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ISSN: 1579-4377 REPLACEMENT OF METHANOL BY ETHANOL ON GALLIC ACID DETERMINATION BY RHODANINE AND ITS IMPACTS ON THE TANNASE ASSAY
Gustavo A. S. Pinto1, Sonia Couri2, * , Elisabeth B. Gonalves2
2 1 Embrapa Agroindstria Tropical Fortaleza/CE. Embrapa Agroindstria de Alimentos - Av. das Amricas, 29501 Guaratiba, Rio de Janeiro Brazil,

KEYWORDS gallic acid determination, tannase, Aspergillus niger, solid state fermentation. ABSTRACT The replacemente of methanol by ethanol, due to high toxicity of methanol, brought no significant alteration in gallic acid determination. Ethanol, as solvent, promoted an increase in stability of the colored complex between gallic acid and rhodanine. The results showed that the determination of gallic acid by rhodanine provides a good alternative as a reliable tannase assay.

INTRODUCTION Tannase is responsible for the hydrolysis of ester and depsidic bonds in hydrolysable tannins, as tannic acid. This enzyme finds applications in food, beverage, feed, chemistry and pharmaceutical industries (13). Tannase is produced by bacteria, yeast and filamentous fungi (4-6). Many authors report that the main tannase producers are species of Aspergillus and Penicillium (2, 7-9). However, Bajpai and Patil (3) found a Fusarium solani with great synthesis capacity. 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 (3) and reduction of antinutritional effects of tannins in animal feed (Pinto et al, 2001). The first methods for tannase assay were based on the titration of the gallic acid released during the hydrolysis of tannic acid by the enzyme, meanwhile accurate determination of the end point was not possible. Iibuchi et al. (10) reported an UV-spectrophotometric method employed by different authors, in its original or modified forms (11). These methods were based on the decrease of absorbance at 310 nm of a solution of substrate, due to the hydrolysis of ester and depsidic bonds. Haslam and Tanner (12), critics of Iibuchi method and similars, developed a visible spectrophotometric assay that utilizes p-nitrophenil esters of gallic acid, however the unavailability of these substrates prevents its wide acceptance (6). The
* Corresponding author. Embrapa Agroindstria de Alimentos - Av. das Amricas, 29501 Guaratiba, Rio de Janeiro Brazil, CEP: 23020-470, Tel: 55-21-410-7434, Fax: 55-21-410-1090, Email: scoury@ctaa.embrapa.br

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estimation of tannase by gas-chromatography (13) or HPLC (1, 14) measures the gallic acid released, after the enzyme action. These methods are specific and reproducible (13), but they require sophisticate instrumentation and are high time-consuming (15). Inoue and Hagerman (16) related a method for determination of gallotannins, which involves the formation of a complex between rhodanine and gallic acid, with maximum absorbance at 520 nm. Rhodanine reacts with vicinal hydroxyl groups of gallic acid to form a red complex. The unreacted rhodanine, in basic solution, presents no absorbance at wavelengths higher than 450 nm. In this way the gallic acid-rhodanine complex can be conveniently determined without its interference. Rhodanine reacts with quinines and hydroquinones, but the reaction products absorb at longer wavelengths (17). This method is reliable for quantitative routine analysis, very specific, has no interferences from other plant phenolics, including tannic acid, and presents high sensitivity and precision. Inoues method later was adapted by Skene and Brooker (18) and Sharma et al. (15) for the assay of tannase in bacterium and fungal systens, respectively. In ours previous work for screening of tannase producer strains (PINTO et al., 2001), the activity of this enzyme was measure by decrease of UV absorvance (IIBUCHI et al., 1961 and SANDERSON and COGGON, 1974). However, this method showed some negative points: 1) it didnt express the activity under international system units, once it represents a change of 0.001 Abs unit; 2) a small difference in UV absorbance between products and reagents was observed and 3) a excessive use of chemicals, in special ethanol. The method proposed by Inoue and Hagerman (1984) for gallic acid determination and adapted by Sharma et al. (2001) for tannase activity estimation was selected as the most suitable for routine analysis. However, some changes were introduced in the last method for to reduce cost, hardzarous and laboratory wastes. This article aimed to replace the solvent originally described (methanol), by a less toxic (ethanol), and evaluated the correspondence with Inoues method and its impacts on tannase determination.

MATERIALS AND METHODS Reagents and Solutions Gallic acid, rhodanine and tannic acid were purchased from Sigma Chemical Co., Aldrich, and Vetec, Brazil, respectively. Methanol and ethanol were purchased from Merck, Germany. The commercial tannase-containing enzyme preparation utilized was Biopectinase CT from Quest International Co., Ireland, batch number S9910209.All other reagents were analytical grade. Ethanolic and methanolic rhodanine reagents were stable and could be kept under refrigeration at least for 2 weeks. The buffered (acetate 20mM, pH 5.0) solution of tannic acid had to be prepared freshly, and could be kept for 2 days in refrigerator. The stock solution of gallic acid (100 g/mL, in 0.2 N H2SO4) was stable for 2 weeks at room temperature. The aqueous solution of 0.5N KOH was stored at room temperature.

Gallic acid determination and experimental procedure In a test tube, 0.4 mL of gallic acid containing solution was added to 0.6 mL of a 0.667%wv solution of alcoholic rhodanine reagent. The test tubes were kept at room temperature for 5 minutes and then 0.4 mL of KOH solution was added. After 2.5 minutes, the mixture was diluted to 10.0 mL with distilled water. The absorbance was read at 520 nm. Rhodanine was dissolved in both alcohols. Standard curves for both alcohols were done and compared statiscally. Ten unknown samples of gallic acid were analyzed by both reagents. Random number tables and a random numbers generator (Stagraphics) were used for labeling the samples and for delineating of treatment positions. T-Student, F-Snedecor, as well 2 test, exploratory methods, and regression analysis

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were employed for the analysis of mean of variances, repeatability, behavior of data and of agreement between methods. The commercial tannase preparation was diluted in acetate buffer, pH 5.0, 20mM. A sample of 0.25 mL of tannase solution was added to 5.0 mL of tannic acid solution, homogenized and incubated at 30C. Different concentrations of tannic acid (20 to 200 g/mL) and reaction times (2.5 to 20.0 minutes) were evaluated. One unit of tannase was defined as one micromole of gallic acid formed by minute.

Microorganism In this work was utilized the Aspergillus niger 3T5B8, 11T25A5 and 11T53A9, from EMBRAPA/Food Technology stock collection, selected previously by their potential in tannase synthesis (Pinto et al., 2001).

Fermentations The solid phase of medium was composed by 100,0 g of wheat bran or 98,4 g of wheat bran with 1,6 g tannic acid, to evaluate the effect of addition of 1% of synthesis inductor. 60 ml of 0,91%wv (NH4)2SO4 solution were mixed with solid phase. After homogenization, the medium was autoclaved at 121C by 15 minutes. Solid-state fermentation were carried out in trays (dimensions: 13 x 10 x 3 cm) with 40g of the sterile fermentation medium. The medium was inoculated with 107 spores/g. and incubated at 32 C for 96 hours. Each fermentation was conducted in triplicate. The samples were taken by addition of 100 ml of a sodium acetate buffer 20 mM (pH 4.2) to fermented medium, the suspension was incubated for 1 hour at 320C, for enzyme extraction. The solid residue was separated from the solution by filtration through Whatman N1 filter paper. The samples was storage at 18oC. The enzyme activity was determined in supernant. One unit of tannase was defined as the amount of enzyme liberating 1 mol of gallic acid by minute under assay conditions.

Protein assay Determined according to the Lowry method (19).

RESULTS AND DISCUSSION Volume of the reagents The original method for gallic acid determination by rhodanine, described by Inoue and Hagerman (16), utilized a reagent volumes 2.5 times greater than this work. The methanolic rhodanine standart curves based on original and modified volumes didnt showed a significative difference in the same concentration interval (Data not shown). This fact represent a reduction in the analysis cost and generation of harmful residues.

Replacement of methanol on gallic acid determination The substitution of methanol as rhodanine solvent aimed to reduce the toxicity of solution and the harzaderous for the analist. An important aspect of this replacment is solvent toxicity. Ethanol is not toxic, in contrast methanol is very toxic by inhalation, in contact with skin and if swallowed (SIGMA-ALDRICH,
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2003). In this way the use of ethanol promote operational conditions more safety for the analyst and to the laboratorial environment. Rhodanine methanolic and ethanolic standard curves (Figure 1) presented F1,19 = 7562,38 and F1,19 = 9870,98 (significant p 0.05), respectivily, demonstrating to be representative for the experimental data. Both of them were similar to the calibration curve obtained by Inoue and Hagerman (1988). T-student test demonstrated that both standard curves have similar means (p 0.05), in the same concentration interval (Figure 1). F-Snedecor test showed compatibility between variations of the standard curves (F = 1,000) and variations of solvents (F = 1,054).

0,70 0,60 0,50 Absorbance 0,40 0,30 0,20 0,10 0,00 0,00

0,01

0,02

0,03

0,04

0,05

0,06

0,07

0,08

0,09

0,10

Gallic acid (mg) Methanol Ethanol

Figure 1: Gallic acid calibration curves with alcoholic rhodanine. Gallic acid standards were prepared in 0.2N H2SO4.

The correspondence between the solvents was established on a regression studied of the analysis of ten unkwon samples. Random number tables and a random numbers generator (Stagraphics) were used for labeling the samples and for delineating of treatment positions. The analyst done the determination in blind mode. The results showed in Figure 2. A linear correlation between the values obtained may be observed. The linear effect of regression was significant (F1.9 = 14.37) and there was no significant lack of fit (F1.9 = 0.55). The coefficient of determination was 99,88%. An investigation more refined had not demonstrated improper behaviors. The results of absorbance with ethanol may be written as: Et = 1.016934 Met. This fact shows that there is no difference when using ethanol or methanol as rhodanine solvent.

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120,0 100,0 80,0 60,0 40,0 20,0 0,0 0,0 20,0 40,0 60,0 80,0 100,0 120,0

Gallic acid by ethanolic rhodanine (g/mL)

Gallic acid by methanolic rhodanine (g/mL)

Figure 2: Comparison of gallic acid determination by two standard curves: correspondence between absorbances of ethanolic and methanolic rhodanine.

Table 1: Determination of gallic acid concentrations for ten unknown samples.

Gallic Acid (g/mL) Sample 1 2 3 4 5 6 7 8 9 10 Analysis 1 Methanol 74.2 48.0 108.4 11.9 27.3 13.6 97.5 24.1 55.6 37.5 Ethanol 80.3 48.3 111.0 11.8 26.2 13.2 99.6 24.2 54.6 38.8 Analysis 2 Methanol Ethanol 51.5 53.6 105.3 108.3 22.9 23.9 96.5 96.1 39.8 37.5 28.2 27.8 14.6 13.7 78.8 77.0 12.6 11.6 49.1 49.2

Random number tables and a random numbers generator (Stagraphics) were used for labeling the samples and for delineating of treatment positions. The analyst done the determination in blind mode.

Stability of cromogem complex It was observed that the complex formed in ethanolic medium demonstrated to be more stable than that formed in methanol. Figure 3 shows the comparison of the stability of cromogen in the both solvents. When methanol was used, the formation of the cromogen complex between gallic acid and rhodanine reached its maximum 4 minutes after the final dilution; the absorbance did not change over the next 10 minutes, which is in agreement with Sharma et al. (15) and Inoue and Hagerman (16), after this time the dissociation of the complex was monitored by the absorbance values decrease. In ethanolic medium, the cromogen presented no change in absorbance in 30 minutes. This fact allows more flexibility in routine procedures, once a greater number of assays may be done simultaneously.

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0,320 0,300 Absorbance 0,280 0,260 0,240 0,220 0 5 10 15 Time (minutes) Methanol Ethanol 20 25 30

Figure 3: Stability of cromogen complex between gallic acid and rhodanine in methanol and ethanol. 50 g/mL gallic acid solution was used.

Tannase assay The new rhodanine solvent in the tannase assay was too evalutated. Besides their role as rhodanine solvent, the alcohols have an other function in methods for the determination of tannase activity. Methanol (18) and ethanol (10) are utilized as stoppers of the enzymatic reaction. A commercial tannase solution, 77.8 g/mL of protein, was incubated at different substrate concentrations by 20 minutes. It shows that the lowest substrates concentrations, a linear behavior was observed up to 10 minutes (Figure 4a). In the highest concentrations of tannic acid, this behavior was observed during all time. Figure 4b shows that the gradual increase in substrate concentration did not promote a linear increase in enzymatic activity. This fact demonstrates that the saturation of the enzyme active sites by substrate molecules is occurring.

36 Gallic acid released (mg)

250 Tannase activity (U/mL)

0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0

32 28 24 20 16 12 8 4 0 Reaction time (minutes) 20 120 40 140 60 160 80 180 100 200

200 150 100 50 0 0 20 40 60 80 100 120 140 160 180 200 Tannic acid (g)

Figure 4: Different solutions of tannic acid incubated with a diluted solution of a commercial enzyme, which contained approximately 78 g/mL of protein

Inoue and Hagerman (16) utilized acid hydrolysis to release free gallic acid in solution from tannins. The time required for complete hydrolysis was 26 hours. In the present method, the hydrolysis was carried out by the enzyme. For releasing of gallate, Jean et al. (13), Sharma et al. (16) and Skene and Brooker (18) used methyl gallate as substrate for tannase action. The enzyme activity is only expressed by its esterase
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fraction. Nevertheless, tannase has two distinct activities, esterase and depsidic (6). The utilization of tannic acid as substrate, allows to observe both activities simultaneously. The utilization, of this method for routine analysis of tannase, was confirmed by the results shown in Figure 5. An increase in protein concentration, from 20.0 to 161.1 g/mL of protein, followed by incubation for 10 minutes in a 200 g/mL solution of tannic acid, gives a linear response of activity. The prime factor for an enzymatic assay is to guarantee that all active enzyme molecules are bound to the substrate, once the product formation is linearly dependent of the complex enzyme-substrate. The method proposed here is capable of to determine tannase activity in a large range of protein concentration.

35 Tannase activity (U/mL) 30 25 20 15 10 5 0 0 20 40 60 80 100 120 140 160 180 Protein concentration (g/mL)

Figure 5: Relation between protein concentration and tannase activity measured by the ethanolic rhodanine method. A linear relation was observed - r2 = 0,9989.

Production of tannase by solid state fermentation system The proposed method, at least, was utilized to determination of tannase synthesized by solid substrate fermentations. For this purpose A. niger 3T5B8, 11T25A5 and 11T53A9, selected previously by their potential in tannase synthesis (Pinto et al., 2001) were inoculatedin solid state medium. The results showed that A. niger 3T5B8 was the best producer of this enzyme, with 1,67U.g-1 after 72 hours of fermentation (Table 2).

Table 2: Determination of tannase activity in solid substarte fermentations, with absence and presence of 1% of tannic acid.

3T5B8 TIME (hours) 0 24 48 72 96 0% 0 0 0 0 0 1% 0 0.60 0.88 1.67 0.80

STRAIN 11T25A5 0% 1% 0 0 0 0.43 0 0.64 0 0.44 0. 0.53

11T53A9 0% 1% 0 0 0 0.34 0 0.14 0 0.24 0 0.28

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Distinct authors cited the needed of tannic acid as tannase synthesis inductor (Bajpai & Patil, 1997 and Lekha & Lonsane, 1997). The results, showed in Table 2, corroborated the idea that tannase is an inducible enzyme. In absence of tannic acid, the fermentated media demonstrated no presence of tannase activity. In contrast, when tannic acid was presented in the medium, extracellular tannase activity was detected. This fact was observed for all strains tested.

CONCLUSIONS The proposed method didnt showed significative distinct results to gallic acid determination. It allowed a great decrease in analysis cost and in laboratory harzadorous wastes management. For determination of tannase activity, it demonstrate to be adequate for analysis of several simultaneous samples.

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