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Production of Tannase by various fungal cultures in solid state fermentation of ground nut shell.

Introduction

Groundnut is the major oilseed crop in India accounting for 45% of oilseed area and 55% of oilseed production in the country. Groundnut (Arachis hypogaea L.) is believed to be the native of Brazil to Peru, Argentina and Ghana, from where it was introduced into Jamaica, Cuba and other West Indies islands. It was introduced into India during the first half of the sixteenth century from one of the Pacific islands of China, where it was introduced earlier from either Central America or South America. Now India along with china accounts for half of the world's groundnut production (Table. 1). About 7.5 million hectares groundnut cultivation is put under annually and the production is about 5-6 million Tonnes. 70% of the area and 75% of the production are concentrated in the four states of Gujarat, Andhra Pradesh, Tamil Nadu and Karnataka (Fig. 1). Andhra Pradesh, Tamil Nadu, Karnataka and Orissa have irrigated area forms about 6% of the total groundnut area in India. Grown in tropical and subtropical areas, groundnut thrives between 25-28[degrees]C and ca. 500-mm rainfall in open soil.

Solid-state fermentation (SSF) involves the growth of microorganisms on moist solid substrates in the absence of free flowing water and is an alternative cultivation system for the production of value added products from microorganisms, especially enzymes or secondary metabolites. Agro-industrial residues are generally considered the best substrates for the process, including enzyme production, based on SSF (Ellaiah et al., 2002). Compared with submerged fermentation, the use of SSF presents advantages such as lower power requirements, smaller reactor

volume and high productivity (Bertolin et al., 2001). Castilho et al., (2000) state that the conditions in solid-state fermentation were closer to those found in the natural habitat of filamentous fungi, which were, thus, able to grow better and excrete larger quantities of enzymes. This can be of special interest in those processes where the crude fermented product may be used directly, as the enzyme source. Low capital investment, low waste water output, higher concentration of metabolites obtained and low downstream processing cost (Kumaran, 1997). The major crop residues produced in India are straws of paddy, wheat, millet, sorghum, pulses, oilseed crops, maize stalks and cobs, cotton stalks, jute sticks, sugar cane trash, mustard stalks, etc. The agro-industrial residues like groundnut shells, rice husk, bagasse, cotton waste, coconut shell, and coir pith are used for enzyme production.

Tannin acyl hydrolase commonly called tannase is produced by a number of microorganisms like fungi -Aspergillus, Penicillium, Rhizopus sp, Yeast--Candida sp. and bacteria--Bacillus sp. (9,15) Several agro-industrial waste and by-products such as orange bagasse (Martins et al., 2002), sugar cane bagasse (Silva et al., 2002) wheat bran (Cavalitto et al., 1996) and other food processing waste (Zheng and Shetty, 2000) are effective substrates for depolymerizing enzyme production by solid-state fermentation, which proved to be highly efficient technique in the production of tannase. The major commercial application of this enzyme is in the hydrolysis of gallotannin to gallic acid, which is an intermediate required for the synthesis of an antifolic antibacterial drug trimethoprim (Sitting et al., 1988).Tannase is extensively used in the preparation of instant tea, wine, beer, and coffee--flavored soft drinks and also as additive for detannification of food (Lekha et al., 1993).

This study was taken up with the objective to produce increased quantities of tannase through bioconversion of groundnut shell as a cheaper raw material, using fungal organisms.

Materials and Methods

Raw material

Groundnut shell was collected from a local vegetable oil industry and grinded to obtain 0.5 mm particle size using a standard sieve, and preserved at room temperature. The chemical constituents of uninoculated groundnut shell were analyzed. According to the standard methods described by Ranade et al., (1980).

Microorganisms

Aspergillus flavus, Aspergillus niger Aspergillus oryzae and Penicillium sp. were isolated by primary selection from a naturally contaminated groundnut husk by serial dilution and pour plate techniques. The pure cultures were identified by their morphology and colony characteristics. The organisms were maintained on PDA slants and stored at 4[degrees]C. The slants were freshly made once a month.

Preparation of spore inoculums

Aspergillus flavus, Aspergillus niger, Aspergillus oryzae and Penicillium sp. spore inoculums were prepared by adding 2.5mL of sterile distilled water containing 0.1% Tween 80 to a fully sporulated culture. The spores were dislodged using a sterile inoculation loop under strict aseptic conditions and number of viable spores in the suspension was determined using the plate count method. A volume of 1 mL with concentration of 36x[10.sup.9] spores was used as inoculums.

Production of tannase under SSF

A five gram substrate of groundnut shell powder was taken in 250-mL Erlenmeyer flask and moistened with 5 mL of salt solution. The composition of the salt solution was N[H.sub.4]N[O.sub.3] 0.5%, NaCl 0.1%, MgS[O.sub.4] 7[H.sub.2]O 0.1% and Tannic acid 4% at pH =5.5. The contents were sterilized by autoclaving at 121[degrees]C; 15lbps for 20 min. The cooled sterilized solid substrate was inoculated with 1 ml of the spore inoculum, mixed properly and incubated at 30 [degrees]C for 168 h.

Extraction and analysis of crude enzyme

A 10% mycelial suspension mass from the fermented substrate was prepared in 0.05 M citrate buffer, pH 5.0, and frozen overnight. Acid washed sand, four times the weight of the mycelium, was added, and the mixture was ground in a chilled Pestle&Mortar kept in an ice bath. Crude enzyme was separated from the fermented matter by centrifugation at 8000 rpm at 4[degrees]C for 20 min. The filtrate was collected in bottles and preserved for further studies. The supernatant (mycelia extract) was used for tannase assay.

Purification and characterization

A volume each of 100 ml of crude tannase was taken, added slowly with the various concentration levels (0-40, 40-60 and 60-80 %) of ammonium sulphate. The addition of ammonium sulphate was done under constant stirring at 4 [degrees]C for 30 min and then stirring was continued for another 30 min and then allowed for settlement for 3 h at 4 [degrees]C. The precipitated protein was separated by centrifugation at 8000 rpm at 4 [degrees]C for 20 min. The separated proteins were then dissolved in minimum amount of 0.05 M citrate buffer (pH-5) and refrigerated for further analysis. Precipitated proteins were transferred into a dialysis tube using a micropipette and dialyzed against citrate buffer (0.05 M, pH-5) at 4 [degrees]C. The buffer was stirred gently using a magnetic stirrer to enhance solute exchange. Dialysis was conducted over night and the buffer was changed several times to increase the efficiency of the dialysis.

DEAE Sephadex A-50 chromatography

A Glass column was packed with DEAE Sephadex A-50 and was equilibrated with 0.05 M citrate buffer (pH 5.0). One ml of the dialyzed sample was applied on the column and the elution was done using 0.05 M citrate buffer (pH 5.0). The fractions were monitored and collected. The fractions corresponding to tannase activity were pooled and used for estimation.

Tannase Assay

Tannase was assayed following Sharma et al., (2000) method using gallic acid as a standard. The pink colour developed was read at 520 nm using a spectrophotometer (Shimadzu UV-160A). The enzyme activity was calculated from the change in absorbance. One unit of tannase activity was defined as the amount of enzyme required to liberate one micromole of gallic acid per minute under defined reaction conditions. Enzyme yield was expressed as units/gram dry substrate (U/gms)/min.

[delta]A520 = (Atest--Ablank)--(Acontrol--Ablank)

HPTLC analysis of Flavonoids in fermented substrate

HPTLC (CAMAG) analyses were performed on 0.20 mm-thick silica gel 60 TLC aluminum using ethyl acetate/formic acid/water (6:1:1 v/v)15 as mobile phase. Each of the dried fractions obtained was dissolved in 300 [micro]L methanol. Rutin hydrate and Querectin (95% pure; Sigma), were used as a standards (100 [micro]g mL-1 in methanol). For the analysis of fermented biomass fractions, 5 [micro]L of the samples and 5 [micro]L of the rutin standard were placed on a silica TLC plate using Automated CAMAG TLC Applicator, 8 [micro]L of sample and 3 [micro]L of rutin standard were used for the analysis of fractions. Visualization of the flavonoids and phenolic acids was achieved by spraying the sheets with polyethylene glycol reagent . Typical intense fluorescence in UV light at 365 nm was produced immediately on spraying (flavonoids appeared as orange-yellow bands, whereas phenolic acids formed blue fluorescent zones).Addition of polyethylene glycol solution lowered the detection limit and intensified fluorescence. The detection limit for the flavonoids and phenolic acids was reported as 2.5g by Wagner et al., (1996)

Tannin estimation

The estimation of tannin content was done following the protein precipitation method of Haggerman et al. (1978). Dried leaves were ground finely in methanol and kept overnight at 4 [degrees]C. One ml of extract was taken in a tube and 3 ml of BSA solution was added and kept for 15 min at room temperature. The tubes were centrifuged at 5000g for 10 min, supernatant was discarded and pellet was dissolved in 3ml of SDS-triethanolamine solution. One ml of Fe[Cl.sub.3] solution was added and tubes were kept for 15 min at room temperature for color stabilization. Color was read at 530 nm against the blank.

Protein estimation

During incubation period the protein was estimated following Lowry's method (1962) using bovine serum albumin as a standard.

Effect of incubation time

After inoculation the flasks were incubated at 30 [degrees]C and the enzyme activity was measured after different time periods ranging from 48 h to 168 h.

Results and Discussion

Proximal Analysis of groundnut shell

The chemical constituents of groundnut shell analyzed and are listed in Table 2. The high concentration of starch suggested the possible use of groundnut shell for amylase induction in solid-state conditions.

Tannase production in Solid State fermentation using Groundnut shell

Tannase production by A.niger, A.oryzae, A.flavus, Penicillium sp., is reported in Table. 3. In the present study A.niger had a higher tannase activity when compared to other fungi, in the purified form. In 96th hours all the organisms produced more tannase. (Fig. 1).Previously maximum extracellular tannase and gallic acid production was recorded in 96 h and 120 h by A.niger and Rhizopus oryzae {Cavalitto et al., (1996), Iibuchi et al., (1967) Martins et al., (2000)}

[FIGURE 2 OMITTED]

The A.niger produced tannase was 32.70 U/g/min in the crude form. The crude tannase when precipitated using Ammonium sulphate 60- 80% saturation showed 46.11[+ or -]3 U/g/min of Tannase activity (Fif.2 & Table. 3).After dialysis the enzyme activity was enhanced when compared to the crude enzyme. The dialyzed enzyme was further purified through DEAE-Sephadex A-50 and the eluted fractions showed 121[+ or -]2 U/g/min. Mitchell and Lonsane, 1992 reported that the Production enzyme is often simple, when agro-industrial by-products like wheat bran, rice bran or wheat straw are used as substrate, weight of substrate is low. Hence, enzyme activity is usually very high.

Effect of incubation period

The enzyme production started after 48 h of incubation and increased with time reaching a maximum at 96 h (32.7 U/ml/min).This might be the fungi entered in to its exponential phase. Thereafter, the enzyme production started decreasing (Fig. 3).

[FIGURE 3 OMITTED]

Estimation of protein

The total soluble extracellular protein content reached a maximum at 96h (Fig-4). After studying the extracellular protein content it was found that the organism produced maximum tannase in its exponential phase of growth.(Banrejee et al., 2001).

[FIGURE 4 OMITTED]

Determination of Flavonoids by HPTLC

The conditions used led to a good separation of the peaks which could be identified in the chromatogram (Figure-5), as rutin (Rt=0.63), Gallic acid (Rt=0.07) and Querectin (Rt=0.76). They were identified by comparison with the Standard flavonoids chromatogram (Figure 5,6,&7) and the calibration curves showed linearity in the concentration range used for the standards. The highest content of total flavonoids Rutin--50.85 [micro]g/ml , Gallic acid -1828.97 [micro]g/ml and Querectin--492.81 [micro]g/ml was shown in the Aspergillus niger biomass.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

[FIGURE 7 OMITTED]

Conclusion

From this study it is concluded that A.niger is found to be the most suitable fungi in the bioconversion of groundnut shell to obtain increased quantities of tannase. The partially purified tannase was used to hydrolyse tannic acid for production of gallic acid.

References

[1] Banerjee, D., Mondal, K. C., Pati, B. R.: Production and characterization of extracellular and intracellular tannase from newly isolated Aspergillus aculeatus DBF9. J Basic Microbiol 41, 313 (2001).

[2] Bertolin, T. E.; Costa, J. A. V. and Pasquali, G. D. L. (2001), Glucoamylase production in batch and fed batch solid-state fermentation: effect of maltose or starch addition. J. Microbial. Biotech. 11, 13-16.

[3] Castillo, L. R.; Medronho, R. A. and Alves, T. L. M. (2000), Production and extraction of pectinases obtained by solid-state fermentation of agro industrial residues with Aspergillus niger. Biores. Techno. 71, 45-50.

[4] Cavalitto, S. F.; Arcas, J. A. and Hours, R.A, (1996), Pectinase production profile of Aspergillus foedidusin solid state cultures at different acidities. Biotechnology Letter, 18, 251-256.

[5] Chatterjee, R., Dutta, A., Banerjee, R., Bhattacharya, B. C.: Production of tannase by solid state fermentation. Bioprocess Engg 14, 159 (1996).

[6] Ellaiah, P.; Adinarayana, K.; Bhavan, Y.; Padmaja, P. and Srinivasulu, B. (2002), Optimization of process parameters for glucoamylase production under solid-state fermentation by a newly isolated Aspergillus species. Process Biochem. 38, 615- 620.

[7] Haggerman, A.E., Butler, L.G., 1978. Protein precipitation method for determination of tannins. J. Agric. Food Chem. 26, 809-812.

[8] Iibuchi, S., Y. Minoda and K. Yamada, 1967. Agric. 16. Spencer, C.M., Y. Cai, J.D. Martin, S.H. Graffney, Biol. Chem., 32: 513-518.

[9] Kumaran, S., Sastry, C. A. and Vikineswary, S., World J. Microbiol. Biotechnol., 1997,13, 43-49.

[10] Lekha P K, and Lonsane B K, Chem Microbiol; Technol Lebensm, 1993, 44, 215

[11] Lekha, P. K., Lonsane, B. K.: Comparative titers, location and properties of tannin acyl hydrolase produced by Aspergillus niger PKL104 in solid state, liquid surface and submerged fermentation. Process Biochem 29, 497 (1994).

[12] Lowry, O. H., Rosebrough, N. J., Farr, A., Randall, R.: Protein measurement with Folin-phenol reagent. J Biol Chem 193, 265 (1951).

[13] Martins, E. S.; Silva, R. and Gomes, E. (2000), Solid state production of thermostable pectinases from thermophilic Thermoascus aurantiacus. Process Biochem., 37, 949-954.

[14] Rajakumar, G.S. and S.C. Nandy, 1983. Appl. Enviorn. E. Haslam, 1988. Polyphenolcomplexation-some Microbiol., 46: 525-527.

[15] Ranade, D.R., J.A. Gore and S.H. Godbole (1980). Microbiological aspects of biogas production-Laboratory manual. M.A.C.S. Research Institute, Pune, India.

[16] Sabu, A., Pandey, A., Dvad, M. J., Szakacs, G.: Tamarind seed powder and palm karnel cake: two novel agro residues for the production of tannase under solid state fermentation by Aspergillus niger ATCC 16620. Biores Technol 96, 1223 (2005).

[17] Sharma, S., Bhat, T.K. and Dawra, R.K. (2000). A spectrophotometic method for assay of tannase using rhodanine. AnayticalBiochemistry, 279: 85-89.

[18] Silva, D.; Martins, E. S.; Silva, R. and Gomes, E (2002). Pectinase production from Penicillium viridicatum RFC3 by solid state fermentation using agricultural residues and agro-industrial by-product. Braz. J. Microbial., 33, 318-324.

[19] Sitting M, In Pharmaceutical Manufacturing Encyclopedia, 2nd (eds.). 1988, 282

[20] Wagner.H. and S. Bladt, Plant Drug Analysis, Springer, Berlin 1996, pp. 195-197

[21] Zhen, Z. and Shetty, K (2000), Solid state production of polygalacturonase by Lentinus edodes using fruit processing wastes. Process Biochem. 35, 825-830.

R. Paranthaman (1), R. Vidyalakshmi (2), S. Murugesh (3) and K. Singaravadivel (4)

(1,2,4) Indian Institute of Crop Processing Technology, Thanjavur--613 005, Tamil Nadu, India

(3) SASTRA University, Thanjavur--613 402. Tamil Nadu, India

(1) E.Mail: paranthhu@yahoo.com, (2) Email: onlyvidu@yahoo.co.in, (4) Email: pprcksvel@yahoo.com
Table 1: World groundnut production trend (Source FAO 2003).

                  Production     Harvest area     Yield
Countries           (Mt.)           (Ha)         (Kg/Ha)

China              15,277,455     5,125,400       2,981
India               7,500,000     8,000,000         938
Nigeria             2,699,000     2,782,000         970
United States
 of America        1,879,750        530,950       3,540

Table 2: Chemical constituents of groundnut shell.

Constituents             Compositions

Starch                  28 [+ or -] 5
Hemicellulose           11 [+ or -] 2
cellulose               21 [+ or -] 3
Pectin                 1.0 [+ or -] 0.3
Organic nitrogen       1.3 [+ or -] 0.2
Protein                8.0 [+ or -] 1
Oil and Fat            3.0 [+ or -] 0.5
Coloring matter        4.2 [+ or -] 0.6
C:N ratio               22 [+ or -] 2

Table 3: Tannase production in Groundnut shell.

                           Tannase activity (U/g/min)
S.N
o     Organism               Crude             Ammonium
                                               sulphate

1    Aspergillus niger    32.70 [+ or -] 2   46.11 [+ or -] 3

2    Aspergillus oryzae   28.23 [+ or -] 1   35.76 [+ or -] 1

3    Aspergillus flavus   32.91 [+ or -] 1   49.14 [+ or -] 1

4    Penicillium sp.,     27.02 [+ or -] 1   36.64 [+ or -] 2

      Dialysis                 Column
S.N                        Chromatography
o

1     72.92 [+ or -] 2      121 [+ or -] 2

2     66.48 [+ or -] 3       98 [+ or -] 2

3     61.76 [+ or -] 1       83 [+ or -] 2

4     53.50 [+ or -] 2       73 [+ or -] 2

Figure 1

Statewise Production of Groundnut in India
During 2001-02

Gujarant           38%
Tamilnadu          18%
Andhrapradesh      17%
Kamataka            8%
Maharastra          7%
Rajasthan           4%
Madhya Pradesh      4%
Uttar Pradesh       1%
Orrisa              1%
Others              2%

Note: Table from pie graph.
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Author:Paranthaman, R.; Vidyalakshmi, R.; Murugesh, S.; Singaravadivel, K.
Publication:International Journal of Biotechnology & Biochemistry
Article Type:Report
Geographic Code:9INDI
Date:May 1, 2009
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