Rapid separation and enrichment method of foodborne spores based on dual recognition effect antibiotic-magnetic beads and application

Abstract

This invention belongs to the field of foodborne pathogenic microorganism detection, and relates to a rapid isolation and enrichment method for foodborne spores based on a dual recognition effect using antibiotic-magnetic beads, and its application. The sample to be tested is added to a buffer solution and then mixed with Van-Fe3O4 and Amp-Fe3O4 magnetic beads for incubation. After standing, the magnetic field is used to adsorb the conjugate of the magnetic beads and bacterial vegetative cells onto one side, removing the bacterial vegetative cells. The supernatant is then collected by centrifugation, yielding the enriched product. This invention, by using two antibiotic magnetic beads in combination, achieves rapid isolation and enrichment of bacterial cells and spores in complex microbial systems under the dual recognition effect. The spore isolation and enrichment efficiency can reach 99%, solving the technical problem that existing technologies using single antibiotic magnetic beads cannot achieve complete capture of microorganisms in complex systems. Furthermore, the method provided by this invention does not interfere with or affect the structure and state of the spores. This provides a new approach and method for the rapid isolation and enrichment of foodborne spores in complex systems.

Description

Technical Field

[0001] This invention belongs to the field of foodborne pathogenic microorganism detection, and relates to a method and application for rapid isolation and enrichment of foodborne spores based on antibiotic-magnetic beads with dual recognition effect. Background Technology

[0002] Spores are dormant forms of Bacillus or Clostridium bacteria under nutrient-deficient conditions. Spores are extremely resilient, exhibiting strong resistance to radiation, heat, dryness, extreme pH levels, hydrostatic pressure, and some toxic chemicals. When the external environment becomes suitable for spore growth, they germinate into vegetative bacteria and rapidly multiply, causing food spoilage and even foodborne illnesses, posing a significant safety hazard to consumers. Therefore, rapid detection of foodborne spores is crucial for food safety supervision and management in the medium- and high-temperature meat processing industry. However, in actual sample testing of foodborne spores, interference from the food matrix and coexisting microorganisms is easily encountered. Current rapid detection methods struggle to directly and with high sensitivity and specificity for the rapid detection of foodborne pathogenic spores. Isolating and enriching foodborne spores from complex microbial systems is a critical challenge and key to achieving rapid detection of foodborne spores.

[0003] Currently, enrichment culture and non-selective methods are commonly used for the isolation and enrichment of microorganisms. While enrichment culture yields good enrichment results, it suffers from drawbacks such as cumbersome operation, long processing time, inability to quantify, and difficulty in avoiding interference from other microorganisms, making it unsuitable for the detection of foodborne spores. Common non-selective isolation and enrichment methods include centrifugation and filtration, which rely on the physicochemical properties of the target microorganisms, such as sedimentation coefficient and physical size, to achieve the isolation and enrichment of the target microorganisms. Centrifugation and filtration methods can effectively separate and enrich target substances with large ranges in sedimentation coefficient and physical size, offering advantages such as simple operation and low cost. However, in complex microbial systems, relying solely on differences in particle size and sedimentation coefficient is insufficient to isolate and enrich all foodborne spores. The recovery rate of non-selective isolation and enrichment methods is usually low, affecting the accuracy and sensitivity of subsequent detection.

[0004] In recent years, magnetic separation technology, as a novel selective separation and enrichment method, has been widely used in the pretreatment process for rapid detection of foodborne pathogens. It can optimize or replace traditional separation and enrichment methods to a certain extent, better achieving the separation and enrichment of target microorganisms in complex samples. Among these methods, antigen-antibody reactions are the most common targeting mechanism in magnetic separation; however, antibodies, bacteriophages, and other recognition molecules are costly, require sophisticated operation, and are difficult to prepare. Antibiotics, as broad-spectrum recognition molecules, are stable, readily available, and inexpensive, and have strong binding affinity to bacteria, making them ideal candidates for magnetic separation recognition molecules. The capture ability of different antibiotics for Gram-positive (G+) and Gram-negative (G-) bacteria is closely related to the composition of their outermost structural layer. The cell walls or cell membranes of G+ and G- bacteria contain many sites that specifically target and bind to antibiotics; for example, D-alanyl-D-alanine (D-Ala-D-Ala) in Gram-positive bacteria can be captured by vancomycin (Van). For example, patent CN106957841A discloses a method for rapidly enriching and separating Listeria monocytogenes using vancomycin combined with polyethylene glycol-modified magnetic nanoparticles. The method includes preparing magnetic nanoparticles coupled with polyethylene glycol, vancomycin-coupled polyethylene glycol-coated magnetic nanoparticles, and a composite of polyethylene glycol and vancomycin-modified magnetic nanoparticles to capture Listeria monocytogenes in the sample solution. However, this method using single antibiotic magnetic beads can only capture Listeria monocytogenes in the system, making it difficult to achieve complete capture of microorganisms in complex systems. Summary of the Invention

[0005] To address the technical challenge of using single antibiotic magnetic beads to capture all microorganisms in complex systems, this invention proposes a rapid isolation and enrichment method for foodborne spores based on a dual-recognition effect of antibiotic-magnetic beads, along with its application. Although spores are dormant bacterial cells, their outermost structure is not similar to that of bacteria. This invention combines Van / Fe3O4 and Amp / Fe3O4 magnetic beads, utilizing their multiple recognition effects to remove bacterial vegetative cells from complex microbial systems, achieving rapid isolation and enrichment of spores without affecting their structure and state. This facilitates subsequent rapid detection of foodborne spores.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0007] A rapid isolation and enrichment method for foodborne spores based on antibiotic-magnetic beads with dual recognition effect, comprising the following steps:

[0008] Step 1: Preparation of Fe3O4 nanoparticles. Ferric chloride hexahydrate (FeCl3·6H2O), sodium acetate trihydrate (C2H3O2Na·3H2O), and sodium dodecyl sulfonate (SDS) were dissolved in ethylene glycol. After stirring, the mixture was transferred to a 50 mL microwave synthesis reaction vessel. The reaction was first accelerated by heating at 145–155 °C for 17–22 min, and then heated at 190–210 °C for 35–45 min to facilitate the decomposition and volatilization of ethylene glycol. After cooling to room temperature, the nanoparticles were washed alternately with deionized water and anhydrous ethanol 3–5 times, and then vacuum dried to obtain magnetic Fe3O4 nanoparticles.

[0009] Step 2: Carboxylation treatment of Fe3O4 nanoparticles. 3-Aminopropyltriethoxysilane (APTES), glutaric anhydride, and dimethylformamide (DMF) were sequentially added to a 200 mL round-bottom flask and stirred in a water bath at 29–32 °C for 2.7–3.2 h. Then, Fe3O4 nanoparticles, H2O, and DMF were added sequentially, and stirring continued in a water bath at 29–32 °C for 4.8–5.3 h. After washing 3–5 times with ethanol, the nanoparticles were dried in a drying oven at 60 °C to obtain surface-carboxylated Fe3O4 magnetic nanoparticle powder for later use.

[0010] Step 3: Preparation of Van-Fe3O4 and Amp-Fe3O4 antibiotic magnetic beads.

[0011] (1) Activation of surface carboxylated Fe3O4 magnetic nanoparticles. Take a solution of surface carboxylated Fe3O4 magnetic nanoparticles and add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to activate the nanoparticles at room temperature for 60 min. Wash the nanoparticles with PBS buffer 2 to 3 times for later use.

[0012] (2) Activation of antibiotics. Vancomycin (Van) and penicillin (Amp) were respectively added to EDC solution (10 mg / mL) and NHS solution (10 mg / mL) and activated in the dark for 8-10 min for later use.

[0013] (3) At room temperature, the activated antibiotics (Van and Amp) were incubated with activated surface carboxylated Fe3O4 magnetic nanoparticles for 120 min, washed 3 to 5 times with PBS buffer, and resuspended in 10 mL PBS buffer (0.01 mol / L, pH 7.4) to obtain antibiotic-Fe3O4 magnetic beads, namely Van / Fe3O4 and Amp / Fe3O4 magnetic beads, which were stored at 4 °C for use.

[0014] Step 4, based on the above steps, outlines the specific operational procedure for the rapid isolation and enrichment method of foodborne spores using Van / Fe3O4-Amp / Fe3O4 antibiotic magnetic beads based on the dual recognition effect. This includes: placing a mixed microbial culture in sterile PBS buffer, then adding Van / Fe3O4 and Amp / Fe3O4 antibiotic magnetic beads, and incubating on a shaker at 20–25°C for 40–50 minutes. After incubation, the mixture is placed on a magnetic rack and allowed to stand for 3 minutes. The magnetic field is used to adsorb the magnetic beads and bacterial vegetative cells onto one side, removing the bacterial vegetative cells. The supernatant is then poured off, and the enriched product is collected by centrifugation at 7000 rpm.

[0015] Furthermore, in step 1, the molar ratio of ferric chloride hexahydrate, sodium acetate trihydrate, and sodium dodecyl sulfonate is (9-11):(3-5):(2-4).

[0016] Furthermore, each 25 mL of ethylene glycol in step 1 contains 9–11 mmol of ferric chloride hexahydrate.

[0017] Furthermore, in step 2, the volume ratio of 3-aminopropyltriethoxysilane to dimethylformamide is (1.70–1.78):(50–65), and for every 1.70–1.78 mL of 3-aminopropyltriethoxysilane added, 0.80–0.88 g of glutaric anhydride needs to be added.

[0018] Furthermore, in step 2, the mass-to-volume ratio of 3-aminopropyltriethoxysilane to H2O and DMF is (1.70–1.78):(8–10):(95–105), and for every 1.70–1.78 mL of 3-aminopropyltriethoxysilane added, 1.1–1.3 g of magnetic Fe3O4 nanoparticles need to be added.

[0019] Furthermore, in step 3(1), the concentrations of the surface carboxylated Fe3O4 magnetic nanoparticle solution, the EDC solution, and the NHS solution are 5 mg / mL, 10 mg / mL, and 10 mg / mL, respectively, and the volume ratio of the surface carboxylated Fe3O4 magnetic nanoparticle solution to the EDC solution and the NHS solution is 2 mL:(300~500) μL:(300~500) μL.

[0020] Furthermore, in step 3(1), the concentration of the PBS buffer is 0.01 mol / L and the pH is 7.4.

[0021] Furthermore, in step 3(2), 1.5 mL of EDC solution and 1.5 mL of NHS solution need to be added for every 100 mg Van; 1.5 mL of EDC solution and 1.5 mL of NHS solution need to be added for every 100 mg Amp, with the concentrations of EDC solution and NHS solution being 10 mg / mL and 10 mg / mL, respectively.

[0022] Furthermore, in step 3(3), the concentrations of Van / Fe3O4 antibiotic magnetic beads and Amp / Fe3O4 antibiotic magnetic beads after resuspension are 0.8–1.2 mg / mL.

[0023] Furthermore, in step 4, the concentrations of both bacterial vegetative cells and spores in the mixed microbial solution are 10. 4 CFU / mL, the volume ratio of bacterial vegetative cells to spores is 1:1; the volume ratio of the test sample, buffer solution, Van / Fe3O4 magnetic beads and Amp / Fe3O4 magnetic beads is 1:(7~8):(1~1.5):(1~1.5).

[0024] The application of antibiotic-magnetic beads obtained by the above method in the rapid isolation and enrichment of foodborne spores.

[0025] The present invention has the following beneficial effects:

[0026] Compared to the 78.86% bacterial vegetative cell removal efficiency and 96.42% spore collection efficiency achieved by using Van / Fe3O4 antibiotic magnetic beads alone in complex microbial systems, or the 84.64% bacterial vegetative cell removal efficiency and 95.64% spore collection efficiency achieved by using Amp / Fe3O4 antibiotic magnetic beads alone in complex microbial systems, the method of this invention achieves a dual recognition effect by using Van / Fe3O4-Amp / Fe3O4 antibiotic magnetic beads in combination. This is based on the recognition principle that Van can bind to D-Ala-D-Ala on the surface of Gram-positive bacterial cell walls, and Amp can covalently bind to penicillin-binding proteins (PBPs) on most bacterial cell walls. This enables the rapid separation and enrichment of bacterial cells and spores in complex microbial systems. The obtained spore separation and enrichment efficiency can reach 99.98%, and the bacterial vegetative cell removal efficiency can reach 99.13%. Moreover, this method does not interfere with or affect the structure ("bright white center" oval structure under phase contrast microscopy) and state of the spores. Furthermore, this invention also studies the isolation and enrichment effect of foodborne spores (taking Bacillus cereus as an example) in complex systems of common foodborne pathogens (taking Staphylococcus aureus, Salmonella, Clostridium perfringens, Bacillus subtilis, and Bacillus cereus as examples). It was found that the Van / Amp-Fe3O4 magnetic beads based on the dual recognition effect achieved a 99.22% removal efficiency for bacterial vegetative cells and a 99.16% spore collection efficiency. This demonstrates that the Van / Amp-Fe3O4 magnetic beads prepared in this invention, based on the dual recognition effect, have a good effect on the isolation and enrichment of foodborne spores in complex systems of common foodborne pathogens. This invention provides new thinking and methods for the rapid isolation and enrichment of foodborne spores in complex systems, provides technical support for the rapid detection of foodborne spores, and provides protection for the quality and safety supervision of the meat products industry and consumer health. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram illustrating the preparation principle of Van / Fe3O4 and Amp / Fe3O4 antibiotic magnetic beads in Example 1 of the present invention, wherein (A) preparation of Fe3O4 nanoparticles; (B) carboxylation treatment of Fe3O4 nanoparticles; and (C) preparation of Van-Fe3O4 and Amp-Fe3O4 antibiotic magnetic beads.

[0029] Figure 2This is a flowchart of the rapid isolation and enrichment method for foodborne spores based on Van / Amp-Fe3O4 magnetic beads with dual recognition effect in Embodiment 1 of the present invention.

[0030] Figure 3 This is a scanning electron microscope image of the Fe3O4 magnetic nanoparticles prepared in step 1 of Example 1 of the present invention.

[0031] Figure 4 The images show the characterization of Van / Fe3O4 and Amp / Fe3O4 antibiotic magnetic beads in Example 1 of this invention. (A) and (B) are the UV-Vis absorption spectra of Van / Fe3O4 and Amp / Fe3O4 antibiotic magnetic beads; (C) and (D) are the hysteresis loop characterization results of Van / Fe3O4 and Amp / Fe3O4 antibiotic magnetic beads; and (E) and (F) are the UV-Vis absorption scanning electron microscopy results of Van / Fe3O4 and Amp / Fe3O4 antibiotic magnetic beads.

[0032] Figure 5 The images show a comparison of phase-contrast microscopy results before and after spore isolation and enrichment in a mixed system of Salmonella, Bacillus cereus, and Bacillus cereus spores using Van / Fe3O4, Amp / Fe3O4, and Van / Fe3O4-Amp / Fe3O4 antibiotic magnetic beads in Example 2 of the efficacy test of this invention. (A) is a phase-contrast microscopy image of the mixed microbial system before isolation and enrichment; (B) is a phase-contrast microscopy image of the enriched product after isolation and enrichment using Van / Fe3O4 antibiotic magnetic beads alone; (C) is a phase-contrast microscopy image of the enriched product after isolation and enrichment using Amp / Fe3O4 antibiotic magnetic beads alone; and (D) is a phase-contrast microscopy image of the enriched product after isolation and enrichment using Van / Amp-Fe3O4 magnetic beads in combination.

[0033] Figure 6 The images shown are phase-contrast microscopy, scanning electron microscopy, and Raman spectra of foodborne spores in a complex system of common foodborne pathogens after isolation and enrichment of foodborne spores in the enriched product based on the dual recognition effect of Van / Amp-Fe3O4 magnetic beads in Example 3 of the present invention. (A) is a phase-contrast microscopy image of foodborne spores in the enriched product; (B) is a scanning electron microscopy image of foodborne spores in the enriched product; (C) is a Raman spectrum comparison between Bacillus cereus spores in the enriched product and pure Bacillus cereus spores, where a is the Raman spectrum of Bacillus cereus spores in the enriched product and b is the Raman spectrum of pure Bacillus cereus spores. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] All raw materials used in this invention are commercially available or prepared using conventional methods in the art. In this invention, Van / Amp-Fe3O4 is Van / Fe3O4-Amp / Fe3O4. The solvents in the surface-carboxylated Fe3O4 magnetic nanoparticle solution, EDC solution, and NHS solution are all ultrapure water.

[0036] Example 1

[0037] like Figure 1 and Figure 2 The diagram shows a flowchart illustrating the rapid isolation and enrichment method and application of foodborne spores based on antibiotic-magnetic beads with dual recognition effects, including the following steps:

[0038] Step 1: Preparation of Fe3O4 nanoparticles. 10 mmol of ferric chloride hexahydrate (FeCl3·6H2O), 4 mmol of sodium acetate trihydrate (C2H3O2Na·3H2O), and 3 mmol of sodium dodecyl sulfonate (SDS) were dissolved in 25 mL of ethylene glycol. After stirring, the mixture was transferred to a 50 mL microwave synthesis reaction vessel. The reaction was first accelerated by heating at 150 °C for 20 min, then heated at 200 °C for 40 min to facilitate the decomposition and volatilization of ethylene glycol. After cooling to room temperature, the nanoparticles were washed alternately with deionized water and anhydrous ethanol 3–5 times, and then vacuum dried to obtain magnetic Fe3O4 nanoparticles. Figure 1 As shown in (A).

[0039] Step 2: Carboxylation treatment of Fe3O4 nanoparticles. 1.74 mL of 3-aminopropyltriethoxysilane (APTES), 0.84 g of glutaric anhydride, and 60 mL of dimethylformamide (DMF) were sequentially added to a 200 mL round-bottom flask and stirred in a 30°C water bath for 3 h. Then, 1.2 g of Fe3O4 nanoparticles, 9 mL of H2O, and 100 mL of DMF were added sequentially, and stirring was continued in a 30°C water bath for 5 h. After washing 3–5 times with ethanol, the nanoparticles were dried in a 60°C drying oven to obtain surface-carboxylated Fe3O4 magnetic nanoparticle powder for later use. Figure 1 As shown in (B).

[0040] Step 3: Preparation of Van-Fe3O4 and Amp-Fe3O4 antibiotic magnetic beads. (1) Activation of surface carboxylated Fe3O4 magnetic nanoparticles. Take 2 mL of surface carboxylated Fe3O4 magnetic nanoparticle solution (5 mg / mL, solvent: ultrapure water) and add 400 μL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 10 mg / mL, solvent: ultrapure water) and 400 μL of N-hydroxysuccinimide (NHS, 10 mg / mL, solvent: ultrapure water) at room temperature for 60 min. Wash 2-3 times with 9 mL of PBS buffer (0.01 mol / L, pH 7.4) for later use. (2) Activation of antibiotics. Add 1.5 mL of EDC (10 mg / mL) and 1.5 mL of NHS (10 mg / mL) to 100 mg vancomycin (Van) and 100 mg penicillin (Amp), respectively, and activate for 10 min in the dark for later use. (3) At room temperature, incubate the activated antibiotics (Van and Amp) with activated surface carboxylated Fe3O4 magnetic nanoparticles for 120 min, wash 3-5 times with PBS buffer (0.01 mol / L, pH 7.4), and resuspend in 10 mL of PBS buffer (0.01 mol / L, pH 7.4) to obtain antibiotic-Fe3O4 magnetic beads, namely Van / Fe3O4 and Amp / Fe3O4 magnetic beads, with a concentration of 1.0 mg / mL, and store at 4℃ for use. Figure 1 As shown in (C).

[0041] Step 4: Based on the above steps, the detailed operation flowchart of the rapid isolation and enrichment method for foodborne spores based on the dual recognition effect of Van / Amp-Fe3O4 magnetic beads is as follows: Figure 2 As shown, the main steps include: taking 100 μL of mixed microbial culture (the concentration of both bacterial vegetative cells and spores is 10). 4 CFU / mL (1:1 volume ratio) was added to 750 μL of sterile PBS buffer, followed by the addition of 125 μL Van / Fe3O4 and 125 μL Amp / Fe3O4 antibiotic magnetic beads. The mixture was incubated on a shaker for 45 min to allow the bacterial vegetative cells to fully bind with the Van / Amp-Fe3O4 beads. After incubation, the mixture was placed on a magnetic rack for 3 min. The magnetic field was used to adsorb the binding material between the magnetic beads and bacterial vegetative cells to one side, removing the bacterial vegetative cells. The supernatant was discarded, and the enriched product was collected by centrifugation at 7000 rpm. The sterilization efficiency and spore loss rate were calculated using the plate count method. The isolation and enrichment effect of foodborne spores was observed using phase-contrast microscopy, and the surface structure and morphology were observed using scanning electron microscopy (SEM) to determine if they were affected.

[0042] The Fe3O4 magnetic nanoparticles and Van-Fe3O4 and Amp-Fe3O4 antibiotic magnetic beads obtained in the above steps were characterized by ultraviolet-visible spectrophotometry (UV-Vis), scanning electron microscopy (SEM), and hysteresis loop testing (VSM). The results are as follows: Figure 3 and Figure 4 As shown.

[0043] Depend on Figure 3 Scanning electron microscopy (SEM) images show that the Fe3O4 magnetic nanoparticles have a uniform spherical structure. According to the particle size distribution calculation software Nano Measurer, the average particle size of the Fe3O4 magnetic nanoparticles is about 200 nm.

[0044] exist Figure 4 (A) The UV-Vis spectra of Van-Fe3O4 and Van show the same absorption peak at 280 nm, which preliminarily proves the successful combination of Van and Fe3O4; similarly... Figure 4 (B) In this study, Amp-Fe3O4 and Amp also exhibit the same absorption peak at 235 nm, preliminarily proving the successful binding of Amp and Fe3O4. Figure 4 The hysteresis loop results in (C) and (D) show that the Fe3O4 magnetic nanoparticles have good magnetic saturation strength. After reacting with Van and Amp to form Van / Fe3O4 and Amp / Fe3O4 antibiotic magnetic beads, the magnetic saturation strength is significantly reduced, indicating that Van and Amp are successfully modified on the Fe3O4 surface. Moreover, all curves intersect the origin and no hysteresis occurs, indicating that Van-Fe3O4 and Amp-Fe3O4 antibiotic magnetic beads retain excellent superparamagnetism. Figure 4 SEM results (E) and (F) show that the Van-Fe3O4 and Amp-Fe3O4 antibiotic magnetic beads have a uniform spherical structure, good dispersibility, and are compatible with... Figure 3 Compared with the electron microscopy images of Fe3O4 magnetic nanoparticles, small antibiotic particles can be clearly observed on the surface of Van-Fe3O4 and Amp-Fe3O4 antibiotic magnetic beads, indicating that Van and Amp were successfully coupled to the surface of Fe3O4 magnetic nanomaterials.

[0045] Example 2

[0046] A rapid isolation and enrichment method for foodborne spores based on antibiotic-magnetic beads with dual recognition effect and its application, including the following steps:

[0047] Step 1: Preparation of Fe3O4 nanoparticles. 9 mmol of ferric chloride hexahydrate (FeCl3·6H2O), 3 mmol of sodium acetate trihydrate (C2H3O2Na·3H2O), and 2 mmol of sodium dodecyl sulfonate (SDS) were dissolved in 25 mL of ethylene glycol. After stirring, the mixture was transferred to a 50 mL microwave synthesis reaction vessel. The reaction was first accelerated by heating at 145 °C for 22 min, then heated at 190 °C for 35 min. After cooling to room temperature to facilitate the decomposition and volatilization of ethylene glycol, the nanoparticles were washed alternately with deionized water and anhydrous ethanol 3–5 times, and then vacuum dried to obtain magnetic Fe3O4 nanoparticles. Figure 1 As shown in (A).

[0048] Step 2: Carboxylation treatment of Fe3O4 nanoparticles. 1.70 mL of 3-aminopropyltriethoxysilane (APTES), 0.80 g of glutaric anhydride, and 65 mL of dimethylformamide (DMF) were sequentially added to a 200 mL round-bottom flask and stirred in a water bath at 29 °C for 2.7 h. Then, 1.1 g of Fe3O4 nanoparticles, 8 mL of H2O, and 95 mL of DMF were added sequentially, and stirring was continued in a water bath at 32 °C for 4.8 h. After washing 3–5 times with ethanol, the nanoparticles were dried in a drying oven at 60 °C to obtain surface-carboxylated Fe3O4 magnetic nanoparticle powder for later use. Figure 1 As shown in (B).

[0049] Step 3: Preparation of Van-Fe3O4 and Amp-Fe3O4 antibiotic magnetic beads. (1) Activation of surface carboxylated Fe3O4 magnetic nanoparticles. Take 2 mL of surface carboxylated Fe3O4 magnetic nanoparticle solution (5 mg / mL) and add 300 μL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 10 mg / mL) and 300 μL of N-hydroxysuccinimide (NHS, 10 mg / mL) at room temperature for 60 min. Wash 2-3 times with 8 mL of PBS buffer (0.01 mol / L, pH 7.4) for later use. (2) Activation of antibiotics. Add 1.5 mL of LEDC (10 mg / mL) and 1.5 mL of NHS (10 mg / mL) to 100 mg vancomycin (Van) and 100 mg penicillin (Amp), respectively, and activate in the dark for 8 min for later use. (3) At room temperature, activated antibiotics (Van and Amp) were incubated with activated surface-carboxylated Fe3O4 magnetic nanoparticles for 120 min. After washing 3–5 times with PBS buffer (0.01 mol / L, pH 7.4), the nanoparticles were resuspended in 10 mL of PBS buffer (0.01 mol / L, pH 7.4) to obtain antibiotic-Fe3O4 magnetic beads, namely Van / Fe3O4 and Amp / Fe3O4 magnetic beads, which were stored at 4°C for use. Figure 1As shown in (C).

[0050] Step 4: Based on the above steps, the detailed operation flowchart of the rapid isolation and enrichment method for foodborne spores based on the dual recognition effect of Van / Amp-Fe3O4 magnetic beads is as follows: Figure 2 As shown, the main steps include: taking 100 μL of mixed microbial culture (the concentration of both bacterial vegetative cells and spores is 10). 4 CFU / mL (1:1 volume ratio) was added to 700 μL of sterile PBS buffer, followed by the addition of 100 μL Van / Fe3O4 antibiotic magnetic beads and 100 μL Amp / Fe3O4 antibiotic magnetic beads. The mixture was incubated on a shaker at 20°C for 40 min to allow the bacterial vegetative cells to fully bind with the Van / Fe3O4-Amp / Fe3O4 antibiotic magnetic beads. After incubation, the mixture was placed on a magnetic rack for 3 min. The magnetic field was used to adsorb the binding material between the magnetic beads and bacterial vegetative cells to one side, removing the bacterial vegetative cells. The supernatant was discarded, and the enriched product was collected by centrifugation at 7000 rpm. The sterilization efficiency and spore loss rate were calculated using the plate count method. The isolation and enrichment effect of foodborne spores was observed using phase-contrast microscopy, and the surface structure and morphology were observed using scanning electron microscopy (SEM) to determine if they were affected.

[0051] Example 3

[0052] A rapid isolation and enrichment method for foodborne spores based on antibiotic-magnetic beads with dual recognition effect and its application, including the following steps:

[0053] Step 1: Preparation of Fe3O4 nanoparticles. Ferric chloride hexahydrate (FeCl3·6H2O), 5 mmol sodium acetate trihydrate (C2H3O2Na·3H2O), and 4 mmol sodium dodecyl sulfonate (SDS) were dissolved in 25 mL ethylene glycol. After stirring, the mixture was transferred to a 50 mL microwave synthesis reaction vessel. The reaction was first accelerated by heating at 155 °C for 17 min, then heated at 210 °C for 45 min. After cooling to room temperature to facilitate the decomposition and volatilization of ethylene glycol, the nanoparticles were washed alternately with deionized water and anhydrous ethanol 3–5 times, and then vacuum dried to obtain magnetic Fe3O4 nanoparticles. Figure 1 As shown in (A).

[0054] Step 2: Carboxylation treatment of Fe3O4 nanoparticles. 1.78 mL of 3-aminopropyltriethoxysilane (APTES), 0.88 g of glutaric anhydride, and 50 mL of dimethylformamide (DMF) were sequentially added to a 200 mL round-bottom flask and stirred in a 32°C water bath for 3.2 h. Then, 1.3 g of Fe3O4 nanoparticles, 10 mL of H2O, and 105 mL of DMF were added sequentially, and stirring was continued in a 29°C water bath for 5.3 h. After washing 3–5 times with ethanol, the nanoparticles were dried in a 60°C drying oven to obtain surface-carboxylated Fe3O4 magnetic nanoparticle powder for later use. Figure 1 As shown in (B).

[0055] Step 3: Preparation of Van-Fe3O4 and Amp-Fe3O4 antibiotic magnetic beads. (1) Activation of surface carboxylated Fe3O4 magnetic nanoparticles. Take 2 mL of surface carboxylated Fe3O4 magnetic nanoparticle solution (5 mg / mL) and add 500 μL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 10 mg / mL) and 500 μL of N-hydroxysuccinimide (NHS, 10 mg / mL) at room temperature for 60 min. Wash 2-3 times with 10 mL of PBS buffer (0.01 mol / L, pH 7.4) for later use. (2) Activation of antibiotics. Add 1.5 mL of LEDC (10 mg / mL) and 1.5 mL of NHS (10 mg / mL) to 100 mg vancomycin (Van) and 100 mg penicillin (Amp), respectively, and activate in the dark for 10 min for later use. (3) At room temperature, activated antibiotics (Van and Amp) were incubated with activated surface-carboxylated Fe3O4 magnetic nanoparticles for 120 min. After washing 3–5 times with PBS buffer (0.01 mol / L, pH 7.4), the nanoparticles were resuspended in 10 mL of PBS buffer (0.01 mol / L, pH 7.4) to obtain antibiotic-Fe3O4 magnetic beads, namely Van / Fe3O4 and Amp / Fe3O4 magnetic beads, which were stored at 4°C for use. Figure 1 As shown in (C).

[0056] Step 4: Based on the above steps, the detailed operation flowchart of the rapid isolation and enrichment method for foodborne spores based on the dual recognition effect of Van / Amp-Fe3O4 magnetic beads is as follows: Figure 2 As shown, the main steps include: taking 100 μL of mixed microbial culture (the concentration of both bacterial vegetative cells and spores is 10). 4CFU / mL (1:1 volume ratio) was added to 800 μL of sterile PBS buffer, followed by the addition of 150 μL Van / Fe3O4 and 150 μL Amp / Fe3O4 antibiotic magnetic beads. The mixture was incubated on a shaker at 25°C for 50 min to allow for thorough binding of bacterial vegetative cells with the Van / Fe3O4-Amp / Fe3O4 antibiotic magnetic beads. After incubation, the mixture was placed on a magnetic rack for 3 min. The magnetic field was used to adsorb the binding material between the magnetic beads and bacterial vegetative cells to one side, removing the bacterial vegetative cells. The supernatant was discarded, and the enriched product was collected by centrifugation at 7000 rpm. The sterilization efficiency and spore loss rate were calculated using the plate count method. The isolation and enrichment effect of foodborne spores was observed using phase-contrast microscopy, and the surface structure and morphology were examined using scanning electron microscopy (SEM) to determine if they were affected.

[0057] Example 1 of the effect test

[0058] This test compares the recognition and capture abilities of common antibiotics against Gram-positive and Gram-negative bacteria, ultimately determining the antibiotics with the optimal recognition and capture abilities for each type of bacteria. Common antibiotics included vancomycin (Van), polymyxin B sulfate (PB), ampicillin sodium (Amp), chloramphenicol (CL), erythromycin (EM), kanamycin monosulfate (KM), and nisin.

[0059] (1) Preparation of microbial samples. Staphylococcus aureus and Salmonella were selected as representative strains of Gram-positive and Gram-negative bacteria, respectively. Staphylococcus aureus and Salmonella stored at -80℃ were activated by culturing on LB agar at 37℃ for 18–24 h, and then transferred to LB liquid culture medium for enrichment culture on a shaker at 180 r / min for 18–24 h. The concentration was adjusted to 10⁻⁶ using plate counting. 4 CFU / mL was prepared for use. Staphylococcus aureus and Salmonella were cultured and counted separately on nutrient agar medium.

[0060] (2) Seven different antibiotic magnetic beads, namely Van / Fe3O4, PB / Fe3O4, Amp / Fe3O4, CL / Fe3O4, EM / / Fe3O4, KM / Fe3O4 and Nisin / Fe3O4, were prepared according to the operating steps in Example 1. These seven antibiotic magnetic beads were then subjected to a known concentration of 10... 4The capture efficiency of Staphylococcus aureus and Salmonella at CFU / mL was calculated. The capture efficiency of seven different antibiotic magnetic beads against Staphylococcus aureus (G+ bacteria) and Salmonella (G- bacteria) was verified by plate counting comparison. The results are shown in Table 1. The results showed that Van / Fe3O4 antibiotic magnetic beads had the best capture efficiency against Staphylococcus aureus (G+ bacteria), reaching 97.32%, while Amp / Fe3O4 had the best capture efficiency against Salmonella (G- bacteria), reaching 94.71%. Therefore, Van / Fe3O4 and Amp / Fe3O4, which had the best recognition and capture effects against Gram-positive and Gram-negative bacteria respectively, were selected to further construct dual-recognition effect magnetic beads, realizing a rapid isolation and enrichment strategy for spores from complex microbial systems using dual-recognition effect antibiotic magnetic beads.

[0061] Table 1. Capture efficiency of seven different antibiotic magnetic beads against Staphylococcus aureus and Salmonella.

[0062]

[0063]

[0064] Example 2 of effect test

[0065] This test case aims to determine the removal efficiency of bacterial vegetative cells and the isolation and enrichment effect of foodborne spores in a mixed system of Salmonella, Bacillus cereus, and Bacillus cereus spores, using Van / Fe3O4, Amp / Fe3O4 antibiotic magnetic beads alone and in combination.

[0066] (1) Preparation of microbial samples. Salmonella and Bacillus cereus stored at -80℃ were activated by culturing on LB agar medium at 37℃ for 18–24 h, and then transferred to LB liquid culture medium for enrichment culture on a shaker at 180 r / min for 18–24 h. The concentration was adjusted to 10 by plate counting. 4 CFU / mL was kept on hand. Salmonella and Bacillus cereus were cultured and counted separately on nutrient agar medium. Bacillus cereus spore culture: Single colonies of Bacillus cereus obtained from LB agar medium in the above steps were picked and inoculated into LB liquid medium. After incubation at 150 rpm and 37°C for 18–24 h on a shaker, the colonies were transferred to LB agar medium supplemented with 50 mg / L MnSO4 and then incubated at 37°C for 7 days before centrifugation to collect spores. After pretreatment with water at 80°C for 15 min (which kills the vegetative cells of the bacteria but does not kill the spores and to some extent stimulates spore germination and cell formation), the spore concentration was calculated by culturing on nutrient agar medium.

[0067] (2) The known concentration is 104 Salmonella, Bacillus cereus, and Bacillus cereus spores (CFU / mL) were mixed (volume ratio 1:1:1) and then processed according to step 4 in Example 1. Experimental group 1 used only Van / Fe3O4 antibiotic magnetic beads, experimental group 2 used only Amp / Fe3O4 antibiotic magnetic beads, and experimental group 3 used a combination of Van / Fe3O4 and Amp / Fe3O4 antibiotic magnetic beads. Plate counting was used to verify the removal efficiency of bacterial vegetative cells (G+ and G- bacteria) and spore isolation and enrichment effects of the three antibiotic magnetic bead application methods in the mixed microbial system. Salmonella and Bacillus cereus were cultured and counted on nutrient agar medium. Spore concentrations were pretreated with water at 80℃ for 15 min before being cultured and counted on LB agar medium.

[0068] The bacterial vegetative cell removal efficiency was calculated using formulas (1) to (3). The calculated bacterial vegetative cell removal efficiencies for the three experimental groups are shown in Table 1.

[0069] X1=X2-Y1 Formula (1)

[0070] S1=(C1-X1) / C1 Formula (2)

[0071] S2=Y1 / C2 Formula (3)

[0072] X1 represents the concentration of residual bacterial vegetative cells in the enriched product after separation and enrichment using this method, in units of lg (CFU / mL).

[0073] X2 represents the sum of the concentrations of residual bacterial vegetative cells and spores in the enriched product after separation and enrichment by this method without treatment at 80℃ for 15 min, in units of lg (CFU / mL).

[0074] Y1 represents the concentration of spores in the enriched product after separation and enrichment using this method, and is expressed in units of lg (CFU / mL).

[0075] S1 represents the removal efficiency of bacterial vegetative cells after separation and enrichment by this method, in percentage (%).

[0076] C1 represents the concentration of bacterial vegetative cells in a complex microbial system before isolation and enrichment using this method, expressed in lg (CFU / mL).

[0077] C2 represents the concentration of foodborne spores in a complex microbial system before isolation and enrichment using this method, expressed in lg (CFU / mL).

[0078] Table 1. Comparison of bacterial vegetative cell removal efficiency and spore collection efficiency in complex microbial systems among the three experimental groups.

[0079] Experimental group Experimental group 1 Experimental group 2 Experimental group 3 Bacterial vegetative removal efficiency 78.86％ 84.64％ 99.13％ Spore collection efficiency 96.42％ 95.64％ 99.98％

[0080] The results in Table 1 show that Van / Amp-Fe3O4 magnetic beads have the best removal efficiency of bacterial vegetative cells (G+ and G- bacteria) and spore collection efficiency in mixed microbial systems, both exceeding 99%.

[0081] at the same time, Figure 5 The images show a comparison of phase-contrast microscopy results before and after spore isolation and enrichment in a mixed system of Salmonella, Bacillus cereus, and Bacillus cereus spores using Van / Fe3O4, Amp / Fe3O4, and Van / Amp-Fe3O4 magnetic beads in Example 2 of the efficacy test of this invention. (A) is a phase-contrast microscopy image of the mixed microbial system before isolation and enrichment; (B) is a phase-contrast microscopy image of the enriched product after isolation and enrichment using Van / Fe3O4 antibiotic magnetic beads alone; (C) is a phase-contrast microscopy image of the enriched product after isolation and enrichment using Amp / Fe3O4 antibiotic magnetic beads alone; and (D) is a phase-contrast microscopy image of the enriched product after isolation and enrichment using a combination of Van / Fe3O4 and Amp / Fe3O4 antibiotic magnetic beads. Figure 5 Phase contrast microscopy results of the enriched samples showed that the purity of spores obtained by using Van / Amp-Fe3O4 magnetic beads in experimental group 3 was the best. The spores exhibited an elliptical structure with a bright white center under phase contrast microscopy. Therefore, the method of rapid isolation and enrichment of foodborne spores in mixed microbial systems based on Van / Amp-Fe3O4 magnetic beads with a dual recognition effect is feasible.

[0082] Example 3 of the effect test

[0083] This test example used the antibiotics polymyxin B (PB) and chloramphenicol (CL). Following steps 3 and 4 in Example 2, Van / PB-Fe3O4, Van / CL-Fe3O4, Amp / PB-Fe3O4, Amp / CL-Fe3O4 and PB / CL-Fe3O4 magnetic beads with dual recognition effects were prepared, as well as Van / Amp / CL-Fe3O4 magnetic beads, Van / Amp / PB-Fe3O4 magnetic beads with triple antibiotic combination, and Van / Amp / PB / CL-Fe3O4 magnetic beads with quadruple antibiotic combination, in order to determine the superior isolation and enrichment effect of Van / Amp-Fe3O4 magnetic beads in antibiotic-magnetic beads with dual recognition effects on foodborne spores in complex systems of common foodborne pathogens.

[0084] Common foodborne pathogens (such as Staphylococcus aureus, Salmonella, Clostridium perfringens, Bacillus subtilis, and Bacillus cereus) and foodborne spores (such as Bacillus cereus) were activated and mixed according to the procedures in (1) and (2) of Effect Test Example 1. The removal efficiency of bacterial vegetative cells and the spore collection efficiency were calculated according to formulas (1) to (3) in Effect Test Example 1, and the results are shown in Table 2.

[0085] Table 2 Comparison of the effects of antibiotic-magnetic beads on spore isolation and enrichment in complex systems of common foodborne pathogens.

[0086]

[0087]

[0088] Table 2 shows that Van / Amp-Fe3O4 magnetic beads exhibited the best isolation and enrichment effect on foodborne spores in complex systems containing common foodborne pathogens, maintaining a removal efficiency of over 99% for bacterial vegetative cells and a spore collection efficiency of over 99%. Among these, the triple-combination of Van / Amp / CL-Fe3O4 antibiotic magnetic beads and Van / Amp / PB-Fe3O4... 44 Antibiotic magnetic beads and quadruple therapy using Van / Amp / PB / CL-Fe3O 44 The removal efficiency of antibiotic magnetic beads for bacterial vegetative cells decreased significantly. This may be because the use of antibiotic magnetic beads with low recognition effect on bacterial vegetative cells (PB-Fe3O4 and CL-Fe3O4) reduced the ability of triple and quadruple use of antibiotic magnetic beads to capture bacterial cells. This further verifies that antibiotic magnetic beads with dual recognition effect are the most efficient and cost-effective way to achieve rapid separation and enrichment of spores in complex systems.

[0089] Further research was conducted on the effects of Van / Amp-Fe3O4 magnetic beads based on dual recognition effect on the structure and state of spores isolated and enriched in complex systems of common foodborne pathogens. Figure 6 The images shown are phase-contrast microscopy, scanning electron microscopy, and Raman spectra comparisons of foodborne spores isolated and enriched from a complex system of common foodborne pathogens using Van / Amp-Fe3O4 magnetic beads based on the dual recognition effect, as presented in Example 3 of the efficacy test of this invention. (A) is a phase-contrast microscopy image of the foodborne spores in the enrichment; (B) is a scanning electron microscopy image of the foodborne spores in the enrichment; (C) is a Raman spectrum comparison between Bacillus cereus spores in the enrichment and pure Bacillus cereus spores, where a is the Raman spectrum of Bacillus cereus spores in the enrichment and b is the Raman spectrum of pure Bacillus cereus spores. Figure 6 (A) indicates that, under a phase-contrast microscope, the enriched material after separation and enrichment contains relatively pure foodborne spores with almost no interference from bacterial vegetative cells. For example... Figure 6 As shown in (B), scanning electron microscopy observation of the spores in the isolated spores revealed that the isolated and enriched spores still exhibited an elliptical structure, with no structural damage and no magnetic material particles adhering to the surface. Raman spectroscopy, as a bio-fingerprinting technique, can provide rich information on molecular structure and composition. Raman spectra of Bacillus cereus and pure Bacillus cereus samples were collected and compared, and the results are as follows: Figure 6 As shown in (C), the main characteristic peak (a) of Bacillus cereus spores in the enrichment is basically consistent with the main characteristic peak (b) of pure Bacillus cereus spores without this method in terms of peak position and peak intensity. Therefore, this method, based on the dual recognition effect, has no effect on the structure and state of foodborne spores after isolation and enrichment of foodborne spores in complex systems of common foodborne pathogens by Van / Amp-Fe3O4 magnetic beads.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for rapid isolation and enrichment of foodborne spores based on antibiotic-magnetic beads with dual recognition effect, characterized in that, The steps are as follows: (1) Preparation of Fe3O4 nanoparticles: Ferric chloride hexahydrate, sodium acetate trihydrate and sodium dodecyl sulfonate are dissolved in solvent I, stirred and microwave reaction is carried out. After the reaction is completed, the mixture is cooled to room temperature, washed and dried to obtain magnetic Fe3O4 nanoparticles; the molar ratio of ferric chloride hexahydrate, sodium acetate trihydrate and sodium dodecyl sulfonate in step (1) is (9~11):(3~5):(2~4), and solvent I is ethylene glycol; the microwave reaction in step (1) is carried out as follows: first heat at 145~155℃ for 17~22 min, and then heat at 190~210℃ for 35~45 min; (2) Carboxylation treatment of Fe3O4 nanoparticles: 3-aminopropyltriethoxysilane, glutaric anhydride and dimethylformamide were mixed and stirred for the first time under water bath conditions. Then, the magnetic Fe3O4 nanoparticles obtained in step (1), H2O and DMF were added in sequence. The mixture was stirred for the second time under water bath conditions. After washing, it was dried to obtain Fe3O4 magnetic nanoparticle powder with carboxylation on the surface. The volume ratio of 3-aminopropyltriethoxysilane to dimethylformamide was (1.70~1.78):(50~65). For every 1.70~1.78 mL of 3-aminopropyltriethoxysilane added, 0.80~0.88 g of glutaric anhydride was added. The mass-volume ratio of 3-aminopropyltriethoxysilane to H2O and DMF was (1.70~1.78):(8~10):(95~105). For every 1.70~1.78 mL of 3-aminopropyltriethoxysilane added, 0.80~0.88 g of glutaric anhydride was added. 3-Aminopropyltriethoxysilane requires the addition of 1.1~1.3 g of magnetic Fe3O4 nanoparticles; (3) Preparation of Van / Fe3O4 and Amp / Fe3O4 antibiotic magnetic beads: The surface carboxylated Fe3O4 magnetic nanoparticle powder, Van and Amp obtained in step (2) were activated respectively, and the activated surface carboxylated Fe3O4 magnetic nanoparticle powder was incubated with the activated Van and the activated Amp respectively. After incubation, the powder was washed multiple times with PBS buffer and resuspended in buffer to obtain Van / Fe3O4 antibiotic magnetic beads and Amp / Fe3O4 antibiotic magnetic beads; the activation in step (3) refers to: immersing the surface carboxylated Fe3O4 magnetic nanoparticle powder, Van and Amp in EDC solution and NHS solution respectively for activation; (4) Rapid isolation and enrichment method for foodborne spores: Add the sample to the buffer solution and mix it with Van / Fe3O4 antibiotic magnetic beads and Amp / Fe3O4 antibiotic magnetic beads and incubate. Let it stand, take the supernatant, centrifuge and collect the enriched product, which is the foodborne spore.

2. The method for rapid isolation and enrichment of foodborne spores based on antibiotic-magnetic beads with dual recognition effect according to claim 1, characterized in that: In step (1), the washing process involves alternating between deionized water and anhydrous ethanol for 3 to 5 times.

3. The method for rapid isolation and enrichment of foodborne spores based on antibiotic-magnetic beads with dual recognition effect according to claim 2, characterized in that: The temperature of the first stirring in step (2) is 29~32 ℃, and the stirring time is 2.7~3.2 h.

4. The method for rapid isolation and enrichment of foodborne spores based on antibiotic-magnetic beads with dual recognition effect according to any one of claims 1-3, characterized in that: The temperature of the second stirring in step (2) is 29~32 ℃, and the stirring time is 4.8~5.3 h; the washing reagent in step (2) is ethanol, and the number of washing times is 3~5.

5. The method for rapid isolation and enrichment of foodborne spores based on antibiotic-magnetic beads with dual recognition effect according to claim 4, characterized in that: In step (3), the incubation temperature was room temperature and the incubation time was 120 min; the concentration of PBS buffer was 0.01 mol / L and the pH was 7.4; after resuspension, the concentrations of Van / Fe3O4 antibiotic magnetic beads and Amp / Fe3O4 antibiotic magnetic beads were both 0.8~1.2 mg / mL.

6. The method for rapid isolation and enrichment of foodborne spores based on antibiotic-magnetic beads with dual recognition effect according to claim 1, characterized in that: In step (4), the concentrations of bacterial vegetative cells and spores in the sample to be tested are both 10. 4 The concentration of CFU / mL was 1:1 for bacterial vegetative cells and spores; the volume ratio of the test sample, buffer, Van / Fe3O4 antibiotic magnetic beads and Amp / Fe3O4 antibiotic magnetic beads was 1:(7~8):(1~1.5):(1~1.5); the incubation temperature was 20~25 ℃; the incubation time was 40~50 min; the standing time was 3 min; and the centrifugation speed was 7000 r / min.

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