A probiotic spore site-directed germination microsphere capable of being activated under pathological microenvironment and a preparation method and application thereof

Abstract

The present application relates to a kind of probiotic spore site germination microspheres capable of being activated under pathological microenvironment and its preparation method and application, the probiotic spore site germination microspheres are obtained by dextran hydrogel microspheres D, probiotic spore nanoscale enzyme complex SPzyme and spore nutrition germinant X are wrapped in.The SPzyme is prepared by layer-by-layer coating process, simple operation, low cost, strong stability;Then SPzyme and spore nutrition germinant X are co-encapsulated by microsphere prepared based on microfluidic technology.Probiotic spore can be activated by pathological microenvironment and germinate into active probiotic bacteria under the action of nutrition germinant;The germinated probiotic bacteria can quickly consume the oxygen in colon site, significantly inhibit the growth of harmful bacteria, provide anaerobic environment for probiotic bacteria, positively regulate intestinal flora.The probiotic spore site germination microspheres are used for preparing oral microecological pharmaceutical preparation, have the function of resisting gastrointestinal environment invasion, high-efficiency removal active oxygen, effectively relieving intestinal inflammation in intestinal site.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the field of oral probiotic spore preparations, and particularly to a probiotic spore-germination microsphere that can be activated in a pathological microenvironment, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic, immune-mediated inflammatory disease of the gastrointestinal tract that can lead to more serious and fatal conditions such as disability and colorectal cancer. Globally, the incidence of IBD is on the rise, and the disease is difficult to cure completely, requiring timely and long-term treatment to stop the development of symptoms and related complications, posing a significant challenge to global healthcare systems. Emerging evidence suggests that IBD is associated with intestinal mucosal barrier dysfunction, immune dysregulation, and bacterial microbiota dysbiosis. In the state of enteritis, the immune response is overactive, intestinal epithelial oxygenation increases, and large amounts of inflammatory factors and reactive oxygen species (ROS) are produced. On the one hand, ROS can be utilized by facultative anaerobes, leading to their proliferation. On the other hand, almost all beneficial gut microbiota are obligate anaerobes and cannot survive in oxygen-rich and highly inflammatory conditions. This results in decreased gut microbiota biodiversity, disruption of gut microbiota homeostasis, a reduction in the proportion of symbiotic bacteria such as Firmicutes, and a proliferation of harmful bacteria such as facultative anaerobes like Enterobacteriaceae. Current clinical treatments for IBD primarily focus on improving disease-related symptoms by alleviating intestinal inflammation or suppressing the immune response, including anti-inflammatory drugs, immunomodulators, and monoclonal antibodies. However, these treatments can cause serious complications due to off-target side effects, tissue toxicity, and other unforeseen side effects. More importantly, these approaches often fail to address the root causes of IBD, including damage to the gastrointestinal mucosa, loss of intestinal barrier function, and dysbiosis of the gut microbiota. Therefore, there is an urgent need for a strategy that can both eliminate reactive oxygen species (ROS) and suppress excessive inflammatory responses, while simultaneously effectively inhibiting the aerobic overgrowth of Enterobacteriaceae and restoring gut microbiota homeostasis, thus treating colitis at its root.

[0003] The dormant spores formed by the probiotic Bacillus subtilis have attracted increasing attention due to their ability to resist the harsh gastrointestinal (GI) environment. Furthermore, they can germinate and grow into the probiotic Bacillus subtilis, and with the consumption of oxygen, they can specifically limit the eccentric expansion of aerobic Enterobacteriaceae, increase the abundance of beneficial bacteria, and thus improve the gut microenvironment.

[0004] However, the efficacy of probiotic spores in colitis has some limitations and is not entirely satisfactory. First, the in vivo germination rate of the spores is extremely low, only about 1%, indicating weak germination and survival ability in the pathological environment of colitis, and their ROS scavenging ability is negligible. Furthermore, since the spores mainly germinate in the nutrient-rich areas of the small intestine, there is a lack of control over the germination site in the intestine, which severely limits the therapeutic effect of spores in colitis. Therefore, there is an urgent need for a strategy to optimize the therapeutic effect by improving the targeted germination ability and ROS scavenging activity of probiotic spores at the pathological sites of colitis. Summary of the Invention

[0005] Existing technologies lack strategies to optimize the therapeutic effect of probiotic spores by enhancing their targeted germination ability and ROS scavenging activity at the pathological sites of colitis. Therefore, this invention provides a probiotic spore-targeted germination microsphere that can be activated in a pathological microenvironment, along with its preparation method and application. The probiotic spore-targeted germination microspheres of this invention can be activated by the pathological microenvironment of the disease, promoting efficient and targeted germination of spores in the colon, providing a new approach for the treatment of colitis with probiotic spores.

[0006] The probiotic spore-germination microspheres (SPzyme+X@D) constructed in this invention can be formulated into oral drug preparations for precise targeted regulation and treatment of colitis. In some specific embodiments of this invention, the spore nanozyme complex SPzyme is constructed by in-situ growth of polydopamine-palladium (PDA-Pd) nanozymes on the spore surface; nanozymes possess excellent ROS scavenging capabilities, therefore, nanozyme-encapsulated spores are expected to enhance their ROS scavenging ability and growth activity at pathological sites. In some specific embodiments of this invention, L-alanine (L-Ala), as a common spore germination nutrient, can promote rapid and effective spore germination after targeting the colon, further enhancing the therapeutic effect of spores on colitis. Furthermore, thanks to microfluidic co-delivery technology, this invention encapsulates the spore nanozyme and the spore germination nutrient in dextran microspheres for colon-targeted delivery.

[0007] After targeting the colon, the nanozymes effectively remove reactive oxygen species (ROS) from the colitis site during the slow degradation of the microspheres by colonic glucanase present in the colon, thereby alleviating inflammation. Simultaneously, the degradation of the nanozyme coating exposes probiotic spores. These spores then germinate and grow in the colon under the influence of released L-Ala, rapidly depleting oxygen in the colitis site. This significantly inhibits the spread of harmful Enterobacteriaceae and provides an anaerobic environment for the growth of other beneficial bacteria in the colon, positively regulating the intestinal flora and effectively treating colitis.

[0008] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0009] One of the technical solutions of the present invention provides a probiotic spore-specific germination microsphere that can be activated in a pathological microenvironment. The probiotic spore-specific germination microsphere is obtained by encapsulating a probiotic spore nanoenzyme complex (SPzyme) and a spore nutrient germination agent (X) in dextran hydrogel microspheres (Dextran, D), and is named SPzyme+X@D. The probiotic spore nanoenzyme complex SPzyme is composed of probiotic spores and nanoenzymes.

[0010] Furthermore, the probiotic spores are derived from probiotics, which are selected from one or more of Bacillus subtilis, Bacillus cereus, Bacillus clausti, Bacillus coagulans, Bacillus licheniformis, Bacillus polyfermentans, and Bacillus pumilus.

[0011] Furthermore, the nanozyme is an antioxidant nanomaterial selected from one or more of polydopamine, cerium dioxide, manganese tetroxide, palladium, and platinum.

[0012] In some specific embodiments of this application, the nanozyme is selected from polydopamine and palladium metal.

[0013] Furthermore, the spore nutrient germination agent is a small molecule spore nutrient germination agent, selected from one or more of L-alanine, L-valine, and glucose.

[0014] The second technical solution of the present invention provides a method for preparing the above-mentioned probiotic spore-germination microspheres that can be activated in a pathological microenvironment, comprising the following steps:

[0015] (1) Preparation of probiotic spores: Probiotic bacterial solution in the logarithmic growth phase is inoculated onto a plate culture medium, cultured and purified to obtain probiotic spores;

[0016] (2) Preparation of probiotic spore nanozyme complex (SPzyme): The probiotic spores obtained in (1) are dispersed in nanozyme solution. After stirring, the excess nanozyme solution is washed away with sterile water. The obtained SP@Y is the probiotic spore nanozyme complex (SPzyme).

[0017] (3) Preparation of probiotic spore-specific germination microspheres (SPzyme+X@D) that can be activated in a pathological microenvironment: SPzyme prepared in step (2), methacrylated dextran, spore nutrient germination agent X, and photoinitiator were dissolved in sterile PBS solution to obtain the inner phase. The oil phase solution was used as the outer phase. Uniform monodisperse droplets were prepared by microfluidic device. The microspheres were solidified into microspheres by photocrosslinking reaction to obtain probiotic spore-specific germination microspheres SPzyme+X@D.

[0018] Further, in step (1), the plate culture medium is a spore-producing agar plate culture medium; the culture operation is specifically to culture at 36-38℃ for 48-96h to obtain spores, and to purify them using Histodenz solution or other methods recognized by those in the art to obtain probiotic spores.

[0019] Further, in step (2), the concentration of the nanozyme solution is 0.2–10 mg / mL; the solvent of the nanozyme solution is either Tris-HCl or sterile PBS; and the concentration of the probiotic spores dispersed in the nanozyme solution is 10 mg / mL. 6 ~10 9 CFU / mL; the stirring can be carried out using a magnetic stirrer for 1-2 hours.

[0020] In some specific embodiments of the present invention, the nanozyme solution is a dopamine solution and a Na2PdCl4 solution; the concentration of the dopamine solution is 0.2–2 mg / mL, and the solvent is Tris-HCl with pH = 8.4–8.6; the concentration of the Tris-HCl is 10–20 mM; the concentration of the Na2PdCl4 solution is 1–10 mg / mL, and the solvent is sterile PBS, the concentration of the sterile PBS is 10 mM, and the pH is 7.1–7.3;

[0021] In some specific embodiments of the present invention, probiotic spores are first dispersed in a dopamine solution, magnetically stirred, and then washed with sterile water to obtain SP@PDA; then, SP@PDA is resuspended in a Na2PdCl4 solution, magnetically stirred, and ascorbic acid solution is added (the role of ascorbic acid is to reduce Pd ions to Pd nanoparticles) and stirring is continued, and finally washed with sterile water to obtain the probiotic spore nanoenzyme complex SPzyme; the concentration of the ascorbic acid solution is 1-10 mg / mL, and the solvent is sterile PBS; the concentration of the sterile PBS is 10 mM, and the pH is 7.1-7.3.

[0022] In the final internal phase solution obtained in step (3), the concentration of the dispersed SPzyme is 10. 6 ~10 9 The concentration of the spore germination agent X is 10–100 mM; the concentration of the methacrylated dextran is 80–120 mg / mL; the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) at a concentration of 1–5 mg / mL; and the sterile PBS solution is used as the solvent at a concentration of 10 mM and a pH of 7.1–7.3.

[0023] In step (3), the oil phase solution is selected from any one of paraffin oil, dimethyl silicone oil, and castor oil.

[0024] The third technical solution of the present invention provides an application of the above-mentioned probiotic spore-germinating microspheres that can be activated in a pathological microenvironment in the preparation of oral microecological drug formulations.

[0025] Furthermore, the oral probiotic preparation acts on the intestines;

[0026] Specifically, the probiotic spore-germinating microspheres in the oral probiotic preparation can be activated by the microenvironment of the pathological site, releasing spore nutrient germination agents, activating the probiotic spores, and enabling them to grow into active probiotics. These probiotics rapidly consume oxygen in the pathological site, inhibit the growth of harmful Enterobacteriaceae, and simultaneously provide an anaerobic environment for the growth of other probiotics in the colon, promoting intestinal flora homeostasis. The nanoenzymes in the probiotic spore-germinating microspheres can efficiently remove high concentrations of reactive oxygen species from the disease site, alleviating intestinal inflammation.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] 1) The nanozyme used in this invention has superoxide dismutase (SOD) and catalase (CAT) enzyme activities, which can efficiently remove high concentrations of reactive oxygen species in inflammatory diseases such as enteritis and effectively relieve intestinal inflammation.

[0029] 2) The probiotic spore nanoenzyme complex used in this invention can respond to hydrogen peroxide in the pathological microenvironment in the colon, thereby degrading the dextran hydrogel coating and germinating efficiently into active probiotics under the action of spore nutrient germination agent, thus achieving precise and targeted germination of probiotic spores in the colon disease site.

[0030] 3) Germinating aerobic spore-forming bacteria can rapidly consume oxygen in the colon, significantly inhibit the growth of harmful Enterobacteriaceae, and at the same time provide an anaerobic environment for the growth of other beneficial bacteria in the colon, thus promoting intestinal flora homeostasis.

[0031] 4) The probiotic spore nanoenzyme complex used in this invention is prepared through a layer-by-layer coating process. This method is universal and simple, enabling rapid nanoenzyme modification of probiotic spores with low production costs and strong stability. The microspheres prepared in this invention utilize microfluidic technology, resulting in a simple preparation process that does not require complex mechanical processing and exhibits good monodispersity. Attached Figure Description

[0032] Figure 1 The flowcharts are for the preparation of microspheres for site-directed germination of probiotic spores in Examples 1-3;

[0033] Figure 2 Light microscopic images of probiotic spores from Example 1;

[0034] Figure 3This is a transmission electron microscopy (TEM) characterization image of the probiotic spore nanozyme complex (SPzyme) from Example 1.

[0035] Figure 4 This is a scanning electron microscope (SEM) image of the SPzyme+Ala@D microspheres in Example 1. Detailed Implementation

[0036] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below with reference to specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0038] Example 1: A microsphere for orally delivered Bacillus subtilis spore-specific germination

[0039] The preparation process of this embodiment is referenced. Figure 1 The following steps are included:

[0040] (1) Microbial culture and sample preparation

[0041] The cryopreserved Bacillus subtilis strain (purchased from Beijing Bio-Biotechnology Co., Ltd.) was thawed in a 37°C water bath beforehand. Then, 10 μL of the target bacterial suspension was transferred to a shaker tube containing 1 mL of sterile NB liquid medium and incubated at 37°C with shaking at 200 rpm for 12 h. After the bacterial suspension became turbid, another 10 μL of the target bacterial suspension was transferred to a fresh 1 mL of sterile NB liquid medium. This was repeated 2–3 times to enhance the viability of the probiotics. After activation, the probiotics were inoculated into sterile NB liquid medium at a 1:100 (V / V) ratio and incubated at 37°C with shaking at 200 rpm until the OD600 reached 0.2–0.3, ready for use.

[0042] (2) Preparation of probiotic spores

[0043] The cultured Bacillus subtilis suspension was spread onto spore-producing agar plates and incubated at 37°C for 72 h. Spores were scraped from the agar plates and washed three times with sterile ddH2O. The suspension was then resuspended in 350 μL of 20% Histodesnz, and the suspension was separated into layers on 1 mL of 50% Histodesnz. The plates were centrifuged at 16000 x g for 30 min at room temperature. The purified probiotic spores were collected and washed three more times with sterile ddH2O to obtain the purified probiotic spores.

[0044] The morphology of the purified probiotics was observed under an optical microscope. Figure 2 The images show light microscopic images of the purified probiotic spores. The results indicate that the purified Bacillus subtilis spores are well dispersed and have a rod-shaped or short rod-shaped morphology, which is consistent with the expected morphology.

[0045] (3) Preparation of probiotic spore nanozyme complex (SPzyme)

[0046] Dopamine (DA) was dissolved in Tris-HCl (pH 8.5) solution to a concentration of 1 mg / mL. Probiotic spores were then dispersed in the dopamine solution to a concentration of 10 mg / mL. 8 CFU / mL. Stir magnetically for 1 h, then wash away excess dopamine solution with sterile ddH2O to obtain SP@PDA; resuspend SP@PDA in 3 mg / mL Na2PdCl4, stir magnetically for 1 h, add 3 mg / mL ascorbic acid and continue stirring for 1 h, wash three times with sterile ddH2O to obtain probiotic spore nanozyme complex (SPzyme).

[0047] The morphology of the probiotic spore nanozyme complex was examined under a transmission electron microscope, and the results are as follows: Figure 3 As shown, the deposition of nanozymes on the spore surface can be clearly seen under a transmission electron microscope.

[0048] (4) Preparation of probiotic spore-germination microspheres (SPzyme+Ala@D)

[0049] The internal phase consisted of methacrylated dextran (Dex-GMA, 100 mg / mL), a photoinitiator (LAP, 2.5 mg / mL), and SPzyme (10... 8 The mixture consisted of CFU / mL and spore germination agent (L-Ala, 20mM) dissolved in sterile PBS solution; the outer phase consisted of paraffin oil; monodisperse W / O type emulsion droplets were obtained by microfluidic device and solidified into SPzyme+Ala@D microspheres under laser.

[0050] The SPzyme+Ala@D microspheres and their internal morphology were examined using scanning electron microscopy. The results are as follows: Figure 4As shown, the microspheres contain a large number of SPzymes.

[0051] Example 2: A microsphere for orally delivered Bacillus coagulans spore-specific germination

[0052] The preparation process of this embodiment is referenced. Figure 1 The following steps are included:

[0053] (1) Microbial culture and sample preparation

[0054] The cryopreserved *Bacillus coagulans* strain (purchased from Beijing BioBio Biotechnology Co., Ltd.) was thawed in a 37°C water bath. Then, 10 μL of the target bacterial suspension was transferred to a shaker tube containing 1 mL of sterile NB liquid medium and incubated at 37°C with shaking at 200 rpm for 12 h. After the bacterial suspension became turbid, another 10 μL of the target bacterial suspension was transferred to a fresh 1 mL of sterile NB liquid medium. This was repeated 2–3 times to enhance the viability of the probiotics. After activation, the probiotics were inoculated into sterile NB liquid medium at a 1:100 (V / V) ratio and incubated at 37°C with shaking at 200 rpm until the OD600 reached 0.2–0.3, ready for use.

[0055] (2) Preparation of probiotic spores:

[0056] The cultured Bacillus coagulans suspension was spread onto spore-producing agar plates and incubated at 37°C for 48 hours. Spores were scraped from the agar plates and washed three times with sterile ddH₂O. The suspension was then resuspended in 350 μL of 20% Histodesnz, and the layers were separated onto 1 mL of 50% Histodesnz. The plates were centrifuged at 16000 x g for 30 min at room temperature. The purified probiotic spores were collected and washed three more times with sterile ddH₂O to obtain the purified probiotic spores.

[0057] (3) Preparation of probiotic spore nanozyme complex (SPzyme)

[0058] Dopamine (DA) was dissolved in Tris-HCl (pH 8.5) solution to a concentration of 1 mg / mL. Probiotic spores were then dispersed in the dopamine solution to a concentration of 10 mg / mL. 8 CFU / mL. Stir magnetically for 1 h, then wash away excess dopamine solution with sterile ddH2O to obtain SP@PDA; resuspend SP@PDA in 3 mg / mL Na2PdCl4, stir magnetically for 1 h, add 3 mg / mL ascorbic acid and continue stirring for 1 h, wash three times with sterile ddH2O to obtain probiotic spore nanozyme complex (SPzyme).

[0059] (4) Preparation of probiotic spore-germination microspheres (SPzyme+Ala@D)

[0060] The internal phase consisted of methacrylated dextran (Dex-GMA, 100 mg / mL), a photoinitiator (LAP, 2.5 mg / mL), and SPzyme (10... 8 The mixture consisted of CFU / mL and spore germination agent (L-Ala, 20mM) dissolved in sterile PBS solution; the outer phase consisted of paraffin oil; monodisperse W / O type emulsion droplets were obtained by microfluidic device and solidified into SPzyme+Ala@D microspheres under laser.

[0061] Example 3: A microsphere for orally delivered Bacillus cereus spore-specific germination

[0062] The preparation process of this embodiment is referenced. Figure 1 The following steps are included:

[0063] (1) Microbial culture and sample preparation

[0064] The cryopreserved Bacillus cereus strain (purchased from Beijing BioBio Biotechnology Co., Ltd.) was thawed in a 37°C water bath beforehand. Then, 10 μL of the target bacterial suspension was transferred to a shaker tube containing 1 mL of sterile NB liquid medium and incubated at 37°C with shaking at 200 rpm for 12 h. After the bacterial suspension became turbid, another 10 μL of the target bacterial suspension was transferred to a fresh 1 mL of sterile NB liquid medium. This was repeated 2–3 times to enhance the viability of the probiotics. After activation, the probiotics were inoculated into sterile NB liquid medium at a 1:100 (V / V) ratio and incubated at 37°C with shaking at 200 rpm until the OD600 reached 0.2–0.3, ready for use.

[0065] (2) Preparation of probiotic spores:

[0066] The cultured Bacillus cereus bacterial suspension was spread onto spore-producing agar plates and incubated at 37°C for 48 hours. Spores were scraped from the agar plates and washed three times with sterile ddH2O. The suspension was then resuspended in 350 μL of 20% Histodesnz, and the suspension was separated into layers on 1 mL of 50% Histodesnz. The suspension was centrifuged at 16000 x g for 30 min at room temperature. The purified probiotic spores were collected and washed three more times with sterile ddH2O to obtain the purified probiotic spores.

[0067] (3) Preparation of probiotic spore nanozyme complex (SPzyme)

[0068] Dopamine (DA) was dissolved in Tris-HCl (pH 8.5) solution to a concentration of 1 mg / mL. Probiotic spores were then dispersed in the dopamine solution to a concentration of 10 mg / mL. 8 CFU / mL. Stir magnetically for 1 h, then wash away excess dopamine solution with sterile ddH2O to obtain SP@PDA; resuspend SP@PDA in 3 mg / mL Na2PdCl4, stir magnetically for 1 h, add 3 mg / mL ascorbic acid and continue stirring for 1 h, wash three times with sterile ddH2O to obtain probiotic spore nanozyme complex (SPzyme).

[0069] (4) Preparation of probiotic spore-germination microspheres (SPzyme+Ala@D)

[0070] The internal phase consisted of methacrylated dextran (Dex-GMA, 100 mg / mL), a photoinitiator (LAP, 2.5 mg / mL), and SPzyme (10... 8 The mixture consisted of CFU / mL and spore germination agent (L-Ala, 20mM) dissolved in sterile PBS solution; the outer phase consisted of paraffin oil; monodisperse W / O type emulsion droplets were obtained by microfluidic device and solidified into SPzyme+Ala@D microspheres under laser.

[0071] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A type of microsphere for targeted germination of probiotic spores that can be activated in a pathological microenvironment, characterized in that, The structure of the probiotic spore-germinating microspheres is as follows: The dextran hydrogel microspheres D, and the probiotic spore nanoenzyme complex SPzyme and spore nutrient germination agent X encapsulated therein, are named SPzyme+X@D; The probiotic spore nanoenzyme complex SPzyme consists of probiotic spores and a nanoenzyme coating constructed by in-situ growth on the surface of the probiotic spores; the nanoenzyme is a polydopamine-palladium nanoenzyme; and the spore nutrient germination agent is one or both of L-alanine and L-valine. The probiotic spore nanoenzyme complex responds to hydrogen peroxide in the pathological microenvironment of the colon, which degrades the dextran hydrogel and, under the action of the spore nutrient germination agent, efficiently germinates and grows into active probiotics, thereby achieving precise and targeted germination of probiotic spores in the diseased colon.

2. The microspheres for targeted germination of probiotic spores that can be activated in a pathological microenvironment, as described in claim 1, are characterized in that... The probiotic spores are derived from probiotics, which are one or more of Bacillus subtilis, Bacillus clausti, Bacillus coagulans, and Bacillus licheniformis.

3. A method for preparing probiotic spore-germination microspheres capable of activation in a pathological microenvironment, as described in any one of claims 1 to 2, characterized in that... Includes the following steps: (1) Preparation of probiotic spores: Probiotic bacterial solution is inoculated onto plate culture medium, cultured and purified to obtain probiotic spores; (2) Preparation of probiotic spore nanozyme complex SPzyme: The probiotic spores obtained in (1) were dispersed in dopamine solution, magnetically stirred and washed with sterile water to obtain SP@PDA; SP@PDA was then resuspended in Na2PdCl4 solution, magnetically stirred and ascorbic acid solution was added and stirred for a longer time, and finally washed with sterile water to obtain probiotic spore nanozyme complex SPzyme; (3) Preparation of probiotic spore-specific germination microspheres SPzyme+X@D that can be activated in a pathological microenvironment: SPzyme, methacrylated dextran, spore nutrient germination agent X, and photoinitiator prepared in step (2) were dissolved in sterile PBS solution to obtain the inner phase. The oil phase solution was used as the outer phase. Uniform monodisperse droplets were prepared by microfluidic device. The microspheres were solidified into microspheres by photocrosslinking reaction to obtain probiotic spore-specific germination microspheres SPzyme+X@D.

4. The method for preparing probiotic spore-germination microspheres capable of activation in a pathological microenvironment, as described in claim 3, is characterized in that... In step (1), the probiotic culture is in the logarithmic growth phase; the plate culture medium is spore-forming agar plate culture medium; the plate culture conditions are 36~38℃ for 48~96 h.

5. The method for preparing probiotic spore-germination microspheres capable of activation in a pathological microenvironment, as described in claim 3, is characterized in that... In the final internal phase solution obtained in step (3), the concentration of the dispersed SPzyme is 10. 6 ~10 9 The concentration of the spore germination agent X is 10-100 mM; the concentration of the methacrylated dextran is 80-120 mg / mL; the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate with a concentration of 1-5 mg / mL; and the sterile PBS solution is used as a solvent with a concentration of 10 mM and a pH of 7.1-7.

3.

6. The method for preparing probiotic spore-germination microspheres capable of activation in a pathological microenvironment, as described in claim 3, is characterized in that... In step (3), the oil phase solution is any one of paraffin oil, dimethyl silicone oil or castor oil.

7. The application of the probiotic spore-germinating microspheres that can be activated in a pathological microenvironment as described in claims 1-2, and the probiotic spore-germinating microspheres obtained by the preparation method described in any one of claims 3-6, in the preparation of oral probiotic drug formulations; wherein the oral probiotic drug formulation acts on the intestine.

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