Biological composite material, preparation method and application thereof, and method for removing mycotoxin in liquid-phase food

Abstract

The invention relates to the technical field of composite materials, and discloses a biological composite material as well as a preparation method and application thereof. The biological composite material contains cordyceps taishanensis and a metal organic framework material, the structure of the metal organic framework material is shown as a formula (I), X is selected from Mn, Fe, Ce, Zn and Cu, and M is selected from at least one of Ti, Zr and Hf. In addition, the invention also discloses a method for removing fungaltoxin in a liquid phase. The biological composite material can effectively degrade fungaltoxin possibly existing in liquid-phase food, and the quality of the food is not affected.

Description

Technical Field

[0001] This invention relates to the field of composite material technology, specifically to a biocomposite material, its preparation method and application, and a method for removing fungal toxins from liquid food. Background Technology

[0002] Mycotoxin contamination in the food chain can have significant adverse effects on human health and cause substantial socioeconomic losses. Ochratoxin A (OTA) is a secondary metabolite, primarily produced by Aspergillus and Penicillium fungi, and is widely found in various foods such as grape products and edible oils, posing a serious threat to human health. Because OTA possesses genotoxic, nephrotoxic, carcinogenic, teratogenic, immunosuppressive, and hepatotoxic properties, it poses a potential safety hazard to edible oils and other foods.

[0003] To date, numerous physical and chemical methods have been devised to reduce the risk of mycotoxin contamination in food. However, simple physical adsorption is insufficient for complete detoxification, while chemical detoxification may damage the nutritional and flavor components of edible oils. Microbial enzymatic methods can effectively degrade mycotoxins and have been successfully used in the detoxification process of various food matrices. However, certain inherent limitations, such as sensitivity to environmental conditions, complex purification processes, and difficulties in reuse, limit the large-scale application of natural enzymes. Furthermore, the enzymatic detoxification of mycotoxins in edible oils presents significant challenges because electron transfer and hydrolysis occur minimally in enzymatic reactions in edible oils, resulting in limited enzyme activity.

[0004] Metal-organic frameworks (MOFs) are a class of organic-inorganic hybrid materials with excellent pore structure, adsorption capacity, and enzyme-like catalytic performance, showing great promise in the detoxification of mycotoxins. Due to their tunable metal active sites, MOFs are used as biomimetic nanozyme catalysts in enzymatic reactions, including oxidase and peroxidase reaction systems. Currently, the nanozymes studied for degrading mycotoxins are mainly peroxidase-mimicking enzymes. In the presence of H₂O₂, these nanozyme catalysts can achieve effective removal of mycotoxins based on peroxidase-like activity. However, peroxidase-like nanozymes require destructive H₂O₂ to exhibit catalytic activity, which could have a devastating impact on food quality.

[0005] Therefore, there is an urgent need for a material that can degrade fungal toxins without affecting food quality. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems of limited removal efficiency of mycotoxins by microbial enzymatic degradation and the impact of metal-organic frameworks on food quality in existing technologies. This invention provides a biocomposite material, its preparation method and application, and a method for removing mycotoxins from liquid food. This biocomposite material can effectively degrade mycotoxins that may be present in liquid food without affecting the quality of the food.

[0007] To achieve the above objectives, a first aspect of the present invention provides a biocomposite material comprising fungi and a metal-organic framework material, wherein the structure of the metal-organic framework material is shown in formula (I).

[0008]

[0009] Wherein, X is selected from at least one of Mn, Fe, Ce, Zn and Cu, and M is selected from at least one of Ti, Zr and Hf.

[0010] Preferably, the biocomposite material comprises a substrate formed from Cordyceps militaris and a metal-organic framework material doped in the substrate.

[0011] Preferably, X is Mn and M is Zr.

[0012] More preferably, in the biocomposite material, the content of the metal-organic framework material, calculated as coordination metal, is 0.5-0.8 wt%.

[0013] Preferably, the Cordyceps fungus is Cordyceps militaris.

[0014] A second aspect of the present invention provides a method for preparing a biocomposite material, the method comprising: mixing Cordyceps mycelium with a metal-organic framework material and then molding the mixture, wherein the structure of the metal-organic framework material is shown in formula (I).

[0015]

[0016] Wherein, X is selected from at least one of Mn, Fe, Ce, Zn and Cu, and M is selected from at least one of Ti, Zr and Hf.

[0017] Preferably, the preparation method of the metal-organic framework material includes: in solvent I, X-TCPP, benzoic acid and metal salt I are subjected to a contact reaction I, wherein the metal salt I is selected from at least one of tetravalent zirconium salt, tetravalent titanium salt and tetravalent hafnium salt.

[0018] More preferably, the conditions for the contact reaction I include: a temperature of 100-140°C and a time of 20-28 hours.

[0019] Preferably, the amount of benzoic acid used is 45-60g relative to 1g of X-TCPP, and the amount of metal salt I used is 1-2g.

[0020] Preferably, the preparation method of X-TCPP includes: in solvent II, a porphyrin precursor and a metal salt II undergo a contact reaction II; then in solvent III, the product of the contact reaction II and a base undergo a contact reaction III; wherein the metal salt II is selected from at least one of Mn salt, Fe salt, Ce salt, Zn salt and Cu salt.

[0021] More preferably, the method for preparing the porphyrin precursor includes: in solvent IV, pyrrole and p-formylbenzoate undergoing a contact reaction IV.

[0022] Preferably, the conditions for the contact reaction II include at least: a temperature of 95-105°C and a time of 3-5 hours;

[0023] The conditions for the contact reaction III include at least the following: a temperature of 75-85°C and a time of 10-14 hours.

[0024] The conditions for the contact reaction IV include at least the following: a temperature of 130-150°C and a time of 0.5-1.5 h.

[0025] Preferably, solvent I is DMF, solvent II is N,N-dimethylformamide, solvent III is a mixture of tetrahydrofuran and methanol, and solvent IV is propionic acid;

[0026] The metal salt I is selected from at least one of titanium chloride, zirconium chloride, and hafnium chloride;

[0027] The metal salt II is selected from at least one of manganese nitrate, copper nitrate, ferric nitrate, zinc nitrate, and cerium nitrate;

[0028] The alkali is sodium hydroxide and / or potassium hydroxide.

[0029] More preferably, the metal salt I is zirconium chloride and the metal salt II is manganese nitrate.

[0030] Preferably, the mixing is carried out in water.

[0031] More preferably, the mass ratio of the Cordyceps mycelium to the metal-organic framework material, based on dry weight, is 60-100:1.

[0032] Preferably, the cordyceps mycelium is Cordyceps militaris mycelium.

[0033] A third aspect of the present invention provides a biocomposite material prepared by the preparation method described in the second aspect above.

[0034] The fourth aspect of the present invention provides the application of the biocomposite material described in the first or third aspect above in the removal of fungal toxins.

[0035] Preferably, the fungal toxin is ochratoxin.

[0036] The fifth aspect of the present invention provides a method for removing mycotoxins from liquid food, the method comprising: mixing and incubating the liquid food containing mycotoxins with a biological composite material and then subjecting it to light treatment, wherein the biological composite material is the aforementioned biological composite material.

[0037] Preferably, the incubation is carried out in the dark, and the lighting conditions include at least: xenon lamp irradiation with an irradiation power of 200-400W and an irradiation time of 60-100min.

[0038] Preferably, the fungal toxin is ochratoxin.

[0039] Through the above technical solution, the biocomposite material provided by the present invention contains Cordyceps militaris and metal-organic framework material, and the structure of the metal-organic framework material is restricted to the form shown in formula (I). Through the interaction between Cordyceps militaris and the metal-organic framework material with the structure shown in formula (I), the biocomposite material has a good removal effect on OTA in liquid food and does not affect the quality of liquid food. Attached Figure Description

[0040] Figure 1 This is a schematic diagram illustrating the function of the biocomposite membrane of the present invention;

[0041] Figure 2 This is a transmission electron microscope image of the biofilm in Example 1, where A and B are TEM images at different magnifications, CF is an elemental distribution map of different elements, and G is an EDS spectrum.

[0042] Figure 3 The above are characterization diagrams of the biofilm in Example 1, where A is the N2 adsorption isotherm, B is the Fourier transform infrared spectrum, C is the diffuse reflectance ultraviolet-visible absorption spectrum, and D is the calculated band gap energy.

[0043] Figure 4 Four are SEM images of the biofilms of Comparative Example 1 and Example 1, where A and B are SEM images of the surface of the biofilm of Comparative Example 1, C and D are SEM images of the surface of the biofilm of Example 1, E and F are SEM images of the cross-section of the biofilm of Comparative Example 1, and G and H are SEM images of the cross-section of the biofilm of Example 1.

[0044] Figure 5The graphs show the OTA removal effects of the biofilms in Example 1 and Comparative Example 1. In the graphs, A is the photocatalytic degradation performance graph, B is the removal capacity graph, C is the kinetic graph, and D is the OTA removal efficiency trend graph of PCN-222(Mn) / biofilm after 6 adsorption-desorption cycles.

[0045] Figure 6 This is the total ion chromatogram of OTα and its degradation products. Detailed Implementation

[0046] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0047] As previously stated, a first aspect of the present invention provides a biocomposite material containing Cordyceps militaris and a metal-organic framework material, wherein the structure of the metal-organic framework material is shown in formula (I).

[0048]

[0049] Wherein, X is selected from at least one of Mn, Fe, Ce, Zn and Cu, and M is selected from at least one of Ti, Zr and Hf.

[0050] According to the present invention, in formula (I), a solid line connecting two atoms indicates that the two atoms are connected by a covalent bond, and a dashed line connecting two atoms (or one atom and one ion, or two ions) indicates that the two atoms (or one atom and one ion, or two ions) are connected by a coordinate bond. X is a divalent or trivalent ion, and X is connected to N by a coordinate bond. M is a tetravalent metal ion, and M is connected to O by a coordinate bond. Two adjacent O atoms are also connected by a coordinate bond.

[0051] In formula (I), X can be one or more of Mn, Fe, Ce, Zn, and Cu; M can be one or more of Ti, Zr, and Hf. If M is more than one of Ti, Zr, and Hf, the mass ratio of the multiple metal ions is determined according to the actual situation. When X is more than one of Mn, Fe, Ce, Zn, and Cu, the metal-organic framework material is a mixture; when M is more than one of Ti, Zr, and Hf, the metal-organic framework material can be a mixture or a pure substance.

[0052] During the research process, the inventors unexpectedly discovered that the biocomposite material contains Cordyceps militaris and metal-organic framework materials. By restricting the structure of the metal-organic framework material to the form shown in formula (I), the biocomposite material can effectively remove OTA from liquid food through the interaction between Cordyceps militaris and the metal-organic framework material with the structure shown in formula (I), without affecting the quality of the liquid food.

[0053] According to the present invention, preferably, the biocomposite material comprises a substrate formed from Cordyceps militaris and a metal-organic framework material doped in the substrate. In the preparation process, Cordyceps militaris mycelium and the metal-organic framework material are directly mixed and then molded, so in the resulting biocomposite material, the metal-organic framework material is doped into the substrate formed from Cordyceps militaris.

[0054] Preferably, X is Mn and M is Zr. Using the above-mentioned metal elements as coordinating metals can further improve the synergistic effect between the metal-organic framework material and Cordyceps militaris, thereby further improving the removal effect of the composite material on OTA.

[0055] According to the present invention, the content of the metal-organic framework material, calculated as coordination metal, in the biocomposite material is 0.39-1.1 wt%, and can be 0.39 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, or any value between these values. Preferably, the content of the metal-organic framework material, calculated as coordination metal, in the biocomposite material is 0.5-0.8 wt%. Studies have found that controlling the metal-organic framework material in the biocomposite material within the above range results in a better removal effect of the biocomposite material on OTA.

[0056] Preferably, the Cordyceps fungus is Cordyceps militaris. Using Cordyceps militaris can further improve the removal efficiency of the biocomposite material for OTA.

[0057] A second aspect of the present invention provides a method for preparing a biocomposite material, the method comprising: mixing Cordyceps mycelium with a metal-organic framework material and then molding the mixture, wherein the structure of the metal-organic framework material is shown in formula (I).

[0058]

[0059] Wherein, X is selected from at least one of Mn, Fe, Ce, Zn and Cu, and M is selected from at least one of Ti, Zr and Hf.

[0060] The biocomposite material prepared by the above method has a good removal effect on OTA in liquid food through the interaction between Cordyceps and metal-organic framework material with structure as shown in formula (I), and does not affect the quality of liquid food.

[0061] Preferably, the preparation method of the metal-organic framework material includes: in solvent I, X-TCPP, benzoic acid and metal salt I undergo a contact reaction I, wherein metal salt I is selected from at least one of tetravalent zirconium salt, tetravalent titanium salt and tetravalent hafnium salt. The metal-organic framework material prepared by the above method has a good synergistic effect with Cordyceps militaris, thereby enabling the prepared biocomposite material to have a good removal effect on OTA.

[0062] According to the present invention, X-TCPP can be obtained commercially or prepared using existing methods. The structure of X-TCPP is shown in formula (II).

[0063]

[0064] Preferably, the preparation method of X-TCPP includes: in solvent II, a porphyrin precursor and a metal salt II undergo a contact reaction II; then in solvent III, the product of the contact reaction II and an alkali undergo a contact reaction III; wherein the metal salt II is selected from at least one of manganese salt, iron salt, cerium salt, zinc salt, and copper salt. X-TCPP prepared by the above method exhibits better mixing effects in Cordyceps mycelium, thereby further improving the removal effect of the obtained biocomposite material on OTA.

[0065] According to the present invention, an alkaline solution can be obtained by first mixing alkali and water, and then the alkaline solution can be reacted with the product of contact product II.

[0066] Porphyrin precursors are commercially available or prepared. Preferably, the preparation method of the porphyrin precursor includes: reacting pyrrole and p-formylbenzoate in solvent IV via a contact reaction IV.

[0067] Preferably, the conditions for contact reaction II include at least the following: a temperature of 95-105°C, which can be 95°C, 97°C, 99°C, 101°C, 103°C, 105°C, or any value between these values; and a time of 3-5 hours, which can be 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, or any value between these values. The conditions for contact reaction III include at least the following: a temperature of 75-85°C, which can be 75°C, 77°C, 79°C, 81°C, 83°C, 85°C, or any value between these values; and a time of 10-14 hours, which can be 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, or any value between these values. The conditions for the contact reaction IV include at least the following: a temperature of 130-150°C, which can be 130°C, 135°C, 140°C, 145°C, 150°C, or any value between these values; and a time of 0.5-1.5h, which can be 0.5h, 1h, 1.5h, or any value between these values.

[0068] Solvents II, III, and IV are not specifically limited. In one specific embodiment of the invention, solvent II is N,N-dimethylformamide, solvent III is a mixture of tetrahydrofuran and methanol, and solvent IV is propionic acid. Preferably, in solvent III, the volume ratio of tetrahydrofuran to methanol is 1:0.7-1.4, and can be 1:0.7, 1:0.9, 1:1.1, 1:1.3, 1:1.4, or any value between these values.

[0069] Preferably, the mass ratio of the porphyrin precursor to metal salt II is 1:1.8-3, which can be 1:1.8, 1:2.1, 1:2.4, 1:2.7, 1:3, or any value between these values. The mass ratio of the product of contact reaction II to the base is 1:7.5-8.3, which can be 1:7.5, 1:7.7, 1:7.9, 1:8.3, 1:8.5, or any value between these values. The molar ratio of the pyrrole to the p-formylbenzoate is 1:0.93-1.05, which can be 1:0.93, 1:0.97, 1:1.01, 1:1.05, or any value between these values.

[0070] Preferably, the metal salt II is selected from at least one of manganese nitrate, copper nitrate, ferric nitrate, zinc nitrate, and cerium nitrate, and more preferably manganese nitrate. This can further improve the reaction effect.

[0071] Preferably, the conditions for contact reaction I include: a temperature of 100-140°C, which can be 100°C, 110°C, 120°C, 130°C, 140°C, or any value between these values; and a time of 20-28 hours, which can be 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, or any value between these values. Studies have found that under the above reaction conditions, X-TCPP, benzoic acid, and metal salt I exhibit better reaction performance, thereby increasing the yield of metal-organic framework materials.

[0072] Preferably, the amount of benzoic acid used is 45-60g relative to 1g of X-TCPP, and the amount of the metal salt is 0.5-2.5g. More preferably, the amount of benzoic acid used is 45-60g relative to 1g of X-TCPP, and the amount of the metal salt is 1-2g. The metal-organic framework material prepared under the above conditions exhibits good synergistic effects with Cordyceps militaris, thereby resulting in a better removal effect of OTA by the prepared biocomposite material.

[0073] Preferably, metal salt I is selected from at least one of titanium chloride, zirconium chloride, and hafnium chloride, and solvent I is DMF. Using the aforementioned metal salt I enables the prepared metal-organic framework material to have a better synergistic effect with Cordyceps militaris, thereby resulting in a better removal effect of OTA on the prepared biocomposite material. Further preferably, considering the potential to improve the synergistic effect between the metal-organic framework material and Cordyceps militaris, the metal salt is zirconium chloride.

[0074] Preferably, the p-formylbenzoate can be at least one of methyl p-formylbenzoate, ethyl p-formylbenzoate, propyl p-formylbenzoate and butyl p-formylbenzoate, and is more preferably methyl p-formylbenzoate.

[0075] Preferably, the mixing is carried out in water, which can improve the mixing effect between the metal-organic framework material and Cordyceps militaris. The molding process can be drying.

[0076] Preferably, the mass ratio of the Cordyceps mycelium to the metal-organic framework material, based on dry weight, is 60-100:1, and can be 60:1, 70:1, 80:1, 90:1, 100:1, or any value between these ratios. The Cordyceps mycelium and metal-organic framework material at the above mass ratio exhibit good synergistic effects, thereby enabling the prepared biocomposite material to achieve better removal of OTA.

[0077] Preferably, the cordyceps mycelium is Cordyceps militaris mycelium. Using the above-mentioned cordyceps mycelium in conjunction with metal-organic framework materials results in better synergy, thereby further improving the removal efficiency of the biocomposite material for OTA.

[0078] According to the present invention, Cordyceps militaris can be cultured in any feasible culture medium, without limitation, as long as Cordyceps mycelium can be cultured. In one specific embodiment of the present invention, Cordyceps militaris is prepared by fermentation in potato dextrose medium. The method for preparing potato dextrose medium includes: washing potatoes, peeling them, and cutting them into pieces approximately 1 cm in size. 3 Weigh 200g of the small pieces and boil them in 1000mL of water for 20-30 minutes. Filter the solution through double-layered gauze, collect the filtrate, add water to make up to 1000mL, and then add magnesium sulfate, potassium dihydrogen phosphate, glucose, urea, zinc sulfate, and ferrous sulfate, so that the concentrations are: magnesium sulfate 0.1-1wt%, potassium dihydrogen phosphate 0.1-1wt%, glucose 10-50wt%, urea 1-5wt%, zinc sulfate 0.1-0.5wt%, and ferrous sulfate greater than 0wt% and less than or equal to 1wt%.

[0079] A third aspect of this invention provides a biocomposite material prepared by the preparation method described in the second aspect above. This biocomposite material possesses all the advantages of the material prepared by the above method, which will not be elaborated here.

[0080] The fourth aspect of the present invention provides the application of the biocomposite material described in the first or third aspect above in the removal of fungal toxins.

[0081] The aforementioned biocomposite material has a good removal effect on fungal toxins and does not affect the quality of food.

[0082] The fifth aspect of the present invention provides a method for removing mycotoxins from liquid food, comprising: mixing and incubating the liquid food containing mycotoxins with a biological composite material and then subjecting it to light treatment, wherein the biological composite material is the aforementioned biological composite material.

[0083] Preferably, the incubation is carried out in darkness, and the lighting conditions include at least: xenon lamp irradiation with a power of 200-400W and a duration of 60-100 minutes. Under these conditions, there is a good removal effect on fungal toxins.

[0084] Preferably, the incubation time is 40-80 minutes.

[0085] Preferably, the fungal toxin is ochratoxin. The above method has a better removal effect on ochratoxin.

[0086] Preferably, the concentration of the mycotoxin in the liquid food is 5-20 μg / mL. Controlling the concentration of the mycotoxin in the liquid food under these conditions results in better removal efficiency.

[0087] Preferably, the amount of the biocomposite material used is 3-5 mg relative to 100 μL of liquid food. This reduces the amount of biocomposite material used while ensuring the removal rate.

[0088] According to a particularly preferred embodiment of the present invention, a method for preparing a biocomposite membrane is provided, comprising:

[0089] (1) Pyrrole and methyl paraformylbenzoate were placed in a 250 mL three-necked flask at a molar ratio of 1:0.93-1.05. Propionic acid was added to completely dissolve the organic matter to obtain a mixture. The mixture was placed in an oil bath and heated under reflux at 130-150 °C for 0.5-1.5 h. After cooling to room temperature, anhydrous ethanol was added and the mixture was placed at 4 °C overnight. The mixture was filtered to obtain a solid crude product. The product was washed twice with ethanol and ethyl acetate, respectively. Finally, the solid crude product was dried and purified to obtain a purple porphyrin precursor.

[0090] (2) The obtained porphyrin precursor and manganese nitrate tetrahydrate were dissolved in N,N-dimethylformamide at a mass ratio of 1:1.8-3. The mixture was refluxed at 95-105℃ for 3-5 hours. After cooling to room temperature, water was added. The precipitate was filtered, washed with water, and the resulting solid was dissolved in dichloromethane. Then it was extracted with HCl and water. The organic layer was rotary evaporated to obtain a purple-red powder.

[0091] (3) The obtained purplish-red powder was dissolved in a mixed solution of tetrahydrofuran and methanol (volume ratio 1:0.7-1.4), and then KOH aqueous solution was added. The mixture was heated at 75-85℃ and maintained for 10-14 h, then cooled to room temperature. The resulting precipitate was acidified with 1M HCl solution until no solid was produced. The obtained solid was filtered, washed with a large amount of distilled water, and dried to obtain tetracarboxyporphyrin ligand Mn-TCPP. The mass ratio of purplish-red powder to KOH was 1:7.5-8.3.

[0092] (4) Mn-TCPP, ZrCl4 and benzoic acid were dissolved in DMF by ultrasonication at a mass ratio of 1:45-60:1-2. Then, the mixture was heated at 100-140℃ for 20-28 hours. After cooling to room temperature, the product was washed several times with DMF and acetone, and then vacuum dried to obtain PCN-222(Mn) powder.

[0093] (5) PCN-222(Mn) powder at a mass ratio of 1:60-100 was dispersed in washed Cordyceps mycelium to obtain a biofilm mixture. The biofilm mixture was then placed in a petri dish and transferred to an oven (60°C) for 12 hours to obtain PCN-222(Mn) / biofilm.

[0094] The PCN-222(Mn) / biofilm prepared by the above method exhibits excellent removal efficiency of OTA in liquid food through the interaction between Cordyceps militaris and the metal-organic framework material with the structure shown in formula (I). It can efficiently degrade ochratoxin into non-toxic small molecules without affecting the quality of the liquid food. This avoids the damage to food nutrients caused by the H2O2 required by traditional peroxidase-like nanoenzymes.

[0095] The present invention will be described in detail below through examples. In the following examples, the Cordyceps militaris fungus was obtained from the laboratory of the School of Food and Pharmaceutical Engineering, Nanjing Normal University. The preparation method is as follows:

[0096] Fresh, healthy Cordyceps militaris free from pests and diseases was selected as the isolation material. After cleaning and sterilization, the wild Cordyceps militaris strain was isolated using tissue isolation methods. The outer skin was removed with sterile scissors, and the Cordyceps militaris stroma was cut into small segments. These segments were inoculated onto PDA medium and incubated at 22-26℃ for 48-72 hours. A mother culture with good characteristics was selected as the inoculum. Mycelium was then obtained through culture.

[0097] The fungus *Rhizopus oryzae* was obtained from the laboratory of the College of Food and Pharmaceutical Engineering, Nanjing Normal University. Its preparation method is as follows:

[0098] Samples were taken from decaying wheat straw, and the sample powder was soaked in 50 mL of physiological saline at 20°C, shaking every 2 hours. 100 μL of the sample suspension was evenly spread on nutrient agar plates and incubated at 28°C for 3-7 days. Morphologically typical single colonies were picked and streaked onto nutrient agar plates. Pure cultures were obtained by repeated streaking. A mother culture with good characteristics was selected as the inoculum. Mycelia were then cultured.

[0099] Example 1

[0100] (1) Pyrrole (3.0 g, 0.043 mol) and methyl p-formylbenzoate (6.9 g, 0.042 mol) were placed in a 250 mL three-necked flask, and 100 mL of propionic acid was added to completely dissolve the organic matter to obtain a mixture. The mixture was placed in an oil bath and heated under reflux at 140 °C for 1 h, and then cooled to room temperature. 50 mL of anhydrous ethanol was added and placed at 4 °C overnight. The mixture was filtered to obtain a solid crude product, which was washed twice with ethanol and ethyl acetate, respectively. Finally, the solid crude product was dried and purified to obtain a purple porphyrin precursor.

[0101] (2) The obtained porphyrin precursor (1.0 g) and manganese nitrate tetrahydrate (2.5 g) were dissolved in 100 mL of N,N-dimethylformamide and refluxed at 100 °C for 4 h. After cooling to room temperature, 150 mL of water was added. The precipitate was filtered, washed with water, and the obtained solid was dissolved in dichloromethane. Then it was extracted with HCl and water. The organic layer was rotary evaporated to obtain a purple-red powder.

[0102] (3) Dissolve the obtained purple-red powder (0.85g) in a mixed solution of 60mL tetrahydrofuran and 60mL methanol, then add 60mL KOH-water solution (containing 6.82g KOH), heat at 80℃ for 12h and then cool to room temperature. Acidify the generated precipitate solid with 1M HCl solution until no solid is produced. Filter the obtained solid, wash with a large amount of distilled water and dry to obtain tetracarboxyporphyrin ligand Mn-TCPP.

[0103] (4) Mn-TCPP (50 mg), ZrCl4 (75 mg, CAS No.: 10026-11-6) and benzoic acid (2.7 g) were dissolved by sonication in DMF (15 mL). The mixture was then heated at 120 °C for 24 hours. After cooling to room temperature, the product was washed several times with DMF and acetone, and then dried under vacuum to obtain PCN-222(Mn) powder.

[0104] (5) Disperse 0.25g of PCN-222(Mn) powder in 50g of washed Cordyceps militaris mycelium to obtain a biofilm mixture. Then place the biofilm mixture in a petri dish and transfer it to an oven (60℃) for 12 hours to obtain 20g of PCN-222(Mn) / biofilm.

[0105] The morphological characteristics of PCN-222(Mn) were characterized using transmission electron microscopy, such as... Figure 2 As shown in Figures A and 2B, the obtained PCN-222(Mn) exhibits a spherical structure with an average size of approximately 5 nm. The elemental distribution and EDS spectrum of PCN-222(Mn) are shown below. Figure 2 As shown in CG, all the expected elements C, O, Mn, and Zr can be observed and uniformly distributed in the material, confirming the successful preparation of PCN-222(Mn).

[0106] Figure 3 In sample A, PCN-222(Mn) exhibited a typical type IV N2 adsorption isotherm, which increased significantly at P / P0 = 0.05 and P / P0 = 0.4, indicating the presence of microporous and mesoporous structures. The specific surface area and average pore volume of PCN-222(Mn) were 172.29 m² / s. 2 / g and 0.13cm 3 / g provides sufficient adsorption sites. Furthermore, the pore size of PCN-222(Mn) was calculated to be approximately 1.05 nm.

[0107] The microstructure of PCN-222(Mn) was further investigated using FT-IR spectroscopy. Figure 3 B). The spectrum shows that at 1708 cm⁻¹ -1 and 1403cm -1 There are two spokes at this point, corresponding to the C=O and C=C of the benzene ring, respectively. At 1324 cm... -1 Pyrrole deformation was observed at 1012 cm⁻¹, with the Mn–N peak located at 1012 cm⁻¹. -1 Location. 719cm -1 and 779cm -1 The characteristic peak at that location can be attributed to the CH stretching vibration of the benzene ring.

[0108] The light absorption efficiency of PCN-222(Mn) was investigated using ultraviolet-visible diffuse reflectance spectroscopy (UV-DRS). Figure 3 As shown in Figure C, PCN-222(Mn) exhibits broad and strong visible light absorption in the 200-800 nm range, which can be attributed to Mn-TCPP as a powerful visible light trapping unit. Furthermore, based on the Kubelka-Munk function, the bandgap energy of PCN-222(Mn) is estimated to be 1.57 eV. Figure 3 D). The narrow bandgap energy indicates that PCN-222(Mn) can absorb more visible light, thereby enhancing its photocatalytic activity.

[0109] Example 2

[0110] (1) Pyrrole (3.0 g, 0.043 mol) and methyl p-formylbenzoate (7.4 g, 0.045 mol) were placed in a 250 mL three-necked flask, and 100 mL of propionic acid was added to completely dissolve the organic matter to obtain a mixture. The mixture was placed in an oil bath and heated under reflux at 130 °C for 1.5 h, and then cooled to room temperature. 50 mL of anhydrous ethanol was added and the mixture was placed at 4 °C overnight. The mixture was filtered to obtain a solid crude product, which was washed twice with ethanol and ethyl acetate, respectively. Finally, the solid crude product was dried and purified to obtain a purple porphyrin precursor.

[0111] (2) The obtained porphyrin precursor (1.0 g) and manganese nitrate tetrahydrate (2 g) were dissolved in 100 mL of N,N-dimethylformamide and refluxed at 95 °C for 5 h. After cooling to room temperature, 150 mL of water was added. The precipitate was filtered, washed with water, and the obtained solid was dissolved in dichloromethane. Then it was extracted with HCl and water. The organic layer was rotary evaporated to obtain a purple-red powder.

[0112] (3) Dissolve the obtained purple-red powder (0.85g) in a mixed solution of 50mL tetrahydrofuran and 70mL methanol, then add 60mL of KOH- aqueous solution (containing 7g of KOH), heat at 75℃ for 14h and cool to room temperature, acidify the generated precipitate solid with 1M HCl solution until no solid is produced, filter the obtained solid, wash with a large amount of distilled water and dry to obtain tetracarboxyporphyrin ligand Mn-TCPP.

[0113] (4) Mn-TCPP (50 mg), ZrCl4 (50 mg), and benzoic acid (2.4 g) were dissolved by sonication in DMF (15 mL). The mixture was then heated at 110 °C for 28 hours. After cooling to room temperature, the product was washed several times with DMF and acetone, and then vacuum dried to obtain PCN-222(Mn) powder.

[0114] (5) Disperse 0.25g of PCN-222(Mn) powder in 40g of washed Cordyceps militaris mycelium to obtain a biofilm mixture. Then place the biofilm mixture in a petri dish and transfer it to an oven (60℃) for 12 hours to obtain 16g of PCN-222(Mn) / biofilm.

[0115] Example 3

[0116] (1) Pyrrole (3.0 g, 0.043 mol) and methyl p-formylbenzoate (6.6 g, 0.04 mol) were placed in a 250 mL three-necked flask, and 100 mL of propionic acid was added to completely dissolve the organic matter to obtain a mixture. The mixture was placed in an oil bath and heated under reflux at 150 °C for 1 h, and then cooled to room temperature. 50 mL of anhydrous ethanol was added and placed at 4 °C overnight. The mixture was filtered to obtain a solid crude product, which was washed twice with ethanol and ethyl acetate, respectively. Finally, the solid crude product was dried and purified to obtain a purple porphyrin precursor.

[0117] (2) The obtained porphyrin precursor (1.0 g) and manganese nitrate tetrahydrate (3 g) were dissolved in 100 mL of N,N-dimethylformamide and refluxed at 105 °C for 3 h. After cooling to room temperature, 150 mL of water was added. The precipitate was filtered, washed with water, and the obtained solid was dissolved in dichloromethane. Then it was extracted with HCl and water. The organic layer was rotary evaporated to obtain a purple-red powder.

[0118] (3) Dissolve the obtained purple-red powder (0.85g) in a mixed solution of 70mL tetrahydrofuran and 50mL methanol, then add 60mL of KOH- aqueous solution (containing 6.5g KOH), heat at 85℃ for 10h and then cool to room temperature. Acidify the generated precipitate solid with 1M HCl solution until no solid is produced. Filter the obtained solid, wash with a large amount of distilled water and dry to obtain tetracarboxyporphyrin ligand Mn-TCPP.

[0119] (4) Mn-TCPP (50 mg), ZrCl4 (100 mg), and benzoic acid (3 g) were dissolved by sonication in DMF (15 mL). The mixture was then heated at 130 °C for 20 hours. After cooling to room temperature, the product was washed several times with DMF and acetone, and then vacuum dried to obtain PCN-222(Mn) powder.

[0120] (5) 0.25g of PCN-222(Mn) powder was dispersed in 60g of washed Cordyceps militaris mycelium to obtain a biofilm mixture. The biofilm mixture was then placed in a petri dish and transferred to an oven (60℃) for 12 hours to obtain 24g of PCN-222(Mn) / biofilm.

[0121] Example 4

[0122] PCN-222(Mn) / biofilm was prepared according to the method described in Example 2, except that 30g of washed Cordyceps militaris mycelium was added, resulting in 12g of PCN-222(Mn) / biofilm.

[0123] Example 5

[0124] PCN-222(Mn) / biofilm was prepared according to the method described in Example 3, except that 80g of washed Cordyceps militaris mycelium was added, resulting in 32g of PCN-222(Mn) / biofilm.

[0125] Example 6

[0126] PCN-222(Mn) / biofilm was prepared according to the method described in Example 1, except that 75 mg ZrCl4 was replaced with 61.91 mg TiCl4.

[0127] Example 7

[0128] PCN-222 / biofilm was prepared according to the method described in Example 1, except that 2.5g of manganese nitrate tetrahydrate was replaced with 1.9g of copper nitrate.

[0129] Example 8

[0130] PCN-222 / biofilm was prepared according to the method described in Example 1, except that 2.5g of manganese nitrate tetrahydrate was replaced with 3g of zinc nitrate hexahydrate.

[0131] Example 9

[0132] PCN-222 / biofilm was prepared according to the method described in Example 1, except that 2.5g of manganese nitrate tetrahydrate was replaced with 1.8g of ferrous nitrate.

[0133] Example 10

[0134] PCN-222 / biofilm was prepared according to the method described in Example 1, except that 2.5g of manganese nitrate tetrahydrate was replaced with 4.3g of cerium nitrate hexahydrate.

[0135] Comparative Example 1

[0136] Place 50g of washed Cordyceps militaris mycelium into a petri dish and transfer it to an oven (60℃) for 12 hours to obtain 20g of biofilm.

[0137] Comparative Example 2

[0138] (1) Tetracarboxyporphyrin ligand Mn-TCPP was prepared according to the method described in Example 1.

[0139] (2) 0.13g of Mn-TCPP powder was dispersed in 50g of washed Cordyceps militaris mycelium to obtain a biofilm mixture. The biofilm mixture was then placed in a petri dish and transferred to an oven (60℃) for 12 hours to obtain 20g of Mn-TCPP / biofilm.

[0140] Comparative Example 3

[0141] PCN-222 / biofilm was prepared according to the method described in Example 1, except that Cordyceps militaris mycelium was replaced with Rhizopus oryzae mycelium.

[0142] Test Example 1

[0143] The biofilms obtained in Comparative Example 1 and PCN-222(Mn) / biofilms obtained in Example 1 were characterized by scanning electron microscopy. See [link to relevant documentation]. Figure 4 .exist Figure 4In the examples, AB and EF are biofilms obtained in Comparative Example 1, exhibiting the expected compact microstructure; CD and GH are PCN-222(Mn) / biofilms obtained in Example 1, with relatively rough surfaces and protruding structures on both the surface and cross-section of the biofilm. The corresponding EDS distribution maps show that the pure biofilm is rich in C, N, and O elements. Furthermore, the PCN-222(Mn) / biofilm contains abundant Mn and Zr elements. Clearly, the PCN-222(Mn) / biofilm retains the microstructure of the biofilm, with PCN-222(Mn) particles uniformly distributed and tightly attached to the biofilm matrix.

[0144] Test Example 2

[0145] 0.2 mg of PCN-222(Mn) prepared in Example 1 and 16 mg of the biofilms prepared in the examples and comparative examples were added to 10 μg / mL OTA (400 μL). After incubation in the dark for 60 min, PCN-222(Mn) or biofilms were separated by centrifugation at 12000 g relative centrifugation force (RCF) for 1 min. Then, 0.2 mL of the liquid supernatant was filtered through a 0.22 μm filter and stored at 4 °C for HPLC analysis. The experimental conditions were the same as above for the synergistic mechanism of adsorption, enzyme catalysis, and photocatalysis, except that the solution was irradiated with a 300 W xenon lamp (simulating sunlight) for 80 min after incubation in the dark.

[0146] The OTA concentration was then analyzed by HPLC. The excitation and emission wavelengths were 330 nm and 460 nm, respectively. The analysis was performed under isocratic conditions at a flow rate of 1.0 mL / min, using ultrapure water containing 2% acetic acid and acetonitrile (50:50, v / v) as the mobile phase at a flow rate of 1 mL / min.

[0147] like Figure 5 As shown in AC, the OTA removal efficiencies of the systems treated with pure biofilm in Comparative Example 1, pristine PCN-222 (Mn), and PCN-222 / biofilm in Example 1 were 19%, 89%, and 90%, respectively. PCN-222 / biofilm treatment can improve OTA removal efficiency.

[0148] The recovered biofilm was rinsed several times with ultrapure water and ethanol solution, dried at 60°C for 12 hours, and then the next cycle was performed.

[0149] The results showed that after six consecutive cycles, the degradation rate of OTA remained above 80%. Figure 5 D). The slight decrease in degradation efficiency may be due to matrix adsorption on the biofilm surface and the natural decomposition of the biofilm during the cleaning process.

[0150] The test data are shown in Table 2:

[0151] Table 2

[0152]

[0153]

[0154] Test Example 3

[0155] Degradation products were determined by LC-MS / MS. Chromatographic separation was performed on a C18 column (ACQUITYUPLC BEH, 1.7 μm, 2.1 mm × 50 mm). The injection volume was 10 μL, and the mobile phase consisted of acetonitrile and water containing 0.1% formic acid, with a flow rate of 0.1 mL / min. Mass spectrometry detection was performed on an MS device equipped with an electrospray ionization (ESI) interface. The ion source was operated in negative ionization mode at -4500 V and 500 °C, with a declustering potential of -30 V.

[0156] like Figure 6 As shown, LC-MS was used to identify the degradation products of OTA catalyzed by PCN-222(Mn). It can be seen that OTA can be degraded to OTα by PCN-222(Mn). To investigate the degradation mechanism, OTA releases OTα and L-phenylalanine (L-β-Phe) through the hydrolysis of the OTA amide group. This reaction may be mediated by carboxypeptidase or similar enzymes. OTα was clearly identified as a degradation product of OTA. Compared with OTA, OTα has significantly reduced toxicity. Therefore, PCN-222(Mn) can reduce OTA content, and it has broad application prospects in edible oils.

[0157] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A biocomposite material, characterized in that, The biocomposite material contains Cordyceps militaris and a metal-organic framework, the structure of which is shown in formula (I). Wherein, X is selected from at least one of Mn, Fe, Ce, Zn and Cu, and M is selected from at least one of Ti, Zr and Hf.

2. The biocomposite material according to claim 1, characterized in that, The biocomposite material includes a substrate formed from Cordyceps militaris and a metal-organic framework material doped in the substrate.

3. The biocomposite material according to claim 1 or 2, characterized in that, X is Mn, and M is Zr; Preferably, in the biocomposite material, the content of the metal-organic framework material, calculated as coordination metal, is 0.5-0.8 wt%. Preferably, the Cordyceps fungus is Cordyceps militaris.

4. A method for preparing a biocomposite material, characterized in that, The preparation method includes: mixing Cordyceps mycelium with a metal-organic framework material and then molding the mixture, wherein the structure of the metal-organic framework material is shown in formula (I). Wherein, X is selected from at least one of Mn, Fe, Ce, Zn and Cu, and M is selected from at least one of Ti, Zr and Hf.

5. The preparation method according to claim 4, characterized in that, The preparation method of the metal-organic framework material includes: in solvent I, X-TCPP, benzoic acid and metal salt I are subjected to contact reaction I, wherein metal salt I is selected from at least one of tetravalent zirconium salt, tetravalent titanium salt and tetravalent hafnium salt; Preferably, the conditions for contact reaction I include: a temperature of 100-140°C and a time of 20-28 hours; Preferably, the amount of benzoic acid used is 45-60g relative to 1g of X-TCPP, and the amount of metal salt I used is 1-2g.

6. The preparation method according to claim 5, characterized in that, The preparation method of X-TCPP includes: in solvent II, a porphyrin precursor and a metal salt II undergo a contact reaction II; then in solvent III, the product of the contact reaction II and a base undergo a contact reaction III; wherein the metal salt II is selected from at least one of Mn salt, Fe salt, Ce salt, Zn salt and Cu salt; Preferably, the method for preparing the porphyrin precursor includes: in solvent IV, reacting pyrrole and p-formylbenzoate in a contact reaction IV; Preferably, the conditions for the contact reaction II include at least: a temperature of 95-105°C and a time of 3-5 hours; The conditions for the contact reaction III include at least the following: a temperature of 75-85°C and a time of 10-14 hours. The conditions for the contact reaction IV include at least the following: a temperature of 130-150°C and a time of 0.5-1.5 h; Preferably, solvent I is DMF, solvent II is N,N-dimethylformamide, solvent III is a mixture of tetrahydrofuran and methanol, and solvent IV is propionic acid; The metal salt I is selected from at least one of titanium chloride, zirconium chloride and hafnium chloride, and is more preferably zirconium chloride; The metal salt II is selected from at least one of manganese nitrate, copper nitrate, iron nitrate, zinc nitrate and cerium nitrate, and is more preferably manganese nitrate; The alkali is sodium hydroxide and / or potassium hydroxide.

7. The preparation method according to any one of claims 4 to 6, characterized in that, The mixing takes place in water; Preferably, the mass ratio of the Cordyceps mycelium to the metal-organic framework material, based on dry weight, is 60-100:1; Preferably, the cordyceps mycelium is Cordyceps militaris mycelium.

8. The biocomposite material prepared by the preparation method according to any one of claims 4 to 7.

9. The use of the biocomposite material according to any one of claims 1 to 3 and claim 8 in the removal of fungal toxins; Preferably, the fungal toxin is ochratoxin.

10. A method for removing mycotoxins from liquid food, characterized in that, The method includes: mixing and incubating a liquid food containing mycotoxins and a biocomposite material, followed by light treatment, wherein the biocomposite material is the biocomposite material described in any one of claims 1 to 3 and claim 8; Preferably, the incubation is carried out in the dark, and the lighting conditions include at least: xenon lamp irradiation with an irradiation power of 200-400W and an irradiation time of 60-100min; Preferably, the fungal toxin is ochratoxin.

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