Laccase-Mimicking Cu-Tannic Acid Nanozyme for Zearalenone Detoxification: Mechanism and Application in Corn Oil

TL;DR

A copper-tannic acid nanozyme effectively degrades the harmful mycotoxin zearalenone in corn oil, reducing its toxicity and estrogenic effects.

Contribution

A laccase-mimicking CuTA nanozyme is introduced for detoxifying zearalenone in corn oil with high efficiency and reusability.

Findings

The CuTA nanozyme degrades zearalenone into less harmful hydroxy derivatives.

The nanozyme reduces zearalenone's estrogenic activity by 43.7-fold.

82% zearalenone removal was achieved in corn oil within 12 hours.

Abstract

Zearalenone (ZEN), a prevalent estrogenic mycotoxin found in grains and oils, poses significant health risks due to its endocrine-disrupting properties. This study elucidates the application of a laccase-mimicking copper-tannic acid (CuTA) nanozyme as an effective catalyst for the degradation of ZEN. The CuTA nanozyme was capable of directly catalyzing the oxidation of ZEN, with optimal reaction conditions observed at a pH of 7.0 and temperatures ranging from 37 to 57 °C. The degradation products of ZEN were identified as 13-hydroxy-zearalenone (13-OH-ZEN) and 15-hydroxy-zearalenone (15-OH-ZEN). Furthermore, cytotoxicity assessments demonstrated that the CuTA nanozyme-mediated degradation of ZEN effectively reduced the hepatotoxicity of this mycotoxin. The E-screen bioassay revealed a 43.7-fold reduction in the estrogenic activity of ZEN after CuTA-mediated degradation. In corn oil, the CuTA nanozyme achieved 82% ZEN removal within 12 h and maintained 58% efficiency after four reuse cycles. These results highlight the potential use of the CuTA nanozyme to detoxify ZEN in corn oil.

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1. Introduction

Mycotoxins are secondary metabolites predominantly synthesized by species of Aspergillus, Penicillium, and Fusarium, contaminating about 25% of global agricultural commodities annually [1]. Among these identified mycotoxins, aflatoxins (AFT), deoxynivalenol (DON), and zearalenone (ZEN) pose significant threats to food safety. ZEN, an estrogenic mycotoxin produced by Fusarium species, exhibits documented genotoxic, immunotoxic, teratogenic, carcinogenic, and hepatotoxic effects [2]. Critically, its structural similarity to endogenous estrogens enables competitive binding to estrogen receptors. This disruption of hormonal balance can lead to reproductive disorders in both humans and livestock [3]. Owing to these health implications, the European Food Safety Authority (EFSA) has established strict limits for ZEN exposure, with a threshold of 0.25 μg per kg of body weight per day [4]. Strategies to mitigate dietary exposure to ZEN include pre-harvest prevention and post-harvest mitigation of ZEN contamination. Implementing good agricultural practices (GAP), such as crop rotation, pest and water management, and careful fertilizer use, can effectively manage fungal growth and reduce ZEN production. ZEN contamination in agricultural products continues to be a major issue due to the variety of ZEN-producing fungi and the rising frequency of extreme weather events. Consequently, it is imperative to investigate and develop effective methods for the removal of ZEN from contaminated food commodities.

Current strategies for the decontamination of ZEN primarily encompass physical, chemical, and biological techniques. Physical approaches include selective removal of fungal-infected kernels via optical sorting and density segregation, the employment of mycotoxin adsorbents such as yeast cell walls and clay minerals, and the use of advanced oxidation techniques, including cold plasma and irradiation [5]. Chemical detoxification employs strong oxidizing agents, such as electrolyzed oxidizing water, chlorine dioxide, and ozone, to disrupt the chemical structure of ZEN [6]. However, the extensive application of these physical and chemical approaches faces several challenges, including a lack of specificity, high expenses, nutrient depletion, and the potential for secondary environmental pollution. In contrast, biodegradation using microorganisms or enzymes offers a more efficient and environmentally sustainable solution. Researchers have isolated numerous strains of microorganisms capable of degrading ZEN, including Bacillus licheniformis ZOM-1 [7], Aeromicrobium sp. HA [8], and Gordonia hydrophobica HAU421 [9]. Several enzymes capable of degrading ZEN have also been discovered, including lactonohydrolases [9], peroxidases [10], and laccases [7].

Laccases are multi-copper oxidases that catalyze the oxidation of various phenolic and non-phenolic substrates. Laccases are considered environmentally benign biocatalysts, as they utilize molecular oxygen as the terminal electron acceptor, producing water as the sole by-product [11]. In research by Banu et al. [12], an 81.7% degradation of ZEN in a liquid medium was achieved using purified laccase from Trametes versicolor. Similarly, research on laccase Ery4 from Pleurotus eryngii demonstrated complete degradation of ZEN within 72 h [13]. However, the use of natural laccases is limited by practical challenges, including sensitivity to environmental conditions and high production costs, which restrict their industrial application. Various coordinated strategies are being explored to enhance the applicability of laccase and address existing challenges. Nanomaterial-based artificial enzymes, commonly referred to as nanozymes, are esteemed for their catalytic efficiency, ease of synthesis, cost-effectiveness, and robustness under adverse conditions. Copper-based nanozymes, in particular, mimic the structure and function of natural laccases while offering enhanced stability and cost-effectiveness [14,15]. Recent studies have explored the use of copper-tannic acid (CuTA) nanosheets as laccase mimics, demonstrating their potential in various oxidative reactions [15,16]. However, a comprehensive exploration of the effectiveness and practical applications of laccase-mimicking nanozymes in ZEN degradation remains pending. This study investigates the degradation of ZEN using a CuTA nanozyme, focusing on its catalytic efficiency and the resulting degradation products. Furthermore, we evaluate its practical performance in corn oil, a common ZEN-contaminated commodity, to assess its feasibility for large-scale food safety applications. This study seeks to offer a practical solution for mitigating ZEN contamination in the food supply chain by bridging laboratory research with industrial application.

2. Materials and Methods

2.1. Reagents and Chemicals

ZEN standard (purity  ≥  98%) and ZEN immunoaffinity columns were obtained from Pribolab Co., Ltd. (Qingdao, China). Cupric sulfate pentahydrate (CuSO_4_·5H_2_O), tannic acid (TA), 4-aminoantipyrine (4-AP), and 2,4-dichlorophenol (2,4-DP) were purchased from Macklin Biochemical Technology Co., Ltd. (Shanghai, China).

2.2. Synthesis of Laccase-Mimicking CuTA Nanozyme and Test for Its Ability to Degrade ZEN

The CuTA nanozyme was prepared through an oxidative coupling assembly method with modifications [17]. Briefly, 875 mg of CuSO_4_·5H_2_O and 27 mg of tannic acid were dissolved in 50 mL of ultrapure water. The solution pH was adjusted to 7.4 with 2 M NaOH, followed by incubation at 50 °C for 3 h. The resulting precipitate was collected by centrifugation (10,000× g, 10 min), washed thoroughly with deionized water, and freeze-dried under vacuum. Morphological characterization was performed using transmission electron microscopy (TEM, JEOL JEM-2100, Tokyo, Japan). Laccase-mimetic activity was tested using 2,4-DP as substrate and 4-AP as chromogenic ligand [14]. The reaction system (1 mL) contained 100 μg mL^−1^ of 4-AP, 100 μg mL^−1^ of 2,4-DP, and 1 mg mL^−1^ of CuTA nanozyme. After incubation at 37 °C for 1 h, the formation of a red product was observed. To assess the ZEN degradation capability of the CuTA nanozyme, 10 µg mL^−1^ of ZEN was incubated with 20 mg mL^−1^ of CuTA nanozyme in PBS (pH 7.0) at 37 °C with shaking at 180 rpm for 24 h. Residual ZEN was quantified by HPLC as described in Section 2.4.

2.3. Characteristics of ZEN Degradation by CuTA Nanozyme

A systematic investigation of the catalytic properties of the CuTA nanozyme towards ZEN oxidation was conducted. Degradation kinetics were assessed in 1 mL of reaction volume containing ZEN (10 μg mL^−1^) and CuTA nanozyme (20 mg mL^−1^). Mixtures were shaken at 180 rpm and maintained at 37 °C for time intervals ranging from 1 to 24 h (1, 3, 6, 9, 12, 15, 18, 21, 24 h). The pH dependence of ZEN degradation was evaluated by resuspending the CuTA nanozyme (20 mg mL^−1^) in buffers spanning pH 3.0 to 9.0, followed by incubation with ZEN (10 μg mL^−1^) at 37 °C for 24 h. Temperature effects were examined by incubating CuTA nanozyme (20 mg mL^−1^) with ZEN (10 μg mL^−1^) over a temperature range of 22 °C to 57 °C and at pH 7.0 for 24 h. Additionally, the influence of metal ions on the efficiency of CuTA nanozyme-catalyzed ZEN degradation was tested by the addition of 1 mM of the tested metal ions.

2.4. Quantification of ZEN

ZEN quantification was performed using an HPLC system (S3000, Acchrom Tech, Shanghai, China) equipped with a fluorescence detector (λex = 274 nm, λem = 440 nm). Separation was achieved on a reversed-phase BST Rutin C18 BD column (5 μm, 4.6 × 150 mm) with an acetonitrile-water (60:40, v/v) mobile phase at a flow rate of 1 mL min^−1^. The linearity of the calibration curve was assessed using ZEN standard solutions across the concentration range of 0.05–5 μg mL^−1^ (0.05, 0.10, 0.25, 0.5, 1.0, 2.5, and 5 μg mL^−1^). Following the addition of methanol (1:1, v/v) and vortexing for 30 s, the samples were centrifuged at 12,000 rpm for 10 min. The clarified supernatant was subsequently filtered using a 0.22 μm RC filter and injected into the HPLC system for analysis.

2.5. Identification of ZEN Degradation Products

The ZEN degradation products generated by CuTA nanozyme catalysis were identified using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS; Acquity H-Class UPLC system with Xevo G2-S QTOF, Waters, Milford, CT, USA). Separation was performed on an Acquity HSS T3 C18 column (1.7 μm, 2.1 × 100 mm) at 40 °C. The mobile phase comprised (A) 0.1% formic acid in deionized water and (B) acetonitrile, flowing at 0.3 mL min^−1^. The gradient elution program was as follows: 0–1 min, 5% B; 1–10 min, linear increase to 95% B; 10–13 min, hold at 95% B; 13–14 min, return to 5% B; 14–17 min, hold at 5% B for column re-equilibration. Mass spectrometry analysis was performed in negative electrospray ionization (ESI^−^) mode. Data acquisition covered the m/z range of 50–1000 Da.

2.6. Hepatotoxicity Assessment of ZEN Degradation Products

The hepatotoxicity of ZEN and its degradation products generated by CuTA nanozyme catalysis was assessed using the human hepatocyte cell line L-02 (Tongpai biotechnology Co., Ltd., Shanghai, China). The L-02 cells were routinely cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C in a humidified 5% CO_2_ incubator. A CCK-8 assay was performed to determine cell viability. Briefly, L-02 cells (1 × 10 ^4^ cells/well in 96-well plates) were exposed to 200 μM of either ZEN or its degradation products for 24 h. After treatment, 10 μL of CCK-8 reagent was added to each well, followed by a 1 h incubation and absorbance measurement at 450 nm using a microplate reader (Thermo Fisher Scientific, Delaware, DE, USA). Membrane integrity markers, including lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities in the culture supernatant were measured using commercial assay kits. Intracellular reactive oxygen species (ROS) levels were detected using the fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA). Cells were incubated with 10 μM of DCFH-DA for 30 min at 37 °C, washed with PBS, and imaged using a fluorescence microscope. Fluorescence intensity was quantified using ImageJ software (version 1.8.0). Cell apoptosis and necrosis were analyzed by flow cytometry (CytoFLEX, Beckman Coulter, Brea, CA, USA) using a dual-staining approach with Annexin V-FITC and propidium iodide (PI), following the manufacturer’s protocol. Cells were categorized as viable (Annexin V^−^/PI^−^), early apoptotic (Annexin V^+^/PI^−^), late apoptotic (Annexin V^+^/PI^+^), or necrotic (Annexin V^−^/PI^+^).

2.7. Estrogenicity Assessment of ZEN Degradation Products

The estrogenic activity of ZEN and its CuTA nanozyme-derived degradation products was investigated using the standardized E-screen bioassay with estrogen-sensitive MCF-7 cells (Tongpai biotechnology Co., Ltd., Shanghai, China). The ZEN degradation products were prepared by incubating 100 μM ZEN with 60 mg mL^−1^ CuTA nanozyme in a 10 mL reaction system for 48 h to achieve complete degradation of ZEN. The ZEN degradation products were extracted twice using an equal volume of ethyl acetate. The extracted ZEN degradation products were dried under a nitrogen stream at 40 °C and then dissolved in 10 mL phenol red-free DMEM/F-12 medium containing 10% FBS for subsequent studies. MCF-7 cells were maintained in phenol red-free DMEM/F-12 medium supplemented with 10% charcoal-stripped FBS to eliminate potential estrogenic interference from media components. Prior to experiments, cells were synchronized by serum starvation for 24 h. For the E-screen bioassay, cells were seeded in 96-well plates (3000 cells/well) and allowed to adhere for 24 h before exposure to varying concentrations of ZEN and its degradation products. After 72 h of exposure, cell proliferation was assessed via the CCK-8 assay. The proliferative effect (PE) was calculated using the formula: PE = (A450 test − A450 blank)/(A450 control − A450 blank). Dose-PE curves were fitted using logistic regression (GraphPad Prism v9.0), with the half-maximal effective concentration (EC50) derived from sigmoidal modeling.

2.8. Performance of CuTA Nanozyme for ZEN Elimination in Corn Oil

Corn oil was spiked with ZEN (500 ng mL^−1^) and treated with 2 mg mL^−1^ of CuTA nanozyme at 37 °C with shaking at 180 rpm. Samples were collected at 3, 6, 9, and 12 h. The extraction and purification of ZEN from corn oil were conducted according to the following procedure. Briefly, 10 mL of corn oil was vigorously mixed with 100 mL of acetonitrile/water (9:1, v/v) solution. After centrifugation at 3000 rpm for 5 min, a 10 mL aliquot of the acetonitrile phase was collected and diluted with 40 mL of phosphate buffer solution containing Tween-20. The resulting mixture was then filtered through a 110 mm glass microfiber filter. A 10 mL portion of the filtrate was subsequently passed through an immunoaffinity column (AokinImmunoClean CF ZEN, Aokin, Ansbach, Germany) at a controlled flow rate. The column was washed, and ZEN was eluted with 1 mL of methanol into a glass tube for subsequent quantification by HPLC. For reusability testing, the CuTA nanozyme was recovered by centrifugation and reused for four consecutive cycles.

2.9. Statistical Analysis

Data were analyzed using SPSS V20.0 software. The one-way ANOVA followed by Tukey’s HSD test was applied, with a p-value < 0.05 considered statistically significant.

3. Results and Discussion

3.1. Synthesis of Laccase-Mimicking CuTA Nanozyme and Test for Its Ability to Degrade ZEN

The CuTA nanozyme was synthesized via oxidative coupling assembly. The freeze-dried product exhibited a uniform dark brown appearance (Figure 1A), and transmission electron microscopy (TEM) revealed that the CuTA nanozyme possessed a sheet-like nanostructure (Figure 1B), consistent with previous reports on tannic acid-copper coordination complexes [17]. To confirm the laccase-mimicking activity of the CuTA nanozyme, we employed a chromogenic reaction using 2,4-DP and 4-AP. As shown in Figure 1C, the CuTA nanozyme catalyzed the oxidation of 2,4-DP, resulting in a distinct red product, whereas no color change was observed in the absence of the nanozyme. This confirmed the successful synthesis of a laccase-like catalyst, in agreement with previous studies on copper-based nanozymes [14,15]. The ZEN-oxidizing capability of the CuTA nanozyme was further investigated using HPLC analysis. The retention time for ZEN was 7.5 min, and there was an approximately 80% reduction in the peak area of ZEN following incubation with the CuTA nanozyme (Figure 1D). These findings substantiate the catalytic role of the CuTA nanozyme in the degradation of ZEN.

3.2. Characteristics of ZEN Degradation by CuTA Nanozyme

We further characterized the effects of incubation time, pH, temperature, and metal ions on the degradation of ZEN by the CuTA nanozyme. Figure 2A demonstrates the time-dependent manner of ZEN degradation by the CuTA nanozyme, where the degradation percentage increased with prolonged reaction time. Notably, the maximum degradation rate of 81.6% was attained with 20 mg mL^−1^ of the CuTA nanozyme after incubation for 24 h. The pH-dependent activity of the CuTA nanozyme revealed optimal ZEN degradation at pH 7.0, with a significant decline observed under acidic (pH < 5.0) or strongly alkaline (pH > 8.0) conditions (Figure 2B). This neutral pH preference distinguishes the CuTA nanozyme from fungal laccases, which typically exhibit peak activity at pH 3.0–4.0 [18,19]. The temperature profile indicated that the ZEN degradation efficiency increased from 49% at 22 °C to 94% at 42 °C, remaining stable up to 57 °C (Figure 2C). The optimal temperature for the CuTA nanozyme was comparable to that of the laccase Lac2 (55 °C) from Pleurotus pulmonarius [19], but lower than that of the CotA laccase (80 °C) from Bacillus licheniformis [7]. Metal ion interference represents a critical limitation for natural enzyme catalysis, as these ions may induce structural perturbations at the active site of natural enzymes. Mwabulili et al. [20] documented a >60% reduction in ZEN degradation efficiency by laccase BswLac when exposed to 10 mM of Fe^2+^, Zn^2+^, or Cu^2+^. On the contrary, Qin et al. [21] reported that copper ions acted as redox mediators to boost laccase activity. Our findings reveal a distinct advantage of the CuTA nanozyme. As shown in Figure 2D, the catalytic performance of the CuTA nanozyme remained unaffected by various metal ions. This metal-independent catalytic behavior significantly enhances the suitability of the CuTA nanozyme for deployment in challenging food processing environments, where ion interference typically compromises enzymatic efficacy.

3.3. Identification of ZEN Degradation Products

The degradation products of ZEN by the CuTA nanozyme were analyzed by UPLC-MS/MS to clarify the oxidative transformation pathway. ZEN (C_18_H_22_O_5_) displayed a parent ion at m/z 317.1522 [M-H]^−^, which generated daughter ions at m/z 273.1609, m/z 187.0484, m/z 175.0479, m/z 160.0234 and m/z 131.0559 (Figure 3A). These daughter ions matched the MS/MS fragments of ZEN reported by Yang et al. [22]. Two oxidative degradation products were identified, each exhibiting a mass increase of 16 Da relative to ZEN. The hydroxylation at the C13 position of ZEN generated 13-hydroxy-zearalenone (13-OH-ZEN, m/z 333.1403 [M-H]^−^, C_18_H_22_O_6_), yielding daughter ions at m/z 291.1649, m/z 219.1050, m/z 161.0646 and m/z 149.0641 (Figure 3B). This compound is the main intermediate degradation product of ZEN by laccases [7,23]. Moreover, the hydroxylation at the C15 position of ZEN formed 15-hydroxy-zearalenone (15-OH-ZEN, C_18_H_22_O_6_), which exhibited a parent ion at m/z 333.1349 [M-H]^−^ and produced characteristic daughter ions at m/z 289.1412 and m/z 191.0350 (Figure 3C). The MS/MS profile of 15-OH-ZEN aligned with the results reported by Jia et al. [23] and Qin et al. [24]. Based on MS/MS analysis, the products of the CuTA nanozyme-catalyzed oxidation of ZEN were tentatively assigned as 13-OH-ZEN and 15-OH-ZEN. Further purification of these compounds, along with definitive structural elucidation by nuclear magnetic resonance spectroscopy, is necessary. Hydroxylation at the C13/C15 positions represents a distinct detoxification pathway compared to microbial lactonases, which cleave the macrocyclic structure of ZEN [9]. However, 13-OH-ZEN and 15-OH-ZEN are typically generated through the enzymatic oxidation of ZEN by peroxidases or laccases [18,24]. This structural modification likely reduces the estrogenic activity of ZEN by sterically hindering receptor binding [25,26].

3.4. Hepatotoxicity Assessment of ZEN Degradation Products

The feasibility of the CuTA nanozyme in degrading ZEN is contingent not only upon its catalytic efficiency but also on the biosafety of the resultant metabolites. The liver is the main organ responsible for both the bioactivation and pathological effects of ZEN [27]. Accumulating evidence indicates that ZEN induces hepatotoxicity primarily through oxidative stress-mediated mitochondrial dysfunction and activation of apoptotic pathways [28,29]. This study employed human hepatocytes L-02, known for their robust liver function [30], to evaluate the hepatotoxicity of the degradation products resulting from CuTA nanozyme-catalyzed degradation of ZEN. As depicted in Figure 4A, L-02 cells exposed to 200 μM ZEN exhibited a 47% reduction in viability. Conversely, CuTA-pretreated ZEN (ZEN + CuTA) restored viability to control levels. Hepatocyte damage enhances membrane permeability, causing the leakage of intracellular enzymes such as LDH, ALT, and AST. Thus, the activities of these enzymes are crucial indicators for evaluating cellular damage. As demonstrated in Figure 4B–D, ZEN increased extracellular LDH, ALT, and AST activities by 2.7–3.8-fold, whereas ZEN + CuTA maintained enzyme leakage at levels comparable to the control group, indicating preserved membrane integrity. Intracellular ROS levels in ZEN-treated cells surged 12.8-fold (Figure 4E,F), aligning with ZEN’s documented role in NADPH oxidase activation [31]. In contrast, CuTA-derived ZEN metabolites showed no elevation in ROS, likely due to attenuated redox cycling resulting from steric hindrance introduced by hydroxyl groups [25]. Flow cytometry revealed that ZEN triggered both apoptosis and necrosis (Figure 4G,H). This dual cell death pathway correlates with ZEN’s established activation of p53-dependent mitochondrial apoptosis [29,31]. In contrast, L-02 cells exposed to ZEN degradation products maintained apoptosis and necrosis rates within physiological ranges. Thus, the CuTA nanozyme-catalyzed degradation of ZEN resulted in the detoxification of its hepatotoxic effects. Consistently, Sun et al. [7] observed that ZEN decreased the viability of human gastric mucosa epithelium (GES-1) cells, in contrast to its metabolite 15-OH-ZEN, which showed no significant effect.

3.5. Estrogenicity of ZEN Degradation Products

The endocrine-disrupting effects of ZEN stem from its structural mimicry of 17β-estradiol (E2), enabling competitive binding to estrogen receptors [3]. The E-screen bioassay measures the increased proliferation of target cells in response to estrogenic compounds [32]. The human breast cancer cell line MCF-7, which expresses both ER subtypes (ERα and ERβ), is commonly utilized in the E-screen bioassay [33]. In the present study, the E-screen assay revealed that the degradation products exhibited significantly reduced estrogenic activity compared to ZEN. The proliferative effect (PE) induced by varying concentrations of ZEN and its degradation products was modeled using a logistic equation (Figure 5). The EC50 value for ZEN and the CuTA nanozyme-catalyzed degradation products was 14.3 nM and 624.7 nM, respectively. These findings indicate that the estrogenic activity of ZEN was reduced by 43.7-fold following treatment with the CuTA nanozyme. Consistently, available evidence suggests that 13-OH-ZEN and 15-OH-ZEN are less estrogenic than the parent ZEN molecule. The key reason for their reduced estrogenicity is that hydroxylation at the C13 and C15 positions impairs efficient binding of ZEN to estrogen receptors [25,26]. While 13-OH-ZEN and 15-OH-ZEN are considered less hazardous individually, their behavior in complex biological systems warrants further investigation.

3.6. Performance of CuTA Nanozyme for ZEN Elimination in Corn Oil

ZEN is commonly found in various grains and agricultural products, particularly corn. Contamination can occur at multiple stages, including cultivation, harvesting, storage, transportation, and processing, especially under favorable environmental conditions. During the industrial pressing process, corn gum is particularly susceptible to fungal infection and subsequent ZEN contamination in corn oil. Although some attempts have been made to degrade ZEN, effective strategies for its degradation in corn oil remain limited. Zhou et al. [34] utilized immobilized zearalenone lactonase to degrade ZEN in oil, achieving approximately 30% degradation. Figure 6A depicts the time-dependent efficiency of ZEN elimination from corn oil by the CuTA enzyme. The ZEN content decreased by 17%, 39%, 68%, and 82% after 3, 6, 9, and 12 h, respectively. A notable advantage of nanozymes over biological enzymes is their reusability. In the present study, the removal rates of ZEN in corn oil by the CuTA nanozyme over four reuse cycles were 82%, 79%, 72%, and 58%, respectively (Figure 6B). These results indicate that the CuTA nanozyme demonstrates excellent operational stability across consecutive cycles. While this study indicates that the CuTA nanozyme has potential as a commercial catalyst for ZEN detoxification in corn oil, the performance of the CuTA nanozyme in other complex food matrices requires further validation. Moreover, additional research is necessary to implement this innovative catalyst on a large scale.

4. Conclusions

In summary, this study demonstrated that the CuTA nanozyme, exhibiting laccase-like activity, is capable of directly oxidizing ZEN. UPLC-MS/MS analysis tentatively identified 13-OH-ZEN and 15-OH-ZEN as the degradation products. Cytotoxicity assays demonstrated that the hydroxylation of ZEN by the CuTA nanozyme effectively reduced its hepatotoxicity. The E-screen bioassay detected a 43.7-fold decrease in the estrogenic activity of the ZEN degradation products. The detoxification effects were primarily evaluated in vitro using cell models, while in vivo studies are needed to confirm the reduced hepatotoxicity and estrogenicity in animal systems. Corn oil treated with the CuTA nanozyme showed an 82% reduction in ZEN concentration. Moreover, the CuTA nanozyme demonstrated reusability, maintaining a ZEN degradation efficiency of approximately 58% after four consecutive cycles. Overall, our findings suggest that the CuTA nanozyme is an effective catalyst for the removal of ZEN from corn oil; however, further efforts are necessary to bridge the gap between its laboratory-scale application and industrial-scale implementation.