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Production of tannin acyl hydrolase and HPLC determination of flavonoids from fermented rice bran.

Introduction

Gallic acid, the hydrolytic product of tannin hydrolysis finds application in many fields including pharmaceutical, dye-making, leather and chemical industries (Hadi et al. 1994, Mukherjee and Banerjee 2003). Besides this, gallic acid possesses wide range of biological activities, such as antioxidant, antibacterial, antiviral, analgesic etc. As antioxidant gallic acid acts as an antiapoptotic agent and helps to protect human cells against oxidative damage (Trevino-Cueto, B et al., 2006).Gallic acid is produced by acid hydrolysis of tannic acid but it has cost, yield and low purity disadvantages. Alternatively, gallic acid can be produced by the microbial hydrolysis of tannic acid by tannase (tannin-acylhydrolase B 3.1.1.20), an inducible enzyme, secreted by microorganisms (Pinto et al., 2001).

Tannase is used in the manufacture of instant tea and production of gallic acid, a substrate for chemical synthesis of propyl gallate and trimethoprim which have applications in the food and pharmaceutical industries. Other potential uses of tannase are stabilization of malt polyphenols, clarification of beer and fruit juices (Cantarelli), prevention of phenol-induced madeirization in wine and fruit juices and reduction of antinutritional effects of tannins in animal feed (Pinto et al, 2001). Tannins are polyphenolic secondary metabolites of plants, which form hydrogen bonds in solutions, resulting in the formation of tannin-protein complexes (Sharma et al., 1999). Two groups of tannins are distinguished according to their structures: hydrolyzable and condensed ones (Regerat et al., 1989; Chamkha et al., 2002; Huang et al., 2005). Hydrolyzable tannins are composed of esters of gallic acid (gallotannins) or ellagic acid (ellagitannins) with a sugar core which is usually glucose (Bhat et al., 1998).

India is an agrarian country where 2/3 of its population depend on rice as staple food. On average 4 million tonnes of rice bran, 0.6 million tonnes of rice bran oil and 0.2 million tonnes of deoiled rice bran (DOB) waste are generated annually containing 39 % of cellulose and 9 % of protein (SEA 1996). Therefore, it is felt that there is an urgent need to utilize DOB to convert cellulose into protein for food and feed by solid state fermentation technology. A fermentation process on solid support, which has low moisture content, characterizes Solid State Fermentation (SSF). It is a low-level technology, which uses a reduced reactor volume per unit of converted substrate, where fungi were applied to obtain desirable product. Microbial utilization of agro residue for the preparation of cellulose polymers and production of industrial enzymes, liquid fuels, protein rich food & feed etc is a novel attractive approach to meet immediate demand for food and energy (Purnendu Ghosh & Ajay singh, 1984).

Rice bran is one of the most abundant and locally available agricultural waste which contains variable ingredients such as carbohydrate that maybe used as a carbon and energy source for the growth of fungi in the production of single cell protein (Khan et al., 1992).

In this study, deoiled rice bran, a locally available industrial waste, was used as a substrate for the production Tannase and analysis the phenolic compounds in fermented substrate by using HPLC

Materials and Methods

Substrate

Rice Bran was collected from a local oil industry and grinded to obtain 0.5 mm particle size using a standard sieve.

Microorganisms

Aspergillus niger was isolated by naturally contaminated Rice bran. The culture was maintained on PDA slants and subcultured for every 15 days.

Preparation of spore inoculums

The spore suspension was prepared by adding sterile distilled water containing 0.1 % Tween 80 to a fully sporulated culture. The spores were dislodged using a sterile inoculation loop.

Production of tannase under SSF

A five gram substrate of Rice bran was 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] x 7[H.sub.2]O 0.1 % and Tannic acid 4% at pH =5.5. The cooled sterilized solid substrate was inoculated with 1 ml of the spore inoculum and incubated at 30[degrees]C for 96 h.

Extraction and analysis of crude enzyme

After incubation 0.05 M citrate buffer (pH 5.0) was added to the fermented substrate and homogenized with Mortar and Pestle. The Crude enzyme was separated from the homogenized matter by centrifugation at 8000 rpm at 4[degrees]C for 20 min. The supernatant was collected and estimate the Tannase activity. Tannase was assayed following the method of Sharma et al., 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)

Purification of Tannase

The crude tannase was precipitated by adding Solid Ammonium sulphate (60-80%) and kept for overnight at 4[degrees]C. The precipitated protein was separated by centrifugation at 8000 rpm at 4[degrees]C for 20 min. Then the precipitate was dialyzed against citrate buffer (0.05 M, pH-5) at 4[degrees]C.

HPLC analysis of Flavonoids in fermented substrate Standard preparation

Standard stock solutions of five phenolic compounds were prepared in methanol, at concentrations of 0.420, 0.434, 0.400, 0.402 and 0.402 mg x mL-1 for GA, CA, RU,EA and QU respectively. All standard solutions were filtered through 0.45 mm membrane filter (Millipore), and injected by autosampler.

Sample preparation

The fermented substrate was washed and dried at 60[degrees]C. The dried substrate was grind to fine powder. The extraction was carried out using 0.5 g of powdered substrate with 50 mL of 95% ethanol. The extract was collected and filtered; the filtrate was dried at 50[degrees]C under reduced pressure in a rotary evaporator. The dried crude extract was dissolved in 100 mL of mobile phase. After filtering through a filter paper and 0.45 [mu] membrane filter, the extract was injected into HPLC by autosampler.

HPLC conditions

High performance liquid chromatography (HPLC) of the samples was performed with the HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with two Shimadzu LC-10 ATVP reciprocating pumps, a variable UV-VIS detector (Shimadzu SPD-10 AVP) and a Winchrom integrator (Winchrom). Reverse phase chromatographic analysis was carried out in isocratic conditions using RP C-18 HPLC coloum (250 x 4.6 mm id, particle size 5 [micro]m, Luna 5[micro] C-18 (2), Phenomenex, USA) at 40[degrees]C. Running conditions included injection volume: 5 [micro]l, mobile phase: solvent A [water-acetic acid (25:1, v/v)] to solvent B (methanol). Solvent B was increased to 50% in 4 min and subsequently increased to 80% in 10 min at a flow rate of 1.0 mL/min. Detection wavelength was 280 nm. Samples were filtered through organic solvent compatible membrane filters (Pore size 0.20 [micro]m, Millipore) prior to injection in sample loop. Tannic acid standard was used as internal and external standards. Weerasak., et al.(2007).

Determination of GalloTannin content in fermented Rice bran by HPLC Sample Preparation

One g of fermented biomass was macerated in paste-mortor followed by suspending fine-crushed sample into 5 ml of ethanol-water (80:20, v/v) in glass tubes. The suspension was subjected to ultra-sonication at 60 % duty cycles for 25 min at 4[degrees]C followed by centrifugation at 8000 rpm for 15 min. The clear-greenish supernatant to evaporation under vacuum evaporator. Dried samples were resuspended in 1.0 ml HPLC grade methanol by vortexing and stored at 4[degrees]C for further analysis

HPLC Conditions

The Gallo tannin content was estimated using High Performance Liquid Chromatography conditions, Weerasak., et al.(2007) . Tannin present in the samples was identified by comparing retention time (Rt) of the standards and by the co-injection. Contents of tannin were calculated by comparing peak areas of reference compound with those in the samples run under similar elution conditions.

Results and Discussion

Production of Tannase from Rice bran fermented with Aspergillus niger was shown in Fig-1: The maximum production of was obtained at 96 h of incubation at 30[degrees]C in purified form. Further increasing the incubation period the activity was decreased. It might be tannic acid content in fermented medium decreases sharply and is completely utilized within 96 h of growth. Lewis and Starkey, (1969) reported some microorganisms degrade this compound by producing tannase and play an active role in the soil for nutrient recycling through decomposition of tannin-containing plant materials. In our study we got the maximum activity of 43 U/g/min at 96 h. Previously Deschamps et al.,(1983) found that after 72 h of incubation, the yield of gallic acid was maximum. The enzyme, tannase, hydrolyzes the ester bonds of tannin in the substrate to produce gallic acid and glucose. The initial increase in enzyme activity up to 72 h and its subsequent decrease may be due to catabolite repression Chatterjee R et al (1996).

[FIGURE 1 OMITTED]

Determination of flavonoids by HPLC

The three phenolic compounds, GA, RU and QU, are polar molecules. In the beginning, various proportions of either methanol-water or acetonitrile-water system were used as mobile phases but separation was not satisfactory. The presence of acid in a mobile phase system gave a much better separation for GA, and QU except it could not separate baseline RU and EA. The gradient elution of solvent A [water-acetic acid (25:1 v/v)] and solvent B(methanol) had a significant effect on the resolution of compounds. As a result, solvent gradients were formed, using dual pumping system, by varying the proportion of solvent A [water-acetic acid (25:1, v/v)] to solvent B (methanol). Solvent B was increased to 50% in 4 min and subsequently increased to 80% in 10 min at a flow rate of 1.0 mL/min. Detection wavelength was 280 nm.

[FIGURE 2 OMITTED]

Figure 2 shows that a good separation can be achieved within 15 min using the condition described. Symmetrical, sharp and well-resolved peaks were observed for GA, RU and QU. The elution order and the retention times for GA, RU and QU were 3.325, 5.1 and 5.867 min respectively.

Sample analysis and recovery

The ethanol extracts of Fermented rice bran was analysed and compared with standards. It was found that the extract contained GA 0.338 mg/gm and QU 0.142 mg/gm of wet weight. Rutin could not be identified.

[FIGURE 3 OMITTED]

Determination of GalloTannin content in fermented Rice Bran by HPLC.

The HPLC Result shows that Gallo Tannin content in of fermented rice bran was found to be 67.2 ug / g. The obtained value was compared with standard. The retention time for tannic acid in fermented rice bran was found to be 2.925.

Standard Chromatogram of Gallo tannin standard

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

References

[1] Amarowicz R., Weidner S. 2001. Content of phenolic acids in rye caryopses determined using DAD-HPLC method. Czech J. Food Sci. 19: 201-205

[2] Bhat TK, Singh B, Sharma OP (1998). Microbial degradation of tannins--A current perspective. Biodegradation, 9: 343-357

[3] Chamkha M, Record E, Garcia JL, Asther M, Labat M (2002). Isolation from a shea cake digester of a tannin-tolerant Escherichia coli strain decarboxylating Phydroxybenzoic and vanillic acids. Curr. Microbiol. 44: 341-349.

[4] Chatterjee R, A Dutta, R Banerjee and BC Bhattacharyya. 1996. Production of tannase by solid state fermentation. Bioprocess Eng 14: 159-162

[5] Deschamps AM, G Otuk and JM Lebeault. 1983. Production of tannase and degradation of chestnut tannin by bacteria. J Ferment Technol 61: 55-59

[6] Hadi, T. A., Banerjee, R. and Bhattacharya, B. C., 1994. Optimization of tannase biosynthesis by newly isolated Rhizopus oryzae. Bioprocess Eng., 11, 239-243.

[7] Huang W, Ni J, Borthwick AGL (2005). Biosynthesis of valonia tannin hydrolase and hydrolysis of valonia tannin to ellagic acid by Aspergillus SHL 6. Process Biochem. 40: 1245-1249.

[8] Khan, Y., U. Dahot and Y. Khan, 1992. Single cell protein production by Penicillum javanicum from pretreated rice husk. J. Islam. Acad. Sci., 5: 39-43.

[9] Lewis, J. A. and Starkey, R. L. (1969) Decomposition of plant tannins by some soil microorganisms. Soi lSci.,107,235-241.

[10] Mukherjee, G. and Banerjee, R., 2003. Production of Gallic acid. Biotechnological routes (Part 1). Chimica Oggi chemistry today, 21 (1/2), 59-62.

[11] Pinto, G.A.S.; Leite, S.G.F.; Terzi, S.G.; Couri, S. Brazilian J. Microbiol. 2001,

[12] Pinto, G.A.S.; Leite, S.G.F.; Terzi1, S.Z.; Couri, S. (2001). Selection of tannase producing Aspergillus niger strains Brazilian J. Microbiol., 32, 24-26.

[13] Purnendu Ghosh and Ajay Singh. 1984. Physiochemical and biological treatments for enzymatic/ microbial conversion of lignocellulosic mass. Adv. Appl. Microbiol. 39, 295-333.

[14] Regerat F, Pourrat H, Pourrat A (1989). Hydrolysis by fermentation of tannins from gall nuts. Jalca, 84: 323-328.

[15] Sharma S, Bhat TK, Dawra RK (1999). Isolation, purification and properties of tannase from Aspergillus niger van Tieghem. World J. Microbiol. Biotechnol. 15: 673-677.

[16] Trevino-Cueto, B.; Luis, M.; Contreras-Esquivel, J.C.; Rodriguez, R.; Aguilera, A.; Aguilar, C.N. (2006) Gallic acid and tannase accumulation during fungal solid state culture of a tannin-rich desert plant (Larrea tridentata Cov.) Bioresource Technol., 98 (3), 721-724.

[17] Weerasak Samee and Suwanna Vorarat Simultaneous Determination of Gallic acid, Catechin, Rutin, Ellagic Acid and Quercetin in Flower Extracts of Michelia alba, Caesalpinia pulcherrima and Nelumbo nucifera by HPLC Thai Pharm Health Sci J 2007;2(2):131-137

R. Paranthaman (1), R. Vidyalakshmi (2), J. Indhumathi (3) and K. Singaravadivel (4)

(1) Technical Assistant, Indian Institute of Crop Processing Technology, Ministry of Food Processing Industries, Govt.of.India, Thanjavur-613 005 (TN), India E.Mail: paranthhu@yahoo.com

(2) Indian Institute of Crop Processing Technology, Ministry of Food Processing Industries, Govt.of.India, Thanjavur-613 005 (TN), India Email: onlyvidu@yahoo.co.in

(3) Senior Research Fellow, Indian Institute of Crop Processing Technology, Ministry of Food Processing Industries, Govt.of.India, Thanjavur-613 005 (TN), India Email: jindhu_micro@yahoo.com

(4) Principal Scientist, Indian Institute of Crop Processing Technology, Ministry of Food Processing Industries, Govt.of.India, Thanjavur-613 005 (TN), India Email: pprcksvel@yahoo.com
SPD10Avp (280nm)

Retention                        ESTD
Time        Area       Height    concentration   Units   Name

3.325       1451317    239378    5.0             ug/ul   Gallic Acid
5.100       2945595    386194    5.0             ug/ul   Rutin
5.867       18720032   2030782   5.0             ug/ul   Querectin

              SPD10Avp (280nm)

Retention                                ESTD
Time          Area       Height          concentration

3.475         109855     14066           0.169
                                         0.000 BDL
5.858         133509     12007           0.071

              SPD10Avp (280nm)

                                         Concentration
Retention                                mg/gm
Time          Units      Name            (wet basis)

3.475         ug/ul      Gallic Acid     0.338
              ug/ul      Rutin           0.00
5.858         ug/ul      Querectin       0.142

SPD10Avp (280nm)

Retention                        ESTD
Time        Area       Height    concentration   Units   Name

2.925       31148602   2222096   20.000          ug      Tannic acid

SPD10Avp (280nm)

                 Retention                            ESTD
Samples          Time          Area        Height     concentration

Uninoculated     3.175         4386172     583759     2.808
Fermented        3.075         2093710     193804     1.344
  rice bran

                                                      Concentration
                                                      mg/gm
Samples          Units         Name                   (wet basis)

Uninoculated     ug            Tannic acid            1.40
Fermented        ug            Tannic acid            0.67
  rice bran
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Title Annotation:high performance liquid chromatography
Author:Paranthaman, R.; Vidyalakshmi, R.; Indhumathi, J.; Singaravadivel, K.
Publication:International Journal of Applied Chemistry
Article Type:Report
Date:May 1, 2009
Words:2506
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