Effect of microwave oven processing treatments on reduction of Aflatoxin B1 and Ochratoxin A in maize flour European Journal of Chemistry 10 (3) (2019) 224-227 European Journal of Chemistry View Journal Online View Article Online Effect of microwave oven processing treatments on reduction of Aflatoxin B1 and Ochratoxin A in maize flour Hourieh Alkadi 1,* and Jihad Altal 2 1 Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy, Arab International University, Damascus, Syria h-kadi@aiu.edu.sy (H.A.) 2 Department of Mycotoxins, Directorate of Technical Affairs and Quality Laboratory, Damascus, Syria jat_altal@hotmail.com (J.A.) * Corresponding author at: Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy, Arab International University, Damascus, Syria. Tel: +963.935.497995 Fax: +963.935.497995 e-mail: h-kadi@aiu.edu.sy (H. Alkadi). 10.5155/eurjchem.10.3.224-227.1840 Received: 03 March 2019 Received in revised form: 11 April 2019 Accepted: 16 May 2019 Published online: 30 September 2019 Printed: 30 September 2019 The effect of microwave heat has been evaluated for reduction of Aflatoxin B1 and Ochratoxin A in artificially contaminated maize flour. Contaminated maize flour were heated in microwave for various times at two different power levels. The results suggest that Aflatoxin B1 and Ochratoxin A contamination can be reduced by heating samples using microwave oven. The exposure time to heat appears to have a great effect in reduction both of the toxins in maize flour samples. It is also necessary to pay attention on initial concentrations of Aflatoxin B1 or Ochratoxin A in studied maize flour samples. HPLC Reduction Microwave Mycotoxins Aflatoxin B1 Ochratoxin A Cite this: Eur. J. Chem. 2019, 10(3), 224-227 Journal website: www.eurjchem.com 1. Introduction Poor harvesting practices, improper drying, handling, packaging, storage and transport conditions contribute to fungal growth and increase the risk of mycotoxin production [1]. Moreover, mycotoxins are extremely stable and moderately heat-resistant compounds that remain almost intact after food processing [2]. Mycotoxins are fungal second- dary metabolites found in many plant foodstuffs, particularly in cereals, fruits, nuts, kernels, spices, seeds, etc. animal fodder, and are toxic for humans and animals when ingested or inhaled with dust. The most relevant mycotoxins for food safety are Aflatoxins (AFs), Ochratoxin A (OTA), Patulin, Fumonisins, Zearalenone (ZEN), and Trichothecenes. These mycotoxins are produced by some species from the genera Aspergillus, Penicillium, and Fusarium and have multiple and combined toxic characteristics. These mycotoxins may be carcinogenic, mutagenic, teratogenic, cytotoxic, neurotoxic, nephrotoxic, immunosuppressive, and/or estrogenic [3]. AFs and OTA are the most important naturally occurring mycotoxins in agricultural products [4]. AFs are difurano- coumarins composed from two furans and a coumarin ring [5]. AFs are highly carcinogenic and hepatotoxic and are primarily found in peanuts, maize, nuts, spices, and in milk [6]. Among 18 different types of AFs identified, major members are Aflatoxins B1, B2, G1, G2, M1, and M2 (AFM1 and AFM2 are the hydroxylation products of AFB1 and AFB2, have been identified in milk and dairy products). Aflatoxin B1 (AFB1) is the most abundantly produced and the most toxic followed by G1, B2, and G2. AFB1 is classified by the International Agency of Research on Cancer as Group 1 carcinogen [7-9]. Aflatoxin s (AFs) are very stable and may resist quite severe processes like roasting, extrusion, baking, and cooking. For this reason, they can be a problem in processed foods, such as roasted nuts and bakery products [10]. OTA is a phenylalanyl derivative of a substitute disocoumarin (R)-N-[5-chloro-3,4-dihydro-8-hydroxy-3-met- hyl-1-oxo-1H-2-benzopyran-7-y1)-carbonyl]-L-phenyl alanine. Its name is derived from Aspergillus ochraceus, the musty from which it was first isolated. OTA is the most toxic member of the Ochratoxins. This mycotoxin is structurally similar to the amino acid phenylalanine (Phe). Thus, it has an inhibitory effect on a number of enzymes that use phenylalanine as a substrate, in particular, Phe-tRNA synthetase, which can result in the inhibition of protein synthesis. It is a mitochondrial poison, causing mitochondrial damage, oxidative burst, lipid ABSTRACT RESEARCH ARTICLE KEYWORDS European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2019 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. http://dx.doi.org/10.5155/eurjchem.10.3.224-227.1840 http://dx.doi.org/10.5155/eurjchem.10.3.224-227.1840 https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.10.3.224-227.1840&domain=pdf&date_stamp=2019-09-30 http://www.eurjchem.com/ http://dx.doi.org/10.5155/eurjchem.10.3.224-227.1840 mailto:h-kadi@aiu.edu.sy mailto:jat_altal@hotmail.com mailto:h-kadi@aiu.edu.sy http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.10.3.224-227.1840&domain=pdf&date_stamp=2019-09-30� Alkadi and Altal / European Journal of Chemistry 10 (3) (2019) 224-227 225 Table 1. Effect of microwave heating on Aflatoxin B1 and Ochratoxin A concentration in contaminated maize flour at 50% power of high frequency output of 2450 MHz *. Heating time (min) Aflatoxin B1 (µg/kg) Ochratoxin A (µg/kg) T1AFB1 T2AFB1 T3AFB1 T1OTA T2OTA T3OTA 0.00 300.00±0.01 200.00±0.01 100.00±0.01 300.00±0.01 200.00±0.01 100.00±0.01 2.00 300.00±0.01 200.00±0.01 100.00±0.01 300.00±0.01 200.00±0.01 100.00±0.01 4.00 298.00±0.03 199.00±0.01 99.80±0.06 299.00±0.04 199.30±0.06 100.00±0.09 6.00 289.00±0.08 195.00±0.07 98.34±0.02 292.00±0.01 196.72±0.08 99.25±0.07 8.00 255.00±0.05 178.00±0.04 90.00±0.06 260.00±0.08 185.00±0.09 94.42±0.05 10.00 205.00±0.06 163.00±0.05 74.00±0.09 215.00±0.08 167.00±0.04 86.19±0.07 * Values are mean of 3 replicates (n = 3). peroxidation, and interferes with oxidative phosphorylation. In addition, OTA increases apoptosis in several cell types and OTA has been classified as a possible human carcinogenic (Group 2B) [10,11]. OTA is a stable compound that is not destroyed by common food preparation procedures. Tempe- ratures above 250 °C for several minutes are necessary to reduce the concentration of this toxin [12]. There are many methods for reduction of AFs and OTA such as physical (Cleaning, heating, irradiation, adsorption), chemical (chemical compound, ozonization) and biological (applying bacteria, yeast and nontoxigenic Aspergillus strains) [13]. Many researches interested in reduction of AFs and OTA in food and feed. The present study was planned to assess the effect of microwave oven processing treatments on reduction of Aflatoxin B1 and Ochratoxin A in maize flour as long as maize flour has wide uses and maize is considered one of the most mycotoxins contaminated crops. 2. Experimental 2.1. Materials Acetonitrile and methanol (HPLC-gradient grade) were obtained from Sigma-Aldrich (Germany). Standard AFB1 were obtained from Supelco (Spin). Standard OTA were obtained from Sigma-Aldrich (Germany). Immunoaffinity columns for AFs were broached from Alfa Test (Germany), immunoaffinity columns for OTA were broached from Germany, potassium dihydrogen phosphate from Merck (Germany), iodine, sodium chloride from Panreac (Spin). Maize flour samples were broached from local markets. 2.2. Apparatus HPLC method was performed on a Shimadzu (Kyoto, Japan) liquid chromatography system, equipped with a model LC-20 AT pump and CTO-20A oven. The detector was a fluorescence detector (Shimadzu® RF-10 AXL, Kyoto, Japan) programmed to monitor at 365 nm for excitation and 435 nm for emission. AFB1 and OTA were completely separated using a stainless steel column of dimension (4.6 × 250 mm2) packed with symmetry C18 and 4 μm particle size (Merck, Germany). For the microwave application, a 2450 MHz microwave oven (Wattar, WST-61) was used. 2.3. Chromatographic conditions The chromatographic experiments for determine OTA were carried out by using HPLC-FLD as follows; Mobile phase: Acetonitrile, water and acetic acid (60:39:1, v:v:v), Column oven temperature: 40 °C, Injection volume: 50 μL, Flow rate: 1 mL/min, Detection fluorescence: excitation at 333 nm, emission at 460 nm. The chromatographic experiments for determine AFB1 were carried out by using HPLC-FLD as follows; Mobile phase: Water, acetonitrile, and methanol (60:20:20, v:v:v), Column oven temperature: 40 °C, Injection volume: 50 μL, Flow rate: 1.3 mL/min, Detection fluorescence: excitation at 365 nm, emission at 435 nm. 2.4. Sample preparation and microwave heating The study involved four maize flour samples, analyzed to be free of AFB1 by HPLC. One of the free samples of aflatoxin B1 (AFB1) was considered as control (CAFB1). Three addition treatments, T1AFB1, T2AFB1 and T3AFB1 were artificially conta- minated with pure Aflatoxin of 300, 200 and 100 µg/kg of AFB1. Aflatoxin s were dissolved in 5 mL of methanol of HPLC grade, and mixed with a mount of maize flour for 30 min. control and contaminated maize flour samples were then placed in plastic containers for 1 week. Maize flour samples were analyzed for Aflatoxin after storage. Two maize flour samples (about 100 g) in duplicate of each treatment (CAFB1, T1AFB1, T2AFB1 and T3AFB1) were heated in microwave oven for 2, 4, 5, 6, 8, and 10 min at 50 and 100% power of high frequency output of 2450 MHz. For testing reduction of OTA in maize flour, it was done the same work mentioned above for AFB1. 2.5. Extraction of Aflatoxin B1 from samples AFB1 was extracted from samples of maize flour treatment with microwave oven using 5 g of sodium chloride and 125 mL of methanol:water solution (30:70, v:v); the extract was filtered by filter 0.45 μm (PTFE), then 15 mL of filtrate was added into a conical flask with glass stopper, after that 30 mL of water was added and mixed and filtered the diluted extract through a glass paper [14]. The immunoaffinity column was prepared according to the supplier’s instruction. Afterwards 1 mL of methanol was passed into the immunoaffinity column to elute the AFs, then 1 mL of water was added to the extracts. 2.6. Extraction of Ochratoxin A from samples OTA was extracted from 10 g of sample by using 40 mL methanol:water (80:20, v:v). The extract was filtered, then 4 mL of filtered extract were diluted to 50 mL with phosphate- buffered saline (PBS), and applied to an OTA immunoaffinity column. The column was washed with water and the OTA was eluted from immunoaffinity column with methanol [15]. 3. Results In this research, the validated methods were used and the internal and external quality control experiments were performed. Regarding internal quality control, the accuracy and precision of the methods were verified. For this purpose, AFB1 and OTA recoveries were recorded by analyzing a blank sample, spiked at 4 ng/g for AFB1 and also 3 ng/g for OTA. Recovery rate for AFB1 was 75-80% and the average coeffi- cient of variation was 4.9% and approximately the same for OTA. The AFB1 and OTA level was corrected, according to the recovery value. LOD and LOQ for AFB1 were 0.06 μg/kg and 0.100 μg/kg, respectively. LOD and LOQ for OTA were 0.05 and 0.10 μg/kg, respectively. The linearity in the working standard solutions at three determinations of six concentration levels was reliable, 0.9996 for AFB1 and 0.9998 for OTA. The effect of microwave heating on AFB1 and OTA concentration at 50 and 100% power of high frequency output of 2450 MHz is presented in Table 1 and 2. 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.3.224-227.1840 226 Alkadi and Altal / European Journal of Chemistry 10 (3) (2019) 224-227 Table 2. Effect of microwave heating on Aflatoxin B1 and Ochratoxin A concentration in contaminated maize flour at 100% power of high frequency output of 2450 MHz *. Heating time (min) Aflatoxin B1 (µg/kg) Ochratoxin A (µg/kg) T1AFB1 T2AFB1 T3AFB1 T1OTA T2OTA T3OTA 0.00 300.00±0.01 200.00±0.01 100.00±0.01 300.00±0.02 200.00±0.01 100.00±0.05 2.00 295.00±0.05 198.00±0.02 99.38±0.01 297.15±0.03 199.08±0.01 99.91±0.01 4.00 286.50±0.08 193.64±0.05 97.25±0.03 290.30±0.01 197.34±0.09 99.05±0.01 6.00 230.40±0.09 160.00±0.08 89.46±0.06 242.00±0.07 172.66±0.07 90.94±0.05 8.00 184.00±0.06 142.72±0.09 76.87±0.03 193.67±0.08 150.06±0.08 79.99±0.01 10.00 96.80±0.03 110.34±0.09 60.79±0.03 110.45±0.01 119.00±0.08 62.78±0.09 * Values are mean of 3 replicates (n = 3). Table 3. Effect of microwave heating on aflatoxin B1(%) and ochratoxin A (%) in contaminated maize flour at 50% power of high frequency output of 2450 MHz *. Ochratoxin A% Aflatoxin B1 % Heating time (min) T3OTA T2OTA T1OTA T3AFB1 T2AFB1 T1AFB1 100.00±0.01 100.00±0.01 100.00±0.01 100.00±0.01 100.00±0.01 100.00±0.01 0.00 100.00±0.01 100.00±0.01 100.00±0.01 100.00±0.01 100.00±0.01 100.00±0.01 2.00 100.00±0.09 99.65±0.06 99.67±0.04 99.80±0.06 99.50±0.01 99.33±0.03 4.00 99.25±0.07 98.36±0.08 97.33±0.01 98.34±0.02 97.50±0.07 96.33±0.08 6.00 94.42±0.05 92.50±0.09 86.67±0.08 90.00±0.06 89.00±0.04 85.00±0.05 8.00 86.19±0.07 83.50±0.04 71.67±0.08 74.00±0.09 81.50±0.05 68.33±0.06 10.00 * Values are mean of 3 replicates (n = 3). Table 4. Effect of microwave heating on aflatoxin B1(%) and ochratoxin A (%) in contaminated maize flour at 100% power of high frequency output of 2450 MHz *. Heating time (min) Aflatoxin B1 % Ochratoxin A% T1AFB1 T2AFB1 T3AFB1 T1OTA T2OTA T3OTA 0.00 100.00±0.01 100.00±0.01 100.00±0.01 100.00±0.02 100.00±0.01 100.00±0.05 2.00 98.33±0.05 99.00±0.02 99.38±0.01 99.05±0.03 99.54±0.01 99.91±0.01 4.00 95.50±0.08 96.82±0.05 97.25±0.03 96.77±0.01 98.67±0.09 99.05±0.01 6.00 76.80±0.09 80.00±0.08 89.46±0.06 80.67±0.07 86.33±0.07 90.94±0.05 8.00 61.33±0.06 71.36±0.09 76.87±0.03 64.56±0.08 75.03±0.08 79.99±0.01 10.00 32.28±0.03 55.17±0.09 60.79±0.03 36.82±0.01 59.50±0.08 62.78±0.09 * Values are mean of 3 replicates (n = 3). Also, the percentage of reduction of AFB1 and OTA contamination in artificially contaminated maize flour by microwave heating at medium and high power level for 2, 4, 6, 8 and 10 min is shown in Table 3 and 4. Although, almost all treatment protocols showed some degree of AFB1 and OTA degradation, heating samples at high power level of microwave oven for 8 min and at medium level of microwave oven for 10 min caused the best reduction on level of AFB1 and OTA in tested samples .Effect of microwave heating at 100% power for 10 min on reduction of AFB1 and OTA in artificially contaminated maize flour can be seen in Table 2 and 4, it is noticed to be the higher reduction of AFB1 and OTA as compared to other treatments, but there was some changes in appearance of maize flour .The results in Table 1 and 2 appear that degradation of AFB1 and OTA is also dependent on their initial concentrations in samples, this might be due to availability of more active toxins for destruction during microwave heating. 4. Discussion The results presented in this study have been shown a relatively degradation of AFB1 and OTA. Moreover, the degree of AFB1 and OTA reduction was found to be dependent on the duration of exposure to microwave heating and the initial concentrations of AFB1 or OTA in samples, and the destruction of Aflatoxins AFB1 and Ochratoxin A OTA were found to increase by increasing the power level of microwave oven too. The effect of microwave heating on degradation of AFB1 and OTA was found to be relatively efficient just at longer time exposure and high level power of microwave oven; this could be due to the resistance of most mycotoxins to heat [16]. Also, the results were in agreement with those of Samarajeewa et al. [17] who found that 200 °C was required for degradation of 60% of AFs in olive oils. Also, the results in the present study were relatively in agreement with those of Pluyer et al. [18], they found that microwave heating was equally effective in destroying 48 to 61% of AFB1 in peanuts. There were some agreements with the study which was done by Hussain and Luttfullah, too [7]. Herzallah et al. had studied about of artificially contaminated feeds, and they found that microwave heating was not high efficient to decontaminate of AFs [19]. They explained that the lower efficacy of microwave heating in decontamination of AFs which could be due to the result of shorter heating time, which was 10 min, as well as the feed particle geometrical dimension, which influences decreasing the penetration depth of the microwaves through the feed, this could be due to the AFs of naturally contaminated feeds being embedded within the feed commodities, on the contrary to the feed artificially contaminated with AFs that are not fully embedded within the commodities. Therefore, the natural AFs might be less likely to be degraded with radiation treatments, because they are within the commodity protected from radiation versus being on the surface of feeds when artificially applied [19]. Depending on that, results can be explained the results in the present work. 5. Conclusion It was sum up from the present study that AFB1 and OTA level reduction rate increase with increase in exposure time, microwave oven power level, and the initial concentrations of AFB1 or OTA in studied maize flour samples. Acknowledgements This work was supported by Arab International University, Damascus, Syria Disclosure statement Conflict of interests: The authors declare that we have no conflict of interest. Author contributions: The authors contributed equally to this work. Ethical approval: All ethical guidelines have been adhered. 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.3.224-227.1840 Alkadi and Altal / European Journal of Chemistry 10 (3) (2019) 224-227 227 Sample availability: Samples of the compounds are available. ORCID Hourieh Alkadi http://orcid.org/0000-0003-0832-1012 Jihad Altal http://orcid.org/0000-0001-8488-2132 References [1]. 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Food Protect. 1987, 50(6), 504- 508. [19]. Herzallah, S.; Alshawabkeh, K.; Al-Fataftah, A. J. Appl. Poul. Res. 2008, 17, 515-521. Copyright © 2019 by Authors. This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.3.224-227.1840 http://orcid.org/0000-0003-0832-1012 http://orcid.org/0000-0001-8488-2132 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Materials 2.2. Apparatus 2.3. Chromatographic conditions 2.4. Sample preparation and microwave heating 2.5. Extraction of Aflatoxin B1 from samples 2.6. Extraction of Ochratoxin A from samples 3. Results 4. Discussion 5. Conclusion Acknowledgements Disclosure statement ORCID References PrintField10: PrintField11: PrintField12: PrintField13: PrintField20: PrintField21: PrintField22: PrintField23: