1. Introduction
Pollution of bulk foods by mycotoxin has become a global concern and causes considerable economic loss every year. As mycotoxins with strong toxicity, zearalenone (ZEN) and ochratoxin A (OTA) have extremely extensive distributions [
1]. ZEN is a secondary metabolism product produced by
Fusarium strains and can be detected in cereals like corn, barley, and wheat. Corn is predominantly contaminated by ZEN, and most regions in China are exposed to ZEN pollution [
2]. OTA is produced by
Aspergillus and
Penicillium fungi during storage of cereals and associated products [
3,
4,
5]. Food and food products are substantially contaminated by OTA [
6]. ZEN can cause many symptoms (e.g., nausea, emesis, and diarrhea) in humans and animals, abnormal reproductive functions (e.g., abortions and dead fetuses) of animals, poor growth, immunity inhibition, sterility, and malformation [
7,
8]. OTA is known to be hepatotoxic, nephrotoxic, teratogenic, mutagenic, and carcinogenic [
9].
ZEN and OTA readily pollute most cereal crops with high global yield, such as corn [
10]. Scholars have investigated the pollution of 7.049 mycotoxins in corn, soybean meal, and wheat fodders from 2009 to 2011. The detection rates of ZEN and OTA were 45% and 28%, respectively [
11]. Researchers [
12] who investigated mycotoxin pollution in South Africa in one decade (2007−2016) confirmed that
Fusarium and
Aspergillus fungi are the main pollutants of corn. In Zimbabwe, the ZEN detection rate in corn samples was 15%, and the average ZEN concentration was 110 μg/kg. An investigation revealed that OTA exists from a cold temperate zone to a tropical zone (North America and South America, North Europe and West Europe, and Africa and South Asia) and contaminates cereals and relevant products [
13]. Considering the serious worldwide pollution of foods and food products by mycotoxins, most countries have proposed strict regulations on mycotoxin limits. In China, the ZEN limit in cereal and relevant products is 60 μg/kg, and the OTA limit in cereal and relevant grinding products is 5 μg/kg [
14].
Currently, ZEN and OTA pollution is difficult to control by regional and weather conditions. Moreover, ZEN and OTA have stable properties and are difficult to degrade, thereby posing considerable threats to human and animal health and economic development. Electron beam irradiation (EBI) has been applied in the degradation of mycotoxins in recent years. EBI can adequately maintain the freshness and edible quality of foods without pollution and residues due to it’s the slight temperature rise it causes compared with γ-irradiation [
15]. In addition, no induced radioactivity is involved in the safe dose. EBI is sanitary and safe. Xue et al. studied the influences of 0−5.40 kGy EBI treatment on the gelatinization and the physical and chemical properties of corn flour. They found that the total content of starch and crude fibers in corn flour declined dramatically (
p < 0.05), whereas the moisture and reducing sugar contents increased significantly (
p < 0.05) [
16]. Thus, EBI treatment can influence the physical and chemical properties of corn. According to previous studies, the mice model can be used for the in vivo toxicity assessment of ZEN [
17,
18].
Many studies concerning the influences of EBI treatment on ZEN- and OTA-contaminated corn have been conducted [
19]. However, the influences of EBI treatment on the physical and chemical properties of contaminated corns have not been extensively studied, and no relevant toxicity has been reported. Thus, the present work analyzed the physical and chemical properties of contaminated corn after EBI treatment. In addition, ZEN- and OTA-contaminated corns and contaminated corns after EBI treatment were fed to mice, and toxicity was subsequently assessed to explore the quality changes and safety of contaminated corns following EBI treatment. This study can provide theoretical reference and practical basis for the safety of corn and processed products processed by EBI.
2. Materials and Methods
2.1. Materials and Reagents
ZEN- and OTA-contaminated corn kernels and non-contaminated corn kernels were collected from farmer’s markets in Taixing City, Jiangsu Province, China. ZEN and OTA standard samples (purity ≥ 99.8%) and acetic acid (chromatographically pure) were provided by Bailingwei Technology Co., Ltd. (Beijing, China). Methyl alcohol and acetonitrile (chromatographically pure) were produced by Fisher Scientific Company (Waltham, MA, USA). Ultrapure water (resistance ≥ 18.2 MΩ/cm) was prepared by using the Simplicity UV ultrapure water instrument (Millipore Company, Bedford, MA, USA). Nitrogen (purity ≥ 99.8%) was bought from Wuxi Shinnai Chemical Gas Co., Ltd, Wuxi, China. Other analytically pure reagents were bought from Sinopharm Group Chemical Reagent Co., Ltd.(Shanghai, China) unless otherwise specified.
Blood biochemical reagents were imported from WAKO Pure Chemical Industries, Ltd. (Kanagawa, Japan) with original packaging. ICR (Institute of Cancer Research) male mice (clean level) were bought from Shanghai Slyke Experimental Animal Co., Ltd., Shanghai, China. The license number of animal production is SCXK (Shanghai) 2012−0002, and the animal qualification certificate number is 20150000522095.
Basic and custom-made fodders were provided by Jiangsu Xietong Medicine Bioengineering Co., Ltd., Jiangsu, China. Custom-made fodders consist of 50% corn, 30% maintaining basic mice fodders, 2% salad oil, 10% casein, 2% crude fiber, 3% experimental animal gunk, and 3% calcium bicarbonate.
2.2. Main Instruments and Equipment
High-performance liquid chromatograph (HPLC) 1260 series with fluorescence detector (FLD) and ZORBAX SB C18 chromatographic column (150 mm × 4.6 mm; particle size: 5 μm) were manufactured by Agilent Company (Palo Alto, CA, USA). The EBI accelerator (AB5.0) was manufactured by Wuxi ELPONT Radiation Technological Co., Ltd. (Wuxi, China). X-ray diffractometer (D8) was manufactured by Bruker (China) AXS Co., Ltd., Beijing, China. High-resolution tungsten filament scanning electron microscope (SU3800) was manufactured by Techcomp (China) Scientific Instrument Co., Ltd., Tianjing, China.
2.3. EBI Treatment of Contaminated Corn
Corn kernels (200 g) with different moisture contents (13.9% and 19.1%) were placed in polyethylene bags and flattened to approximately 0.5 cm thickness. In addition, corn kernels were ground into powder and then sieved through a 30-mesh (0.6 mm) screen. An equal amount of the corn flour was placed in polyethylene bags and flattened before EBI treatment. The above samples were radiated under different doses (0, 5, 10, 20, 30, and 50 kGy). The accelerated electron energy, beam flow, and dose rates were set to 5 MeV, 20 mA, and 2 kGy/s, respectively. All radiated samples were tested at 4 °C.
2.4. Determination of OTA and ZEN in Corn and Fodders
ZEN content was tested by GB 5009.209–2016 Test of Zearalenone in Foods [
20]. OTA content was tested by GB 5009.96–2016 Test of Ochratoxin A in Foods [
21].
2.5. Test of Physical and Chemical Properties of Corn
2.5.1. Amylose Content Test
Amylose content was tested by GB/T 15683–2008 Test of Amylose Content in Rice [
22]. Starch content in corn was tested by GB/T 5009.9–2016 Test of Starch in Foods [
23].
2.5.2. Starch Crystallinity Test
Corn flour was kept under constant humidity for 48 h after grinding and sieving. Sheet samples of corn flour were made, and starch was scanned using an X-ray diffractometer within the range of 4°–40°.
2.5.3. Observation of Starch Particles
The extracted corn starch was ground and sieved. After metal spraying, pictures were obtained using a scanning electron microscope. The pictures were magnified by 600 and 2400 times.
2.5.4. Test of Amino Acids in Corn
Approximately 100 mg of corn flour was dissolved into a hydrolysis tube to which 8 mL of 6 mol/L HCL was added and nitrogen was supplied for 3 min. Then, the tube was sealed and hydrolyzed at 120 °C for 2 h. Next, 4.8 mL of 10 mol/L NaOH was added for neutralization, and the mixture was transferred to a 25 mL volumetric flask for constant volume. The supernatant was filtered, centrifuged, and finally transferred to a brown sample bottle for HPLC analysis.
2.6. Mouse Experiment
2.6.1. Animal Grouping and Culture
In this experiment, 48 male ICR mice (clean level) were bought from Shanghai Slyke Experimental Animal Co., Ltd., China. They were randomly divided into six groups according to weight, and eight mice were assigned per group (
Table 1).
All experimental animals were cultured with a barrier system in the Animal Experimental Center of Jiangnan University with the following conditions: indoor temperature of 22 ± 2 °C, relative humidity of 50%−60%, and day–night alternation hours of 12 h/12 h. The mice ate food and drank water freely and were fed with fodder daily. Every week, water was changed twice, and padding was changed once.
This study was approved by the Ethical Committee of Jiangnan University and the Laboratory Animal Management Committee of Jiangsu Province, China (Approval No. 2110748). All animal experiments were conducted in compliance with the standard ethical guidelines under the control of the aforementioned ethical committees.
2.6.2. Serum and Routine Blood Biochemical Examination
The mice were fasted one night before the blood samples were collected from their eyeballs. The samples were placed in centrifuge tubes with an anticoagulant. The blood samples were centrifuged for 10 min at a rate of 3.000 r/min to obtain the surface blood plasma. The samples were reacted in a reaction cup under 37 °C. Various blood routine indexes were tested using an automatic biochemical analyzer.
2.6.3. Organ Index and Histopathological Examination
The mice were sacrificed by breaking their necks. The livers, kidneys, spleens, and testes were collected and weighted. These organs were rinsed with normal saline and dried with a piece of filtering paper. Some liver and kidney tissues were cut and fixed in 4% neutral formaldehyde solution. The pathological changes of the tissues were observed under a TEM after HE dyeing.
2.7. Data Processing
All experimental procedures were conducted according to the principles of green analytical chemistry [
24,
25]. The results are expressed as mean ± standard deviation. Data were analyzed by the SPSS 16.0 software. The inter-group difference was analyzed by one-way analysis of variance.
p ≤ 0.05 indicates significant difference.
4. Conclusions
The influences of EBI treatment on the physical and chemical properties of mycotoxin-contaminated corn were directly manifested by the reduced amylose content and starch crystallinity. Starch particles were broken, and starch appearance was destroyed along with the reduction of essential and total amino acid contents. Toxicity assessment revealed no significant differences among the ICR groups in terms of body weight, liver weight, and organ index. Blood indexes such as ALT, AST, TBIL, BUN, and SCr of mice fed with contaminated corn increased significantly (p < 0.05), whereas the TP and ALB decreased significantly (p < 0.05). Liver histopathological tissue lesions were observed, but no histopathological tissue lesion was found in the kidneys. Feeding mice with corn fodders after EBI treatment can improve blood biochemical indexes significantly. The resulting blood indexes were similar to those of normal mice, and no evident liver histopathological changes were noted. EBI treatment can influence some physical and chemical properties of contaminated corn, relieve the toxicity of mycotoxin in corn effectively, and increase the utilization of contaminated corn. This study provides theoretical reference and practical basis for the safety of EBI in treating mycotoxins in grains and the subsequent processing of grains in the future.