Structures of Degradation Products and Degradation Pathways of

High-voltage atmospheric cold plasma (HVACP) is a novel nonthermal decontamination technology that has potential for use in the food industry. In this...
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Structures of Degradation Products and Degradation Pathways of Aflatoxin B1 by High-Voltage Atmospheric Cold Plasma (HVACP) Treatment Hu Shi,† Bruce Cooper,‡ Richard L. Stroshine,† Klein E. Ileleji,*,† and Kevin M. Keener§ †

Department of Agricultural and Biological Engineering, ‡Bindley Bioscience Center, Purdue University, West Lafayette, Indiana 47907-2093, United States § Department of Food Science and Human Nutrition, Iowa State University, Ames, Iowa 50011-1061, United States ABSTRACT: High-voltage atmospheric cold plasma (HVACP) is a novel nonthermal decontamination technology that has potential for use in the food industry. In this study, HVACP was applied to treat pure aflatoxin B1 (AFB1) powder on a glass slide. AFB1 was degraded by 76% using a 5 min HVACP treatment in air having 40% relative humidity. The degradation products of AFB1 were separated, and their molecular formulas were elucidated using liquid-chromatography time-of-flight mass spectrometry (HPLC−TOF-MS). Six main degradation products were observed. The structures of the degradation products were further clarified via orbitrap mass spectrometry by means of fragmentation of the parental ions. Two degradation pathways were proposed on the basis of the structure of the degradation products. Among the six degradation products, two were ozonolysis products of AFB1. The appearance of the other four degradation products indicates that AFB1 was degraded by other reactive species besides ozone that were generated during HVACP treatment. Reactive oxygen gas species are suggested as the major agents for aflatoxin degradation during HVACP treatment. Two degradation pathways of AFB1 by HVACP treatment were proposed. One pathway involves reactions in which H•, OH•, CHO• radicals are added. The other involves epoxidation by HO2• radicals and oxidation of AFB1by the combined effects of the oxidative species OH•, H2O2, and O3. According to the structure− bioactivity relationship of AFB1, the bioactivity of the AFB1 samples subjected to HVACP treatment is significantly reduced because of the disappearance of the C8C9 double bond in the furofuran ring in all of the major degradation products as well as the modification of the lactone ring, cyclopentanone, and the methoxyl group. KEYWORDS: aflatoxin B1, high-voltage cold plasma (HVACP), degradation products, orbitrap

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INTRODUCTION Aflatoxins are mycotoxins produced by the fungal species A. (Aspergillus) flavus, A. parasiticus, and A. notimus.1 Aflatoxins are highly toxic, immunosuppressive, mutagenic, and carcinogenic to humans and animals.2,3 Among the aflatoxins that are found, aflatoxin B1 (AFB1) is the most potent teratogen, mutagen, and heptocarcinogen. It has been identified as a class 1 carcinogen by the International Agency for Research on Cancer.4 Varieties of agricultural products, such as corn, peanuts, pistachios, and cottonseed are susceptible to aflatoxin contamination. Prevention of contamination by toxigenic fungi is the most rational and cost-effective approach to reducing the risks associated with the presence of aflatoxin. However, it is not always possible with current agronomic and storage practices, especially when the environmental conditions are favorable for the growth of toxigenic fungi.5 Therefore, detoxification has gained importance in order to salvage aflatoxin-contaminated grains and safeguard the grain industry from food safety concerns and economic losses. Detoxification methods include cleaning and sorting, use of food additives, treatment with ozone, and treatment with electrolyzed oxidizing water.5−8 Lately, much attention has been paid to cold (nonthermal) plasma as a novel microbial decontamination technology for the food industry. It has the advantages of high efficiency and short treatment time, it leaves no residue, and it has a low overall impact on the quality of treated food products.9−11 In addition to © 2017 American Chemical Society

microbial degradation, the effectiveness of cold plasma technology in degradation of mycotoxins was recently studied by investigators using different approaches of plasma generation. It was reported that, in a model system, the mycotoxins AFB1, DON, and NIV were successfully removed by 5 s of treatment with microwave-induced argon plasma.12 Nitrogen gas plasma generated by a static induction thyristor efficiently degraded AFB1 to one-tenth of its original level within 15 min.13 Lowtemperature radio-frequency plasma degraded 88% of AFB1 within 10 min.14 In another study, multiple mycotoxins produced by Fusarium, Aspergillus, and Alternaria species were effectively degraded by high-voltage atmospheric cold plasma (HVACP) treatment.15 HVACP is a cold plasma technology that generates cold plasma by means of dielectric barrier discharge (DBD). As opposed to other types of cold plasma technology, HVACP treats the samples at atmospheric pressure thereby eliminating the need for vacuum equipment. In addition, HVACP has the advantage of high adaptability. Different geometries and varieties of gases can be applied in conjunction with the treatment.10 HVACP treatment of aflatoxin has been shown to be highly efficient in treatment of hazelnuts where 70% of the total Received: Revised: Accepted: Published: 6222

April 6, 2017 June 17, 2017 June 23, 2017 June 23, 2017 DOI: 10.1021/acs.jafc.7b01604 J. Agric. Food Chem. 2017, 65, 6222−6230

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Journal of Agricultural and Food Chemistry aflatoxin was degraded by 12 min of HVACP treatment.16 In previous work by several of the authors of this paper, 10 min of HVACP treatment reduced the aflatoxin level in a sample of corn by 82%.17 These results indicate that HVACP is a very promising technology for degrading mycotoxins. However, none of these studies on HVACP treatment of aflatoxin included identification of the degradation products, and the potential toxicity of the degradation products was not discussed. Thus, there is a lack of understanding of the reaction mechanisms involved with HVACP treatment.15−17 This information is essential for a more in-depth understanding of the HVACP process, and this information is the basis for developing future applications. Clarification of the reaction mechanism of HVACP treatment is difficult, as hundreds of reactive species can be generated during the HVACP process including electrons, radicals, ions, ozone, and other reactive oxygen or nitrogen species.18 Thus, this study is a continuation of previous work on treating aflatoxincontaminated corn using HVACP.17 Its purposes are to clarify the structures of AFB1 degradation products, to elucidate the reaction mechanism and degradation pathway of AFB1, and to identify reactive gas species that are responsible for AFB1 degradation. The toxicity of the degradation products is also discussed on the basis of their structures. In this study, pure AFB1 powder was placed on a glass slide and was treated using HVACP. The degradation products were separated, and their chemical formulas were elucidated using liquid-chromatography time-of-flight mass spectrometry (HPLC−TOF-MS), which has previously been used as an effective tool in analyzing aflatoxin degradation products.19−21 The structures of the reaction products were further investigated using orbitrap mass spectrometry. This is a new technology used for high-resolution mass spectrometry and has the features of miniature design, high-speed detection, and excellent quantification. It can be used for both reaction product identification and molecular structure characterization.22,23

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placed inside a translucent polypropylene box (Grainger Inc., Lake Forest, IL, U.S.A.). Boxes were sealed inside a high-barrier Cryovac B2630 film in order to prevent leakage of the filled gas as well as to contain the gas species that were generated. The air used as a fill gas (78% N2, 22% O2) was purchased from a local gas supplier and had a certificate of analysis. The gas in the tank had a relative humidity (RH) of 5%. In order to increase the RH to 40%, the working gas was passed through a water bubbler. The gas flow rate and water depth were adjusted, and the humidity of the exiting air was measured with a psychrometer (Extech Instruments Inc., Nashua, NH, U.S.A.). The final RH of the humidified air was 40 ± 3%. The storage bags containing AFB1 on the glass slide were filled with the working gas and were purged three to five times for 2 min to ensure purity of the gas in the bag. HVACP treatments were conducted utilizing the HVACP system (Phenix Technolgies, Accident, MD, U.S.A.) shown in Figure 1. This is patented technology developed by Dr. Keener while he was working at Purdue University.24 The parameters for the experimental set-up are shown in Table 1. The temperature of the plasma was measured using an

Table 1. Parameters Describing the HVACP System

a

parametera

value

powder/frequency applied voltage bag size (L × W) compartment box size (L × W × H) gap distance gas temperature

200 W / 50 Hz 90 kV 35.56 cm × 26.82 cm 27.31 cm × 17.78 cm × 4.45 cm 4.44 cm ≈40 °C

L: length, W: width, H: height.

infrared thermometer (Omega Engineering Inc., Bridgeport, NJ, U.S.A.).The AFB1 samples were treated for 1, 2, or 5 min. For each treatment time, the experiment was conducted in triplicate. After treatment, the AFB1 samples were stored in their sealed bag at room temperature for 24 h. This storage period allows the generated plasma to decompose so that the gas in the bag is once again air. It has been demonstrated that the majority of aflatoxin degradation occurs during the HVACP treatment time rather than during the storage period.17 HPLC−MS Analysis. The HVACP-treated and untreated AFB1 samples were carefully rinsed multiple times with 1 mL of a 50% ethanol aqueous solution to extract the AFB1 and degradation products. The extracts then were transferred to an Eppendorf tube (2.0 mL) and were stored at −5 °C in a freezer before being subjected to HPLC analysis with mass spectrometry. HPLC−MS data on the AFB1 degradation products were obtained on a time-of-flight (TOF) instrument system (Agilent Technologies, Santa Clara, CA, U.S.A.) equipped with an 1100 series binary solvent delivery system and an autosampler. Chromatography was performed on a 2.1 × 150 mm Waters Xterra C18 column with a particle size of 3.5 μm. The injection volume was 10 μL, and the flow rate was 300 μL/min. The mobile phase was a gradient prepared from distilled water with 0.1% formic acid (component A) and acetonitrile with 0.1% formic acid (component B). Gradient elution started with 10% B for 1 min, after which B was increased linearly to 95% in 20 min and subsequently kept isocratic for 1 min. The proportion of B was then decreased to 10% in 1 min and kept isocratic for 7 min. The total run time was 30 min. The MS was run with positive electrospray ionization (ESI), and the data were collected over the range of 75−1000 m/z. High mass accuracy was ensured by infusing lock mass calibrants corresponding to 121.0508 and 922.0098 m/z. HPLC−MS−MS Analysis. The same chromatographic conditions were used for the HPLC−MS−MS analysis as described in the HPLC− MS section above. Tests were performed using a Thermo LTQ Orbitrap XL mass spectrometer. The analysis used positive polarity electrospray ionization. High mass accuracy fragmentation data were acquired using the data-dependent scanning mode. Fourier transform-based mass spectrometry (FTMS), with a resolution of 60 000 and a range of 50−1100 m/z was used for full scan analysis. An FTMS resolution of 7500 was used for MS−MS data acquisition in the collision induced dissociation (CID) mode. The five most intense ions were acquired with

MATERIALS AND METHODS

Chemicals and Reagents. Aflatoxin B1 (2,3,6a,9a-tetrahydro-4methoxy-cyclopenta[c]furo[2′,3′,4,5]furo[2,3-h]chromene-1,11-dione; C17H12O6, purity >98%) was purchased from Cayman Chemicals Inc. (Ann Arbor, MI, U.S.A.). Chloroform and 200-proof ethanol were obtained from the campus laboratory store. AFB1 powder was dissolved in chloroform and serially diluted to a concentration of 50 μg/mL, and the AFB1 standard solution was stored at −5 °C in a freezer prior to being used in the tests. Treating AFB1 with HVACP. Figure 1 is a schematic of the experimental set-up for HVACP treatment of AFB1 which was placed on a glass slide. Standard AFB1 solution in chloroform (100 μL; 50 μg/mL) was pipetted onto a glass slide. This was followed by a wait time of 2 h to allow the chloroform to fully evaporate. Thus, about 5 μg of aflatoxin in powder form was subjected to HVACP treatment. The glass slide was

Figure 1. Schematic of experimental setup for HVACP treatment of aflatoxin in corn. 6223

DOI: 10.1021/acs.jafc.7b01604 J. Agric. Food Chem. 2017, 65, 6222−6230

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Figure 2. Chromatograms of AFB1 untreated (5 μg/mL) in 50% ethanol solution (A) and AFB1 sample treated by HVACP in ambient air for 5 min (B). a minimum signal of 1000, an isolation width of 2, a normalized collision energy of 35 eV, a default charge state of 1, an activation Q of 0.250, and an activation time of 30 ms.

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HVACP treatment, which is consistent with our previous observation on the aflatoxin degradation kinetics in corn by HVACP treatment.17 About 76% of the AFB1, as determined from the areas of the peaks, was degraded after 5 min of HVACP treatment. Molecular Formulas of Degradation Products. As an aid to identifying the molecular formulas of the degradation products of AFB1, data for both AFB1 and its HVACP degradation products are summarized in Table 2. The data included were retention time, proposed formula, experimental mass, mass error, double bond equivalent (DBE), and the score. (The overall score ranges from 0 to 100%, with a score closer to 100% being better.) Compared with the theoretical mass obtained from the proposed molecular formula, the mass determined by TOF-MS experiments had less than a 6 ppm error. The results showed that since the product masses were accurately determined, the elemental composition could be determined by considering all possible elemental compositions. Because AFB1 was treated by HVACP in a pure system, AFB1 and its degradation products could only be composed of four elements: carbon, oxygen, hydrogen, and nitrogen. The molecular formulas were proposed on the basis of the products’ observed isotope distribution patterns and their exact mass by using MassHunter Qualitative Analysis software (Agilent Technologies, Santa Clara, CA, USA). As an example, the molecular formulas and several possible molecular compositions of product 2 were proposed as C17 H14 O7, C18H10N4O3, and C16H8N7O2 with scores of 95, 89, and 90%, respectively. Since C17 H14 O7 has the highest score, it is the most likely to be the

RESULTS AND DISCUSSION

Formation of AFB1 Degradation Products with HVACP Treatment Time. Figure 2 shows the total ion chromatograms of the untreated AFB1 sample and of a sample treated by HVACP in ambient air for 5 min. Only one peak appeared for the AFB1 sample without HVACP treatment, whereas seven relatively large peaks (including the AFB1 peak) were observed for the HVACP treated sample. This is a good indication that the AFB1 was degraded by the HVACP treatment. Six major degradation products are shown in Figure 2B. The retention time and peak shape revealed satisfactory separation of the degradation products except for products 4 and 5, whose retention times were nearly the same so that their peaks were not completely separated. Figure 3 shows the change in the response value for AFB1 and degradation products (P1−P6) with increasing HVACP treatment time in air. As treatment time increased, AFB1 was gradually decomposed. All degradation products gradually increased, except for degradation product P2, which increased in the first 2 min of HVACP treatment and then decreased with longer HVACP treatment time. The decrease in P2 suggests that it might be an intermediate reaction product that was further converted into other degradation products. The coefficients of variance (COV) for measurements of AFB1 and degradation products were between 7% and 14%. The reduction in AFB1 level followed an exponential decay trend during the 5 min 6224

DOI: 10.1021/acs.jafc.7b01604 J. Agric. Food Chem. 2017, 65, 6222−6230

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Figure 3. Relative change in response value of AFB1 and degradation products 1 through 6 (P1−P6) for AFB1 samples with increasing HVACP treatment time in air. (Note: AFB1 and P2 are normalized so that they can be plotted on the same scale.)

furofuran ring (products 1−6), the cyclopentenone (products 1, 6) and the methoxy group (product 1). Reaction Mechanism and Degradation Pathway of AFB1 by HVACP Treatment. There are three possible agents of cold plasma treatment that could act on food-borne pathogens. They are generated heat, ultraviolet (UV) radiation, and reactive gas species.25 Since HVACP treatment is a nonthermal process, the temperature of the sample during HVACP treatment is around 40 °C, which is well below the temperature (ca. 260 °C) required for thermal decomposition of AFB1 in powder form.26 Thus, the contribution of heat generated during HVACP treatment is considered to have a negligible effect on aflatoxin degradation. During HVACP treatment, UV light was emitted with peaks at 316, 337, and 357 nm due to the N2 species transition.17 However, the emission intensity of UV light during HVACP is less than 50 μW/cm2.27 This is much lower than the UV intensity (1820−13 200 μW/cm2) required for effective degradation of aflatoxin.28 Thus, the effect of UV emission is also considered to be insignificant. This conclusion is further corroborated by the observation that the degradation products of HVACP treatment are different from the three identified major degradation products of AFB1 subjected to UV radiation.28 Thus, it is postulated that the reactive gas species generated during plasma treatment is responsible for aflatoxin degradation. For HVACP treatment, when air was applied as the gas inside the bag reactive oxygen and nitrogen species were generated, which can degrade food-borne pathogens, pesticides, allergens, and other undesirable chemical compounds.29−32 The authors believe that the reactive oxygen species, rather than the nitrogen species, plays a major role in aflatoxin degradation during HVACP treatment. There are two reasons. First, much lower degradation of aflatoxin was achieved in pure nitrogen gas than in air, and there are lower concentrations of ozone and other oxidizing species generated in nitrogen gas than in air.17 The second reason is that we did not find the nitrogen molecule in the degradation products of AFB1 during HVACP treatment. The authors expected to see it in the products if it had played a

Table 2. Proposed Formulas for AFB1 and Its Degradation Products Obtained Using LC−TOF−MS proposed products

retention time (min)

proposed formula

observed mass (m/z)a

diffb (ppm)

DBEc

score (%)

1 2 3 4 5 6 AFB1

10.1 11.0 11.5 11.9 12.0 12.5 13.4

C16 H16 O6 C17H14O7 C14 H12 O5 C14 H10 O6 C17 H12 O7 C19 H18 O8 C17 H12 O6

305.1028 331.0820 261.0772 275.2560 329.0666 375.1085 313.0716

−1.98 −1.67 −5.92 −3.62 −4.01 −2.93 −2.52

9 11 9 10 12 11 12

98.12 98.7 90.32 96.24 92.00 96.09 99.1

a

The m/z of [M + H]+. bDiff = mass difference. cDBE = double bond equivalents.

molecular formula. Another indicator is the double bond equivalent (DBE) values of the degradation products, which should be similar to that of AFB1. The AFB1 consists of 17 carbon atoms and 12 hydrogen atoms with a DBE value of 12. The DBE values of the molecular formulas, C17H14O7, C18H10N4O3, C16H8N7O2 are 11, 16, and 14.5, respectively. Since the molecular formula and DBE of C17H14O7 are the most similar to AFB1, it is the most likely of the three to be the molecular formula. Proposed Structures for Degradation Products. To elucidate the structure of the six degradation products of AFB1, the degradation products were further analyzed by orbitrap MS−MS to determine the exact masses of the fragmentation ions. This enabled postulation of their most probable parental structure and the structure of the reaction products of AFB1. On the basis of the accurate masses of the parent ions and fragments obtained from MS−MS, the analysis of the structures of the six degradation products are shown in Figure 4. The structures of the AFB1 degradation products by HVACP are summarized in Figure 5. The structures of the six degradation products (P1−P6) are similar to the structure of AFB1. The degradation by HVACP resulted in the modification of the AFB1 6225

DOI: 10.1021/acs.jafc.7b01604 J. Agric. Food Chem. 2017, 65, 6222−6230

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Figure 4. Orbitrap MS−MS spectra and proposed fragmentation (insets) of degradation products of AFB1 by HVACP.

Figure 5. Proposed structure of degradation products (P1−P6) of AFB1 produced by HVACP treatment.

during HVACP treatment has been supported by this study. Two aflatoxin degradation products, product 1 (C16H16O6) and product 2 (C17H14O7) have been shown to be major ozonolysis products of the treatment of AFB1 by aqueous ozone.19,20

significant role in degrading aflatoxin. One of the major reactive species generated by HVACP is ozone.33−35 It has been shown that it effectively degrades aflatoxins in seeds, grains, and foods.36−38 The effect of generated ozone on aflatoxin degradation 6226

DOI: 10.1021/acs.jafc.7b01604 J. Agric. Food Chem. 2017, 65, 6222−6230

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Journal of Agricultural and Food Chemistry Four other new major degradation products were identified, indicating that the degradation of AFB1 by HVACP treatment involves new pathways and reactive oxygen gas species other than ozone. On the basis of the structure of the six degradation products of AFB1 produced by HVACP treatment, two degradation pathways were proposed as shown in Figure 6 and Figure 7.

Figure 7. Second degradation pathway of AFB1 by HVACP treatment.

further hydrogenated to form the degradation product C16H17O6 (m/z 305.1028). The second branch of the reaction is the addition of an aldehyde group (CHO) to form the intermediate product, C19H15O8 (m/z 371.0767). Next, the carbonyl groups in the lactone ring and cyclopentanone of this intermediate product were hydrogenated to form the degradation product, C19H19O8 (m/z 375.1056). From the first degradation pathway of AFB1, the crucial reactive agents are the hydrogen atom (H) and the hydroxyl radical (OH•), which were generated in the HVACP system by the breakdown of water molecules.39 These two species are responsible for hydration and hydrogenation to form new degradation products. Another reactive agent generated by HVACP is the aldehyde (CHO•) radical, which is formed in the HVACP system when carbon dioxide (CO2) is present in the used gas.40 The second pathway mainly involves epoxidation and oxidation reactions. The first branch is the formation of degradation product C17H13O7 (m/z 329.0666) through epoxidation of the terminal double bond of AFB1. The epoxidation reaction could be attributed to the hydroperoxyl radical (HO2•) that is generated during HVACP treatment. Its concentration increases when the relative humidity of the air is higher.41 HO2• is one type

Figure 6. First degradation pathway of AFB1 by HVACP treatment.

The first pathway is from AFB1 to degradation products C17H15O7 (m/z 331.0821), C16H17O6 (m/z 305.1028), and C19H19O8 (m/z 375.1056). The second pathway is from AFB1 to degradation products C14H13O5 (m/z 261.0755), C14H11O6 (m/z 275.0549), and C17H13O7 (m/z 329.0666). The first degradation pathway primarily involves addition reactions in which a water molecule (H2O), hydrogen molecule (H), or aldehyde group (CHO) is added to AFB1. The first branch reaction starts with the addition of water molecules (hydration reaction) to the C8C9 double bond at the furan ring of AFB1 to form the degradation product C17H15O7 (m/z 331.0821). The methoxy group (−OCH3) of AFB1 was cleaved to form the intermediate product C16H13O6 (m/z 301.0712), and the carbonyl groups of the intermediate product C16H13O6 were 6227

DOI: 10.1021/acs.jafc.7b01604 J. Agric. Food Chem. 2017, 65, 6222−6230

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of the aflatoxin structure is essential for toxic and carcinogenic activities, and the double bond in the terminal furan ring is a notably important determinant of toxic and carcinogenic potency.49 As shown in Figure 5, all of the six proposed major degradation products of AFB1 lost their double bond, and the degradation products were different from AFB1 by further modification of the furofuran ring (products 1−6), lactone ring (products 1, 6), cyclopantenone (products 1, 6), or the methoxyl structure (product 1). Therefore, on the basis of the relationships between chemical structure and biological activity reported in the literature, the authors predict that the toxicity, carcinogenicity, and mutagenicity of the HVACP degradation products will be substantially less than those attributes of AFB1. This has been confirmed by earlier research on treating aflatoxin B1 with gaseous and aqueous ozone. In those studies, the toxicity of the AFB1 degradation products was greatly reduced or disappeared for the modified structures.51,52 Biological tests of toxicity of treated aflatoxin samples were not conducted in both previous studies of HVACP treatment or in this study.15−17 For other types of cold plasma technologies, including microwave-induced argon plasma and radio-frequency-induced nitrogen plasma, biological tests have demonstrated markedly reduced toxicity in treated samples. This is a good indicator that the cold plasma treatment will also reduce toxicity. Nevertheless, the authors recommend that additional bioactivity tests be conducted in the future to ensure that HVACP treated samples are safe for animals and humans. Testing could be conducted using duckling, Ames, or cell model tests.

of peroxyl radical that reacts with double bonds and leads to epoxide formation.42 In the second branch, the furofuran ring of AFB1 is cleaved, and the degradation product C14H11O5 (m/z 261.0755) is formed. Further oxidation of this product leads to formation of another degradation product, C14H11O6 (m/z 275.0549). Several oxygen-containing reactive species, such as atomic oxygen (O), the hydroxyl radical (OH•), hydrogen peroxide (H2O2), and ozone (O3) have been found to be present during HVACP treatment.27,35,43 Since the concentration of these species during HVACP treatment was not quantified in this study, it is unclear which specific species reacted with AFB1. It is more likely the degradation of AFB1 is through the combined action of these species, since they coexisted during the treatment and are interconvertible. For example, H2O2 and OH• can be formed by decomposition of O3 with a water molecule. Also, H2O2 can be formed by the combining of two OH• groups. The specific contribution from each species during HVACP treatment depends on the relative humidity and the composition of the used gas. For HVACP treatment in dry air, O3 is considered the major reactive species. However, in humid air higher concentrations of OH• and H2O2 are generated.35,44,45 It has been reported that under humid conditions or when in a liquid matrix, OH• and H2O2 are the major reactive species that achieve decontamination of organic compounds and microbes.32,45 In this study, the relative humidity of air during HVACP treatment was at an intermediate level (40%). Therefore, it is expected that degradation of AFB1 was through simultaneous reactions with OH•, H2O2, and O3. In summary, the second pathway involves an epoxidation reaction by HO2• radicals and oxidation reactions through the combined effects of the oxidative species OH•, H2O2, and O3. Since different plasma generation methods, such as microwave-induced argon plasma, nitrogen-gas plasma by a staticinduction thyristor, radio-frequency cold plasma in air, and HVACP, generate plasma with different profiles of reactive species, their degradation of aflatoxin should involve different reaction mechanisms. However, the degradation products and reaction mechanisms of aflatoxin degradation associated with microwave-induced argon plasma and with nitrogen plasma generated by a static-induction thyristor have not yet been clarified by the investigators in their studies.12,13 Thus, the authors of this paper cannot compare the reaction mechanisms of these plasma technologies to HVACP. Five major aflatoxin degradation products from radio-frequency cold plasma were identified. They are all different from the degradation products by HVACP treatment.14 A similarity to this study is that the radio-frequency cold plasma was generated with air as the applied gas. The OH• and H• radicals were generated and were also reported as major reactive species for aflatoxin degradation by radio frequency.14 In order to make additional comparisons of the efficacy and mechanisms of the different cold plasma technologies, the profiles and concentrations of the generated gas species would have to be determined using optical emission and absorption spectroscopy. Toxicity Analysis of the Degradation Products. Since the discovery of aflatoxin in the early 1960s, the toxicology of aflatoxins have been widely studied.46,47 The relationship of structure-to-bioactivity (toxicity, carcinogenicity, and mutagenicity) of aflatoxins has been elucidated.48,49 AFB1 has an optimal molecular structure for acute toxicity, mutagenicity, and carcinogenicity. Any changes in the furofuran ring, the lactone ring, the cyclopantenone, or the methoxyl structure would result in a marked reduction in biological activity.50 The furofuran moiety

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AUTHOR INFORMATION

Corresponding Author

*K.E.I.: Phone: 765-494-1198; E-mail: [email protected]; Fax: 765-496-1115. ORCID

Hu Shi: 0000-0002-5577-931X Klein E. Ileleji: 0000-0002-7377-9671 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by a 2010 Team Award from the NC-213 Anderson Research Grant Program. The project was entitled Reduction of Mycotoxin Levels in Distillers Grains. The authors are grateful for the assistance with the HVACP system provided by Ms. Jean Jensen and Zifan Wan, research scientists from Purdue’s Food Science Department.

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REFERENCES

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Journal of Agricultural and Food Chemistry

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DOI: 10.1021/acs.jafc.7b01604 J. Agric. Food Chem. 2017, 65, 6222−6230

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DOI: 10.1021/acs.jafc.7b01604 J. Agric. Food Chem. 2017, 65, 6222−6230