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Article

Enzymatic Oxidation of Aflatoxin M1 in Milk Using CotA Laccase

by
Yongpeng Guo
1,
Hao Lv
1,
Zhiyong Rao
1,
Zhixiang Wang
1,*,
Wei Zhang
1,
Yu Tang
2 and
Lihong Zhao
2,*
1
College of Animal Science and Technology, Henan Agricultural University, Zhengzhou 450046, China
2
College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
*
Authors to whom correspondence should be addressed.
Foods 2024, 13(22), 3702; https://doi.org/10.3390/foods13223702
Submission received: 22 October 2024 / Revised: 15 November 2024 / Accepted: 15 November 2024 / Published: 20 November 2024
(This article belongs to the Special Issue Advances in the Detection, Characterization and Reduction of Mycotoxins in Food)
Browse Figures
Figure 1
<p>Enzymatic properties and kinetics of CotA laccase-mediated AFM<sub>1</sub> oxidation. Effect of pH (<b>A</b>), temperature (<b>B</b>), and metal ions (<b>C</b>) on CotA laccase-mediated AFM<sub>1</sub> oxidation. (<b>D</b>) Michaelis–Menten plot of CotA laccase-catalyzed AFM<sub>1</sub> oxidation.</p> "> Figure 2
<p>Identification of CotA laccase-mediated AFM<sub>1</sub> oxidation products. (<b>A</b>) HPLC chromatograms of AFM<sub>1</sub> and CotA laccase-mediated AFM<sub>1</sub> oxidation products. (<b>B</b>) Mass spectra analysis of AFM<sub>1</sub> and CotA laccase-mediated AFM<sub>1</sub> oxidation products. (<b>C</b>) The reaction scheme for AFM<sub>1</sub> oxidation by CotA laccase.</p> "> Figure 3
<p>Molecular docking analysis of AFM<sub>1</sub> with CotA laccase. (<b>A</b>) The two-dimensional interaction model of AFM<sub>1</sub> with CotA laccase. (<b>B</b>) The three-dimensional interaction model of AFM<sub>1</sub> with CotA laccase.</p> "> Figure 4
<p>Evaluation of the cytotoxic effects of AFM<sub>1</sub> and its oxidation products. (<b>A</b>) Viability of L-02 cells following exposure to 100 μΜ of AFM<sub>1</sub> and CotA laccase-catalyzed AFM<sub>1</sub> oxidation products. (<b>B</b>) LDH activity. (<b>C</b>,<b>D</b>) Apoptosis rate of L-02 cells. Different letters denote statistically significant differences between groups (<span class="html-italic">p</span> &lt; 0.05).</p> "> Figure 5
<p>Elimination of AFM<sub>1</sub> in milk by CotA laccase. Effect of CotA laccase amount (<b>A</b>) and incubation time (<b>B</b>) on AFM<sub>1</sub> degradation rate in skim milk and whole milk.</p> ">
Versions Notes

Abstract

:
Aflatoxin M1 (AFM1) in milk poses a significant threat to human health. This study examined the capacity of Bacillus licheniformis CotA laccase to oxidize AFM1. The optimal conditions for the CotA laccase-catalyzed AFM1 oxidation were observed at pH 8.0 and 70 °C, achieving an AFM1 oxidation rate of 86% in 30 min. The Km and Vmax values for CotA laccase with respect to AFM1 were 18.91 μg mL−1 and 9.968 μg min−1 mg−1, respectively. Computational analysis suggested that AFM1 interacted with CotA laccase via hydrogen bonding and van der Waals interactions. Moreover, the oxidation products of AFM1 mediated by CotA laccase were identified as the C3-hydroxy derivatives of AFM1 by HPLC-FLD and UPLC-TOF/MS. Toxicological assessment revealed that the hepatotoxicity of AFM1 was substantially reduced following oxidation by CotA laccase. The efficacy of CotA laccase in removing AFM1 in milk was further tested, and the result showed that the enzyme agent achieved an AFM1 removal rate of 83.5% in skim milk and 65.1% in whole milk. These findings suggested that CotA laccase was a novel AFM1 oxidase capable of eliminating AFM1 in milk. More effort is still needed to improve the AFM1 oxidase activity of CotA laccase in order to shorten the processing time when applying the enzyme in the milk industry.

1. Introduction

Milk is commonly recognized as a fundamental food product that provides a comprehensive range of readily accessible and bioavailable nutrients crucial for human growth, development, and health maintenance. Moreover, the consumption of milk products is correlated with a decreased risk of cardiovascular diseases, notably stroke [1]. However, the food hygiene of milk products is often compromised by aflatoxin M1 (AFM1), which has been a great concern for consumers. AFM1, known as a milk toxin, is the hydroxylated form of aflatoxin B1 (AFB1), which is generated by the hepatic mitochondrial cytochrome P450 enzymes and excreted in the milk, feces, and urine of lactating animals after the consumption of an AFB1-contaminated diet. AFM1 appears in dairy cow milk within 12 h of the first AFB1 administration and clears from milk after a 72 h AFB1 withdrawal period [2,3]. The carry-over rate of dietary AFB1 to milk AFM1 in dairy cattle ranges between 0.3 and 6.2% depending on the health conditions, milk yield, feed type, and level of contamination [4]. AFM1 remains stable during various milk processing methods, including sterilization, pasteurization, and fermentation, and its presence has been documented throughout the entire dairy supply chain, encompassing products such as milk powder, cheese, and yogurt [5]. The prevalence of AFM1 in milk poses significant health risks, particularly for vulnerable populations such as infants and the elderly, who may be more susceptible to its harmful effects. Although AFM1 has been found to be 10-fold less carcinogenic than AFB1, IARC continues to classify it as a class 1 carcinogen [6]. Given the potential health risks of AFM1, over 60 countries have developed guidelines for the maximum residue level of milk AFM1. Among these, 34 countries, including China, have set the limit at 0.5 µg L−1 in fluid milk. In contrast, the European Union has adopted a more stringent maximum level at 0.05 µg L−1 for AFM1 in raw milk and milk intended for the production of dairy products.
The development of efficient and environmentally sustainable methods to reduce AFM1 contamination in milk is urgently needed. Milk contamination can be reduced either indirectly, by preventing dairy animals’ dietary exposure to AFB1, or directly, by eliminating AFM1 from contaminated milk. The implementation of good agricultural practices (GAPs), such as crop rotation, soil management, insect damage control, the selection of fungal-resistant crop varieties, and timely harvesting, is helpful for the inhibition of mold infection and AFB1 generation [7]. Unfortunately, these pre-harvest prevention strategies are not consistently sufficient to produce AFB1-free crops. To mitigate the carry-over of feed-derived AFB1 to milk AFM1, dairy farming commonly employs organic and inorganic adsorbents like yeast cell wall extracts [8], calcium montmorillonite clay [9], and hydrated sodium calcium aluminosilicates [10], which can bind AFB1 within the gastrointestinal tract of animals. Moreover, several reports documenting the capacity of yeasts and lactic acid bacteria to bind AFM1 in contaminated milk are available [11,12,13]. Despite favorable outcomes of microbial cells in milk AFM1 elimination, the commercialization of adsorption-associated technologies still has limitations, like the instability of microbial cell–AFM1 complexes, the increase in microbial loads, and the loss of nutritional value [14].
In recent years, research emphasis has gradually shifted toward the microbial and enzymatic degradation of aflatoxins in food commodities, which transforms aflatoxins into less harmful metabolites, while preserving the palatability and nutritional value of food and feed. A number of AFB1-degrading fungal and bacterial strains have been identified, including Pleurotus eryngii [15], Ganoderma sinense [16], Myroides odoratimimus [17], Pseudomonas aeruginosa [18], Cellulosimicrobium funkei [19], and Bacillus licheniformis [20]. Several studies have also documented that AFB1 can be degraded by laccase [21,22], peroxidase [23,24], dipeptidyl peptidase [25], and F420H2-dependent reductase [26]. However, the capacity and application potential of these microorganisms and enzymes for degrading AFM1 have been rarely studied. In this study, we expanded upon previous findings that demonstrated the capacity of CotA laccase to directly oxidize AFB1 without the need for redox mediators, resulting in the formation of aflatoxin Q1 and epi-aflatoxin Q1 [22]. We further investigated the enzymatic characteristics of CotA laccase in oxidizing AFM1. CotA laccase-mediated AFM1 oxidation products were identified. Additionally, we evaluated the toxicity of AFM1 oxidation products using hepatocytes L-02. The interaction between CotA laccase and AFM1 was investigated by molecular docking simulation. Furthermore, the efficacy of CotA laccase in removing AFM1 in both skim milk and whole milk was evaluated for the first time. This work is expected to contribute to the advancement of enzyme-based strategies to mitigate AFM1 contamination in milk products.

2. Materials and Methods

2.1. Materials and Reagents

CotA laccase was expressed and purified from Escherichia coli Rossta (DE3) bearing recombinant expression vector pET31b-CotA as described previously [22]. AFM1 was purchased from Sigma-Aldrich (Shanghai, China). AFM1 immune-affinity columns were obtained from Clover Technology Group, Inc. (Beijing, China). Ultrahigh-temperature-treated (UHT) whole milk and skim milk were purchased from a local supermarket.

2.2. Enzymatic Characteristics of CotA Laccase for Oxidizing AFM1

The enzymatic characteristics of CotA laccase in the oxidation of AFM1 were characterized. To assess the impact of pH on AFM1 oxidation, a reaction mixture comprising 1 μg mL−1 of AFM1 and 0.1 U mL−1 of CotA laccase was incubated at 37 °C across a range of pH conditions (pH 4.0 to 9.0) for 12 h. The impact of temperature on AFM1 oxidation was assessed by incubating 1 μg mL−1 of AFM1 with 0.1 U mL−1 of CotA laccase across a temperature range of 30 to 80 °C for 30 min. Additionally, the influence of metal ions on CotA laccase-mediated AFM1 oxidation was evaluated by pre-incubating 0.5 U mL−1 of CotA laccase with 10 mM of different metal ions at 37 °C and pH 7.0 for 10 min. Subsequently, AFM1 (1 μg mL−1) was introduced, and the mixture was incubated at 37 °C for an additional 30 min. The Michaelis–Menten kinetics of CotA laccase-mediated AFM1 oxidation was analyzed at 37 °C and pH 7.0. Initial reaction velocities were determined by quantifying the reduction in AFM1 concentration in 30 min following the introduction of 0.1 U mL−1 of CotA laccase. The experimental data were fitted to the Michaelis–Menten equation to derive the Km and Vmax values with Graphpad Prism 7.0.

2.3. AFM1 Concentration Determination by HPLC

Chromatographic analysis was conducted utilizing a high-performance liquid chromatography (HPLC) system (Shimadzu LC-10 AT, Shimadzu, Tokyo, Japan), which was equipped with a post-column photochemical derivatization unit and an RF-20A fluorescence detector (Shimadzu, Tokyo, Japan). The separation process employed an isocratic method using a reverse-phase column (DIKMA, C18, 5 μm, 150 × 4.6 mm). The mobile phase consisted of acetonitrile, methanol, and water in a volumetric ratio of 24:8:68. The excitation and emission wavelengths were set at 365 nm and 435 nm, respectively. The flow rate was maintained at 1.0 mL min−1, and the injection volume was 20 μL.

2.4. UPLC-TOF/MS Analysis of CotA Laccase-Mediated AFM1 Oxidation Products

UPLC-TOF/MS was applied to identify CotA laccase-mediated AFM1 oxidation products. Chromatographic separation was performed using an Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm) (Waters, Milford, MA, USA). The mobile phase, delivered at a flow rate of 0.3 mL min−1, comprised a binary solvent system of water and methanol. The elution gradient was as follows: 0–10 min, 5% to 50% methanol; 10–11 min, 50% to 95% methanol; 11–13 min, 95% methanol; and 13–15 min, 95% to 5% methanol. The sample injection volume was set at 2 μL. Mass spectral data were acquired using a Waters Xevo G2-XS QTOF mass spectrometer (Waters, Milford, MA, USA). The TOF-MS was conducted in ESI positive mode. The mass spectrometry parameters were set as follows: ion source temperature at 350 °C, nitrogen gas flow rate at 10 L min−1, capillary voltage at 3000 V, and scan range from m/z 50 to 1000.

2.5. Homology Modeling and Molecular Docking

The three-dimensional structure of B. licheniformis ANSB821 CotA laccase was constructed with the X-ray crystal structure of B. subtilis 168 CotA laccase (PDB entry 2WSD) [27] as a template with the SWISS-MODEL sever. The three-dimensional structure of AFM1 was generated using ChemBioDraw 2014. Molecular docking was conducted using MOE Dock in MOE v2014.0901. The docking procedure adhered to the “induced fit” protocol, which permitted the side chains of the receptor pocket to adjust in response to ligand conformations, while maintaining a positional constraint.

2.6. Cytotoxicity Evaluation of AFM1 Oxidation Products

Human fetal hepatocyte cell line L-02 obtained from Tongpai Biotechnology Co., Ltd. (Shanghai, China) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum and 100 U mL−1 of penicillin and streptomycin. Cell viability was assessed using a CCK-8 assay kit (Biosharp, Beijing, China). L-02 cells were inoculated in a 96-well plate (1 × 104 cells each well), and treated with 100 μM of AFM1 and CotA laccase-catalyzed AFM1 oxidation products for 24 h. Afterwards, 10 μL of CCK-8 reagent was introduced into each well, followed by incubation at 37 °C for 1 h. The absorbance value at 450 nm was taken with a microplate reader to assess cell viability. The extracellular lactate dehydrogenase (LDH) activity and cell apoptosis rate of L-02 cells were also measured after exposure to 100 μM of AFM1 and CotA laccase-catalyzed AFM1 oxidation products for 24 h. LDH activity was determined using a commercially available kit (Biosharp, Beijing, China). The rate of cellular apoptosis was assessed utilizing the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (Uelandy, Suzhou, China).

2.7. Performance of CotA Laccase in Degrading AFM1 in Milk

The experiments regarding the elimination of AFM1 in whole milk and skim milk by CotA laccase were carried out in 100 mL conical flasks. Milk samples were spiked with AFM1 at a concentration of 2.0 ng mL−1. To study the effect of the amount of CotA laccase on AFM1 oxidation, CotA laccase was added to 20 mL of spiked milk to reach a final concentration ranging from 0.1 to 2.0 U mL−1. The control was prepared with sodium phosphate buffer in place of CotA laccase. The samples were incubated at 37 °C for 12 h in a water bath. The effect of incubation time on AFM1 oxidation was determined at 3, 6, 12, 18, and 24 h, respectively, with 1.0 U mL−1 of CotA laccase at 37 °C. All experiments were repeated three times. The concentration of AFM1 in milk was measured with HPLC. Briefly, the milk samples were centrifuged at 6000 rpm for 20 min. Then, 10 mL of supernatant was collected and applied to an AFM1 immune-affinity column at a steady flow rate of 1 mL min−1. The column was washed twice with 10 mL of ultrapure water. AFM1 was eluted from the column with 2 mL of methanol. The eluent was evaporated to dryness at 40 °C under a N2 stream and reconstituted in 1 mL of ultrapure water before loading in the HPLC system. Validation parameters for the determination of AFM1 concentration in milk with the HPLC-FLD method are summarized in Table S1.

3. Results and Discussion

3.1. Enzymatic Properties of CotA Laccase for Oxidizing AFM1

Laccases, a class of multicopper oxidases, possess the ability to oxidize a diverse array of aromatic compounds [28]. Due to their versatility, stability, and broad substrate specificity, numerous applications of laccases have been developed within the food industry over recent decades, including roles in baking, fruit juice clarification, wine and beer stabilization, and sugar beet pectin gelation [29]. Our previous study found that CotA laccase could serve as a novel aflatoxin oxidase [22].
In the current study, we further characterized the enzymatic properties and kinetics of CotA laccase in the oxidation of AFM1. The influence of pH, temperature, and metal ions on CotA laccase-catalyzed AFM1 oxidation was initially examined. The optimal pH range for CotA laccase in oxidizing AFM1 was observed at pH 7.0 to 9.0, with the oxidation rate remaining above 86%, and the highest AFM1 oxidation rate of 94% was obtained at pH 8.0 (Figure 1A). The substrate-dependent optimal pH is a characteristic feature of laccases. Previously, the optimal pH values for B. licheniformis CotA laccase in the oxidation of 2, 2′-Azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine, 2,6-dimethoxyphenol, and AFB1 were determined to be 4.2, 6.8, 7.2, and 8.0, respectively [22]. Therefore, it is essential to determine the optimal pH for the oxidation of various substrates using CotA laccase. The effect of temperature on the oxidation of AFM1 by CotA laccase is illustrated in Figure 1B. The oxidation rate of AFM1 increased from 22% to 86% as the temperature rose from 30 °C to 70 °C, and then dropped to 60% at 80 °C. This observation aligns with the known characteristics of Bacillus CotA laccases, which are recognized as thermophilic enzymes with optimal activity within a temperature range of 50 °C to 70 °C [30]. The influence of various metal ions on the CotA laccase-mediated oxidation of AFM1 was examined, as depicted in Figure 1C. The presence of K+, Li+, and Ba2+ exhibited minimal impact on AFM1 oxidation by CotA laccase, whereas Mn2+ and Ca2+ at 10 mM caused a more than 15% reduction in the AFM1 oxidation rate. Moreover, Cu2+, Zn2+, Co2+, and Ni2+ could strongly inhibit the AFM1-oxidizing activity of CotA laccase. Indeed, the inhibitory effects of Cu2+, Zn2+, Co2+, and Ni2+ are commonly observed in laccases, potentially due to their interaction with the electron transport system of the enzymes [31,32].
The kinetic parameters of CotA laccase with respect to AFM1 were determined by fitting the experimental data to the Michaelis–Menten plot (Figure 1D). The coefficient of determination (R2) was 0.9897, indicating an excellent fit of the model to the data. The Km and Vmax values were calculated to be 18.91 μg mL−1 and 9.968 μg min−1 mg−1, respectively. This strong correlation of the Michaelis–Menten model further implies that AFM1 is a novel substrate of CotA laccase.

3.2. Identification of CotA Laccase-Mediated AFM1 Oxidation Products

CotA laccase-mediated AFM1 oxidation products in sodium phosphate buffer were characterized by HPLC-FLD and UPLC-TOF/MS analysis. The oxidation of AFM1 resulted in the formation of two products with retention times earlier than that of AFM1 (Figure 2A). The oxidation products were further characterized by MS analysis, as shown in Figure 2B and Figure S1. The two products showed the same molecular ion peak at m/z 345.03 ([M + H]+), m/z 367.01 ([M + Na]+) and m/z 382.98 ([M + K]+), corresponding to the formula of C17H12O8, which suggested the addition of a single oxygen to AFM1 (C17H12O7). Aflatoxins can be degraded by several mechanisms, such as epoxidation, hydroxylation, dehydrogenation, and reduction. Research by Loi et al. [21] suggested that treating aflatoxins with Lac2 laccase led to cleavage of the lactone ring, whereas Alberts et al. [33] found that pure laccase of T. versicolor changed the double bond of the furan ring. Moreover, Liu et al. [34] reported that both the furan ring and lactone ring were abolished after treatment by Bacillus pumilus superoxide dismutase.
Our previous study confirmed that CotA laccase catalyzed the C3-hydroxylation of AFB1, leading to the formation of a pair of epimers, AFQ1 and epi-AFQ1 [22]. Based on HPLC-FLD and UPLC-TOF/MS analysis in this study, the same degradation pathway was proposed for AFM1, as shown in Figure 2C. The two transformation products were named aflatoxin N1 (AFN1, 3S) and epi-aflatoxin N1 (epi-AFN1, 3R). Further study is necessary to confirm their chemical structures using nuclear magnetic resonance spectroscopy.

3.3. Interaction of AFM1 with CotA Laccase by Molecular Docking

A docking simulation study was conducted to examine the interaction of AFM1 with CotA laccase. The docking score for the binding mode of AFM1 with CotA laccase was calculated to be −5.50 kcal mol−1 (Figure 3). The binding free energy was negative, reflecting that AFM1 oxidation by CotA laccase was spontaneous. The main interaction observed between AFM1 and CotA laccase was hydrogen bonding. Specifically, the two carbonyl oxygen atoms of the lactone group in AFM1, acting as hydrogen bond acceptors, formed hydrogen bonds with the backbone of Gly321 and Gly322, respectively. Additionally, AFM1 provided good van der Waals interaction with Ile260, Ile318, Cys320, and Ile417. Liu et al. [35] also reported that hydrogen bonding was the primary driving force for the binding between aflatoxins (AFB1, AFB2, AFG1, and AFG2) and Trametes sp. C30 laccase. The in silico investigation conducted by Dellafiora et al. [36] indicated distinct binding configurations of AFB1 and AFM1 within the catalytic sites of laccases derived from Trametes versicolor. The hydroxyl group on the difuran moiety of AFM1 enabled a more profound penetration into the catalytic pocket of T. versicolor laccases, attributed to the establishment of an additional hydrogen bond within the AFM1–laccase complex. However, we found that the simulated binding model of AFM1 to CotA laccase was the same as the AFB1-CotA interaction model [22], which was in agreement with the finding in this study that AFB1 and AFM1 underwent same molecular modification by CotA laccase.

3.4. Hepatotoxicity Evaluation of AFM1 Oxidation Products

The liver, being the main organ for mycotoxin metabolism, is primarily impacted by AFM1 toxicity. A previous study has documented that AFM1 exposure results in liver damage, characterized by poor cytoplasmic integrity, inflammatory infiltration, sinusoidal dilation, and nuclear irregularities [37]. Thus, this study assessed the cytotoxicity of AFM1 oxidation products using hepatocytes L-02. As shown in Figure 4A, the viability reduced remarkably following exposure to 100 μΜ of AFM1 for 24 h. Conversely, treatment with 100 μΜ of CotA laccase-mediated AFM1 oxidation products did not notably impact cell viability. The detrimental impact of AFM1 on L-02 cells was corroborated by a notable increase in the extracellular LDH activity of the AFM1 group (Figure 4B). LDH, a cytoplasmic enzyme, is rapidly released into the culture medium upon cytomembrane leakage, serving as an indicator of cell damage. Consistent with the result of cell viability, no significant difference was observed in extracellular LDH activity between the group treated with AFM1 oxidation products and the control group. Furthermore, flow cytometry analysis indicated a significant increase in cell apoptosis rate in the AFM1 group compared to the control, whereas the AFM1 oxidation products did not induce cell apoptosis (Figure 4C,D). Collectively, these findings suggest that the CotA laccase-mediated oxidation of AFM1 effectively detoxifies its hepatotoxic effects.

3.5. Performance of CotA Laccase to Degrade AFM1 in Milk

The performance of CotA laccase to eliminate AFM1 in milk was further investigated. Whole milk and skim milk spiked with AFM1 at a concentration of 2 ng mL−1 were subjected to treatment with CotA laccase. The impact of the amount of CotA laccase on AFM1 elimination is shown in Figure 5A. The AFM1 removal percentage increased with the increase in CotA laccase from 0.1 to 2.0 U mL−1, and the maximum removal rates were 83.5% and 65.1% for skim milk and whole milk, respectively.
As summarized in Table 1, several research efforts have been dedicated to the enzymatic elimination of AFM1 in milk. Marimón Sibaja et al. [38] reported that commercial horseradish peroxidase (HRP) could reduce AFM1 content in UTH milk by 65.0%. Kerstner et al. [39] used crude extract of peroxidase from rice bran to eliminate AFM1 in milk and achieved the removal percentage of 71.2%. However, H2O2 was required for the oxidation of AFM1 by peroxidases. With respect to large-scale milk processing, CotA laccase has the advantage of directly oxidizing AFM1 by using O2 as an electron acceptor. The food matrix can exert a significant influence on the performance of enzymes in food processing. The AFM1 elimination efficiency of CotA laccase was significantly reduced by fat and fat-soluble vitamins in whole milk.
The impact of incubation time on the elimination of AFM1 is given in Figure 5B. The removal percentage of AFM1 in milk by CotA laccase increased with increasing incubation time, which reached 80.9% and 59.3% for skim milk and whole milk, respectively, at 37 °C after 24 h. Procedures possessing high applicable potential for the elimination of AFM1 in milk should meet a set of criteria including safety, rapidity, and selectivity [43]. The negative impact of CotA laccase treatment on the nutritive value, organoleptic profiles, and technological properties of milk should be further investigated.

4. Conclusions

This study presents the first characterization of the enzymatic properties and kinetics of CotA laccase in oxidizing AFM1. The optimal oxidation of AFM1 by CotA laccase was observed at pH 8.0 and 70 °C. Molecular docking simulations suggested that AFM1 could interact with CotA laccase via hydrogen bonding and van der Waals interactions. The CotA-AFM1 complex formed two hydrogen bonds via the carbonyl oxygen atoms and the Gly321 and Gly322 residues. Moreover, HPLC-FLD and UPLC-TOF/MS analysis indicated that CotA laccase oxidized AFM1 to its C3-hyhroxy derivatives AFN1 and epi-AFN1. Furthermore, the oxidation of AFM1 catalyzed by CotA laccase was observed to significantly reduce its cytotoxicity in hepatocytes. Subsequently, CotA laccase was employed to eliminate AFM1 in milk, with the removal percentage being influenced by factors such as milk type, CotA laccase concentration, and incubation duration. It was shown that 83.5% of AFM1 in skim milk and 65.1% of AFM1 in whole milk were eliminated by 2 U mL−1 of CotA laccase at 37 °C after 12 h. This study highlights the AFM1 oxidase activity of CotA laccase. Nonetheless, further research is necessary to increase the AFM1 removal efficiency of CotA laccase in milk by enzyme engineering technology.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods13223702/s1, Figure S1: (A) Selected ion chromatography of AFM1; (B) Selected ion chromatography of CotA laccase-mediated AFM1 oxidation products. Table S1. Validation parameters for the determination of AFM1 concentration in milk with HPLC-FLD method.

Author Contributions

Conceptualization, Y.G. and L.Z.; data curation, Y.G.; formal analysis, Y.T.; funding acquisition, Y.G.; investigation, W.Z.; project administration, Y.G., H.L. and Y.T.; supervision, L.Z.; validation, H.L. and Z.R.; visualization, Z.W.; writing—original draft, Y.G.; writing—review and editing, Z.W. and L.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Science and Technology Project of Henan Province (242102111006), China Postdoctoral Science Foundation (2023M730998), and Young Talents Fund of Henan Agricultural University (30501327).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article and Supplementary Materials; further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Foods 13 03702 g001
Figure 1. Enzymatic properties and kinetics of CotA laccase-mediated AFM1 oxidation. Effect of pH (A), temperature (B), and metal ions (C) on CotA laccase-mediated AFM1 oxidation. (D) Michaelis–Menten plot of CotA laccase-catalyzed AFM1 oxidation.
Figure 1. Enzymatic properties and kinetics of CotA laccase-mediated AFM1 oxidation. Effect of pH (A), temperature (B), and metal ions (C) on CotA laccase-mediated AFM1 oxidation. (D) Michaelis–Menten plot of CotA laccase-catalyzed AFM1 oxidation.
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Figure 2. Identification of CotA laccase-mediated AFM1 oxidation products. (A) HPLC chromatograms of AFM1 and CotA laccase-mediated AFM1 oxidation products. (B) Mass spectra analysis of AFM1 and CotA laccase-mediated AFM1 oxidation products. (C) The reaction scheme for AFM1 oxidation by CotA laccase.
Figure 2. Identification of CotA laccase-mediated AFM1 oxidation products. (A) HPLC chromatograms of AFM1 and CotA laccase-mediated AFM1 oxidation products. (B) Mass spectra analysis of AFM1 and CotA laccase-mediated AFM1 oxidation products. (C) The reaction scheme for AFM1 oxidation by CotA laccase.
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Figure 3. Molecular docking analysis of AFM1 with CotA laccase. (A) The two-dimensional interaction model of AFM1 with CotA laccase. (B) The three-dimensional interaction model of AFM1 with CotA laccase.
Figure 3. Molecular docking analysis of AFM1 with CotA laccase. (A) The two-dimensional interaction model of AFM1 with CotA laccase. (B) The three-dimensional interaction model of AFM1 with CotA laccase.
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Figure 4. Evaluation of the cytotoxic effects of AFM1 and its oxidation products. (A) Viability of L-02 cells following exposure to 100 μΜ of AFM1 and CotA laccase-catalyzed AFM1 oxidation products. (B) LDH activity. (C,D) Apoptosis rate of L-02 cells. Different letters denote statistically significant differences between groups (p < 0.05).
Figure 4. Evaluation of the cytotoxic effects of AFM1 and its oxidation products. (A) Viability of L-02 cells following exposure to 100 μΜ of AFM1 and CotA laccase-catalyzed AFM1 oxidation products. (B) LDH activity. (C,D) Apoptosis rate of L-02 cells. Different letters denote statistically significant differences between groups (p < 0.05).
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Figure 5. Elimination of AFM1 in milk by CotA laccase. Effect of CotA laccase amount (A) and incubation time (B) on AFM1 degradation rate in skim milk and whole milk.
Figure 5. Elimination of AFM1 in milk by CotA laccase. Effect of CotA laccase amount (A) and incubation time (B) on AFM1 degradation rate in skim milk and whole milk.
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Table 1. Summary of enzymatic elimination of AFM1 in milk.
Table 1. Summary of enzymatic elimination of AFM1 in milk.
EnzymeOriginReaction ConditionsElimination RateReference
HRPAmoracia rusticana0.015 U mL−1 HRP, 0.08% H2O2, 5 ng mL−1 AFM1, 30 °C for 8 h65.0%[38]
RBPRice bran0.015 U mL−1 HRP, 0.08% H2O2, 5 ng mL−1 AFM1, 4 °C for 24 h71.2%[39]
SODBacillus pumilus1 U mL−1 SOD, 2 μg mL−1 AFM1, 40 °C for 24 h26.0%[34]
CATBacillus pumilus1 U mL−1 CAT, 2 μg mL−1 AFM1, 40 °C for 12 h47.2%[40]
POD1Bacillus pumilus1 U mL−1 POD1, 2 μg mL−1 AFM1, 35 °C for 12 h22.4%[41]
POD2Bacillus pumilus1 U mL−1 POD2, 2 μg mL−1 AFM1, 35 °C for 12 h25.6%
POD3Bacillus pumilus1 U mL−1 POD3, 2 μg mL−1 AFM1, 35 °C for 24 h24.3%
LacTrametes versicolor20 mg mL−1 Lac, 0.5 ng mL−1 AFM1, 25 °C for 80 min32.0%[42]
CotA Bacillus licheniformis2 U mL−1 CotA, 2 ng mL−1 AFM1, 37 °C for 12 h83.5% for skim milk;
65.1% for whole milk		This study
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MDPI and ACS Style

Guo, Y.; Lv, H.; Rao, Z.; Wang, Z.; Zhang, W.; Tang, Y.; Zhao, L. Enzymatic Oxidation of Aflatoxin M1 in Milk Using CotA Laccase. Foods 2024, 13, 3702. https://doi.org/10.3390/foods13223702

AMA Style

Guo Y, Lv H, Rao Z, Wang Z, Zhang W, Tang Y, Zhao L. Enzymatic Oxidation of Aflatoxin M1 in Milk Using CotA Laccase. Foods. 2024; 13(22):3702. https://doi.org/10.3390/foods13223702

Chicago/Turabian Style

Guo, Yongpeng, Hao Lv, Zhiyong Rao, Zhixiang Wang, Wei Zhang, Yu Tang, and Lihong Zhao. 2024. "Enzymatic Oxidation of Aflatoxin M1 in Milk Using CotA Laccase" Foods 13, no. 22: 3702. https://doi.org/10.3390/foods13223702

APA Style

Guo, Y., Lv, H., Rao, Z., Wang, Z., Zhang, W., Tang, Y., & Zhao, L. (2024). Enzymatic Oxidation of Aflatoxin M1 in Milk Using CotA Laccase. Foods, 13(22), 3702. https://doi.org/10.3390/foods13223702

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