Method for detecting nutritional cells and endophytic spores of probiotic bacillus based on'dual differential accumulation 'SERS (Surface Enhanced Raman Scattering) strategy

Abstract

The invention belongs to the technical field of microbiological detection, and relates to a method for detecting nutritional cells and endophytic spores of probiotic bacillus based on a'dual differential accumulation 'SERS (Surface Enhanced Raman Scattering) strategy. According to the method, a'dual differential accumulation 'SERS strategy is constructed in a mode of combining Fe3O4 (at) AgNPs-Van magnetic beads and an AuNPs nano-filter paper nano-array substrate, rapid separation and enrichment of vegetative cells and endophytic spores of the probiotic bacillus are realized, meanwhile, 'cliff type' signal difference is formed by SERS signals of the vegetative cells and the endophytic spores, SERS spectrum analysis is carried out, and the SERS spectrum analysis result is obtained. A PCA analysis method and a PLSA-DA analysis method are combined to carry out classification identification analysis and significant difference characteristic substance analysis, and an SERS spectrum digital quantitative model is constructed. According to the method, the separation effect on the probiotic bacillus nutrient cells and the endophytic spores reaches 98% or above, and the lowest detection limit is 101 CFU / mL. And a technical support is provided for rapid separation, enrichment and detection of nutritional cells and endophytic spores of probiotic bacillus in an actual sample.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial detection, and particularly relates to a method for detecting vegetative cells and endogenous spores of probiotic bacillus based on a "double differential accumulation" SERS strategy. Background Art

[0002] Probiotics mainly include lactic acid bacteria (such as lactobacillus and bifidobacterium), yeast and probiotic Bacillus. Probiotic Bacillus can produce many metabolites with antibacterial activity, rich in protease, lipase and amylase active substances, which can increase the conversion rate of fermented food matrix and effectively improve the intestinal ecology of the human body. It has a very wide range of application value in scientific fields such as food. Probiotic Bacillus coagulans, Bacillus licheniformis and Bacillus subtilis have always been considered as "generally recognized as safe (GRAS)" strains and have been used as probiotics to relieve enteritis and diarrhea. In recent years, they have emerged in the field of fermented foods and are widely used in biotechnology and fermentation.

[0003] Probiotic Bacillus generally undergoes two forms: vegetative cells and endospores. Endospores are dormant forms formed by vegetative cells in adverse environments or when nutrients are deficient. They can resist extreme environmental factors such as high temperature, strong acid and bile, and can survive in the gastrointestinal tract (GIT) of humans and animals as probiotics. Endospores germinate in the intestines as vegetative forms, which can metabolize and produce probiotic substances such as lactic acid, antibacterial agents, and digestive enzymes. In recent years, probiotic Bacillus has been used as a food additive in the form of endospores in the industrial production of coffee, jelly, pasta, etc. Although it has a large market scale as a probiotic, there is still a lack of rapid qualitative and quantitative methods for endospores to monitor the quality of such products. Vegetative cells are the key to influencing and determining the generation of flavor substances in some fermented foods, and have the functional characteristics of metabolizing complex flavor compounds and secreting rich enzyme systems. Probiotic Bacillus can solve the problems of single flavor and long fermentation time of fermented products caused by the lack of certain functional genes and insufficient metabolic capacity of existing fermentation agent strains, and also has great potential in improving the safety of fermented products. The fermentation process is an extremely complex and natural process of microbial colony action. Monitoring the number of microorganisms during the fermentation process is the key to determining the yield and quality of fermented foods. When using probiotic Bacillus for fermentation production, it is necessary to monitor the growth metabolism and bacterial cell accumulation of endogenous Bacillus. Vegetative cells will produce endogenous spores under conditions of nutrient scarcity in the late fermentation period, affecting the fermentation effect. Whether it is the application of endogenous spores of probiotic Bacillus as nutritional enhancers or the application of vegetative cells as fermentation agents, there is an urgent need for rapid qualitative identification and digital quantitative analysis of endogenous spores and vegetative cells in complex matrices.

[0004] Probiotic Bacillus is usually analyzed qualitatively and quantitatively by plate counting combined with 16S rDNA, which has a long culture cycle, a delayed result, and cannot distinguish endogenous spores from vegetative cells. It is also possible to quantitatively determine endogenous spores and qualitatively analyze them by heating and killing vegetative cells according to the heat resistance of endogenous spores, but heating will promote the mutual transformation of vegetative cells and endogenous spores, with large errors and time-consuming and cumbersome operation, which is not suitable for on-site real-time detection. In order to overcome the above problems, it is urgent to develop a detection method with short analysis time and high sensitivity. Raman spectroscopy (Raman spectra) analysis method analyzes the scattering spectrum with different frequencies from the incident light to obtain information on molecular vibration and rotation, and is applied to molecular structure research. Raman spectroscopy technology can realize label-free, in-situ rapid detection of microbial species, and combining precious metal nanomaterials as substrates (such as precious metals such as gold and silver) can solve the shortcomings of weak detection signals and achieve significant signal enhancement. SERS, as a biological fingerprint recognition technology, can provide rich molecular structure and composition information to meet the rapid detection needs of probiotic Bacillus. Magnetic bead sorting technology uses magnetic microbeads to specifically bind to cells to achieve the purpose of magnetic bead separation, purification and enrichment of cells in a magnetic field. As an important cell separation and purification technology, it has broad development prospects in the separation and enrichment of microorganisms. Patent 202310249614.2 discloses a method for rapid separation and enrichment of foodborne spores based on dual recognition effect antibiotics-magnetic beads. By combining two antibiotic magnetic beads, Van-Fe3O4 magnetic beads and Amp-Fe3O4 magnetic beads, the rapid separation and enrichment of bacterial cells and spores in complex microbial systems is achieved under the action of the dual recognition effect. The resulting spore separation and enrichment efficiency can reach 99%, providing new ideas and new means for the rapid separation and enrichment of foodborne spores in complex systems.

[0005] Therefore, using the above technology to construct a rapid qualitative identification and digital quantitative analysis method for endogenous spores and vegetative cells can provide effective technical support for the dynamic real-time monitoring and tracking of probiotic Bacillus during the fermentation process and the quality control of fermented foods. Summary of the invention

[0006] In order to solve the above problems, the present invention proposes a method for detecting vegetative cells and endogenous spores of probiotic Bacillus based on the "double differential accumulation" SERS strategy.

[0007] The technical solution of the present invention is achieved in this way:

[0008] A Fe3O4@AgNPs-Van magnetic beads, the steps are as follows:

[0009] (1) Preparation of Fe3O4@AgNPs magnetic beads: 3-aminopropyltriethylsilane (APTES) was added to the ethanol dispersion of Fe3O4 nanoparticles and mixed evenly, washed, dispersed in AgNO3 solution and mixed evenly, then added with NaBH4 solution and mixed evenly, washed, and obtained magnetic Fe3O4@AgNPs;

[0010] (2) Preparation of Fe3O4@AgNPs-Van magnetic beads: Magnetic Fe3O4@AgNPs were added to MUA ethanol solution for ultrasonic reaction. The Fe3O4@Ag-MUA magnetic microspheres obtained by the reaction were dissolved in ultrapure water. EDC aqueous solution and vancomycin solution were added for ultrasonic reaction to obtain Fe3O4@AgNPs-Van magnetic beads, which were stored in PBS buffer for later use.

[0011] Preferably, in the above step (1), the concentration of the ethanol dispersion of Fe3O4 nanoparticles is 0.0025-0.0035 g / mL, the concentration of the AgNO3 solution is 0.1-0.2 mol / L, the concentration of the NaBH4 solution is 0.3-0.5 mol / L, and the volume ratio of APTES, the ethanol dispersion of Fe3O4 nanoparticles, the AgNO3 solution and the NaBH4 solution is (0.1-0.3):(1-3):(1-3):(0.5-1).

[0012] Preferably, in the above step (2), the mass ratio of Fe3O4@AgNPs, MUA, EDC and Van is (0.08-0.12):(0.040-0.050):(8-12):(4-6); the concentration of MUA ethanol solution is 20 μM, the concentration of EDC aqueous solution is 5-10 mg / mL, and the concentration of Van solution is 5-10 mg / mL.

[0013] An AuNPs nanofilter paper nanoarray substrate is characterized in that the preparation steps are as follows: placing a nanofiltration membrane in an APTES aqueous solution for hydrophilic treatment, washing, and then immersing it in an AuNPs solution for reaction to obtain an AuNPs nanofilter paper nanoarray substrate.

[0014] Preferably, the volume concentration of the APTES aqueous solution is 5%, the hydrophilic treatment time is 1 to 2 hours, and the reaction time in the AuNPs solution is 3 to 4 hours.

[0015] Based on the above-mentioned Fe3O4@AgNPs-Van magnetic beads or AuNPs nanofilter paper nanoarray substrate, it is used to rapidly detect vegetative cells and endogenous spores of probiotic Bacillus.

[0016] A method for rapidly detecting vegetative cells and endogenous spores of probiotic Bacillus, comprising the following steps:

[0017] a. Dilute the sample to be tested with sterile water 4 to 6 times to obtain a mixed bacterial solution and incubate with PBS buffer containing the above-mentioned Fe3O4@AgNPs-Van magnetic beads to form magnetized microorganisms;

[0018] b. The magnetized microorganisms are adsorbed by a magnet on one side, and the resulting precipitate is component 1. The supernatant is centrifuged and separated to obtain component 2;

[0019] c. Resuspend component 1 and component 2 in 0.5-1.5 mL sterile water, take 10-20 μL and drop it on the AuNPs nanofilter paper nanoarray substrate according to claim 4 or 5, collect SERS spectra, and distinguish vegetative cells and endogenous spores according to the SERS signal intensity.

[0020] Preferably, the concentration of the PBS buffer containing Fe3O4@AgNPs-Van magnetic beads in the above step a is 10mM; the volume ratio of the mixed bacterial solution to the PBS buffer containing Fe3O4@AgNPs-Van magnetic beads is (4-6):(3-5); the shaking incubation time is 8-10min; the centrifugal speed in step b is 7000-8000r / min.

[0021] Preferably, the acquisition parameters of the SERS spectrum in the above step c are: the excitation light source is a 532.8 nm helium-neon laser, a 100x objective lens, an integration time of 30 s, a cumulative number of 3 times, a maximum laser intensity of 25 mW, and a Raman spectrum scanning range of 400-1800 cm -1 .

[0024] Preferably, in the above step c, the components with strong SERS signals are vegetative cells, and the components with weak SERS signals are endogenous spores.

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

[0026] 1. The present invention combines Fe3O4@AgNPs-Van magnetic beads and AuNPs nanofilter paper nanoarray substrate for the first time, constructing a "double differential accumulation" SERS strategy, which can achieve rapid separation and enrichment of vegetative cells and endogenous spores of probiotic Bacillus, while making the SERS signals of vegetative cells and endogenous spores form a "cliff-like" signal difference, and conduct SERS spectrum analysis, combine PCA and PLSA-DA analysis methods to perform classification and identification analysis and significant difference characteristic material analysis, and construct a SERS spectrum digital quantitative model. It provides technical support for the rapid separation, enrichment and detection of vegetative cells and endogenous spores of probiotic Bacillus in actual samples, and escorts the industrial production application of probiotic Bacillus and the dynamic real-time monitoring of the fermentation process.

[0027] 2. The capture and separation effect of Fe3O4@AgNPs-Van magnetic beads on mixed samples of vegetative cells and endogenous spores reached more than 98%, and the minimum detection limit was 10 1 CFU / mL. The practical application of the separation and enrichment of probiotic Bacillus vegetative cells and endogenous spores in liquid culture medium (TSB and DSM culture medium), milk and sausage samples was verified. Compared with the traditional plate counting method, the results showed that 10 6 The average recovery rates of B. coagulans endospores, B. licheniformis endospores and B. subtilis endospores at CFU / mL concentration in liquid culture medium spike experiments were above 97%, and the average recovery rates in milk and sausages were above 95%. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 Flowchart of the “double differential accumulation” SERS strategy for rapid separation and identification of probiotic vegetative cells and endospores.

[0030] Figure 2 Characterization diagram of Fe3O4@AgNPs-Van magnetic beads; (A) is the SEM image of Fe3O4, (B) is the SEM image of Fe3O4@AgNPs, (C) is the SEM image of Fe3O4@AgNPs-Van magnetic beads, (D) is the hysteresis curve of Fe3O4@AgNPs-Van magnetic beads, (E) is the XRD image of Fe3O4@AgNPs-Van magnetic beads, (F) to (I) are EDS element mapping images of Fe3O4@AgNPs-Van magnetic beads.

[0031] Figure 3Phase contrast microscopy and SEM images of mixed samples of vegetative cells and endogenous spores of three probiotic Bacillus species; (A) is a phase contrast microscopy image of a mixed sample of B. coagulans vegetative cells and endogenous spores, (B) is a SEM image of B. coagulans vegetative cells after capture and separation using Fe3O4@AgNPs-Van magnetic beads, (C) is a SEM image of B. coagulans endogenous spores after capture and separation using Fe3O4@AgNPs-Van magnetic beads, (D) is a phase contrast microscopy image of a mixed sample of B. licheniformis vegetative cells and endogenous spores, and (E) is a SEM image of Fe3O4@AgN (F) is the SEM image of B.licheniformis vegetative cells after capture and separation using Ps-Van magnetic beads, (G) is the phase contrast microscope image of a mixed sample of B.subtilis vegetative cells and endogenous spores after capture and separation using Fe3O4@AgNPs-Van magnetic beads, (H) is the SEM image of B.subtilis vegetative cells after capture and separation using Fe3O4@AgNPs-Van magnetic beads, and (I) is the SEM image of B.subtilis endogenous spores after capture and separation using Fe3O4@AgNPs-Van magnetic beads.

[0032] Figure 4 Phase contrast microscopy images of endogenous spores obtained after Fe3O4@AgNPs-Van magnetic beads were used to separate the probiotic Bacillus mixed sample; (A) and (B) are phase contrast microscopy images of B. coagulans endogenous spores in different fields of view, (C) and (D) are phase contrast microscopy images of B. licheniformis endogenous spores in different fields of view, and (E) and (F) are phase contrast microscopy images of B. subtilis endogenous spores in different fields of view.

[0033] Figure 5 Characterization diagrams of AuNPs and AuNPs nanofilter paper nanoarray substrates; (A) is the US-vis spectrum of AuNPs, (B) is the particle size distribution diagram of AuNPs, (C) is the TEM image of AuNPs, (D) is the SEM image of the AuNPs nanofilter paper nanoarray substrate, (E) is the enhancement effect of the "double differential accumulation" SERS strategy on the vegetative cells of three probiotic Bacillus species, and (F) is the enhancement effect of the "double differential accumulation" SERS strategy on the endogenous spores of three probiotic Bacillus species.

[0034] Figure 6The average SERS spectral analysis and 50-time spectral reproducibility color barcode of 3 probiotic Bacillus vegetative cells and endogenous spores; (A) is the average SERS fingerprint of 3 probiotic Bacillus vegetative cells, (B) is the color barcode of 50-time spectral reproducibility of 3 probiotic Bacillus vegetative cells, (C) is the average SERS fingerprint of 3 probiotic Bacillus endogenous spores, and (D) is the color barcode of 50-time spectral reproducibility of 3 probiotic Bacillus endogenous spores.

[0035] Figure 7 It is a qualitative and quantitative model; (A) is the PCA analysis diagram of 6 microbial samples, (B) is the screening result of the difference characteristic peaks of the SERS average fingerprint spectrum based on PLSDA analysis, (C) is the enhancement effect diagram of the "double differential accumulation" SERS strategy on B. coagulans vegetative cells, (D) is the digital quantitative model of B. coagulans vegetative cells; (E) is the enhancement effect diagram of the "double differential accumulation" SERS strategy on B. coagulans endogenous spores, and (F) is the digital quantitative model of B. coagulans endogenous spores.

[0036] Figure 8 is a digital quantitative model; wherein (A) is a diagram showing the enhancement effect of the “double differential accumulation” SERS strategy on B.licheniformis vegetative cells, (B) is a digital quantitative model of B.licheniformis vegetative cells, (C) is a diagram showing the enhancement effect of the “double differential accumulation” SERS strategy on B.licheniformis endogenous spores, (D) is a digital quantitative model of B.licheniformis endogenous spores, (E) is a diagram showing the enhancement effect of the “double differential accumulation” SERS strategy on B.subtilis vegetative cells, (F) is a digital quantitative model of B.subtilis vegetative cells, (G) is a diagram showing the enhancement effect of the “double differential accumulation” SERS strategy on B.subtilis endogenous spores, and (H) is a digital quantitative model of B.subtilis endogenous spores. DETAILED DESCRIPTION

[0037] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0039] The flow chart of the "double differential accumulation" SERS strategy for rapid separation and identification of probiotic vegetative cells and endogenous spores in the present invention is as follows: Figure 1 As shown in the figure, step 1 is a schematic diagram of the synthesis of Fe3O4@AgNPs-Van magnetic beads, step 2 is a schematic diagram of the synthesis of AuNPs nanofilter paper nanoarray substrate, and step 3 is the application of Fe3O4@AgNPs-Van magnetic beads and AuNPs nanofilter paper nanoarray substrate in the separation and detection of probiotic Bacillus vegetative cells and endogenous spores.

[0040] The method proposed in the present invention is applicable to the detection of all probiotic Bacillus, and is described below by taking Bacillus coagulans, Bacillus licheniformis and Bacillus subtilis as examples.

[0041] 1. Materials and Methods

[0042] 1.1 Bacterial strains

[0043] The strains Bacillus coagulans, Bacillus licheniformis and Bacillus subtilis were purchased as laboratory standard samples.

[0044] 1.2 Preparation of vegetative cells and endospores

[0045] Bacillus coagulans, Bacillus licheniformis and Bacillus subtilis stored in a -80°C refrigerator were activated by culturing with NA agar medium at 37°C for 18 to 24 hours, and transferred to NB liquid culture medium for enrichment culture in a shaker at 180 r / min for 18 to 24 hours, and washed three times with sterile water to obtain nutrient cells of the three probiotic Bacillus.

[0046] 0.1 mL of the nutrient cell enrichment solution of Bacillus coagulans and Bacillus licheniformis was inoculated into 10 mL of TSB medium (containing 50 mg / L MnSO4), and 0.1 mL of Bacillus subtilis was inoculated into 10 mL of DSM medium for promoting endospore growth. The culture was shaken at 37°C for 7 to 10 days. Sterile glass balls were placed in the culture medium to facilitate the formation and detachment of endospores. The endospore solution after culture was heated at 80°C in a water bath for 20 minutes to destroy the nutrient cells, and the endospore suspension was repeatedly washed 3 times by centrifugation with sterile water (7012 rpm, 20 minutes, 4°C). Endospores were detected by phase contrast microscopy with a 100x oil lens, and the endospores washed by centrifugation were resuspended in sterile water and stored at 4°C for later use.

[0047] 1.3 Test equipment

[0048] UV-visible spectrophotometer (UV-2600; Shimadzu), laser particle size analyzer (Mastersizer3000), X-ray powder diffractometer (X-ray diffraction, XRD, Cu target, λ=0.1541 nm, accelerating voltage 40 kV, current 30 mA), vibrating sample magnetometer (VSM, LakeShore 7404), scanning electron microscope (SEM, JSM-IT100; Jeol) and transmission electron microscope (TEM, JEOL JEM-2100plus LaB6).

[0049] 1.4 Data Processing

[0050] Excel 2010, Origin 8.0, SPSS16.0 and LabSpec 6.0 software were used to analyze and process the data.

[0051] Example 1

[0052] The separation, enrichment and detection method of probiotic Bacillus vegetative cells and endogenous spores based on double differential accumulation SERS strategy is as follows:

[0053] 1. The preparation method of Fe3O4@AgNPs-Van magnetic beads is as follows:

[0054] Firstly, Fe3O4NPs were synthesized by a one-step hydrothermal method, and then their surface was amino-modified by APTES. Finally, with the help of gold-ammonia bonds, AgNPs and Van were orderly bound to the surface of magnetic beads to obtain Fe3O4@AuNPs-Van magnetic beads with both target affinity and high magnetic responsiveness.

[0055] (1) Preparation of Fe3O4. Fe3O4NPs were synthesized by a one-step hydrothermal method. Specifically, 1.35g FeCl3 and 3.6g sodium acetate (CH3COONa) were added to 40mL ethylene glycol (C2H6O2) at room temperature. After magnetic stirring for 1h, the resulting solution was transferred to a reactor and heated to 200℃ for 6h. The reactor was then cooled to room temperature to obtain Fe3O4 nanoparticles. Fe3O4NPs were collected by a magnet and washed three times with anhydrous ethanol and ultrapure water respectively. Finally, the synthesized Fe3O4 nanoparticles were dried in a vacuum oven at 60℃ for 12h and packaged in clean 5mL EP tubes for storage at 4℃.

[0056] (2) Preparation of Fe3O4@AgNPs. Weigh 0.15 g of Fe3O4 particles and disperse them in 60 mL of anhydrous ethanol by ultrasonic. Add 600 μL of 3-aminopropyltriethylsilane (APTES) under rapid mechanical stirring. Continue stirring for 4 h, then magnetically separate the APTES-modified Fe3O4 and wash it twice with anhydrous ethanol. Then disperse it in 60 mL of 0.1 mol / L AgNO3 solution, stir for 15 min, and add 30 mL of 0.4 mol L -1 NaBH4 was added to reduce the adsorbed Ag+ to nano-Ag. After all the NaBH4 was added, stirring was continued for 60 min. The mixture was magnetically separated again and finally washed three times with ultrapure water to obtain magnetic Fe3O4@AgNPs.

[0057] (3) Preparation of Fe3O4@AgNPs-Van magnetic beads. 0.1 mg of Fe3O4@AgNPs microspheres were added to 10 mL of MUA ethanol solution (20 μM), and after ultrasonic reaction for 2 h, they were washed twice with ethanol and ultrapure water to remove unreacted MUA. The obtained Fe3O4@Ag-MUA magnetic microspheres were dissolved in 10 mL of ultrapure water, and 1 mL of EDC aqueous solution (10 mg / mL) and 1 mL of vancomycin solution (5 mg / mL) were added in sequence and ultrasonic reaction was continued for 2 h. The final product, Fe3O4@AgNPs-Van microspheres, was enriched with an external magnet, washed twice with ultrapure water, and stored in PBS buffer (10 mM, pH 7.4) for later use.

[0058] 1.1 Characterization of Fe3O4@AgNPs-Van magnetic beads

[0059] In order to confirm the successful preparation of Fe3O4@AgNPs-Van magnetic beads and their structure, size and morphology, Fe3O4, Fe3O4@AgNPs and Fe3O4@AgNPs-Van magnetic beads were characterized by SEM, XRD, nanoparticle size analyzer, vibrating sample magnetometer, X-ray powder diffractometer, and transmission electron microscopy. The results are shown in Figure 2. Figure 2shown.

[0060] From the SEM results, we can see that the surface of Fe3O4 nanoparticles is smooth, the particle size is uniform and the average particle size is about 100nm ( Figure 2 A). Under the action of APTES and NaBH4, silver ions are rapidly reduced on the surface of Fe3O4 nanoparticles, eventually forming a continuous rough silver shell that completely covers the magnetic core ( Figure 2 B). The average particle size of Fe3O4@AgNPs magnetic beads increases to about 110nm, so the thickness of the silver shell formed by Fe3O4 nanoparticles is about 5nm, but the particle size distribution of Fe3O4@AgNPs-Van magnetic beads is basically consistent with that of Fe3O4@AgNPs ( Figure 2 C). Figure 2 D shows the change of magnetic properties of Fe3O4 before and after modification. At room temperature, the hysteresis curves of Fe3O4, Fe3O4@AgNPs and Fe3O4@AgNPs-Van magnetic beads intersect with the origin, and no hysteresis occurs, indicating that all products have superparamagnetism, and Fe3O4@AgNPs-Van magnetic beads still have excellent magnetic properties. The gradual decrease of magnetic saturation intensity also shows the successful coating of non-magnetic components outside the Fe3O4 core. Figure 2 E shows the XRD patterns of Fe3O4 and Fe3O4@Ag magnetic microspheres. It can be clearly seen from the figure that the diffraction peaks (2θ) of Fe3O4 are distributed at 30°, 35.4°, 43°, 56.9°, and 62.5°, corresponding to the (112), (211), (220), (303), and (224) crystal planes of the anti-crystalline tip structure of Fe3O4, respectively. Four new diffraction peaks appeared in the XRD pattern of Fe3O4@Ag, and their 2θ values ​​were located at 38.3°, 44.2°, 64.5°, and 77.4°, respectively, corresponding to the (111), (200), (220), and (311) crystal planes of the silver cubic crystal structure. The results of the XRD test show that the silver shell was successfully reduced on the Fe3O4 surface. Figure 2 (F)-(I) show the EDS element spectrum scanning results of Fe3O4@AgNPs-Van magnetic beads. It can be seen that the core of Fe3O4@AgNPs-Van magnetic beads is composed of Fe elements (green), the outer shell is composed of a large amount of Ag elements (blue), and the outermost layer has a uniform distribution of N elements (red). Only the vancomycin modified in the outermost layer contains N elements, so the N signal appearing in the spectrum scan results also proves the successful modification of vancomycin. Therefore, Fe3O4@AgNPs-Van magnetic beads were confirmed to be successfully prepared.

[0061] 1.2 Capture and separation effect of Fe3O4@AgNPs-Van magnetic beads on mixed samples of vegetative cells and endogenous spores

[0062] In order to test the binding ability of Fe3O4@AgNPs-Van magnetic beads with vegetative cells and endogenous spores and the capture and separation effect, we took mixed samples of vegetative cells and endogenous spores of three probiotic Bacillus species as the research objects and verified them by phase contrast microscopy, SEM images and plate counting. The results are shown in Figure 3 , Figure 4 And as shown in Table 1.

[0063] Depend on Figure 3 (A)-(C) show that after using Fe3O4@AgNPs-Van magnetic beads, the mixed sample of B.coagulans vegetative cells and endogenous spores formed a tightly bound stable complex on the surface of B.coagulans vegetative cells, while the surface of B.coagulans endogenous spores remained smooth, and no nanoparticles were observed to be attached to the surface. The experimental results show that Fe3O4@AgNPs-Van magnetic beads can bind to the surface of B.coagulans vegetative cells in large quantities, but cannot attach to endogenous spores. Similarly, similar phenomena also exist in the capture and separation of vegetative cells and endogenous spores of B.licheniformis and B.subtilis using Fe3O4@AgNPs-Van magnetic beads. This is because the terminal end of the muramyl pentapeptide (D-Ala-D-Ala) of the bacterial cell wall component of B. coagulans, B. licheniformis and B. subtilis vegetative cells can form a hydrogen bond complex with the carboxyl group in vancomycin and bind firmly. Figure 3 (D)-(I). Phase contrast microscopy images of endogenous spores obtained after separation of probiotic Bacillus mixed samples using Fe3O4@AgNPs-Van magnetic beads ( Figure 4 ), indicating that Fe3O4@AgNPs-Van magnetic beads can separate endogenous spores from vegetative cells in mixed samples. Combined with the plate counting method, the separation effect of Fe3O4@AgNPs-Van magnetic beads on vegetative cells and endogenous spores of three probiotic Bacillus can reach more than 98% (Table 1).

[0064] Table 1 Separation efficiency of Fe3O4@AgNPs-Van magnetic beads for vegetative cells and endogenous spores in composite microbial systems

[0065]

[0066] Take 1 mL of mixed microbial culture (V 营养细胞 :V 芽孢 =1:1, where the concentrations of vegetative cells and endospores are both 10 3CFU / mL) in 5 mL of sterile water, then add 4 mL of Fe3O4@AgNPs-Van magnetic beads, shake and incubate on a shaker for 8 to 10 minutes to allow the vegetative cells to fully combine with the Fe3O4@AgNPs-Van magnetic beads to obtain magnetized microorganisms. The sample was placed on a magnetic rack for 3 minutes, and the magnetic field was used to separate the vegetative cells and spores by adsorbing the magnetized vegetative cell conjugate on one side. Further, the supernatant was centrifuged at 7000 r / min to collect pure spores.

[0067] According to the following formula, the plate counting method was used to calculate the number of vegetative cells and endogenous spores in the composite microbial system by Fe3O4@AgNPs-Van magnetic beads, and the separation efficiency of the two was calculated according to the counts.

[0068] Endospore separation efficiency = lg S1 / lg S0×100 Formula (1)

[0069] Separation efficiency of vegetative cells = lg (S2-S3) / lg S2×100 Formula (2)

[0070] Where S0 represents the initial concentration of endospores;

[0071] S1 represents the concentration of endogenous spores in the supernatant after treatment with Fe3O4@AgNPs-Van magnetic beads, and the nutrient cells that may exist were killed by treatment at 80 °C for 15 min before plate counting;

[0072] S2 represents the initial concentration of vegetative cells, CFU / mL;

[0073] S3 represents the microbial concentration of the Fe3O4@AgNPs-Van magnetic beads and nutrient cells complex, CFU / mL.

[0074] 2. Preparation of AuNPs Nanofilter Paper Nanoarray Substrate

[0075] Preparation of AuNPs. Dilute and mix 5 mL of 1% HAuCl4 solution with 142 mL of deionized water, heat to boiling, quickly add 17 mL of 34 mM trisodium citrate solution, and keep boiling for 10 min after the solution turns red. After the solution cools to room temperature, dispense into airtight light-proof glass bottles and store at 4°C.

[0076] Nanofiltration membrane materials mainly include polyamide (PA) and sulfonated covalent organic framework (COFs). The nanofiltration membrane is placed in 5% APTES for hydrophilic treatment for 2 hours, then taken out and washed three times with ultrapure water. APTES solution can silanize the surface of the nanofiltration membrane. The methoxy group in APTES is very easy to hydrolyze. The silanol (Si-OH) produced by hydrolysis combines with the hydroxyl group of the nanofiltration membrane in the form of hydrogen bonds, assembling APTES on the surface of the nanofiltration membrane, exposing the amino group (-NH2) at the end. The treated nanofiltration membrane is placed in the AuNPs solution, and a strong chemical interaction occurs between the AuNPs and the exposed amino group of APTES, which triggers the self-assembly of AuNPs on the surface of the nanofiltration membrane film. AuNPs can be adsorbed on the nanofiltration membrane. After 4 hours, it is taken out to obtain the AuNPs nanofiltration paper nanoarray substrate material.

[0077] 2.1 Characterization of AuNPs nanofilter paper nanoarray substrate

[0078] Figure 5 (A) shows the UV-visible absorption spectrum of AuNPs sol. The maximum absorption peak of the surface plasmon resonance (SPR) of AuNPs is located at 518nm, which is within the normal absorption peak wavelength range of AuNPs. As shown in Figures (B) and (C), the particle size distribution of AuNPs is between 40 and 120nm. The TEM image can clearly observe the specific surface microstructure of the object to be tested. Almost all AuNPs are spherical in structure and the particle size distribution is relatively uniform. The particle sizes of 200 nanoparticles are counted and the average particle size of AuNPs is calculated to be about 78nm. Figure 5 (D) The SEM image of the AuNPs nanofilter paper nanoarray substrate shows that the distribution of AuNPs on the surface of the nanofilter paper is relatively uniform and dense, ensuring the overall uniformity of the substrate. The field coupling response of closely adjacent nanoparticles produces a strong "hot spot", which gives the substrate a higher SERS signal enhancement ability.

[0079] The comparison results of the enhancement effect of the Raman signal enhancement strategy established in the present invention on the vegetative cells and endogenous spores of three probiotic Bacillus are as follows: Figure 5 (E), (F) and Table 2. Figure 5(E) and (F) show that in the Raman detection without enhanced substrate materials, the Raman spectrum intensity of the vegetative cells and endogenous spores of probiotic bacillus is very low, and it is difficult to quickly identify different vegetative cells and endogenous spores; using Fe3O4@AgNPs-Van magnetic beads and AuNPs nanofilter paper nanoarray substrates for Raman signal enhancement, the Raman signals of the vegetative cells and endogenous spores of the three probiotic bacilli are significantly enhanced, and the characteristic peaks with weak signals can also be displayed in the SERS spectrum. The increase in Raman characteristic peaks is more conducive to distinguishing and identifying different vegetative cells and endogenous spores. According to the peak intensity changes of the most significant characteristic peak bands in the SERS spectra of the vegetative cells and endogenous spores of the three probiotic bacilli, the method of the present invention is calculated according to Formula 1 to enhance the Raman spectra of the vegetative cells and endogenous spores of the three probiotic bacilli. The results are shown in Table 2. After calculation, the maximum EF of the vegetative cells of the three probiotic bacilli by this method is 1.24×10 4 The maximum EF for endogenous spores of three probiotic Bacillus species was 1.40×10 3 . This is mainly because in the present invention, only the AuNPs nanofilter paper nanoarray substrate plays a role in signal enhancement when the endogenous spores are signal enhanced. The plasma coupling between the Fe3O4@AgNPs-Van magnetic beads on the vegetative cells and the metal particles of the AuNPs nanofilter paper nanoarray substrate forms an electromagnetic field enhancement, which produces additional "hot spots", resulting in a "cliff-like" signal difference between the SERS signals of the vegetative cells and the endogenous spores. So far, the strategy in the present invention has achieved the separation of vegetative cells and endogenous spores and the "cliff-like" difference in SERS signal intensity. The "double differential accumulation" SERS strategy has laid a good foundation for the rapid separation and identification of vegetative cells and endogenous spores of different probiotic Bacillus.

[0080] Table 2 Comparison of the enhancement effect of this method on probiotic Bacillus vegetative cells and endogenous spores

[0081]

[0082] Note: C RS =10C SERS .

[0083]

[0084] I RS and I SERS They are respectively the spectral intensity values ​​of the detected object collected before and after using the "double differential accumulation" SERS strategy of the present invention.

[0085] 3. Sample Preparation and Raman Spectrum Collection

[0086] The vegetative cells and endogenous spores of probiotic Bacillus after Fe3O4@AgNPs-Van magnetic beads separation were resuspended in 1 mL of sterile water, and 10 μL of the dilution was dripped on the AuNPs nanofilter paper nanoarray substrate, and the SERS spectra were collected directly using a LabRam HR laser confocal Raman spectrometer (Horiba Scientific) to prevent the formation of "coffee rings" from affecting the detection effect. Before the detection, the Raman spectrum of silicon was calibrated at 520.7 cm -1 The acquisition parameters were set as follows: the excitation light source was 532.8 nm helium-neon (He-Ne) laser, the objective lens was 100 times, the integration time was 30 s, the accumulation times were 3 times, the laser intensity was set to the maximum intensity of 25 mW, and the Raman spectrum scanning range was set to 400-1800 cm -1 . 50 spectra were randomly collected for each sample.

[0087] 3.1 Tentative assignment of SERS spectra

[0088] All spectra were recorded and preprocessed using the instrument's own software LabSpec 6.0, which removed background and corrected baselines. The average SERS spectra and two-dimensional mapping of the vegetative cells and endogenous spores of three common probiotic Bacillus species are shown in Figure 2. Figure 6 As shown, the specific characteristic peaks and their corresponding tentative attributions are listed in detail in Table 3. Figure 6 As shown in (a) and (c), we found that the SERS signal intensities of the vegetative cells of the three probiotic Bacillus species collected based on the "double differential accumulation" SERS strategy were significantly higher than the SERS signals of the endogenous spores (nearly 10 times), which intuitively distinguished the vegetative cells from the endogenous spores. -1 (CS stretch model ofcysteine)、776~782cm -1 (Cytosine or uracil(nucleic acids))、996~1006cm -1 (Phenylalanine(proteins))、1211~1235cm -1 (CH2 stretching, Amide III (protein)), 1003~1010cm -1 (Phenylalanine(proteins))、1125~1147cm -1 (PO2 - stretching(nucleicacid))、1334~1345cm -1(Adenine (nucleic acids)) and 1430~1460cm -1 (CH deformation (lipids)) There are 8 Raman characteristic peaks in total, and the peak intensities are different. Most of these SERS Raman shifts are related to some typical chemical bonds and vibration forms of DNA / RNA, proteins and lipids. This is because vegetative cells have common basic structures, such as cell walls and cell membranes, but the composition and types of proteins, lipids and nucleic acids vary from species to species. The vegetative cells of the three probiotic Bacillus species have their own unique characteristic peaks (see Table 1 for details). These spectral shift differences are mainly caused by the different composition of the test object itself and the different SERS signal response levels. These signal differences make it easy to distinguish the vegetative cells of different species of probiotic Bacillus species.

[0089] Ca 2+ -DPA is abundant in the endogenous spores of probiotic Bacillus, accounting for 15-25% of their dry weight. In the SERS spectra of the endogenous spores of three probiotic Bacillus, Ca 2+ -DPA has a dominant Raman vibration peak number and peak intensity, which is the main marker for distinguishing spores from bacteria. Its Raman vibration peak position is approximately 656cm -1 、783cm -1 、856cm -1 1003~1010cm -1 、1311~1346cm -1 1390~1395cm -1 、1447~1452cm -1 and 1577~1579cm -1 Band. Ca 2+ The difference in -DPA content resulted in different peak numbers and intensities of the three endospores (as shown in Table 3). These differences indicate that the Ca content in each spore 2+ -DPA content and structure are significantly different, and can also be used as a basis for the classification and identification of endospores of different species.

[0090] In addition, three endospores were found at 585-597 cm -1 、684~703cm -1 、734~747cm -1 、819~822cm -1 、1125~1147cm -1 、1311~1346cm -1 and 1664~1666cm -1There are 6 Raman characteristic peaks with obvious differences in peak intensity. These Raman vibration peaks are respectively attributed to Carbon hydrates, CS stretch model of cysteine, NAG, NAM (glycosidic ring mode), OPO stretching (DNA / RNA), PO2 - stretching (nucleic acid), Adenine (nucleic acids) and Amide I (unsaturated lipid). These vibration peaks mainly come from proteins, lipids and nucleic acids. The spore coat is a capsid protein formed by hydrophobic proteins located outside the spore cortex. The protein content in the spore coat accounts for 50-80% of the total volume of the spore. The spore cortex is composed of peptidoglycan, which is similar to the peptidoglycan of the bacterial vegetative cell wall. The cortical peptidoglycan is also polymerized by acetylglucosamine (N-acetylglucosamine, NAG) and acetylmuramic acid (Nacetyimuramic, NAM). NAG, NAM (glycosidic ring mode) may come from the cortex of the spore. The main components of the spore inner membrane include phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The Raman vibration peak of nucleic acid PO2 - It is likely to extend symmetrically from the phosphate backbone. The differences in the positions and intensities of the Raman vibration peaks of the three endogenous spores are shown in Tables 3 and Figure 6 This strategy can not only make the SERS signals of vegetative cells and endospores form a "cliff-like" differentiation, but also reflect the differences between different vegetative cells and different endospores.

[0091] Then, we used color barcodes to study the SERS characteristic bands of vegetative cells and endogenous spores of three probiotic Bacillus species. Each sample was scanned 50 times with reproducibility. The SERS spectral color barcodes were as follows: Figure 6 (B) and (D). The x-axis represents the Raman shift, and the y-axis represents 50 spectra randomly detected from different samples. The color and line width in the barcode represent the relative intensity and peak width of the peak. The strong color difference in the SERS spectral color barcode can intuitively and effectively distinguish different types of probiotic Bacillus vegetative cells and endogenous spores. Figure 6 The barcodes of each characteristic peak in (B) and (D) are very uniform, reflecting the high reproducibility of the detection. The significant characteristic peak positions (785, 660, 1450 and 1680 cm -1 ) calculated the corresponding relative standard deviations, and the RSDs were all below 7%, confirming the high detection reproducibility of the "double differential accumulation" SERS strategy proposed in the present invention.

[0092] Table 3 Tentative assignments of SERS spectra of vegetative cells and endogenous spores of three probiotic Bacillus species

[0093]

[0094] Example 2

[0095] Construction of a qualitative and quantitative model for the rapid separation, enrichment and detection of probiotic Bacillus vegetative cells and endogenous spores based on the "double differential accumulation" SERS strategy:

[0096] In order to better realize the visual qualitative classification and identification of different probiotic Bacillus vegetative cells and endogenous spores, the PCA analysis method is used to convert the complex spectral data into a three-dimensional scatter plot, so as to intuitively observe the subtle differences between similar spectra and to distinguish the relationship between different types of microbial samples. PCA analysis was performed on 50 samples of 3 probiotic Bacillus vegetative cells and endogenous spores to evaluate the differences between the SERS spectra of 6 microbial samples collected based on the "double differential accumulation" SERS strategy proposed in this invention. The analysis results are shown in Figure 2. Figure 7 (A) As shown. Figure 7 (A) It can be seen that PCA analysis can clearly cluster the six microbial samples into six independent clusters, each cluster is independent and non-repetitive, and each spore can be distinguished from each other. The variance contribution rate of PC1 is 56.8%, the variance contribution rate of PC2 is 23.5%, and the variance contribution rate of PC3 is 13.1%. The cumulative contribution rate is 93.4%, which basically represents all the spectral information. Furthermore, the two-dimensional data of the main characteristic peaks of the SERS spectra of the six microbial samples collected were imported into the MetaboAnalyst6.0 website, and the mathematical model was established using orthogonal partial least squares discriminant analysis (PLS-DA). The main different chemical components and marker metabolites between the two were screened with the variable importance projection VIP value > 1 as the standard. The larger the VIP value (VIP > 1), the more significant the contribution of the variable to the construction of the PLS-DA model, which is the spectral characteristic peak and differential structural substance with significant differences between the groups.

[0097] Table 4 Screening results of SERS average fingerprint difference peaks based on PLSDA analysis

[0098]

[0099] Figure 7 (B) and Table 4 show the top 30 differential characteristic peaks and differential substances with VIP greater than 1 in the SESR spectra of the six microbial samples, which are mainly attributed to the band 1430-1460cm -1 (CH deformation(lipids),Ca2+ -DPA)、1611~1614cm -1 (AmideⅡ(protein))、1283~1288cm -1 (-CH2deformation(lipid))、1311~1346cm -1 (Adenine(nucleic acids))、1123~1147cm -1 (PO2 - stretching(nucleic acid))、1211~1236cm -1 (CH2 stretching, Amide III(protein)) and 966~1006cm -1 (Phenylalanine(proteins),Ca 2+ -DPA) These Raman shift bands are important difference characteristic peaks reflecting the SERS spectra of the six microbial samples, and contribute significantly to classification and identification.

[0100] The concentrations were 10 1 CFU / mL~10 6 CFU / mL of B. coagulans vegetative cells were collected by SERS spectrum acquisition using the method constructed by the present invention, 1451 cm -1 The logarithm of the concentration of B. coagulans vegetative cells and the relationship between 1451 cm -1 The linear relationship between the SERS peak intensities at Figure 7 As shown in (C) and (D). Figure 7 (C) shows that the vegetative cells of B. coagulans are at 1451 cm -1 The SERS intensity at 10 1 cfu / mL, B. coagulans vegetative cells at 1451cm -1 The obvious characteristic peaks can still be observed at 1451 cm / mL, so the detection limit of this method for B. coagulans is less than 10 CFU / mL. The logarithmic values ​​(log C) of different concentrations of B. coagulans vegetative cells are used as the horizontal axis, and the strongest characteristic peak at 1451 cm / mL is used as the horizontal axis. -1 The peak intensity at y = 122393.19x-110543.51 was used as the ordinate, and the digital quantitative equation of B. coagulans was established. 2 =0.99( Figure 7 (D)). Figure 7As shown in (E) and (F), the digital quantitative equation for B. coagulans endospores is y = 12188.72x - 8908.86, R 2 =0.99.

[0101] Table 5 Digital quantitative equations for SERS spectra of endogenous spores and vegetative cells of three probiotic Bacillus species

[0102]

[0103] Similarly, the digital quantitative models of vegetative cells and endospores of B. licheniformis and vegetative cells and endospores of B. subtilis are shown in Figure 2. Figure 8 As shown in Table 5. It can be seen that the "double differential accumulation" SERS strategy proposed in the present invention has good quantitative analysis capabilities for the vegetative cells and endogenous spores of the three probiotics, and the minimum detection limit can reach 10 1 CFU / mL.

[0104] Implementation effect example

[0105] Actual sample spike recovery test of the rapid separation, enrichment and detection method of probiotic Bacillus vegetative cells and endogenous spores based on double differential accumulation SERS strategy:

[0106] Since the cultivation and purification of endogenous spores are difficult and challenging, we evaluated the practical applicability of this method by the separation and purification efficiency of endogenous spores in mixed samples. This method was used for the detection of probiotic Bacillus in liquid culture medium (TSB and DSM culture medium), milk and sausage samples. The samples were processed as follows: 10 mL of liquid culture medium, milk and sausage suspension (V 香肠 :V 无菌水 =1:9) sample was centrifuged at 6000 rpm for 5 min, repeated 3 times, large particles were removed and the supernatant was retained. Then, 2 mL of mixed microbial culture (V 营养细胞 :V 芽孢 =1:1, where the concentrations of vegetative cells and endospores are both 10 6CFU / mL) were added to 4mL of the above supernatant, and then 4mL of Fe3O4@AgNPs-Van magnetic bead solution was added, and incubated on a shaker for 8-10min to allow the vegetative cells to fully bind to the Fe3O4@AgNPs-Van magnetic beads. The sample was placed on a magnetic rack for 3min, and the conjugate of Fe3O4@AgNPs-Van magnetic beads and vegetative cells was adsorbed on one side. Further, the supernatant was poured out, and the supernatant was centrifuged at 7000r / min to collect pure endogenous spores. The enrichment near the magnet was the conjugate of Fe3O4@AgNPs-Van magnetic beads and vegetative cells. The vegetative cells and endogenous spores separated by Fe3O4@AgNPs-Van magnetic beads were resuspended in 1mL of sterile water, and 10μL of the dilution was dropped on the AuNPs nanofilter paper nanoarray substrate, and the SERS spectrum was collected, and the results were compared with the traditional plate counting method.

[0107] Table 6 Evaluation results of the practical applicability of this experimental design method for the separation and enrichment of endogenous spores

[0108]

[0109] The results are shown in Table 6. 6 The average recovery rate of B.coagulans endogenous spores, B.licheniformis endogenous spores and B.subtilis endogenous spores in liquid culture medium spiked experiments at CFU / mL concentrations reached more than 97%, and the average recovery rates in milk and sausages reached more than 95%. The study showed that the method is suitable for efficient separation and enrichment of B.coagulans endogenous spores, B.licheniformis endogenous spores and B.subtilis endogenous spores from different types of complex environmental media, providing technical support for the separation and enrichment of endogenous spores of probiotic Bacillus in actual samples, and is of great significance for the high-density culture of endogenous spores of probiotic Bacillus and the industrial production of probiotic foods containing endogenous Bacillus.

[0110] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Fe3O4@AgNPs-Van magnetic bead, characterized in that: The preparation steps are as follows: (1) Preparation of Fe3O4@AgNPs magnetic beads: APTES was added to the ethanol dispersion of Fe3O4 nanoparticles and mixed, washed, dispersed in AgNO3 solution and mixed, then added with NaBH4 solution and mixed, washed, and obtained magnetic Fe3O4@AgNPs. (2) Preparation of Fe3O4@AgNPs-Van magnetic beads: Magnetic Fe3O4@AgNPs were added to MUA ethanol solution for ultrasonic reaction. The Fe3O4@Ag-MUA magnetic microspheres obtained by the reaction were dissolved in ultrapure water. EDC aqueous solution and vancomycin solution were added for ultrasonic reaction to obtain Fe3O4@AgNPs-Van magnetic beads.

2. The Fe3O4@AgNPs-Van magnetic beads according to claim 1, characterized in that: In the step (1), the concentration of the ethanol dispersion of Fe3O4 nanoparticles is 0.0025-0.0035 g / mL, the concentration of the AgNO3 solution is 0.1-0.2 mol / L, the concentration of the NaBH4 solution is 0.3-0.5 mol / L, and the volume ratio of APTES, the ethanol dispersion of Fe3O4 nanoparticles, the AgNO3 solution and the NaBH4 solution is (0.1-0.3):(1-3):(1-3):(0.5-1).

3. The Fe3O4@AgNPs-Van magnetic beads according to claim 1, characterized in that: In the step (2), the mass ratio of magnetic Fe3O4@AgNPs, MUA, EDC and vancomycin is (0.08-0.12): (0.040-0.050): (8-12): (4-6); the concentration of MUA ethanol solution is 20 μM, the concentration of EDC aqueous solution is 5-10 mg / mL, and the concentration of vancomycin solution is 5-10 mg / mL.

4. An AuNPs nanofilter paper nanoarray substrate, characterized in that: The preparation steps are as follows: the nanofiltration membrane is placed in an APTES aqueous solution for hydrophilic treatment, washed, and then immersed in an AuNPs solution for reaction to obtain an AuNPs nanofiltration paper nanoarray substrate.

5. The AuNPs nanofilter paper nanoarray substrate according to claim 4, characterized in that: The volume concentration of the APTES aqueous solution is 5%, the hydrophilic treatment time is 1-2 h, and the reaction time in the AuNPs solution is 3-4 h.

6. Use of the Fe3O4@AgNPs-Van magnetic beads according to any one of claims 1 to 3 and the AuNPs nanofilter paper nanoarray substrate according to claim 4 or 5 in separating and detecting vegetative cells and endogenous spores of probiotic Bacillus.

7. A method for rapid detection of vegetative cells and endogenous spores of probiotic Bacillus, characterized in that: Here are the steps: a. The sample to be tested is diluted 4 to 6 times with sterile water to obtain a mixed bacterial solution and incubated with PBS buffer containing the above-mentioned Fe3O4@AgNPs-Van magnetic beads to form magnetized microorganisms; b. The magnetized microorganisms are adsorbed by a magnet on one side, and the resulting precipitate is component 1. The supernatant is centrifuged and separated to obtain component 2; c. Resuspend component 1 and component 2 in 0.5-1.5 mL sterile water, take 10-20 μL and drop it on the AuNPs nanofilter paper nanoarray substrate according to claim 4 or 5, collect SERS spectra, and distinguish vegetative cells and endogenous spores according to the SERS signal intensity.

8. The method for rapid detection of vegetative cells and endogenous spores of probiotic Bacillus according to claim 7, characterized in that: The concentration of the PBS buffer containing the Fe3O4@AgNPs-Van magnetic beads in step a is 10 mM; the volume ratio of the mixed bacterial solution to the PBS buffer containing the Fe3O4@AgNPs-Van magnetic beads is (4-6):(3-5); the shaking incubation time is 8-10 min; and the centrifugal speed in step b is 7000-8000 r / min.

9. The method for rapid detection of vegetative cells and endogenous spores of probiotic Bacillus according to claim 8, characterized in that: The acquisition parameters of the SERS spectrum in step c are: the excitation light source is 532.8 nm helium-neon laser, 100x objective lens, integration time is 30 s, accumulation times are 3 times, maximum laser intensity is 25 mW, Raman spectrum scanning range is 400-1800 cm -1 .

10. The method for rapid detection of vegetative cells and endogenous spores of probiotic Bacillus according to claim 9, characterized in that: In the step c, the components with strong SERS signals are vegetative cells, and the components with weak SERS signals are endogenous spores.

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