A method for synthesizing a composite material of Cu-TMA metal-organic framework encapsulated spores.

Abstract

This invention relates to the field of biomaterial preparation, specifically to a method for synthesizing a composite material of spores encapsulated by the metal-organic framework material Cu-TMA, comprising the following steps: Bacillus velezensis strain preserved in glycerol is inoculated into LB liquid medium and then placed in a full-temperature shaking incubator for culture; sterilized solid culture medium is dispensed into petri dishes, overnight cultured bacterial solution is added to the petri dishes and spread evenly, then the dishes are placed face up to allow the culture medium to adsorb the bacterial solution, and then inverted in an incubator for culture; mature spores are collected and washed, and then the treated spores are used as carriers to synthesize spore@Cu-TMA composite material through in-situ self-assembly of Cu-TMA on the spore surface. The composite material prepared by this invention has advantages such as high laccase activity, environmental friendliness, and low production cost, and the ABTS oxidative free radical obtained by oxidation using this composite material can maintain stability for more than 20 days, which can significantly improve the detection efficiency of antioxidants.

Description

Technical Field

[0001] This invention relates to the field of biomaterial preparation, and in particular to a method for synthesizing a composite material in which a metal-organic framework material Cu-TMA encapsulates spores. Background Technology

[0002] Metal-organic frameworks (MOFs) are porous, coordinated crystalline materials that self-assemble from metal ions (or metal clusters) and organic ligands through coordination chemistry. They possess advantages such as high porosity, high loading capacity, large specific surface area, and diverse structures, and are widely used in gas storage and separation, food detection, sensing, and catalysis. The porosity of MOFs also facilitates the diffusion of various organic and inorganic substances to their binding sites, while the metal ions can act as coenzymes, thereby enhancing the activity of immobilized enzymes. Although MOF nanomaterials exhibit efficient adsorption and catalysis, they suffer from poor stability in aqueous solutions, are prone to aggregation, and are difficult to separate, which limits their large-scale regeneration and reuse. To overcome these shortcomings, researchers have attempted to immobilize MOF nanoparticles on mesoporous or macroporous matrices to form composite materials, aiming to reduce or even eliminate these problems.

[0003] Microorganisms are widely distributed in nature. They are small in size, have a large specific surface area, porous cell walls, and exhibit chemical and biological diversity. Furthermore, they are low-cost and environmentally friendly to cultivate, making them suitable as carriers for assembling various materials and preparing composite materials with specific functional properties. Spores are oval or round, thick-walled, and highly resistant dormant bacterial bodies produced by bacilli and clostridium under external pressures (such as temperature increases or decreases, nutrient deficiencies, or excessive accumulation of harmful metabolites). Research has found that spore coat protein A (CotA) from Bacillus velezensis not only possesses laccase activity but also exhibits good thermal stability. However, its laccase activity is not high. Therefore, it is particularly important to develop a novel and efficient antioxidant analysis method based on spores, utilizing the laccase activity of the thermally stable CotA protein, and combining it with a high-performance immobilization material that can effectively promote the laccase activity of CotA protein. Summary of the Invention

[0004] The purpose of this invention is to provide a method for synthesizing a composite material in which the metal-organic framework material Cu-TMA encapsulates spores, in order to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a method for synthesizing a composite material of Cu-TMA metal-organic framework material encapsulating spores, comprising the following steps:

[0007] (1) Prepare LB liquid culture medium and autoclave it. Inoculate the glycerol-preserved Bacillus velezensis strain into the sterilized LB liquid culture medium and then place it in a full-temperature shaking incubator for activation. Activate it twice in a row.

[0008] (2) Prepare LB solid medium, sterilize it and dispense it into petri dishes. After it cools down, take the overnight culture solution and add it into the petri dishes. Then place it upright so that the culture medium can absorb the culture solution. Then invert it and incubate it in an incubator.

[0009] (3) Collect the mature spores and wash them;

[0010] (4) First, resuspend 0.75g of spores (wet weight) in Cu(CH3COOH)2·H2O solution. Then, slowly add H3BTC solution dropwise to the above solution and carry out the reaction in an incubator. After the reaction is completed, centrifuge the material, discard the supernatant, and then repeatedly centrifuge and wash the supernatant with deionized water until there is no foam. Add deionized water to resuspend the material and calculate the mass concentration for subsequent use.

[0011] (5) X-ray diffraction (XRD), scanning electron microscopy (SEM), and zeta potential characterization analysis of the pretreatment of composite materials before submission for testing.

[0012] In the above-mentioned method for synthesizing a composite material of Cu-TMA metal-organic framework encapsulated spores, as a preferred embodiment, the cleaning step in step (3) includes:

[0013] Step 1: Pour 5-10 mL of deionized water into a culture dish containing mature spores, gently scrape the spores from the culture dish with a spreader, and put the collected spores into a beaker;

[0014] Step 2: Filter the collected spores through a 150-250 mesh filter cloth;

[0015] Step 3: Centrifuge the filtered spores at 11000-13000 rpm for 3 minutes.

[0016] Step 4: Place the centrifuged spores on ice and pre-cool for 8-12 minutes;

[0017] Step 5: Place the pre-frozen spores on ice for sonication. The sonication conditions are: power 28%-32%, time 2-4s, interval 2-4s, sonication time 8-12min.

[0018] Step 6: Wash the spores again with deionized water after the ultrasound is finished, and set the centrifugation parameters as in Step 3 until the supernatant is clear and transparent.

[0019] In the above-mentioned method for synthesizing a composite material of Cu-TMA metal-organic framework encapsulated spores, as a preferred embodiment, in step (4), Cu(CH3COOH)2·H2O (copper acetate, 87.5mM) is prepared by weighing 0.4367g and dissolving it in 25mL of NaAc-HAc buffer (200mM, pH 2-9); in step (4), H3BTC (trimethylammonium phosphate, 25mM) is prepared by weighing 0.1313g and dissolving it in 25mL of NaAc-HAc buffer (200mM, pH 2-9).

[0020] Compared with existing technologies, the present invention provides a method for synthesizing a composite material of Cu-TMA metal-organic framework material encapsulating spores, which has the following technical advantages: This composite material has advantages such as being environmentally friendly and having low production costs. Furthermore, by fully utilizing the advantages of microorganisms (spores) and MOF nanomaterials, the composite material overcomes the shortcomings of free enzymes, such as difficulty in reusing and poor stability, as well as the tendency of MOF materials to aggregate and be difficult to recycle in aquatic environments. In addition, direct immobilization using spores avoids cumbersome experimental operations such as enzyme protein separation and purification, and also overcomes the defect of easy degradation and inactivation of proteins in vitro. It also exhibits high laccase activity and can be used to prepare efficient and stable ABTS. ·+ Free radicals (stable for more than 20 days) are used for the detection of antioxidants.

[0021] This invention is based on spores and utilizes the laccase activity of the thermally stable CotA protein. It combines a high-performance immobilization material (Cu-TMA) that can effectively promote the laccase activity of CotA protein. By self-assembling Cu-TMA on the spore surface, a spore@Cu-TMA composite material with high laccase activity is obtained, which provides the possibility for preparing efficient and stable ABTS·+ and can be further applied to the field of antioxidant activity analysis. Attached Figure Description

[0022] Figure 1 The SEM and EDX characterization results of the spore@Cu-TMA composite material of the synthesis method of the metal-organic framework material Cu-TMA encapsulated spore provided in the embodiment of the present invention are as follows: A and C are scanning electron microscope images of spores, B and D are scanning electron microscope images of spore@Cu-TMA composite material, and E, F, G and H are schematic diagrams of the distribution results of carbon, oxygen, nitrogen and copper elements, respectively.

[0023] Figure 2A schematic diagram showing the optimization of the amount of spore@Cu-TMA composite material used in the synthesis method of a composite material containing spores encapsulated by a metal-organic framework material Cu-TMA provided in an embodiment of the present invention.

[0024] Figure 3 This is a schematic diagram showing the optimization of the reaction time of the spore@Cu-TMA composite material, which is a method for synthesizing a spore-encapsulated composite material of metal-organic framework material Cu-TMA provided in an embodiment of the present invention.

[0025] Figure 4 This is a schematic diagram showing the optimized pH of the spore@Cu-TMA composite material reaction, which is a method for synthesizing a spore-encapsulated composite material of metal-organic framework material Cu-TMA provided in an embodiment of the present invention.

[0026] Figure 5 This is a schematic diagram showing the optimized reaction temperature of the spore@Cu-TMA composite material, which is a method for synthesizing a spore-encapsulated composite material of metal-organic framework material Cu-TMA provided in an embodiment of the present invention.

[0027] Figure 6 This is a schematic diagram of the ABTS resilience of the spore@Cu-TMA composite material, which is a method for synthesizing a composite material of Cu-TMA metal-organic framework material provided in an embodiment of the present invention.

[0028] Figure 7 A schematic diagram of laccase activity detection of a spore@Cu-TMA composite material, which is a method for synthesizing a spore-encapsulated composite material of metal-organic framework material Cu-TMA provided in an embodiment of the present invention.

[0029] Figure 8 ABTS for the synthesis method of a composite material of Cu-TMA metal-organic framework material encapsulating spores provided in the embodiments of the present invention ·+ Schematic diagram of free radical stability monitoring;

[0030] Figure 9 A schematic diagram illustrating the interference analysis of different metal cations in the synthesis method of a composite material of Cu-TMA metal-organic framework material encapsulating spores provided in an embodiment of the present invention;

[0031] Figure 10 This is a schematic diagram illustrating different anion interferences in the synthesis method of a composite material containing Cu-TMA metal-organic framework encapsulated spores provided in an embodiment of the present invention.

[0032] Figure 11 A schematic diagram of the spectral analysis of a composite material of Cu-TMA metal-organic framework material encapsulating spores in the wavelength range of 300-700 nm before and after ABTS oxidation, provided in an embodiment of the present invention.

[0033] Figure 12 A schematic diagram illustrating the detection range analysis of glutathione, Trolox, and caffeic acid in a method for synthesizing a composite material of Cu-TMA metal-organic framework material encapsulating spores, provided in an embodiment of the present invention.

[0034] Figure 13 This diagram illustrates the spiked recovery rate study of glutathione, Trolox, and caffeic acid in a method for synthesizing a composite material of Cu-TMA metal-organic framework material encapsulating spores, as provided in an embodiment of the present invention. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] Unless otherwise specified, the term "parts" in this invention refers to parts by weight.

[0041] Example 1

[0042] Preparation and optimization of physicochemical conditions of spore-based Cu-TMA composite materials;

[0043] I. Preparation of spore@Cu-TMA composite material

[0044] Includes the following steps:

[0045] (1) Inoculate the Bacillus velezensis strain preserved in glycerol into LB liquid medium, and then place it in a full-temperature shaking incubator for activation. Activate twice in a row. The LB liquid medium is prepared as follows: 5g sodium chloride, 5g tryptone, 2.5g yeast extract, and finally add 500mL deionized water. The medium needs to be autoclaved at 121℃ for 20min.

[0046] (2) Prepare LB solid medium, sterilize it and dispense it into petri dishes. After it cools down, take the overnight culture solution and add it into the petri dishes. Then place it upright so that the culture medium can absorb the culture solution. Then invert it and incubate it in an incubator.

[0047] (3) Inject the matured spores into deionized water, collect the spores, and wash the collected spores. The washing steps include:

[0048] Step 1: Pour 5-10 mL of deionized water into a culture dish containing mature spores, gently scrape the spores from the culture dish with a spreader, and put the collected spores into a beaker;

[0049] Step 2: Filter the collected spores through a 200-mesh filter cloth;

[0050] Step 3: Centrifuge the filtered product at 12000 rpm for 3 minutes.

[0051] Step 4: Place the centrifuged spores on ice to pre-cool for 10 minutes;

[0052] Step 5: Place the pre-cooled spores on ice for sonication. The sonication conditions are: power 30%, time 3s, interval 3s, sonication time 10min.

[0053] Step 6: Wash the spores again with deionized water after the ultrasound, and set the centrifugation parameters as in Step 3 until the clear liquid is clear and transparent.

[0054] (4) First, resuspend 0.75g of spores (wet weight) in Cu(CH3COOH)2·H2O solution. Then, slowly add H3BTC solution dropwise to the above solution in portions. The reaction is carried out in an incubator. After the reaction is completed, centrifuge the sample, discard the supernatant, and then repeatedly centrifuge and wash the sample with deionized water until there is no foam in the supernatant. Resuspend the material in deionized water and calculate the mass concentration for subsequent use. Cu(CH3COOH)2·H2O solution (87.5mM): Weigh 0.4367g and dissolve it in 25mL NaAc-HAc buffer (200mM, pH 7); H3BTC solution (25mM): Weigh 0.1313g and dissolve it in 25mL NaAc-HAc buffer (200mM, pH 7).

[0055] (5) Characterization and analysis of composite materials: X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive X-ray (EDX) elemental qualitative analysis were performed. The pretreatment before submission included: taking an appropriate amount of spores@Cu-TMA composite material, centrifuging at 12000 rpm for 2 min and discarding the supernatant, washing the precipitate twice with anhydrous ethanol, and then drying it in an oven at 60℃ for 2 h.

[0056] The results are as follows Figure 1 As shown, images A and C are scanning electron microscope (SEM) images of the spores. It can be observed from the images that the spore surface is rough and wrinkled. Images B and D are SEM images of the spore@Cu-TMA composite material. A comparison between the two clearly shows that a layer of Cu-TMA material is uniformly attached to the spore surface. Subsequently, further verification was performed using EDX elemental qualitative analysis. The results showed that carbon (Figure E), oxygen (Figure F), nitrogen (Figure G), and copper (Figure H) were uniformly distributed throughout the spore surface. The above results indicate that the spore@Cu-TMA composite material was successfully synthesized.

[0057] II. Optimization of Factor Physicochemical Conditions

[0058] (1) Optimization of the dosage of spore@Cu-TMA composite material

[0059] Based on the mass concentration of the composite material, wet weight groups of 2.5 mg, 5.0 mg, 10.0 mg, 20.0 mg, 30.0 mg, and 40.0 mg were set up, with three replicates for each group. After centrifugation (12000 rpm, 20 s), the supernatant was discarded. Then, 796 μL of NaAc-HAc buffer (200 mM, pH 4) was added to each precipitate to resuspend it. 4 μL of ABTS (100 mM) was then added to each tube, and the reaction was carried out at 25 °C and 180 rpm for 35 min. After the reaction was completed, the reaction solution was separated by centrifugation (12000 rpm, 20 s). After zeroing at a wavelength of 414 nm with NaAc-HAc buffer (200 mM, pH 4), 50 μL of the reaction solution from each group was taken and 350 μL of NaAc-HAc buffer (200 mM, pH 4) was added to measure its absorbance.

[0060] The results are as follows Figure 2 As shown, when the amount of spore@Cu-TMA composite material is between 2.5 μg and 10.0 μg, the increase in absorbance is significant with increasing material amount. When the amount is between 10.0 μg and 30.0 μg, the increase in absorbance with increasing material amount is less than in the first half. When the amount is between 30.0 μg and 40.0 μg, the increase in absorbance is almost zero, and equilibrium is reached at 30.0 μg. This indicates that when the amount of material is 30.0 μg, ABTS can be completely oxidized within 35 min. Therefore, an amount of 30.0 μg of spore@Cu-TMA composite material is sufficient for the experimental requirements.

[0061] (2) Optimization of reaction time

[0062] Groups with reaction times of 5 min, 10 min, 20 min, 30 min, 40 min, and 50 min were set up. 30.0 μg of spore@Cu-TMA composite material was added to each group. Then, 796 μL of NaAc-HAc buffer (200 mM, pH 4) was added to each precipitate to resuspend it. 4 μL of ABTS (100 mM) was added to each tube and the tubes were placed in a shaker at 25 °C and 180 rpm. After the test time, the tubes were centrifuged (12000 rpm, 20 s). 50 μL of the supernatant was taken and 350 μL of NaAc-HAc buffer (200 mM, pH 4) was added. The absorbance was measured at 414 nm and recorded.

[0063] like Figure 3 As shown, the absorbance value continuously increases with increasing reaction time from 5 to 30 minutes, but after 30 minutes, the absorbance value approaches zero with increasing reaction time, indicating that the spore@Cu-TMA composite material can completely oxidize ABTS to ABTS within 30 minutes.·+ Since the free radicals are present, a reaction time of 30 minutes is sufficient to meet the experimental requirements.

[0064] (3) Optimization of reaction pH

[0065] Prepare NaAc-HAc buffer (200 mM) with pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0. Add 30.0 μg of spore@Cu-TMA composite material (wet weight) to a 1.5 mL EP tube, then add 796 μL of the above buffer solutions of different pH values ​​to resuspend the precipitate, and add 4 μL of ABTS (100 mM). Then place the tube in a shaker at 25 °C and 180 rpm for 30 min. After the reaction, centrifuge (12000 rpm, 20 s), take 50 μL of the reaction solution, add 350 μL of NaAc-HAc buffer (200 mM, pH 4), and measure and record the absorbance at 414 nm.

[0066] like Figure 4 As shown, the absorbance reaches its peak at pH 4, meaning that pH 4 is the optimal pH for the reaction.

[0067] (4) Optimization of reaction temperature

[0068] The reaction temperatures were set at 20℃, 25℃, 30℃, 35℃, 45℃, 55℃, 65℃, and 75℃. 30.0 μg of spore-@Cu-TMA composite material (wet weight) was added to each EP tube, followed by 796 μL of NaAc-HAc buffer (200 mM, pH 4) to resuspend the spores. The tubes were then preheated in a water bath for 5 min. 4 μL of ABTS (100 mM) was added to each tube, and the reaction was carried out for 30 min. After centrifugation (12000 rpm, 20 s), 50 μL of the reaction solution was collected, and 350 μL of NaAc-HAc buffer (200 mM, pH 4) was added. The absorbance was measured at 414 nm and recorded.

[0069] like Figure 5 As shown, when the temperature is between 20℃ and 30℃, the absorbance increases slowly with increasing temperature. When the temperature is between 30℃ and 75℃, the absorbance begins to drop sharply with increasing temperature, reaching its lowest point at 65℃. When the temperature increases further, the change in absorbance is not significant. Therefore, the material has high laccase activity when the temperature is between 20℃ and 35℃, and the activity reaches its highest point at 30℃, but the increase is not significant.

[0070] The study investigated the resilient properties of ABTS on spore-@Cu-TMA composites, such as... Figure 6As observed, after the addition of the composite material, ABTS was catalytically oxidized, and the absorbance value increased steadily. After 5 minutes of reaction, centrifugation was performed to remove the composite material, and the absorbance value hardly changed. After 8 minutes, the composite material was reintroduced, and the absorbance value increased steadily again, indicating that ABTS formed ABTS. ·+ The process can be controlled at any time by removing or re-adding the composite material, while the oxidation of ABTS catalyzed by CotA protein solution or laccase cannot achieve the same effect;

[0071] Solution preparation

[0072] NaAcHAc buffer (200mM, pH 4): Accurately weigh 16.406g of NaAc (sodium acetate, Mw = 82.03), dissolve it in 800mL of deionized water, then add 12mL of glacial acetic acid to adjust the pH to 4, and finally make up to 1L with a volumetric flask.

[0073] ABTS (100mM): Accurately weigh 0.055g of ABTS and add 1mL of NaAc HAc buffer (200mM, pH 4) to dissolve completely. Then wrap it in aluminum foil and store it in a refrigerator at 4°C away from light.

[0074] Example 2

[0075] Laccase activity assay of spore@Cu-TMA composite material

[0076] Weigh out specific amounts of spore@Cu-TMA composite material and pure spores, respectively, and prepare a suspension with a concentration of 1 mg / mL using NaAc-HAc buffer (200 mM, pH = 4). Add 1 μL of ABTS stock solution to each well of the ELISA plate. Add 100 μL of the spore@Cu-TMA composite material and pure spore suspension to each well as the experimental group. A blank control group is also set up, where the same volume of NaAc-HAc buffer is added to the ELISA plate containing ABTS stock solution. OD is measured every 10 seconds after material addition. 414nm The enzyme activity was measured for 400 seconds and then calculated according to the formula.

[0077]

[0078] V 总 : Total volume of the reaction;

[0079] V 酶 The volume of the added spores or composite materials;

[0080] ε: Absorption coefficient: 3.6 × 10⁴ mol -1 L cm -1 ;

[0081] OD: The difference between the material's absorbance and the background absorbance;

[0082] t: Reaction time (min);

[0083] The catalytic activity of spore-@Cu-TMA composite material and pure spores for ABTS was analyzed using ABTS as a substrate. The results are as follows: Figure 7 As shown, compared with the laccase activity of pure spores (9.811 U / mg), the catalytic activity of spores@Cu-TMA increased by 1.82 times (17.811 U / mg), indicating that the encapsulation of Cu-TMA can enhance the laccase catalytic activity of spores to a certain extent. Preparation of ABTS (100mM) stock solution: Accurately weigh 0.055g of ABTS and add 1mL of NaAc-HAc buffer (200mM, pH 4) to dissolve completely. Then wrap with aluminum foil and store in a refrigerator at 4°C in the dark.

[0084] Example 3

[0085] Preparation and stability monitoring of ABTS oxidative radicals

[0086] ABTS and the composite material were placed in a shaker at 30°C and 180 rpm for 30 minutes, then centrifuged. The mixture containing ABTS was then... ·+ The reaction solution was filtered through a 0.22 μm filter membrane and transferred to a 50 mL centrifuge tube wrapped in aluminum foil. It was then stored at 4°C for later use. ABTS was then... ·+ 20 μL of the free radical solution was diluted with 580 μL of NaAc-HAc buffer (200 mM, pH 4), and its absorbance was measured at 414 nm. The absorbance changes were continuously recorded, and monitoring was stopped when the absorbance decreased by approximately 10% from the initial value. Figure 8 As shown, the absorbance began to decrease significantly on day 13, and by day 21, the absorbance had decreased by 12.04% compared to day 1. The absorbance on day 23 had decreased by 13.64% compared to day 1. ABTS obtained by oxidation using spore@Cu-TMA composite material... ·+ Free radicals without adding any ABTS ·+ When a protective agent (such as ATP) prevents the disproportionation reaction, its relative activity decreased by only 13.64% over 23 days, compared to ABTS obtained by the HRP-H2O2 system. ·+ The ABTS prepared in this experiment had a shelf life of only 2 days. ·+ The storage time was increased by 11.5 times. In summary, this method can prepare ABTS with high stability. ·+ This lays a solid foundation for high-throughput determination of antioxidant capacity;

[0087] Example 4

[0088] Antioxidant activity analysis of different antioxidants

[0089] The experiment investigated the effects of common metal cations and anions found in food and blood as interfering substances on the reaction system. Solutions with different concentrations of interfering substances were prepared and the composite material was resuspended in them. The absorbance was measured at 414 nm. The results are as follows: Figure 9 and 10 As shown, within the allowable error range, copper ions and manganese ions have a significant impact on the enzyme activity of the composite material. The enzyme activity of the material increases with the increase of copper ion concentration, but gradually decreases with the increase of manganese ion concentration. After removing copper ions and manganese ions, the remaining cations and anions have almost no effect on the reaction system within the error range. It can be seen that the composite material has a strong resistance to ion interference.

[0090] The spectra of ABTS before and after oxidation were analyzed in the range of 300–700 nm, such as... Figure 11 As shown, before ABTS is oxidized (shown by the black line in the figure), it only has an absorption peak at 340 nm. After ABTS is oxidized (shown by the green line in the figure), it not only has an absorption peak at 340 nm, but also new absorption peaks at 414 nm and 645 nm. The change in the absorption peak at 414 nm is particularly significant. Therefore, the absorption peak at 414 nm is used to represent ABTS. ·+ The absorption peak position of oxidized ABTS was observed. When an antioxidant (such as caffeic acid) was added, the trend of the spectral line remained unchanged; only the absorbance decreased (as shown by the blue line). This indicates that the absorption peak at 414 nm was achieved using oxidized ABTS. ·+ It is feasible to analyze the activity of antioxidants. Preparation of caffeic acid (5mM) standard solution: Weigh 0.009g of caffeic acid, dissolve it in 10mL of anhydrous ethanol, wrap it with tin foil and set aside.

[0091] Therefore, under optimal experimental conditions, the scavenging rate of different concentrations of Trolox was investigated, such as... Figure 12 As shown, at 1.0×10 -2 μmol L -1 Up to 180.0 μmol L -1 Within the range, ABTS ·+ The free radical scavenging rate and the concentration of Trolox showed a linear relationship, with the linear equation being Y = 0.95579 - 0.00359X and a correlation coefficient of 0.9974. Using three times the standard deviation as the judgment criterion, the limit of detection (LOD) for Trolox using this method was calculated to be 7.39 × 10⁻⁶. -3 μmol L -1Preparation of Trolox (5mM) standard solution: Accurately weigh 0.125g of Trolox reagent using an analytical balance, dissolve it in anhydrous ethanol, and finally dilute to 100mL in a brown volumetric flask.

[0092] The antioxidant capacity of different types of antioxidants, including hydrophilic antioxidants (GSH: reduced glutathione) and lipophilic antioxidants (caffeic acid), was further investigated, and the results are as follows: Figure 12 As shown, the detection linear range of GSH is 1.0 × 10⁻⁶. -2 ~140.0 μmol L -1 The linear regression equation is Y = 0.95516 - 0.00316X(R). 2 =0.9919), LOD is 8.899×10 -3 μmol L -1 The linear range for the detection of caffeic acid is 1.0 × 10⁻⁶. -2 ~130.0 μmol L -1 The linear regression equation is Y = 0.90288 - 0.0057X(R). 2 =0.9958), LOD is 4.83×10 -3 μmol L -1 Preparation of GSH (5mM) standard solution: Accurately weigh 0.154g of GSH, dissolve it in NaAc-HAc buffer (200mM, pH 4), and bring the volume to 100mL in a brown volumetric flask.

[0093] For 0.05 μmol L -1 Eight sets of repeatability tests were performed on caffeic acid, and the relative standard deviation (RSD) of the clearance rate was calculated to be 1.67%, indicating that the method has good repeatability.

[0094] The spiked recoveries of the three antioxidants were calculated under the same experimental conditions after reconstituted glutathione, caffeic acid, and Trolox standard solutions at the same concentrations using distilled water and methanol solutions. Figure 13 It can be seen that the spiked recoveries of glutathione are between 95.29% and 101.11%, the spiked recoveries of Trolox are between 97.43% and 99.38%, and the spiked recoveries of caffeic acid are between 92.57% and 93.62%. The spiked recoveries of the three samples are all in the range of 90% to 110%, indicating that the method of this experiment can be used for the detection of actual samples.

[0095] Conclusion: This experiment demonstrates that the outer wall protein CotA, which possesses bacterial laccase activity on the surface of Bacillus velezensis spores, can catalyze the oxidation of ABTS to form ABTS. ·+ Cationic free radicals, and then the organic compound pyromellitic acid with its cofactor copper ions, self-assemble a metal-organic framework on the spore surface to enhance the laccase activity of CotA protein, thereby establishing a spore-based metal-organic framework composite sensor for antioxidant capacity analysis.

[0096] This study employed a "post-additional" approach to analyze the antioxidant activity of antioxidants, using a spore@CU-TMA composite material instead of traditional laccase to catalyze the ABTS reaction, forming blue-green ABTS. ·+ This method has many advantages, such as: easy spore cultivation and acquisition, no need for CotA protein purification, wide application range and reusability of the composite material, and simple recovery steps. Furthermore, the ABTS prepared by this method... ·+ Compared to free radicals prepared by traditional methods, cationic free radicals exhibit significantly improved stability, which makes high-dose ABTS more suitable. ·+ The long-term preservation of free radicals has been achieved, which can meet the needs of long-term high-throughput screening of antioxidants. This method does not require expensive instruments and has low experimental costs. At the same time, this method has been successfully used to detect some actual antioxidant samples, indicating that this method has a very broad market application prospect.

[0097] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for synthesizing a composite material of metal organic framework material Cu-TMA encapsulating spores, characterized in that, Includes the following steps: (1) Prepare LB liquid medium, inoculate the glycerol-preserved Bacillus velezensis strain into LB liquid medium, and then place it in a full-temperature shaking incubator for activation, and activate it twice in a row; (2) Prepare LB solid medium, sterilize it and dispense it into petri dishes. After it cools down, take the overnight cultured bacterial solution and add it to the petri dishes. Then place it upright so that the culture medium can absorb the bacterial solution. Then invert it in the incubator and culture until the spores mature. (3) Collect and clean the mature spores, and then perform ultrasonic treatment; (4) First use a concentration of 87.5 mM Resuspend 0.75 g of wet spores in the solution, then add 25 mM solution. The solution was slowly added dropwise to the above solution in portions, and the reaction was carried out in an incubator at 25-30℃ for 30 minutes. After the reaction, the mixture was centrifuged, the supernatant was discarded, and the supernatant was repeatedly washed with deionized water until no foam was visible in the supernatant. solution and The solvent for all solutions was 200 mM NaAc-HAc buffer solution at pH 7.

2. The method of synthesis of claim 1, wherein, The cleaning and ultrasonic treatment in step (3) include: Step 1: Pour 5-10 mL of deionized water into a culture dish containing mature spores, gently scrape the spores from the culture dish with a spreader, and put the collected spores into a beaker; Step 2: Filter the collected spores through a 150-250 mesh filter cloth; Step 3: Centrifuge the filtered spores at 11000-13000 rpm for 3 min. Step 4: Place the centrifuged spores on ice and pre-cool for 8-12 minutes; Step 5: Place the pre-cooled spores on ice for sonication. The sonication conditions are: power 28%-32%, sonication time 2-4 s, interval time 2-4 s, and total sonication time 8-12 min. Step 6: Wash the spores again with deionized water after the ultrasound is finished, and set the centrifugation parameters as in Step 3 until the supernatant is clear and transparent.

3. The method of synthesis of claim 1, wherein: In step (4) The solution is an 87.5 mM copper acetate solution, prepared as follows: Weigh 0.4367 g of copper acetate and dissolve it in 25 mL of 200 mM, pH 7 NaAc-HAc buffer solution; the step (4) in... The solution is a 25 mM pyromellitic acid solution, which is prepared by weighing 0.1313 g of pyromellitic acid and dissolving it in 25 mL of 200 mM, pH 7 NaAc-HAc buffer solution.

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