Application of atmospheric cold plasma for zearalenone detoxification in cereals: Kinetics, mechanisms, and cytotoxicity analysis

Graphical abstract

Highlights

• •ACP is an efficient method for ZEN detoxification in cereals.
• •The degradation rate of ZEN was 96.18 % after 30-W ACP treatment for 180 s.
• •Four possible ZEN degradation products were identified after ACP treatment.
• •ACP significantly reduced the cytotoxicity of ZEN both in vitro and in vivo.
• •ACP produced four major degradation product via oxidative destruction of C=C in ZEN.

Abstract

Introduction

Zearalenone (ZEN) is one of the most widely contaminated mycotoxins in world, posing a severe threat to human and animal health. Atmospheric cold plasma (ACP) holds great penitential in mycotoxin degradation.

Objectives

This study aimed to investigate the degradation efficiency and mechanisms of ACP on ZEN as well as the cytotoxicity of ZEN degradation products by ACP. Additionally, this study also investigated the degradation efficiency of ACP on ZEN in cereals and its effect on cereal quality.

Methods

The degradation efficiency and products of ZEN by ACP was analyzed by HPLC and LC-MS/MS. The human normal liver cells and mice were employed to assess the cytotoxicity of ZEN degradation products. The ZEN artificially contaminated cereals were used to evaluate the feasibility of ACP detoxification in cereals.

Results

The results showed that the degradation rate of ZEN was 96.18 % after 30-W ACP treatment for 180 s. The degradation rate was dependent on the discharge power, and treatment time and distance. Four major ZEN degradation products were produced after ACP treatment due to the oxidative destruction of C C double bond, namely C₁₈H₂₂O₇ (m/z = 351.19), C₁₈H₂₂O₈ (m/z = 367.14), C₁₈H₂₂O₆ (m/z = 335.14), and C₁₇H₂₀O₆ (m/z = 321.19). L02 cell viability was increased from 52.4 % to 99.76 % with ACP treatment time ranging from 0 to 180 s. Mice results showed significant recovery of body weight and depth of colonic crypts as well as mitigation of glomerular and liver damage. Additionally, ACP removed up to 50.55 % and 58.07 % of ZEN from wheat and corn.

Conclusions

This study demonstrates that ACP could efficiently degrade ZEN in cereals and its cytotoxicity was significantly reduced. Therefore, ACP is a promising effective method for ZEN detoxification in cereals to ensure human and animal health. Future study needs to develop large-scale ACP device with high degradation efficiency.

Introduction

Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin produced by a fungus of the genus Fusarium [1], [2]. For the first time, it was isolated from maize infected with Fusarium [3]. ZEN was frequently detected in different grains and their products, such as rye, rice, sesame, flour, soybeans, beer, and corn oil [4], [5], [6], [7]. In addition to lowering food quality, ZEN is dangerous for both human and animal health [8]. Animal sterility and reproductive disorders can result from ZEN's competitive binding to the mammalian estrogen receptor due to its structure being like that of naturally occurring oestrogens [9]. Moreover, it has been observed that ZEN binds to estrogen receptors in the mammary glands, raising the risk of human breast cancer [19], [20]. In addition, it has been verified to induce immunotoxicity, hepatotoxicity, nephrotoxicity, hematopoxicity, and genotoxicity [10]. Therefore, there is an urgent need to develop safe and effective ZEN detoxification methods.

Given that the chemical structure of ZEN is quite stable, it is difficult to eliminate or destroy ZEN by the traditional food processing methods [11], [12]. Currently, the main methods used to eliminate ZEN from food include biological chemical and physical methods (such as microwave treatment, ozonation, ultraviolet (UV) irradiation, gamma irradiation, etc.) [13]. However, these methods have boundedness, such as a few chemical treatments that could leave unknown compounds that affect food quality. Biological treatment methods, represented by enzyme treatment, are proprietary and not widely available. The physical methods such as irradiation treatment that require approval before they can be put into use [14], [15], [16].

Atmospheric cold plasma (ACP) is an ionized gas rich in ions, electrons, UV light, free radicals, reactive oxygen and nitrogen species (RONS), and other reactive species, and was the fourth state of matter to exist. ACP includes superoxide (^•O₂⁻), atomic oxygen (O), ozone (O₃), oxygen (¹O₂), hydroxyl radicals (^•OH), nitric oxide radical (^•NO), and hydrogen peroxide (H₂O₂) [17], [26], [33]. Given that the RONS in ACP have high oxidation capacity and there are few harmful by-products produced during ACP treatment process, ACP has become an emerging efficient and eco-friendly non-thermal food decontamination technology [18]. Numerous researches have evidenced the effectiveness of ACP in food sterilization [21], [22], [49], [50]. Recently, ACP has also shown great potential in the post-harvest control of mycotoxin contamination [23]. ACP could effectively eliminate multiple mycotoxins, including deoxynivalenol (DON), ZEN, aflatoxins (AFs), ochratoxin A(OTA), fumonisins (FBs), T-2 and HT-2 toxins [25], [34], [46], [51], [52], [53].

Although several previous literatures have reported the degradation effect of ACP treatment on ZEN [39], [61], [62], there is a lack of information on the degradation products of ZEN after ACP treatment and the in vivo toxicity of ZEN following ACP treatment. Additionally, limited information is available on the effect of ACP treatment parameters on ZEN degradation and its potential detoxification in cereals. Herein, this study was aimed to investigate the degradation efficiency of ACP on ZEN at different treatment parameters and its degradation products. The cytotoxicity of ZEN degradation products after ACP treatment was also assessed. Meanwhile, the degradation efficiency of ACP on ZEN in cereals and its effect on cereal quality was explored to evaluate the feasibility of ACP detoxification in cereals.

Materials and methods

Chemicals and reagents

ZEN mycotoxin standards (CAS No. 17924-92-4, MW: 318.36, purity > 98 %) was purchased from Aladdin (Shanghai, China). ZEN power was dissolved in acetonitrile and dilute the concentration to 1000 g/mL, the ZEN standard solution was stored at −20 °C. Corn and wheat were bought from a nearby grocery store. Every chemical utilized were of chromatographic grade.

ACP device and production

Fig. 1A displayed the ACP device’s schematic diagram. In the hexagon mesh elements, the ACP showed a homogeneous discharge (Fig. 1B). An in-depth explanation of the device may be read in an earlier article. [31]. As shown in Fig. 1C, the low-frequency power supply produced a sinusoidal high voltage with alterable frequency. The plasma was tuned for a 7 kHz discharge frequency in this study. The temperature during the ACP generation was measured with a thermal imaging camera (FLIR C3-X, USA) [21]. The temperature of sample table surface during ACP treatment was 25.2 °C (Fig. 1D).

Fig. 1.

Experimental information: (A) A schematic diagram of ACP device. (B) Picture of electrical discharge. (C) Voltage and the current. (D) The thermographic measurement of plasma discharges.

ACP characterization

AvaSpec-ULS4096CL-EVO Fiber Optic Spectrometer (Avantes, USA) was used to measure optical emission spectroscopy (OES) of ACP in the 200–1100 nm region [31]. The UV intensity of the ACP was measured by the handheld laser power and energy meter (VEGA, OPHIR, USA). The O₃ detector was used to determine the amount of O₃ present in the chamber. (NGP5-50-O₃, China) [22]. The temperature and humidity sensor (HC2-SE, Rotronic, Zurich, Switzerland) was used to measure the chamber's relative humidity and temperature during the ACP discharge procedure [22]. The Fourier transform infrared (FTIR) spectroscopy (Bruker, Tensor II) was used to detect the gaseous components produced by surface plasma in the wavenumber range of 500–3000 cm⁻¹ [65].

ACP degradation of ZEN

ZEN solution (10 μL) was dropped onto the glass slides (size: 10 × 10 mm) and leaved to dry for 20 min in the dark to allow the solution to evaporate. The glass slides were placed on the lifting platform and treated for five exposure times (1, 3, 5, 7, and 9 min) with the ACP generated by different discharge powers (10, 20, and 30 W). Under 30-W discharge power and 3-min treatment time, the distance between the sample surface and the electrode plate was respectively adjusted to 1, 2, 3, and 5 mm to evaluate the effect of discharge distance on ACP degradation. Meanwhile, under 30-W discharge power and 1-mm discharge distance, ZEN with different concentrations (5, 10, 15, and 20 μg/mL) were treated by ACP to evaluate the effect of ZEN concentration on ACP degradation. Immediately after treatment, the glass slides were placed in centrifuge tubes containing 1 mL of mobile phase solution (acetonitrile (ACN): water = 55: 45, v/v) for recovery [39].

ACP treatment of ZEN

The recovered ZEN solution from the ACP treatment was passed through a 0.22 μm nylon filter (Jinteng, China) using a disposable syringe, and the filtered solution was sealed into a 2 mL vial for high-performance liquid chromatography (HPLC) (E2695, Waters, USA) analysis [63]. The column used for HPLC was a C18 column (150 × 4.6 mm, 5 μm) (Waters, USA) with a column temperature of 30 ± 5 °C and a sample solution injection volume of 30 μL. The mobile phase was ACN: water (55: 45, v/v) at a flow rate of 1 mL/min. The fluorescence detector (E2475, Waters, USA) was used (excitation wavelength at 274 nm, emission wavelength at 440 nm). ZEN after 30-W ACP treatment was re-dissolved by the mobile phase, and 1 mL of derivatization solution (2.5 g of aluminum chloride dissolved in 50 mL of methanol) was added and detected using a fluorescence spectrophotometer.

ZEN degradation kinetics

The kinetic model of ZEN degradation efficiency at different ZEN concentration was first-order kinetic model as follows [60],

$$\ln \frac{c(ZEN,0)}{c(ZEN,T)}=k_{\mathit{ZEN}}T$$

where T (min) represented the period of ACP treatment; k_ZEN was the reaction rate constant; and c_{(ZEN, 0)} (μg/mL) represented the content of ZEN at T = 0; c_{(ZEN, T)} (μg/mL) was the content of ZEN at time T.

$$\ln \frac{c(ZEN,0)}{c(ZEN,D)}=k_{\mathit{ZEN}}D$$

where c_{(ZEN, 0)} (μg/mL) was the content of ZEN at D = 0; c_{(ZEN, D)} (μg/mL) was the content of ZEN at distance D; D (mm) was the ACP treatment distance; k_ZEN was the reaction rate constant.

Determination of ZEN degradation products by ACP

Liquid Chromatograph Mass Spectrometer (LC-MS/MS) (Thermo Fisher Scientific, USA) was used to investigate the degradation products of ZEN following ACP treatment [39]. The chromatographic column was BEH C18 (Waters, USA), and the mobile phases were (A) 0.1 % formic acid aqueous solution (v/v) and (B) ACN. The gradient elution conditions for the mobile phase were as follows: 0–0.1 min 95 % A and 5 % B, 0.1–12 min 5–55 % B, 12–15 min 55–100 % B, and 15–17 min 100–5 % B. The flow rate was 0.3 mL/min. 30 °C was the column temperature. The injection volume was 10 μL. The Electrospray Ionization (ESI) source performed the mass spectrometry detection. ESI was used for mass spectrometry, and the mode was ESI⁺. The mass spectrometer's entire mass scan range (m/z) was from 150 to 1500. Mass spectral data were processed and analyzed by Thermo Scientific Xcalibur and Compound Discoverer analysis software.

L02 cell viability assay

The human normal liver cells (L02) were purchased from Procell (Wuhan, China). L02 cells were cultured in RPMI-1640 (Hyclone, Logan, UT, USA) containing 10 % lethal bovine serum (FBS, South America) at 37 °C under 5 % CO₂ [64]. The cytotoxicity of ZEN and ACP-treated ZEN was analyzed by cell counting kit-8 (CCK8, Beyotime, Shanghai, China). The L02 cells suspensions at concentration 3 × 10³ /mL (100 □L) were inoculated into 96-well plates and incubated in a cell incubator for 24 h. The ACP-treated ZEN was re-solubilized by 10 □L of dimethyl sulfoxide (DMSO) and diluted to 30 □g/mL using RPMI-1640. The same volume of DMSO was used as the blank control. After the supernatant was removed, ZEN with different treatment times were added to 96-well plates with 100 □L per well and continued to incubate for 48 h. 10 □L of CCK-8 reagent was added into each well and incubated for 1 h. After that, the optical density (OD) value at 450 nm was measured by an enzyme marker (Spectrophotometer 3020, Thermo Fisher Scientific, USA).

$$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{\mathrm{O}\mathrm{D}\mathrm{o}\mathrm{f}\mathrm{A}\mathrm{C}\mathrm{P}-\mathrm{Z}\mathrm{E}\mathrm{N}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}-\mathrm{O}\mathrm{D}\mathrm{o}\mathrm{f}\mathrm{m}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{u}\mathrm{m}\mathrm{a}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{e}}{\mathrm{O}\mathrm{D}\mathrm{o}\mathrm{f}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}-\mathrm{O}\mathrm{D}\mathrm{m}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{u}\mathrm{m}\mathrm{a}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{e}}\times 100\%$$

LIVE/DEAD fluorescence assay

Cytotoxicity was further evaluated using Calcein Acetoxymethyl Ester (Calcein AM) / Propidium Iodide (PI) fluorescence assay (C2015, beyotime). After the L02 cells were treated by ZEN or Pla-ZEN, the medium was discarded. Then, 1 mL of Calcein AM/PI assay working solution was added, and the L02 cells were incubated in the cell incubator for 30 min under light protection. Using a fluorescence microscope (BX53, Olympus), the Calcein AM and PI fluorescence was examined following incubation. Whereas PI exhibits red fluorescence (Ex/Em = 535/617 nm), calcein AM exhibits green fluorescence (Ex/Em = 494/517 nm).

Animal study

Six-week-old male C57BL/6J mice were utilized as the animal model, and the mice were randomly divided into three groups: control (CK), ZEN, and ACP-treated ZEN (Pla-ZEN) groups (n = 8 per group). Throughout the experimental period, the mice in all groups had regular diet, excretion, activity, and without deaths or abnormalities. ZEN was dissolved in DMSO and corn oil. The mice in the ZEN group were given 40 mg/kg∙bw of ZEN by gavage for 21 consecutive days, with an equal volume of solvent given to the CK mice and an equal volume of the ACP-treated ZEN products given to the treated mice. All mice were fed and watered ad libitum throughout the experimental period and mice weight were measured every other day during gavage. Then, the mice were euthanized on day 22 after blood collection.

Ethics statement

All experiments involving animals were conducted according to the ethical policies and procedures approved by the local animal ethics committee of Zhengzhou University (Approval no: ZZU-LAC20220114).

Bioindicator detection

Mice's blood was drawn before to execution, and serum was extracted using centrifugation at 3000 rpm for 20 min at 4 °C. Serum was used to identify the biochemical markers.

Histopathological measurement

Mouse colon, liver, and kidney tissues were formalin-fixed, embedded using paraffin, and prepared into tissue sections with a thickness of 4 μm. For hematoxylin eosin (H&E) staining [64], the tissues were deparaffinized using xylene and gradient ethanol, and stained with hematoxylin solution, which was then rinsed by the running water for 5 min, washed with 1 % ethyl hydrochloride solution for 30 s, rinsed by water for 30 s. After that, the tissues were stained by 0.5 % eosin for 3 min and then washed by the distilled water for 30 s. Then, the tissues were dehydrated in 50 %, 70 %, 100 %, and 100 % ethanol and then transparent in a transparency agent. Finally, the sections were sealed with the resin sealer.

For periodic acid-schiff stain (PAS), the tissue sections were deparaffinized using dimethylbenzene and gradient ethanol, and treated with 1 % periodic acid for 10 min. After that, the tissues were rinsed with water and incubated with Schiff's reagent for 10 min, and then rinsed in tap water for a few minutes to develop the color, and the nuclei were stained with hematoxylin for 2 min. Then, the tissues were dehydrated in 50 %, 70 %, 100 %, and 100 % ethanol, transparent in a transparency agent, and finally sealed with the resin sealer. Analysis of glomerular diameter in mice kidney tissue and crypt depth in colon tissue was performed by special computer software, Pannoramic Viewer (3D Histech, Hungary).

Construction of ZEN artificially contaminated cereal model and ACP treatment

The Wang et al. [31] technique was used to cultivate Fusarium graminearum (PH-1). After the culture was finished, the mycelium in the suspension was removed by filtration with sterile gauze, and the spore suspension was diluted to control the spore concentration at 10⁶ CFU/mL. Add 2 mL of spore suspension to the sterilized wheat and corn kernels and shake well. Incubate in 28 °C incubator for 7 d. After the incubation, 5 g of wheat seeds, corn seeds, wheat flour, and corn flour were placed on the sample table and treated by 30-W ACP for 5, 10, 15, and 20 min.

Quantification of ZEN in wheat and corn

The ACP-treated wheat and corn were ground into powder using a pulverizer, and 2.5 g of the powder was weighed and added to 10 mL of ACN: water (84:16, v/v). The sample extract was quickly filtered and purified with a PriboFast M260 (Pribolab, China) column into a collection tube, then the sample was filtered through a 0.22 □m nylon filter into a 2 mL vial and analyzed by HPLC as described in 2.5.

Determination of color

The colors of wheat and corn flour were measured by a colorimeter (CS-10, Hangzhou Caipu Technology Co., Ltd., China) using the CIE (L*a*b*) system, according to the instructions. The measurement parameters were L (L = 0 [black] and L = 100 [white]), a (a = greenness and + a = redness), and b (−b = blueness and + b = yellowness).

Determination of pasting properties

The pasting properties of ACP-treated wheat and corn were determined by a rapid viscosity analyzer (RVA-TecMaster, Perten, Sweden).

Determination of gluten content

The gluten index, wet gluten content, dry gluten content and gluten water absorption of wheat was analyzed using a gluten quantity and quality determination system (Glutomatic 2200, Perten, Sweden) according to the instructions.

Statistical analysis

At least three replications of each experiment were performed. Data were analyzed using analysis of variance (ANOVA) with Tukey’s post hoc test with a confidence level at P < 0.05 using SPSS 22.0 statistical software. Additionally, the paired-sample t test was also used and the significant difference was expressed as n.s., P > 0.05; *, P < 0.05; ^**, P < 0.01; ^***, P < 0.001; ^****, P < 0.0001.

Results and discussion

O₃, UV, and reactive species in ACP

As shown in Fig. 2A, the temperature inside the discharge chamber was increased from 28.86 to 37.07 °C, and the humidity was decreased from 21.38 % to 17.83 % after 20-min ACP treatment. The temperature change was due to the molecules collided during ACP discharge process. The excess vibrational energy was transferred to the surrounding environment in the form of heat energy and thus increasing the temperature inside the discharge chamber. The ionization of free water molecules in the air during the discharge process leads to a decrease in the relative humidity of the discharge chamber [27].

Fig. 2.

Active species produced during ACP discharges. (A) Changes in temperature and humidity within the chamber. (B) Changes in UV intensity and O₃ concentration (n = 3). (C) Optical OES of ACP at different discharge power. (D) FTIR spectra of ACP reactive gases at 30 W.

O₃ was one of the main active substances produced during the discharge process of ACP [22]. Many studies have shown that O₃ could degrade various mycotoxins [45] Fig. 2B represented the changes of O₃ concentration during 20-min ACP discharge. The O₃ concentration was immediately increased to 0.62 ppm at the first 30 s, and then reaching the maximum concentration of 0.7 ppm. The outcomes showed that during the discharge process, the ACP using air as the working gas generated O₃.

The UV intensity at 365 and 420 nm both exhibited a pattern of first increasing and then dropping, as seen in Fig. 2B. At the beginning of ACP discharge, the UV intensity at 365 and 420 nm was instantaneously increased to 3.5 and 5.3 μW/cm², respectively. After 20-min ACP discharge, the UV intensity at 365 and 420 nm was decreased to 0.3 and 4 μW/cm², respectively.

The reactive species in ACP at different discharge power was measured by OES. The excited nitrogen species, NO_γ-system, and excited OH (A → X) band dominated the emission spectra, as Fig. 2C illustrates [30], [48]. The experimental results agree with the study by Wang et al [31]. Although the major RONS obtained at different discharge power were the same, their intensity was increased with the discharge power. The results showed that ^•OH and NOx were produced during the ACP discharge. The ^•OH were produced primarily by the dissociation of water molecules by energetic electrons or long-lived plasma species in ACP [22], [31], indicating the existence of water vapor in the discharge chamber, which was consistent with the detection of relative humidity.

Fig. 2D shows the FTIR measurements under air conditions. There are abundant O₃ and N_xO_y generated by ACP. Among them, NO₂ had the highest absorbance value. Similar results were reported by wang et al [65]. The FTIR result is consistent with the detection of O₃ and OES, indicating that O₃, ^•OH, NO, NO₂, N₂O, N₂O₅ as well as nitrogen molecules and ions are produced during the ACP discharge process.

Effect of ACP on ZEN degradation efficiency at different treatment parameters

Fig. 3A depicted the impact of ACP therapy on ZEN elimination at several power levels. With the increase of ACP discharge power, the elimination rate of ZEN became higher. The degradation rate of ZEN reached 96.08 % when treated at 30 W for 3 min. Similar results were obtained by Wang et al. [32] in their study of ACP degradation of AFB₁. The OES result (Fig. 2C) indicates that when the discharge power rose, the concentration of RONS in ACP increased as well, leading to a greater degrading efficiency. Other researchers also reported similar results. [33], [34].

Fig. 3.

Degradation efficiency of ZEN after ACP treatment (n = 3). (A) ACP treatment with different discharge power. (B) ACP treatment at different time. (C) ACP treatment at different treatment distance. (D) ZEN at different initial concentration (E) Three-dimensional fluorescence spectra of ZEN after ACP treatment. Different letters indicate significant differences (P < 0.05) among different samples.

Further research was conducted at a discharge power of 30 W for 3 min to examine the impact of ACP treatment duration on ZEN degradation. The degradation rate of ZEN rose dramatically with the expansion of the ACP treatment duration, as Fig. 3B illustrates. The ZEN degradation by ACP at different treatment time was fitted by a first-order kinetic model with R² > 0.9. Shi et al.'s study [24] produced similar outcomes, increasing the elimination of AFs from 57.15 % to 90.48 % during a period spanning from 1 to 30 min. Furthermore, the degradation of ZEN was also evaluated by three-dimensional fluorescence spectroscopy. As shown in Fig. 3E, two fluorescent bands were observed in the ZEN solution at EX/EM = 275/420 and 337/420 before treatment. The intensity of these fluorescence bands was gradually decreased with the treatment time, which was consistent with HPLC result.

Irradiation distance was also one of the critical factors in determining the degradation effect of ACP on mycotoxins. The reduce rate of ZEN was greatly reduced with an increase in irradiation distance, as Fig. 3C illustrates (P < 0.05). At 1 mm, ZEN's elimination rate was 91.79 %; at 10 mm, it dropped to 63.74 %, respectively. The ZEN degradation by ACP at different treatment distance was fitted by a first-order kinetic model with R² > 0.9. Similar results were obtained in ACP degradation of AOH, AME, and AFB₁ [35], [36]. It's possible that the simultaneous activity of active species with short and extended lifespans at close irradiation distances caused the mycotoxin degradation. On the other hand, because short-lived active species are quenched, only long-lived active species can break down mycotoxins across long radiation distances. [34], [37], [40].

The degradation rate of ZEN as a function of initial ZEN quantity was illustrated in Fig. 3D. It could be seen that the initial ZEN concentration does not significantly affect the elimination rate of ZEN in the selected concentration range (5–20 μg/ml) (P > 0.05). The outcome was in line with Zhang et al.'s [38] findings, which indicated that when DON was initially concentrated in the range of 0.5–5 μg/mL, there was no discernible variation in the degrading efficiency of DON by ACP. The reason for this phenomenon was that the energy of ACP was sufficient to disrupt the molecular structure of mycotoxins independent of the concentration of mycotoxins.

Degradation products of ZEN after ACP treatment

ln order to investigate ZEN degradation products following ACP treatment and propose the possible degradation pathway, ZEN after 0, 60, 120, and 180 s of ACP treatment was analyzed by LC-MS. By comparing the total ion chromatography plots of ZEN before and after ACP treatment, four major degradation products of ZEN were generated after ACP treatment. Fig. 4A illustrated the change pattern of ionic abundances of four ZEN degradation products following ACP treatment. With the increase of ACP treatment time, the ionic intensity of the four degradation products were all initially increased at the first 60 s, and then decreased during 180-s treatment. To further determine the structures of the four degradation products of ZEN by ACP, the fragmentation of ZEN degradation products' parent and secondary ions. Table S1 listed the chemical formula, mass-to-charge ratio, and retention duration for ZEN and its degradation products.

Fig. 4.

ZEN degradation products by ACP and their possible degradation pathways. (A) Mass chromatograms of ZEN degradation products. (B) Proposed mechanism for ACP degradation.

O₃ was detected in the ACP system by FTIR (Fig. 2), which is a strong oxidant that can react with the most reactive C C double bond in ZEN via an addition reaction, leading to the breakage of the double bond and the formation of a 1,2,3-trioxane cyclic compounds [41]. The intermediates were further oxidized in the presence of O₃ to form C₁₈H₂₂O₇ (P1)_, which was consistent with the results reported by Zheng et al [39]. Meanwhile, a portion of ZEN could collide with ^•OH to produce C₁₈H₂₂O₆ (P3-1). Strong oxidizing substances in the ACP system such as ^•OH and ¹O₂ could further react with P3-1, leading to the breakage of the double bond and the formation of the C₁₈H₂₆O₅ intermediate. The RONS in ACP combined with the olefinic double bond to form aldehydes at the ends of the double bond, thus generating P1. P1 continued to be oxidized by RONS, and the aldehyde site away from the benzene ring underwent an oxidation reaction to form a carboxylate group to produce P2. Due to the electrophilic effect of RONS, the OH-1 and OH-3 on the benzene ring of the degradation product P2 were destroyed to form P3-2. With further oxidation, P3-2 was demethylated to form P4.

Besides RONS, ACP can also generate UV light, and heat. ZEN is a thermally stable mycotoxin, which would not be affected by the temperature increase caused by ACP treatment [28]. The strongest UV intensity generated by ACP discharge was up to 6.4 μW/cm², which was far below the intensity required for mycotoxin degradation [29]. Therefore, the oxidative attack caused by RONS in ACP was responsible for ZEN degradation rather than the UV light and heat in this study.

Reduction of in vitro cytotoxicity of ZEN by ACP

The change in the structure of ZEN may affect the toxicity of ZEN. The liver is an important target organ for ZEN upon entry into the organism. The cytotoxicity of ZEN after ACP treatment was investigated by using the normal human hepatocytes (L02 cells). As shown in Fig. 5A, ZEN had no cytotoxicity on L02 until its concentration was higher than 20 μg/mL. The cell viability of L02 was deceased to 0.46 after 30 μg/mL ZEN treatment, which concentration was selected for the cytotoxicity assay of ACP-treated ZEN. The cytotoxicity of ZEN was significantly decreased after ACP treatment in a time-dependent manner (P < 0.05) (Fig. 5B). The cell viability of ZEN after 180-s ACP treatment was increased to 99.76 % compared with ZEN without ACP treatment, indicating that the degradation products of ZEN after ACP treatment were non-toxic to L02 cells. Additionally, the live/dead fluorescence test was used to confirm ZEN's cell viability following ACP treatment. The green fluorescence of the Calcein AM picture rose dramatically with the length of the ACP treatment period, as seen in Fig. 5C. In contrast, the red fluorescence of PI image was weakened. The merged image of no treatment (0 s) was dominated by red fluorescence with almost no green fluorescence, while the combined images at 180 s were dominated by green fluorescence with virtually no red fluorescence, indicating that ACP could reduce ZEN cytotoxicity, which was consistent with the result of CCK-8 assay. Previous findings have also shown that ACP treatment could significantly reduce the cytotoxicity of AFB₁, OTA, DON, and NIV [42], [66], [25].

Fig. 5.

ACP treatment reduces the cytotoxicity of ZEN. (A) Effect of different concentration of ZEN on the viability of L02 cells (n = 6). (B) Effect of ZEN on L02 cell viability after ACP treatment at different time. (C) Fluorescence microscopy assessment of live (green)/dead (red) cells. Different letters indicate significant differences (P < 0.05) among different samples. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Mitigation of in vivo cytotoxicity of ZEN by ACP

Upon oral exposure, ZEN is rapidly absorbed through the gastrointestinal tract and circulates in the bloodstream, affecting various organs like the liver, kidneys, intestines, adipose tissue, and reproductive organs [67]. The liver is the primary organ affected by ZEN, and both short-term and long-term exposure can lead to liver lesions and increase the risk of liver cancer [68]. Additionally, ZEN is immunotoxic and nephrotoxic, which may disrupt the stability of endocrine system, impair intestinal health, and cause metabolic disorders. Even low-dose exposure to ZEN can result in various pathological changes in the body [67]. To further verify the in vivo cytotoxicity of ACP-treated ZEN, the mice were administered ZEN or ACP-treated ZEN by gavage for 21 days. As shown in Fig. 6B, the body weight of mice in CK and Pla-ZEN groups was increased steadily with time, while the body weight in ZEN group was increased slowly and even began to decrease at day 9. The body weights of the CK and ZEN groups differed significantly, suggesting that the mice's development was negatively impacted by ZEN administration. Additionally, Wan et al. [44] noted that a reduction in food intake and body weight growth were significant indicators of mycotoxin in mice. However, there was no discernible change in body weight between the Pla-ZEN and CK groups, suggesting that ACP might mitigate the detrimental effects of ZEN on the development of mice.

Fig. 6.

(A) Schematic diagram of ZEN in vivo toxicity evaluation in mice after ACP treatment (n = 8). (B) Changes in body weight of mice. The concentration of (C) high-density lipoprotein cholesterol (HDL-C), (D) low-density lipoprotein cholesterol (LDL-C), (E) total cholesterol (TC), (F) triglycerides (TG), (G) alanine aminotransferase (ALT), (H) aspartate aminotransferase (AST), (I) alkaline phosphatase (ALP), (J) total protein (TP), (K) albumin (ALB), (L) UREA, and (M) creatinine (CREA) in mice after 21 days of continuous gavage. The significant difference between ZEN or Pla-ZEN treated samples and the control (CK) is expressed as *P < 0.05, **P < 0.01, and ***P < 0.001.

As shown in Fig. 6C-F, the high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and triglycerides (TG) value was increased in the ZEN group. In contrast, no significant differences existed in the CK and Pla-ZEN groups of mice. The occurrence of type 2 diabetes was closely related with the conventional lipid measurement index [57], [58]. Alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin (ALB), aspartate aminotransferase (AST), and total protein (TP) were essential markers in the clinical assessment of liver injury [54], [56]. As shown in Fig. 6G-K, ALT, AST, and ALP indices were significantly elevated in ZEN group. The Pla-ZEN and CK groups did not significantly vary in these metrics. A slight decrease in TP and ALB content could be detected in ZEN group compared with CK or Pla-ZEN group. The results indicated that ZEN ingestion caused hepatocellular damage in mice. UREA and creatinine (CREA) are important indicators for assessing the renal function [55]. As shown in Fig. 6L-M, the values of UREA and CREA were significantly elevated in ZEN group mice. In contrast, there was no significant change in the Pla-ZEN group mice compared to those in the CK group mice. The results indicated that long-term intake of ZEN caused abnormal liver and kidney function and adversely affected blood lipid levels, while the mice's health was not significantly impacted by ZEN treatment with ACP.

The histological structure of liver, kidney, and colon was observed by H&E staining. As shown in Fig. 7A, compared with CK mice, mice in ZEN group showed significant hepatocellular swelling. Some hepatocytes exhibited nuclear crumpling, irregular nuclear morphology, and small reddish particles in the cytoplasm. The renal glomeruli were atrophied, and the depth of colonic crypts was shallow. Statistics of 20 glomerular diameters and 5 crypt depths in 5 randomly selected mice further demonstrated that ZEN significantly shortened the glomerular diameters and colonic crypt depths (Fig. 7C-D). In contrast, tissue damage in the liver, kidney and colon was significantly attenuated in the Pla-ZEN group. These results indicated that long-term intake of ZEN caused significant damage to mice's liver, kidney, and colon tissues, while the in vivo toxicity of ZEN was significantly reduced after ACP treatment.

Fig. 7.

ACP treatment reduces liver, kidney, and colon damage in ZEN. (A) Hematoxylin eosin (H&E) and (B) periodic Acid-Schiff stain (PAS) staining analysis of mouse liver, kidney, and colon tissues. (C) Changes in glomerular diameter (n = 20). (D) Variation in depth of colonic crypts (n = 5). The significant difference is expressed as *P < 0.05, **P < 0.01, and ***P < 0.001, and not significant difference is expressed as ^nsP > 0.05 among the control (CK), ZEN, and Pla-ZEN group.

Histology generally uses PAS to detect glycogen and other polysaccharide substances [59]. As shown in Fig. 7B, the glycogen deposition in hepatocytes and a decrease in the number of cup cells in the colon were found in the ZEN group, which was consist with H&E staining. The results revealed that ZEN could cause damage to the hepatocytes of mice, resulting in the accumulation of glycogen and induction of insulin resistance, which was corresponded to the changes in serum HDL-C, LDL-C, TG, and TC in the mice of the ZEN group. In addition, given that the function of cup cells was mainly to secrete mucin, the decrease of cup cells may lead to an impaired intestinal mucosal barrier [43]. All of results were responsible for the slow weight gain or weight loss in mice of the ZEN group.

Overall, the in vivo and in vitro toxicity of ZEN was significantly reduced by ACP treatment, which also indicates that the degradation products of ZEN are virtually non-toxic or slightly toxic. Of course, the specific toxicity of each degradation product needs to be verified by further experiments.

ACP degradation of ZEN in cereals

To further evaluate the practical application of ACP in mycotoxin degradation to ensure food safety, the elimination rate of ZEN on wheat and corn by ACP and its effect on the quality of wheat and maize were also researched. As depicted in Fig. 8A-B, the degradation rate of ZEN in wheat and corn were both significantly increased with ACP treatment time (P < 0.05). Following a 20-minute duration of ACP treatment, the degradation rate of ZEN in wheat kernels and flour was 24.12 % and 50.55 %, respectively. The degradation of ZEN in corn kernels and flour was 31.53 % and 58.07 %, respectively. Notably, in both wheat and maize, there was a discernible variation in the rate of mycotoxin degradation between grains and flour, indicating that ACP had a matrix effect on the degradation of mycotoxins. The ventral grooves in wheat or maize kernels could protect mycotoxins from the active species in ACP. In addition, the flour had a larger contact area compared to the cereal grains, thus the active species in ACP could react more adequately with ZEN in flour. Furthermore, the degradation rate of ZEN in corn was higher than that in wheat, which may be due to the different food characteristics such as water activity, water content, and food size [36], [47].

Fig. 8.

Degradation efficiency of ZEN in (A) wheat and (B) corn after ACP treatment (n = 3). Different letters indicate significant differences (P < 0.05) among different treatment time for the same cereal state.

Color is the most direct influence on the appearance of food. Therefore, the color of wheat and maize was examined after ACP treatment. The L*, a* and b* represents the degree of brightness, green/red, and blue/yellow of sample, respectively. As shown in Table 1, there was almost no significant different in color between untreated and ACP-treated cereals. Besides, the b* value of wheat was significantly decreased after 15 and 20-min ACP treatment (P < 0.05). It was possible that the strongly oxidizing species in ACP can react with pigments in wheat, thus altering the stability of carotenoids and lutein [78], [79]. Therefore, ACP treatment had little effect on the color of cereal color.

Table 1.

Effect of ACP treatment on the color of wheat and corn.

Food type	Wheat			Corn
Treatment time	L*	a*	b*	L*	a*	b*
0 min	87.99 ± 1.92a	5.76 ± 0.22a	14.23 ± 0.10a	88.50 ± 0.10b	4.14 ± 0.19a	31.98 ± 0.87b
5 min	87.58 ± 0.94a	5.59 ± 0.28a	15.31 ± 0.82a	90.22 ± 0.29ab	4.23 ± 0.21a	32.78 ± 1.58ab
10 min	88.83 ± 1.00a	5.45 ± 0.32a	14.11 ± 1.09a	89.78 ± 0.55ab	4.44 ± 0.38a	33.03 ± 1.51ab
15 min	87.36 ± 1.75a	5.22 ± 0.44a	12.43 ± 0.22b	89.93 ± 1.02ab	4.56 ± 0.22a	32.89 ± 0.13ab
20 min	88.46 ± 0.93a	5.16 ± 0.54a	12.12 ± 0.70b	89.64 ± 0.80ab	4.18 ± 0.26a	33.27 ± 1.33ab

Data are presented as means ± SD (n = 3). Different letters indicate significant differences (P < 0.05) among different treatment time.

Starch is an important index to evaluate grain quality, which is often determined by pasting characteristics. The pasting capabilities are a crucial criterion for choosing thickening and binder in the industrial area [70]. The pasting characteristics of wheat and corn flour before and after ACP treatment are shown in Table 2. The peak viscosity, through viscosity, breakdown, final viscosity, and setback of wheat and corn were both significantly increased during the first 10 min and then decreased with the ACP treatment time extended to 20 min (P < 0.05), while the pasting temperature was remarkedly increased with ACP treatment time (P < 0.05). The results demonstrate that ACP treatment at different time has different effects on the pasting properties of wheat and corn starch. The breakdown and setback values are indicators of the thermal stability and retrogradation of starch [75], [76], both of which were altered after ACP treatment, indicating that ACP can influence the thermal stability and retrogradation of wheat and corn flour to some extent.

Table 2.

Pasting properties of wheat and corn after ACP treatment at different time.

Food type	Treatment time	Peak viscosity (cp)	Trough viscosity (cp)	Breakdown (cp)	Final viscosity (cp)	Setback (cp)	Pasting Temp (°C)
Wheat	0 min	1547 ± 78ab	1101.5 ± 78.5ab	445.5 ± 51.5c	2063.5 ± 103.5b	961.5 ± 13.5b	86.4 ± 0.36b
	5 min	1658.5 ± 189.5a	1168.5 ± 108.5a	490 ± 80b	2165.5 ± 362a	997.5 ± 154a	86.7 ± 0.27ab
	10 min	1689.5 ± 226.5a	1176 ± 150a	513.5 ± 77a	2174.5 ± 217a	998.5 ± 67a	86.2 ± 0.26b
	15 min	1469.5 ± 196.5bc	1047 ± 158ab	422.5 ± 36d	1955.5 ± 270bc	908.5 ± 111bc	87.2a
	20 min	1415.5 ± 54.5c	990 ± 43c	425.5 ± 10d	1883.5 ± 36bc	893.5 ± 8c	87.3 ± 0.04a
Corn	0 min	1178.5 ± 32b	1062 ± 12b	116.5 ± 22.5b	2107.5 ± 89.5b	1045 ± 33b	77.425 ± 0.2b
	5 min	1390.5 ± 54a	1214.5 ± 39a	176 ± 16a	2433.5 ± 78a	1219 ± 46a	78.2 ± 0.04a
	10 min	1316.5 ± 54a	1192.5 ± 125ab	124 ± 25b	2035.5 ± 92b	843 ± 166bc	77.4b
	15 min	1175 ± 169b	1058 ± 121b	117 ± 48b	1857 ± 53c	799 ± 131c	77.9 ± 0.5ab
	20 min	977.5 ± 40.5c	879.5 ± 18c	98 ± 22c	1585 ± 68d	705.5 ± 50d	78.7 ± 0.6a

Data are presented as means ± SD (n = 3). Different letters indicate significant differences (P < 0.05) among different treatment time.

Proteins are important nutrients in cereals [74]. The changes in gluten content of wheat flour before and after ACP treatment are shown in Table 3. The results showed that the gluten index and content of wheat was initially increased during the first 5 min and then decreased with the extension of ACP treatment time compared with the untreated samples (P < 0.05), indicating that ACP treatment has a dose–effect on the gluten content. The decreased gluten content after long-time ACP treatment was probably due to the destruction of the non-covalent and S-S bonds among protein molecules by RONS in ACP [71], [72]. Similar with ACP treatment, the long-time treatment of O₃ also caused the proteins to split molecularly [73]. The wheat with low gluten content was more suitable for making pasta products with specific gluten content requirements [77].

Table 3.

Gluten index and content of wheat flour after ACP treatment at different time.

Treatment time	Gluten Index	Wet gluten content (%)	Dry gluten content (%)	Gluten water absorption rate (%)
0 min	75.35 ± 0.11a	34.32 ± 0.09b	8.76 ± 0.08b	294.32 ± 3.98a
5 min	76.93 ± 0.82a	36.55 ± 0.23a	9.33 ± 0.12a	291.35 ± 5.19a
10 min	73.87 ± 0.32b	33.21 ± 0.12bc	9.28 ± 0.05a	282.31 ± 12.82b
15 min	72.19 ± 0.61bc	32.90 ± 0.21bc	8.32 ± 0.09c	275.99 ± 16.37c
20 min	71.21 ± 0.79bc	28.89 ± 0.09d	7.51 ± 0.26d	260.63 ± 11.93d

Data are presented as means ± SD (n = 3). Different letters indicate significant differences (P < 0.05) among different treatment time.

Taken together, the above results indicate that ACP treatment could efficiently degrade ZEN both in wheat and corn, which didn’t significantly affect the color of wheat and corn, while altered the pasting property and gluten content of wheat and corn flour to some extent. The degradation efficiency of ACP is influenced by various factors, such as treatment time, power, distance, contact area, and food type [69]. Future study needs to optimize ACP operating parameters to improve ACP degradation efficiency in cereals and meanwhile minimize ACP effects on cereal quality. Additionally, the ACP equipment used in this study is a laboratory-scale plasma device. Therefore, it is also urgent to develop large-scale ACP device to facilitate the application of ACP detoxification in cereals.

Conclusion

In summary, this study demonstrate that that ACP has great potential to degrade ZEN in cereals and eliminate ZEN cytotoxicity both in vitro and in vivo. ACP produced four major degradation products through oxidative destruction of C C in ZEN. During the process of ACP degradation, the pasting property and gluten content of wheat and corn flour were altered to some extent. Therefore, future study needs to optimize ACP operating parameters to improve ACP degradation efficiency in cereals and meanwhile minimize ACP effects on cereal quality. Furthermore, the large-scale ACP device with high degradation efficiency needs to be developed to improve the feasibility of ACP detoxification in cereals.

CRediT authorship contribution statement

Mengjie Liu: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft. Junxia Feng: Funding acquisition, Investigation, Resources, Software. Yongqin Fan: Data curation, Formal analysis, Methodology, Validation. Xudong Yang: Methodology, Resources, Software. Ruike Chen: Formal analysis, Methodology, Software. Cui Xu: Data curation, Investigation, Software. Hangbo Xu: Investigation, Supervision, Validation. Dongjie Cui: Investigation, Supervision, Visualization. Ruixue Wang: Visualization, Writing – review & editing. Zhen Jiao: Funding acquisition, Supervision, Writing – review & editing. Ruonan Ma: Conceptualization, Funding acquisition, Project administration, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary material

The following are the Supplementary data to this article: