Kinetic, Logistic, Box-Behnken model and statistical procedure for media optimization of tannase producing bacteria from Tannery Effluent and its application in Glue and gelatine production for pharma applications

 

Puja Kumari, Shatabdi Sen, Suneetha V*

Department of Biotechnology, VIT University, Vellore-632014, Tamil Nadu, India

 

ABSTRACT:

A key enzyme tannase enables the degradation of gallotannins, a type of hydrolysable tannins. A wide variety of microorganisms such as yeasts, fungi and bacteria leads to the production of tannase. Among these the tannase producing bacteria were commonly reported in dissipated water from paper, leather and forestry industries. In this study we will discuss about diverse bacteria that were isolated from the tannery effluent of several industries and its screening, characterization and the production of extracellular tannase. The production of extracellular tannase is highly influenced by Media Optimization and hence wide range of pH and temperature is utilized for the production of tannase and the optimal activity was found to be at 45°C and at a pH 6.5. The placket-Burman Logistic, Kinectic and Box-Behnken models were employed to optimize the media for tannase producing bacteria. In submerged culture (SMC) and solid-state (SSC) the induction and repression pattern of tannase production can be observed and the results presented that SSC has higher potential to minimize catabolite repression. Assessment of the effect of various additives on tannase activity was also done and thus among the various metal salts tested, the enzyme was found to be strongly inhibited byHgCl₂ followed by ZnCl2 and MnCl2. Again it was also found that EDTA and b- mercaptoethanol had inhibitory effect where as the detergents (Tween-20, -60 and -80 as well as SDS) did not affect the activity of the enzyme¹. The recovery of glue and gelatin from   tannery effluent was also studied and for that a three step hydrolysis process is best suited for maximum recovery of collagen hydrolysate followed by the glue and gelatine production from it. It was also reported in accordance with the antibiotic sensitivity test (Abst) that the bacteria were very sensitive for streptomycin.

 

 

 

 

INTRODUCTION:

Among the secondary metabolites, tannins existing in the leaves, bark as well as stem, of the plant domain play vital role in the defence mechanism of the plant and protect them from microbial attacks. Polyphenolic compounds such as Tannins are the 4th most abundant plant element after hemicelluloses, cellulose and lignin and are available in varying molecular weight. An inducible and hydrolytic enzyme, Tannase or tannin acyl hydrolase (EC, 3.1.1.20) acts as a catalyst to allow the production of Gallic acid and tannic acid from hydrolysable tannins and gallic acid esters. Different group of microorganism such as yeasts, bacteria and fungi is involved in the production of Tannase. Bacteria of the Lactobacillus and Bacillus genus and filamentous fungi of the Penicillium and Aspergillus genus were involved in the production of the enzyme tannase. Tannase making is found to be higher in presence of crude tannin compared to pure tannic acid that is used as a substrate. Also among different concentration of crude tannin, 0.5 % (w/v) induces maximum production of the enzyme².It has been reported that both undefined media and defined (synthetic) can be utilized for production of tannase. In case of defined media the only source of carbon is tannic acid. It is also reported that simple sugars are easier to metabolize than tannic acid, hence several nitrogen sources has been employed in the defined media for the optimum growth of the tannase producing bacteria. Some of the important nitrogen sources involve ammonium di-hydrogen phosphate, sodium nitrate, ammonium chloride, ammonium sulphate, monosodium glutamate, and ammonium oxalate and glutamic acid^3.

 

In case of undefined media, natural compounds such as chestnut extract, tannin extract enriched with a tannin source are mostly employed. Several inert carriers such as polyurethane foam, impregnated with tannic acid-containing medium have been found to enhance tannase producing bacteria to a great extent.

 

It was observed that enzyme production by many tannase producing bacteria was directly related to tannic acid degradation and Gallic acid formation. Gallic acid was accumulated in the medium up to 36 h, and after that its amount was found to be declined. Due to its high molecular weight, tannic acid itself is impermeable to the cell membrane, but it can break into glucose and Gallic acid by the production of tannase. Initially, organisms accumulated glucose as an easily available source of carbon for rapid growth and Gallic acid remained in the culture broth. Gallic acid was ultimately utilized by the organism in the latter phase of its growth. After the depletion of both products - glucose and Gallic acid, the organism entered into the stationary phase⁴

 

Fig.1. Chemical structure of tannic acid with the ester and depside bonds indicated. “n” is the number of galloyl groups which can vary from 0-4⁵.

In our study tannery solid waste, chrome shaving dust has been characterized and subjected to biochemical treatment by using several proteolytic enzyme and mild alkalis. Full chrome having chrome content 3.42 % and low chrome having chrome content 1.37 % obtained from cow wet blue leather can be used as raw materials for extraction of glue and gelatin.

 

ig 2. Tannery Effluent

 

MATERIALS AND METHODS:

Microorganisms produce tannase which can be done by different methods like liquid surface, submerged, solid state cultures and modified solid-state cultures. The main advantages of solid state cultures include lower production costs, simplicity, low wastewater and high enzyme yield. Recovery of extracellular enzymes and process control are the main advantages of submerged culture.

 

Materials and chemicals required:

Tannic acid, Hi-Media (Microbiological media) and high methodical grade chemicals were used.

 

Selection of tannase producing bacteria:

Soil samples and tannery effluent sludge were taken from the tannery effluent outlets of various industries. Minimal media was prepared using various salts and tannic acid. The salts and nitrogen sources essential for tannase production include sodium nitrite, potassium chloride, magnesium sulphate, glutamic acid, ammonium oxalate, monosodium glutamate, and ammonium sulphate and potassium hydrogen phosphate. The minimal medium was re-suspended with 1 gram of tannery effluent soil. In case of Klebsiellapneumonia under controlled growth condition of about 37°C and pH 6.0 and at 150 rpm for 24 hours microbial growth can be observed. Serial dilution of the cultures was carried out with sterile saline solution and nutrient agar plates were used for spread plating (Jayaraman et al; 2011). Tannic acid utilizing bacteria were observed as separate colonies on the nutrient agar plates. 1% tannic acid was used as sole carbon source in the minimal media that was used for screening of the tannase utilizing bacteria. For further tannase production, only those exhibiting maximum zone of clearance were considered and systemic classification of microorganism’s as well as16S rRNA gene sequence homology were utilized for further characterization of the bacteria. Specific activity of tannase was expressed as U/mg protein and can calculated by dividing the enzyme units with protein content.

 

Specific activity = Enzyme activity (U/ml)/Protein (mg/ml)

 

Fig 3.Quadrant Streaking          Fig 4. Zone of clearance (Plate assay)

 

Kinetic models for growth kinetics:

The most widely used unstructured models to describe cell growth are the Monod kinetic model and Logistic growth model. Monod Model Monod model is the first model used to represent the growth kinetics of microorganisms. Monod model relates the specific growth rate and the concentration of the limiting substrate and is described by (1). The relationship of specific growth rate to substrate concentration often assumes the form of saturation kinetics.

              m_m S

m= ----------------------     (1)

             K_S +S

 

Monod model relates the growth rate to the concentration of a single growth limiting substrate [m = f(s)] via two parameters, the maximum specific growth rate (mm) and the saturation constant for substrate (KS). Since growth is a result of catabolic and anabolic enzymatic activities, these processes, i.e., substrate utilization or growth associated product formation can also be quantitatively described on the basis of growth models

 

Logistic growth model:

The logistic growth model is a substrate independent model. The logistic model states that the rate of growth of the cell is proportional to the cell mass concentration present at any time. When the cell mass reaches the stationary phase there is no growth and hence the rate becomes zero. The growth rate thus depends on how far the cell mass concentration is away from the stationary phase.

 

dX

------------- =kX                             (2)

dt

 

Equation (2) implies that the growth rate increases with an increase in cell mass concentration and is independent of the substrate concentration. In reality, a hyperbolic link governs the growth of the cell and the equation is given by,

 

dX

---------- =k(1-X /X_s ) X                                    (3)

dt

Let β = 1/ Xs, then (3) become                   

dX

---------- = k (1-b)X                                           (4)

dt

 

Box–Behnken model for media optimization:

A generally utilized3^k-p fractional  factorial outline is Box–Behnken, in light of the fact that it considers more trial focuses (permitting then more degrees of opportunity, which suggests a more exact analysis)than the typical fractional factorial, yet not exactly the full factorial configuration. This kind of outline is an accumulation of statistical methods for outlining tests, building models, assessing the impacts of various factors and seeking ideal states of contemplated elements for attractive reactions. For instance Granato et al. (2010) used a 3² design to develop a soy-based guava dessert where guava juice and soy protein were the autonomous variables, and the reactions were the tangible properties and physicochemical qualities of such items. The creators acquired huge RSM models and reasoned that RSM was a sufficient methodology for demonstrating the physicochemical parameters and the level of preferring of richness of treats.

 

For three-level factorial designs (3k), the mathematical model used to describe the relationship between factors and the response variable is linear:

 

Y =β₀ + β₁ x₁+ β₂ x₂ + β₁₂ x₁ x₂+ β₁₁ x₁²+ β₂₂ x₂²+ ε

 

The Box-Behnken can be utilized during the optimization of the media for the tannase producing bacteria only when the number of variables is less. At the point when there are numerous variables to be tried, the Plackett–Burman configuration might be a fabulous choice, once it has been broadly used to create process conditions and to permit the comprehension of the impacts of different physicochemical, biochemical and tactile variables utilizing a least number of tests. The Plackett–Burman configuration is broadly utilized as a part of food research since it permits the screening of principle components from a substantial number of variables that can be held in the further advancement process (Siala et al., 2012). For instance during the optimization of the media for the tannase producing bacteria we  utilized a Plackett– Burman configuration to dissect the impact of different conditions identified with the piece of the medium, inoculums size and temperature of maturation, totalling 11 autonomous variables. The authors confirmed that monopotassium phoshphate (KH2PO4), pH, and temperature were the three most critical variables; at that point they a Box–Behnken configuration of RSM was utilized to advance tannase production by the tannase producing bacteria.

 

Media optimization by Plackett-Burman design of experiments:

It’s a design tool that is employed to help increase production of tannase by the tannase producing bacteria. It is a design of experiments, one statistical way of analyzing data. The Plackett- Burman model basically looks like a matrix, has variables across and runs downs. It is used to study the effects of design parameters on the system states so that intelligent design decisions can be made. Design was meant to improve the quality control process. Experiments used to find upper and lower control limits of a certain variable. Refining of the process was done to find influencing factors. It allows efficient estimation and improves the quality of the product (tannase) in a less expensive way⁶. In Plackett- Burman: Columns represent factors and specify level to set for factors. Rows contain process runs followed by post-processing of results. We can use mostly any variable that that has an effect on another variable. The more variables we have the better for we can predict changes more accurately. Starting with factors such as temperature, pressure, etc. and how they affect a certain variable (yield, percentage etc.).More factors we have the more information that can be determined factors. We can label these factors f1 through fn.¹⁹

 

The Plackett–Burman factorial design was employed in this study of optimization of the media to correlate dependent and independent variables using the following polynomial model:

 

Y =A₀ + A₁ X₁+ A₂ X₂+ A₃ X₃+ ------------+ A_n X_n

 

Where Y is the response, A₀ the constant and A₁ to A_n are the coefficients of the response values.

 

Tannase enzyme assay:

Initially the enzyme has to be extracted and purified. Cell free medium forms the basis for the purification of the enzyme tannase. Firstly ammonium sulphate utilized for the saturation of the cell free medium and precipitation is carried out at 4ºC for 6 hrs. Removal of precipitate is done and then the saturation of the supernatant is carried out with ammonium sulphate, and for the precipitation of the remaining enzyme it is kept overnight. 20 mL and1 mM citrate buffer is used for the dissolution of the precipitate and again more purification is carried out by Cellulose ion exchange column chromatography (DEAE). At a flow rate of 0.5 mL/min the elution is carried out using the concentration gradient of NaClin 50 mM citrate buffer  at pH = 6.0 from 0 to 0.2 M  and later analysis of the fractions were done for tannase activities, and used for analysisSDS-PAGE^7,20

 

In case of the assay of tannase enzyme from Rhodococccus, the mixture of the reaction consisting of0.1 mL of the enzyme extract and 0.3 mL of tannic acid (0.7% (w/v) in 0.2 M citrate buffer (pH 6.0) .and then incubation is carried out for 20 min at 30 degree Celsius. Addition of 3 mL of bovine serum albumin solution is doneto station the enzymatic reaction -The precipitation of residual tannic acid byBSA (1 mg/mL). Later centrifugation is carried out at 9000×g for 15 min at 4ºC and the residue is dissolved in 2 mL of SDS-triethanolamine and after that 1 mL of FeCl3 reagent was added and a holding period of 15 min was done for stabilization of colour. The enzyme activity was calculated from the change in absorbance⁸.

 

∆ A520 = (Atest – Ablank) – (Acontrol – Ablank)

The absorbance is measured at 530 nm. One unit of tannase activity is said to be the amount of tannic acid that can be hydrolyzed by 1 mL of enzyme minute of reaction.

 

Table 1. Reported tannase producing Bacteria

 

Effect of pH, temperature and agitation

For Klebsiella pneumonia, In MMT broth at a pH range of (4.0 - 8.0) the influence of pH on tannase production were observed and the temperature was maintained at 37°C at 150 rpm. Quantification of growth and tannase activity was done after 28 hours and the temperature was varied between 30 to 60°C at150 rpm and pH 6 to observe the influence of temperature on the growth of the tannase producing^9,10.Speedvariation between 50 to 250 rpm at 37°C and at pH 6.0 are done to observe the influence of agitation on tannase production¹⁸.

 

In case of Rhodococcus, By varying pH range from 4.0 to 10.0 we can observe the effect of pH on the activity of the enzyme using sodium- phosphate buffer (pH 6.0-8.0, citrate- buffer (pH 3.0-5.0), and glycine NaOH buffer (pH 9.0-10.0).Between temperatures 15 to 65ºC the effect of temperature can be obtained¹¹

 

Table: 2 Effect of the Incubation temperature on tannaseactivity¹²

 

Table: 3 Effect of Incubation periods on tannaseactivity¹²

 

Effect of metal ions and salts:

Enzyme activity is highly affected by the metal ions and needs  pre-incubation with metal ions Ca2+, Hg2+, Fe3+, Co2+, Mg2+, Cu2+ in citrate buffer (pH 6.0) utilizing various water soluble salts within the concentration range of  1 and 10 mM.(Kar et al., 2003; Selwal et al., 2010)

 

Table 4: Effect of Metal Salts on tannase Activity

 

Table 5: Effect of Other Additives on tannase Activity

 

For tannase productionthe effects of various salts (1 mM) were tested. The salts included were sodium thiosulphate, calcium carbonate, ammonium sulphate, ammonium ferrous sulphate, ammonium molybdate, ammonium nitrate, ammonium chloride, ammonium azide, ammonium oxalate, cuprous chloride, ammonium carbonate, zinc chloride, barium chloride, magnesium chloride, barium chloride, ferrous chloride and calcium chloride. 

 

HPLC and SDS-PAGE for estimation for tannase and gallic acid:

The molecular mass of various proteins were determined using the standard protein mixture of 220, 97, 66, 45, 30, 20 and 14 kDa^16,17. The properties of the purified tannase analysis were carried out by electrophoresis SDS-PAGE.

 

By using High performance thin layer chromatography (HPTLC) system the produced Gallic acid was analyzed. Chamber containing developing solvent (acetic acid:butanol: water in 1:4:1 ratio) were used for developing plates. Saturation of the chamber was done prior to plate development for 20 min. The plate after development was scanned at 280 nm^13,14. The analysis of the result was done using HPTLC Win CATS 1.4.4.6337 software.The following equation from the chromatogram was used for determining the Retention value (Rf).

 

Rf = Distance moved by the compound /Distance moved by the solvent front

 

Antimicrobial activity determination:

The antimicrobial activity of the tannase producing bacteria was determined according to the diffusion method on nutrient agar with holes. The bacteria were surface inoculated with 0.1 mL suspension P.syringaepv. Tomato Ro was added to the nutrient medium in a ratio of 1/100 after cooling to 45-48 degree Celsius .Four holes, 7mm in diameter, were cut in the Petri plates, and 30 micro litre of the tannase producing bacteria were dripped into each of them. The Petri plates were brought to the room temperature for 30 min, and incubated at 28 degree Celsius for 72 hr. The antimicrobial activity of the tannase producing bacteria was determined according to the sizes of the sterile zones (diameter in mm).

 

Glue and gelatine Extraction:

MgO and CaO can be used for mild alkaline medium whereas trypsin, pepsin and proteinase K can be used as proteolytic enzymes to degrade the chrome shavings. A three step hydrolysis process is best suited for maximum recovery of collagen hydrolysate followed by the glue and gelatin production from it. The collagen hydrolysate is mostly extracted through vacuum filtration and is preserved at 4 ºC in an incubator.

 

RESULTS AND DISCUSSION:

Characteristics of the strains obtained from tannery effluents

It was found that many strains of bacteria were present in the tannery effluents which produce tannase. Few bacterial sources of tannase are Bacillus sp, Corneybacterium, Pseudomonas, Klebsiella, Lactobacillus, Citrobacter sp, Enterococcus, Rhodococcus etc. Morphological characterization indicated that most of the tannase producing bacteria are gram negative, non motile, capsulated, rod shaped bacteria.

 

RAW MATERIAL

LIME TREATMENT

FILTRATION

STOCK PROCESSING

DECOLOURISATION

CONCENTRATION

 

 

Fig 5: Glue

 

Effect of carbon and nitrogen sources

Table 4. Effect of carbon sources on tannase enzyme activity (Bradoo et al; 2005)

 

Table 5. Effect of Nitrogen sources on tannase enzyme activity ¹³

 

Application of tannase enzyme and optimal condition for its synthesis

Main applications of tannase are increase of instantaneous tea, acorn wine and Gallic acid production. Also, tannase is being used as clarifying agent in juice coffee and flavoured beverage. The practice of bacterial strains for production of tannase possesses the advantage of a short period time of fermentation and extracellular enzyme. Optimal conditions of fermentation and tannase production are different between bacterial species. Hence, the better culture conditions should be identified for each bacterium. Due to the growing demand of tannase and its potential applications in pharmaceuticals, food and chemical industries, it is essential to separate new bacterial strains capable to produce high amounts of enzyme (Enemour and Odibo, 2009; Sabu et al., 2006; Mondal et al., 2001b; Vinod et al., 2009; Mahapatra and Banerjee, 2009; Batra and Saxena, 2005).

 

Figure 6: Effect of Reaction Time on Tannase Activity (Jayaraman et al; 2011)

 

Figure 7: Effect of Substrate Concentration (Jayaraman et al; 2011)

 

 

Glue and gelatine production

Chrome Shaving Dust is one of the major tannery solid wastes and it contains significant substance mainly collagen protein to a judicious content, it was treated to harvest value added product “Protein Hydrolysate or Collagen Hydrolysate” by using chemical and biochemical methods. A biochemical method here denotes the chemical treatment first and then enzymatic treatment of chrome shaving dust. Mild alkaline treatment using CaO and MgO was carried out in the first step of chemical treatment and lastly enzymatic treatment was followed for the biochemical treatment of chrome shaving dust. Proteolytic enzymes e.g., Trypsin, Pepsin and Proteinase K were used under mild alkaline condition to extract glue and gelatine from chrome shavings¹⁷

 

Figure8: Statistical procedure used to analyse results from a design of experiment

 

 

Fig 9: Gallo tannins biodegradation pathway

 

Leather is a important substance that mainly contain collagenous protein

Collagen hydrosylate is the main source of glue and gelatin

In the process of hydrolysis breaking down of the molecular bonds between individual collagen strands using combinations of acids, heat, and enzymes or alkalis takes place

Glue and gelatin are used as digestible, in the manufacture of ice cream, in the confectionery industry and photographic materials

 

Glue and gelatine assay:

1. Hydroxyproline:

Hydroxyproline is very abundant in collagen and absent in other proteins, except elastin. Its measurement provides a means to assay for gelatin when all other protein is present.

Procedure:

Put a 3 mm square sample in a test tube; add 0.015ml of NaOH. Heat the test tube containing the mixture in a boiling water bath for ten minutes and then allow it to cool. Add 0.05ml of CuSO₄and 0.025mlof H₂O₂. It should be kept in mind that the peroxide does not touch the upper part of the test tube. Shake the test tube until foaming decreases and place again in boiling water bath for another five minutes. Cool the tube and add 0.125ml of H₂SO₄and 0.1ml of reagent. Results: Appearance of a rose-red or pink colour within ten minutes shows the existence of animal glue or gelatine¹⁷.

 

2. Tannin Test:

Reagent: 2% of tannic acid. Procedure: Remove sample to watch glass or microscope slide, first wet, and then heat to induce extract. Remove the slide from heat and add a drop of tannic acid to the sample extract. Results: Appearance of Dirty-white precipitate indicates the presence of gelatine. Interference: Starch can interfere with the formation of precipitate.

 

DISCUSSION:

Incubation temperature in the scope of 25-40°C has been utilized with most procedures being directed at around 30°C for bacterial system (Yamada et al., 1968; Rajkumar and Nandy, 1983; Lekha and Lonsane, 1994). Comparative pattern was seen in bacterial concentrates as well. Kumar et al. (1999) reported greatest tannase activity at 30°C by Citrobacter freundii while Das Mohapatra et al. (2006) reported most extreme tannase generation at 35°C by Bacillus lichiniformis KBR6. Mondal et al. (2001) reported an ideal temperature of 40°C for their strain Bacillus cereus KBR9, while tannase creation was ideal at 30°C if there should arise an occurrence of Lactobacillus sp. ASR-S1 (Sabu et al., 2006). Selwal et al. (2010) have reported an ideal temperature of 37°C for their culture Pseudomonas aeruginosa III B 8914.

 

The ideal starting medium pH seems, by all accounts, to be between 4.5-7.0 for SmF and 5.0-6.5 for SSF for the majority of the fungal culture (Van de Lagemaat and Pyle, 2006). If there should be an occurrence of bacterial culture, Selwal et al. (2010) have reported an ideal pH of 5.5 for their culture Pseudomonas aeruginosa III B 8914, while Mondal et al. (2001) reported an ideal pH of 5.0 for their strain Bacillus cereus KBR9. For Bacillus lichiniformis KBR6, Mondal and Pati (2000) have reported the ideal pH as 5.0. Ayed and Hamdi (2002) reported most extreme tannase generation at pH 6.0 for Lactobacillus plantarum. Selwal et al. (2010) have observed that underlying medium pH of 7 was ideal for their strain Psuedomonas aeroginosa. Belur et al. (2010) have observed that underlying medium pH of 6.0 was the ideal for their strain Serratia ficaria DTC.

 

If there should arise an occurrence of bacteria, the greatest efficiency was accounted for with in 24 h of brooding. Greatest tannase movement was seen at the stationary period of development (24 h) by Selwal et al. (2010) for their way of life Pseudomonas aeruginosa III B 8914, Ayed and Hamdi (2002) for their culture Lactobacillus plantarum and Mondal et al. (2001) for their culture Bacillus cereus KBR9. Same laborers reported greatest tannase creation at the late exponential period of development (21 h) in Bacillus lichiniformis KBR6 (Mondal et al., 2000; Mondal and Pati, 2000). Kumar et al. (1999) reported greatest tannase profitability in the early exponential stage (6 h) by Citrobacter freundii. Deschamps et al. (1983) have recorded ideal creation after 6 h of incubation in their fermenter for Bacillus plumilus, Bacillus polymyxa, Corynebacterium sp. furthermore, Klebisella pneumoniae. Belur et al. (2010) have reported most extreme compound generation in the late stationary period of development , which was at 24th hour of hatching in the event of Serratia ficaria DTC. Selwal et al. (2010) additionally have reported the comparable pattern if there should be an occurrence of their separate Pseudomonas aeroginosa IIIB 8914. Accordingly writing audit recommends that ideal conditions can change extensively contingent upon the microorganisms and kind of maturation.

 

ACKNOWLEDGEMENT:

We want to express our sincere gratitude to our Honorable Chancellor Dr G. Viswanathan for his support and encouragement and special thanks to Dr. Sekar Viswanathan and Sri G.V. Selvam for their constant motivation to do research and also tannery industries from Rani pet.

 

CONCLUSION:

Novel applications of tannase can be developed. Ethanol production from agro industries are of great importance. Phenols generated from lignin through prior treatment of the agro wastes. Phenols are known to inhibit the hydrolytic activity of cellulase. These phenol compounds can be rapidly degraded by tannase and thus subsequently lead to the increase in the catalytic activity of cellulase. In humans identification of Staphilococcus lugdunensis can be carried out by the tannase gene and their respective enzyme activity and can be employed to prevent the colon cancer. Therapeutic applications of tannase enzyme involve generation of novel molecules, such as prunioside A with anti-inflammatory activity. Other major uses of tannase are found in the manufacture of laundry detergents in the form of additive and homogenization of the tannins preparation for good grade leather tannins in the leather industry. Tannin is an anti-nutrient that is present in tea, since tannins form complexes with digestive enzymes, proteins and starch and reduces their nutritive value and hence is an undesirable ingredient in tea. Therefore tannase, an enzyme capable of hydrolyzing the ester bonds in hydrolysable tannins can be employed for the production of tannin free tea.

Future perspective

In spite of the many applications of tannase, industrial-scale manufacture of tannase is very limited, mostly because of its high cost of production. As per our knowledge, very few firms manufacture and sell tannase, and therefore the cost of this enzyme is often greater than that of other industrial-grade enzymes, even from the same dealer.

 

Hence, many changes have been done to improve the manufacture systems. It includes the screening for novel tannase-producing microbial strains, the use of new fermentation systems, the optimization of media and culture conditions, and the synthesis of the enzyme by recombinant microbes.

 

Many researchers have recently carried out various experiments for novel sources of tannase. These researches have been made to find out microbes able to produce larger enzymatic titer value or enzymes with required characteristics, such as greater stability at a broad range of pHand temperature.

 

Plant design for tannase production

Conventionally, industrial production of tannase was done completely in SmF systems (submerged fermentation). But, in the recent years a number of researches have shown the advantages of SSF (solid state fermentation) for the synthesis of tannase and other related enzymes. These benefits include higher degrees of activity, extracellular nature of the enzyme, increased productivity, and increased stability to temperature and pH changes. Moreover, the SSF allows the manufacture of more compact reactors with a smaller amount ofenergy requests and it also cause less harm to the environment.

 

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19.     Aguilar, C.N., Rodrguez, R., Gutierrez-Sanchez, G., Augur, C., Favela-Torres, E., Prado Barragan, L.A., Ramırez-Coronel, A. y Contreras-Esquivel, J.C. Microbial Tannases: Advances and Perspectives. Applied Microbiology and Biotechnology,2007,Vol 76, 47-59

20.     Deschamps, Production of tannase through submerged fermentation of tannin containing plant extract by Bacillus licheniformis KBR6.Polish journal of Microbiology, 2006,Vol 55,No 4,297-301

 

 

 

 

Accepted on 17.05.2017           © RJPT All right reserved