Nano-engineered bacillus subtilis as well as preparation method and application thereof

Abstract

The invention relates to nano-engineered bacillus subtilis as well as a preparation method and application thereof, and belongs to the technical field of biology. Aiming at the technical problems of low probiotic delivery efficiency, insufficient regulation and control of enteropathogenic bacteria and single ROS (reactive oxygen species) removal in the prior art, a ternary composite system of chitosan-coated bacillus subtilis bridged WO3 (at) PDA nanoparticles is constructed. The preparation method comprises the following steps: carrying out auto-polymerization on dopamine hydrochloride and WO3 nanoparticles to form a WO3 (at) PDA core-shell structure; the method comprises the following steps: coating bacillus subtilis with chitosan through pH regulation to form BSCS; and finally, preparing the BSCS-coated WO3-coated PDA complex microbial inoculant through electrostatic interaction bridging. Experiments show that the preparation can relieve inflammatory symptoms of mice with DSS-induced colitis and effectively realize intestinal micro-ecological reprogramming. The invention provides a novel nano-probiotic synergistic treatment system for IBD treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to nano-engineered Bacillus subtilis and its preparation method and application Background Art

[0002] Inflammatory bowel disease (IBD) is a common chronic relapsing disease, mainly including Crohn's disease (CD) and ulcerative colitis (UC), which affects the colon and rectum. In recent years, the incidence and prevalence of IBD have been continuously increasing, becoming a global public health problem. Intestinal inflammation leads to macrophage activation, secreting excessive reactive oxygen species (ROS). These ROS trigger an inflammatory cascade reaction by continuously activating NF-κB and promote the proliferation of facultative anaerobic bacteria as electron acceptors, resulting in the occupation of the ecological niche by Enterobacteriaceae, jointly causing intestinal barrier dysfunction and intestinal flora dysbiosis. Therefore, clearing excessive ROS to stabilize the immune microenvironment at the inflammatory site is crucial for reshaping the intestinal microecology. Research shows that biomaterials such as polyphenolic antioxidants and nanozymes can effectively scavenge ROS and relieve oxidative stress by reacting with ROS or enzyme-like catalysis. However, the single ROS scavenger treatment strategy has poor effects

[0003] The introduction of probiotics can play a role in regulating intestinal homeostasis by inhibiting pathogens and regulating intestinal flora imbalance. Bacillus subtilis (BS), as a promising IBD treatment strain, can produce short-chain fatty acids in the intestine, repair the intestinal mucosal barrier, inhibit pathogenic bacteria, and restore intestinal flora homeostasis, etc. However, the treatment strategy of oral probiotics is limited by the effective transport and colonization of probiotics at the inflammatory site and their sensitivity to the harsh gastrointestinal environment, which limits their viability and retention time in the intestine

[0004] To improve the therapeutic effect of probiotic-assisted intestinal microecological reprogramming in IBD, it is necessary to perform protective oral delivery of probiotics and specifically regulate pathogenic bacteria to expand the ecological niche of probiotics and protect them from competitive inhibition by pathogenic bacteria. As conditional pathogens, Enterobacteriaceae produce a large amount of toxins in the inflamed intestine and competitively inhibit the colonization and growth of orally administered probiotics. The strategy of treating IBD by reprogramming the intestinal microecology with orally administered probiotics depends on the effective regulation of pathogenic bacteria. Tungstate can inhibit the overgrowth of Enterobacteriaceae by selectively inhibiting the molybdenum-cofactor-dependent microbial respiratory pathway by substituting tungsten ions for molybdenum in nitrate reductase, without affecting beneficial bacteria. This strategy can regulate the dysbiosis in IBD, but the unfavorable distribution and uncontrollable pharmacokinetics of tungstate limit its application. Tungsten oxide nanoparticles (WO3 NPs) have advantages in reshaping the intestinal microecology of the inflamed colon, but tungsten ions are released prematurely under acidic conditions, which may have an adverse effect on body organs.

[0005] Therefore, it is very important to incorporate a pathogenic bacteria metabolic reprogramming module into the protective probiotic delivery system to simultaneously meet inflammation-targeted promotion of probiotic colonization, which is also a key scientific issue that should be considered in the design of the IBD intestinal microecological reprogramming system. Excess ROS produced in the inflamed intestine not only oxidatively damages the intestinal epithelial barrier but also acts as a danger signal molecule to promote the transformation of M2-phenotype macrophages into M1-phenotype and activate an uncontrollable inflammatory cascade. Polydopamine (PDA), as a polyphenolic polymer material, has unique redox capabilities and can provide electrons to inhibit ROS radicals. Polydopamine nanoparticles (PDA NPs) have effective antioxidant capabilities to scavenge superoxide anion (·O2-), hydroxyl radical (·OH), and DPPH, and exhibit a concentration-dependent intracellular ROS scavenging effect. The acid-resistant property of PDA and the strong adhesion and easy modification properties of catechol groups make it widely used in the development and application of surface coatings and composites of different materials. Summary of the Invention

[0006] In view of this, the present invention adopts the strategy of using chitosan (CS) as the intermediate layer. As a cationic polysaccharide, chitosan carries a positive charge and can coat the surface of Bacillus subtilis. Negatively charged WO3@PDA nanoparticles can be bridged with chitosan-coated Bacillus subtilis through electrostatic interaction. By using the protection of chitosan coating and WO3@PDA nanoparticles, the survival rate of Bacillus subtilis in the gastrointestinal environment can be improved.

[0007] One of the purposes of the present invention is to provide a nano-engineered Bacillus subtilis, the second purpose is to provide a preparation method of the nano-engineered Bacillus subtilis, and the third purpose is to provide an application of the nano-engineered Bacillus subtilis in the preparation of IBD drugs.

[0008] To achieve the above purposes, the present invention provides the following technical solutions:

[0009] The present invention provides a nano-engineered Bacillus subtilis, which is composed of WO3 nanoparticles wrapped by PDA and Bacillus subtilis wrapped by chitosan;

[0010] Furthermore, the preparation method of the nano-engineered Bacillus subtilis is as follows:

[0011] S1: Synthesize WO3 nanoparticles wrapped by PDA

[0012] The WO3 nanoparticles are dispersed in a Tris buffer solution containing dopamine hydrochloride to prepare the WO3@PDA nanoparticles;

[0013] S2: Synthesize Bacillus subtilis wrapped by chitosan

[0014] Chitosan is dissolved in acidic PBS, and the Bacillus subtilis bacterial solution is added and stirred. The pH value of the solution is adjusted to deposit chitosan on the surface of Bacillus subtilis to prepare the chitosan-wrapped Bacillus subtilis;

[0015] S3: Synthesize Bacillus subtilis bridging WO3@PDA

[0016] The WO3@PDA prepared in step S1 and the chitosan-wrapped Bacillus subtilis prepared in step S2 are mixed and stirred in acidic PBS, and the nano-engineered Bacillus subtilis BSCS@WO3@PDA is collected after centrifugation;

[0017] Preferably, in step S1, the concentration ratio of dopamine hydrochloride to WO3 nanoparticles is 1:2, and the pH of the Tris buffer solution is 8.5;

[0018] Preferably, in step S2, the chitosan solution concentration is 2 mg / mL, the bacterial solution concentration is (1 - 9) x 10 9 CFU / mL, and the solution pH value is 6.5;

[0019] Preferably, in step S3, the ratio of WO3@PDA to chitosan-wrapped Bacillus subtilis is 1 mg: 1 x 10 9 CFU, and the pH of the PBS buffer solution is 6.0;

[0020] Further, the application of nanoengineered Bacillus subtilis in the preparation of drugs;

[0021] Preferably, the application of nanoengineered Bacillus subtilis in the preparation of drugs for the treatment of IBD.

[0022] The beneficial effects of the present invention are as follows:

[0023] The present invention prepares a nanoengineered Bacillus subtilis, which has broad-spectrum ROS scavenging performance and can alleviate the oxidative stress of colitis; at the same time, it can selectively inhibit pathogenic Enterobacteriaceae and play a role in reprogramming the intestinal flora metabolism; and it can protectively transport Bacillus subtilis to smoothly reach the colon inflammation site, regulate the intestinal flora, enhance the intestinal barrier, and then relieve intestinal inflammation. The specific advantages are as follows:

[0024] 1. Improve the delivery efficiency and survival rate of probiotics

[0025] Through the dual protection structure of chitosan coating (BSCS) and WO3@PDA bridging. The cationic property of chitosan neutralizes the erosion of probiotics by gastric acid; the modification of PDA-based nanoparticles on the surface of probiotics provides a strategy for their delivery in a complex gastrointestinal environment as a protection module for probiotics.

[0026] Enhanced intestinal targeting: The ROS-responsive degradation property of PDA enables WO3@PDA to release tungsten ions in the high-ROS environment of the inflamed colon.

[0027] 2. Achieve dual regulation of the inflammatory microenvironment

[0028] The core-shell structure of WO3@PDA and the ROS scavenging ability of PDA act synergistically. PDA scavenges excessive ROS and alleviates oxidative stress. WO3@PDA releases tungsten ions at the inflammation site and selectively inhibits Enterobacteriaceae. The acid-resistant property of PDA and the strong adhesion and easy modification properties of catechol groups help PDA form a composite structure with WO3 NPs to construct WO3@PDA core-shell nanoparticles and reduce the release of tungsten ions under acidic conditions;

[0029] 3. Reduce the risk of systemic toxicity, the core-shell structure and ROS-responsive degradation mechanism of WO3@PDA. The PDA coating reduces the premature release of tungsten ions in the gastric acid environment (pH 1-3) and increases the accumulation in the inflamed colon, avoiding the adverse distribution in normal tissues.

[0030] This system simultaneously has the ability to scavenge excess ROS in the inflammatory environment and selectively regulate Enterobacteriaceae, and is expected to effectively treat IBD. The present invention also provides a preparation method of the nanoengineered Bacillus subtilis, which has a simple preparation method, mild reaction conditions and low cost. It has broad application prospects.

[0031] Other advantages, objectives, and features of the present invention will, to some extent, be elaborated in the subsequent description, and to some extent, will be obvious to those skilled in the art based on the study of the following text, or can be learned from the practice of the present invention. The objectives and other advantages of the present invention can be achieved and obtained through the following description. Brief Description of the Drawings

[0032] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be described in detail preferably with reference to the accompanying drawings, where:

[0033] Figure 1 is the Zeta potential diagram;

[0034] Figure 2 is the transmission electron microscopy (TEM) diagram;

[0035] Figure 3 is the diagram of the change in mouse body weight;

[0036] Figure 4 is the diagram of the change in mouse symptom score;

[0037] Figure 5 is the anatomical diagram of the mouse colon;

[0038] Figure 6 is the statistical chart of the mouse colon length. Detailed Description of the Embodiments

[0039] The following illustrates the embodiments of the present invention through specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. The details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the diagrams provided in the following examples only illustrate the basic concept of the present invention schematically. Without conflict, the following examples and the features in the examples can be combined with each other.

[0040] Among them, the accompanying drawings are only for illustrative purposes, showing only schematic diagrams, not actual diagrams, and should not be construed as a limitation to the present invention; in order to better illustrate the embodiments of the present invention, some components in the accompanying drawings will be omitted, enlarged, or reduced, which do not represent the dimensions of the actual product; for those skilled in the art, it is understandable that some well-known structures and their descriptions in the accompanying drawings may be omitted.

[0041] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components; in the description of the present invention, it should be understood that if there are terms such as "upper", "lower", "left", "right", "front", "rear", etc. indicating the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, it is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, the terms describing the positional relationship in the accompanying drawings are only for illustrative purposes and cannot be construed as a limitation of the present invention. For those of ordinary skill in the art, the specific meanings of the above terms can be understood according to specific circumstances.

[0042] The main reagents and instruments in the following examples: tungsten trioxide (WO3) nanoparticles (nanopowder, Sigma-Aldrich, USA), PBS buffer, dopamine hydrochloride (analytical pure, Shanghai Aladdin Biochemical Technology Co., Ltd.), chitosan, tris(hydroxymethyl)aminomethane, LB broth, agar powder (analytical pure, Beijing Solarbio Science & Technology Co., Ltd.), Bacillus subtilis (ATCC6051, China Baosai Plasmid Strain Co.), absolute ethanol (analytical pure, Chongqing Chuandong Chemical Co., Ltd.); ultrasonic cleaner (SB-4200D, Ningbo Xinzhi Biotechnology Co., Ltd.), magnetic stirrer (LC-MSB-HD, Shanghai Lichen Bangxi Instrument Technology Co., Ltd.), constant temperature shaker (THZ-98AB, Shanghai Yiheng Scientific Instrument Co., Ltd.), high-speed refrigerated centrifuge (3-18K, Sigma-Aldrich, USA), particle size analyzer (ZS90, Malvern, UK).

[0043] Preparation of WO3 nanoparticles (WO3@PDA) encapsulated with PDA in Example 1

[0044] WO3@PDA was synthesized by the hot solvent method. Using Tris buffer with pH = 8.5 as the reaction solution, 1 mg / mL of WO3 nanoparticles were added. After ultrasonic dispersion, 0.5 mg / mL of dopamine hydrochloride was added, and the mixture was stirred at 1200 rpm at room temperature for 2 hours. The reaction solution changed from milky white to black. The reaction solution was collected, centrifuged at 11000 rpm for 10 minutes by a high-speed centrifuge, washed twice with absolute ethanol, and finally collected with absolute ethanol and stored at 4°C.

[0045] Preparation of Bacillus subtilis (BSCS) encapsulated with chitosan in Example 2

[0046] Pick a single colony of BS from the agar plate coated with BS into the LB liquid medium, and shake culture at 37°C and 200 rpm on a constant temperature shaker for 14 hours to make the OD of the bacterial solution 600 = 1.0, and the bacterial solution concentration was calculated to be 2x10 9CFU / mL. Pipette 5 mL of the bacterial solution, centrifuge at 6000 rpm for 5 minutes, add 10 mL of a 2 mg / mL chitosan solution with a pH of 6.0, stir at 1200 rpm for 30 minutes, then centrifuge at 6000 rpm for 5 minutes, and wash twice with PBS to obtain BSCS.

[0047] Example 3. Preparation of Bacillus subtilis bridged with WO3@PDA (BSCS@WO3@PDA)

[0048] Collect 10 mg of the WO3@PDA particles prepared in the first step, disperse them in 10 mL of a PBS buffer with a pH of 6.0, add the BSCS prepared in the second step, stir at 1200 rpm for 2 hours, after the reaction is completed, centrifuge at 6000 rpm, discard the supernatant, resuspend the precipitate with PBS, and collect to obtain BSCS@WO3@PDA.

[0049] Example 4. Characterization of nanoengineered Bacillus subtilis

[0050] The obtained system was characterized by a nanoparticle size analyzer and a transmission electron microscope, and the results are shown in Figure 1 and Figure 2 . After being wrapped with PDA, the potential of the WO3 particles changed. Combining with the obvious core-shell structure in TEM, it can be proved that the wrapping of PDA was successful. After the BS was wrapped with chitosan, the potential changed from negative to positive, and an obvious coating was also visible in the TEM image, indicating the successful preparation of BSCS. Finally, after BSCS was connected to WO3@PDA, the potential changed from positive to negative again. Combining with the enrichment of particles on the bacterial surface in TEM, it proved the successful preparation of BSCS@WO3@PDA.

[0051] Example 5. Application of nanoengineered Bacillus subtilis

[0052] The prepared nanoengineered probiotics were used for oral treatment of a DSS-induced colitis mouse model. After 7 days of feeding with 2.5% DSS to establish a colitis model, BS, WO3@PDA, and BSCS@WO3@PDA were intragastrically administered starting from the third day of modeling for a total of 7 days. During the treatment process, the body weight changes, soft stools, and bloody stools of the mice were monitored daily. After the treatment was completed, the mice were sacrificed, the colon tissues were collected, and corresponding efficacy evaluations were carried out. The specific results are as shown in Figure 3 and Figure 4 , BSCS@WO3@PDA can effectively relieve symptoms such as weight loss, soft stools, and rectal bleeding caused by DSS. As shown in Figure 5 and Figure 6 , BSCS@WO3@PDA can restore the colon shortening caused by DSS. Therefore, BSCS@WO3@PDA can effectively relieve colitis in mice and has application potential in the treatment of IBD.

[0053] The BSCS@WO3@PDA of the present invention and the control group were comparatively analyzed in the treatment of DSS-induced colitis mouse model as follows:

[0054] 1. Significantly relieve weight loss

[0055] In the DSS model group, the body weight of mice continued to decline due to intestinal inflammation, while the decline in body weight in the treatment group was significantly less than that in the group of BS alone or WO3@PDA. This indicates that the composite system effectively alleviated intestinal inflammation in mice and slowed down the disease progression through synergistic effects (probiotic colonization + oxidative stress regulation).

[0056] 2. Improve intestinal symptoms

[0057] Symptom scores (soft stools, bloody stools) showed that the scores in the BSCS@WO3@PDA group were significantly lower than those in the control group. The specific manifestations were as follows:

[0058] Reduction of soft stools: It indicates that the intestinal water absorption function is restored and the inflammatory exudation is reduced;

[0059] Relief of bloody stools: It reflects the repair of intestinal mucosal damage and the inhibition of the release of inflammatory factors (such as TNF-α, IL-6);

[0060] This effect is attributed to WO3@PDA scavenging ROS and inhibiting the NF-κB pathway, while the chitosan and WO3@PDA coatings protect probiotics and enhance the intestinal barrier repair ability.

[0061] 3. Restore colon length

[0062] The colon in the DSS model group was significantly shortened (caused by inflammatory fibrosis and edema), while the colon length in the BSCS@WO3@PDA group was close to normal. Scanning electron microscopy showed that nanoparticles were enriched on the surface of probiotics, and it was speculated that they played a role through the following mechanisms:

[0063] Inhibit the proliferation of pathogenic bacteria: WO3 releases tungsten ions to selectively inhibit Enterobacteriaceae, reduce toxin production and ecological occupancy.

[0064] Promote probiotic colonization: The chitosan and WO3@PDA coatings improve the survival rate of Bacillus subtilis in the gastric acid environment and enhance its ecological occupancy in the colon.

[0065] Antioxidant protection: PDA scavenges ROS, reduces oxidative damage, and prevents the inflammatory cascade reaction.

[0066] The technological breakthroughs of the present invention are as follows:

[0067] 1. Multi-mechanism synergistic treatment

[0068] Traditional IBD therapies mostly target single targets (such as anti - inflammation or probiotic supplementation), while BSCS@WO3@PDA integrates: ROS scavenging (the antioxidant activity of PDA), pathogenic bacteria inhibition (WO3 selectively regulates the metabolism of Enterobacteriaceae), and probiotic delivery protection (chitosan and PDA enhance gastric acid tolerance). The three work together to break the "inflammation - dysbiosis" vicious cycle and achieve microecological reprogramming.

[0069] 2. Targeted delivery and controlled - release design

[0070] ROS - responsive release: WO3@PDA is stable in acidic environments, reducing the premature release of tungsten ions in gastric acid. Meanwhile, the PDA shell degrades in the high - ROS environment of the inflamed colon, releasing tungsten ions at a specific site and reducing systemic toxicity.

[0071] Electrostatic bridging and directional adsorption: Negatively charged WO3@PDA is enriched on the positively charged BSCS surface through electrostatic interactions, ensuring the co - localization of nanoparticles and probiotics to the inflamed site.

[0072] 3. Breaking through the bottleneck of probiotic application

[0073] The oral survival rate of probiotics alone is low, while the chitosan and WO3@PDA coating strategies significantly improve the survival rate of Bacillus subtilis in simulated gastric juice. Combining with the role of WO3@PDA in selectively inhibiting Enterobacteriaceae, it creates an "ecological vacuum" for probiotic colonization and achieves long - term regulation of the microbiota.

[0074] In summary, through nano - engineering design, the present invention integrates three functional modules of antioxidant, antibacterial, and probiotic protection to achieve multi - target synergistic treatment of IBD, and the effect is verified to be significantly better than that of single components in animal models.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the present technical solution, and they should all be covered by the scope of the claims of the present invention.

Claims

1. Nanoengineered Bacillus subtilis, characterized in that: The nanoengineered Bacillus subtilis is composed of WO3 nanoparticles encapsulated by PDA and Bacillus subtilis encapsulated by chitosan.

2. The preparation method of the nano-engineered Bacillus subtilis according to claim 1, characterized in that, The steps are as follows: S1: Synthesize WO3 nanoparticles encapsulated by PDA The WO3 nanoparticles are dispersed in Tris buffer containing hydrochloric acid dopamine to prepare the WO3@PDA nanoparticles; S2: Synthesize Bacillus subtilis encapsulated by chitosan Chitosan is dissolved in acidic PBS, and the Bacillus subtilis bacterial solution is added and stirred. The pH value of the solution is adjusted to deposit chitosan on the surface of Bacillus subtilis to prepare the Bacillus subtilis encapsulated by chitosan; S3: Synthesize Bacillus subtilis bridging WO3@PDA The WO3@PDA prepared in step S1 and the Bacillus subtilis encapsulated by chitosan prepared in step S2 are mixed and stirred in acidic PBS, and the nanoengineered Bacillus subtilis BSCS@WO3@PDA is collected after centrifugation.

3. The preparation method of the nano-engineered Bacillus subtilis according to claim 2, characterized in that: In step S1, the concentration ratio of hydrochloric acid dopamine to WO3 nanoparticles is 1:2, and the pH of the Tris buffer is 8.

5.

4. The preparation method of the nano-engineered Bacillus subtilis according to claim 2, characterized in that: In the step S2, the concentration of the chitosan solution is 2 mg / mL, the concentration of the bacterial solution is (1 - 9) x 10 9 CFU / mL, and the pH value of the solution is 6.

5.

5. The preparation method of the nano-engineered Bacillus subtilis according to claim 2, wherein: In the step S3, the ratio of WO3@PDA to the chitosan-coated Bacillus subtilis is 1 mg: 1 x 10 9 CFU, and the pH of the PBS buffer is 6.

0.

6. Use of the nanoengineered Bacillus subtilis according to claim 1 in the preparation of a drug.

7. Use of the nanoengineered Bacillus subtilis according to claim 1 in the preparation of a drug for treating IBD.

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