FREITAS-SILVA, O.

NOGUEIRA, R.

ROCHA, M. W.

SOARES, C.

VENÂNCIO, A.

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  12309:30-10:00  AQUEOUS OZONE, A FRIENDLY METHOD FOR AFLATOXINS DEGRADATION  Otniel Freitas-Silva1,2*, Mariana Wagner Rocha3, Célia Soares1, Regina Nogueira1, Armando Venâncio1  1IBB — Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.  2 EMBRAPA Food Technology. Av das Américas, 29501, 23.020-470, Rio de Janeiro, Brazil.; 3Food Science and Technology Department, Center of Agricultural Sciences, Federal University of Santa Catarina, Post Office Box 476, 88.304-001, Florianopolis, SC, Brazil  * Tel: (+351) 253 604 400     ofreitas@deb.uminho.pt  Background:  Aflatoxins (AF) are highly toxic and hepatocarcinogenic metabolic compounds produced by Aspergillus species such as A. flavus, A. parasiticus and A. nomius during infection and colonization of food raw materials as cereals, pulses and tree nuts and their by products. Several chemical methods have been shown to be effective on removing aflatoxins; however, they need to fulfill many characteristics to be approved by regulators agencies or, in some cases, they are still too expensive to be feasible on an industrial scale. One of the agents with great potential to reduce mycotoxins is ozone. It is effective against many mycotoxins and leaves no toxic residues after treatment. Aim: to evaluate the use of aqueous ozone on degradation of AFB1, AFB2, AFG1 and AFG2.  Materials and Methods: Water saturated with ozone was prepared by bubbling gas, generated by passing extra-dry oxygen through an air-cooled corona discharge generator (Model CD-COM-HF-4) for 10 minutes (with the power generator at 100%; gas flow of 25 L / hours) in a bottle with 1000 mL of Milli-Q water, at 3 ºC. A concentrated ozone solution with c.a. 20 mg/L was obtained and was diluted with ozone demand free water. An aflatoxins stock solution (AFB1 and AFG1 – 2 mg/L each and AFB2 and AFG2 - 0.5 mg/L each) was used and it was diluted with ozone demand free water to the final working concentration. The degradation of mycotoxins by aqueous ozone was tested at 5 defined aqueous ozone levels: 0 (Control), 0.1, 1.0, 10 and 20 mg/L. A volume of 20 μL of each aflatoxin solution was applied into 2 mL vials, containing 200 μl of each aqueous ozone solution or ozone demand free water (Control) and left reacting for 30 minutes. To stop the reaction, 1200 μL of the mobile phase (see below) was added. Five replicates at each condition were made. Samples were analyzed using a HPLC equipped with a Jasco FP-920 fluorescence detector at 365 and 435 nm (excitation and emission, respectively), using photochemical post-column derivatization (PHRED unit). Chromatographic separation was performed on a reverse phase C18 column (Waters Spherisorb ODS2, 4.6 x 250 mm, 5 μm), fitted with a pre-column with the same stationary phase. The mobile phase used was pumped at 1.0 mL/min and consisted of an isocratic program as follows: water:acetonitrile:methanol (3:1:1, v/v). The injection volume was 100 μL.   124Results and Discussion: AFB1 and AFG1 were more sensitive to ozone treatment than AFB2 and AFG2, since ozone acts preferentially against unsaturated compounds, by what may be classified by an electrophilic attack. The higher sensitivity of AFB1 and AFG1 is due to the 8,9 double bond forming the vinyl ether at the terminal furan ring (Mackenzie et al, 1998; Proctor et al, 2004), which is not present in AFB2 and AFG2. These results reinforce the relevance of this degradation strategy, since the more toxic aflatoxins (AFB1 and AFG1) (IARC, 2002; Proctor et al, 2004) are also the most sensitive ones to ozone. The highest degradation level for AFB1 was 100%. The effective degradation of AFB2, AFG2 and AFG1 was 8.06, 15.96 and 98.49%, respectively. As expected, the maximum degradation occurred at the highest ozone level (20 μg/mL). Conclusion: Aqueous ozone can be used as a treatment for AF decontamination. Ozone foaming ability allows it to be trapped in a better way in water, since water is an industrial vehicle for washing and cleaning processes. However, gaseous ozone was found to be more effective then aqueous ozone, in the degradation of AFB1 from dried figs (Zorlugenç et al., 2008).  References: Akbas, M.Y., Ozdemir, M. 2006. Effect of different ozone treatments on aflatoxin degradation and physicochemical properties of pistachios. J. Sci.Food Agric. 86: 2099-2104. IARC, 2002. Monographs on the Evaluation of Carcinogenic Risks to Humans Some Traditional Herbal Medicines, Some Mycotoxins, Naphthalene and Styrene. Volume 82. 590 pages.http://monographs.iarc.fr/ENG/Monographs/vol82/index.php McKenzie, K.S. et al. 1998. Aflatoxicosis in turkey poults is prevented by treatment of naturally contaminated corn with ozone generated by electrolysis. Poultry Science. 77: 1094-1102. Proctor, A.D., et al. 2004. Degradation of aflatoxins in peanut kernels/flour by gaseous ozonation and mild heat treatment. Food Add. Contam. 21 (8): 6–793. Prudente, A.D. and King, J.M. 2002. Efficacy and safety evaluation of ozone to degrade aflatoxin in corn. J. Food Sci. 67(8): 2866-2872. Samarajeewa, U. et al. 1990. Detoxification of aflatoxins in foods and feeds by physical and chemical methods. J. Food Protect. 53(6): 489-501. Zorlugenç, B., et al. 2008. The influence of gaseous ozone and ozonated water on microbial flora and degradation of aflatoxin B1 in dried figs. Food Chem. Toxicol., 46, 3593-3597. Acknowledgements: The authors thank Dr Scussel, V.M. from Food Science and Technology Department of the Federal University of Santa Catarina, SC, Brazil, for suggestions, comments and for Rocha, M.W. supervision during the Brazil-Portugal student exchange. Célia Soares was supported by a grant from Fundação para a Ciência e Tecnologia (reference SFRH/BD/37264/2007) 

Aqueous ozone, a friendly method for aflatoxins degradation.

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