Production of tannin acyl hydrolase and HPLC determination of flavonoids from fermented rice bran.
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 188.8.131.52), 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
Rice Bran was collected from a local oil industry and grinded to obtain 0.5 mm particle size using a standard sieve.
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.
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.
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
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]
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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: email@example.com
(2) Indian Institute of Crop Processing Technology, Ministry of Food Processing Industries, Govt.of.India, Thanjavur-613 005 (TN), India Email: firstname.lastname@example.org
(3) Senior Research Fellow, Indian Institute of Crop Processing Technology, Ministry of Food Processing Industries, Govt.of.India, Thanjavur-613 005 (TN), India Email: email@example.com
(4) Principal Scientist, Indian Institute of Crop Processing Technology, Ministry of Food Processing Industries, Govt.of.India, Thanjavur-613 005 (TN), India Email: firstname.lastname@example.org
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|
|Date:||May 1, 2009|
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