presence of tannin–degrading microorganisms in the gut of two herbivores, plateau pikas (Ochotona curzoniae) and root voles (Microtus oeconomus). Tannase activity in the feces of wild plateau pikas and root voles increased from June to August, corresponding to the increase in condensed and hydrolyzable tannin concentrations in plants during this period. Using intragastric tannic acid infusion, we found that tannase activities were significantly higher following administration of three concentrations of tannic acid than that in the control group for root voles. In contrast, for plateau pikas, animals administered tannic acid exhibited lower tannase activity in low–dose and high–dose groups than that in the control group. Tannase activities in root voles were higher than those in plateau pikas among different months and in different tannic acid infusion groups. Three tannin–degrading anaerobic strains were isolated from the cecum of plateau pikas (termed A1, A2, and A3) and root voles (termed M1, M2, and M3). 16 s rDNA sequencing assigned isolates A1 and M1 to the genus Enterobacter, A2 and M2 to the genus Shigella, and A3 and M3 to the genus Staphylococcus. These data provided the first evidence of microorganisms with tannin–degrading activity in the gut of plateau pikas and root voles. We speculated that both animals could degrade tannins in the gut, but that root voles would have a greater ability to process tannins than plateau pikas.

Tannase is an important enzyme widely used in the production of food, feed, pharmaceuticals, beverages, leather and cosmetics, but its industrial application is still limited because of its high production cost. Agri-industrial residues are generally rich in tannins and can be used as low-cost substrates for tannase production. In this study, selected agri-industrial residues were evaluated as potential substrates for enhanced tannase production by a locally isolated fungal strain via solid-state fermentation. Among all four agri-industrial residues tested, rice bran, a nutrient-rich substrate with a low tannin level, showed the highest tannase activity during solid-state fermentation, followed by brewer's rice, spent coffee ground and desiccated coconut residue. Spent coffee ground contained the highest tannin content (155 mg tannin/g substrate), but exhibited poor fungal growth, possibly because of the high tannin content in spent coffee, which might have inhibited fungal growth. However, the incorporation of small amounts of spent coffee ground (0.5% tannic acid equivalent, TAE) into rice bran enhanced tannase activity by 1.8-fold and 3-fold in comparison to rice bran used as single substrate or with the addition of tannic acid, respectively, with 260.39 U/g tannase being produced. The use of nutrient- and tannin-rich agri-industrial residues, such as rice bran and spent coffee ground, enhances tannase activity, suggesting that such low-cost substrates can be used in industrial tannase production.