Ligand Screening for Enzyme Immobilization Enables Efficient Removal of Aflatoxin B1 in Continuous Flow System

TL;DR

This study improves enzyme stability and activity for removing a harmful toxin from edible oils using a new material design.

Contribution

A ligand screening strategy was used to optimize enzyme immobilization on ZIFs, significantly enhancing activity and stability.

Findings

Lac@ZIFs(IM) achieved 93% AFB1 degradation in 4 hours, 21 times more efficient than free laccase.

Lac@ZIFs(IM) showed 6.6 times higher activity than Lac@ZIF-8 after 20 hours in a continuous flow system.

Using imidazole as a ligand increased enzymatic activity by 2.16 times compared to ZIF-8.

Abstract

Aflatoxin B1 (AFB1) contamination is a significant issue for the safety of edible oils. Biodegradation of mycotoxins represents a green and efficient approach. However, enzymes exhibit low catalytic activity and stability under harsh conditions, leading to rapid deactivation in edible oils. Zeolitic imidazolate frameworks (ZIFs) possess high specific surface areas, tunable pore sizes, and excellent thermal stability. Immobilizing enzymes on ZIFs can address the problem of enzyme inactivation during application. Although the stability of the enzyme can be enhanced after immobilization, the overall enzymatic activity remains limited. To overcome the issues of low catalytic activity and poor cycling stability associated with enzymes immobilized on ZIF-8 using 2-methylimidazole (2-mIM) as the ligand, this study optimized the ZIF structure through a ligand screening strategy. Both encapsulation efficiency and cycling stability were enhanced. This research found that the activity of Lac@ZIFs(IM), which uses imidazole (IM) as the ligand, was 2.16 times that of Lac@ZIF-8. The degradation efficiency of AFB1 reached 93% within 4 h in edible oil using Lac@ZIFs(IM) as the catalyst, which was 21-fold higher than that of free laccase. Lac@ZIFs(IM) exhibited excellent activity in the continuous flow system. After 20 h of continuous reaction, the activity of Lac@ZIFs(IM) was 6.6 times that of Lac@ZIF-8. This study provides a novel approach for the efficient enzymatic degradation of mycotoxins.

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Full text

1. Introduction

Edible vegetable oils are indispensable in daily diets [1,2,3,4,5]. Mycotoxin contamination is a major safety concern for edible oils. Potent carcinogenic mycotoxins such as AFB1, ZEN, and DON are frequently detected in peanut oil. To ensure edible oil safety [6,7,8,9,10,11,12,13,14,15], developing highly efficient and specific AFB1 degradation technologies is crucial. Enzymatic degradation of AFB1 in edible oils offers advantages such as high specificity and mild reaction conditions. Laccase is one of the most widely used enzymes for mycotoxin degradation [16,17,18,19,20,21,22,23,24]. Laccase exhibits significant degradation activity toward AFB1 in the presence of mediators [25,26,27,28,29,30,31]. However, laccase is prone to inactivation and exhibits poor stability in edible oils. Furthermore, electron transfer and hydrolysis reactions are nearly impossible to proceed in edible oils, limiting their application [32,33,34,35].

Enzyme immobilization technology [36,37,38,39,40,41] offers an effective approach to enhance enzyme stability and activity in edible oils. ZIFs, favored for their mild synthesis conditions and excellent biocompatibility, are widely used in enzyme immobilization [42,43,44,45]. ZIF-8, which is formed by the coordination of zinc ions with 2-mIM, is the commonly used carrier for immobilized enzymes [46,47]. Recently, strategies such as defect engineering and ligand screening have been developed for applications in gas adsorption and separation, environmental remediation, and catalysis. The pore size of MOFs and the microenvironment of enzyme catalysis can be tuned by ligand screening. However, the mass transfer resistance and restricted enzyme conformation caused by the microporous structure of ZIF-8 lead to poor enzyme activity. Additionally, enzyme leakage or carrier structural damage often occurs during cycle reactions [48,49,50].

Immobilized enzymes employed in continuous flow systems represent a cutting-edge technology for pollutant treatment and green transformation. This approach delivers high process efficiency, favorable mass and heat transfer, and elevated spatiotemporal yield in biocatalysis. Additionally, enzymes exhibit resilience to harsh conditions and readily recover and are reused in the continuous-flow systems. This approach enables reaction-separation coupling, facilitating automation and scale-up. Reported applications include environmental remediation (e.g., degradation of hydrogen peroxide, antibiotics, phenols), food processing (e.g., cold fog prevention in brewing), and biocatalytic synthesis (e.g., microfluidic chips/enzyme membrane reactors for fine chemical synthesis) [51,52,53,54,55,56]. However, the activity and stability of enzymes in the continuous flow systems suffer from substrate diffusion limitations.

In this study, by modulating the ligand structure, the crystal morphology, pore size, and surface chemistry of ZIFs were effectively adjusted to optimize the enzyme catalytic performance. We systematically selected a series of imidazole ligands with varying substituents and spatial structures, and synthesized a range of Lac@ZIFs composites in situ under mild conditions. Using imidazole ligands as the organic ligand, we created a ZIF with larger pores than ZIF-8. The encapsulation efficiency, relative enzyme activity, and cycling stability of Lac@ZIFs were evaluated. Experimental results demonstrated that the AFB1 degradation rate of Lac@ZIFs(IM) in peanut oil was 22 times higher than that of free laccase. After 20 h of cyclic operation in the continuous flow system, the relative activity of Lac@ZIFs(IM) was 6.6 times that of Lac@ZIF-8. This study provides a novel strategy for constructing highly efficient and stable biocatalysts for mycotoxin degradation.

2. Results and Discussion

2.1. Synthesis of ZIFs and Lac@ZIFs

The organic ligand of ZIFs was screened for constructing a suitable carrier for laccase immobilization to degrade AFB1 in edible oils. Considering the coordination environment and formation mechanism of ZIFs, 2-methylimidazole (Ligand-2) was selected as the standard. A series of structurally analogous imidazole-based ligands was used for synthesizing ZIFs, including imidazole (IM, Ligand-1), 2-methylimidazole (2-mIM, Ligand-2), 2-ethylimidazole (2-eIM, Ligand-3), 2-propylimidazole (2-pIM, Ligand-4), 2,4-dimethylimidazole (2,4-dmIM, Ligand-5), 1H-imidazole-2(3H)-thione (2-tIM, Ligand-6), and 2-methylthiazole (2-mTZ, Ligand-7). Under identical external conditions, a series of Lac@ZIF composites incorporating different ligands was synthesized using a constant metal-to-ligand molar ratio of 1:20.

The synthesized composites were systematically analyzed. To determine the optimal detection wavelength for the Bradford assay, the absorbance of Bovine Serum Albumin (BSA) standard solutions at varying concentrations was measured across different wavelengths. As shown in Figure 1a, the wavelength exhibiting the maximum absorbance change was identified as 595 nm, which was subsequently used to generate the protein standard curve presented in Figure 1b. A comparison of two precursor mixing strategies was investigated. Lac@ZIF composites were synthesized by either premixing the laccase with the ligand solution or with the zinc ion solution for 5 min prior to reaction. The results (Figure 1c) indicate that premixing the enzyme with the metal ions led to significantly higher immobilization efficiency. This enhancement is attributed to the enrichment of positively charged zinc ions on the enzyme surface. These ions may act as nucleation sites for ZIF-8 crystal growth, thereby facilitating the encapsulation of laccase within the framework [57]. The enzyme loading capacity and encapsulation efficiency of laccase within ZIFs synthesized with different ligands were calculated (Figure 1d). While the Ligand-4 system achieved the highest encapsulation efficiency (76.36%), its enzyme loading was relatively low. A comprehensive consideration of both parameters suggests that the Lac@ZIFs complexes formed with Ligand-1, Ligand-3, and Ligand-5 are the most favorable for laccase immobilization. The lower encapsulation observed for Ligand-6 and Ligand-7 may be due to the presence of sulfur atoms within the imidazole rings, which could alter the spatial configuration of the ZIFs and consequently hinder the encapsulation process.

The morphology and microstructure of Lac@ZIF composites formed with different ligands were further analyzed using transmission electron microscopy (TEM) [58], as shown in Figure 2. Composites derived from Ligand-1 and Ligand-2 exhibited well-defined polyhedral crystal structures with uniform particle sizes. This observation indicates that both imidazole and 2-methylimidazole can effectively support the stable formation of a zeolitic imidazolate framework. Specifically, the composite prepared with Ligand-1 (imidazole) displayed larger particle dimensions, suggesting a relatively slow nucleation rate, which allowed for extended crystal growth periods and, consequently, the development of larger particles. In contrast, the composite synthesized with Ligand-2 (2-methylimidazole) featured smaller particles, a result attributed to faster nucleation leading to shorter crystal growth times and the formation of finer particles. In comparison, composites formed with Ligands-3 through 7 presented morphologies that were ambiguous and predominantly amorphous in character. This suggests that the nucleation and crystal growth processes were significantly hindered with these ligands, preventing the development of a well-defined crystalline structure. A comprehensive evaluation considering encapsulation efficiency, enzyme loading capacity, and crystal morphology reveals that Ligand-1 offers distinct advantages over Ligand-2. The Lac@ZIFs synthesized using Ligand-1 not only possess a desirable crystalline architecture but also demonstrate concurrently high values for both encapsulation efficiency and enzyme loading [59].

2.2. Activity Test of Lac@ZIFs

To further evaluate the impact of different ligands on the Lac@ZIF composites, the enzymatic activities of composites modified with various ligands were measured [60]. After adding laccase and its precursors to the substrate for activity assay, the absorbance spectra were scanned as shown in Figure 3a. The most significant change in absorbance was observed at a wavelength of 415 nm, indicating that subsequent activity measurements can be conducted at this wavelength. As shown in Figure 3a, scanning spectroscopy was performed on substrates for activity testing across different precursors. The effects of varying concentrations of the precursors (zinc ions and IM ligand) on enzyme activity were then investigated (Figure 3b,c). The results indicate that zinc ions had a relatively minor inhibitory effect on laccase activity, causing a reduction of approximately 30% compared to the free enzyme, with little variation across the tested concentration range. In contrast, IM exerted a significantly stronger inhibitory effect, reducing the relative activity by about 60%. Furthermore, the inhibitory effect was concentration dependent, with higher ligand concentrations leading to greater activity loss. The above results indicate that ligand screening is a viable strategy to mitigate the negative impact on enzyme activity.

The enzymatic activities of Lac@ZIFs synthesized with the different ligand series are presented in Figure 3d. While composites derived from Ligand-6 and Ligand-7 exhibited relatively high residual activity, their practical application is limited by their low encapsulation efficiency, low enzyme loading capacity, and amorphous structure, which may fail to provide adequate protection for the enzyme under specific operational conditions. Notably, Ligand-1 (IM) residual activity is 38.19%, and Ligand-2 (2-mIM) residual activity is 17.64%. The composite synthesized with Ligand-1 (IM) demonstrated an activity approximately 2.16-fold that of the Ligand-2 (2-mIM) counterpart, representing the optimal performance among the series. This superior activity aligns well with its favorable encapsulation efficiency and well-defined crystalline morphology observed previously. This correlation suggests that the Ligand-1 system not only facilitates effective enzyme immobilization structurally but also that the larger particle size resulting from slower nucleation kinetics may contribute to enhanced enzymatic performance [61,62].

Among the tested ligands, Ligand-1 demonstrated superior performance in the activity assays. Consequently, the Lac@ZIF composite synthesized with Ligand-1 was selected for further characterization [63]. The results of these characterizations are presented in Figure 4. Scanning electron microscopy (SEM) images of pristine ZIF-8 (Figure 4a) and the Ligand-1 derived the Lac@ZIF composite (Figure 4b) reveal uniform particles with an average size of approximately 100 nm. TEM further confirms that the particles possess a regular polyhedral morphology, consistent with the typical structure of ZIF-8. This indicates the successful formation of a stable, porous framework suitable for enzyme encapsulation and immobilization. TEM–energy dispersive spectroscopy (TEM-EDS) analysis of the Lac@ZIF composite (Figure 4c) detected the presence of four primary elements: Zn, C, O, and N. The coexistence of these elements, particularly the nitrogen and oxygen signatures, provides direct evidence for the successful incorporation of laccase into the composite structure. Fourier transform infrared spectroscopy (FTIR) was employed to compare the free enzyme, pristine ZIF-8, and the Lac@ZIFs composite (Figure 4d). A characteristic absorption peak at 1654 cm^−1^, attributed to the C=O stretching vibration within the laccase structure, is present in both the free laccase and the Lac@ZIFs spectra but absent in the spectrum of pristine ZIF-8. This observation confirms the successful loading of laccase within the ZIF-8 framework. Finally, the X-ray diffraction (XRD) pattern of the Lac@ZIF composite (Figure 4e) matches the standard diffraction peaks of ZIF-8. This indicates that the encapsulation of laccase did not alter the inherent crystalline structure of the ZIF-8 host material [64].

Characterization of the porous structure of both ZIFs and the Lac@ZIFs composite synthesized with Ligand-1 (Figure 5a). Following the encapsulation of laccase, the specific surface area and total pore volume decreased significantly from 1638 to 1497 m^2^/g and from 0.608 to 0.558 cm^3^/g, respectively. The pore size distribution curve derived using the SF method (Figure 5b) revealed a bimodal distribution in the Lac@ZIFs composite. Furthermore, the average pore size increased from 1.9 nm for the ZIFs to 2.2 nm after enzyme encapsulation, indicating a slight pore expansion upon the introduction of laccase. The reduction in specific surface area and pore volume stems from the enzyme’s dimensions exceeding the ZIFs’ pore size, resulting in lattice distortion upon enzyme loading [65]. This has a negligible impact on substrate diffusion. The increase in average particle size confirms successful enzyme loading within the ZIFs, whilst the larger pore size facilitates mass transfer for substrates. Overall, enzyme loading enhances substrate diffusion [66].

2.3. Cyclic Activity Test of Lac@ZIFs

To evaluate the reusability of the synthesized materials, consecutive cycling experiments were conducted for eight runs using the Lac@ZIF composites synthesized with Ligand-1 and Ligand-2. The XRD patterns and SEM images of the composites after 1, 4, and 8 cycles are presented in Figure 6a and Figure 6b, respectively. The XRD patterns reveal characteristic diffraction peaks of ZIF-8 at angles such as 7.3°, 10.4°, 12.7°, and 16.5°, corresponding to the (011), (002), (112), and (022) crystal planes, respectively [67]. With increasing cycle numbers, the intensity of these characteristic peaks gradually weakened. SEM observations indicate that the composites underwent slight structural collapse, resulting in a partial loss of their original crystalline architecture. Notably, after eight cycles, the composite derived from Ligand-1 exhibited less structural degradation compared to that from Ligand-2. As shown in Figure 6c, the enzymatic activity of both materials gradually declined with repeated use. For the Lac@ZIF composite formed with Ligand-1, the relative activity remained above 80% during the first five cycles, demonstrating good initial stability. However, a more pronounced decline in activity commenced after the sixth cycle. This accelerated deactivation is likely associated with the leaching of active components. Despite this, the material retained a significant level of reusability, maintaining 62.99% of its initial activity after eight cycles. In contrast, the composite synthesized with Ligand-2 maintained its activity above 80% only for the first three cycles. A sharp decrease in activity was observed from the fourth cycle onward, culminating in a residual activity of merely 33.04% after eight cycles. Therefore, considering both the initial activity advantage and the retained enzymatic activity over cycles, the overall activity of the Lac@ZIFs composite synthesized with Ligand-1 after 8 cycles of regeneration is approximately four times that of the composite prepared with Ligand-2.

The results from the activity assays and XRD characterizations are mutually corroborative. Compared to the traditional Ligand-2 (2-mIM) and other imidazole-based structural analogs investigated, Ligand-1 (IM) demonstrates distinct advantages as a ligand [68]. It effectively ensures a high laccase loading capacity while maintaining the structural integrity of the ZIFs framework. Consequently, the resulting Lac@ZIFs composite exhibits superior encapsulated enzyme activity and enhanced cycling stability. These combined properties make it a more promising material for applications requiring high enzymatic stability.

2.4. Biodegradation of AFB1 in Edible Oil Catalyzed by Lac@ZIFs

Existing studies have demonstrated that laccase can catalyze the oxidation of AFB1 to AFQ1 in aqueous solutions. Toxicological analyses indicate that AFQ1 exhibits negligible toxicity to human hepatocytes. However, due to its low redox potential, laccase demonstrates limited detoxification efficiency against non-phenolic compounds. To address this limitation, mediators are employed to function as an “electron shuttle” for the indirect oxidation of substrates. In this process, laccase catalyzes the reduction of oxygen to water while generating oxidized mediators. These mediators, characterized as long-lived radicals, subsequently facilitate the oxidation of non-phenolic compounds.

ABTS is the most commonly used mediator, but it is not permitted for addition to food. Considering the safety of edible oils, phenolic food additives TBHQ (an antioxidant for oils and fats) and TA (a processing aid in oil refining) were investigated as mediators in the AFB1 degradation process (Figure 7b). When TA was used as a mediator, laccase activity was significantly enhanced, whereas no notable improvement was observed with TBHQ as the mediator. Using 0.5 mM TA as the mediator, the degradation rate of AFB1 reached 53.03% within 12 h, which is 2.46 times and 12.53 times that achieved with ABTS and TBHQ at the same concentration, respectively. Tannic acid is a food additive, as stated in GB 2760-2014. Therefore, TA was subsequently used as the mediator for degrading AFB1 in peanut oil [69].

Detoxification experiments were conducted in edible oil contaminated with AFB1 (200 μg/kg). When Lac@ZIFs(IM) was used as the catalyst, the degradation rate of AFB1 reached 74.7% in 80 min and 93% within 4 h. In contrast, when employing free laccase as a catalyst, AFB1 degradation reached only 4.23% within 4 h, likely due to enzyme denaturation in the oil (Figure 7c).

To further investigate the operational stability of Lac@ZIFs synthesized with ligand-1 (IM), activity tests were performed on Lac@ZIFs(IM) and Lac@ZIF-8. The immobilized enzymes in a continuous flow system were tested for activity every 5 h (Figure 7d). The results showed that after 20 h, Lac@ZIF-8 retained only 10.6% relative activity, whereas Lac@ZIFs(IM) maintained 68.1% relative activity, confirming the superior stability of Lac@ZIFs(IM) in AFB1 degradation. In a continuous flow system, the degradation rate of AFB1 in edible oils by Lac@ZIFs(IM) was one to two orders of magnitude higher than that of free laccase previously reported [70]. The experiments demonstrate that Lac@ZIFs(IM) is an efficient and stable catalyst for degrading mycotoxins in edible oils. Moreover, through a continuous flow system, large-scale degradation can be achieved, indicating promising prospects for practical application.

3. Conclusions

In this study, a ligand screening strategy was successfully employed to optimize Lac@ZIFs composites. The catalytic performance of Lac@ZIFs(IM) for AFB1 degradation in edible oil was significantly enhanced compared to Lac@ZIF-8 synthesized with 2-methylimidazole as the ligand. The nucleation and growth processes of ZIF crystals were effectively modulated when using imidazole as the ligand. This study demonstrated that synthesizing ZIFs with different organic ligands not only altered the crystal morphology of the composite but, more importantly, regulated the pore size distribution of the composite, creating a microenvironment that was more conducive to mass transfer for the bulky substrates of laccase and potentially enhancing multiple interactions between the carrier and enzyme surface. The optimized carrier structure improved immobilization efficiency and enzyme loading capacity. The higher relative embedded activity indicated that the new carrier environment better preserved the native conformation of laccase and the accessibility of its catalytic active sites, thereby reducing activity loss due to the immobilization. In the AFB1 degradation reaction in edible oil, Lac@ZIFs(IM) achieved an efficiency 22-fold that of the free laccase. After 20 h of continuous circulation in a flow-through system, the relative activity of Lac@ZIFs(IM) was 6.6-fold that of Lac@ZIF-8, demonstrating the superior stability of Lac@ZIFs(IM). This study optimized the ZIF structure through a ligand screening strategy, providing a novel pathway for the environmentally friendly degradation of mycotoxins in edible oils.

4. Materials and Methods

4.1. Materials

Laccase (Aspergillus), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), tan-nicacid(TA), and tert-butylhydroquinone(TBHQ) were purchased from Sigma Aldrich, Saint Louis, MO, USA. Imidazole (IM), 2-methylimidazole (2-mIM), 2-ethylimidazole (2-eIM), 2-propylimidazole (2-pIM), 2,4-dimethylimidazole (2,4-dmIM), 1H-imidazole-2(3H)-thione (2-tIM), and 2-methylthiazole (2-mTZ) were purchased from Shanghai Bide Pharmatech Co., Ltd. (Shanghai, China). The Bradford protein assay kit, phosphate buffer (PB, 0.1 mol/L, pH 7.0), Tris-HCl buffer (0.1 mol/L, pH 7.0), and ethylenediaminetetraacetic acid (EDTA, 0.5 mol/L, pH 8.0) solution were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Analytical-grade ethanol, zinc acetate, and phosphate buffer (PB) were sourced from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). AFB1 standard (purity > 99%) was purchased from Fermentek (Jerusalem, Israel). AFB1 standard stock solution (1 mg/mL) was prepared in acetonitrile and stored at 20 °C for future use. Peanut oil samples were obtained via extraction using an oil press (purity > 99%). The peanuts originated from the Huayu No. 25 cultivar developed by the Shandong Provincial Peanut Research Institute (Jinan, China). High-performance liquid chromatography (HPLC)-grade methanol and acetonitrile were purchased from Tedia Company, Inc. (Fairfield, OH, USA) and were filtered through 0.22 μm nylon 6 microporous membranes prior to use.

4.2. Synthesis of ZIFs and Lac@ZIFs

The ligand solutions (IM, 2-mIM, 2-eIM, 2-pIM, 2,4-dmIM, 2-tIM, and 2-mTZ) at specific concentrations (2 mol/L), zinc acetate solution (0.35 mol/L), and laccase solution (5 mg/mL) were prepared. In total, 1.4 mL of ligand solution was added, and a magnetic bead was introduced. Simultaneously, 400 μL of zinc acetate solution and 200 μL of Lac solution (which was replaced with 200 μL of deionized water when ZIF-8 was prepared) were added to a centrifuge tube. The mixture was premixed for 5 min and then transferred to the vial. The reaction was conducted at 400 rpm for 30 min at room temperature. After the reaction was completed, the product was centrifuged and washed twice with deionized water. The washed product was freeze-dried to obtain the final ZIFs or Lac@ZIFs. The protein loading was determined by the Bradford method.

$$[eqn]$$ $$[eqn]$$

where m represents the mass of the laccase initially added; C and V, respectively, correspond to the final laccase concentration and volume in the suspension. M is the weight of the final lac@ZIF-8 composite product.

4.3. Protein Concentration Assay

Protein concentration was determined using the Bradford method. Twenty microliters of protein solution were added to a 96-well plate, followed by 200 μL of 1 × 250 staining solution. After a 4 min reaction, protein concentration was measured using an enzyme-linked immunosorbent assay reader (Tecan Spark, Tecan Group Ltd., Zurich, Switzerland).

4.4. Activity Testing

The enzyme activity of the Lac@ZIFs complex was determined using ABTS as the reaction substrate. The powder was weighed and dissolved in Tris-HCl buffer (50 mmol/L, pH 7.0), with a final concentration of 0.25 mmol/L. In total, 950 μL of the substrate solution was added to 1 mL of the quartz cuvette, and then 50 μL of the enzyme (0.1 mg/mL) solution was added to initiate the reaction. The absorbance of the reaction system at 415 nm changed over time using a Shimadzu UV-2550 ultraviolet–visible spectrophotometer (Kyoto, Japan). The reaction temperature is 25 °C. The change in absorbance was recorded over a 15 s period. All results were compared after conversion according to the dilution ratio.

4.5. Cyclic Activity Assay

The Lac@ZIFs complex was resuspended and centrifuged at 12,000 rpm for 5 min to complete one cycle. After each cycle, the sample was freeze-dried, and its enzyme activity was determined using the method described above.

4.6. Characterization

The microscopic morphology of the Lac@ZIF-8 complex was observed using field emission SEM (JSM 7401, Japan Electron Optics Ltd., Tokyo, Japan). The powder-like compound was evenly applied onto the conductive adhesive and subjected to gold spraying treatment. Observation was performed under an acceleration voltage of 5 kV. The microscopic morphology of the Lac@ZIF-8 complex was also observed using a high-resolution TEM (JEOL M-2010, Japan Electron Optics Ltd., Tokyo, Japan), and energy dispersive X-ray spectroscopy (EDS) was performed to determine the elemental composition of the sample. The test acceleration voltage was set at 120 kV. Physical adsorption and desorption tests (BET) were conducted to explore the influence of specific surface area and pore volume on the embedding and activity of enzymes. XRD (Bruker D8 Advance, Bruker Corporation, Mannheim, Germany) patterns of Lac@ZIF-8 composites were obtained using an X-ray diffractometer (Cu Kα, λ = 0.154 nm), with a test range of 5° to 50° and a scanning speed of 2°/min to study the crystal structure of the composites. The powder samples of the composite were mixed with spectrally pure KBr and pressed into tablets. Infrared spectra of the composite material were acquired using a Fourier transform infrared spectrometer (Nicolet 6700, Thermo Fisher Scientific, Waltham, MA, USA) to investigate its chemical composition.

4.7. Extraction and Determination of AFB1 in Edible Oils

The AFB1 extraction and determination in peanut oil followed the Chinese National Standard (GB5009.96-2016) [71]. Weigh 5 g (±0.01) of peanut oil sample into a 50 mL centrifuge tube. Add 20 mL of methanol–water extraction solvent (volume ratio 7:3) and shake at 2500 r/min for 10 min. Centrifuge at 3500× g and 10 °C for 10 min. Transfer the supernatant through a 0.22 μm nylon microporous membrane filter into a sample vial for subsequent analysis. The aflatoxin B1 (AFB1) content in the sample was determined using high-performance liquid chromatography (Agilent 1260, Agilent Technologies, Inc., Santa Clara, CA, USA), equipped with a fluorescence detector and Agilent C18 column (250 × 4.6 mm, 5 μm particle size). Analytical conditions were as follows: injection volume 20 μL; mobile phase acetonitrile-water (55:45 v/v); flow rate 0.6 mL/min; column temperature 40 °C; excitation wavelength 365 nm; emission wavelength 440 nm. The degradation efficiency of AFB1 in edible oils was calculated using Formula (3).

$$[eqn]$$

where C1 and C2 stand for the concentrations of AFB1 in the untreated and treated peanut oil.

4.8. Lac@ZIFs Mediated Biodegradation of AFB1 in Edible Oil

Contaminated edible oil was prepared by artificially adding AFB1 to peanut oil at a final concentration of 200 μg/kg. One gram of the contaminated edible oil was added to a sample vial along with 1 mL of mediator (0.5 mM), followed by 1 mg of Lac@ZIFs or an equivalent amount of free enzyme.

4.9. Cyclic Degradation Activity Testing

Cyclic degradation activity was tested in a continuous flow apparatus. TA was added at a ratio of 1 mL/g to edible oil containing 200 μg/kg AFB1, then pumped into a continuous-flow system loaded with either Lac@ZIFs(IM) or Lac@ZIF-8 for degradation. Enzyme activity was measured every five hours to evaluate the cyclic performance of the immobilized enzymes.