SciELO - Scientific Electronic Library Online

vol.45 número1Comparison of Cr(VI) adsorption onto polymer-based magnetic hybrid adsorbents by nonlinear isotherm and kinetic modelsAnalysis of chitosan/polyvinylpyrrolidone (structure, ftir, electrostatic potential, homo/lumo orbitals) using computational chemistry índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Latin American applied research

versión On-line ISSN 1851-8796

Lat. Am. appl. res. vol.45 no.1 Bahía Blanca ene. 2015


Abatement of ochratoxin a from contaminated wine and grape juice by activated carbon adsorption

N. D. Martínez, A. M. Rodríguez, R. B. Venturini, A. R. Gutiérrez and D.L. Granados

Instituto de Ingeniería Química, Univ. Nac. de San Juan, 5400 San Juan, Argentina.

Abstract— The ochratoxin A (OTA) is an organic and toxic for man compound, generated by fungi usually present in the vineyards. This compound can be found in grape and its derivative products.
In this article, activated carbon from grape stalk was used as an adsorbent to reduce the concentration of OTA in wine and grape must. The adsorption tests were carried out following the same procedure for wine and must. This procedure consisted in adding OTA to the samples; three aliquots were taken from them, putting each one in contact with different amounts of activated carbon. In each of the tests, the concentration of OTA was determined at 30, 60 and 120 minutes by High Performance Liquid Chromatography (HPLC).
Satisfying results were obtained, reducing OTA levels by 80% in wine and 96% in must, when 100 mg of carbon were used during 2 hours of contact.
The influence of contacting with activated carbon on the color of wine was analyzed by spectrophotometry. Neither of the analyzed samples showed a significant variation on color.

Keywords— Ochratoxin A; Wine; Grape Juice; Activated Carbon; HPLC.


The agro industry involving grape and its derivatives is strongly related to the economic development of the province of San Juan, Argentina. Among the commercialized products, wine and grape juice are the ones for which the province is known on an international level. San Juan's wine production represents 19% of the total production in the country. Also it is the main exporter of concentrated must in Argentina. Nowadays, 21 grape juice concentration plants are currently functioning in the province, including the biggest one in South America, according to data provided by the Ministerio de Ciencia y Tecnología de la República Argentina (2007).

For the international commercialization of wine and concentrated grape juice, the products must meet very high quality standards, according to the current legislations of the importing countries. There are at least 99 countries that have regulations for mycotoxins (87% of the world's population) (FAO, 2003).

One of the most important requirements involves the amount of mycotoxins, secondary metabolites which even in small quantities can potentially contaminate a large number of food products, including grape juice and wine. In certain concentrations, these compounds are toxic for man and can produce a clinical-pathological state known as micotoxicosis (Ruiz and Font, 2007).

One of the main micotoxins is ochratoxin A (OTA). The International Agency for Research on Cancer (2011) lists OTA as possibly carcinogenic to humans (Group 2B). The ochratoxin A is an organic compound (Fig. 1) generated by fungi like Penicillium verrucosum, Aspergillus ochraceus and Aspergillus niger (Ravelo Abreu et al., 2011), which can be normally found in grapes, especially in periods in which the rainfall exceeds the normal average values of precipitation. The production of this mycotoxin is influenced by the value of the activity of water (between 0.80 and 0.90), the temperature (between 277 and 310 K, depending on the microorganism), the use of pesticides, the conditions of transportation of fresh grapes, the conditions of fermentation, etc. (Ravelo Abreu et al., 2011).

Fig. 1. Molecule of Ochratoxin A.

The OTA has nephrotoxic, hepatotoxic, neurotoxic, teratogenic and immunotoxic properties when it is incorporated to the human organism in high concentrations (López de Cerain and Soriano, 2007). This toxin can be found in fresh grape and, therefore, in wine and grape juice produced from contaminated fruit. In most countries, the limit of tolerance for OTA is 2 μg/l for wine and must (European Commission, 2006; Ministerio de Economía de la República Argentina, 2013).

Once the presence of OTA is detected in wine and must, the only way of eliminating is through a thermal treatment, due to the fact that this toxin degrades at 373 K (Gimeno and Martins, 2003), temperature at which the product is also degraded. So, it becomes necessary to find alternative methods that produce OTA abatement, maintaining the product integrity. Some publications have been found concerning its elimination using activated carbons (Gambuti et al., 2005; Az3Oeno, 2007; Espejo and Armada, 2009).

Activated carbon is a material with high adsorbent efficiency due to the high specific surface it develops and its microporous structure, with slit-like cells (Rodríguez-Reinoso, 2002).

At the Instituto de Ingeniería Química at Universidad Nacional de San Juan, a technique for producing activated carbons of vegetable origin has been developed, including carbon from grape stalk. A number of researches have been conducted about the adsorbent properties of these carbons, obtaining very good results (Deiana et al., 2002; Martínez et al., 2009). For the development of this work, activated carbon was obtained from grape stalk. This raw material is the ligneous skeleton that remains after the grains were extracted from the grape cluster. It results as a residue of the wine, raisin and juice grape industries and was used as an adsorbent to low the concentration of OTA in wine and grape juice.

The analysis to determine OTA levels were conducted in LAPRIQ (Laboratorio de Análisis de Productos Regionales de Ingeniería Química), an accredited laboratory by the Norm IRAM (2005) by the Organismo Argentino de Acreditación (OAA). The range of accreditation contemplates OTA determination in wine and grape juice by a self-conducted validated method based in High Performance Liquid Chromatography (HPLC), a mandatory method in Argentinean regulations. Color analysis by U-V Spectrophotometry where conducted to detect any variations in the colorimetric characteristics of the wines involved in the tests.


A. Preparation of grape stalk activated carbon

Grape stalk from a local industry was used as a raw material to prepare the activated carbon. To obtain carbon, a protocol described in previous work was followed (Deiana et al., 2002; Martínez et al., 2009; Tancredi et al., 1996). This material was subjected to carbonization in a batch process using a retort-like stainless steel reactor in an inert nitrogen atmosphere, with a heating rate of 1.4 K/min, from room temperature up to 773 K, and keeping the final temperature for 2 hours. The obtained coal was leached with HCl 5% (w/v), to reduce the content of sodium and potassium. Then, it was dried in oven at 378 K for 24 hours and subsequently, a physical activation with water vapor was made using an electric furnace. The carbon was heated to 1123 K in nitrogen flow. When the final temperature was reached, the nitrogen current was substituted by steam water as activating agent, at a flowing rate of 1.7 g/g of carbonized matter per hour, during 105 minutes.

After that, the carbon was cooled under nitrogen atmosphere and ground to ASTM mesh size 30 (Heicoin, 2014).

B. Characterization

Textural parameters tests were determined by nitrogen adsorption-desorption isotherms, using an Autosorb-1-Quantachrome equipment. Samples of 0.100 g were oven-dried at 378 K during 24 hours and outgassed at 473 K under vacuum for 10 hours. The final pressure was less than 10-4 mbar. The specific surface area and pore size distributions were estimated by BET (Brunauer et al., 1938; Greg and Sing, 1982). The specific total pore volume was determined from the adsorption isotherm at the relative pressure of 0.99, converted to liquid volume assuming a nitrogen density of 0.808 g/cm3. The specific micropore volume was measure by Dubinin-Radushkevich model (Rouquerol et al., 1999).

In order to determine the net surface charge, the point of zero charge (pHPZC) was measured by mass titration method following the procedure detailed by Noh and Schwartz (1990). This value indicates the pH at which are equal the positive and negative charges on the surface. Additionally, the pH of activated carbon in distilled water was measured.

Trials to determine acidity and basicity were made in order to gauge the concentration of acid and basic superficial groups (Noh and Schwarz, 1990). Ash was measured under ASTM 2866-94 (2004).

C. Adsorption tests

To carry out adsorption tests, samples of grape juice and wine 100% Syrah were contaminated with OTA from R-Biopharm Rhône standard.

From contaminated samples of juice and wine were taken, respectively, three aliquots of 200 ml in an Erlenmeyer flask. Each one of them was contacted with 25, 50 and 100 mg of activated carbon, with continuous agitation at room temperature. From each one test, three samples of 30 ml were extracted at 30, 60 and 120 minutes. At all times the pH of the solution was measured to determine if a change in it could change the conditions of adsorption. PH meter ADWA 1000 was used. This equipment was submitted to pH in water interlaboratory test to verify its performance in proficiency testing.

The extracted aliquots were centrifuged for 1 minute to remove the particles of carbon. 20 ml of supernatant were retreated and the other 10 ml of residue were turned over into the Erlenmeyer flask so as to minimize changes in the amount of activated carbon present in the tested sample.

Supernatant 20 ml were subjected to analysis of OTA. The method of determination has been validated as owner one, having qualified the IRAM 301-ISO/IEC 17025 with this scope. Samples were submitted to the extraction process with clean-up OCHRAPREP ® immunoaffinity columns, and further elution with methanol. OTA was measured by Perkin Elmer Series 200 HPLC (High Performance Liquid Chromatography), with fluorescence detector.

D. Wine color index assessment

Given the importance of color variation in wines, for each wine sample 100% Syrah contaminated with OTA and treated with activated carbon, Color Index (CI) was measured using a 6100 MAPADA PC double beam UV-visible spectrophotometer.

These measurements were performed according to Resolution C9/08 INV (Instituto Nacional de Vitivinicultura, 2008), which sets for red wines more than 500 color units (CI) with a tolerance of ±10% from 2009.


A. Characterization

Nitrogen adsorption-desorption isotherms for the grape stalk activated carbon is shown in Fig. 2.

Fig. 2. Nitrogen adsorption-desorption isotherms (77K).

Figure 3 shows the pore size distributions analyzed using the BJH method. The textural parameters modeled from the adsorption data and physicochemical characteristics are summarized in Table 1.

Fig. 3. Pore size distribution.

Table 1. Textural and physicochemical characteristics of the activated carbon.

The obtained values show that the activated carbon has a high surface area and a total pore and micropore volume which make it suitable to be a good adsorbent. In spite of it is essentially a highly microporous materials, the isotherm presents such a hysteresis loop indicating a considerable presence of mesoporous. The BJH pore size distribution shows important mesoporosity in the range of 40-90 Å.

In respect of the physicochemical parameters, carbon surface is strongly basic, consistent with the pH results and the value of pHPZC. In previous work (Deiana et al., 2002) it was determined high amount of sodium and potassium, consistent with the high quantity of ash obtained, even after washing with hydrochloric acid. Activated carbons are of amphoteric nature (Rodríguez-Reinoso, 2002). This implies the coexistence of acid and basic groups on its surface. The basicity is associated with the presence of structures type chromene, quinone or g-pyrone (Fig. 4) and with delocalized p electrons in the basal plane on the surface. The acid groups are related to the existence of structures such as carboxyl, carbonyl, phenol and lactone. According to the obtained results, basic groups predominate in the activated carbon prepared to this work.

Fig. 4. Attributed structures on surfaces of activated carbons with basic behavior.

B. Adsorption tests

In trials for wine, the obtained OTA concentration in the initial contaminated sample was 3.29 g/l. The pH of the solutions at all times and in all samples kept at 3.5 pH units, independent of the amount of activated carbon added. The normal pH of a red wine is between 3.3 and 3.6. The presence of carbon did not change the natural acidity of the sample. In grape juice trials, the OTA concentration obtained in the initial contaminated sample was 2.62 g/l and the measured pH was 3.4.

Figure 5 shows the obtained chromatogram corresponding to the untreated sample of wine and, in the same graphic, the chromatograms for tests treated with 50 mg of activated carbon at 30, 60 and 120 minutes of contact. The similar obtained chromatograms for grape juice, with the same operation conditions, are showed in Fig. 6. In both, Figs. 5 and 6, some symbols were used in order to identify each one of line.

Fig. 5. HPLC chromatograms for OTA-wine tests with 50 mg of activated carbon.

Fig. 6. HPLC chromatograms for OTA- grape juice tests with 50 mg of activated carbon.

Figure 7 shows the OTA concentration drop for wines in function of time and the amount of activated carbon used in the assay. In all cases, the lowering of OTA was strongly marked. A larger amount of activated carbon leads to a greater activity of the adsorbent material so that the concentration of the mycotoxin continuously decreases from the initial value to the maximum value at 120 minutes.

Fig. 7. Curves of OTA abatement in wine with different amounts of activated carbon.

The adsorption behavior in grape juice was similar to the wine as seen in Fig. 8. In this graphic the curves of OTA concentration versus time contact with activated carbon are shown. In Figs. 9 and 10 it can see a graph showing the percentage abatement from baseline to that obtained after 120 minutes of contact, for both products. In the first one, wine samples treated with 25, 50 and 100 mg of carbon, falling toxin concentration reached 56, 75 and 88% respectively to the end time.

Fig. 8. Curves of OTA abatement in grape juice with different amounts of activated carbon.

Fig. 9. OTA abatement percentage in wine.

Fig. 10. OTA abatement percentage in grape juice.

The percentage abatement is defined as:

In the other one, the percentage abatement of OTA in grape juice is presented. The adsorption behavior was similar in both cases, to the wine, as seen in Figs. 9 and 10. In the first one, the curves of OTA concentration versus time contact with activated carbon are shown. In the other one, the abatement of the mycotoxin in percentage relative to the initial concentration is presented. In the samples treated with 25, 50 and 100 mg of activated carbon, OTA concentration falling reached 76, 91 and 96.7% respectively to the end time. All treated grape juice samples show a similar behavior, slightly better, to those of wine, presenting a marked lowering of OTA, with a maximum value at 120 minutes.

The curves obtained for both products are regular and its shape suggests that the adsorption could follow over time, becoming asymptotic at a value which could be determined by the amount of active sites on the carbon surface or the availability of free entry micropores, considering that it is a carbon with a high content in pores smaller than 2 nm.

When a carbon with large amount of basic surface groups is in an acid medium, (e.g., pH 3.6 for wine and 3.4 for grape juice) has a predominantly positive load, corresponding to the loss of hydrogen atom with its electron in the chromene group and the loss of an electron of basal oxygen. Probably these positive charges act as liaisons with the OTA molecule.

Other researchers (Gambuti et al., 2005; Az3Oeno, 2007; Espejo and Armada, 2009) report similar results for similar amounts of adsorbent. In those works mineral coals with mesoporous and longer exposure times were reported. In no case was reached more than 75% reduction of OTA

C. Color Index

In Table 2, the results of assays for determination of the color index (CI) are shown. The symbol A corresponds to the absorbance; the associated number refers to the corresponding wavelength. Measurements were made at wavelengths of 420 and 520 nm. The initial sample of wine had an IC 908 color units. The values obtained indicate no significant variation in this parameter in any of the treated samples, even when used increasing amounts of activated carbon.

Table 2. Wine Color Index results.

Gambuti et al. (2005) reported no change in the color of red wine used in the tests, in agreement with the results obtained in this work. In other works changes in color that reach 9% are reported (Az3Oeno, 2007; Espejo and Armada, 2009).


The adsorption on activated carbon is a good method to decrease or eliminate ochratoxin A (OTA) present in wines and grape juice without altering the color characteristics. Simultaneously, a residue of the wine industry, this is grape stalks, is used as a raw material for producing the activated carbon.

The behavior of activated carbon is the expected one, according to the exposure time and the amount of adsorbent used. That is, the activity of the adsorbent increases with both, the increase in exposure time and the weight of carbon added.

In all cases, the curves tended to an asymptote constant with time. This constant value denotes the adsorption limit, which depends on the weight of activated carbon used. This conduct is probably due to the progressive disappearance of active sites and/or plugging of micropores.

1. ASTM-Standard Test Method for Total Ash Content of Activated Carbon, D2866-94 (2004).
2. AZ3 Oeno SLL, "Charbon Gota,", España (2007).
3. Brunauer, S., P. Emmett and E. Teller, "Adsorption of gases in multimolecular layers," J. Am. Chem. Soc., 60, 309-19 (1938).
4. Deiana, A.C., L.M. Petkovic, H.S. Silva, E.W. Aguilar, M.F. Sardella and R.B. Venturini, "Residuos y subproductos para la obtención de briquetas de Carbón Activado", VI Curso-Taller Iberoamericano de Adsorbentes y Catalizadores para la Protección Ambiental, Redes Temáticas VC y VC - CYTEDVenezuela, 9 (2002).
5. Espejo, F.J. and S. Armada, "Effect of activated carbón on ochratoxin A reduction in Pedro Ximenez sweet winw made from off-vine dried grapes," European Food Research and Technology, 229, 255-262 (2009).
6. European Commission, "Commission Regulation (EC) N° 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs", Official Journal of the European Union, 364, 5-24 (2006).
7. FAO, Agricultural Department, Reglamentos a nivel mundial para las micotoxinas en los alimentos,, Document Repository FAO (2003).
8. Gambuti, A., D. Strollo, A. Genovese, M. Ugliano, A, Ritieni and L. Moio, "Influence of enological practices on ochratoxin A concentration in wine," American Journal of Enology and Viticulture, 56, 155-162 (2005).
9. Gimeno, A. and M. Martins, Micotoxinas y Micotoxicosis en Animales y Humanos, Special Nutrients Inc., USA, 1-160 (2003).
10. Greg, S. and K. Sing, Adsorption, Surface area and Porosity, Harcoury Brace & Co. Publishers, USA (1982).
11. Heicoin, %20TEST%20SIEVES.pdf (2004).
12. Instituto Nacional de Vitivinicultura, "Nuevo índice de color para vinos tintos nacionales," Ministerio de Agricultura, Ganadería y Pesca de la República Argentina, Boletín Oficial, 31421, 3 (2008).
13. International Agency for Research on Cancer, IARC classifies Radiofrequency Electromagnetic Fields as possibly carcinogenic to humans, Press release, 208, (2011).
14. IRAM 301-ISO/IEC 17025:2005, 4th Ed. (2005).
15. López de Cerain, A. and J. Soriano, "Ocratoxina A," Micotoxinas en alimentos, Ediciones Díaz de Santos, España, 201-218 (2007).
16. Martínez, N.D., R.B. Venturini, H.S. Silva, J.E. González and A.M. Rodríguez, "Copper on Activated Carbon for Catalytic Wet Air Oxidation," Materials esearch, 12, 45-50 (2009).
17. Ministerio de Ciencia y Tecnología de la República Argentina, COFECYT, Debilidades y desafíos tecnológicos del sector productivo vitivinícola,, Argentina (2007).
18. Ministerio de Economía de la República Argentina, INFOLEG (Centro de Documentación e Información), (2013).
19. Noh, J. and J. Schwarz, "Effect of HNO3 treatment on the surface acidity of activated carbons," Carbon, 28, 675-682 (1990).
20. Ravelo Abreu, A., C. Rubio Armendáriz, A.J. Gutiérrez Fernández and A. Hardisson de la Torre, "La ocratoxina A en alimentos de consumo humano: revisión," Nutr Hosp., 26:6, 1215-1226 (2011).
21. Rodríguez-Reinoso F., "Carbons," Handbook of Porous Solids, Germany, 13, 1766-1827, (2002).
22. Rouquerol, F., J. Rouquerol and K. Sing, "Adsorption by powders & porous solids," Principles, Methodology and Applications, Academic Press, UK (1999).
23. Ruiz, M.J. and G. Font, "Toxicidad y evaluación de riesgos," Micotoxinas en alimentos, Ediciones Díaz de Santos, España, 15-27 (2007).
24. Tancredi, N., T. Cordero, J. Rodríguez-Mirasol and J. Rodríguez, "Activated carbon from uruguayan eucalyptus wood," Fuel, 75, 1701-1706 (1996).

Received: January 28, 2014.
Accepted: June 29, 2014.
Recommended by Subject Editor: M. Luján Ferreira