Probiotic spore fixed-point germination microsphere capable of being activated in pathological microenvironment as well as preparation method and application of probiotic spore fixed-point germination microsphere

Abstract

The invention relates to a probiotic spore fixed-point germination microsphere capable of being activated in a pathological microenvironment as well as a preparation method and application of the probiotic spore fixed-point germination microsphere. The probiotic spore fixed-point germination microsphere is obtained by wrapping a probiotic spore nano-enzyme compound SPzyme and a spore nutrient germinator X with dextran hydrogel microspheres D. Wherein the SPzyme is prepared through a layer-by-layer coating process, the operation is simple, the cost is low, and the stability is high; and then co-encapsulating the SPzyme and the spore nutrient germinating agent X by virtue of microspheres prepared on the basis of a micro-fluidic technology. The probiotic spores can be activated by the pathological microenvironment and germinate into active probiotics under the action of the nutrient germinating agent; the germinated probiotics can rapidly consume oxygen in the colon part, the growth of harmful bacteria is remarkably inhibited, an anaerobic environment is provided for the probiotics, and the intestinal flora is positively regulated. The probiotic spore fixed-point germination microsphere is used for preparing an oral micro-ecological medicinal preparation, and has the functions of resisting gastrointestinal tract environment invasion, efficiently removing active oxygen and effectively relieving intestinal inflammation at the intestinal tract part.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, specifically to the field of oral probiotic spore preparations, and in particular to a probiotic spore fixed-site germination microsphere capable of being activated under a pathological microenvironment, and a preparation method and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic gastrointestinal immune-mediated inflammatory disease that can further lead to more serious and fatal diseases such as disability and colorectal cancer. Globally, the incidence of IBD is on the rise, and the disease is difficult to completely cure. Timely and long-term treatment is required to prevent the development of disease symptoms and related complications, which poses a huge challenge to the world's health care system. Emerging evidence shows that IBD is associated with intestinal mucosal barrier dysfunction, immune dysfunction, and bacterial microbiome dysbiosis. In the state of enteritis, the immune response is overactive, intestinal epithelial oxygenation increases, and a large number of inflammatory factors and reactive oxygen species (ROS) are produced. On the one hand, ROS can be used by facultative anaerobes to cause their massive proliferation. On the other hand, almost all beneficial intestinal microorganisms are obligate anaerobes and have difficulty surviving under conditions of sufficient oxygen and high inflammation, which leads to a decrease in the biodiversity of the intestinal flora, a disruption of the intestinal flora homeostasis, a decrease in the proportion of commensal bacteria such as Firmicutes, and a large proliferation of harmful bacteria such as facultative anaerobic bacteria such as Enterobacteriaceae. Existing clinical therapies for IBD mainly focus on improving disease-related symptoms by alleviating intestinal inflammation or suppressing immune responses, including anti-inflammatory drugs, immunomodulators, and monoclonal antibodies. However, these treatments may cause serious complications due to off-target side effects, tissue toxicity, and other unexpected side effects. More importantly, these approaches often fail to address the root causes of IBD, including gastrointestinal mucosal damage, loss of intestinal barrier function, and dysbiosis of intestinal symbiotic microbiota. Therefore, there is an urgent need for a strategy that can both scavenge ROS and inhibit excessive inflammatory responses, while also being highly effective in inhibiting aerobic overgrowth of Enterobacteriaceae, restoring intestinal flora homeostasis, and treating colitis from the root.

[0003] Dormant spores formed by the probiotic Bacillus subtilis have attracted increasing attention due to their ability to resist the harsh gastrointestinal (GI) environment. In addition, they can germinate and grow into probiotic Bacillus subtilis, which, along with the consumption of oxygen, can specifically limit the dysbiotic expansion of aerobic Enterobacteriaceae and increase the abundance of beneficial bacteria, thereby improving the intestinal microenvironment.

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

[0005] The prior art lacks a strategy that can optimize the therapeutic effect of probiotic spores by improving their targeted germination ability and ROS scavenging activity in the pathological site of colitis. The present invention provides a probiotic spore fixed-site germination microsphere that can be activated under a pathological microenvironment and a preparation method and application thereof. The probiotic spore fixed-site germination microsphere of the present invention can be activated by the disease pathological microenvironment, promote the fixed-site efficient germination of spores in the colon, and provide a new idea for the treatment of colitis with probiotic spores.

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

[0007] After targeting the colon, the colon glucanase in the colon slowly degrades the microspheres, and the nanozyme can effectively remove ROS in the colitis site, thereby alleviating inflammation. At the same time, after the nanozyme coating is degraded, the probiotic spores are exposed, and then the probiotic spores germinate and grow at the colon site under the action of the released L-Ala, and quickly deplete the oxygen in the colitis site, thereby significantly inhibiting the expansion of harmful bacteria of the Enterobacteriaceae family, and providing an anaerobic environment for the growth of other probiotics in the colon, positively regulating the intestinal flora, and effectively treating colitis.

[0008] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

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

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

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

[0012] In some specific embodiments of the present 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-specific germination microspheres that can be activated under a pathological microenvironment, comprising the following steps:

[0015] (1) Preparing probiotic spores: inoculating a probiotic bacterial liquid in a logarithmic growth phase onto a plate culture medium, culturing and purifying to obtain probiotic spores;

[0016] (2) Preparation of probiotic spore nanozyme complex (SPzyme): dispersing the probiotic spores obtained in (1) in the nanozyme solution, stirring, and washing away the excess nanozyme solution 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 under pathological microenvironments: The SPzyme prepared in step (2), methacrylated dextran, spore nutrient germination agent X, and photoinitiator are dissolved in a sterile PBS solution to obtain an inner phase, and the oil phase solution is used as the outer phase. Monodisperse droplets of uniform size are prepared by a microfluidic device; and the droplets are solidified into microspheres by a photocrosslinking reaction to obtain probiotic spore-specific probiotic spore-specific germination microspheres SPzyme+X@D.

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

[0019] Furthermore, in step (2), the concentration of the nanozyme solution is 0.2 to 10 mg / mL; the solvent of the nanozyme solution is any one of Tris-HCl or sterile PBS; the concentration of the probiotic spores dispersed in the nanozyme solution is 10 6 ~10 9 CFU / mL; the stirring can be performed by a magnetic stirrer, and the stirring time is 1 to 2 hours.

[0020] In some specific embodiments of the present invention, the nanozyme solution is a dopamine solution and Na 2 PdCl 4 solution; the concentration of the dopamine solution is 0.2 to 2 mg / mL, and the solvent is Tris-HCl with a pH of 8.4 to 8.6; the concentration of the Tris-HCl is 10 to 20 mM; the Na 2 PdCl 4 The concentration of the solution is 1-10 mg / mL, 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 embodiments of the present invention, the probiotic spores are first dispersed in a dopamine solution, magnetically stirred, and then washed with sterile water to obtain SP@PDA; SP@PDA is then resuspended in Na 2 PdCl 4 In the solution, after magnetic stirring, ascorbic acid solution is added (the role of ascorbic acid is to reduce Pd ions into Pd nanoparticles) and stirring is continued, and finally washed with sterile water to obtain the probiotic spore nanozyme 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 internal phase solution finally obtained in step (3), the concentration of the SPzyme after dispersion is 10 6 ~10 9 CFU / mL; the concentration of the spore nutrient 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) with a concentration of 1-5 mg / mL; the sterile PBS solution is used as a solvent with 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-specific germination microspheres that can be activated under a pathological microenvironment in the preparation of an oral probiotic drug preparation.

[0025] Furthermore, the oral microecological drug preparation has an action site of the intestinal tract;

[0026] Specifically, the probiotic spore-specific germination microspheres in the oral probiotic drug preparation can be activated by the microenvironment of the pathological site, release spore nutrient germination agents, activate probiotic spores, and make them grow into active probiotics, quickly consume oxygen in the pathological site, inhibit the growth of harmful bacteria of the Enterobacteriaceae family, and at the same time provide an anaerobic environment for the growth of other probiotics in the colon, thereby promoting the homeostasis of intestinal flora; the nanozymes in the probiotic spore-specific germination microspheres can efficiently remove high concentrations of reactive oxygen in the disease site and relieve intestinal inflammation.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

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

[0029] 2) The probiotic spore nanozyme complex used in the present invention can respond to hydrogen peroxide in the pathological microenvironment in the colon, and then the glucan hydrogel coating is degraded and efficiently germinates and grows into active probiotics under the action of the spore nutrient germination agent, thereby achieving precise and targeted germination of probiotic spores in the colon disease site.

[0030] 3) The germinated aerobic Bacillus can quickly consume oxygen in the colon, significantly inhibiting the growth of harmful bacteria of the Enterobacteriaceae family, while providing an anaerobic environment for the growth of other probiotics in the colon and promoting the homeostasis of intestinal flora.

[0031] 4) The probiotic spore nanozyme complex used in the present invention is prepared by a layer-by-layer coating process, the preparation method is universal and simple, and the nanozyme modification of probiotic spores can be quickly achieved, the production cost is low, and the stability is strong. The microspheres prepared by the present invention rely on microfluidic technology, the preparation process is simple, no complex mechanical processing process is required, and the monodispersity is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a flow chart for preparing the probiotic spore fixed-site germination microspheres of Examples 1 to 3;

[0033] Figure 2 This is a light microscopic image of the probiotic spores of Example 1;

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

[0035] Figure 4 This is a scanning electron microscope characterization image of SPzyme+Ala@D microspheres in Example 1. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below in conjunction with 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 form. It should be noted that for those of ordinary skill in the art, several variations and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0037] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified.

[0038] Example 1 A Bacillus subtilis spore-specific germination microsphere for oral delivery

[0039] The preparation process of this embodiment refers to Figure 1 , including the following steps:

[0040] (1) Bacterial culture and sample preparation

[0041] The frozen Bacillus subtilis strain (purchased from Beijing Biobo Biotechnology Co., Ltd.) was pre-dissolved in a 37°C water bath, and then 10 μL of the target bacterial suspension was transferred to a shaking tube containing 1 mL of sterile NB liquid culture medium, and cultured at 200 rpm in a 37°C constant temperature incubator for 12 hours. When the bacterial solution becomes turbid, 10 μL of the target bacterial solution is again transferred to a new 1 mL of sterile NB liquid culture medium. Continuously subculture 2 to 3 times to enhance the activity of the probiotics. After activation, inoculate in sterile NB liquid culture medium at an inoculum size of 1:100 (V / V), and culture in a 37°C constant temperature incubator at 200 rpm with shaking until OD600 is 0.2-0.3, and set aside.

[0042] (2) Preparation of probiotic spores

[0043] The cultured Bacillus subtilis liquid was spread on a spore-forming agar plate and cultured in a 37°C constant temperature incubator for 72 h. The spores on the agar plate were scraped and sterilized with ddH 2O and washed three times. Then resuspended with 350 μL 20% Histodenz, layered on 1 mL 50% Histodenz, and centrifuged at 16000 x g for 30 min at room temperature. Purified probiotic spores were collected and then washed with sterile ddH 2 O was washed three times to obtain purified probiotic spores.

[0044] The morphology of the purified probiotics was observed under an optical microscope. Figure 2 This is a light microscopy picture of the purified probiotic spores. The results show that the purified Bacillus subtilis spores have good dispersibility and are rod-shaped or short rod-shaped, which is in line 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, and then probiotic spores were dispersed in the dopamine solution to a concentration of 10 8 CFU / mL. Stir magnetically for 1 h and then use sterile ddH 2 O to wash away the excess dopamine solution to obtain SP@PDA; resuspend SP@PDA in 3 mg / mL Na 2 PdCl 4 After magnetic stirring for 1 h, 3 mg / mL ascorbic acid was added and continued stirring for 1 h. 2 O was washed three times to obtain the probiotic spore nanozyme complex (SPzyme).

[0047] The morphology of the probiotic spore nanozyme complex was detected under transmission electron microscopy. 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-specific germination microspheres (SPzyme+Ala@D)

[0049] The internal phase consisted of methacrylated dextran (Dex-GMA, 100 mg / mL), photoinitiator (LAP, 2.5 mg / mL), SPzyme (10 8 CFU / mL) and spore nutrient germination agent (L-Ala, 20 mM) dissolved in sterile PBS solution; the external phase is composed of paraffin oil; monodisperse W / O type emulsion droplets are 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 by scanning electron microscopy. Figure 4As shown, it can be seen that the microspheres contain a large amount of SPzymes.

[0051] Example 2 A Bacillus coagulans spore-specific germination microsphere for oral delivery

[0052] The preparation process of this embodiment refers to Figure 1 , including the following steps:

[0053] (1) Bacterial culture and sample preparation

[0054] The frozen Bacillus coagulans strain (purchased from Beijing Biobo Biotechnology Co., Ltd.) was pre-dissolved in a 37°C water bath, and then 10 μL of the target bacterial suspension was transferred to a shaking tube containing 1 mL of sterile NB liquid culture medium, and cultured at 200 rpm in a 37°C constant temperature incubator for 12 hours. When the bacterial solution becomes turbid, 10 μL of the target bacterial solution is again transferred to a new 1 mL of sterile NB liquid culture medium. Continuously subculture 2 to 3 times to enhance the activity of the probiotics. After activation, inoculate in sterile NB liquid culture medium at an inoculum of 1:100 (V / V), and culture in a 37°C constant temperature incubator at 200 rpm with shaking until OD600 is 0.2-0.3, and set aside.

[0055] (2) Preparation of probiotic spores:

[0056] The cultured Bacillus coagulans was spread on a spore-forming agar plate and cultured in a 37°C constant temperature incubator for 48 h. The spores on the agar plate were scraped and sterilized with ddH 2 O and washed three times. Then resuspended with 350 μL 20% Histodenz, layered on 1 mL 50% Histodenz, and centrifuged at 16000 x g for 30 min at room temperature. Purified probiotic spores were collected and then washed with sterile ddH 2 O was washed three times to obtain 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, and then probiotic spores were dispersed in the dopamine solution to a concentration of 10 8 CFU / mL. Stir magnetically for 1 h and then use sterile ddH 2 O to wash away the excess dopamine solution to obtain SP@PDA; resuspend SP@PDA in 3 mg / mL Na 2 PdCl 4 After magnetic stirring for 1 h, 3 mg / mL ascorbic acid was added and continued stirring for 1 h. 2O was washed three times to obtain the probiotic spore nanozyme complex (SPzyme).

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

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

[0061] Example 3 A Bacillus cereus spore-specific germination microsphere for oral delivery

[0062] The preparation process of this embodiment refers to Figure 1 , including the following steps:

[0063] (1) Bacterial culture and sample preparation

[0064] The frozen Bacillus cereus strain (purchased from Beijing Biobo Biotechnology Co., Ltd.) was pre-dissolved in a 37°C water bath, and then 10 μL of the target bacterial suspension was transferred to a shaking tube containing 1 mL of sterile NB liquid culture medium, and cultured at 200 rpm in a 37°C constant temperature incubator for 12 hours. When the bacterial solution becomes turbid, 10 μL of the target bacterial solution is again transferred to a new 1 mL of sterile NB liquid culture medium. Continuously subculture 2 to 3 times to enhance the activity of the probiotics. After activation, inoculate in sterile NB liquid culture medium at an inoculum size of 1:100 (V / V), and culture in a 37°C constant temperature incubator at 200 rpm with shaking until OD600 is 0.2-0.3, and set aside.

[0065] (2) Preparation of probiotic spores:

[0066] The cultured Bacillus cereus was spread on a spore-forming agar plate and cultured in a 37°C constant temperature incubator for 48 h. The spores on the agar plate were scraped and sterilized with ddH 2 O and washed three times. Then resuspended with 350 μL 20% Histodenz, layered on 1 mL 50% Histodenz, and centrifuged at 16000 x g for 30 min at room temperature. Purified probiotic spores were collected and then washed with sterile ddH 2 O was washed three times to obtain 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, and then probiotic spores were dispersed in the dopamine solution to a concentration of 10 8 CFU / mL. Stir magnetically for 1 h and then use sterile ddH 2 O to wash away the excess dopamine solution to obtain SP@PDA; resuspend SP@PDA in 3 mg / mL Na 2 PdCl 4 After magnetic stirring for 1 h, 3 mg / mL ascorbic acid was added and continued stirring for 1 h. 2 O was washed three times to obtain the probiotic spore nanozyme complex (SPzyme).

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

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

[0071] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and 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 present invention should be within the scope of protection of the present invention.

Claims

1. A probiotic spore-specific germination microsphere that can be activated under a pathological microenvironment, characterized in that: The structure of the probiotic spore fixed-site germination microspheres is: Dextran hydrogel microspheres D, and their encapsulated probiotic spore nanozyme complex SPzyme and spore nutrient germination agent X, named SPzyme+X@D; The probiotic spore nanozyme complex SPzyme is composed of probiotic spores and nanozymes.

2. The probiotic spore-specific germination microspheres that can be activated under pathological microenvironment according to claim 1, characterized in that: The probiotic spores are from probiotics, and the probiotics are one or more of Bacillus subtilis, Bacillus cereus, Bacillus clausii, Bacillus coagulans, Bacillus licheniformis, Bacillus multifermentans or Bacillus pumilus.

3. The probiotic spore-specific germination microspheres that can be activated under pathological microenvironment according to claim 1, characterized in that: The nanozyme is an antioxidant nanomaterial, and the antioxidant nanomaterial is one or more of polydopamine, cerium dioxide, manganese tetraoxide, palladium metal or platinum metal.

4. The probiotic spore-specific germination microspheres that can be activated under pathological microenvironment according to claim 1, characterized in that: The spore nutrient germination agent is a small molecule spore nutrient germination agent, which is one or more of L-alanine, L-valine or glucose.

5. The method for preparing a probiotic spore-specific germination microsphere capable of being activated under a pathological microenvironment as claimed in any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Preparing probiotic spores: inoculating a probiotic bacterial liquid into a plate culture medium, culturing and purifying to obtain probiotic spores; (2) Preparation of probiotic spore nanozyme complex SPzyme: dispersing the probiotic spores obtained in (1) in a nanozyme solution, stirring, and washing away excess nanozyme solution with sterile water to obtain SP@Y; namely, the probiotic spore nanozyme complex SPzyme; (3) Preparation of probiotic spore-specific germination microspheres SPzyme+X@D that can be activated under pathological microenvironments: The SPzyme prepared in step (2), methacrylated dextran, spore nutrient germination agent X, and photoinitiator are dissolved in a sterile PBS solution to obtain an inner phase, and the oil phase solution is used as the outer phase. Monodisperse droplets of uniform size are prepared by a microfluidic device; and the droplets are solidified into microspheres by a photocrosslinking reaction to obtain probiotic spore-specific probiotic spore-specific germination microspheres SPzyme+X@D.

6. The method for preparing a probiotic spore-specific germination microsphere capable of being activated under a pathological microenvironment according to claim 5, characterized in that: In step (1), the probiotic bacterial liquid is in the logarithmic growth phase; the plate culture medium is a spore-forming agar plate culture medium; and the plate culture conditions are 36-38° C. for 48-96 hours.

7. The method for preparing a probiotic spore-specific germination microsphere capable of being activated under a pathological microenvironment according to claim 5, characterized in that: In step (2), the concentration of the nanozyme solution is 0.2-10 mg / mL; the solvent of the nanozyme solution is any one of Tris-HCl or sterile PBS; the pH of the Tris-HCl is 8.4-8.6, and the concentration is 10-20 mM; the pH of the sterile PBS is 7.1-7.3, and the concentration is 10 mM; The concentration of the probiotic spores dispersed in the nanozyme solution is 10 6 ~10 9 CFU / mL; Each stirring time is 1 to 2 hours.

8. The method for preparing a probiotic spore-specific germination microsphere capable of being activated under a pathological microenvironment according to claim 5, characterized in that: In the internal phase solution finally obtained in step (3), the concentration of the SPzyme after dispersion is 10 6 ~10 9 CFU / mL; the concentration of the spore nutrient germination agent X is 10-100 mM; the concentration of the methacrylated dextran is 80-120 mg / mL; the photoinitiator is phenyl-2,4,6-trimethylbenzoyl lithium phosphinate, with a concentration of 1-5 mg / mL; the sterile PBS solution is used as a solvent, with a concentration of 10 mM and a pH of 7.1-7.

3.

9. The method for preparing a probiotic spore-specific germination microsphere capable of being activated under a pathological microenvironment according to claim 5, characterized in that: In step (3), the oil phase solution is any one of paraffin oil, dimethyl silicone oil or castor oil.

10. Use of the probiotic spore site-specific germination microspheres that can be activated under a pathological microenvironment as described in claims 1 to 4, and the probiotic spore site-specific germination microspheres obtained by the preparation method according to any one of claims 5 to 9 in the preparation of oral probiotic ecological drug preparations; the oral probiotic ecological drug preparations act on the intestine.

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