Tannase producing organisms

Indian black berry known as Jamun is a minor fruit which contains high amount of tannin. Extraction and clarification of juice is quite difficult due to its pulp nature. Tannase a membrane bound enzyme is added to clarify the Jamun juice which helps to obtain high yield. The target area of the work is to build up the procedure for streamlining of process factor to obtain Jamun juice, utilizing tannase (strain: Aspergillus ficcum). Physicochemical parameters (clarity, colour change, polyphenol, turbidity, protein, TSS and yield) were analyzed at a temperature range between (30C – 50C), with (0.01% – 0.1% w/v) concentration and time orbit of (40-120 min). Coefficient of determination (R) value more than 0.9 have been used to measure the prominent differences in the response characteristics. Clarified juice was obtained at 0.05% enzyme concentration at 40C for 80 minutes.

Tannase production under solid -state fermentation was investigated using isolated Aspergillus oryzae . Among all agro industrial waste material evaluated, cashew husk supported maximum tannase production. The metabolic processes of microorganisms are influenced by changes in parameters like Temperature, pH, incubation time, humidity etc., which are very specific for a particular organism. Microbia l synthesis of enzymes in a SSF process are also affected by factors like particle size of substrate, water content, relative humidity, type and size of inoculum, control of temperature, period of cultivation, etc. Biotransformation of cashew husk tannin t o gallic acid by SSF is also influenced by all the factors affecting tannase production, since the synthesized enzyme causes the breakdown of tannin to gallic acid and glucose.

The presence of high tannin content in fruit juices and other beverages generates bitterness and haze formation with a concomitant deterioration of their quality. This problem could be circumvented by treating these food products with the enzyme tannase (tannin acyl hydrolase EC 3.1.1.20), which contains the ability to hydrolyze the ester and depside linkages present in hydrolyzable tannins. In the present study, a low molecular weight (38.20 kDa) tannase purified from Klebsiella pneumoniae KP715242 was used for clarification and detannification of orange juice, beer, and tea cream solubilization. The initial tannin content of orange juice was reduced to 42.45% upon treatment with 4.5 U/mL of purified tannase at 35°C for 3.5 h. Beer haze was clarified by incubating the colloidal suspension with the purified enzyme. Under the optimum condition of temperature (30°C), incubation time (3.5 h), and enzyme concentration (4.5 U/mL), the initial tannin content of beer was reduced to 46.47%. Tea cream solubilization was achieved by treating the tea extract with varying enzyme concentrations at different time intervals and temperatures. The maximum reduction in the tea tannin content (55%) was observed at 35°C when the enzyme was incubated for 3 h at a concentration of 6.0 U/mL. The results of this investigation could be further exploited at an industrial scale in the detannification of fruit juice, beer, and tea.

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 "cajá" (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 (cajá) and mango wastes as substrates for tannase production by filamentous fungi.

The juice extracted from cashew apples contains tannins that must be reduced due to their astringency and anti-nutritional properties. This study investigated reducing the tannins in cashew apple juice by mixing gelatin at 0.0 to 1.0% (w/v) with the juice for 5 to 15 min. Response surface methodology was used to simulate tannin concentration, turbidity, and weight of precipitate as a function of gelatin concentration and mixing time. All models were significant (P<0.01) and residuals exhibited normal distribution. Increasing the gelatin concentration decreased tannins and juice turbidity. Optimal conditions for minimum concentration of tannins and turbidity were obtained by adding gelatin at 0.67% (w/v) for 15 min. Cashew apple juice with the tannins reduced could be used in the subsequent production of wine and bioethanol.

Tannins are groups of phenolic compounds that cause astringency and turbidity in fruit juices. Many factors had an influence on the concentration of tannins in fruit juices, including cultivar, parts and maturity stages, as well as pH and viscosity of juices. Many methods were investigated for tannins removal from fruit juices. Physical methods included heat treatment and membrane filtration. Chemical methods were based on clarifying agents included polysaccharides (starch and chitosan), proteins (gelatin and casein) and synthetic polymers (polyvinyl polypyrolidone). The enzymatic method was based on the application of enzyme tannase to hydrolyze tannins into soluble compounds. Tannins removal efficiency of each method depended on fruit juice, type and parameters of the method. Downstream process should be considered especially for application of clarifying agents.

Indian black berry known as Jamun is a minor fruit which contains high amount of tannin. Extraction and clarification of juice is quite difficult due to its pulp nature. Tannase a membrane bound enzyme is added to clarify the Jamun juice which helps to obtain high yield. The target area of the work is to build up the procedure for streamlining of process factor to obtain Jamun juice, utilizing tannase (strain: Aspergillus ficcum). Physicochemical parameters (clarity, colour change, polyphenol, turbidity, protein, TSS and yield) were analyzed at a temperature range between (30C – 50C), with (0.01% – 0.1% w/v) concentration and time orbit of (40-120 min). Coefficient of determination (R) value more than 0.9 have been used to measure the prominent differences in the response characteristics. Clarified juice was obtained at 0.05% enzyme concentration at 40C for 80 minutes.

Thermophilic tannase from Enterobacter cloacae MTCC 9125 was purified using two-step purification strategy comprising of ultrafiltration and ion-exchange chromatography. A purification fold of 8.47 with 33.1% yield was obtained. The apparent molecular mass of tannase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 31 71 kDa. The temperature of 50 1C and pH 5.5 were found to be optimum for tannase activity. Tannase retained almost 50% activity after 2 and 4 h of incubation at its optimum pH (pH 5.5) and temperature (50 1C), respectively. K m of the enzyme for tannic acid was found to be 0.00337 M with V max 3.401 U/ml. Effects of several metal salts, chelators, surfactants, and typical enzyme inhibitors on tannase activity were evaluated. Inhibition by n-bromosuccinic acid and phenylmethylsulfonyl fluoride (PMSF) indicated that tryptophan and serine or cysteine residues play an important role in maintaining the active conformation of the enzyme. Among the divalent cations, Mg 2 þ , Zn 2 þ and Mn 2 þ were found to be activators of the enzyme whereas Fe 2 þ , Ba 2 þ and Cu 2 þ acted as inhibitors. Surfactant such as Tween 20, Tween 80, Triton X-100 and SDS resulted in considerable loss of enzyme activity.

Tannase is an enzyme that hydrolyzes esters and lateral bonds of tannins, such as tannic acid, releasing glucose and gallic acid and stands out in the clarification of wines and juices. Fungi of the genera Aspergillus and Penicillium are excellent producers of this enzyme. The search for fungi that produce high levels of tannase as well as new substrates for the enzyme production by the SSF is required. The objectives of this study were to evaluate the production of tannase by Aspergillus and Penicillium species through SSF using leaves and agroindustrial waste barbados cherry and mangaba fruit as substrate, select the best producer, optimize production, characterize the crude enzyme extract, and apply it the clarification of grape juice. Selecting the best producer was performed by planning Placket-Burman and RSM. P. montanense showed highest activity with 41.64 U/mL after 72 h of fermentation residue using barbados cherry, with 3.5% tannic acid and 70% moisture. The enzyme showed the highest activity at pH 9.0 and 50 ∘ C. The tannase of P. montanense was stable over a wide pH range and temperature and, when applied to grape juice, showed higher efficiency by reducing 46% of the tannin content after incubation 120 m.

Guava juice is known to be rich in antioxidant activity due a high level of vitamins A and C. However, tannins present in the guava juice form tannin–protein complexes that affect the utilization of vitamins and minerals and inhibit digestive enzymes. Beside this, bitterness and cloudiness are the other major problems of juice industries. The present study aimed to utilize a low-cost substrate (tea residue) for the production of tannase and its application in detannification of guava juice. Solid-state fermentation (SSF) was evaluated to produce tannase from Aspergillus niger. Maximum tannase (1.86 U=g dry substrate) production was observed at 30 °C after 96 hr of the incubation period. The optimum pH of the moistening agent was found to be 5.0. Partially purified enzyme using ammonium sulfate precipitation was subjected to guava
juice treatment at a level of 0.5, 1.0, and 2.0% for 30 and 60 min. Decrease in tannic acid content of guava juice was found to be 17.60, 29.04, and 44.38% after 30 min and 40.59, 53.69, and 59.23% after 60 min, respectively.